SI21661A - Procedure for evaluation of fraction of hybrid cells and cell products by means of confocal microscopy and autoclavable electrofusion chamber - Google Patents

Abstract

The subject of the invention belonging to the filed of biomedicine technology is a procedure of evaluation of the fraction of hybrid cells and cell products - hybridoms - in a cell vaccine, which comprises measurement of surface area of colocalized pixels on a picture taken by a multichannel confocal microscope, as well as an autoclavable electrofusion chamber intended for multiple use in preparation of hybridom cells. The procedure for quantification of fraction of hybrid cells enables the evaluation of a hybrid cell fraction by measuring surface areas of colocalised pixels and simultaneous visualisation and documentation of the procedure. The applicability of the procedure is wide also for any cases where fractions of one or several cell types or structures on a picture are determined. The subject of invention is also a respective autoclavable electrofusion chamber, more precisely, a chamber for the fusion of biological cells in the electrical field. The invention refers to the structure of the electrofusion chamber which is intended for multiple application. The chamber is composed of an upper part (1), lower part (2), glass plate (4) with electrodes, o-ring, cover and nut, which all can be autoclaved separately and put together as a sterile electrofusion chamber very easily in a sterile particle-free chamber. Besides, it is possible to change the size of the electrofusion surface by changing individual parts of the electrofusion chamber and thus fuse various volumes of cell suspensions in the chamber. The electrofusion chamber is made of biocompatible materials and is suitable for preparation of hybrid cells for human vaccines.

Description

PROCEDURE FOR EVALUATION OF SHARE OF HYBRID CELLS AND WHOLE PRODUCTS BY CONFOCAL MICROSCOPY AND

AUTOCLAWABLE ELECTROFUSION CHAMBER

The subject matter of the invention within the field of biomedical technology is a method for evaluating the proportion of hybrid cells and cell products of hybridomas in a cell vaccine, comprising measurements of the surface area of colocalized pixels in an image taken with a multi-channel confocal microscope and autoclavable electrofusion chamber for multiple use hybridoma cells. The invention belongs to class C12N13 / 00 of the international patent classification.

A technical problem that the present invention successfully solves is a process for the reliable and rapid determination of the percentage of hybrid cells in the fusion between two samples of fluorescently labeled cells and the construction of a suitable fusion chamber made of biocompatible materials for human use.

In the field of biomedical technology, there has been a great interest recently in the fusion of dendritic and tumor cells for the preparation of vaccines for antitumor immunotherapy. Dendritic cells are immunoactive cells that have the ability to present antigens on their surface. Tumor cells, however, are antigen donors. Fusion of dendritic and tumor cells yields hybrid cells that express specific tumor antigens and are immunostimulatory (Gong et al., PNAS, 97 (6), 2000; Kugler et al., Nature Medicine, 6 (3), 2000; Orentas et al. al., Cellular lmmunology 213, 4-13 (2001); Zhang et al., World J Gastroenterol., 9 (3) 2003).

io There are two major sets of problems in this field: the construction of a device for the reproducible preparation of hybrid cells by electro-fusion, and in this respect the determination of the efficiency of cell fusion.

Assessment of the efficiency of fusion and the formation of hybrid cells is crucial for the use of hybrid cells in research and in therapeutic applications, and the method according to the invention allows for the rapid and accurate determination of the efficiency of fusion. The precision of the method is determined by the use of microscopy, and the speed of the procedure is the same as that used in flow cytometry procedures, a method that has hitherto been a commonly used method for determining the proportion of fused cells in cellular vaccines. A fundamental problem with the known flow cytometry procedure is insensitivity, which contributes to the relatively high proportion of so-called false positives. The method of the present invention, which successfully remedies this defect, was introduced by confocal microscopy and is not only useful for solving a specific problem, that is, for determining the proportion of hybridomas in a cell vaccine, but its utility is wider. It can be used in all cases where we measure either the proportions of cells or other structures in a preparation that are labeled with different fluorescently labeled probes.

Electro-fusion is the process by which the action of an electric field on cells in suspension results in the fusion of two or more cells into a new cell (a hybrid cell). The hybrid cell has the physiological properties of both fusion partners. Two methods are used to prepare cell hybridomas in the studies mentioned above: fusion using polyethylene glycol and electrofusion.

Electrofusion gives a significantly higher percentage of fused cells than the polyethylene glycol fusion method (Orientas et al., 2001).

The electrofusion process takes place in three stages (Zimmermann U,. Rev Physiol Biochem Pharmacol. 1986; 105: 176-256). In the first stage, the cells that are put into the electro-fusion process come in close contact with each other. Cells must be in electrically conductive medium. Dielectrophoresis is mostly used to establish close contact between cells. This process results in the movement, migration and orientation of cells in a non-homogeneous alternating electric field. The cells between the electrodes are placed in pearl chains in the direction of maximum electric field density. In the next stage (fusion), the cells are exposed to a short intense electric pulse (or several pulses), usually a DC pulse, which causes the permeabilization of the cell membrane and then the merging of the two membranes at the point where the cells are in close contact. In the third stage (post-alignment), the fusion product is stabilized. Using dielectrophoresis, the cells remain in close contact. The fused cells are rounded up into a hybrid cell.

The electrofusion process takes place in an electrofusion chamber, which basically has 5 spaces for suspending the cells and at least one pair opposite the placed electrodes, so that the cell suspension during the electrofusion process is between the electrodes.

The electrodes are connected to the power supply (AC or DC power source).

The electrofusion chamber intended for the preparation of vaccines must be capable of fusing a sufficiently large volume of cell suspension and must allow reproducibility of the electrofusion process.

Kugler et al. (2000) used Biorad's electroporation cuvette to prepare vaccines in the aforementioned article. The camphor is basically designed to generate a homogeneous electric field. As mentioned earlier, the condition for successful electrofusion is the dielectrophoretic arrangement of cells between two electrodes. To optimize this electrofusion phase, Kugler et al. (2000) used a drop of paraffin wax (dielectrical wax) applied to one side of an electroporation cuvette. Such a solution makes it difficult to standardize and repeat the electrofusion process. The chamber so adapted is difficult to clean or re-sterilize and therefore not suitable for reuse.

The types of commercially available electrofusion chambers are mainly intended for single use e.g. Microslide 450 (for electrodes has two stainless steel tubes mounted on the glass surface, creating a divergent electric field) and Microslide 453 (two stainless steel bars (or gold) mounted side by side and creating a homogeneous electric field) or 454 Meander Fusion Chamber (Genetronics , Inc., ΒΤΧ Instruments, San Diego, CA). Even with these types of chambers, the problem is the reproducibility of the fusion process, since the fusion space is not limited and it is difficult to achieve the same cell density, depending on the surface of the exposed electrodes.

Another problem encountered with single-use chambers is the inaccuracy in electrode fabrication, which also affects the poor reproducibility of the fusion process. Because they are relatively inexpensive, io disposable chambers are mostly made of materials that are not biocompatible.

The electrofusion chamber intended for the preparation of vaccines must also ensure the sterility of the fusion product. All parts of the cam that come into contact with the cell suspension must be sterile. The fusion must take place in an closed system that prevents any possible contamination during the fusion process. The commercially available disposable electrofusion chambers are either non-sterile or pre-sterilized, usually with gamma rays, and are useful for research purposes only.

From all of the above, it is clear that there is a need for a new type 20 electrofusion chamber that will allow the production of sufficient large quantities of hybrid cells to be prepared for the preparation of vaccines. The design solution must be such as to enable reproducibility of the electrofusion process, ensure sterility of the electrofusion process and meet standards for humane use (biocompatibility of materials), economical and environmentally friendly.

There are several problems with the preparation of hybrid cells (hybridomas) for vaccines.

- the problem of fusion of large and different volumes of cell suspension;

- providing a sufficiently large percentage of the fused cells; sterility (and pyrogenicity);

biocompatibility of materials that come into contact with cell suspension;

- the reduction of the cost of preparing vaccines with a reusable chamber design and for the rapid evaluation of the proportion of hybrids already in the fusion chamber itself.

Known methods for determining hybridomas have thus far been evaluated for fusion efficiency using flow cytometry that is faster than microscopic procedures. The use of a microscope enables visualization and documentation of individual cells. Hybrid cells are separated from non-hybrid cells at the microscopic level. Examination of cells by classical microscopy is a time-consuming method. So far, microscopes have been used solely for visualization, and evaluating the effectiveness of fusion with a microscope would be time consuming and subjective.

The simplest type of cam consists of a microscope glass, to which two parallel wires are attached. This type of cell is used to approximate the optimum electric field that results in dielectric arrangement and cell fusion. A droplet of cells was suspended between the wires and individual fusion rates were observed under the microscope. This type of cam allows fusion of only a small volume of cells. Sterility is also a problem.

Patent documents such as e.g. U.S. Pat.No. No. 4,441,972, U.S. Pat. Pat.No. No. 4,578,168, U.S. Pat. Pat.No. 4.764, 473 U.S. Pat. Pat.No. 2002164776 solve the problem of fusion of large volumes of cell suspension and the problem of implementation (configuration) of electrodes that generate a non-homogeneous electric field.

U.S. Pat. No. No. 4,441,972 describes a type of cam where the fusion space w is defined by a disk-shaped upper and lower electrode plate. At least one of the electrodes has several grooves arranged concentrically on the surface. A homogeneous electric field is created at the tips of these. The design of the cam is mechanically complex and requires fine tuning of the parallel electrodes. The space between the parallel electrodes is difficult to clean. It is unlikely that this system would allow the fusion of large quantities of cells.

U.S. Pat.No. No. 4,578,168 describes a chamber where several flat mesh wire electrodes are mounted. The laminate structure in the cassette allows the installation of a series of spacers and mesh wire electrodes. This embodiment enables the fusion of cell suspensions in large volumes for multiple use, including multiple sterilization. However, a bad feature of this cell is that the cells are trapped within the network structure and therefore difficult to remove after the fusion process is performed.

U.S. Pat.No. No. 4,764,473 describes a cam having a tapered cylindrical core around which double wire helix wire electrodes are wound, and a container following the cylindrical core in shape. The narrow space between these two parts is the space for cell suspension. Chamomile allows the fusion of smaller volumes of cell suspension (optim. 0.3 ml), but its disadvantage is that it is not autoclavable.

Document US2002164776, or its equivalent EP1245669, emphasizes that the chamber enables the processing of larger cell volumes. The electrodes are laminar and continuous and are configured io to generate a non-homogeneous field. The electrodes have a surface of electrically conductive material and are wavy. Any el. conductive materials: carbon-enriched plastics or semiconductors. Therefore, this technology is suitable for the production of molds by injection molding into the molds, which is ideally suited for the preparation of disposable and used molds. It is also suitable for radiation sterilization.

US 4804450 describes a cell fusion device having a fusion space containing a pair of electrodes. They are arranged so that they have the same dimensions (height) to minimize the inhomogeneity of the electric field.

With U.S. document Pat.No. No. 4,832,814. is a device consisting of a thin layer-film of electrodes applied by etching to an optically transparent bottom layer with a channel defining the boundaries with photocopolymer material applied as a laminate between the top optically transparent and the bottom panel.

At the top is an optical panel that closes the channel and has an opening through which cells can be inserted into the channel. The device can be made using optical tools. During this process, a product is created that is already sterile due to the nature of the work. Also, the volume of the oyster can be arbitrarily varied by changing the cavities in the laminate where the cells are placed. The electrodes are either chromium or a mixture of indium and tin oxides, which is difficult to reconcile with biocompatibility. However, it is a good feature of such a cell that it allows determination of the proportion of hybridomas by microscopy, which is the object of the present invention.

The process for evaluating the fraction of hybrid cells and cell products by confocal microscopy according to the invention is a rapid process for quantifying the fraction of hybrid cells that result from cell fusion. The procedure is based on the use of a confocal microscope. According to the process of the invention, a large number of cells can be quickly and reliably analyzed. Each cell sample is labeled with fluorescent dye. Cells are vitally colored. For different is samples, dyes with different emission spectra (eg red and green) are used. After labeling, the cells are arranged by dielectrophoresis in a high-frequency alternating electric field so that they are contacted. The cells are then fused (eg with an electric shock). Examination of single hybrid cells under a confocal microscope is based on double fluorescence of hybrid cells.

2o Determine the proportion of hybrid cells in images taken with a confocal microscope by measuring the area occupied by dual-fluorescence pixels. This surface is compared with all pixels that indicate only single fluorescence sections. Electronic image noise is eliminated by two-dimensional image filtering. Before comparing the intensities of different image channels, adjust the contrast of each channel so that pixel intensities occupy all available range. We then determine a blank intensity value that separates the background image intensities from the signal intensity of the fluorescently labeled cells. We use the empty intensity value to binarize the image. In binarization, an image that has intensities of the entire area is converted to a two-level image. The computer program counts all pixels with above-average intensity. The ratio of the number of pixels having an above-average intensity for both channels to the number of pixels having an above-average intensity for at least one channel represents the proportion of hybrid cells with respect to all cells.

Furthermore, the autoclavable electrofusion chamber according to the invention has the following characteristics:

The electrodes are charged to the glass through an optical process mask, with a thickness of metal biocompatible coating, up to half the diameter of the cells. The edge of the electrodes, which are 50 to 350 micrometers apart, creates a non-homogeneous semicircular field, which is crucial for dielectrophoresis between two or more bands, at

2o plane of the laid electrode bands. The electrode strips are paired so that they are parallel or applied at angles to create an electric field gradient, so as to allow optimal fusion parameters to be determined in one experiment in one suspension. The electric field gradient is encoded in x, y coordinates due to the electrode deposition. This allows the optimal fusion conditions to be analyzed by analyzing the proportions of the fusion cells in different regions of the plane having an electrode-applied surface;

The construction of the chamber is designed to allow simple (sterile) disassembly and assembly, autoclaving and to allow the use of biocompatible materials. The method of autoclaving (or dry sterilization) sterilization has several advantages over sterilization by gamma or ethylene oxide;

by changing the fusion chamber portions, parts labeled EL.B having differently sized central openings allow the fusion of differently large volumes of cell suspensions. Cells, however, can be quickly and easily removed, which is an advantage over USP4832814;

a special connector solution enables quick assembly and disassembly of the fusion chamber.

The invention will be explained in more detail on the basis of an embodiment and figures, of which:

Figure 1 two samples of different cells, indicated by two different ones fluorescent dyes taken with confocal a microscope; Figure 2 hybrid double-fluorescence cell, with intensity chart 5 green and red fluorescence for three adjacent cells; Figure 3 graph of the correlation between counting cells with double fluorescence and by measuring the area occupied by dual-fluorescence pixels; Figure 4 comparison of the results of the analysis of the samples with the flow cytometer with the results obtained by the method according to the invention with 10 confocal microscope; Figure 5 schematic of an electrofusion cam - top view - none top plan view; Figure 6 sketch of a longitudinal cross-section of an electrofusion chamber through the axis A-A; 15 Figure 7 sketch of glass panel with electrodes - top view (not in scale); Figure 8 sketch of the connection to the power source (AC or DC) - camera view from the side (Fig. 8A) or from above (Fig 8B); 20 Figure 9 guides for screws and nuts; Figure 10 drawing of all parts of the camel.

The process for evaluating the proportion of hybrid cells and cell products by confocal microscopy according to the present invention will be explained in more detail below.

Cellular vaccines prepared by fusion of dentritic (antigen 5 presenting) cells and tumor cells are increasingly at the forefront of stimulating tumor immune rejection in almost all animals studied (Scott-Taylor et al., 2000; Biochemica et Biophysica Acta, 1500; 265-279). Quantification of the fraction of hybrid cells is important in the development of electrofusion procedures. To date, researchers have used flow cytometry (Jaroszeski et al., 1994, Analytical Biochemistry, 216, 271-275). However, various microscopic techniques were used for visualization and documentation, which were time consuming and, due to subjectivity, unsuitable for accurate quantification (Gottfried et al., 2002; Cancer lmmunity, 2, 15266).

is The developed method according to the invention enables the estimation of the proportion of hybrid cells by analyzing the surface area on images taken with a confocal microscope. With this procedure, a large number of cells can be analyzed after electrofusion. Two samples of different cells were labeled with two different fluorescent dyes. Figures 1 and 2 were taken using a confocal microscope in two ways:

i) we counted the cells with double fluorescence and calculated the percentage of these cells with respect to all the cells labeled;

ii) We measured the area occupied by dual-fluorescence pixels.

Another way was much faster than cell counting. The results of both procedures have a high degree of correlation, as can be seen in Figure 3. The same samples were also analyzed with a flow cytometer. These results also correlate with the results obtained by the new method with a confocal microscope (Figure 4), but the correlation coefficient is low.

The average percentage of hybrid cells determined by a confocal microscope was twice less than the average percentage of hybrid cells determined by flow cytometry. This difference is probably due to the greater selectivity of the new confocal microscope procedure. The flow-through cytometer can also detect aggregates of non-merged, glued cells and identify them as hybrid cells.

Fluorescence dyes were used for the fluorescence labeling of cells: green 5-chloromethyl fluorescein diacetate (CMFDA, 7 μΜ) and red 6-chloromethyl benzoyl amino tetramethylrhodamine (CMTMR, 5 μΜ).

We used an Eppendorf Multiporator® screwdriver electroporator. The volume of the camel was 250 pl. We also used our own planar chamber of the invention, with a volume of 5 ml (Figure 10). Cell contact was achieved by dielectrophoresis. An alternating electric field (280 V / cm, 2 MHz) was brought in for 30 seconds. Electrofusion was caused by a high-voltage shock (1600-2800 V / cm) for 30 ps, then re-introduced an alternating electric field (280 V / cm, 2 MHz) for 30 seconds. Cell suspension was analyzed by confocal microscope and flow cytometry.

The percentage of hybrid cells was determined by counting yellow - double fluorescent cells. The percentage was calculated with respect to all cells counted in suspension.

The percentage of hybrid cells was also determined by analyzing the confocal image in 5 terms of measuring the area occupied by pixels with colocalized red and green fluorescence. The percentage was calculated with respect to all fluorescence pixels. An empty value was determined that separated the background intensities from the signal intensities of the green and red cells. The blank value was 41 of all 255 levels of intensity, corresponding to 16% of maximum intensity. The same io blank value was used to determine the double-fluorescence hybrid cells. The electronic noise that would cause the colocalized pixels to be overestimated was removed by Gaussian filtering.

After the cell fusion process was completed, the cell suspension was analyzed by a confocal microscope. We used a Zeiss is 510 confocal microscope with a Plan-Neofluoar lens (20x, NA = 0.5). CMFDA fluorescence was excited with an argon laser at 488 nm and CMTMR with a He / Ne laser at 543 nm. The fluorescents of CMFDA and CMTMR were separated by BP 505530 nm and LP 560 nm emission filters. The percentage of fused cells was first determined by counting the cells in the captured image. Figure 2 shows a picture with a hybrid cell.

To confirm that this is a hybrid double-fluorescence cell, we drew a line diagram of the green and red fluorescence intensities for the three adjacent cells (the profile drawn in the diagram is covered by an arrow in the figure). The dashed line indicates green fluorescence and the solid line indicates red fluorescence. The left and right cells are single-fluorescence, the center and the hybrid cell are double-fluorescence. The meter is 100 gm. The average proportion of hybrid cells determined by counting in all experiments was 4.1 ± 0.3%, (n = 47), significantly higher than the proportion of virtual hybrid cells in the control experiment without electrofusion (0.2 + 0.1%, n = 12).

The same images were also analyzed according to the faster procedure described by measuring the surfaces of colocalized pixels. Figure 3 shows the results of estimating the proportion of hybrid cells both ways using a confocal microscope. The results were statistically significantly correlated (R = 0.9; P <0.001, n = 59). The fraction of the io surface area of the colocalized pixels therefore represents the fraction of hybrid cells in suspension. By measuring the surface area of the colocalized pixels, the average proportion of hybrid cells was calculated to be 4.3 ± 0.3% (n = 47), which was not significantly different from the proportion of hybrid cells determined by counting (4.1 ± 0.3%, n = 47 ).

The proportion of hybrid cells determined by a confocal microscope was also compared to that determined by the classical flow cytometer method. The diagram in Figure 4 shows that the proportions of hybrid cells are determined by measurements of the surface area of colocalized pixels on confocal images (abscissa) and flow cytometer (ordinate), correlated (black circles: fusion of PC-12 cells and DC cells; white circles: identical cells without fusion).

The relatively small correlation coefficient (R = 0.3; P <0.05) is probably due to the higher selectivity of the confocal microscope and the indiscriminate flow cytometry method. The average proportion of hybrid cells determined by a confocal microscope (surface measurements: 4.3 ± 0.3; counting of hybrid cells: 4.1 ± 0.3, n = 47) is less than the proportion determined by flow cytometry ( 9.1 ± 0.8, n = 47), as the flow cytometer can detect cell aggregates as hybrid cells. The proportions of hybrid cells determined by all the methods used are significantly (P <0.001) greater than the proportions determined for control cells where no electrofusion was used.

To eliminate the shortcomings of existing electrofusion chambers, we have developed a new type of electrofusion chambers. The presented cam is intended for multiple use, ie. chamomile, which is compact but lightweight, durable, easy to clean and maintain and also allows to determine optimal io fusion conditions and fusion rates with confocal microxop. The chamber can be sterilized (without losing quality materials) by autoclaving. Autoclaving sterilization has several advantages over other methods of sterilization, in particular it is the simplest and cheapest method of sterilization. The cost of autoclaving is minimal. Also, the purchase of an autoclaving unit is incomparably cheaper than the installation of an ethylene oxide sterilization unit or gamma rays (Co-60, Cs-137, radioisotopes, accel. Electrons). Ethylene oxide is toxic and potentially carcinogenic. The reusable electrofusion chamber according to the invention has six separate parts, which can be autoclaved separately and very easily assembled into a sterile electrofusion chamber in a dust-free sterile chamber.

The adaptive electrofusion chamber according to the invention is adapted for the fusion of different cell suspension volumes. The camphor is designed so that by changing one part of the electrofusion chamber, which has differently large central openings, we change the size of the electrofusion surface, ie. the effective length of the electrodes in contact with the cell suspension. Solves the problem of purchasing chamomile for different cell volumes. Not only is repeated use economical, but it is also consistent with reducing the environmental burden of its destruction, which is also a problem with single use chambers.

The invention also relates to the dimensions and shape of electrodes for uniform treatment of cells throughout the fusion surface. The thickness of the electrodes and the distance between the electrodes is defined with a tolerance of ± 0.9 nm. The depth of the electrodes is smaller than the radius of the cells suspended in the electrodes, so that a non-homogeneous electric field is generated for the dielectrophoretic arrangement of cells in the region of maximum electric field density, which is a condition for successful fusion. In addition, the subject of the invention is the placement of electrode strips that are either arranged in parallel or at an angle to create an electric field gradient that is different in different fields with the x, y coordinates of the glass substrate of the bottom of the electrofusion chamber.

The connection to the electrodes can be detached from the electrodes, unlike some designs where the connectors are fixed to the electrodes. The problem is solved by the spring contacts that are established when assembling the cam. The solution

2o makes it easier to clean and maintain the sterility of the oyster.

Due to the proposed construction of the fusion chamber, it is possible to autoclave individual parts, which are at the same time made of biocompatible materials, to meet the conditions for human use.

5 and 6 show a structural solution of an electrofusion chamber according to the invention, which consists of the following parts: the lower part 1 of the fixed-hole chamber, the upper part 2 with an opening whose diameters may be different, glass panes 4 are arranged between the two portions. with electrodes (shown in Figure 7), an O-ring placed in the draft 3, which is made in the lower side of the upper part 2 of the cam. The lower part 1 and the upper part 2 of the casing of the casing are connected by four screws (the design of the holes for the screw holes fixed in the lower part 2 of the casing is shown in Figures 9A and 9B). Figure 9C shows a cross-section of a nut. The contact plate 5 (Figures 8A, 8B) io is attached to the upper part 1 of the chamber with the same screws that secure all parts of the chamber. The chamber is assembled by placing the bottom of the chamber 2 on the substrate, a glass panel 4 with electrodes is placed in a special slot with slot 3, then the upper part of the chamber 1 is placed on four screws and the contact plate 5 on two in one part. screws. With three to four turns of nuts is, all the components of the chamber are fastened.

The upper part 1 of the cam serves as a glass plate support 4 with electrodes (Figure 10). For ease of installation of the glass panel 4 with the electrodes, the upper part 1 on the upper side has a slot-groove (Figure 10) for the glass panel 4 with the electrodes, which extends along the entire length of the upper part 1, but must be so narrower than the upper part 1 that at the corners of the upper 1 space for mounting the screws. The size of the glass panel 4 with the electrodes corresponds in dimension to the size of the slot 3 in the upper part 1.

The lower part 2 rests on the glass panel 4 with electrodes and is of the same external dimensions as the upper part 1A. Both parts 1 and 2 serve as two parts of a single housing. The lower part 2 has a central circular opening. In our version, the opening is round, but it can also be of different shapes. The lower part 2 has a draft 3. In the draft 3, an O-ring is used to seal the space for suspending the cells in the surface adjacent to the glass plate 4 with the electrodes. Complete sealing of the cell suspension space is achieved by means of four screws mounted asymmetrically at the corners of the upper part 1 and extending axially through the four openings at the corners of the lower part 2 so that io can be used to tighten the glass plate 4 with the electrodes between the upper parts by means of nuts. 1 and bottom 2. The screws are positioned asymmetrically to allow the bottom 2 to be correctly oriented at all times. The screws are threaded off at the tips for quick screwing in the nut. The size of the cell suspension space is determined by the size of the central and circular opening in the lower portion 2, wherein the bottom of the cell suspension space is formed by a glass plate 4 with electrodes, and the side wall represents the circumference of the central circular opening. The size of the central circular aperture is arbitrary, depending on the volume of suspension desired in the electrofusion process. For this purpose, it is practical to make several lower parts 2 with different sizes of the central circular aperture and to change them according to the volume of cell suspension that we want to prepare.

The electrofusion chamber according to the invention has a lid with a holder (Figure 10). The lid shall have a straight edge, at least 2 mm thick, which corresponds in dimension to the circumference of the central circular opening in the lower part 2. Through the bottom of the lid, thin holes for equalizing pressure and air outlet extend through the capillaries. The bottom of the lid is flat and fits into the cell suspension and expels any air bubbles through the capillaries, which contributes to the reproducibility of the fusion process.

Figure 7 shows a blueprint of the electrode glass pane. The electrodes are designed in such a way that the electrically conductive substance is charged to the glass by a photo process. The thickness of the electrodes is less than or equal to half the diameter of the cell (0.5 to 4.5 pm). The tolerance level is ± 0.9 nm. The distance between io electrodes is from 50 to 350 pm. The electrodes can be arranged as a circular fusion field with intercalary bands of parallel electrodes. Alternatively, the electrodes may be angled to create an electric field gradient in different areas of the surface at the x, y coordinates of the glass pane.

Figures 5, 8 and 10 show the design of the electrode connection to the AC source or D.C. The lower part 2 has asymmetrical side, which covers the location of the contacts on the glass panel with the electrodes, spring contacts that are made when assembling the electrofusion chamber according to the invention.

The outer dimensions of the chamber are such that it can be placed in the frame of a microscopic table. The opening in the upper part 1 and the glass plate 4 allow the fusion process to be observed without the risk of the fusion material becoming contaminated.

The electrofusion chamber for the preparation of hybridomas for human use must be sterile and pyrogen-free. All parts of the electrofusion chamber are made of biocompatible materials that are autoclavable ie. withstand the temperature of autoclaving (from 120-130 ° C) and are not sensitive to moisture.

The electrofusion chamber intended for the preparation of vaccines must also be resistant to de-oxygenation. The depyrogenation process of the individual portions of the chamber is performed (before steam sterilization) in a dry sterilizer for 1 hour at 200 ° C or 30 minutes at 250 ° C, or by incubation overnight in 0.1 M NaOH solution.

io The procedure for evaluating the fraction of hybrid cells and also for optimizing the conditions of electrofusion (electrode strips performed at an angle) can be performed on the electrofusion chamber.

The process for evaluating the proportion of hybrid cells and cell products by confocal microscopy according to the invention successfully solves the technical problems posed.

So far, researchers have reported successful cell fusions with a relatively high proportion of hybrid cells (up to 30%). A flow cytometer was commonly used to quantify the proportion of hybrid cells (Hayashi et al., 2002; Clinical lmmunology, 104, 14-20; Gong et al., 2000, Away Natl Acad

Sci USA, 97,5011; Gong et al., 2000, Journal of Immunology, 165, 170511; Akasaki et al., 2001, Journal lmmunotherapy, 24, 106-113). It has also been reported that determining the proportion of hybrid cells with a flow cytometer is not sufficient, as cell aggregates may contribute to the over-measured proportion of hybrid cells (Hayashi et al., 2002). The process of the invention successfully solves this problem. Estimating the proportion of hybrid cells by measuring the surface area of colocalized pixels is fast, specific to hybrid cells, and allows the process to be visualized and documented simultaneously.

Compared to conventional confocal microscopy, estimation of the percentage of hybrid cells is rapid. The time required for analysis of a single sample is equal to the time required for analysis by flow cytometry. The same number of cells in each sample can be analyzed as in flow cytometry. No additional method is needed to visualize and document the analysis.

The confocal microscope procedure is more selective than flow cytometry, as flow cytometry can erroneously detect the aggregates of non-merged, glued cells as hybrid cells. The procedure for estimating the proportion of hybrid cells by surface analysis on images taken with a confocal microscope is sensitive and rapid, and is therefore suitable for the evaluation of large cell samples.

Also, the new construction of the autoclavable electrofusion chamber for carrying out the process according to the invention allows its multiple use, and the chamber itself is compact but lightweight, durable, easy to clean and maintain. The camphor can also be very easily sterilized.

Claims (22)

PATENT APPLICATIONS 1. A method for evaluating the proportion of hybrid cells and cell products by confocal microscopy, 5, characterized in that each sample of cells is labeled with fluorescence dye, after labeling the cells are edited by dielectrophoresis in a high-frequency alternating electric field so that they are touched, followed by examination of individual hybrid cells under a confocal microscope, io based on double fluorescence of hybrid cells, determining the proportion of hybrid cells in images taken with a confocal microscope by measuring the area occupied by those pixels having double fluorescence; to compare this area with all pixels that denote only single fluorescence sections, adjusting the contrast of a single channel before comparing intensities from different image channels so that pixel intensities occupy all available range, and then determine the intensity threshold that separates the background intensities of the image the signal intensity of the fluorescently labeled cells; to use the intensity threshold value to binarize the image, where in binarization The 20 image, which has intensities of the entire area, is converted to a two-tier image, and then all pixels with above-average intensity are counted with a computer program. A method for evaluating the proportion of hybrid cells and cell products by confocal microscopy according to claim 1, characterized in that the cells are stained vitally. Process for evaluating the proportion of hybrid cells and cell products by confocal microscopy according to claims 1 and 2, characterized in that dyes with different emission spectra of io (eg red and green) are used for different samples. Method for evaluating the proportion of hybrid cells and cell products by confocal microscopy according to claims 1 to 3, characterized in that two-dimensional image filtering is used to eliminate the electronic noise of the image, which would cause an overestimation of colocalized pixels. 5. A method for evaluating the proportion of hybrid cells and cell products by confocal microscopy according to claims 1 to 4, 20, characterized in that the same methods can be used to evaluate the proportion of populations in cell suspension for other purposes in biomedical biotechnology. An autoclavable electrofusion chamber according to claims 1 to 5, characterized in that it consists of an upper part (1), a lower part (2), a glass panel (4) with electrodes and connectors to the AC or DC signal source, 5 O-rings, cap and nut. Autoclavable electrofusion chamber according to claim 6, characterized in that the upper part (1) and the lower part (2) are of the same outer dimensions and have a centrally circular orifice, act as halves of the common housing and are connected in four places asymmetrically, for always proper assembly. Autoclavable electrofusion chamber according to claims 6 and 7, characterized in that the groove (3) has the same dimensions as the glass panel (4), such that the glass plate fits into the groove (3) and has a lower part (2) an O-ring groove (3) is installed on the underside, of dimensions corresponding to different dimensions in the central opening of the lower part (2), and the groove (3) has the shape of an O-ring dimension. An autoclavable electrofusion chamber according to claims 6 to 8, characterized in that the size of the central circular opening in the lower part (2) determines the size of the electrofusion surface. An autoclavable electrofusion chamber according to claim 9, 5, characterized in that the size of the central circular aperture in the lower part (2) is arbitrary and determines the volume of the electro-fusate and thus the number of cells in the fusion. Autoclavable electrofusion chamber according to claims 6 to 10, io characterized in that the upper part (1) has four screws and the screws at the top have been removed screws for fast threading of the nut and that the lower part (2) has the same axis four openings through which screws extend. is Autoclavable electrofusion chamber according to claims 6 to 11, characterized in that the parts are made of autoclavable biocompatible materials. An autoclavable electrofusion chamber according to claim 12, 20, characterized in that the parts are made of biocompatible materials according to ASTM standards. Autoclavable electrofusion chamber according to claims 6 to 13, characterized in that it has several pairs of parallel electrodes, the distances between the electrodes and their depths being defined with a tolerance of ± 0.9 nm. An autoclavable electrofusion chamber according to claim 14, characterized in that the depth of the electrodes, compared to the diameter of the cells in suspension between the electrodes, is such as to generate a non-homogeneous field. An autoclavable electrofusion chamber according to claim 14, characterized in that the electrode depth is <cell radius. is Autoclavable electrofusion chamber according to claim 14, characterized in that the depth of the electrodes is not more than 0.5 to 4.5 pm. 18. Autoclavable electrofusion chamber according to claims 6 to 17, 20, characterized in that the lower part (2) has asymmetrical sides, which cover the location of the contacts on the glass panel with the electrodes, spring-loaded electrical contacts, which are established when the electrofusion chamber is assembled. Autoclavable electrofusion chamber according to claims 6 to 18, characterized in that the lid has a straight, at least 2 mm wide edge, which dimensionally corresponds to the circumference of the central opening of the lower part (2) and has a flat bottom that rests on the suspension of cells through which holes (capillaries) pass through (in) 5 which can be blown out of the air and has a holder. An autoclavable electrofusion chamber according to claims 6 to 19, characterized in that its parts are very easy to assemble into a functional electrofusion chamber and that the electrofusion chamber is very easy to disassemble into the components of the electrofusion chamber. 21. Autoclavable electrofusion chamber according to claim 6 to 20, characterized in that 15 that it can be mounted in the frame of a microscopic table. An autoclavable electrofusion chamber according to claims 14 to 18, characterized in that the electrodes are angled to create a gradient of 20 electric fields in different areas of the glass plate surface. Autoclavable electrofusion chamber according to claims 6 to 22, characterized in that it allows the optimal fusion conditions to be analyzed by analyzing the proportions of the fusion cells in different regions of the plane having the electrodes deposited on the glass surface.