WO2005063961A2

WO2005063961A2 - Electrofusion chamber and method for the quantification of hybridoma yields

Info

Publication number: WO2005063961A2
Application number: PCT/SI2004/000048
Authority: WO
Inventors: Marko Kreft; Sonja Grilc; Igor Poberaj; Mateja Gabrijel; Helena Haque Chowdhury; Robert Zorec
Original assignee: Celica, D.O.O.
Priority date: 2003-12-30
Filing date: 2004-12-28
Publication date: 2005-07-14
Also published as: WO2005063961A3; SI21661A

Abstract

The subject of the invention in the field of biomedical technology is the evaluation procedure for the determination of the yield of hybrid cells - hybridomas in a cell vaccine and cell products, which consists of measurements of the area of co-localized pixels of an image recorded by multichannel confocal microscopy and by using a reusable autoclavable electrofusion chamber to be used in the preparation of hybrid cells. The quantification of the yield of hybrid cells is based on measurements of the area of co-localized pixels and allows simultaneous visualization and documentation of the procedure. This approach has a wider applicability, for example also in cases in which a fraction of one or multiple cell types are present in a suspension or a structure. The subject of this invention is also a corresponding autoclavable electrofusion chamber, more precisely, a chamber for the subject of the invention in the field of biomedical technology is the evaluation procedure for the determination of the yield of hybrid cells - hybridomas in a cell vaccine and cell products, which consists of measurements of the area of co-localized pixels of an image recorded by multichannel confocal microscopy and by using a reusable autoclavable electrofusion chamber to be used in the preparation of hybrid cells. The quantification of the yield of hybrid cells is based on measurements of the area of co-localized pixels and allows simultaneous visualization and documentation of the procedure. This approach has a wider applicability, for example also in cases in which a fraction of one or multiple cell types are present in a suspension or a structure. The subject of this invention is also a corresponding autoclavable electrofusion chamber, more precisely, a chamber for fusion of biological cells in electric field. The invention brings a, new construction of a re-usable electrofusion chamber.

Description

CELICA, D.O.O. Ljubljana

A PROCEDURE OF EVALUATING THE YIELD OF HYBRIDOMA CELLS AND CELL PRODUCTS BY USING CONFOCAL MICROSCOPY AND AN AUTOCLAVABLE ELECTROFUSION CHAMBER

The subject of this biomedical technology invention is a procedure for the evaluation of the yield of hybridoma cells and cell products - hybridomas in a cell vaccine - which consists of measurements of the area of co-localized pixels in an image recorded by multichannel confocal microscopy and an autoclavable reusable electrofusion chamber for the production of hybridoma cells. The invention belongs to the class C12N 13/00 of the international patent classification. This invention solves the technical problem of accurate, robust and rapid determination of the yield of hybridoma cells, cells that are formed by fusion of two cells into one, and cell products in which cells of different types/states are present. This new procedure requires a fusion chamber, which was invented here and consists of the construction of a dedicated reusable autoclavable electrofusion chamber made of biocompatible materials to be used in the production of hybridoma cells and other cell products to be used in human and veterinary medicine. In the field of biomedical technology there is a great interest to understand and to manipulate the fusion of dendritic cells with tumour cells to be used in antitumour immunotherapy. Dendritic cells are powerful immunoactive antigen presenting cells. On the other hand, tumour cells function as antigen donors. Fusion of tumour and dendritic cells into hybrid cells provides a system for specific presentation of tumour specific antigens and have an immunostimulative action (Gong et al., PNAS, 97 (6), 2000; Kugler et al., Nature Medicine, 6 (3), 2000; Orentas et al., Cellular Immunology 213, 4-13 (2001); Zhang et al., World J Gastroenterol., 9(3)2003). There are two major requirements that need addressing: On the one hand a device for robust and reproducible production of hybrid cells with electrofusion and on the other hand the corresponding procedure for the determination of the yield of hybrid cells in a cell suspension. The quantification of the effectiveness of the fusion procedure and the making of hybrid cells is of crucial importance for the use of hybrid cells in research and in therapy. The invented procedure provides means for accurate and rapid determination of the yield in the electrofusion process. The accuracy of this approach is determined by the properties of confocal microscopy. The procedure is as fast as flow cytometry, a commonly used standard method for the determination of the fraction of fused cells in cell vaccines. The key disadvantage of the flow cytometry approach is it's relative insensitivity, which contributes to the relatively large fraction of false positive hybrid cells. The procedure submitted for invention rectifies this key problem by using confocal microscopy. Furthermore, the newly invented procedure is not only applicable to solving the specific problem of determining the yield of hybridoma cells in a cell vaccine, but may be used more widely in any application in which measurements of a fraction of a certain cell type/state in a structure or preparation are required. To perform these measurements different cells are labelled by different fluorescent labels. Electrofusion is a process which employs the action of external electric field to merge two or more cells into one cell, a hybrid cell. The new hybrid cell is thought to gain physiological properties of the cells that took part in the electrofusion process. Currently there are two popular methods for the preparation of hybrid cells: fusion by the use of polyethilen glycol and electrofusion. The latter approach generates a significantly higher yield of fused cells in comparison to the approach with polyethilen glycol (Orientas et al., 2001 ). The electrofusion process consists of three steps (Zimmermann U,. Rev Physiol Biochem Pharmacol. 1986; 105:176-256). In the first one (alignment), cells undergoing electrofusion establish close contacts. For this cells need to be placed in an electrically conductive medium. For the establishment of close contact between cells dielectrophoresis is used, which generates movement and orientation of cells in a non-homogeneous AC electric field. Cells align into chains (»pearl chains«) in the direction of the higher density of the electric field. Once cells are aligned, the next step of electrofusion process (fusion) follows. Here, cells are exposed to a very short pulse (or pulses) of electric field, usually this is a DC field which generates membrane permeabilization which is followed by membrane resealing and fusion of two or more cell membranes at the point of contact between cells. In the third step (post-alignment) the fusion product reaches stabilisation. Here dielectrophoresis is used to keep cells in contact for a longer period of time to allow the rounding of the fused cells. The whole process of electrofusion takes place in the electrofusion chamber, which consists of a space into which the cell suspension is placed, and of at least a pair of electrodes, between which the cell suspension is exposed to the electric fields. The electrodes are connected to an AC/DC power source. The electrofusion chamber to be used for the production of cell vaccines should allow the placement of a relatively large volume of cell suspension and should generate reproducible fusion products. Kugler et al. (2000) use the BioRad electroporation cuvette for the preparation of cell vaccines. This electrofusion chamber is designed to generate a homogeneous electric field. However, as already discussed, one of the requirements for successful electrofusion is the dielectrophoretic alignment of cells between the electrodes. To meet this requirement Kugler et al. (2000) use a droplet of parafin wax (dielectrical wax), which is placed on one side of the electroporation cuvette. The disadvantage of this solution is that the placement of the droplet of wax cannot be standardized and this impairs the reproducibility of electrofusion process. Moreover, such an electrofusion chamber cannot be reused since cleaning and sterilization may pose a significant problem. The commercially available electrofusion chambers are mainly designed for single use (for example Microslide 450 [electrodes are represented by two tubes of stainless steel placed onto the glass surface, which generates a divergent electric field] and Microslide 453 [two bars of stainless steel or gold are placed in parallel and form a homogeneous electric field] or 454 Meander Fusion Chamber [Genetronics, Inc., BTX Instruments, San Diego, CA]. In these chambers the reproducibility of the fusion process is reduced by the fact that the space dedicated for electrofusion is not limited, which prevents the establishment of a reproducible cell density along the electrode surfaces. The other disadvantage of chambers intended for single use is the imprecision inherent to the production of electrodes, which further reduces the reproducibility of the electrofusion process. Another disadvantage of single-use electrofusion chambers is that because of the pressure of keeping their cost as low as possible, the materials selected for their construction are usually not biocompatible. The electrofusion chamber to be used for the preparation of cell vaccines must be designed to allow the sterility of the fusion product. Therefore, all parts of the chamber that come into contact with cells should be sterile. Fusion must be performed in a closed system or in a sterile environment, e.g. a sterile hood. Commercially available electrofusion chambers for single use are either supplied in non-sterile or in pre-sterilized conditions (usually by gamma rays) and are therefore used mainly for research purposes. From the above discussion it appears that a new design of the electrofusion chamber is required, which would allow the production of a relatively large volume of cell vaccines with a relatively high fraction of hybrid cells. Moreover, the construction of such chamber should allow reproducibility of fusion, should be easily cleaned and sterilized, should conform to the standards for use in human medicine (biocompatibility of materials used in the making of the electrofusion chamber) and should be economical and environment friendly. To summarize, a number of hurdles and problems are associated with the production of hybrid cells (hybridomas) to be used in cell vaccine production, but the following are outstanding: - The problem od exposing relatively large volumes of cell suspensions to electrofusion; - Establishing a relatively large percent of fused cells; Sterility (and apyrogenicity); - Biocompatibility of materials of the chamber that come into contact with the cell suspension; - Cost reduction of vaccine production through the design of a reusable electrofusion chamber allowing a rapid evaluation of the yield of hybridomas in the chamber itself.

Currently employed procedures that are used to asses the yield of hybridomas have been based mainly on the use of flow cytometry, which provides a reading more rapidly that conventional microscopy examination. On the other hand the advantage of microscopy approach is that it allows the visualization and documentation of single cells. Hybrid cells can be easily distinguished from non-fused cells by a microscope inspection. However the inspection of cells by classical microscopy is a time-consuming exercise. Therefore, microsopy was predominantly used in the past to document the fusion process by showing examples of fused cells, but was not employed to assess quantitatively the yields of hybrid cells in the cell suspension due to the time-consuming effort and due to the relatively high subjectivity of the results. The simplest version of the electrofusion chamber consists of the microscope slide onto which two wires, functioning as electrodes, are attached. Such a chamber is typically used to optimize the parameters of electrofusion (alignment). A droplet of cell suspension is placed between the electrodes and the movement of cells is observed under the microscope. Unfortunatelly, such a chamber can only be used for electrofusion on a relatively small volume of cell suspension under non-sterile conditions. Patent documents, such as for example: U.S. Pat.No. 4,441 ,972, U.S. Pat.No. 4,578,168, U.S. Pat.No. 4.764, 473, U.S. Pat.No. 2002164776 solve the problem of fusion of a relatively large volume of cell suspension and the problem of electrode configuration, which generate a non-homogeneous electric field. U.S. Pat. No. 4,441 ,972 describes a chamber type in which the fusion space is defined by upper and lower electrode plates shaped as a disc. At least one of the electrodes has concentrically placed grooves on its surface. At the apex of these a non-homogeneous electric field is formed. The construction and assembly of the chamber is complex and requires precise parallel positioning of electrodes. In addition, the space between the electrodes is difficult to clean and the system is not tailored for exposure of relatively large volumes of cell suspensions to fusion. U.S. Pat.No. 4,578,168 describes a chamber type in which there are multiple plates of wire mesh electrodes. The laminate construction in a cassette permits the placement of spacers between wire mesh electrodes. A relatively large volume of cell suspension can be exposed to electrofusion with this type of chamber. Moreover, such a chamber can be reused and sterilized. The disadvantage, however, is that cells may be trapped in the tortuous structure of the wire mesh electrodes, which may pose a cleaning problem after the fusion procedure. U.S. Pat.No. 4,764,473 describes a chamber type with a conical cylindrical core, around which a pair of wire electrodes is coiled in a double helix. In addition to this core a vessel fits on top of the helical core. The narrow space between the core and the vessel is filled with cell suspension of an optimal volume of around 0.3 ml. A disadvantage of this chamber is that it can not be sterilized easilly. The document US2002164776 or it's equivalent EP1245669 emphasizes that the chamber allows the processing of larger volumes of cell suspensions. Electrodes are in the form of a laminate and are configured to generate a non-homogeneous electric field. The surface of electrodes is made from electrically conductive material shaped into a waveform. For this any electrically conductive material can be used: i.e. carbon enriched plastics or semiconductor materials. Therefore, the ideal technology of manufacturing these chambers for mainly single use is the injection of mass into moulds shaped according to the chamber. Such chambers can be easily sterilized by irradiation. Document US 4804450 describes a device for cell fusion, characterized by a fusion space in which there is a pair of electrodes. However, these are placed symmetrically equidistantly to minimize the non-homogeneity of the electric field. Document U.S. Pat.No. 4,832,814. describes a device consisting of a thin layer of film electrodes, which are deposited by the etching technique onto an optically transparent ground layer with a channel, which determines the boundaries with the photocopolymer material which is deposited in a laminate type fashion between the upper optically transparent plate and the ground plate. The channel is covered on top by an optical plate, which has an opening through which cell suspension can be introduced into the chamber. This device can be made with optical tools, therefore the end product is already sterilized due to the technology of manufacture. The volume of such a chamber can be changed by changing the dimensions of the space in the laminate into which cell suspension is introduced. Electrodes consist of chromium or a mixture of indium and tin oxides which is not easy to harmonize with biocompatibility requirements. However, an advantage of such a chamber is that it allows the determination of the yield of hybridomas by microscopy, which is also the subject of the present invention. The evaluation procedure of the yield of hybrid cells and cell products by using confocal microscopy represented in this invention is a rapid procedure to quantify the fraction of hybrid cells after electrofusion. By the invented procedure one can accurately and rapidly analyse a large number of cells. Samples of cells are labelled by fluorescent vital dyes. Different samples of cells are labelled by dyes with distinct emission spectra (i.e. red, green). Following cell labelling by fluorescent dyes, cells are aligned by dielectrophoresis in a high frequency alternating electric field to establish close contacts between cells. Then the fusion process is triggered by exposing the cells to a short high intensity electric pulse. The identification of hybrid cells by confocal microscopy is based on dual fluorescence of single cells. The yield of hybrid cells is determined on confocal micrograph images by measuring the area of pixels exhibiting dual fluorescence. This area is compared to the area of the image in which pixels exhibit single fluorescence. Electron noise is eliminated from the image by two-dimensional filtering of the image. Before the intensities of distinct optical channels are compared, the contrast of the image is set in such a way that the distribution of pixel intensities fits into the whole range of available intensities. Then the threshold value is determined, which is used to binarize the image. By this means all the pixel intensity levels are reduced to two levels only. The computer software counts all the pixels with intensity higher than the threshold. The ratio between the number of suprathreshold pixels in each of the optical channels and the number of suprathreshold pixels in at least one channel represents the yield of hybrid cells in comparison to all cells in the cell suspension. The autoclavable electrofusion chamber of this invention has further characteristics: Electrodes are deposited on glass by an optical procedure using a mask. The thickness of the deposited biocompatible metal is equal to approximately the radius of cells under study. The distance between electrodes is from 50 do 350 micrometers. They generate a non-homogeneous half-circular electric field which is essential for dielectrophoresis. Electrode bands are deposited on glass in parallel or at an angle, which generates a gradient of electric field. The gradient of electric field is space coded in x, y coordinates on the surface of the glass plate and can be used to determine optimal fusion parameters in a single experiment on one cell suspension. This allows the analysis of optimal electrofusion conditions by determining the yields of hybridomas in different areas of the glass plate covered by electrodes. - The electrofusion chamber construction is designed to allow easy and sterile assembly, disassembly, sterilization of each of the parts or of the whole assembly in one piece by an autoclave, and it can be made by using biocompatible materials. The mode of sterilization by autoclave (or dry sterilization) has several advantages over the use of sterilization by gamma ray irradiation or by ethylenoxide; By exchanging parts of the electrofusion chamber marked EL.A, which have different diameters of the central round opening, different volumes of cell suspensions can be exposed to the electrofusion process. Cells can be easily removed from the chamber, which is an advantage in comparison to the patent USP4832814; Spring loaded connectors allow rapid assembly and disassembly of the electrofusion chamber. This invention is presented in detail on the basis of the accomplished example and the Figures which show as follows:

Figure 1 Two samples of different cells, labelled by two different fluorescent dyes, recorded by confocal microscope; Figure 2 Hybrid dually fluorescent cell, with an intensity diagram of red (dotted line) and green (continuous line) fluorescence for three neighbouring cells; Figure 3 Correlation between the yields of hybridomas determined by counting hybridomas and by measurements of areas of pixels on the image which exhibit dual fluorescence; Figure 4 Comparison of the results of analysis obtained by flow cytometry with those obtained by the procedure obtained by the invention employing confocal microscopy; Figure 5 Diagram of the electrofusion chamber - view from above - without the cover (»top plan view«); Figure 6 A schematical representation of the longitudinal crossection of the autoclavable electrofusion chamber, along the axis A-A; Figure 7 A schematical representation of the glass plate carrying electrodes - view from above; Figure 8 A schematical representation of the connecting block for the connection of the power source (AC or DC) - view from the side (Figure 8A) or from above (Figure 8B); Figure 9 Inlays with holes for bolts and nuts; Figure 10 Photograph of all parts of the autoclavable electrofusion chamber.

Evaluation procedure for the quantification of the yield of hybridoma cells and cell products by confocal microscopy as proposed by this invention will be explained in detail as follows. Cell vaccines, prepared by exposing dendritic cells (antigen presenting cells) and tumour cells are gaining interest, since it has been shown that they elicit tumour rejection in almost all animal species studied (Scott-Taylor et al., 20O0, Biochemica et Biophysica Acta, 1500, 265-279). The development of procedures of electrofusion strongly depends on quantification of the yield of hybrid cells. To meet this end flow cytometry was extensively used (Jaroszeski et al., 1994, Analytical biochemistry, 216, 271-275). To document the presence of hybrid cells microscopy was usually used, but was not employed to quantitate the fraction of hybrid cells because of the subjectivity and the time-consuming nature of this approach (Gottfried et al., 2002, Cancer Immunity, 2, 15-266). The procedure developed in this invention allows quantitative assesment of the yield of hybrid cells by analysing the images recorded by confocal microscopy. This procedure makes analysis of a large number of cells after electrofusion practicable. Two samples of cell populations were labelled by different (red and green) vital fluorescent dyes. Figures 1 and 2 have been obtained by confocal microscopy and have been analysed by two methods: i) The number of cells exhibiting dual fluorescence was counted by systematic scanning of the image and the fraction of these cells, representing hybrid cells, was expressed in percents relative to all labelled cells; ii) We measured the area of pixels exhibiting dual fluorescence.

The latter method is much more rapid than the former one. The results of both methods show a high degree of correlation, as shown by Figure 3. The same samples were also analysed by flow cytometry, which yielded similar results as the new method based on confocal microscopy (Figure 4). However, the correlation coefficient was rather low. The average percentage of hybrid cells determined by confocal microscopy was two times lower than the average percentage of hybrid cells determined by flow cytometry. The discrepancy is very likely due to the different selectivity of the two methods used. Measurements by flow cytometry detect as hybrid cells also aggregates of unfused cells. To label cells we used two vital fluorescent dyes: green 5-chloromethyl fluorescein diacetate (CMFDA, 7 μM) and red 6-chIoromethyl benzoyl amino tetramethylrhodamine (CMTMR, 5 μM). We used the Eppendorf Multiporator® with the helical electrofusion chamber, the volume of which was 250 μl. In addition we used also the planar autoclavable electrofusion chamber, which is the subject of this invention, with a volume of 5 ml (Figure 10). Contacts between cells were obtained by dielectrophoresis, by exposing them for 30 seconds to an AC field of 280 V/cm, 2 MHz. The fusion was triggered by applying a short pulse of 30 μs (1600-2800 V/cm). Then we applied an AC field of 280 V/cm, 2 MHz for 30 seconds. The cell suspension was analysed by confocal microscopy and flow cytometry. The percentage of hybrid cells was determined by counting yellow - dually fluorescent cells and all cells expressing single fluorescence. The percentage of hybrid cells was also determined by measurements of the area of pixels exhibiting green, red and green and red fluorescence in confocal images. The percentage of hybrid cells is represented by the number of dually fluorescent pixels in relation to all fluorescing pixels. By setting a threshold value (41 out of 255 intensity levels; 16% of maximal intensity) the intensity of the background was separated form the signal intensity of red and green pixels, respectively. The same threshold was used to analyse pixels exhibiting dual fluorescence. Electronic noise of the image was eliminated by Gaussian filtering. Following the electrofusion, the cell suspension was placed on the stage of the confocal microscope for analysis (Zeiss 510 with objective Plan-Neofluoar (20x, NA = 0,5). To excite the fluorescence of CMFDA, an argon laser line of 488 nm was used, whereas to excite the fluorescence of CMTMR, a He/Ne laser line of 543 nm was used. The emission fluorescence of CMFDA and CMTMR was separated with BP 505-530 nm and LP 560 nm emission filters. The percentage of fused cells was first determined by counting cells on the recorded image. Figure 2 (inset) shows an example of a hybrid cell, exhibiting dual fluorescence, which was verified by inspection of the line intensity profile of green and red fluorescence. Dotted line indicates green fluorescence, filled line red fluorescence. The left and right cells on Figure 2 (inset) exhibit single fluorescence, while the middle hybrid cell exhibits dual fluorescence. The bar represents 100 μm. The average percentage of hybrid cells, determined by counting cells, was in all experiments 4.1 ± 0.3% (n=47), which is significantly higher than the percentage of apparent hybrid cells determined in control non- electrofused samples (0.2 ± 0.1%, n=12). The same images were also analysed by the new procedure, as proposed for invention, of measuring areas of co-localized pixels in the image. Figure 3 shows the results of the assessment of the yied of hybrid cells by the two methods employing confocal microscopy. The results are correlated (R=0.9; P<0.001 , n=59). The fraction of area of co-localized pixels therefore represents the fraction of hybrid cells in the cell suspension. Calculation of the percentage of hybrid cells by measurements of the co-localized pixel area resulted in an average fraction of hybrid cells of 4.3 ± 0.3% (n=47), which is not significantly different from the fraction of hybrids determined by the counting of cells (4.1 ± 0.3%, n= 47). The fraction of hybrid cells determined by confocal microscopy was also compared with the fraction determined by the classical flow cytometry approach. The diagram in Figure 4 shows on the abscissa the area measurements of co-localized pixels on confocal images (X-axis) versus the measurements obtained by flow cytometry (Y-axis) (black circles: fusion of PC-12 and dendritic DC cells; white circles: the same cells without exposure to electrofusion). The relatively low correlation coefficient (R = 0.3; P < 0.05) is very probably due to higher relative selectivity of the confocal microscope and to the relatively high nonselectivity of the flow cytometry approach. The average percentage of hybrid cells determined by two confocal microscopy approaches (pixel area measurements: 4.3 ± 0.3; counting hybrid cells: 4.1 ± 0.3, n=47) was significantly lower than the percentage determined by flow cytometry (9.1 ± 0.8, n=47). The fractions of hybrid cells determined by all the methods used were significantly different (P<0.001) from the fractions of apparent hybrids in controls, non-electrofused cell suspensions. To bypass the disadvantages of existing electrofusion chambers, we developed a new type of electrofusion chamber. The proposed chamber is reusable and can be used for multiple electrofusions. The construction is compact, sufficiently light-weight, durable, designed to be easily cleaned and sterilized and allows the determination of optimal electrofusion conditions and the yield of hybrid cells with the confocal or a suitable fluorescence microscope. An autoclave can be used to sterilize the chamber, which has several advantages over other methods of sterilization, but the main one is that it represents a simple and inexpensive way of sterilization. The acquisition of a system for sterilization by an autoclave is relatively inexpensive in comparison to other methods such as those employing gamma irradiation, ethylenoxide and others (Co-60, Cs-137, radioisotopes ). Moreover, the ethylene oxide method is potentially carcinogenic and toxic. The autoclavable electrofusion chamber as presented in this invention consists of six separate parts, which can be autoclaved separately and which can be assembled in a sterile hood into a functional chamber. The electrofusion chamber can be adapted for fusion of different volumes of cell suspensions. The construction of the chamber, one part of it exhibits different diameters of the central round opening of the top part of the chamber (EI.A), allows the electrofusion surface to be modified and therefore permits electrofusion of different volumes of cell suspensions. Thus the problem of keeping multiple copies of an electrofusion chamber is solved. The reusable electrofusion chamber is thus economical, but also reduces the environmental burden of the accumulation and safe destruction of chambers designed for single use. The invention applies also to the dimensions and shape of the electrodes, which are designed for uniform exposure of cells along the whole glass surface to the electric fields. The thickness of electrodes is defined with the tolerance of ± 0.9 nm. The thickness of electrodes is smaller than the radius of cells under study in suspension, which allows the generation of a non- homogeneous electric field between the electrodes for the alignment of cells during dielectrophoresis. In addition, the layout of electrodes on the surface of the glass plate is also a subject of invention. The electrodes are placed either in parallel or at an angle to generate a gradient of electric field, the intensity of which is different at different locations along the surface of the glass electrofusion surface and can be identified by distinct x, y coordinates on the glass plate. Unlike other versions of commercially available chambers, in the chamber subject to this invention, the power supply is connected to the electrodes on the glass plate with removable connectors, which are characterized by spring- loaded contacts which press onto the surface of electrodes on the glass plate upon the assembly of the chamber. This provides an ideal solution for cleaning and sterilization of the chamber. The construction of the chamber, as proposed in this invention, allows separate sterilization of the constituent parts of the chamber. Moreover, the chamber parts are made of biocompatible materials to comply with requirements for use in humans. Figures 5 and 6 show the constructional design of the electrofusion chamber as proposed in this invention, characterized in that the chamber consists of the following parts: the bottom part EI.B of the chamber with a fixed diameter central round opening, and the top part of the chamber EI.A with a central round opening the diameter of which can be changed. Between the two parts of the chamber there is a glass plate with optically deposited electrodes (Figure 7). The O-ring is placed in the groove U, which is surrounding the central opening on the inner side of the top part EI.A of the chamber. The parts EI.A and EI.B of the chamber are joined together by four bolts (the making of metal inlays with holes on part EI.A. for bolts which are fixed into the bottom part of the chamber EI.B is shown on Figures 9A and B). Figure 9C displays the cross section of the nut. The electrical contact unit (Figure 8A, B) is fixed to the top part of the chamber EI.A by the same bolts used to join the bottom and the top parts, EI.A and EI.B, respectively. The chamber assembly starts by placing the bottom part EI.B on a flat horizontal surface, then the glass plate with deposited electrodes is placed in the specially machined frame on the inner side of EI.B, then the top part of the chamber EI.A is positioned on top by allowing the bolts to fit into holes on the upper part EI.A. At this moment the electrical contact unit is placed in the position defined by the two bolts. At the end the nuts are placed on bolts and three to four turns are required to fasten the nuts which hold the unit together. The bottom part EI.B serves as a holder of the glass plate with deposited electrodes (Figure 10). The placing of the glass plate is guided by the specially designed frame machined into the body of the bottom part EI.B. The glass plate with deposited electrodes fits into the frame on the upper part of the bottom part EI.B. The top part EI.A is placed over the glass plate and has the same outside dimensions as the bottom part EI.B. Parts EI.A and EI.B serve as two halves of a unit. In the center of both parts there is a central round (but the opening need not be round - it can be any shape) opening. The surface of the top part EI.A facing the interior of the whole chamber assembly is characterized by a groove (U) around the central round opening into which an O-ring fits. By using the nuts, the contact between the glass surface and the O-ring gets tighter and thus delimits the space into which the cell suspension is placed and prevents a leak into the area of the glass plate devoid of deposited electrodes. The bolts are placed asymetrically to direct the assembly of the chamber in the correct orientation at any time. The tops of the bolts are rounded to facilitate the placing of nuts on the bolts and to ease the fastening of the nuts and quick assembly of the chamber. The size of the fusion volume into which the cell suspension is placed is determined by the round central opening of the upper part EI.A; the bottom of this space is formed by the glass plate in the middle of the whole chamber assembly. The diameter of the central opening can vary and thus determine the volume of the cell suspension that is exposed to the electrofusion process. Therefore, several top parts EI.A are made with several diameters, which can be used as required for the experiment and work in progress. The electrofusion chamber as proposed by this invention consists also of a cover with a holder (Figure 10). The cover is characterized by a flat, at least 2 mm thick rim, which fits into the central round opening of the upper part EI.A. Tiny capillary-like holes through the cover are used to equilibrate the air pressure when the cover is placed on top of the cells in the fusion space of the chamber. The bottom of the cover is flat. Figure 7 shows the top view of the glass plate with the layout of electrodes deposited optically on the surface of glass. The thickness of electrodes is smaller or equal to the radius of the cells exposed to electrofusion (0.5 do 4.5 μm). The electrodes are deposited onto the glass surface with tolerance of ± 0.9 nm. The distance between electrodes is 50 to 350 μm. Electrode layout forms a round fusion area in which electrodes are placed in an intercalar fashion. The ribbons of electrodes are placed in parallel or at an angle, which generates a gradient of electric field, which differs as a function of x, y coordinates on the surface of glass plate. Figures 5, 8 and 10 show how the electrodes are connected to the AC/DC power supply via the connector block. On the side of the top part EI.A there is an area dedicated to the placing of the connector block. Electrical contact between the electrodes and the leads connecting to the power supply is made by spring-loaded contacts. This mode of establishing electrical connection upon the assembly of the electrofusion chamber is the subject of the proposed invention. Outer dimensions of the whole electrofusion chamber assembly fit onto the stage of the microscope. Therefore the round openings of the fusion chamber permit direct visualization of the electrofusion process. The electrofusion chamber to be used in the preparation of hybridomas must be sterile and meet the standards of apyrogenicity. All parts of the electrofusion chamber as proposed for this invention are made from biocompatible materials, which can be sterilized in an autoclave (120-130°C) and are not sensitive to moisture. The design of the electrofusion chamber as proposed by this invention permits the exposure of the chamber to the process of depyrogenization (dry heat for 1 hour at 200°C or 30 min at 250°C, or overnight incubation in 0.1 M solution of NaOH). The optimization of electrofusion parameters and the analysis of the yield of hybridomas can be performed in the electrofusion chamber on the stage of a microscope (electrode ribbons are placed at an angle). The evaluation procedure for the quantitative assesment of hybrid cells and cell products by confocal microscopy as proposed in this invention succesfully solves all the considered technical requirements and problems. The survey of literature reveals that yields of hybridomas as high as 30% have been reported in the past. For this flow cytometry was used as the standard method (Hayashi et al., 2002, Clinical Immunology, 104, 14-20; Gong et al., 2000, Proc Natl Acad Sci U S A, 97, 5011 ; Gong et al., 2000, Journal Immunology, 165, 1705-11 ; Akasaki et al., 2001 , Journal Immunotherapy, 24, 106-113). There are also reports indicating that the use of flow cytometry in the determination of the yield of hybridomas may report erroneous results since cell agregates may contribute to the overestimation of hybrid cells (Hayashi et al., 2002). The proposed invention solves this problem. The assessment of the yield of hybrid cells by measuring the area of co-localized pixels is as rapid as flow cytometry, but it also permits direct visualization and inspection of individual hybridomas, which is essential for the documentation of the process. The time required for the analysis of the yield of hybrid cells of a sample is comparable to flow cytometry. Moreover the number of cells taken into account in the analysis is similar to that taken by flow cytometry, but requires no additional approaches to visualize and document the yield of hybrid cells. In comparison to flow cytometry the procedure of determining the yield of hybridomas by confocal microscopy, the subject of this invention is more selective, since flow cytometry poorly discriminates cell aggregates form true hybrid cells. In contrast the procedure of assessing the yield of hybridomas by measuring the image area of dually fluorescent pixels is a sensitive and rapid approach. In addition, the new design of the reusable autoclavable electrofusion chamber as proposed by this invention represents a device with a number of advantages such as: easy sterilization and cleaning, robust and light construction, simple assembly and disassembly and its manufacture from biocompatible materials. For: CELICA, D.O.O. Ljubljana

Claims

PATENT CLAIMS

1. Evaluation procedure for the determination of the yield of hybridoma cells and cell products with confocal microscopy, characterized in that each sample of cells is labelled by fluorescent dyes, then cells are arranged by dielectrophoresis in a high frequency AC electric field in an electrofusion chamber which leads to the establishment of contacts between cells and the formation of hybrid cells, which are visualized under the confocal microscope by dual fluorescence microscopy. The yield of hybrid cells is determined in images obtained by the confocal microscope by measuring the pixel area which exhibits dual fluorescence in comparison with pixel area exhibiting fluorescence of a single wavelength. Image contrast in each of the channels is set in such a way that pixel intensities attain values in the whole range of available intensities of the imaging device. Then the threshold intensity is determined in order to discriminate between the signal and the background and the threshold is used to binarize the image which transforms the image into a two level image. Then the software determines the number of pixels relative to the background considering the threshold value.

2. Evaluation procedure of the yield of hybridoma cells and cell products by confocal microscopy according to Claim 1 , characterized in that cells are labelled by vital dyes.

3. Evaluation procedure of the yield of hybridoma cells and cell products by confocal microscopy according to Claims 1 and 2, characterized in that for different samples, dyes with distinct emission spectra are employed (i.e. red and green).

4. Evaluation procedure of the yield of hybridoma cells and cell products by confocal microscopy according to Claims 1 to 3, characterized in that for the elimination of noise in the image, which contributes to the overestimation of co-localized pixels, a two-dimensional filtering is used.

5. Evaluation procedure of the yield of hybridoma cells and cell products by confocal microscopy according to Claims 1 to 4, characterized in that the same methods can be used for the evaluation of fractions of cell populations in cell suspensions also in other applications in biomedical biotechnology.

6. Autoclavable electrofusion chamber, according to Claims 1 to 5, characterized in that it consists of a top part (EI.A), a bottom part (EI.B), a glass plate with electrodes with connectors to an AC or DC power source, an O-ring, a cover and nuts.

7. Autoclavable electrofusion chamber, according to Claim 6, characterized in that the top part (EI.A) and the bottom part (EI.B) are of equal external dimensions and that both have a central round (or any other shape) opening, and that both parts represen halves that assemble into a common housing, fastened together at four asymmetrically positioned points to assure correct assemblage.

8. Autoclavable electrofusion chamber, according to Claims 6 and 7, characterized in that the bottom part of the chamber (EI.B) has on the internal side a machined frame of dimensions fitting the glass plate and that the top part of the chamber (EI.A) has a round groove (U) to fit an O-ring on its inner side around the round opening.

9. Autoclavable electrofusion chamber, according to Claims 6 to 8, characterized in that the central round opening in both halves of the fusion chamber determines the size of the area to be used for electrofusion.

10. Autoclavable electrofusion chamber, according to Claim 9, characterized in that the central round opening in both halves of the fusion chamber can be of any reasonable size (diameter), which determines the volume of the electrofusion suspension and by this the number of cells that are exposed to the electrofusion process.

11. Autoclavable electrofusion chamber, according to Claims 6 to 10, characterized in that the bottom half of the chamber (EI.B) has four bolts mounted on top of which the points are machined to ease the mounting of the respective nuts, and that on the top part of the chamber (El. A) there are four holes positioned to fit the four bolts of the bottom half of the chamber, through which the four bolts are placed upon the mounting of the both halves of the electrofusion chamber.

12. Autoclavable electrofusion chamber, according to Claims 6 to 11 , characterized in that all the materials used to make the chamber are autoclavable and biocompatible.

13. Autoclavable electrofusion chamber, according to Claim 12, characterized in that the biocompatible materials are in agreement with ASTM standards.

14. Autoclavable electrofusion chamber, according to Claims 6 to 13, characterized in that on the glass plate multiple pairs of parallel electrodes are located. The distances between electrodes and their thickness are defined with the tolerance of ± 0.9 nm.

15. Autoclavable electrofusion chamber, according to Claim 14, characterized in that the thickness of electrodes is selected in comparison to the diameter of cells in the suspension, to generate a non-homogeneous electric field.

16. Autoclavable electrofusion chamber, according to Claim 14, characterized in that the thickness of electrodes < radius of cells.

17. Autoclavable electrofusion chamber, according to Claim 14, characterized in that the maximal thickness of electrodes is 0.5 to 4.5 μm.

18. Autoclavable electrofusion chamber, according to Claims 6 to 17, characterized in that the bottom part of the chamber (EI.B) has asymmetrically on the side which covers the area of contacts on the glass plate, spring loaded electrical contacts, which form upon the assemblage of the electrofusion chamber.

19. Autoclavable electrofusion chamber, according to Claims 6 to 18, characterized in that it consists of a flat cover with an at least 2 mm wide rim, which fits into the round central opening of the top part (EI.A) of the electrofusion chamber, and that it has a flat surface to establish a tight contact with the glass plate. The rim of the cover which is placed on top of the cell suspension also has a small hole (capillary, v) through which air bubbles may be removed from the cell suspension. The cover also consists of a holder to enable placing the cover on top of the cell suspension

20. Autoclavable electrofusion chamber, according to Claims 6 to 19, characterized in that parts of the electrofusion chamber can be easily assembled into a functional unit and the electrofusion chamber can be easily disassembled.

21. Autoclavable electrofusion chamber, according to Claims 6 to 20, characterized in that it can be mounted into a frame to fit the microscope table.

22. Autoclavable electrofusion chamber, according to Claims 14 to 18, characterized in that the electrodes on the glass plate are positioned at an angle, which is used to generate a gradient of electric field, coded by x,y coordinates of the glass plate plane.

23. Autoclavable electrofusion chamber, according to Claims 6 to 22, characterized in that it allows te analysis of optimal fusion conditions by the analysis of the yield of hybrid cells in different regions of the area of the glass plate covered by the electrodes.

For: CELICA, D.O.O. Ljubljana