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The use of electrical charge to produce cell-cell contact prior to electrofusion
h [electronic resource] /
by Jyothi Fernandex.
[Tampa, Fla] :
b University of South Florida,
ABSTRACT: From previous studies it has been demonstrated that the fusion of tumor cells with antigen-presenting cells generates hybrids that are known to induce anti-tumor immunity. With the advancement of scientific research and medicine, the need to produce cell-cell hybrids for cancer immunotherapy and for various other applications is substantial. Among the many methods used to generate these hybrid cells, electrofusion is a technique that is more widely used and recognized as a method to efficiently produce hybrids. Electrofusion requires two steps. In the first step, cells are brought into close adjacent contact either by a mechanical method like centrifugation or by dieletrophoresis using alternating current (AC). The second step includes the reversible breakdown and fusion of cell membranes induced by high voltage direct current (DC) pulses. The goal of this investigation was to study the use of electrical charge to bring cells into close contact with one another in the cell ^contact stage prior to delivering high voltage fusion pulses. The possibility of achieving considerable cell-cell contact was tested in two separate electrical systems. In the first system B16 murine melanoma cancer cells were subjected to a range of direct current (DC) voltages between 4 V/cm and 40 V/cm. With the use of DC from a small power source the response of the cells was tested in multiple fusion chambers consisting of two or four electrodes. The configurations of the chambers were varied by changing the distance between the electrodes, the thickness, material and type of coating on the electrodes. In the second system the movement of cells in the presence of corona charge was studied. B16 cells in a culture dish were confined by a circular grounded electrode and subjected to corona discharge for known periods of time. Application of corona charge (positive or negative) facilitated the contact of cells in the annular region between the two circular electrodes. After series of ^tests, final designs for fusion chambers to be used with DC and with corona were developed. Cell contact achieved with the DC fusion chamber was not substantial enough to produce a significant amount of fusion yield. The fusion chamber designed to be used with corona on the other hand produced exceptional cell contact results consequentially generating fusion yields as high as 40%.
Thesis (M.A.)--University of South Florida, 2005.
Includes bibliographical references.
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Adviser: .Mark Jaroszeski, Ph.D.
x Chemical Engineering
t USF Electronic Theses and Dissertations.
The Use Of Electrical Charge To Produce Cell-Cell Contact Prior To Electrofusion by Jyothi Fernandes A dissertation submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering Department of Chemical Engineering College of Engineering University of South Florida Major Professor: Mark Jaroszeski, Ph.D. Richard Gilbert, Ph.D. Michael VanAuker, Ph.D. Date of Approval: July 20, 2005 Keywords: corona, dc contact, cell fusion, melanoma cells, fusion chamber Copyright 2005, Jyothi Fernandes
DEDICATION To my parents, who have always stoo d behind me in everything I have done.
ACKNOWLEDGMENTS I would like to immensely thank my a dvisor Dr. Mark Jaroszeski for his exceptional guidance and constructive inputs du ring the course of my research. I thank Dr. Richard Gilbert and Dr. Michael VanAuker for serving on my masterÂ’s thesis committee. I would also like to thank Dr. Andrew Hoff, Niraj Ramachandran and my colleagues in the Gene and Drug Delivery lab fo r their valuable sugge stions and all their help with the corona apparatus. I am grat eful to Dr. Arun Kumar and Mr. Abdur Rehman for their help during the course of my resear ch. Finally I would lik e to thank my family and friends for their encouragement and for helping me stay the course during times when the end seemed unreachable.
i TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v ABSTRACT vii CHAPTER 1: INTRODUCTION 1 1.1 Methods of Cell Fusion 1 1.2 Electrofusion 2 1.2.1 Dieletrophoresis 4 1.2.2 Other Methods of Cell Contact 8 CHAPTER 2: BACKGROUND AND MOTIVATION 10 2.1 Corona 10 2.2 Key Concepts on the Generation of Corona 10 2.3 Applications of Corona 14 2.4 Motivation behind Using Corona as a Cell Cont act Method 15 CHAPTER 3: RESEARCH GOALS 16 CHAPTER 4: MATERIALS AND METHODS 18 4.1 Cell Preparation 18 4.1.1 Cell Line and Culture Methods 18 4.1.2 Cell Counting 19 4.2. Cell Staining 20 4.2.1 Stock Solution of Dyes 20
ii 4.2.2 Staining Technique 21 4.2.3 Fluorescent Microscopy 21 4.3 Media for Electrofusion 22 4.4 DC Cell Contact Apparatus 22 4.4.1 DC Power Source 22 4.4.2 Chambers Used for DC Experimentation 24 22.214.171.124 Contact Chamber with Copper Wires 24 126.96.36.199 Contact Chamber with Stainless Steel Strips 25 188.8.131.52 Contact Chamber with Stainless Steel Wires 25 184.108.40.206 Contact Chambers with Coated Stainless Steel Wires 26 220.127.116.11 DC Fusion Chamber Design 27 4.5 Corona Apparatus 28 4.5.1 Corona Generator 28 4.5.2 Corona Experimental Setup 29 4.5.3 Fusion Chambers Investigated with Corona 32 CHAPTER 5: RESULT S AND CONCLUSIONS 34 5.1 Effect of DC on Cell Movement 34 5.2 Final DC Fusion Chamber 37 5.3 Conclusion and Summary of DC Contact Experiments 39 5.4 Calibration of the Corona Generator 40 5.5 Investigation of Corona Induced Cell Contact 42 5.6 Effect of Combined Negative and Positive Corona Treatment 46 5.7 Determination of Fusion Conditions for B16 Cells 49 5.8 Determination of Time Required for Cell Contact 52 5.9 Effect of Corona Induced Contact on Cell Viability 53 5.10 Results with a Different Cell Line 54
iii CHAPTER6: SUMMARY AND RECOMMENDATIONS 57 6.1 Summary 57 6.2 Recommendations 58 REFERENCES 60 APPENDICES 64 Appendix A: Data for Calibration of the Corona Generator 65 Appendix B: Determination of Time Required for Cell Contact 66 Appendix C: Effect of Corona on Cell Viability 68
iv LIST OF TABLES Table 1.1 Parameters Used for Elect rofusion of Different Cell Types 7 Table 5.1 Determination of Fusion Conditions 50 Table A.1 Data for Calibration of Corona Generator 65 Table B.1 Fusion Yield for Differe nt Corona Contact Ti mesExperiment 1 66 Table B.2 Fusion Yield for Different Cor ona Contact TimesExperi ment 2 67 Table C.1 Experimental Data Showing % Difference in Cell Viability 68
v LIST OF FIGURES Figure 1.1 Individual Cell in a Uniform Electric Field 4 Figure 1.2 Individual Cell in a Non-Uniform Field Unde rgoing Dieletrophoresis 5 Figure 1.3 Mutual Dieletrophoresis a nd Pearl Chain Formation of Cells 5 Figure 1.4 Electrofusion of B16 Tumor Ce lls and Dendritic Cells from BALB/c mice 6 Figure 2.1 Typical Voltage -Current Curve of a DC Corona Discharge 13 Figure 4.1 DC Circuit 23 Figure 4.2 DC Power Source a nd Voltage Divide Control 23 Figure 4.3 DC Contact Chamber with Semi-Circular Copper Electrodes 24 Figure 4.4 DC Contact Chamber w ith S-Shaped Copper Electrodes 25 Figure 4.5 DC Contact Chamber with Stainless Steel Strip Electrodes 25 Figure 4.6 DC Contact Chamber with St ainless Steel Circular Electrodes 26 Figure 4.7 DC Contact Chamber with Parallel Stainless Steel Electrodes 26 Figure 4.8 DC Fusion Chamber Design 27 Figure 4.9 Bottom View of the Corona Generator 29 Figure 4.10 Side View of the Corona Generator 29 Figure 4.11 Experimental Setup of Corona Apparatus 30 Figure 4.12 Virtual Interface of the LabVIEW Program 31 Figure 4.13 Corona Chamber with Parallel Electrodes 32
vi Figure 4.14 Corona Contact Chambe r with Circular Electrodes 33 Figure 5.1 Bubble Formation at Negative Electrode 36 Figure 5.2 Cell Movement in DC Environment 38 Figure 5.3 Plot of Applied Volta ge versus Charge Collected 41 Figure 5.4 Effect of Grounded Electr odes in Corona Contact Chamber 43 Figure 5.5 Aggregation of Cells in Gr ounded Circular Contact Chamber (100x) 45 Figure 5.6 Aggregation of Cells in Gr ounded Circular Contact Chamber (400x) 45 Figure 5.7 Effect of Successive Treatment of Positive and Negative Corona on Cells 47 Figure 5.8 Different Sectional Views of the Circular Chamber after Treatment 48 Figure 5.9 Fluorescent Pictures of Fused B16 Cells 51 Figure 5.10 Plot of % Fusion Yield versus Corona Treatment Time 53 Figure 5.11 Plot of % Decrease in Viability versus Corona Treatment Time 54 Figure 5.12 Aggregation of NT2 Cells after Corona Treatment 55 Figure 5.13 Corona Induced C ontact of NT2 Cells and B16 Cells 56
vii THE USE OF ELECTRICAL CHAR GE TO PRODUCE CELL-CELL CONTACT PRIOR TO ELECTROFUSION Jyothi Fernandes ABSTRACT From previous studies it has been demons trated that the fusion of tumor cells with antigen-presenting cells genera tes hybrids that are known to induce anti-tumor immunity. With the advancement of scientific research and medicine, the need to produce cell-cell hybrids for cancer immunotherapy and for various other applic ations is substantial. Among the many methods used to generate these hybrid cells, electrofusion is a technique that is more widely used and r ecognized as a method to efficiently produce hybrids. Electrofusion requires two steps. In the first step, cells are brought into close adjacent contact either by a mechanical method like centrifugation or by dieletrophoresis using alternating current (AC). The second step includes the reversible breakdown and fusion of cell membranes induced by high voltage direct current (DC) pulses. The goal of this investigation was to study the use of electrical charge to bring cells into close contact with one another in the cell contact stage prior to delivering high voltage fusion pulses. The possibility of achieving considerable cell-cell contact was tested in two separate electrical systems. In the first system B16 murine melano ma cancer cells were subjected to a range
viii of direct current (DC) voltage s between 4 V/cm and 40 V/cm. With the use of DC from a small power source the response of the cells was tested in multiple fusion chambers consisting of two or four electrodes. The conf igurations of the chambers were varied by changing the distance between the electrodes, the thickness, ma terial and type of coating on the electrodes. In the second system the movement of cells in the presence of corona charge was studied. B16 cells in a culture dish were confined by a circular grounded electrode and subjected to corona discharge for known peri ods of time. Application of corona charge (positive or negative) facilitated the contact of cells in the annular region between the two circular electrodes. After series of tests, final designs fo r fusion chambers to be used with DC and with corona were developed. Cell contact achieved with th e DC fusion chamber was not substantial enough to produce a significant amount of fusion yield. The fusion chamber designed to be used with corona on the other hand produced exceptional cell contact results consequentially generati ng fusion yields as high as 40%.
1 CHAPTER 1: INTRODUCTION 1.1 Methods of Cell Fusion Cell to cell fusion is a process that ha s been used to produce many different types of cell hybrids for use in various scien tific applications over the years. The first few attempts of cell fusion were carried out in vitro either using chemical fusogens or inactivated virus. Studies using the viral fu sogen called the Â‘SendaiÂ’ virus were first shown in 1977 . Apart from viral fu sogens, polyethylene glycol (PEG), its derivatives and lysolecithin [2, 3] are some of the chemical agents that have been used to promote cell hybridi zation. Among all the chemical fusogens present, PEG is currently more commonly used and is very functional in produc ing cell hybrids used in cancer research and immunotherapy [4, 5 and 6]. Fusion of cells in the presence of el ectricity or electrofusion is another technique that has over time proved to be a comparatively efficient method of producing higher yields of vi able hybrids. Several earl ier studies have shown the advantages of using electrof usion over chemical and viral fusogens [7, 8 and 9]. Some of the main limitations of using ch emical and viral agents for cell fusion as stated by Zimmermann et al  are listed below:
2 The optimum fusion conditions for a set of species have to be predetermined empirically as they vary from species to species. The number of cells to be fused cannot be pre-selected. The process of fusion between any two cells of different species cannot be viewed under the microscope. A loss of intracellular substances is us ually observed and this could affect the viability of the hybrids. The presence of exogenous reagents pr esent during the fusion process may in some instances have a toxic effect on the cells. The method of electric field induced fusion overcomes most of the above stated disadvantages of using chemical and viral fusogens. A favorable amount of investigation and improvement on the pr ocess of electrofusion has led to its popularity as a fusion method and is used in many research as well as practical applications. 1.2 Electrofusion Since the first few published observations of cell-cell electrofusion in the late 1970Â’s [7, 11] its application has grown from generating so matic cell hybrids  and homokaryon production  to production of tumor cell/d endritic cell hybrids for cancer immunotherapy [10-14]. In fact, elect rofusion has had a great impact on the advancement of research in the area of can cer immunotherapy. In the last decade, the use of tumor cell/dendritic cell hybrids to produce therapeutic cancer vaccines has
3 increased significantly. Dendritic cells (D C) are unique among antigen presenting cells in their ability to induce antigen speci fic T cell (immune cell) responses to tumor cells very efficiently . There are many ongoing clinical studies initiated by companies such as Genzyme and Dendreon th at are testing the use of dendritic cell based hybrid vaccines. In most of these clinical studies the method used for the production of these cell hybrids is primarily electrofusion [13, 18]. Fundamentally electrofusion is a two st ep process. The first step is the creation of tight intercellular contact be tween the cells. The second step is the reversible electrical membrane breakdown of the contacting surf aces. After contact, reversible breakdown (or fusion) is achi eved by delivering 3-8 high voltage pulses generated by a pulse generator. The high voltage ranges between 900 V/cm 2000 V/cm and the pulse duration is within th e range of 20-100 s depending on the type of cells to be fused. High voltage fusion pulse s are delivered to the cells in contact by the means of electrodes and this fusion step is similar regardle ss of the application. The method of achieving cell-ce ll contact however differs wi th different electrofusion techniques. From the first few published pa pers on electrofusion through the most recent, the method used to bring cells in to tight membrane contact is primarily dieletrophoresis. Other cell contact methods have been i nvestigated over the last few years [8, 9 19, 20], but the process of dielet rophoresis is by far still the most popular method for contacting cells.
4 1.2.1 Dielectrophoresis Dielectrophoresis is essentially the move ment of neutral particles (in this case cells) in a non uniform electric field. In the case of electrofusion this field is generated by a source of alternating current (AC). Most cells in suspension usually do not come into close contact with one anothe r due to a net negative charge on the outer membrane surface. During the process of di eletrophoresis, the cells develop a mutual attraction to each other as they beco me dipoles in the AC field . An individual cell in the presence of a uniform electric field gets polarized but is still under the influence of a field that is equal on all sides. There is no net force acting on the (neutral) cell and hence motion in any direction will not occur. Figure 1.1 shows an individual cell in a uniform electric field. Figure 1.1 Individual Cell in a Uniform Electric Field . In a non uniform electric field, the fiel d on both sides of the cell is unequal. As a result there is net force acting on the cell and it undergoes translational motion towards the region of highest field in tensity. This phenomenon of directional movement towards the region of highest field intensity is called dieletrophoresis. The + -+
5 direction of dieletropho resis is independent of the polar ity of the field. If the polarity of the electrodes is switched the cell will still move towards the region of highest field intensity as depicted in figure 1.2 Figure 1.2 Individual Cell in a Non-Uniform Field Undergoi ng Dieletrophoresis . Cells that are moving during dieletrophor esis translate to the region of high field intensity and tend to be in the vicin ity of other polarized cells. Hence, they encounter an enhancement of the local field divergence and will tend to move towards the neighboring cell as the field strength will be stro nger at that cell. This effect is called mutual dieletrophoresis . Figure 1.3 Mutual Dieletrophoresis and Pearl Chain Formation of Cells  + -+ + ++ + + + + -
6 As a result of this mutual dieletrophore sis, cells in an al ternating current will be attracted to each other as they over come the weaker electrostatic repulsion between neighboring cell membranes. This attraction of the cells towards the region of high field intensity and towards each othe r leads to the formation of Â‘pearl chainÂ’ of cells which is a characteristic response of cells in an AC field(Figure 1.3). The dieletrophoretic force creates flat parallel c ontact between the cells in the pearl chain and thus a tight membrane contact is achieved in the AC alignment step. Figure 1.4 Electrofusion of B16 Tumor Cells and Dendritic Cells from BALB/c Mice.  The process of electrofusion as shown by Siders et al  is shown in figure 1.4 (A) Equal numbers of B16 tumor cells and dendritic cells from BALB/c mice were mixed in a waxed electroporation cuve tte. (B) The mixture was then subjected
7 to an alignment AC pulse to promote ce ll to cell contact by production of Â‘pearl chainÂ’ of cells (C) The cells were then pul sed with DC fusion pulse to cause cell membrane fusion. The two important parameters in diel etrophoresis are frequency and amplitude of the AC field. The cells will line up in pearl chains only at certain frequencies and this frequency varies from cell type to cell type . The optimum choice of frequency for positive dieletrophoresis is wi thin the range of 10 kHz and 80 MHz.  for most cells. The amplitude of the AC field is usually within the range of 100400V/cm . A list of typical values for the dieletrophoretic field used in some of electrofusion studies is given in Table 1.1. Table 1.1 Parameters Used for El ectrofusion of Different Cell Types Cell Type Cell Alignment Fusion Pulse Friend Erythroleukemia Cells  AC: 100 V/cm, 2 MHz Square pulse(SP): 2kV/cm, 20s NIH 3T3 Cells  AC: 400-700 V/cm, 1 MHz SP: 7 kV/cm, 50s GL261 Glioma Cells, Murine Dendritic Cells  AC: 150 V/cm, 1MHz SP: 1200V/cm, 25s Human Lymphoblasts, Mouse Lymphoblasts  AC: 800 V/cm, 100KHz SP: 3.3 kV/cm 20s 2 Pulses
8 Dieletrophoresis and pearl-chain forma tion usually have to be performed in a non-conducting medium as the presence of el ectrolytes leads to problems of Joule heating . This causes turbulence and disr uption of the pear-cha ins and hinders the cell alignment process. The limitation of using a non-conducting media is one of the drawbacks of using dieletrophoresis as a cell-cell contact method as the nonconducting media are not physiologically bala nced and may alter cellular integrity. Furthermore, even with specially built cham bers only a limited number of cells can be treated with the associated technology 1.2.2 Other Methods of Cell Contact There have been other methods propos ed for improving cell-cell contact and the efficiency of the overall fusion pro cess. The technique of mono layer cell cultivation of anchorage dependant cells have been researched by Finaz et al  and Blangero et al . This cell-cell contact technique can be used for electrofusion only if the cells to be fused are adherent. Met hods using centrifugation with subsequent or simultaneous pulse application to the cells have also been used . Centrifugation can be used for adherent as well as non-adhe rent cells but it must be carried out in such a manner so as to avoid damage of the electrical ly treated cells. Chemical methods of cell-cell contact include the use of avidin-biotin complex. The limitations of these chemicals are that they sometimes tend to leave certain foreign molecules on the cell surface which may aff ect the fusion process. A few mechanical methods of ce ll-cell contact have been intr oduced. Jaroszeski et al  introduced a specially created multilaye r fusion chamber that facilitated cell-cell
9 contact with the use of mechanical force. This chamber can be adapted for use with different cell types. In 2002, Ramos et al  published studies showing electrofusion of a monolayer of packed cells obtained on a biocompatible filter by well-controlled filtration. Although these mechanical methods have proved to be quite efficient in generating fusion hybrids they require th e use of specially constructed fusion chambers and other equipment. From the above discussed methods it is quite apparent that the process of cellcell contact could benefit from a few improve ments. While most of the methods have been used to produce fusion hybrids efficien tly, each method has its own limitations. Additionally, if one method can be used for certain cell type s it might not prove to be practical for another cell type. This investigation of the use of elect rical charge as a method to bring cells into contact is an effort to find a more generalized protocol for efficient cell-cell contact and subsequent electr ofusion. For this purpose, th e effect of corona charge and electrical charge generated by dir ect current (DC) on cell movement were studied. Both these electrical systems allowe d the use of cells susp ended in PBS. This eliminated the concern of using a non-physiolo gically balanced fusion media as in the case of dieletrophoresis. Furthermore if cel l-cell contact is ach ieved, these systems can be used to produce fusion hybrids in greater numbers ir respective of the adherence properties of the cells. These pot ential advantages were the key motivation to study these two systems for the purpos e of achieving ce ll-cell contact.
10 CHAPTER2: BACKGROUND AND MOTIVATION 2.1 Corona Corona is a self-sustained gas discha rge that is generated by strong electric fields associated with small diameter wire s, needles or sharp edges on an electrode . During corona discharge, a curre nt develops between two high-potential electrodes in a neutral fluid lik e air. This sustained current is produced by ionizing the fluid to create plasma around one electrode. The ions generated in the plasma-process act as the charge ca rriers to the other electrode. Co rona discharge usually involves two asymmetric electrodes, one highly curved (such as the tip of a needle, or a narrow wire) and one of low curvature (such as a plate, or the ground). Corona may be positive, or negative. This is determined by the polarity of the voltage on the highlycurved electrode . 2.2 Key Concepts on the Generation of Corona When high voltage is passed through a c onductor in air, it cau ses ionization of the air around it creating a plasma. Plasma of ten referred to as the Â‘Fourth State of MatterÂ’ is nothing but a gas in its ionized state. A gas becomes a plasma when the addition of heat or other energy causes a si gnificant number of at oms to release some
11 or all of their electrons. Th e remaining parts of those atoms are left with a positive charge, and the detached negative electrons are free to move about. Those atoms and the resulting electri cally charged gas are said to be "ionized." When enough atoms are ionized to significantly affect the electrical characteristics of the gas, it is a plasma . It is this plasma that is responsib le for sustaining the generation of charged particles. Air can be broken down in any of the following 4 ways: glow discharge, corona discharge, sparks a nd arcs. A glow discharge is a cold discharge that generally has a desired effect and is used in neon lamp s, signs and in fluorescent tubes. Sparks are a type of electrical breakdown caused as result of high voltage and very low current as in the case of static el ectricity. Corona is a type of break down where in charged particles are created by ionizing humid air us ing a high electric field. It is an audible and lu minous electric discharge that occurs from very sharp or pointed object or electrodes when the el ectric field attains a very high value. In all these cases of electrical brea kdown, if the charged atoms or particles created are in an electric field they will be accelerated towards one electrode to complete the circuit and constitute a curre nt. The discharge also depends on factors like temperature, relative humidity, pre ssure, chemical composition of the gas. Electron avalanches are the building bl ocks of all true gas discharges . J.S.Townsend was the first to study electron av alanches and their vital role in gaseous discharge. He postulated the theory of Â‘ionization by collisionÂ’ that causes the electrical breakdown of air [ 30]. The formation of a corona discharge relies heavily on the establishment of an of electron avalanche.
12 The initiation of a corona discharge depends on the availability of initiating electrons and a sufficient amount of sustaining electrons to maintain the process of discharge. It has been estimated that a pproximately 20 ion-electron pairs per cubic centimeter-second are produced by naturally o ccurring radiation. This is an adequate number of electrons to initi ate the corona process  The positively charged ions when created will be either attracted very strongly towards or away from the highly curved electrode and the electrons will be attracted in the opposite direction. The direction of motion will depend on the polarity of the applied voltage and this usually prevents the regrouping of the electron and positive ion. The high-energy ions or el ectrons created in the initi al ionization process get accelerated in the electric fi eld and attain enough energy to collide with neutral air molecules and ionize those atoms. This pr oduces a chain reaction where in additional ions and free electrons are accelerated in the field causing additional ionizations. This chain reaction which results in the generati on of a large number of electrons and ions from a single event is referred to as aval anche breakdown or elec tron avalanche . In a corona discharge the electrons and ions produced move toward the positive and negative ions respectively. This movement of electrons and ions constitutes a flow of electric current thr ough the gas. If the polarity of the voltage applied to the wire electrode is positive, the positive ions will flow toward the grounded plate or electrode while the electrons flow to the wi re. If the polarity of the wire electrode is reversed the direction of the flow of ions and electrons will be reversed accordingly . Hence, if the curv ed electrode is positive with respect to the flat electrode, positive corona di scharge is obtained and vice versa.
13 Corona charge produced is a function of the applied voltage. The electrical characteristic of a corona discharge is usually described by a voltage-current (V-I) curve as shown in Figure 2.1 . Figure 2.1 Typical Voltage-Current Curve of a DC Corona Discharge A sufficiently high voltage is requir ed to ionize the air and start an avalanche.The minimum voltage at which the production of corona i ons is initiated is called the corona inception voltage The electric field at initiation depends on the ionization potential of the gas, the mean fr ee path of gas molecules, and the size and surface condition of the high voltage electrode . In the stable corona region secondary ions produced sustain the ionization process. In this region, an increase in the applied voltage causes an increase in the current and a stable discharge is produced. Once the voltage is raised suffici ently high, a spark discharge is produced instead of a stable corona discharge. This is the spark over point limit.
14 Although the basic mechanism by wh ich both the posit ive and negative corona is discharged is common, the voltage ranges for positive corona discharge is slightly different than that for negative corona discharge. The inception voltage for negative corona is around the same as that for positive corona, though the spark over point voltage is much lower. Corona disc harges can be detected in numerous ways. The most obvious way is by the hissing sound that it makes and by a weak bluish glow of visible light that it produces. It can also be detected by charge collecting and measuring devices. 2.3 Applications of Corona Currently corona discharges are effec tive tools for various applications. They are commonly used in commercial electrosta tic devices like photoc opiers, air ionizers and electrostatic precipitators for air pollu tion control [32, 33]. The free-radicals and ions generated in corona reactions can be used to scrub the air of certain noxious products, through free-radical and ion reactions, and can be used to produce ozone . Corona discharges ar e also used for high voltage contact print photography called Kirlian photography . Other applications of corona discharge include treatment of polymer films a nd fabrics , treatment of semi-conductor devices  and treatment of fruits and vegetables in or der to reduce decay and increase shelf-life [37, 38]. A few studies have been proposed to test the benefits of using corona in immunotherapy and medical research. One such study was carried out by Yagi and Yamaguchi to test the effects of corona discharge on the growth of body mass and tumor in rats .
15 2.4 Motivation behind Using Corona as a Cell Contact Method T he key reason behind investigating cor ona as a cell-cell contact method was the low range of current produced (A) due to the ions and electrons. This range of microamperes of current could be app lied to cells without damaging the cell membrane and the constituents of the cells. Furthermore, the use of corona discharge on the cells does not affect the choice of electrofusion medium. As discussed in the previous chapter some of the traditional cell contact methods required the use of a non-physiologically balanced fusion media. Wi th the use of corona as a cell contact method this concern was eliminated. The abil ity to view the electrofusion process and hybrids produced under the microscope as well as the possibility of producing hybrids in greater quantities were ot her incentives that encourag ed this study on the use of corona to enhance cell-cell contact for electrofusion.
16 CHAPTER 3: RESEARCH GOALS Over the past 20 years, there has been a great increase in growth of research in the area of cancer prevention and treatment. This has led to the development of many innovative cancer treatments and has also led to the rise of many investigational studies in the area of cancer immunology. The increase in the use of cell hybr ids in some of the new investigational immunotherapy treatments is the key motiv e behind generalizing a method to make these hybrids. Ongoing clinical trials have paved the way for an increase in demand of tumor/dendritic cell hybrids. As ca n be noticed from published research, electrofusion is the primary method used to produce fused cells. Even with the increased demand for these hybrid cells, th ere have been no major modifications in the electrofusion process since its first few applications. Dieletrophoresis of cells in an AC field is still the most popular met hod used to achieve cell-cell contact even though its non-physiological medium require ments are known to have drawbacks. This investigation is a step towa rds finding a novel cell contact method that eliminates some of the drawbacks of us ing traditional methods, by using electrical charge. As discussed in the previous chapte rs, the use of electrical charge (from DC or produced by corona discharge) over AC a nd some of the other cell contact methods
17 has many advantages. In an attempt to exploi t some of these potential advantages, this study was designed with the following specific aims: To determine whether DC or corona di scharge can be used as a method to achieve tight intercellular contact. To find out the feasibility of usi ng a DC or corona contact process followed by high voltage DC pulses to induce fusion. To determine the conditions required to achieve appropriate cell-cell contact. To uncover the limitations (if any) of using both of these methods of cell contact. To finalize a fusion chamber design th at will incorporate the use of both, cell-cell contact and cel l electrofusion to pr oduce high hybrid yields.
18 CHAPTER 4: MATERIALS AND METHODS 4.1 Cell Preparation 4.1.1 Cell Line and Culture Methods B16-F10 murine melanoma cells (ATCC #CRL-6475: American Type Culture Collection, Rockville, MD) were used for the majority of the experimental work done in this study. The cell line was grown in McCoyÂ’s Medium (Cellgro Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (Cellgro Mediatech, Inc.) and 0.05mg/ml of gentamicin (Cellgro Mediatech, Inc.). The cells were cultured under sterile conditions in 75 cm2 polystyrene canted neck flasks (Corning Incorporated, Corning, NY) and were incubated in 5% CO2 at 37o C (CO2 Water Jacketed Incubator, Forma Scientific, Inc., OH). B16F10 cells were grown as adherent monolayers and required medium renewal and/or sub-culturing every 2-3 da ys. Before sub-culturing, cell monolayers were washed three times with DulbeccoÂ’s Phosphate-Buffered Saline (DPBS 1X w/o Ca and Mg; Cellgro Mediatech, Inc.) suppl emented with 0.05mg/ml of gentamicin. Cells were detached using 0.25% trypsinEDTA (Sigma Chemical Co., St. Louis, MO). Cells that were difficult to detach were placed in the incubator at 37C for
19 approximately one minute to facilitate disp ersal. The trypsin-EDTA was neutralized with growth media prior to aspirating the cells. Whenever required, a portion of the aspirated cells were sub-cultured with a ratio of 1:12. All sub-culturing was carried out under sterile conditions in a biological safety cabinet (Class II A/B3 Biological Safety Cabinet, Forma Scientific). In order to validate cell contact re sults achieved with co rona discharge the NT2 (NTERA-2 cl.D1, ATCC #CRL-6475: Am erican Type Culture Collection, Rockville, MD) cell line was used for a few experiments. These NT2 cells were cultured and harvested with the same media and by the same methods as the B16 cells. 4.1.2 Cell Counting Harvested cells were prepared for counting by washing with DPBS three times. Cells were centrifuged (5810R, E ppendorf, Westbury, NY) at 220 x g for 5 minutes at 20oC and suspended in approximately 5ml of DPBS for each wash. A sample of the cells was then diluted in 0.9% sodium chloride (APP, Schaumburg, IL) and 0.4% trypan blue stain (Cellgro Mediat ech, Inc.). Trypan blue penetrates the membranes of the dead cells and causes th em 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: No. of cells/ ml = cells counted per mm2 x dilution (if used) x 10,000 Where, 10,000 is the conversion factor for 0.1 l to 1ml
20 The percent viability of the cells was also determined after counting. Only those cell cultures that were 85% 100% viable were used for experimentation. 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 a fluorescent microscope. 4.2.1 Stock Solution of Dyes Stock solutions of fluorescent dyes we re prepared in advance using the procedure discussed by Jaroszeski et al [ 24, 25]. The fluorescent dyes used for this study were 5-(and-6)-(((4-chloromethyl) benzoyl)amino) tetramethylrhodamine (CMTMR; Molecular Probes, Eugene, OR) a nd 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes). Both dyes were supplied by the manufacturer in 1mg aliquots. Stock solutions of 5mM concen tration of both dyes were prepared in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO). CMTMR (MrW 554) stock solution was made by mixing the supplied 1 mg aliquot of CMTMR with DMSO to yield a final volume of 3 60l. Correspondingly CMFDA (MrW 465) stock solution was made by mixing the supplied 1 mg ali quot of CMFDA with DMSO to yield a final volume of 430l. Both dyes were easily dissolved in DMSO at room temperature. The DMSO stock solutions were divided into single-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 helps to avoid
21 freeze-thaw cycles of the stock solutions, and hence increases shelf life and ensures consistent results. 4.2.2 Staining Technique For all experiments that utilized stai ned cells, B16F10 cells harvested from a flask were sub-cultured in 2 separate flasks with the same ratio. After 2-3 days the cells reached the desired confluence and were ready to be stained. One aliquot of each CMTMR and CMFDA was removed from st orage and was defrosted to room temperature. All staining was carried out under sterile conditions in a biological safety cabinet. The growth media in both th e flasks was reduced to 7ml, just enough to cover the monolayer of cells on the fl asks. One flask was stained in 120l of CMTMR and the other flask was stained in 40l of CMFDA. The cultures were then incubated at 37oC for two hours. After the incuba tion time was completed the cells were harvested and counted by the regular method using a hemacytometer. 4.2.3 Fluorescent Microscopy A fluorescent microscope (Leica DM IL Leica, West Germany) was used to observe the contact of CMTMR and CMFDA stained cells as well as dual fluorescing fusion cells. Under fluorescent light there was a clear visual distinction between the fused and un-fused cells. The un-fused CMTMR st ained cells appeared red, the un-fused CMFDA stained cells appeared green and th e fusion products of the two were easily
22 distinguished by their orange/y ellow color. Apart from the difference in color, the fusion cells were larger in si ze and were irregularly shaped. 4.3 Media for Electrofusion The media in which cell-cell electrofusi on was conducted in most experiments was DPBS (Cellgro, Mediatech, Inc.). Th e harvested cells (either stained or unstained) were counted and DPBS was then added to the cell solu tion to adjust the concentration of cells as per the requirement of the experiments. 4.4 DC Cell Contact Apparatus 4.4.1 DC Power Source Cell-cell contact was inves tigated using DC from a regulated power supply (model K18S60, Acopian, Easton, PA). In orde r to regulate the amount of voltage flowing to the constructed fusion chambers a voltage divider was added to the circuit. The circuit used for all the DC experiments is shown in figure 4.1
23 Figure 4.1 DC Circuit Figure 4.2 DC Power Source and Voltage Divide Control
24 4.4.2 Chambers Used for DC Experimentation For all of the cell contact experiments, the cell contact chamber consisted of two electrodes attached to either polystyrene petri dishes or glass microscope slides. The configurations of the chamber were varied by changing the material of the electrodes, their geometrical configurati ons or by coating them with different materials. 18.104.22.168 Contact Chamber with Copper Wires Four inch long copper wires were bent to form two circul ar electrodes and were attached to a polystyrene petri di shes using epoxy (ITW Devcon, Danvers, MA). Two types of configurations of copper electrodes were tested. One chamber consisting of semi-circular copper elect rodes (Figure 4.3) and another chamber consisting of S-shaped copper electrodes (Figure 4.4). Figure 4.3 DC Contact Chamber with Semi-Circular Copper Electrodes
25 Figure 4.4 DC Contact Chamber with S-Shaped Copper Electrodes 22.214.171.124 Contact Chamber with Stainless Steel Strips Stainless less steel strips of 4mm widt h were attached to a microscopic slide to form a fusion chamber. The distant be tween the stainless steel electrodes was 3mm. Figure 4.5 DC Contact Chamber with Stainless Steel Strip Electrodes 126.96.36.199 Contact Chamber with Stainless Steel Wires Contact chambers consisting of stainle ss steel wire electrodes were prepared by attaching stainless steel wires (Type 304V Small Parts Inc., Miami Lakes, FL) of 0.038 inch diameter to petri dishes with e poxy. Two main configurations of this type
26 electrode material were studi ed. In one, the electrodes were semi-circular (Figure 4.6as in the case the copper electrodes discusse d above) and in the other, two straight electrodes were attached para llel to each other (Figur e4.7). A distance of 5mm was maintained between the two parallel electrodes. Figure 4.6 DC Contact Chamber with Stainless Steel Circular Electrodes Figure 4.7 DC Contact Chamber with Pa rallel Stainless Steel Electrodes. 188.8.131.52 Contact Chambers with Coat ed Stainless Steel Wires Using the parallel configuration of th e stainless steel electrodes shown in Figure 4.7, 8 other variations of the same ch amber were created by coating either one or both electrodes with wax (Sealing Wax, Yaley Enterprises, Redding, CA), varnish (Delta Technical Coatings, Whittier, CA), silicone conformal coating (Techspray,
27 Amarillo, TX) or Teflon tubing (Small Parts Inc) In all of the 8 chambers, a distance of 5mm was maintained between the parallel stainless steel wires. 184.108.40.206 DC Fusion Chamber Design After investigating the cell contact prope rties with the previously discussed chambers, a final design for a DC fusion chamber was decided (Figure 4.8). This consisted of 2 stainless steel electrodes (Small Parts Inc.) attached to a glass microscopic slide. One of the electrodes was covered with Teflon tubing (Small Parts Inc). Also attached to this chamber were 2 additional electrodes made of flat stainless steel strips. All four of the electrodes were mounted on top of a microscope slide to form a cavity to place the media containing the cells. The cavity measured 4 mm x 6 mm and had an approximate height of 1.5 mm. Figure 4.8 DC Fusion Chamber Design
28 4.5 Corona Apparatus 4.5.1 Corona Generator The corona generator (Figure 4.9 and 4.10) consists of a corona generating element that emitted ions from a 25mm diamet er hole in a stainless steel ground plate. The wire plate geometry of the corona generating element consisted of 9 needles (stainless steel acupuncture needles, gauge no 30,SGAMAC, China) that were contained within a central hol e in a circular white tefl on body. Eight of the needles were arranged in a circle of 9mm diameter with the ninth needle in the center. The height of the needles was adjusted to a hei ght of 6.8mm from the base of the central hole in the teflon body. The circ ular ground plate was attached to the base of the white teflon body which was mounted on a mi cromanipulator. The micromanipulator enabled the corona element to be lowere d to a convenient distance of 8.0 Â– 9.0mm from the cells attached to th e bottom of a petri dish for exposure. It would also allow it to be raised to expose new set of cells. All the corona generating needles in this element had a common connection to the vol tage output of a hi gh voltage DC power supply.
29 Figure 4.9 Bottom View of the Corona Generator Figure 4.10 Side View of the Corona Generator 4.5.2 Corona Experimental Setup The corona experimental setup was composed of the corona generator, a charge collecting plate, an electromet er (model 6517A, Keithley Instruments Inc.,
30 OH), a high voltage power supply (CZE 2000, Spellman High Voltage Electronics, Hauppauge NY), a data acquisition card ( DAC) (PCI 6036 E, National Instruments, Austin, TX), a computer (Dell Dimension 2400, Intel P4, Dell Inc, TX) and LabVIEW (LacVIEW 7, National Instrument s, TX) computer software. Figure 4.11 diagrams the instrument setup from the power supply to the computer. Figure 4.11Experimental Setup of Corona Apparatus The power supply used in the experi ment was a programmable Spellman CZE 2000 and was controlled by the data ac quisition card (DAC). The DAC was connected to the power supply with the he lp of an SCB 68 pin accessory. A program was written in LabVIEW to control the coro na generation. The program basically let the user enter the input voltage, curren t and time for corona generation. The instrumentation also had a temperature and humidity probe that read the temperature and humidity during the experiments. Both these parameters were also read by the
31 program so that the user could monitor the effect of these two conditions on the corona generation process. Figure 4.12 show s the virtual interface of the LabVIEW software. Figure 4.12 Virtual Interface of the LabVIEW program The power supply had a reversible polarity. This meant that the output could be adjusted between positive and negative polarity. The polarity was changed using a signal sent through the data acqui sition card. The high voltage was connected to the positive lead of the corona ge nerator. The ground was connected to the negative lead of the corona generator as well as the ground from the electrometer.
32 The electrometer was used to meas ure the current output of the corona generator prior to all the experiment tests. One end of an input cable terminated at a 3-slot male triax connector that attached to the electrometer. The other end of the cable had two alligator clip s; the input high was connected to the metal collecting plate while the input low was conne cted to the common negative lead. 4.5.3 Fusion Chambers Inve stigated with Corona Two configurations of fusion chambers were investigated for use with the corona generator. The first chamber consis ted of two parallel stainless steel wires attached to the bottom of a petri dish at a distance of 0.5cm apart. Figure 4.13 shows the simple setup of the chamber. Figure 4.13 Corona Chamber with Parallel Electrodes After observing the cell contact properti es of this chamber when used with corona, a final chamber was designed. This final chamber (Fig4.15) consisted of a circular outer stainless steel wire and an inner circular stainle ss steel plate having a thickness of 3mm. The centra l plate was connected to an electrical wire from the bottom of the chamber. Both the outer stai nless steel electrode and the central plate
33 electrode could be connected to a ground source during co rona treatment and to the electroporator during electrofusion. Figure 4.14 Corona Contact Chambe r with Circular Electrodes
34 CHAPTER5: RESULTS AND DISCUSSION 5.1 Effect of DC on Cell Movement The use of DC for inducing cell contact wa s first initiated as a result of the visual observations of move ment of cells in a DC field between two copper electrodes. B16 cells suspended in PBS wh en placed in a petri dish between two copper electrodes connected to a low voltage DC source showed some translational movement towards or away from one of th e electrodes. In order to exploit this translational movement to enhance cellcell contact a number of different cell-cell contact chambers were de signed and investigated. The first set of DC cell contact e xperiments which were carried out in chambers with semi-circular (Figure 4.3) or s-shaped copper (Figure 4.4) electrodes showed some cell movement but problems of cell death and toxici ty were extensive due to oxidation of copper B16 cells suspended in PBS were adjusted to a concentration of 5x105 cells/mL of PBS and were placed in the chambers. Through the copper electrodes the cells were expos ed to voltages rang ing between 1.002.00 V. Although slight cell movement was obs erved in the vicinity of the positive electrode, the formation of a green film in the area surr ounding the negati ve electrode led to pervasive cell death. Th is green film formed due to the characteristic oxidation of Cu (Copper) to Cu+2 proved to be lethal to the cells.
35 In order to overcome the problem of copper oxidation th e copper electrodes were replaced by stainless steel strips (Figure 4.5). Since some cell movement was observed in the earlier set of chambers the distance between the electrodes was reduced to 3mm in order to exploit the small distance the cells traveled. All other experimental conditions were kept constant. The applica tion of the same amount of voltage led to bubble and froth formation at the negative electrode which resulted in heavy cell death. The voltage range was further widened an d very low voltages (from 0.012.00) were tested. At a voltage less th an 1 V bubble formation was significantly reduced but no cell movement in any direction was observed. A new set of fusion chambers were th en designed to furt her investigate the appropriate amount of DC voltage requi red to move B16 cells without causing substantial cell damage. The contact cham bers were increased in size and the electrode shape was change d from strips to thin stainless steel wires (Figure4.6). In order to compensate for the increase in ch amber size, the concen tration of the cells was increased to 2 x 106 cells/mL. This was done to ensure substantial cell-cell contact with very little move ment of the cells. The stainles s steel wires were arranged in a semi-circular style similar to th e chamber with the copper electrodes. Investigation of this chamber with volta ge ranging from 0.012.00V did not show any significant in cell movement. The cham ber was then modified by replacing the semi-circular electrodes with two straight stainless steel electrode s kept parallel to each other (Figure 4.7). Keepi ng all the other experimental conditions constant this chamber configuration was tested for cell movement at the same voltage range of 0.01-2.00V. At a voltage range between 1.5 and 2.0 V an in cell movement towards
36 the positive electrode was observed. At th e negative electrode there was still a considerable amount of cell death due to bubble formation and frothing of the fusion media at this electrode (Figure 5.1). Figure 5.1 Bubble Formation at the Negative Electrode The froth or bubble formation on the ne gative electrode was attributed to Joule heating. Joule heating is the increase in temperature of a conductor as a result of resistance to an electrical current flowi ng through it. In previous studies involving electrofusion [42, 10, 5] the problem of joule heating was eliminated by coating one of the electrodes with wax. In an effort to re-create the same effect in our system a number of different coatings were teste d. The same parallel el ectrode configuration was maintained and new chambers were designed in which one or two electrodes were coated with wax, varnish, silicone conformal coating or Teflon tubing. Chambers in which one or both of the elec trodes were coated with wax, varnish and
37 silicone conformal coating did not show any substantial decrease in cell death. The electrodes with Teflon tubing however did no t show any visual signs of frothing and bubble formation. The chamber in which onl y one stainless steel electrode was covered with Teflon tubing proved to be most suitable for cell contact with DC. Substantial cell movement and signs of high cell viability was observed around both electrodes at voltages ev en as high as 15 V. 5.2 Final DC Fusion Chamber Bearing in mind all the findings of the previous experiment s, a protocol and chamber design for DC enhanced cell c ontact and subsequent electrofusion was finalized. The fusion chamber consisted of two stainless steel electrodes (one of which was covered with Teflon tubing) for DC cell contact and two other electrodes for effective electrofusion of cells in contact (Figure 4.8). For a cell conc entration of 2 x 106 cells/mL a voltage of about 15 V ( 30 V/cm) furnished considerable cell movement. The recorded current at this voltage for this system was between 14.2916.05 mA. Although cells appeared to move toward s both electrodes within a span of 2-4 minutes the cell movement did not appear to significantly enhance cell-cell contact. Figure 5.2 shows the cells moving upwards towards the positive electrode.
38 A) Cells at in the first 1-2 sec of DC Application B) Cells in after 1 min of DC Application C) Cells after 3 min of DC Application Figure 5.2 Cell Movement in DC Environment As can be seen in Figure 5.2 C the cell contact achieved afte r 3 minutes of DC is not is not as exceptional as that achieve d through some of the previously discussed methods. This is further strengthened by th e fact that electrofusion results obtained by high voltage pulses after DC c ontact were extremely low. The primary cause of low cell-cell to contact even with substantial cell movement is attributed to the settling of so me B16 cells in the first instant they are
39 placed in the chamber. As can be observed in Fig 5.2 A, a substantial fraction of cells have already settled on the bottom of the cham ber. These settled cells are not affected by the DC current. Only the cells that re main suspended in PBS are pulled towards either electrode under the influe nce of electrostatic forces. 5.3 Conclusion and Summary of DC Contact Experiments The quick settling of the B16 cells at the bottom of the chamber was due to surface interaction of the cells and the glass slide on which the chamber was built. As a result of either static friction or elect rostatic attraction the cells were instantly attracted to the surface of the glass slide. Even at a voltage of 40 V/cm the force applied by the DC current was not adequa te enough to overcom e these attractive forces. In an attempt to increase the ce ll movement a number of surface treatments were investigated. Prior to DC contact the glass slide was connected to a ground source in order to drain any re sidual charge that might cause the cells to get attracted to the glass surface. To counteract the sta tic frictional forces, the glass slide was treated with hydrophobic coatings like RAIN -X, silicone conformal coating and Teflon film. All these coatings did not appe ar to significantly d ecrease the number of settled cells in the chamber. After assessment of all the different DC chamber configurations the final DC fusion chamber design exhibits the highest potential for enabling cell-cell contact. Although cell-cell contact obtaine d with the final chamber is not extensive, a few improvements in chamber design may help at tain better cell contact and subsequently
40 higher fusion yields. Investig ation of different non-ioni c hydrophobic coatings to treat the surface of the DC fusion chamber might be the next step towards further improving the fusion yields with th is type of fusion chamber design. 5.4 Calibration of the Corona Generator Prior to corona experimentation the cor ona generator was calibrated. This was done to determine how much charge would be generated by corona discharge at different voltages. The quantity of corona charge emitted was determined by a collector plate that was connected to an electrometer. The electrometer helped to monitor the current generated by the discha rge for a specific a pplied voltage at a given time. With the help of the microman ipulator the distance between the corona generator and the charge collecting plate was adjusted to 0.8cm. This enabled the measurement of charge affecting the cells in the corona contact chamber that was kept at the same distance. For a particular voltage and polarity the corona generator emitted a specific charge. This emitted charge remained approximately constant as long as the dimensions of the corona generating appara tus (temperature, humidity, height of the needles, distance from collecting plate, etc) were unchanged. Figure 5.3 shows the plot of the applied voltage versus the ch arge collected for both positive and negative corona.
41 Figure 5.3 Plot of Applied Volta ge versus Charge Collected All corona experiments were carried out after the generator was calibrated once (Table 5.1). In between experiments, th e corona generator was tested using the charge collecting plate to ensure that th e same amount of current was emitted for a particular voltage range. Calibration of the instrument also allowed for the maintenance of constant experimental conditi ons. In the event that the needles of the generator or the height of the needles were changed the generator could be recalibrated and the voltage could be adjusted to the corresponding limit that emitted the same amount of charge. Applied Voltage vs Charge Collected0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 -20.00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 Current (A/-A) Voltage (kV) Positive Corona Negative Corona
42 5.5 Investigation of Corona Induced Cell Contact The effect of corona discharge on cells suspended in PBS was first tested in a petri dish kept at a distance of 6.74mm from the corona generato r. For these Initial Experiments the cell concentration was adjusted to 2 x 106 cells/mL and effect of corona on cell movement was tested at diffe rent time intervals. The applied voltage was in the range of 6.5kV for negative co rona and 7kV for positive corona. Although some cell contact was observed with pos itive or negative corona in one or two experiments, for the most part no si gnificant cell contact was achieved. A design for a corona contact chamber was proposed for further investigation of the effect of corona on cell movement. The same experiments were then carried out in the corona contact chamber (Figure 4.13) with parallel electrodes. Due to the presence of the electrodes in the center of the chamber, the distance between the chamber and the generator had to be incr ease to 8mm.The application of positive corona at 7 kV or negative corona at 6.5 kV did not produce any substantial cell contact even when tested with long corona exposure times (Figure 5.4-A). In order to thoroughly test the use of the designed chamber the electrodes were connected to a ground source. Keeping the cell concentr ation constant B16 cells suspended in PBS were treated with positive corona discharge at 7 kV for 5 minutes. Due to the effect of positive corona discha rge the cells grouped to form small random clusters (Figure 5.4-B). Within these cluste rs adjacent cells were pushed closed to each and appeared to be in contact. Switc hing polarities and tr eating the same volume
43 of cells to negative corona at 6.5 kV for the same amount of time, provided the same results (Figure 5.4-C). A) Positive Corona Treatment with no Electrodes Grounded B) Positive Corona Treatment with Grounded C) Negative Corona Treatment with Grounded Electrodes Electrodes Figure 5.4 Effect of Grounded Electr odes in Corona Contact Chamber
44 While a cell contact was achieved by th is method, the lack of an explanation for the irregular pattern in the formation of the cell contact clusters left room for further investigation and developm ent of the contact chamber. In order to match the radial output of the corona generator a circular contact chamber was designed (Figure 4.14) to be used to treat cells with corona discharge. The circular chamber consisting of two con centric electrodes had an annular gap that was between 3-4mm wide and was used to treat cell solution volumes of 150170 L. Treatment of cells with positive corona for 5 minutes at 6.5kV with grounded electrodes produce exceptional cel l-cell contact in certain s ections of the chamber. After 5 minutes of corona tr eatment a large number of cells were driven into contact with each other due to aggregation of th e cells towards the center of the annular region between the electrodes. Figures 5.5 a nd 5.6 show the aggregation of cells due to positive corona discharge in the grounde d circular corona c ontact chamber at 40x and 100x magnification respectively. The ce ll contact results achieved with positive corona in the grounded circul ar contact chamber were reproduced when the same volume of cells were treated with negative corona at 6.5 kV for the same amount of time. The switch in polarity did not ch ange the cell contact results obtained.
45 Figure 5.5 Aggregation of Cells in th e Grounded Circular Contact Chamber (100x) Figure 5.6 Aggregation of Cells in the Grounded Circular Contact Chamber (400x)
46 5.6 Effect of Combined Negative and Positive Corona Treatment For investigation purposes experiment s were conducted in which cells were subjected to 5 minutes of positive corona at 6.5kV and then 5 minutes of negative corona at 6 kV successively. As compared to treating cells with only one type of charge this method produced outstanding cel l contact results a ll over the corona contact chamber. The order in which the polarities were run did not make a difference in cell contact results achieved. After successive treatment of positive corona followed by negative corona or nega tive corona followed by positive corona almost all the cells were pushed towards th e outer edges of the annular region. Figure 5.7-A shows the stained cells in the corona chamber when left without treatment for a period of 5 minutes. Figure 5.7-B shows the gro uping of cells into small clusters after treatment with 5 minutes of positive corona at 6.5 kV. Similar results were obtained with treatment of negative corona for the same amount of time. Figure 5.7-C shows the results obtained with combined successive treatment with positive corona for 5 minutes at 6.5kV and then 5 minutes of nega tive corona at 6kV. As can be observed in the figure, the cells are in contact on th e outer edge of the annular region between the electrodes.
47 A) B16 Cells in Circular Corona Chamber prior to Treatment B)After Treatment of 5 minutes of Positive C) After Treatment of 5 minutes of positive corona followed by 5 minutes of negative corona Figure 5.7 Effect of Successive Treatment of Positive and Negative Corona on Cells Another advantage of this successive treatment is that aggregation of cells occurs all around the chamber and is not confin ed to certain sections as in the case of single polarity corona treatment. Figure 5.8 shows the different sections of the chamber after treatment with of 5 minutes of positive corona at 6.5kV followed by 5 minutes of negative corona at 6kV.
48 A)Top B) Bottom A) Left B) Right Figure 5.8 Different Sectional Views of the Circular Chamber after Treatment This type of successive corona treatment provided the best cell contact results with the current design of the circular corona contact chamber only when the electrodes were connected to a ground s ource. After cell contact was achieved the electrodes of the contact chamber were c onnected to an elect roporator to induce electrofusion. The generation of successful electrofusion re sults further substantiated the exceptional cell contact produced by this method.
49 5.7 Determination of Fusion Conditions for B16 Cells: The appropriate conditions for cell fusion are a very important aspect in the production of hybrid cells. For any fusion chamber to operate successfully, the application of suitable pulse parameters should produce substantial fusion yield accompanied by low cell damage. These pulse parameters usually vary with different fusion chamber designs. In order to determine the best conditions for cell fusion in the circular corona fusion chamber, a round of experiments were conducted in which stained B16 cells were subjected to a range of fusion c onditions after sufficient cell contact was achieved with corona. Table 5.1 shows the ch ange in different fusion parameters and its corresponding effect on cell fusion yiel d and cell damage. In these experiments both fusion yield and cell damage were ex amined by visual observation under the microscope. The fused cells were distinguish ed from the un-fused red or green cells by their characteristic yellow/o range color, irregular obl ong shape or large size. Cell death was distinguished by the visual di sintegration of the cell membranes, the formation of a mucous film or by the leak age of stain to the surrounding media.
50 Table 5.1 Determination of Fusion Conditions Electrofusion Parameters Corona Contact Conditions Field Strength (V/cm) Pulse Width ( sec) Number of Pulses Fusion Yield Visible Cell Damage 5 minutes of (+)ve corona at 7kV followed by 5 minutes of (-)ve corona at 8kV 3000 100 6 Low Fusion Yield Extremely High Cell Damage 5 minutes of (+)ve corona at 7kV followed by 5 minutes of (-)ve corona at 8kV 3000 25 6 Low Fusion Yield High Cell Damage 5 minutes of (+)ve corona at 7kV followed by 5 minutes of (-)ve corona at 8kV 2500 100 10 High Fusion Yield Moderate Cell Damage 5 minutes of (+)ve corona at 7kV followed by 5 minutes of (-)ve corona at 8kV 2500 50 10 Moderate Fusion Yield Moderate Cell Damage 5 minutes of (+)ve corona at 7kV followed by 5 minutes of (-)ve corona at 8kV 2500 25 10 Low Fusion Yield Low Cell Damage For all the experiments the cell conc entration was kept constant at 2 x 106 cells/mL and the same corona contact conditio ns were used prior to fusion. As can be observed from Table 5.5 the conditions th at provide optimum fusion yield after sufficient contact is ach ieved are 10 pulses of 100 sec pulse width at 2500 V/cm. Figure 5.9 shows typical fusion results obtai ned after corona contact for 5 minutes and 10 fusion pulses of 100 sec pulse width at 2500 V/cm. The fused cells can be easily distinguished from the un-fused cells by their distinctive yellow/orange color and irregular large shapes.
51 Figure 5.9 Fluorescent pictures of Fused B16 Cells.
52 5.8 Determination of Time Required for Cell Contact To economize the use of the corona contact chamber and to prevent decrease in cell viability the minimum amount of treatment time needed to be determined. A set of experiments were conducted to determine the minimum amount of time required for substantial corona induced contact. As discussed in section 5.6 considerable cell contact was achieved with successive treatment of positive corona followed by negative corona (or vice versa) for equal amounts of time. In order to determine the effect of treatment times, B16 cells were treated with equal intervals of positive and negative corona successively for 5, 4, 3 and 2 minutes. Prior to treatment the cells were stained with CMTMR and CMFDA dyes and were mixed to a get a final concentration of 2x106 cell/mL. After significant contact was achieved with corona treatment, the ce lls were fused using 11 high voltage pulses of 100 sec pulse width at 2500V/cm. Under a fl uorescent microscope the fused cells which were visibly distingui shed from the un-fused cells by their characteristic irregular oblong shape or large size were counted using a hemacytometer. Figure 5.10 shows a plot of the average percent yield ob tained for different contact times. As can be observed, yield increased as corona c ontact time increased. This indicates that a higher level of contact was achieved as th e corona treatment time was increased.
53 % Yield vs Contact Time 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 2+23+34+45+5 Contact TimeAverage % Yield Figure 5.10 Plot of % Fusion Yiel d versus Corona Treatment Time 5.9 Effect of Corona Induced Contact on Cell Viability The use of electric charge for any type of cell treatment can sometimes have an adverse effect on cell viabi lity. If the intensity of char ge or the amount of corona treatment time did affect cell viability in the current corona c ontact system it would result in poor fusion yields. It was therefore essential to determine the effect of just corona charge on cell viability in the circular corona contact chamber. For this purpose cells were treated successively with equal intervals of positive and negative corona at the maximum possible charge (6.5 kV for positive corona and 7kv for negative corona) for 5, 4, 3 and 2 minutes. The cells were not subjected to electrofusion pulses as this experiment was aimed at determining the effect of onl y corona treatment on cell viability. After corona treatment the cells were extracted fr om the chamber, diluted with 0.9% saline and 0.4% trypan blue and counted 3 times in a hemacytometer.
54 A plot of the percent decrease in cell vi ability after corona treatment is shown in Figure 5.11. As can be observed the decrea se in cell viability for highest corona contact time is only 6%. Although longer co rona treatment times have a higher decrease in viability, this decrease in cell viability is quite negligible when compared to some of the other methods of cell contact. 0 2 4 6 8 10 12 14 16 18 20 5+54+43+32+2 Positive + Negative Corona Treatment (mins)% Decrease in Viability Figure 5.11 Plot of % Decrease in Viability versus Corona Treatment Time 5.10 Results with a Different Cell Line After determining the effect of corona on cell viability and its feasibility in producing substantial fusion yields, it was im perative to find out if corona discharge could be used to induce cell contact in other t ypes of cells. For this purpose the effect of corona treatment on NT2 cells was tested in th e circular corona contact chamber. Prior to experimentation the cells were harvested, counted and adjusted to a concentration of 2 x 106 cells/ml (as in the case of B16 cells). The cells were treated with positive corona at 6.5 kV for 5 minutes followed by negative co rona for 5 minutes. On combined corona
55 treatment of the NT2 cells w ith the circular electrodes gr ounded, a significant amount of cell contact was achieved. The NT2 cells aggregat ed in the centre or on the outer edges of the annular region between the electrodes. Figure 5.12 shows the aggregation of NT2 cells after combined positive and negative corona treatment in the circular corona contact chamber. Figure 5.12 Aggregation of NT2 Ce lls after Corona Treatment In order to determine if contact of two different types of cells could be achieved simultaneously in the circular corona contact chamber, a set of experiments was conducted with both NT2 cells and B16 cells. The NT2 cells were stained green with CMFDA and the B16 cells were stained red CMTMR fluorescent dyes. Equal volumes of both stained cells were mixed t ogether prior to placement in the chamber. Treatment with 5 minutes of positive corona at 6.5kV followe d by 5 minutes of negative corona at 6kV
56 induced a significant amount of cell contact between the two different types of cells. Figure 5.13-A shows the stained cells prior to corona treatment and Figure 5.13-B shows the cells in contact after combined corona treatment. A) NT2 cells and B16 cells prior to corona treatment B) NT2 cells and B16 Cells after combined corona Treatment Figure 5.13 Corona Induced Contact of NT2 Cells and B16 Cells From these set of experiments we conc luded that corona induced contact can be achieved for not only B16 cells but other types of cells as well. Thus, corona discharge can be used as an effective way to bring cel ls into close adjacent contact for the purpose of electrofusion.
57 CHAPTER 6: SUMMARY AN D RECOMMENDATIONS 6.1 Summary In an attempt to investigate the use of electrical charge as a cell contact method this study led to the developmen t of two simple fusion chambers. One incorporates the use of DC and the other in corporates the use of corona discharge for the purpose of achieving cell-cell co ntact prior to electrofusion. The final DC chamber design demonstr ates a high potential for achieving considerable amounts of cel l-cell contact with furthe r surface treatment of the chamber. The inability of the electric charge generated by DC to overcome the surface interactions between the cells and chamber surface is a functional limitation of this chamber. Further i nvestigation of cell and glass surface interactions might be the next step towards further improving th e cell contact results and increasing fusion yields for this DC fusion chamber design. The circular chamber designed for use with corona discharge produces outstanding cell-cell contact and consequent ially provides fusion yields as high as 40%. From all experimental observations obtai ned during the course of this study it is clear that a substantial am ount of cell contact and subs equent fusion results are obtained when cells are treated in the circular corona chamber with grounded electrodes by treatment with least 4 minutes of positive corona followed by 4 minutes
58 of negative corona (or vice versa). Furthe rmore unlike other elec trofusion chambers the large amount of cell contact obtained with this chamber is accompanied by a minor decrease in cell viability (about 6%). As can be seen from the last set of experiments this method of inducing cell cont act can be used for cell lines other than B16 cells. Some other important advantag es of using this chamber design for electrofusion are the ability to use physiol ogically balanced fusion media (PBS in this case), the ability to view the cell cont act and electrofusion process under the microscope and the capacity to produce hi gher fusion yields in large samples. 6.2 Recommendations As an effort to try and determine th e reason behind cell aggregation due to successive corona treatment, an attempt to was made to measure the current in the chamber during corona treatment with the he lp of a micrometer. These tests did not return any tangible results. During the proce ss of corona contact the corona discharge was in a way causing some flow of charge through the PBS that was perhaps turning the cells into dipoles and cau sing them to get attracted to each other. But current measurements of PBS or of the grounded elec trodes (with the help of resistors) did not return any results. It was unclear as to whether th e failure to obtain current readings was due to the inability of the micrometer to measure a current that was lower than its scope or because the volume of the cell solution was too small to get any sizeable readings. The investigation of a suitable method or device to measure the current in the chamber during the treatment of corona is perhaps the first step towards providing a
59 hypothesis for the peculiar aggregation of ce lls in this system. The presentation of a mechanism for the flow of cells in the chamber can help in further optimizing the chamber to yield better ce ll contact results and he nce higher fusion yields.
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65 Appendix AData for Calibrati on of the Corona Generator Table A.1Calibration Data for the Corona Generator: Experimental Conditions Temperature Range: 22.7C Â– 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
66 Appendix BDetermination of Time Required for Cell Contact Table B.1 Fusion Yield for Different Cell Contact TimesExperiment 1 Volume in Fusion Chamber: 150 L Temperature Range: 22.6C Â– 22.9C Relative Humidity: 55.8.4 % 56.4 % Distance from Ground plate: 0.8cm Contact Time Fusion Conditions No. of Fused Cells No. of UnFused Cells % Yield 5 min of (+)ve corona at 6.5kV followed by 5 min of (-)ve corona at 6kV 2500 V/cm, 100 s, 11 pulses 100 178 36 % 4 minutes of (+)ve corona at 6.5kV followed by 4 minutes of (-)ve corona at 6kV 2500 V/cm, 100 s, 11 pulses 128 296 30 % 3 minutes of (+)ve corona at 6.5kV followed by 3 minutes of (-)ve corona at 6kV 2500 V/cm, 100 s, 11 pulses 44 247 15% 2 minutes of (+)ve corona at 6.5kV followed by 2 minutes of (-)ve corona at 6kV 2500 V/cm, 100 s, 11 pulses 15 200 7%
67 Appendix B (continued) Table B.2 Fusion Yield for Different Cell Contact Times-Experiment 2 Volume in Fusion Chamber: 150 L Temperature Range: 22.6C Â– 22.9C Relative Humidity: 55.8.4 % 56.4 % Distance from Ground plate: 0.8cm Contact Time Fusion Conditions No. of Fused Cells No. of UnFused Cells % Yield 5 min of (+)ve corona at 6.5kV followed by 5 min of (-)ve corona at 6kV 2500 V/cm, 100 s, 11 pulses 152 227 40 % 4 minutes of (+)ve corona at 6.5kV followed by 4 minutes of (-)ve corona at 6kV 2500 V/cm, 100 s, 11 pulses 103 219 32 % 3 minutes of (+)ve corona at 6.5kV followed by 3 minutes of (-)ve corona at 6kV 2500 V/cm, 100 s, 11 pulses 57 258 18% 2 minutes of (+)ve corona at 6.5kV followed by 2 minutes of (-)ve corona at 6kV 2500 V/cm, 100 s, 11 pulses 20 233 8%
68 Appendix CEffect of Corona on Cell Viability Table C.1 Experimental Data Showi ng % Difference in Cell Viability Contact Time Initial Cell Viability (%) Viability After Corona Treatment (%) Average Cell Viability After Corona Treatment % Decrease in Cell Viability 84.96 84.31 5 min of (+)ve corona at 6.5kV followed by 5 min of (-)ve corona at 6kV 90.31 83.96 84.41 6.53% 83.45 81.89 4 minutes of (+)ve corona at 6.5kV followed by 4 minutes of (-)ve corona at 6kV 86.25 83.6 82.98 3.79% 79.56 79.87 3 minutes of (+)ve corona at 6.5kV followed by 3 minutes of (-)ve corona at 6kV 81.43 80.5 79.98 1.78% 77.7 79.67 2 minutes of (+)ve corona at 6.5kV followed by 2 minutes of (-)ve corona at 6kV 79.91 80.61 79.32 0.74%