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Cellular electrofusion utilizing corona fields and DC pulse technology

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Title:
Cellular electrofusion utilizing corona fields and DC pulse technology
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Language:
English
Creator:
Stein, Joshua
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University of South Florida
Place of Publication:
Tampa, Fla.
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Subjects

Subjects / Keywords:
Direct current
Cell-cell contact
Cell fusion
NT2 cells
Fusion chamber
1:1 fusion
Dissertations, Academic -- Biomedical Engineering -- Masters -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Cell fusion is an important technique that is used in the field of medicine and biomedical research. For instance, fusion can be used to create hybridomas and novel types of secretory hybrid cells. It may also be used to engineer cultured insulin-secreting pancreatic B-cell lines for the treatment of diabetes. Historically, the applications listed above have been accomplished by a number of methodologies including dielectrophoresis, centrifugation, polyethylene glycol (PEG) and viral fusion proteins. However, these approaches often fail to produce the desired results due to poor cell viability, lack of 1:1 fusion, and use of non-physiological environments. It is proposed that the application of an electrical field generated by corona charge (corona fields) and subsequent treatment with direct current (DC) pulse technology will overcome these deficiencies. Isolated and pre-labeled neuronally committed human teratocarcinoma (NT2) cells in monoculture or co-culture, were seeded in chambers, constructed in the laboratory, and allowed to adhere to the chamber bottom prior to corona treatment. A corona generator, also constructed in the laboratory, was used to expose cells to positive and negative electrical charges to induce cell-cell contact. The cells were then pulsed with DC voltage to induce fusion. During the experiments, cells were photographed sequentially to record cell movement/contact and fusion. The project was designed to identify optimal corona-based electrofusion parameters for viable, 1:1 cell fusion. Optimal results for cell-cell contact were obtained using a cell density of 2.35 times ten to the fourth power cells per microliter Dulbecco's Modified Eagle Medium (DMEM) in a grounded circular plate corona chamber following at least 3 minutes of settling time. Corona charges from (+) 6.1 kilivolt and (-) 5.5 kilivolt potentials were determined as being most favorable for cell movement and viability. Fusion was best achieved by first exposing either a circular or square ungrounded corona chamber configuration to 3 minutes (+) corona charge followed by 3 minutes (--) corona charge; disturbing the cells in the chamber with mechanical force; and then exposing them to 8-15 sequences of a 2,500 Volts per centimeter DC pulse at 100 microseconds.
Thesis:
Thesis (M.S.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
System Details:
System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Joshua Stein.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 76 pages.

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University of South Florida
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aleph - 001914445
oclc - 176630831
usfldc doi - E14-SFE0001980
usfldc handle - e14.1980
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SFS0026298:00001


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ABSTRACT: Cell fusion is an important technique that is used in the field of medicine and biomedical research. For instance, fusion can be used to create hybridomas and novel types of secretory hybrid cells. It may also be used to engineer cultured insulin-secreting pancreatic B-cell lines for the treatment of diabetes. Historically, the applications listed above have been accomplished by a number of methodologies including dielectrophoresis, centrifugation, polyethylene glycol (PEG) and viral fusion proteins. However, these approaches often fail to produce the desired results due to poor cell viability, lack of 1:1 fusion, and use of non-physiological environments. It is proposed that the application of an electrical field generated by corona charge (corona fields) and subsequent treatment with direct current (DC) pulse technology will overcome these deficiencies. Isolated and pre-labeled neuronally committed human teratocarcinoma (NT2) cells in monoculture or co-culture, were seeded in chambers, constructed in the laboratory, and allowed to adhere to the chamber bottom prior to corona treatment. A corona generator, also constructed in the laboratory, was used to expose cells to positive and negative electrical charges to induce cell-cell contact. The cells were then pulsed with DC voltage to induce fusion. During the experiments, cells were photographed sequentially to record cell movement/contact and fusion. The project was designed to identify optimal corona-based electrofusion parameters for viable, 1:1 cell fusion. Optimal results for cell-cell contact were obtained using a cell density of 2.35 times ten to the fourth power cells per microliter Dulbecco's Modified Eagle Medium (DMEM) in a grounded circular plate corona chamber following at least 3 minutes of settling time. Corona charges from (+) 6.1 kilivolt and (-) 5.5 kilivolt potentials were determined as being most favorable for cell movement and viability. Fusion was best achieved by first exposing either a circular or square ungrounded corona chamber configuration to 3 minutes (+) corona charge followed by 3 minutes (--) corona charge; disturbing the cells in the chamber with mechanical force; and then exposing them to 8-15 sequences of a 2,500 Volts per centimeter DC pulse at 100 microseconds.
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PAGE 1

Cellular Electrofusion Utilizing Coro na Fields and DC Pulse Technology by Joshua Stein A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biomedical Engineering Department of Chemical Engineering College of Engineering University of South Florida Co-Major Professor: Mark Jaroszeski, Ph.D. Co-Major Professor: Don Cameron, Ph.D. Richard Gilbert, Ph.D. Date of Approval: April 9, 2007 Keywords: direct current, cell-cell contact cell fusion, NT2 cells, fusion chamber, 1:1 fusion Copyright 2007, Joshua Stein

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DEDICATION To my parents, who have always stood behind me in everything I have done.

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ACKNOWLEDGEMENTS I would like to express my utmost apprec iation for all of the guidance, support, and practical inputs that both Dr. Mark Jaro szeski and Dr. Don Cameron have provided me over the course of my thesis project. I would also like to th ank Dr. Richard Gilbert for serving on my master’s thesis committee as well as offering additional information and knowledge. I am very grateful for all of the valuable advice and suggestions I had received from Niraj Ramachandran and my co lleagues in the Gene a nd Drug delivery lab. Finally, I would like to thank all of the pe ople who are close to me in my life for providing me with strength, encouragement, an d the drive to continue my research when the finish line seemed unattainable.

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i TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT vi CHAPTER 1: INTRODUCTION 1 1.1 Cell Fusion Applications 1 1.2 Methods of Cell Fusion 2 1.2.1 Viral Fusion Proteins 2 1.2.2 Polyethylene Glycol (PEG) 3 1.2.3 Centrifugation 3 1.2.4 Electrofusion 4 1.2.4.1 Dielectrophoresis 6 1.2.5 Other Cell Fusion Methods 7 CHAPTER 2: BACKGROUND AND MOTIVATION 9 2.1 Corona Charge 9 2.2 Concepts for the Generation of Corona 9 2.3 Corona Applications 13 2.4 Motivation for Using Corona Fields as a Means for Cell Contact 14 CHAPTER 3: RESEARCH GOALS 16 CHAPTER 4: MATERIALS AND METHODS 19 4.1 Cell Preparation 19 4.1.1 Cell Line and Culture Methods 19 4.1.2 Cell Counting 21 4.2 Cell Staining 22 4.2.1 Stock Solution of Dyes 22 4.2.2 Staining Technique 23 4.2.3 Fluorescent Microscopy 24 4.3 Media for Electrofusion 25

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ii 4.4 Corona Apparatus 25 4.4.1 Corona Generator 25 4.4.2 Corona Experimental Setup 27 4.4.3 Fusion Chambers Investigated with Corona 29 4.5 DC Cell Fusion Apparatus and Experimental Setup 32 CHAPTER 5: RESULTS AND DISCUSSION 34 5.1 Effect of Corona Charge on Suspended Cells vs. Non-Suspended Cells 34 5.2 Combined Negative and Positive Corona Treatment vs. Separate Treatment with Either Positive or Negative Charge 35 5.3 Order of Combined Negative and Positive Corona Treatment 36 5.4 Effect of Grounding Variable Electrodes Under Corona Treatment 40 5.5 Determination of Optimal Corona Treatment Duration 46 5.6 Fusion Experiments with Circular Corona Chamber Containing Electrodes 48 5.7 Results with Different Geometric Corona Chamber Configurations 53 5.7.1 Investigation of Corona Treatment 53 5.7.1.1 Square Chamber without Electrodes 53 5.7.1.2 Circular Chamber without Electrodes 55 5.7.1.3 Square Chamber with Square Electrodes 56 5.7.2 Fusion Analysis 60 5.7.2.1 Verification of Hybridized Cell Viability 66 CHAPTER 6: CONCLUSION AND RECOMMENDATIONS 67 6.1 Conclusion 67 6.2 Recommendations 68 REFERENCES 70 APPENDICES 75 Appendix A: Data for Calibration of the Corona Generator 76

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iii LIST OF TABLES Table 5.1 Determination of Optimal Fusion Parameters for Circular Corona Chamber with Circular Electrodes 50 Table 5.2 Determination of Optimal Fusion Parameters for Square Chamber 66 Table A.1 Data for Calibration of the Corona Generator 76

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iv LIST OF FIGURES Figure 1.1 Model of Sharp Electrod e Containing a Negative Corona Potential (a) and Plot of Distance from Sharp Electrode vs. Strength/Size of Electric Field (b) 12 Figure 1.2 Model of Sharp Electrod e Containing a Positive Corona Potential (a) a nd Plot of Distance from Sharp Electrode vs. Strength/ Size of Electric Field (b) 13 Figure 4.1 Human Neuronally Committed Teratocarcinoma Cell Line (NT2, 100X, Phase Contra st with a Green Filter) 19 Figure 4.2 Hemocytometer at 40X 22 Figure 4.3 Scope View of a Hemocytometer at 100X 22 Figure 4.4 NT2 Monohybrid Under Fluorescent Microscopy (400X) 24 Figure 4.5 Bottom View of Corona Generator 26 Figure 4.6 Side View of Corona Generator 27 Figure 4.7 Corona Experimental Setup 28 Figure 4.8 LabVIEW Computer Prog ram for Corona Generation 29 Figure 4.9 Circular Corona Chambe r with Circular Electrodes 30 Figure 4.10 Circular Corona Chamber without Electrodes 31 Figure 4.11 Square Corona Cham ber without Electrodes 31 Figure 4.12 Square Corona Chambe r with Square Electrodes 31 Figure 4.13 ECM 830 Electroporation DC Pulse Generator 33 Figure 4.14 ECM 800 Electro Cell Ma nipulation Instrument 33

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v Figure 5.1 Successive Corona Treatments of Positive Polarity Followed by Negative Polarity for Up to Six Minutes Each (40X) 37 Figure 5.2 Successive Corona Treatmen ts of Negative Polarity Followed by Positive Polarity for Up to Six Minutes Each (40X) 39 Figure 5.3 Corona Treatment of Circular Corona Chamber Configuration with On ly Outer Electrode Grounded (40X) 42 Figure 5.4 Corona Treatment of Circular Corona Chamber Configuration with On ly Inner Electrode Grounded (40X) 44 Figure 5.5 Corona Treatment of Circular Corona Chamber Configuration with No Elect rodes Grounded (40X) 45 Figure 5.6 Corona Treatment of Ci rcular Corona Chamber with Both Electrodes Grounded fo r Determination of Optimal Corona Treatment Duration (40X) 48 Figure 5.7 NT2 Cells Before and Af ter DC Treatment Using Optimal Corona Exposure Parameters in a Circular Corona Chamber Configuration (40X) 51 Figure 5.8 NT2 Cells Following DC Pulse (100X) 52 Figure 5.9 Effect of Mechanical Di sturbance on NT2 Cells After Corona Treatment in a Square Corona Chamber 54 Figure 5.10 Effect of Mechanical Di sturbance on NT2 Cells After Corona Treatment in a Circular Corona Chamber 56 Figure 5.11 Successful Cell Contact Using Optimal Corona Parameters in a Grounded Square Chamber 58 Figure 5.12 Resulting NT2 Monohybrids in a Square Corona Chamber After Successive Corona Treatment, Mechanical Disturbance, and 2500 DC Volts/cm 62 Figure 5.13 NT2 Monohybrids Once Transf erred to a Petri Dish for Validation of Fusion 64

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vi CELLULAR ELECTROFUSION UTILIZING CORONA FIELDS AND DC PULSE TECHNOLOGY Joshua Stein ABSTRACT Cell fusion is an important technique that is used in the field of medicine and biomedical research. For instance, fusion can be used to create hyb ridomas [1] and novel types of secretory hybrid cells. It may also be used to e ngineer cultured insulin-secreting pancreatic B-cell lines for the treatment of di abetes [2]. Historically, the applications listed above have been accomplished by a number of methodologies including dielectrophoresis, centrifugati on, polyethylene glycol (PEG ) and viral fusion proteins. However, these approaches often fail to produce the desired results due to poor cell viability, lack of 1:1 fusion, and use of non-phys iological environments. It is proposed that the application of an electrical field ge nerated by corona charge (corona fields) and subsequent treatment with direct current (DC) pulse technology will overcome these deficiencies. Isolated and pre-labeled neuronally committed human teratocarcinoma (NT2) cells in monoculture or co-culture, were seeded in chambers, constructed in the laboratory, and allowed to adhere to the cham ber bottom prior to corona treatment. A corona generator, also constr ucted in the laboratory, was used to expose cells to positive and negative electrical charge s to induce cell-cell contact. The cells were then pulsed

PAGE 10

vii with DC voltage to induce fusion. During the experiments, cells were photographed sequentially to record cell movement/contact and fusion. The project was designed to identif y optimal corona-based electrofusion parameters for viable, 1:1 cell fusion. Optimal results for cell-cell contact were obtained using a cell density of 410 35 2 cells/l Dulbecco’s Modified Eagle Medium (DMEM) in a grounded circular plate corona chamber fo llowing at least 3 minutes of settling time. Corona charges from (+) 6.1 kilivolt and (-) 5.5 kilivolt potentials were determined as being most favorable for cell movement and vi ability. Fusion was best achieved by first exposing either a circular or square ungr ounded corona chamber configuration to 3 minutes (+) corona charge followed by 3 minut es (–) corona charge; disturbing the cells in the chamber with mechanical force; and then exposing them to 8-15 sequences of a 2,500 Volts/cm DC pulse at 100 microseconds.

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1 CHAPTER 1: INTRODUCTION 1.1 Cell Fusion Applications Fusion between biological cells is an important procedure that is frequently used in the field of medicine and biomedical resear ch. The applications for cell fusion have grown significantly since its disc overy in the late 1970’s [3]. For instance, it has grown from generating somatic cell hybrids [4] a nd homokaryon production to the production of tumor cell/dendritic cellular hybrids for cancer immunotherapy [5]. In addition, fusion applications have also progressed from creat ing hybridomas and novel types of secretory hybrid cells to engineering cultured insulinsecreting pancreatic B-cell lines for the treatment of diabetes. In recent years, [6] fu sion techniques have b een utilized to create novel hybrids for the facilitation of drug delivery. Cell fusion has even been investigated to be used as a means for bioengineering nove l heterohybridized cell constructs for cell transplantation therapies [2]. Additionally, many of the ongoing clini cal studies initiated by companies such as Genzyme and Dendreon ar e using a form of fusion as their primary method for producing cell hybrids [7]. In es sence, it is quite easy to visualize how influential this methodology is in the field of medicine and how promising it can be for the future of biomedical research.

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21.2 Methods of Cell Fusion There are many different methods currently be ing utilized in scientific studies to bioengineer viable hybridized cellular construc ts. While a variety of these methods are currently being used (some more popular than others ), each respective method has its own deficiencies. This dilemma prompts the need to investigate a novel fusion technique that may overcome these deficiencies. 1.2.1 Viral Fusion Proteins The first few studies involving ce llular fusion were performed in vitro using either an inactivated virus or chemical fusogen. Inac tivated particles from the Sendai virus were used to induce nuclei and micr onuclei into a cell’s cytoplas m [3]. Unfortunately, many limitations existed which restricted the feasib ility of producing a pre-determined quantity of viable hybridized cells on a consistent basi s. The main limitations for using chemical and viral agents, such as the Sendai virus, as stated by Zimmermann [8 ] are listed below: The desired number of fused cells cannot be pre-selected. The process of cellular fusion between two different cell species cannot be viewed under a microscope. The cell viability is jeopardized by a loss of intracellular substances. There is a presence of exogenous reagents during the fusion process, which in some cases may be toxic to the cells. The optimal fusion conditions for a set of species have to be pre-determined empirically as they vary from species to species.

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31.2.2 Polyethylene Glycol (PEG) Apart from viral fusogens, chemicals su ch as polyethylene glycol (PEG), its derivatives and lysolecithin [9] have been us ed to promote cell hybridization, although at a low frequency [10]. A major drawback fo r using PEG based methods is that the PEG has been shown to be cytotoxic to cells and can subsequently generate cell debris which can be taken up by unhybridized cells rendering the identification of true hybrids difficult [11]. 1.2.3 Centrifugation Centrifugation is another method that ha s been used as a means for achieving tight intracellular contact as part of cellula r fusion procedures. One way of performing this has been to centrifuge the cells and then apply direct current (DC) pulses to induce reversible electrical membrane breakdown of the contacting surfaces [12]. The alternate way of performing this takes a dvantage of the relatively lo ng lived (minutes) fusogenic state that exists after the cells have been pulsed. So, cells are first pulsed and then centrifuged into contact with each other [12, 13]. These methods have matched the success of traditional methods (polyeth ylene glycol, viral fusion proteins, dielectrophoresis, etc.). One drawback, part icularly of the second centrifugation method, is that there is no control to insure that th e cells contact each other in the polar regions that have induced membrane defects. Anot her drawback of using centrifugation as a means for cellular contact is that there is al so no control for achie ving a desired fusion ratio, because there is no way of predic ting how many cells will fuse with their counterparts.

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41.2.4 Electrofusion The methodology involving the electrical treatment of living cells and nonliving membrane vesicles in a manner that w ill induce fusion (electrofusion) has been investigated for quite some time. The ear liest published observation of this phenomenon was in 1983 [14]. The experiment was conducte d by Bouchard and Teissie. It involved growing hamster ovary cells in monolayers on a Petri dish followed by exposure to square wave electric pulses to induce th e formation of a large amount of fused mammalian cells. As demonstrated in Boucha rd’s study as well as later studies [15], electrofusion was effective in producing a higher yield of viable hyb rid cells than the other methodologies discussed above. Unfo rtunately, during the earlier years, the mechanism of electrofusion was not comp letely understood. However, as time progressed, a number of scientis ts helped to contribute addi tional knowledge to this novel methodology which opened the door for further op timization. For instance, a couple of investigations conducted by Zi mmermann revealed that elec trofusion in strongly hypoosmolar or isotonic solutions could enhance hybridoma production [16, 17]. In addition, Teissie was able to demonstrate that electrofu sion is a two step process consisting of the creation of cell contact and subsequent re versible electropermeabilization [18]. Furthermore, a scientist from Rockville, MD (Sowers) was not only able to provide convincing evidence that elec trofusion yield is partially controlled by biologically relevant membrane factors [19], but that fu sion of dissimilar membra ne partners depends on additive contributions from each of the two different membranes [20]. There have been many practical applications over the years that have established the use of electrofusion since the first observa tion. For instance, electrofusion has been

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5 used for the production of monoclonal antibodie s [21], transfer of membrane components [22-24], and the production of hybrid cells [25-27]. As discussed above, electrofusion was disc overed to be a two step process. The first step is the creation of tight intracellula r contact between the cells. The second step is the application of very brief but intense DC pulses to the cells resulting in a temporary membrane destabilization of the contacting surf aces. During this destabilization period, molecules that ordinarily would not be able to enter the cell’s membrane can gain access to the cytosol for a time that is on the orde r of minutes after electr ical treatment ceases [28]. In addition, if the cells remain in c ontact during their destab ilized/fusogenic state, membrane fusion can occur. Once tight cellu lar contact has been obtained, reversible permeabilization (or fusion) can be achieved by delivering 6-15 fusogenic DC pulses to the cells by way of electrodes. The field strength ranges from 900 V/cm 3000 V/cm and the interval between pulses can range anywhere from 10 – 100s depending on the type of cells being fused. The me thod of delivering pulses to the cells in order to induce cellcell hybridization is similar in all of the known fusion met hodologies regardless of the application. However, the method of achievi ng cell-cell contact prior to electrofusion is where the methods differ. Unfortunately, forcin g cells into contact w ith each other is the most challenging aspect of elec trofusion due to a variety of limitations which conversely leaves room for improvements.

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61.2.4.1 Dielectrophoresis Dielectrophoresis is curren tly one of the most popular methods for obtaining close intracellular contact prior to fusion. Dielec trophoresis involves the directional movement of cells (neutral particles) towards the region of highest fi eld intensity in a non-uniform electric field [29]. The fiel d that is created during the pr ocess of dielectrophoresis is generated by a source of alte rnating current (AC). Cells normally do not attract one another due to their net negativ e outer surface charge. However, they become dipoles in the AC field and are forced into contact with each other due to a ne t force resulting from the non-uniform AC field. Th e cells undergo translational movement towards the region of highest field intensity when exposed to the non-uniform fi eld. In addition, the cells move into close proximity of each other in thei r polarized state, causing them to attract to each other (due to an enhancement of the local field divergence and increase in field strength near the cell). The localized increas e in field strength ne ar each cell is strong enough to overcome the weaker electrostatic repulsions generated from the outer cell membranes, thereby resulting in the form ation of pearl chai ns of cells [30]. One of the limitations of dielectrophores is as a means for achieving cellular contact is that the cells need to be placed in a non-conducting medium. The presence of electrolytes in conductive me dia leads to Joule heating, turbulence and subsequent disruption of the pearl chains. This hinders the cell alignmen t process [31]. On the other hand, using a non-physiologic medium coul d jeopardize the cellu lar integrity and viability. Additionally, anothe r limitation with using the as sociated technology is that only a small number of cells can be treated.

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71.2.5 Other Cell Fusion Methods There have been some other methods deve loped in an attempt to further optimize cell-cell contact for electrofusion. The te chnique of producing hybr idomas by using laser radiation was investigated by Onkohci N. and Itagaki H. [32, 33]. In this study, the investigators were able to focus pulse lase r beams on the contacting surfaces of select target cells and cut small perforations for mutual communication between the cytoplasm. Although this method proved to be very eff ective for producing hybridomas from a small number of cells, including fragile cells, the technology di d not show the ability to produce a large number of hybrids at one time Another fusion technique involving the cultivation of a monolayer of anchorage depe ndent cells was investigated by Finaz [34] and Teissie [35]. The main problem associated with this technology is that it requires anchorage dependent cells. It is evident from the cell-cell fusion methods described above that there is significant room for technological improvement Many of the techniques listed above have proven to be reasonably efficient in generating hybrids; however, each respective technology has its own limiting factors. Additiona lly, even if a technique is said to be satisfactory for generating hybrids from a specific set of cells, it does not equate to that technique being suitable for ot her cell types. Furthermore, some methods provide very positive results for generating viable hybridize d cells on a consistent basis, but the associated technology may not have the ability to either produce viable hybridomas in mass quantity at one time or a small select target of viable hybrids. Exploring the prospect of us ing electrical charges to induce cellular contact would be a good starting point towards finding a more generalized protocol for efficient cell-cell

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8 contact and subsequent electrofusion. Theref ore, the use of corona charge (a type of electrical charge) as a means for achieving ce llular contact prior to DC mediated fusion should be investigated. Successful developmen t of this technology would most likely be translated to produce select qua ntities of hybrids in a contro lled environmen t irrespective of the cells adherent properties.

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9 CHAPTER 2: BACKGROUND AND MOTIVATION 2.1 Corona Charge Corona charge is a self-sustained current that is generated by st rong electric fields that are associated with a highly curved elec trode containing a high potential gradient. Corona charge may be positive or negative, depending upon the voltage applied to the highly curved electrode [36]. Corona di scharge usually involves two asymmetric electrodes; a highly curved electrode (such as the tip of a needle, or a small diameter wire) and an electrode of low curvature (such as a plate, or the ground) [36]. During the process of corona discharge, a current is produced between the two asymmetric electrodes in a neutral fluid (u sually air). This current is produced by ionizing the fluid to form plasma around the highly curved el ectrode [36]. The ions created from the plasma formation event help to close the circuit by carrying the charge to the other electrode. 2.2 Concepts for the Generation of Corona When an object with a sharp tip become s charged it has a very high potential gradient nearby. As a result, the neutral flui d (air in this case) around that sharp tip has a much higher gradient than elsewhere. If a su fficiently high voltage is applied to the sharp object, the potential gradient may become larg e enough at a point in the fluid so as to

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10 ionize the fluid, thereby cr eating plasma around the sharp ti p [36]. From general physics [37], plasma is nothing more than a gas in its ionized state. When the fluid becomes ionized, the electrons are stri pped from the atoms (leaving each atom with a net positive charge) and the ions are free to move about. The free electric charges that are present make the plasma electrically conductive so th at it responds strongly to electromagnetic fields. The electrons that are freely moving throughout the plasma will continually collide with neutral atoms located outside the plasma to initiate further electron dissociations. These newly ionized atoms will th en help to seed further events such as this. This process is known as an electron avalanche. Both positive and negative corona discharges rely heavily on electron avalanches [36]. Ion species that are created from an electron avalanche will naturally attract them selves to the low curved electrode (ground, where they are neutralized), thus completing the circuit and sustaining the current flow. If conditions of the geometry and gradient are such that th e ionized region continues to grow instead of gradually coming to a halt at a certain distance, a completely conductive path may be formed. The result is a mome ntary spark or continuous arc. This event essentially follows the same mechanism presen ted during a lightning st rike. As with the case of a lightning strike, the general notion is that a spark or continuous arc is simply a flow of current from negative to positive [38]. In the case of corona discharge, it is the ion species that are created from the electr on avalanche that carry the current to the ground. If sufficient voltage is passed through a highly cu rved electrode containing a negative corona potenti al, the free flowing electrons an d secondary elec trons in free space will accelerate more quick ly towards the opposite low curved electrode/ground due to the increase of the repulsive force, resulting in a completely conductive path or spark.

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11 In the case of a highly curved electrode containing a positive corona potential, the ionization region will greatly increase when a high enough voltage is passed through the highly curved electrode, resul ting in the immediate cascade of an electron avalanche and the formation of a completely conductive path. This process is more gradual and occurs at a higher potential than the process involving a highly curved electrode with a negative potential. This is due to the simple fact th at the electrons are closer to the low curved electrode in the negative highly curved elec trode process than they are with the positive highly curved electrode proce ss and would thus require less voltage to reach the ground. As mentioned in the preceding section, there are two different types of corona charge: positive and negative. The type of corona charge is dependent upon the polarity of the highly curved electrode. If the highly cu rved electrode is positive with respect to the flat electrode, then there is a positive coro na potential. If the highly curved electrode is negative with respect to the flat electrode then there is a negative corona potential. The physics between positive and negative corona s differ. This is a result of the great difference in mass between electrons and positively charged ions, as well as the verity that only the electrons have the ability to undergo a certain degree of ionizing inelastic collisions. When using a pointed electrode with a negative potential during corona discharge, the strong electric field located ne ar the highly curved/pointed electrode will generate a force that will act upon the free el ectrons pushing them away from the sharp electrode [39]. These free electrons contribute to the ionization process by facilitating the dissociation of more electr ons (secondary electrons), th ereby creating an electron avalanche in the direction away from the sh arp electrode. Since the electric field decreases as a function of distance from the poi nted electrode, the electrons that continue

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12 to move away from it will eventually arrive in a region that lacks the necessary energy for ionization [39]. As a result, the free elect rons will drift slowly in space creating a negative space charge, and will easily attach to neutral oxygen molecules. The lack of electrons near the sharp electrode (or presence of the negative space charge), due to the presence of the repulsive force, drastically reduces the ionization region (plasma region) and the ionization process eventually stops. The ionization process restarts when the negative ions reach the positive electrode [39]. As a result, negative corona is observed as bursts of ionization. Figure 1.1 depict s the motion of the electrons (a) and the reduction of the ionization region (b) when us ing a sharp edge with a negative corona potential. Figure 1.1 Model of Sharp Elec trode Containing a Negative Co rona Potential (a) and Plot of Distance from Sharp Electrode vs. St rength/Size of Electric Field (b) [39] When the pointed electrode switches polar ity to a positive corona potential, the free electrons will accelerate towards the point an d cause further ionization. The result is a positive space charge. In addition, the flux of electrons towards the high potential electrode drastically increases the ionization region, extending it all the way to the other electrode. Thus, in contrast to the sharp edge at negative potential, the ionization is enhanced by the space charge, and not decreased [39]. It is important to note that even

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13 though the ionization region is increased, ther e are far fewer free electrons in free space with a positive corona than there are with th e negative corona configuration. However, since the electrons in the positive corona case are heavily concentrated close to the surface of the curved electrode (in a region of high-potential gradient), the electrons have a very high energy. In contrast, for the nega tive corona case, many of the electrons are located in the outer, lower field areas. As a result, a hissing sound is typically associated with the positive corona. In addition, the pos itive corona will emit a bluish/white color due to the generation of secondary ions desc ribed above. Figure 1.2 below illustrates the motion of the electrons (a) and the extension of the ionization regi on when using a sharp edge with a positive corona potential. Figure 1.2 Model of Sharp Electr ode Containing a Positive Co rona Potential (a) and Plot of Distance from Sharp Electrode vs. St rength/Size of Electric Field (b) [39] 2.3 Corona Applications Corona discharges are currently used in a wide variety of comm ercial and industrial applications. They are commonly used to gene rate charged surfaces for the application of electrostatic copying or photocopying [36]. Th ey have been known to be used as air ionizers for possible health be nefits [36]. Corona discha rges are also used for high

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14 voltage contact print photography called Kirlia n photography [36]. Ot her applications of corona discharges are scrubbing particles from air in air-conditioning systems [36]. They accomplish this by removing the particulate matter from the air stream and then passing the charged stream through a comb of altern ating polarity, to deposit the charged particles onto oppositely charged plates [36]. In addi tion, the free radicals and ions generated from corona reactions can be used to scr ub the air of certain noxious products [36]. Corona discharge can also be utilized for th e manufacturing of ozone [36]. A few studies have examined using corona for immunothe rapy and biomedical research. One such study by Kwark and Lee involved a real-time corona discharge imaging system as a future biomedical imaging device [40]. 2.4 Motivation for Using Corona Fiel ds as a Means for Cell Contact There were five key determining factors for investigating the use of corona discharge as the methodology for obtaining tight intracellu lar contact prior to electrofusion. One such factor was the low current (A) due to the ions and elect rons. With this range of current, the discharge could be applied without compromising cell viability and integrity. Furthermore, the use of corona discharge as a means for achieving cell-cell contact does not directly affect the choice of electrofusi on medium. As discussed in the previous chapter, some of the more traditional cel l contact methods required the use of a nonphysiologically balanced medium. Thus, using corona charge would help rid the concern involved with this discrepancy. In addition, the ability to view the electrofusion process under a microscope and the ability to move the cells in monolayers helped to provide further incentive towards using this methodol ogy. Moving the cells in monolayers would

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15 enable us to control the desired quantity of intracellular contact for the approximation of a 1:1 fusion event. Lastly, the possibility of having the technological flexibility to either create hybrids in great quantities or to target a small amount of cells for hybridization is a tremendous enticement for using corona discha rge as a method for achieving cell contact.

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16 CHAPTER 3: RESEARCH GOALS Over the past 10 years, there has been a gr eat increase in the growth of research surrounding cell transplantati on therapy as a means for al leviating the devastating symptoms for diseases including stroke, Alzheimer’s disease, sp inal cord injury, cirrhosis of the liver, factor 8 hemophilia, Type I diabetes, and Parkinson’s disease. Parkinson’s disease remains one of the fo remost health issues world-wide, and although many advances have been made to treat the symptoms of this devastating disease, little has been accomplished in actu ally curing the disease. In recent times, the transplantation of isolated NT2 cells (dopamine producing neuronally committed teratocarcinoma cell line) into the Parkinsonian host has shown a 100% success rate in Parkinsonian patients that received NT2 allogr afts [41]. As promising as this is, major problems persist. To allow for prolonged NT2 engraftment, there is a need to continue the chronic use of immunosuppr essive medications introduci ng significant side effects that range from sustained disc omfort to devastating life threatening infections. Recently, a number of studies have investigat ed the idea of using Sertoli cells in allograft cell transplantation protocols in order to achieve prolonged NT2 engraftment in the absence of systemic immune suppression. Sertoli cells are terminally differentiated cells found in mammalian testes that provide a dynamic trophic factor-rich microenvironment for developing spermatids in a sequestered testicular compartment

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17 devoid of blood and lymphatic vascul ature. They also play an esse ntial role in preventing the individuals from rejecting th eir own highly antigenic mature germ cells [42]. It is this localized immunoprotection (provided by Sert oli cells) that has prompted such investigations. One such study was examined by Willing and Cameron [43], where they were able to show that isolated Sertoli cells (iSCs) can create a testis-like immune privileged site outside of the testis a nd that alloand xenogenic neurons can be transplanted there (by co-transplantati on with iSCs) without requiring systemic immunosuppression of the host rat. They al so concluded that iS Cs induce localized immunoprotection. It is, however, unclear how iSCs immunoprotect cell and tissue grafts, although a number of theories have be en offered [42, 44, 45]. In general, the ability of iSCs to cause a significant reduction or even elimination of alloor xenograft rejection appears to be related to their close proximity to the co-transplanted cells and tissues. This localized effect, while in close juxtaposition, suggests that there is a need for the Sertoli cells to maintain close contact with the co-transplanted cell or tissue graft, possibly by fusion. In order to obtain the desired cell therapy for Parkinson’s, the Sertoli cells would have to be fused with the NT2 cells in a way so as to not affect their viability, integrity or functional ity. This idea suggests the inves tigation of a prot ocol that would produce the heterohybrids on a 1: 1 basis in mass quantity, w ith the hope that we would be able to isolate and subculture the hybridized cell with the desired genetic characteristics (local immune protec tion and secretion of dopamine). This investigation is a step towards finding a novel cell contact method that will grant us the greatest opportunity to wards obtaining 1:1 hybridizati on in great quantity without the deficiencies experienced when using traditi onal methods. As discussed in the

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18 previous chapter, the use of electrical char ge produced by corona discharge has many advantages over some of the other more traditi onal cell contact methods In an attempt to exploit some of these potential advantages this study was designed with the following specific aims: To determine whether corona discharge can be used as a method for achieving tight intracellul ar contact. To provide evidence of ab ility to control cellular movement of the cells in monolayers while suspended in a ph ysiologically balanced medium. To determine optimal corona-based pa rameters for approximation of a 1:1 hybridization event. To provide evidence of ab ility to generate large qua ntities of hybridized cells and attempt to use optimal corona-based parameters to generate 1:1 fusion. To determine and confirm that corona does not compromise the hybrid cell viability, integrity, and functionality. To finalize a fusion chamber desi gn and electrofusion method that will incorporate the use of both cell-cell c ontact and cell electrofusion to produce high hybrid yields.

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19 CHAPTER 4: MATERIALS AND METHODS 4.1 Cell Preparations 4.1.1 Cell Line and Culture Methods Neuronally committed dopaminergic human teratocarcinoma cells (NTera-2 cl.D, or NT2; ATCC #CRL-1973: American Type Culture Collection, Rockville, MD) were used throughout the experimental work perf ormed in this study (Figure 4.1). The cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM) (Cellgro Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (FBS) (Cellgro Mediatech, Inc.) and 0.05 mg/ml of gentamicin (C ellgro Mediatech, Inc.). The cells were cultured under aseptic conditions in 21 cm2 polystyrene petri dishes (Corning Incorporated, Corning, NY) and were incubated in 5% CO2 at 37C (CO2 Water Jacketed Incubator, Forma Scientific, Inc., OH). Figure 4.1 Human Neuronally Committed Teratocarcinoma Cell Line (NT2, 100X, Phase Contrast with a Green Filter)

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20 NT2 cells were grown as adherent monolayers and required medium renewal and/or sub-culturing every 3-4 days. NT2 cells are non-ter minally differentiated cells that require sub-culturing at about 90% confluence to insure the recovery of a large quantity of cells and to prevent contact inhibi tion from occurring. Before sub-culturing, cell monolayers were washed three times w ith DMEM supplemented with 0.05 mg/ml of gentamicin. Cells were detached from the dish using a non-enzymatic cell dissolution solution (Cell Stripper; Cellgro Mediatech, Inc. ). In order to further facilitate cell detachment, the cells were placed in the inc ubator at 37C for approxi mately 2-3 minutes. The Cell Stripper solution was neutralized with supplemented DMEM (containing 10% FBS and 0.05 mg/ml gentamicin) following in cubation and prior to aspirating the suspended cells. Once the cells were recove red from the dish, they were centrifuged for 5 minutes at g220and 20C in a 50 ml conical tube Following centrifugation, the supernatant was aspirated, and the cells were re-suspended in DMEM solution supplemented with 0.05 mg/ml gentamicin. The sequence of cen trifugation followed by re-suspension was repeated twice to wash the cells. The NT2 cells were then subcultured with a ratio of either 1:4 or 1:5 depending upon whether the cells were to be subcultured 3 or 4 days later again.

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214.1.2 Cell Counting Harvested cells were prepared fo r counting by washing with Dulbecco’s Phosphate-Buffered Saline (DPBS 1X w/o Ca and Mg; Cellgro Mediatech, Inc.) three times. Cells were centrifuged (58 10R, Eppendorf, Westbury, NY) at g220 for 5 minutes at 20C and suspended in approximately 5 ml of DPBS for each wash. A sample of the cells was then diluted in 0.9% sodi um chloride (APP, Schaumburg, IL) and 0.4% trypan blue stain (Cellgro Medi atech, Inc.). Trypan blue penetrates the membranes of the dead cells and causes them to turn blue. A hemacytometer (Hausser Scientific, Horsham, PA) was used to count viable and non-viable cells at 100x using light microscopy. The concentration of the cells was determined using the following formula: 000 10 # #2 D mm Cells mL Cells Where D = dilution (if used) and 10,000 = conversion factor for 0.1 l to 1 ml The percent viability of the cells was also determined after counting. Only those cell cultures that were 85% 100% viable were used for experimentation. Figure 4.2 and 4.3 show the hemacytometer used for coun ting, and a microscopic view of a 1 mm2 square (counting space) on th e hemacytometer respectively.

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22 Figure 4.2 Hemacytometer [46] Figure 4.3 Scope View of a at 40X Hemacytometer at 100X[46] 4.2 Cell Staining In some experiments the cells were stai ned to assist with visual distinction between fused cells and non-fused ce lls under fluorescent microscopy. 4.2.1 Stock Solution of Dyes Stock solutions of fluorescent dyes were prepared in advance using the procedure discussed by Jaroszeski [47, 48]. The fluorescent dyes used fo r this study were 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetram ethylrhodamine (CMTMR (red fluorescent dye); Molecular Probes, Eugene, OR) and 5chloromethylfluorescein diacetate (CMFDA (green fluorescent dye); Molecular Probe s). Both dyes were supplied by the

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23 manufacturer in 1 mg aliquots. Stock solutio ns of 5 mM concentra tion of both dyes were prepared in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO). CMTMR (MrW 554) stock solution was made by mixing the supplie d 1 mg aliquot of CMTMR with DMSO to yield a final volume of 360 l. Correspondingly CMFDA (MrW 465) stock solution was made by mixing the supplied 1 mg aliquot of CMFDA with DMSO to yield a final volume of 430 l. Both dyes were easily dissolved in DMSO at room temperature. The DMSO stock solutions were divided into si ngle-use aliquots (usually 3 aliquots for CMTMR and 5 or 6 aliquots for CMFDA) and stored at -20C, protected from light. This division into single-use aliquots avoided freeze-thaw cycles of the stock solutions in order to increase shelf life a nd ensure consistent results. 4.2.2 Staining Technique For all experiments that used stained ce lls, NT2 cells were harvested from two 21 cm2 polystyrene cell petri dishes. These cultu res were 3-4 days old and the cells had reached the desired confluence (90%). One aliquot of each CMTMR (75 l) and CMFDA (50 l) were removed from storage and defrosted to room temperature. All staining was performed under aseptic conditions in a biological safety cabinet. The growth media in both petri dishes was re duced to 6 ml (just enough to cover the monolayer of cells adhered to the petri dishes). On e dish was stained with 75 l of CMTMR while the other dish was stained with 50 l of CMFDA. The cultures were then incubated at 37C for two hours. After incu bation, the cells were then harvested and counted by using a hemacytometer.

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244.2.3 Fluorescent Microscopy A fluorescent microscope (Leica DM IL Leica, West Germany) was used to visualize the contact between CMTMR and CMF DA stained cells, as well as the resulting dual fluorescing (fused) cells following direct current (DC) application. Under fluorescent light ther e was a clear visual distin ction between the fused and un-fused cells. The un-fused CMTMR stained cells appeared red, the un-fused CMFDA stained cells appeared green and the fusion pr oducts of the two were easily identified due to their yellowish/orange color and irregu lar shape (larger in size and non-spherical circumference). Figure 4.4, below, show s the resulting NT2 monohybrid (white arrow) when two NT2 cells stained with both CM TMR and CMFDA fuse. The result is a yellowish/orange, irregularly shaped cell. Figure 4.4 NT2 Monohybrid Under Fluorescent Microscopy (400X)

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254.3 Media for Electrofusion The media in which cell-cell electr ofusion was conducted in was DMEM (Cellgro, Mediatech, Inc.) s upplemented with 0.05 mg/ml gentamicin. The media was not supplemented with fetal bovine serum. To elaborate, the presence of serum would act as a blockade by preventing the cell memb ranes from contacting each other. The harvested cells (either stained or unstained ) were counted and the DMEM supplemented with 0.05 mg/ml gentamicin was then adde d to the cell solution to adjust the concentration of cells as per the requirement of the experiments. 4.4 Corona Apparatus 4.4.1 Corona Generator The corona generator (Figure 4.5 and 4.6 below) consisted of a corona generating element that emitted ions from a 25 mm diam eter hole in a stainless steel ground plate. The wire plate geometry of the corona genera ting element consists of 9 needles (stainless steel acupuncture needles, gauge no. 30, SGAM AC, China) that were contained within a circular white teflon disk. The teflon disk was placed within a central hole of a larger white cylindrical teflon body. Eight of the needles were arranged in a circle of 9 mm diameter with the ninth needle located in the center. The height of the needles was adjusted to a height of 6.8 mm from the base of the central hole in the cylindrical teflon body. A circular ground plate was attached to the base of the cylindrical white teflon body, which was mounted on a micromanipulat or. The micromanipulator enabled the corona generator to be lowered to a convenien t distance of 8.0 – 9.0 mm from the cells in

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26 preparation for corona exposure. It could also be raised in preparation for exposure of a new set of cells. In essence, the micromani pulator could be adjusted to move anywhere in 3-dimensional space. This was advantage ous for the purpose of being able to simply position the corona element over a dish of cells located under a microscope in order to observe the cells during corona exposure. Th e entire set of corona generating needles in this element had a common connection to the voltage output of a high voltage DC power supply. Figure 4.5 Bottom View of Corona Generator

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27 Figure 4.6 Side View of Corona Generator 4.4.2 Corona Experimental Setup In order to investigate the effect of cor ona application on cell-cell contact, various pieces of equipment were used to not only generate the corona discharge but view the resulting cellular movement and hybridization. In order to mani pulate and control the intensity level of the discharge, computer so ftware was installed and utilized to help manage the equipment used in the experiment. A corona generator was connected to a high voltage power supply (CZE 2000, Spellman High Voltage Electronics, Hauppauge, NY) in order to generate the corona discharge. The generator had both positive an d negative leads. The positive electrical wire connected the nine acupunc ture needles of the generato r to the high voltage supply, whereas the negative electrical wire connected the ground plat e (located on the bottom of

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28 the white teflon body) to the ground. Add itionally, the generator was attached to a micromanipulator so that it could be suspended above the chamber containing the cells. The chamber was placed on the microscope stage so that cell movement could be observed during corona application. Furthermore, the microscope had capability to simultaneously examine both red and green fluorescence which made it possible to view the formation of hybrid cells. The input voltage, current and duration for corona treatment was keyed into a program that was written in LabVIEW so ftware (LabVIEW 7, National Instruments, TX) on a computer (Dell Dimension 2400, Dell Inc. TX) that controlled the entire system. The computer and software used a data acquisition card (DAC) (PCI 6036 E, National Instruments, Aus tin, TX) to control th e power supply using the user input parameters. Figure 4.7 shows a diagram of the entire instrument system. Figure 4.8 illustrates the LabVIEW computer software for corona generation as seen on the computer monitor. Figure 4.7 Corona Experimental Setup Corona Generator S p ellman Power Su pp l y SCB 68 Pin Accessory Computer with DAC

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29 Figure 4.8 LabVIEW Computer Pr ogram for Corona Generation A reversible polarity switch was installed in the electrical line between the data acquisition card and the power supply to enable the user to switch from positive to negative corona (or vice versa) The polarity was manipulated from a signal sent through the DAC. In order to measure the temperat ure and humidity of the area surrounding the chamber during corona treatment, separate hum idity and temperature probes were used. The probes were placed next to the chamber during the experi ment to measure the effect of these two parameters on the corona generation process. 4.4.3 Fusion Chambers Investigated with Corona Four separate fusion chambers were i nvestigated for use with the corona generator. The first chamber (Figure 4.9) cons isted of a circular outer stainless steel wire and an inner circular stainless steel plate having a thic kness of 3 mm. The central plate was connected to an electrical wire from th e bottom of the chamber. Both the outer stainless steel electrode and inner stainless st eel electrode could be connected to a ground source during corona treatment and to th e DC electroporator during electrofusion.

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30 Figure 4.9 Circular Corona Cham ber with Circular Electrodes After observing the cell contact and fu sion properties when applying corona discharge and subsequent DC pulses us ing this chamber, two more chamber configurations were investigat ed in order to better optimiz e the fusion properties. The next two fusion chambers that we examined were a circular corona chamber without electrodes (Figure 4.10) and a square corona chamber without electr odes (Figure 4.11). The circular corona chamber without electrodes matched the physical characteristics of the first fusion chamber investigated (See Figure 4.9), but this new chamber lacked the circular inner stainless steel electrode and the outer stainless steel el ectrode. The square corona chamber consisted of ei ght plastic square chambers m ounted onto a plastic slide. Only four of the eight plastic wells as shown in Figure 4.11 were used during the investigation. The multiple chambers allowe d for multiple fusion trials to be performed at the same time for comparison.

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31 Figure 4.10 Circular Corona Chamber Figure 4.11 Square Corona Chamber without without Electrodes Electrodes A final chamber (Figure 4.12) was investigated in order to provide additional control on the intracellular contact during corona treatment. This chamber was identical to the square corona chamber stated above with one modification; two square stainless steel square plates were fitted against two of the walls of the chamber. Both square plates/electrodes could be connected to a ground source during corona treatment and then to the DC electroporator during electrofusion. Figure 4.12 Square Corona Cham ber with Square Electrodes

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324.5 DC Cell Fusion Apparatus and Experimental Setup For experiments involving the application of fusogenic pulses, an electroporation DC pulse generator (Figure 4.13) (ECM 830 BTX Molecular Delivery Systems, Harvard, MA) was used to obtain NT2 m onohybridization. In this setup the positive and negative wires were used to connect the DC generator to the electrodes of a fusion chamber for the transfer of direct current to the cells. In other experiments involving the use of fusion chambers without electrodes, an ECM 800 pu lse generator (Figure 4.14) was used to obtain NT2 monohybridization. In these cases, two stainless steel electrodes mounted on the end of a handle and connect ed to the ECM 800 were manua lly placed into the corona chamber for the transfer of direct current to the cell suspension.

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33 Figure 4.13 ECM 830 Electropora tion DC Pulse Generator Figure 4.14 ECM 800 Electro Ce ll Manipulation Instrument A) ECM (Electro Cell Manipulation) 800 Generator B) ECM (Electro Cell Manipulation) 800 Generator with Manual DC Pulse Generator (MPG) Connected C ) Manual DC Pulse Generator

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34 CHAPTER 5: RESULTS AND DISCUSSION 5.1 Effect of Corona Charge on Sus pended Cells vs. Non-Suspended Cells In order to conduct a complete investiga tion for utilizing and optimizing corona discharge as a means for achieving intracellula r contact, it was necessary to first gain insight as to the type of forces, in terms of magnitude and location in free space, that are present during corona treatment. Observation of cellular behavior exhibited under direct corona exposure, when in free suspension or affixed to the chamber floor, would help to provide valuable knowledge on the mechanisms ac ting in the suspension (not just at the surface). By gaining this insight, the probab ility of predicting th e cell movement and thus optimizing the system for cell-cell contact would greatly increase. NT2 cells were placed into a circular corona chamber containing circular electrodes (See Figure 4.9) at a concentration of 610 2 cells/175 l. After introducing the cell suspension into the chamber, the cells were allowed to settle under the influence of gravity for a brief period (1-2 min.) to insu re that a portion of the cells (not all) in suspension had settled to the chamber floor, affixed themselves, and were no longer in free suspension. In order to visually deci pher between the suspended cells and the nonsuspended cells, the chamber was mechanical ly perturbed following the settling time. The cells in suspension would move in response to the disturbance, while the nonsuspended cells would not move. The outer an d inner electrodes were both connected to

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35 a ground source during this experiment. The ce lls were then exposed to 5 minutes of positive corona followed by 5 minutes of negative corona. The cells had to be inspected using a microscope during corona treatment in order to analyze the a ffinity or divergence in cellular behavior exhi bited between the suspende d and non-suspended cells. Observations indicated that there was in fact a diverging effect for corona discharge on suspended vs. non-suspended cells The suspended cells moved rapidly on the surface towards the inner circular elec trode, while the non-suspended cells rolled slowly along the chamber floor in the same direction. This observation simply suggests that the electric field and the charges associ ated with it increase from the chamber floor to the suspension surface. As a result, it might be more beneficial, for the purposes of optimizing this methodology for cell-cell contac t, to continue forward with the corona treatment of non-suspended cells being that it was more gradual and therefore easier to control. 5.2 Combined Negative and Positive Corona Treatment vs. Separate Treatment with Either Positive or Negative Charge To obtain further knowledge of the effect of corona treatment on NT2 cells, a set of preliminary experiments were arranged to observe cellular movement resulting from various corona of both polarities. NT2 cells were delivered into the circul ar corona chamber containing circular electrodes at a concentration of 610 2 cells/175 l and allowed 3 minutes to settle as described above. At this time, approximately 95% of the cells had gravitated to the chamber floor. Both electrodes were connected to a ground source via electrical wires.

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36 Three separate experiments were perfor med that used either positive corona, negative corona, or a combination treatment of positive followed by negative corona. In each test, cell movement and behavior were ob served for the desire d characteristics of achieving tight intracellular c ontact in the annular space between the two circular electrodes. These tests were repeated 3 tim es each for reproducibility. In addition, all three trials were executed with th e same concentration of cells (610 2 cells/175 l), at the same voltage (6.1 kilivolts for positive pol arity, 5.5 kilivolts for negative polarity), and for the same amount of time (10 minutes). In the first test, the NT2 cells were treated with 10 minutes of positive cor ona polarity at 6.1 kilivolts (kV), in the second test the NT2 cells were treated with 10 minutes of ne gative corona polarity at 5.5 kV, and in the third test the NT2 cells were treated with 5 minutes of positive corona at 6.1 kV followed by 5 minutes of negative corona at 5.5 kV. The results showed that the combination treatment was the most successful. 5.3 Order of Combined Negative and Positive Corona Treatment The order of the polarity (positive then nega tive or negative then positive) applied to the cells during the combination corona tr eatment was analyzed using the same corona chamber as in section 5.2 above. The NT2 cel ls were loaded into the corona chamber while suspended in DMEM, supplemented with 0.05 mg/ml gentamicin, at a concentration of 610 2 cells/175 l. The cells were then al lowed to settle for three minutes prior to corona application so that approximately 95% of the cells were attached to the bottom of the chambe r. Next, the non-suspended NT2 cells were exposed to alternating positive and negative charge in the following manner. First, 1 minute positive

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37 corona was applied followed immediately by 1 minute nega tive corona exposure (both electrodes grounded). Corona trea tment was briefly suspended to allow for pictures to be taken with the intent of obtaining photograp hic evidence of cellular movement. This cycle of 1 minute of positive corona, 1 minute of negative corona, and photographs was performed a total of six times. The result wa s a cascade of pictures (Figure 5.1) showing the direction of cellular movement and the location of intracellular contact during 6 minutes of positive followed by 6 minutes of negative corona treatment. The aforementioned steps (6 cycles of alterna ting positive and negative charge involving photographs in between each cycle) were repeat ed a second time, but this time the cells were treated with 1 minute negative corona followed by 1 minute positive corona for each cycle (Figure 5.2). Figure 5.1 Successive Corona Treatments of Positive Polarity Followed by Negative Polarity for Up to Six Minutes Each (40X) A) Before Corona Application B) After 1 Cycle of 1 Minute Positive Corona Followed by 1 Minute Negative Corona

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38 Figure 5.1 Continued C) After 2 Cycles of 1 Minute Positive Corona Followed 1 Minute Negative Corona D) After 3 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona E) After 4 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona F) After 5 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona G) After 6 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona

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39 Figure 5.2 Successive Corona Treatments of Negative Polarity Followed by Positive Polarity for Up to Six Minutes Each (40X) A) Before Corona Application B) After 1 Cycle of 1 Minute Negative Corona Followed by 1 Minute Positive Corona C) After 2 Cycles of 1 Minute Negative Corona Followed by 1 Minute Positive Corona D) After 3 Cycles of 1 Minute Negative Corona Followed by 1 Minute Positive Corona E) After 4 Cycles of 1 Minute Negative Corona Followed by 1 Minute Positive Corona F) After 5 Cycles of 1 Minute Negative Corona Followed by 1 Minute Positive Corona

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40 Figure 5.2 Continued The results revealed (Figures 5.1 and 5. 2) that the order in which the different polarities were applied gave a similar resu lt. In both experiments, the NT2 cells gradually gravitated towards one another in the middle space between both electrodes to form “small islands” of intracellular contact. Hence, any order of corona polarity would be adequate for the purpose of this investigation. 5.4 Effect of Grounding Variable Electrodes Under Corona Treatment The effect of either grounding electrode s on the corona cham ber or not grounding the electrodes was observed. This was done by performing four separate experiments in which the cells were prepared as discussed in Chapter 4, placed into the circular corona chamber with circular electrodes and treated with corona discharge while A) grounding both electrodes, B) groundi ng only the outer el ectrode, C) grounding only the inner electrode and D) not grounding e ither electrode. The reason for analyzing the effect of G) After 6 Cycles of 1 Minute Negative Corona Followed by 1 Minute Positive Corona

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41 grounding versus not grounding is th at the experimentation woul d help to provide further insight as to what the electri cal fields might look like within the suspension, as well as providing key information for the direction in which the forces are applied. In each of the four experiments discussed above, NT2 cells were harvested, counted, and placed into the circular corona chamber containing circular electrodes at a concentration of 610 2 cells/175 l. Results for Trial A (grounding both electrodes) were already obtained from Section 5.2. In Trial B, only the outer electrode was connected to a ground using a wire. The NT2 cel ls were then treated with 5 minutes of positive corona followed by 5 minutes of nega tive corona. A se quence of photographs was taken to record movement while under coro na discharge (Figure 5. 3). The cells were forced into contact near the inner electrode. Since the charge on th e inner electrode was not displaced to a ground source, there wa s an accumulation of charge resulting in cellular attraction. Movement of cells close to an electrode is extremely unfavorable due to the arcing effects associated with the el ectrodes during the applic ation of fusogenic DC pulses. The arcing could potentially jeopardi ze cellular integrity and/or viability. In addition, electrochemical products that ma y form at the electrodes during DC pulse application could adversely affect cell viabilit y. For Trial C, only the inner electrode was connected to a ground source. Photographic evidence of the cellular movement (Figure 5.4) revealed that intracellular contact occurs much like Trial A (in the middle space between the two electrodes), a lthough cell-cell contact is of a smaller qu antity and occurs at a more gradual pace. This result is unfavorable because there might not be enough intracellular contact to obtain a large quantity of hybrids during electrofusion. Finally, in Trial D, neither electrode wa s connected to a ground source A sequence of photographs

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42 (Figure 5.5) was taken during Trial D to once again record cellular movement and behavior. As can be seen below, these photographs illustrate little to no cellular movement during corona application. Any motion at all was simply a result of the electrostatic repulsions/attractions between the cells and the ions present in the media. One can speculate that the evidence presented in Trial D points to the notion that in order for there to be any cellular movement there must be a current present within the solution that acts on the cells forcing them to a certain direction. The current is generated by the transport of charge from one or bot h of the electrodes to a ground source. Figure 5.3 Corona Treatment of Circular Corona Chamber Configuration with Only Outer Electrode Grounded (40X) A) Before Corona Application B) Trial B: After 1 Cycle of 1 Minute Positive CoronaFollowed b y 1 Minute N e g ative Corona

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43 Figure 5.3 Continued C) Trial B: After 2 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona D) Trial B: After 3 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona E) Trial B: After 4 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona F) Trial B: After 5 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona G) Trial B: After 6 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona

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44 Figure 5.4 Corona Treatment of Circular Cor ona Chamber Configuration with Only Inner Electrode Grounded (40X) A) Trial C: Before Corona Application B) Trial C: After 1 Cycle of 1 Minute Positive Corona Followed by 1 Minute Negative Corona C) Trial C: After 2 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona D) Trial C: After 3 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona F) Trial C: After 5 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona E) Trial C: After 4 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona

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45 Figure 5.4 Continued Figure 5.5 Corona Treatment of Circular Corona Chamber Configuration with No Electrodes Grounded (40X) G) Trial C: After 6 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona A) Trial D: Before Corona Application B) Trial D: After 1 Cycle of 1 Minute Positive Corona Followed by 1 Minute Negative Corona

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46 Figure 5.5 Continued In summary, grounding both electrodes (T rial A) provided th e most favorable conditions for the purpose of this investig ation. Not only do th e cells aggregate in desirable quantities at a moderately controlle d pace, but they contact each other in the middle space between the inner and outer elec trodes, thereby preventing the risk of experiencing the deleterious effects of ar cing and electrochemicals during subsequent electrofusion. 5.5 Determination of Optimal Co rona Treatment Duration In order to determine optimal conditions for cellular contact that were favorable for the generation of a large quantity of 1: 1 hybrids, it was necessary to first define exactly what level of intracellu lar contact would be consider ed optimal for the purpose of this investigation. Optimal cell contact condi tions were defined as those conditions that would provide the greatest probability for ge nerating a large quantity of hybridized cells C) Trial D: After 2 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona D) Trial D: After 3 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona

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47 on a 1:1 fusion basis. In order to insure that there would be a large quantity of 1:1 hybrids generated, it was determined that there must be moderate sized cell islands consisting of tight intracellu lar contact present in the corona chamber following corona treatment and prior to electrofusion. NT2 cells were suspended in DMEM media (supplemented with 0.05 mg/ml gentamicin), and pipetted into the co rona chamber at a concentration of 610 2 cells/175 l. The cells were then allowed to settle for 3 minutes by gravity to assure a uniform monolayer of cells in the chamber. The experiment in this section made use of the optimal conditions and parameters determined from the aforementioned sections in this chapter (combined corona polarity treatment in corona chamber with both electrodes connected to a ground source). In this experiment, NT2 cells were treated with six sequences of corona discharge. After each sequence (One sequence = 1 minute positive corona followed by 1 minute negative corona ), a photograph was taken to observe the cell-cell contact properties (Figure 5.6). The particular sequence that matched the conditions for containing mode rate sized cell islands cons isting of tight intracellular contact was determined to be the optimal corona treatment duration. As can be seen from Figure 5.6 below, the application of 3 cycles was optimal. Application of corona treatment for 2 mi nutes on each polarity was not enough time, because there was an insufficient amount of contact. As a result, there would not be enough hybrids generated during electrofusion. On the other hand, applying corona treatment for 4 minutes on each polarity gene rated too much intracellular contact and would not be good in virtue of obtaining hybrids on a 1:1 basis.

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48 Figure 5.6 Corona Treatment of Circular Corona Chamber with Both Electrodes Grounded for Determination of Optimal Corona Treatment Duration (40X) 5.6 Fusion Experiments with Circular Coro na Chamber Containing Electrodes The fusion experiments conducted over the course of this investigation were designed to produce NT2 monohy brids at a 1:1 ratio by using optimal parameters determined from the aforementioned experiments. A) Corona Treatment of 2 Minutes Positive Polarity Followed by 2 Minutes Negative Polarity B) Corona Treatment of 3 Minutes Positive Polarity Followed by 3 Minutes Negative Polarity C) Corona Treatment of 4 Minutes Positive Polarity Followed by 4 Minutes Negative Polarity

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49 NT2 cells were independently stained with 50 l CMFDA (green fluorescent dye) and 75 l CMTMR (red fluorescent dye) and prepared for use in the chamber as described in Chapter 4. Aliquots of NT2 cells consisting of 610 2 cells/175 l DMEM (supplemented with 0.05 mg/ml gentamicin) were pipetted into the corona chamber (circular configuration containi ng circular stainless steel electrodes). The cells were then allowed to settle for 3 minutes to insure that approximately 95% of the cells have confined themselves to the chamber floor. Th is resulted in the formation of a uniform cellular monolayer. Next, the cells were subjected to 3 minutes of positive corona followed by 3 minutes of negative corona. Afte r corona application, the electrodes were connected to the DC electroporator in prepara tion for electrofusion. The cells were then pulsed with direct current pulses. Five separa te electrofusion trials were performed. For the five trials, the cells were pulsed with six or more pulses of 750 Volts/cm, 1000 Volts/cm, 2000 Volts/cm, 2500 Volts/cm and 3000 Volts/cm (all using pulse durations of 100 sec). In each of the five trials, little (< 1% ) to no cell fusion was obtained, and cell viability ranged from little to no cell dama ge (750 – 2000 Volts/cm) to extreme cellular damage (3000 Volts/cm). The aforementioned tria ls were then repeated for 2 minutes of positive corona followed by 2 minutes of negative corona, and 4 minutes of positive corona followed by 4 minutes of negative corona respectively. These additional trials yielded very similar results to the first se t of experiments involving 3 minutes of positive corona followed by 3 minutes of negative corona The data recorded for the set of fusion experiments conducted in this sect ion is shown in Table 5.1.

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50 Table 5.1 Determination of Optimal Fusion Parameters for Circular Corona Chamber with Circular Electrodes Figure 5.7 depicts the NT2 cells staine d with CMFDA and CMTMR in the corona chamber prior to electrofusion, as well as the pre-labeled NT2 cells, with a lack of yellow NT2 monohybrids, in the corona chamber following 6 pulses of 2500 Volts/cm DC at 100 sec respectively. Figure 5.8 is a high er magnification (100X ) of the NT2 cells following electrofusion.

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51 Figure 5.7 NT2 Cells Before and After DC Treatment Using Optimal Corona Exposure Parameters in a Circular Cor ona Chamber Configuration (40X) A) After 3 Minutes of Positive Polar ity Followed by 3 Minutes of Negative Polarity, but Prior to DC Electrofusion (40X) B) Following 6 DC pulses of 2500 Volts/cm at 100 sec intervals (40X)

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52 Figure 5.8 NT2 Cells Following DC Pulse (100X) The inability to fuse may have been a direct result of obtaining a non-uniform DC field, due to the circular geometry of the chamber. For this r eason, one would suspect that it might be beneficial to investigate al ternative corona chamber geometries, such as a square. Despite this unexpected shortcom ing, an interesting phenomenon was observed during electrofusion that provided additional promise for the investigation. During the electrofusion experiments, mechanical pert urbations were delivered to the chamber, which subsequently caused the cells to re lease from the chamber floor and gravitate towards one another in free suspension. The reason for this sudden intracellular attraction has not yet been explained, but this unexpected phenomenon prompted an investigation for the effect of mechanical disturbance on the cells later in the study. NT2 cells following 6 DC pulses of 2500 Volts/cm at 100 sec intervals (100X)

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535.7 Results with Different Geometric Corona Chamber Configurations The following set of experiments was desi gned to investigate the effect of mechanical perturbation on NT2 cells following corona application in alternative corona chamber geometries. 5.7.1 Investigation of Corona Treatment 5.7.1.1 Square Chamber without Electrodes NT2 cells were harvested, counted and delivered at a concentration of 610 2 cells/175 l DMEM (supplemented with 0.05 mg/ml ge ntamicin) into th e square corona chamber (cells were pipetted into only one of the squares) illustrated by Figure 4.11. Unlike the previous corona chamber inve stigated, this chamber did not contain electrodes. The cells were a llowed to settle for 3 minutes and were then exposed to 3 minutes of positive corona discharge followed by 3 minutes of negative corona discharge. As expected, results revealed little to no cellular movement, being that there was no connection to a ground source. However, after applying mech anical distur bance to the chamber, the cells exhibited a fascinating be havior; the cells releas ed from the chamber floor and gravitated towards the periphery (cha mber walls) in every direction, therefore leaving a void space in the middle of the chamber. Figure 5.9 below illustrates the cellular peripheralization that resulted from mechanical disturbance of the square chamber.

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54 Figure 5.9 Effect of Mechanical Disturban ce on NT2 Cells After Corona Treatment in a Square Corona Chamber This method of mechanical perturbation following corona treatment, while difficult to quantify and control, resulted with extremely tight intracellular contact. After A) Left Wall of Square Chamber Following Corona Treatment and Prior to Mechanical Perturbation B) Bottom Wall of Square Chamber Following Corona Treatment and Prior to Mechanical Perturbation VC) Void Space (V) Creat ed in the Middle of the Square Chamber From Cellular Peripheralization Fo llowing Mechanical Disturbance

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55 achieving tight intracellular cont act in a square corona chamber, it was plausible to obtain NT2 monohybrids in the square corona chamber by utilizing th e electrofusion techniques discussed in the preceding section. Unlike th e circular geometry investigated in the previous experiments, the square chamber was able to achieve a uniform DC field throughout the chamber while undergoing electrofusion. Thus, using a square configuration for the corona chamber provide d a great chance for obtaining cell fusion. 5.7.1.2 Circular Chamber without Electrodes The effect of mechanical disturbance on NT2 cells in a circular corona chamber (without electrodes) was examined in order to determine if chamber geometry had any effect on cell movement when expe riencing mechanical disturbance. NT2 cells were harvested, counted and delivered into the circular corona chamber illustrated in Figure 4.10 at a concentration of 610 2 cells/175 l DMEM (supplemented with 0.05 mg/ml gentamicin). The cells were then allowed 3 minutes of settling time prior to corona treatment. Once again, the cells were treated with 3 minutes of positive corona followed by 3 minutes of negative corona and the chamber was subsequently disturbed. Results revealed th at the cells exhibited the same movement as observed in the square corona chamber. Thus, it was concluded that the chamber geometry had no effect on cell movement fo llowing mechanical disturbance. Instead, cellular peripheralization was a ttributed largely to charge buildup on the chamber wall (there were no electrodes present to funne l the charge to a ground source), which subsequently attracted the positively/negatively charged cells. Figure 5.10 illustrates the cellular peripheralization from mechanical disturbance of the circular chamber.

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56 Figure 5.10 Effect of Mechanic al Disturbance on NT2 Cells Af ter Corona Treatment in a Circular Corona Chamber 5.7.1.3 Square Chamber with Square Electrodes Since it had been demonstrated from the previous section that it was possible to obtain tight intracellular contact in a squa re chamber using corona irradiation and subsequent mechanical perturbation, the next step was to attempt to control the level of cell-cell contact by adding electrodes to th e current methodology. The reason for the addition of grounded electrodes to the existing chamber was that it was considered more plausible with the electrode technology to su ccessfully quantify or approximate the ratio of fusion that resulted from subsequent electrofusion. NT2 cells were prepared in accordance w ith the protocol discussed in Chapter 4 and pipetted into the square corona chamber (only one of the squares) containing two stainless steel square electrodes (F igure 4.12) at a concentration of 610 2 cells/175 l A) Middle of Circular Corona Chamber Following Corona Treatment and Prior to Mechanical Perturbation C) Void Space (V) Creat ed in the Middle of the Circular Chamber from Cellular Peripheralization Fo llowing Mechanical Disturbance V

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57 DMEM (supplemented with 0.05 mg/ml gentamic in). Once, the cells were allowed 3 minutes of settling time, they were treated with alternating positive and negative corona charges by the same protocol as discussed in Chapter 5, Section 3. Six cycles were completed, with each cycle representing an additional 1 minute of positive corona followed by 1 minute of negative corona. Cor ona application was briefly suspended in between each cycle to allow for photographs to be taken to record cell movement. This procedure was repeated for evidence of reprodu cibility. The first set of results (Figure 5.11 below) revealed a very positive outcome: the cells gradually formed tight cell islands in the annular space betw een the two electrodes before eventually migrating to the chamber walls in the form of tight intracellu lar aggregates. Unfortunately, these results could not be reproduced in subsequent trials and therefore, the square chamber with electrodes must be further optimized and investigated before using the chamber to acquire hybrid cells during subs equent electrofusion. The reason for why the square corona chamber with electrodes was not able to achieve reproducible results has not yet been determined. Although, as a result, there is room for fu ture investigations involving the optimization of the square chamber with electrodes so that it can be used as a novel method to achieve desired cell-cell contac t and to quantify fu sion ratios during subsequent electrofusion.

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58 Figure 5.11 Successful Cell C ontact Using Optimal Corona Parameters in a Grounded Square Chamber A) Before Corona Application B) After 1 Cycle of 1 Minute Positive Corona Followed by 1 Minute Negative Corona C) After 2 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona D) After 3 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona

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59 Figure 5.11 Continued E) After 4 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona F) After 5 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona G) After 6 Cycles of 1 Minute Positive Corona Followed by 1 Minute Negative Corona

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605.7.2 Fusion Analysis The square corona chamber (Figure 4. 11) discussed in Section 5.7.1.1 was investigated for the purpose of obtaining NT2 monohybrids. The idea was to use a combination treatment involving corona a pplication (3 minutes of positive corona followed by 3 minutes of negative corona (or in reverse)) and subsequent mechanical perturbation followed by exposure with fu sogenic DC pulses in order to obtain hybridization. As discussed in the prev ious text, the aforementioned combination treatment of corona application and mechanical disturbance resulted in tight intracellular contact. Tight intracellular contact in addi tion to the application of a uniform DC field (due to the square geometry of the chambe r) proved to be the right combination for the formation of NT2 monohybrids. Two cultures of NT2 cells were prep ared (grown, stained, harvested, and counted) in accordance with the protocols discu ssed in Chapter 4. The two cultures were pre-labeled with CMFDA and CMTMR respective ly and pipetted into the square corona chamber (only one of the squares in Figure 4.11) at a total concentration of 610 2 cells/175 l DMEM (supplemented with 0.05 mg/ml gentamicin) (individual concentration of 610 1 cells/87.5 l DMEM each). The pre-labeled NT2 cells (red and green stained) were then allo wed 3 minutes of settling time to insure that approximately 95% of the cells had gravitated to the cham ber floor. Once again, the NT2 cells were treated with 3 minutes of positive corona followed by 3 minutes of negative corona and the chamber was subsequently disturbed. Af ter the cells had achieved tight intracellular contact from cellular periphera lization, they were exposed to 8 fusogenic DC pulses of 2500 Volts/cm at 100 sec intervals. The fusogenic pul ses were applied using the hand

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61 held electrode and ECM 800 pulse generator shown in Figure 4.14. The electrode was placed into the chamber containing the pre-la beled green and red NT 2 cells so that the electrodes were in direct contact with the ch amber floor prior to DC delivery. Then, the fusion parameters were entered into the EC M 800 and direct current was subsequently passed through the suspension containing the cell s. The fusion parameters used for this investigation were selected because they we re determined (when comparing with other parameters (Table 5.1)) from the experiment in Section 5.6 as being most optimal for cellular hybridization an d cell viability. As can be seen from Figure 5.12 (below), the pre-labeled NT2 cells accumulated between the MPG’s electrodes and fuse d to form NT2 monohybrids. The NT2 monohybrids (Figure 5.12) were di stinguished from the un-fus ed green or red cells by their characteristic yellowish/orange color, irregular oblong shape or large size. The presence of these NT2 monohybrids further valida ted the theory that th e cells did not fuse in the circular chamber because they di d not experience a unifo rm DC field during electrofusion.

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62 Figure 5.12 Resulting NT2 Monohybrids in a S quare Corona Chamber After Successive Corona Treatment, Mechanical Disturbance, and 2500 DC Volts/cm A) NT2 Cells Accumulating Between the Electrodes as Illustrated by the White Dotted Lines Following Electrofusion (40X) B) Pre-labeled NT2 Cells in the Square Corona Chamber Following DC application (100X) C) Fused NT2 Monohybrids in the Square Corona Chamber Following DC application (100X) D) Fused NT2 Monohybrid in the Square Corona Chamber Following DC application (400X)

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63 Figure 5.12 Continued In order to further verify that the NT 2 Monohybrids were in fact fused cells and not the result of the two fluor escent dyes bleeding together, all of the NT2 cells in the corona chamber were transferred to a petri dish. As illustrated by the photographs taken in Figure 5.13, the yellowish/orange irregular ly shaped cells observed in the corona chamber had the same appearance and morphol ogy in the petri dish, which validated that these cells were in fact NT2 monohybrids. E) Fused NT2 Monohybrids Following DC Application (400X)

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64 Figure 5.13 NT2 Monohybrids Once Transferred to a Petri Dish for Validation of Fusion A) NT2 Cells in Petri Dish Following 2500 Volts/cm Fusogenic DC Pulse Application (40X) B) NT2 Cells in Petri Dish Following 2500 Volts/cm Fusogenic DC Pulse Application (Monohybrids indicated by white arrows) (100X) C) NT2 Monohybrids (Yellowish/Orange Cells Indicated by White Arrows) in Petri Dish Following Electrofusion (400X) D) NT2 Monohybrids (Yellowish/Orange Cells Indicated by White Arrows) in Petri Dish Following Electrofusion (400X)

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65 Figure 5.13 Continued For comparison purposes, the experiment c onducted in this section was repeated three more times using different fusion parameters each time: 15 pulses of 2500 Volts/cm, 8 pulses of 3000 Volts/cm and 15 pu lses of 3000 Volts/cm. Table 5.2 below provides a summary of the results observed for cell viability and fusion in a square corona chamber when using the aforementi oned electrofusion parameters and optimal cell contact techniques (3 or 4 cycles of alternating corona polarities followed by mechanical perturbation). E) NT2 Monohybrid (Indicated by White Arrow) ( 400X ) F) NT2 Monohybrids (Indicated by White Arrows) ( 100X )

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66 Table 5.2 Determination of Optimal Fusion Parameters for Square Chamber 8 pulses15 pulses8 pulses15 pulses # of cycles (1 min. (+), followed by 1 min. (-) and vice versa 3 ~20% Fusion, Minimal Cell Damage ~40% Fusion, Large Cell Damage ~10% Fusion, Very Large Cell Damage ~5% Fusion, Severe Cell Damage 25003000 Voltage (V/cm) 5.7.2.1 Verification of Hybridized Cell Viability Following electrofusion, cell death was di stinguished by the visual membrane disintegration or by stain leaking from the cy tosol into the surroundi ng media. Visual evidence with microscopy revealed that the NT2 monohybrids generated during this investigation did not sh are any of the above ch aracteristics for cell de ath. In order to obtain even further confirmation that these hyb ridized cells were viable, the cells were centrifuged, washed, re-suspended in supplem ented DMEM (with 0.05 mg/ml gentamicin and 10% fetal bovine serum) and subsequently cultured for a period of 2-3 days. At the end of this period, the cells were visually in spected for mitotic activity. As a result, the NT2 cells continued to show the ability to differentiate and grow, therefore providing confirmation of the notion that thes e hybridized cells were viable.

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67 CHAPTER 6: CONCLUSION AND RECOMMENDATIONS 6.1 Conclusion In an attempt to investigate corona di scharge and subsequent direct current electrofusion as a method fo r obtaining tight intracellu lar contact and cell-cell hybridization respectively, a set of optimal parameters or conditions were determined. Early in the investigation, it was demonstrat ed that not only can cel ls be placed into a physiologic-based media during corona treatm ent, but their level of contact can be controlled if the cells are placed into a circ ular chamber containing electrodes that are connected to a ground source. Even though this condition would most likely be best for obtaining 1:1 based hybridization, the geomet ry of the chamber was not favorable for achieving cellular fusion. However, later in the study it was demonstrated that by mechanically disturbing the chamber followi ng corona treatment, tight intracellular contact could be obtained in bot h a circular and square shaped chamber. This discovery provided promise for obtaining large quantitie s of hybridized cells because unlike the circular chamber, the square chamber turned out to be more favorable for generating hybridized cell constructs during electrofusi on. The reason for this difference has not been determined yet. One reason may be that the circular geometry resulted in an uneven distribution of charge during el ectrofusion due to the presence of a non-uniform DC field, charge was evenly distributed over the en tire chamber in the square geometry.

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68 The square chamber used in the investig ation produced outstanding cell-cell contact when using the combination treatment of co rona discharge and m echanical perturbation discussed above. In addition, when the aggreg ated cells were treated with 8-15 fusogenic DC pulses of 2500 Volts/cm at 100 sec intervals (following corona treatment and mechanical disturbance), the chamber pr ovided fusion yields as high as 40%. Throughout the investigation, it was clear that in order to obtain a substantial amount of cell contact (in the form of monolayers) an d subsequent fusion, the cells needed 3 minutes of settling time followed by corona tr eatment of at least 3 minutes of positive corona followed by 3 minutes of negative coro na (or in reverse). Furthermore, unlike other electrofusion methodologies it appears that corona di scharge and subsequent DC application, as described in the last se t of experiments, does not compromise the hybridized cells’ viability, integr ity. Future studies, however, are necessary to verify this observation. 6.2 Recommendations An attempt was made during the investiga tion to obtain 1:1 based fusion by using a square corona chamber that contained grounde d electrodes in order to better control the level of intracellular contact. Unfortunately, these trials did not return any favorable results. For an undetermined reason, the cells were simply not induced to gravitate towards each other while under corona discharge. It was unclear as to whether this was a result of the placement of the electrodes on th e chamber, the geometry of the electrodes used, or the chamber itself. The investigation of a suitable method or device that will not only induce cell-cell contact in a controlled environment but induce cellular fusion on a

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69 near 1:1 basis is perhaps the first step toward s optimizing this technol ogy. In addition, a presentation of the mechanism or characteri zation of the electric fields present in the chamber during corona discharge can help in further optimizing the equipment to provide better cell contact and higher fusion yields. The ultimate goal would be to apply this technology to different cell types in orde r to engineer novel heterohybridized cell constructs that exhibit combined characterist ics not observed in either of the cells being fused. Cell constructs de signed in this way could be utilized in a number of cell therapies, such as therapeutic cell transplantation.

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70 REFERENCES 1. Li, L.H.; Hensen, M.L.; Zhao, Y.L.; Hui, S.W. Electrofusion Between Heterogeneous-Sized Mammalian Cells in a Pellet: Potential Applications in Drug Delivery and Hybridoma Formation. Biophys. J. 1996, 71, 479-486. 2. Mclenaghan, N.H.; Flatt, P.R. E ngineering Cultured Insulin-Secreting Pancreatic B-Cell Lines. J. Mol. Med. 1999, 77, 235-243. 3. Ege, T.; Krondahl, U.; Ringertz, N.R. Introduction of Nuclei and Micronuclei into Cells and Enucleated Cytoplas ms by Sendai Virus Induced Fusion. Exp. Cell Res. 1974, 88, 428-432. 4. Finaz, C.; Lefevre, A.; Teissie, J. Electrofusion. A New, Highly Efficient Technique for Generating Somatic Cell Hybrids. Exp. Cell Res. 1984, 150, 477-482. 5. Sukhorukov, V.L.; Resuss, R.; Endter, J.M.; Fehrmann, S.; KatsenGloba, A.; Gessner, P.; Steinbach, A.; Muller, K.J.; Karpas, A.; Zimmermann, U.; Zimmermann, H. A Biophysical Approach to the Optimization of DendriticTumour Cell Electrofusion. Biochem. Biophys. Res. Commun. 2006, 346, 829839. 6. Hui, S.W. The Application of Electropor ation to Transfect Hematopoietic Cells and to Deliver Drugs and Vaccines Transc utaneously for Cancer Treatment. Technol. Cancer Res. Treat. 2002, 1, 373-384. 7. Valone, F.; Small, E.; MacKenzie, M. ; Burch, P.; Lacy, M.; Peshwa, M.V.; Laus, R. Dendritic Cell-Based Treatment of Cancer: Closing in on a Cellular Therapy. The Cancer Journal 2001,7, S53-S61. 8. Zimmermann, U; Vienken, J. Electric Field-Induced Cell-to-cell Fusion. J Membrane Biol 1982, 67 165-182. 9. Halfer, Carlotta ; Petrella, Lucia. Cell Fusion Induced by Lysolecithin and Concanavalin A in Drosophila Melanogaster Somatic Cells Cultured in Vitro. Experimental Cell Research. 1976, 100, 399-404

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71 10. Hayashi, T.; Tanaka, H.; Tanaka, J.; Wang, R. ; Averbook B.J.; Cohen, P.A.; Shu, S. Immunogenicity and Therapeutic Efficacy of Dendritic-Tumor Hybrid Cells Generated by Electrofusion. Clin. Immunol. 2002, 104, 14-20. 11. Cheong, S.C.; Blangenois, I.; Franssen, J.D.; Servais, C.; Phan, V.; Trakatelli, M.; Bruyns, C.; Vile, R.; Velu, T.; Br andenbruger, A. Generation of Cell Hybrids Via a Fusogenic Cell Line. J. Gene Med. 2006, 8, 919-928. 12. Jaroszeski, M.; Gilbert, R.; Perrott, R.; He ller, R. Enhanced Effects of Multiple Treatment Electrochemotherapy. Melanoma Research 1996, 6, 427-433. 13. Heller, R.; Jaroszeski, M.; Atkin, A. ; Moradpour, D.; Gilbert, R.; Wands, J.; Nicolau, C. In Vivo Ge ne Electroninjection and E xpression in Rat Liver. Fed. Europ. Biochem. Soc. 1996, 389, 225-228. 14. Bouchard, Claude; Teissie, Justin. Homokaryon production by Electrofusion: A Convenient Way to Produce a Large Number of Viable Mammalian Fused Cells. Biochem. Biophys. Res. Commun. 1983, 114, 663-669. 15. Ramos, C.; Teissie, J. Electrofusi on: A Biophysical Modification of Cell Membrane and a Mechanism in Exocytosis. Biochem. 2000, 82, 511-518. 16. Schmitt, J.J.; Zimmermann, U. Enhanced Hybridoma Production by Electrofusion in Strongly Hypo-Osmolar Solutions. Biochem. Biophys. Acta. 1989, 983, 42-50. 17. Vienken, J.; Zimmermann, U. An Improved Electrofusion Technique for Production of Mouse Hybridoma Cells. FEBS Lett. 1985, 182, 278-280. 18. Teissie, J.; Rols, M.P. Fusion of Mammalian Cells in Culture is Obtained by Creating the Contact Between Cells Af ter their Electropermeabilization. Biochem. Biophys. Res. Commun. 1986, 140, 258-266. 19. Sowers, A.E. Evidence that Electrofus ion Yield is Controlled by Biologically Relevant Membrane Factors. Biochem. Biophys. Acta. 1989, 985, 334-338. 20. Sowers, A.E. Electrofusion of Dissim ilar Membrane Fusion Partners Depends on Additive Contributions from Each of the Two Different Membranes. Biochem. Bioiphys. Acta. 1989, 985, 339-342. 21. Heller, L.C.; Pottinger, C.; Jaroszeski, M.J.; Gilber, R.; Heller, R. In Vivo Electroporation of Plasmids Encoding GMCSF and Interleukin-2 into Existing B16 Melanomas combined with Elec trochemotherapy Induces Long Term Antitumor Immunity. Melanoma Research 2000, 577-583.

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72 22. Somiari, S.; Glasspool-Malone, J.; Drabic k, J.J.; Gilbert, R.A.; Heller, R.; Jaroszeski, M.J.; Malone, R.W. Theory and In Vivo Application of Electroporative Gene Delivery. Mol. Therapy 2000, 2, 178-187. 23. Heller; Gilbert; Jaroszeski. Clinical Applications of Electrochemotherapy. Advanced Drug Delivery Reviews 1999, 35, 119-129. 24. Jaroszeski, M.J.; Gilbert, R.; Nicolau, C.; Heller, R. In Vivo Gene Delivery by Electroporation. Advanced Drug Delivery Reviews 1999, 35, 131-137. 25. Hyacinthe; Jaroszeski; Dang; Coppola; Ka rl; Gilbert; Helle r. Electrically Enhanced Drug Delivery for the Treatment of Soft Tissue Scarcoma. Cancer 1999, 85, 409-417. 26. Mir, L.M.; Glass, L.F.; Sersa, G.; Te issie, J.; Domenge, C.; Miklavcic, D.; Jaroszeski, M.J.; Orlowski, S.; Reintgen, D.S.; Rudolf, Z. Effective Treatment of Cutaneous and Subcutaneous Malignant Tumors By Electrochemotherapy. Br. J. of Cancer 1998, 32, 53-64. 27. Heller, R.; Jaroszeski, M.J.; Reintgen, D. ; Puleo, C.; DeConti, R.; Gilbert, R.; Glass, L.F. Treatment of Cutaneous and Subcutaneous Tumors with Electrochemotherapy using In tralesional Bleomycin. Cancer 1998, 83, 148157. 28. Lewis, F.; Glass, M.; Jaroszeski, M.J.; Gilbert, R.; Reintgen, D.S.; Heller, R. Intralesional Bleomycin-Mediated Elect rochemotherapy in 20 Patients with Basal Cell Carcinoma. J. Am. Acad. Derm. 1997, 37, 596-599. 29. Jaroszeski, M.J.; Gilbert, R.; Heller, R. Successful Treatment of Hepatomas with Electrochemotherapy in a Rat Model. Biomchem. Biophys. Acta. 1997, 1334, 15-18. 30. Abidor, I.G.; Sowers, A.E. Kinetics and Mechanism of Cell Membrane Electrofusion. Biophy. J. 1992, 61, 1557-1569. 31. Zimmermann, U; Vienken, J. Electric Field-Induced Cell-to-cell Fusion. J Membrane Biol 1982, 67 165-182. 32. Ohkohchi, N.; Itagaki, H.; Doi, H.; Taguchi, Y.; Satomi, S.; Satoh, S. New Technique for Producing Hybridom a by Using Laser Radiation. Lasers Surg. Med. 2000, 27, 262-268. 33. Itagaki, H.; Doi, H.; Ohkohchi, N.; Sa tomi, S. Development of New Cell Fusion Technique by Laser Device and A pplication to Bio-medical Field. Nippon Rinsho. 1997, 55, 2780-2787.

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73 34. Finaz, C.; Lefevre, A.; Teissie, J. Electrofusion: A Ne w Highly Efficient Technique for Generating Somatic Cell Hybrids. Exp Cell Res 1984, 150, 477482. 35. Bouchard, Claude; Teissie, Justin. Ho mokaryon Production by Electrofusion: A Convenient Way to Produce Large Number of Viable Mammalian Fused Cells. Biochemical and Biophysical Research Communications 1983, 114 663-339. 36. Corona discharge < http://en.wikipedia.org/wiki/Corona_discharge >. 37. Plasma (physics) http://en.wikipedia.org/wiki/Plasma_%28physics%29 38. Boating-Lightning Protection http://www.cdc.gov/nasd/docs/d000001-d000100/d000007/d000007.html 39. http://blazelabs.com/1-intro.asp 40. Kwark, C.; Lee, C.W. Experimental Study of a Real-time Corona Discharge Imaging System as a Future Biomedical Imaging Device. Med. Biol. Eng. Comput. 1994, 32, 283-288. 41. Betchen, S.A.; Kaplitt, M. Future and Current Surgical Therapies in Parkinson’s Disease. Lippincott Williams and Wilins, Inc. 2003, 16, 487-493. 42. Willing, A.E.; Cameron, D.F.; Sanberg, P.R. Sertoli Cell Transplants: Their Use in the Treatment of Neurodegenerative Disease. Molecular Medicine Today 1998, 4, 471-477. 43. Willing, A.E.; Orthber, A.I.; Saporta, S.; Anton, A.; Sinibaldi, S.; Poulos, S.G.; Cameron, D.F.; Freeman, T.B.; Sanberg, P.R. Sertoli Cells Enhance the Survival of Co-transplan ted Dopamine Neurons. Brain Research 1999, 822, 246-250. 44. Emerich, D.F.; Hemendinger, R.; Halberstadt, C.R. The Testicular-derived Sertoli Cell: Cellular Immunoscience to Enable Transplantation. Cell Transplant 2003, 12, 335-349. 45. Halberstadt, C.; Emerich, D.F.; Gores, P. Use of Sertoli Cell Transplants to Provide Local Immunoprotection for Tissue Grafts. Expert Opin. Biol. Ther. 2004, 4, 813-825. 46. http://www.nexcelom.com/Prod_Cellometer_Disposable.asp

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74 47. Jaroszeski, M. J.; Gilbert, R.; Heller, R. Detection and Quantitation of Cell-Cell Electrofusion Products by Flow Cytometry. Analytical Biochemistry, 1994 216, 271-275. 48. Jaroszeski, Mark J.; Heller, Rich ard. Flow Cytometry Protocols. Methods in Molecular Biology 1998, Humana Press, 91 149-157.

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75 APPENDICES

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76Appendix A: Data for Calibration of the Corona Generator Table A.1 Data for Calibration of the Corona Generator Experimental Conditions Temperature Range: 22.7C – 23.0C Relative Humidity: 55.4 % 57.4 % Height of Needles: 6.81mm Applied Voltage (kV) Charge on Collector Plate For Positive Corona(A) Charge on Collector Plate For Negative Corona(A) 3.0 0.00 0.01 3.5 0.00 0.75 4.0 0.02 2.45 4.5 0.02 5.26 5.0 3.59 10.32 5.5 7.35 16.04 6.0 11.86 23.81 6.5 16.89 32.98 7.0 25.28 44.20 7.5 36.70 56.40 8.0 59.00 70.30 8.5 86.80 9.0 104.50 9.5 109.50