WO1986002382A1

WO1986002382A1 - Medium for the production of viable, fused cells

Info

Publication number: WO1986002382A1
Application number: PCT/US1985/002037
Authority: WO
Inventors: Ulrich Zimmermann
Original assignee: Gca Corporation
Priority date: 1984-10-12
Filing date: 1985-10-11
Publication date: 1986-04-24
Also published as: GB8613803D0; DE3437469A1; GB2179368A

Abstract

In order for a medium for the production of viable, fused cells by means of field-induced electrical fusion whereby the medium exhibits a pH value compatible for the cells to be fused and comprises a largely isotonic aqueous solution of non-electrolytes and electrolytes and the ratio of concentration of non-electrolytes to electrolytes in the solution is at least 10:1, to be improved to such an extent that clearly higher yields of viable, i.e. division-capable, fused cells are obtainable, it is proposed that the non-electrolyte part be composed essentially of at least one multiply substituted derivative of one or more of the basic chemical building blocks cyclohexane, tetrahydrofuran and tetrahydropyran, whereby the cyclohexane derivatives exhibit at least two hydroxyl groups and the tetrahydrofuran as well as the tetrahydropyran derivatives exhibit at least one hydroxyl group and one amino group as substituents on the ring and/or on an aliphatic side chain, and that the isotonic property of the solution be constantly guaranteed by the overall osmolarity of the electrolyte and non-electrolyte parts.

Description

MEDIUM FOR THE PRODUCTION OF VIABLE- FUSED CELLS

BACKGROUND OF THE INVENTION

The invention relates to a medium for the production of viable, fused cells by means of field-induced electrical fusion whereby the medium exhibits a pH value compatible for the cells to be fused and comprises a largely isotonic aqueous solution of non-electrolytes and electrolytes with the ratio of concentration of non-electrolytes to electrolytes in the solution being at least 10:1.

A medium of the initially described kind may be composed, for example, of an aqueous solution and boundary layers of ^"higherπmolecular susbtances, such as proteins, which accumulate on surfaces inside a fusion chamber.

The production of fused cells can be broken down into three phases: an orientation phase, the actual fusion processes and a healing phase of the fused cells.

In the orientation phase, the cells which are to be fused are oriented, for example, along lines of force. Particularly suitable for this purpose is the dielectrophoretic effect which is based on polarization phemomena in the cells in the electric field and brings the cells to a certain contact distance from each other. The electric field is built up by a high-frequency alternating voltage in the range of approximately 1 MHz in order to minimize electrolysis phemonema in the medium. The prevention of electrolysis phenomena in the stage prior to fusion is -2-

extremely important because the products of electrolysis have a toxic effect on the cells, particularly when the cellular membranes are in a state of increased permeability.

The actual fusion process is initiated by a field pulse which increases the permeability of the cellular membranes in the contact area between two cells to such an extent that pores or penetrations occur in the membranes through which an exchange of the cytoplasm of the two cells can take place. The a v)litude of the field pulse or the required field strength is inversely proportional to the radius of the cells under treatment according to the Laplace equation (see Biochimica et Biophysica Acta 694, 227 to 277; equation (5)) so that for small cells high field strengths and for large cells low field strengths are necessary in order to produce the penetrations or pores in the membranes. Directly after the field pulse, the field conditions orienting the cells are maintained for a short time in order to facilitate the fusion of the cellular membranes. Also of importance in this regard is the conductivity of the medium or fusion solution which must be so limited that there is no disturbing generation of heat, as a result of which there would be convection currents which would adversely affect the orientation of the fusing cells in relation to each other.

After the alternating field has been switched off, there usually follows a period lasting up to 30 minutes and longer in which the healing of the cellular membranes is normally supported by temperature increase. -During this time, the newly fused cell is still rather sensitive to ambient influences since the permeability of the cellular membranes is still at an increased level and, therefore, the selection mechanism of the membranes is partially inoperative.

Currently, fusion technology is at a level at which cells can be fused with high yields (e.g. 40% and above). However, the viability of the newly produced cells is extremely low with all hitherto known processes and associated media or solutions, i.e. the fused cells are usually not capable of division, so that although a fusion process provides a large number of fused cells, only a few individual cells are capable of division.

It is therefore a principal object of the present invention to create a medium in which clearly improved yields of viable, i.e. division-capable, fused cells are obtainable.

A further object of the prsent invention is to apply the medium according to the invention for the production of viable cells in a modified fusion process such that the survival rate of the fused cells is improved.

A still further object of the invention is the application of a medium according to the invention for the production of viable, fused cells for the non-damaging collection and orientation of the cells before and after the application of the field pulses.

SUMMARY OF THE INVENTION

A medium for the production of viable, fused cells by means of field-induced electrical fusion is provided whereby the medium exhibits a pH value compatible for the cells to be fused and comprises a largely isotonic aqueous solution of non-electrolytes and electrolytes and the ratio of concentration of non-electrolytes to electrolytes in the solution is at least 10:1. In order to allow the medium to produce clearly higher vields of viable, i.e. division-capable, fused cells the non-electrolyte part is composed essentially of at least one multiply substituted derivative of one or more of the basic chemical building blocks cyclohexane, tetrahydrofuran and tetrahydropyran, whereby the cyclohexane derivatives exhibit at least two hydroxyl groups and the tetrahydrofuran as well as the tetrahydropyran derivatives exhibit at least one hydroxyl group and one a ino group as substituents on the ring and/or on an aliphatic side chain, and that the isotonic property of the solution be constantly guaranteed by the overall osmolarity of the electrolyte and non-electrolyte parts.

The medium of the present invention as described above may be utilized in a modified fusion process so that the survival rate of the fuse cells is improved. To achieve such an improved survival rate the cells, after being fused, are transferred into an aftertreatment medium, the overall osmolarity of the components of which guarantees an

property of the solution. In order to apply a medium, of the type described above, before and after the application of the field pulses, an electric alternating field with a frequency below 100 kHz is used.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present invention a medium of the initially described kind is provided in which the non-electrolyte part is composed essentially of at least one multiply substituted -5-

derivative of one or more of the basic chemical building blocks cyclohexane, tetrahydrofuran and tetrahydropyran whereby the cyclohexane derivatives exhibit at least two hydroxyl groups and the tetrahydrofuran as well as the tetrahydropyran derivatives exhibit at least one hydroxyl group and one amino group as substituents on the ring and/or on an aliphatic side chain whereby the isotonic property of the solution is constantly guaranteed by the overall osmolarity of the electrolyte and non-electrolyte parts.

Since the non-electrolyte concentration for many cell strains must be up to one mole and above so that the medium is isosmolar, a critical selection of the non-electrolytes is of great importance particularly as regards their toxic properties with respect to the cell whose cellular membrane has increased permeability. One of the principal requirements for these non-electrolytes is that they must have little or no adverse effect on the enzymatic activities of the cell's own enzymes. This is not the case with hitherto used non-electrolytes, such as mannitol. According to the invention, a wide range of substances are sutiable as non-electrolytes, for example, 1, 4 cyclohexanediol, scyllitol, as well as esters of quinic acid and shikimic acid, as well as, for example, amino sugars and their derivatives.

Preferred cyclohexane derivatives carry an amino group on the ring. Other preferred cyclohexane derivatives exhibit six hydroxyl groups on the ring, for example, inositol.

Suitable non-electrolytes for the medium for the production of viable cell hybrids are tetrahydrofuran and tetrahydropyran derivatives which exhibit an amino group on the ring, particularly amino sugars, such as glucosamine or galactrsamine.

Tetrahydrofuran derivatives with a furanoside structure and tetrahydropyran derivatives with a pyranoside structure can likewise be advantageously added to the medium as non-electrolytes.

Calcium and magnesium salts as electrolytes in the medium have a stabilizing effect on the fusion yields, so that there is good reproducibility of the fusion method. Particularly noteworthy in this respect is the ratio of concentration of calcium and magnesium ions since, in addition to the absolute ionic concentrations, this ratio has a regulating effect on the energy onversion and metabolism of the cell. It is practical to employ calcium/magnesium ratios between 1:2 and 1:10 whereby the range between 1:4 and 1:6 is to be preferred in particular.

A further increase in the yields of living, fused cells is obtainable by optimizing the electrolyte part of the fusion medium. Preferably, the ele trolyte part consists of calcium and magnesium salts which may be chlorides or acetates. Important in this connection is the total ionic strength of the medium surrounding the cell because this has an effect on the adhesion properties of the cellular membrane to other surfaces (J. Cell Seci. j>3, 133 to 124 (1983)). This effect was able to be applied to the adhesion of two cellular membranes by adding, in particular, calcium magnesium salts of a defined total ionic strength to the solution. Calcium concentrations in the range between 0.05 mM and 0.2 mM and magnesium concentrations between 0.05 mM and 1 mM have proven favorable for the fusion medium. Magnesium concentrations in the range between 0.05 mM and 0.6 mM have a particulrly favorable effect on the fusion yields.

It is practical to buffer the solution in the case of a physiological pH value. Particularly suitable for this are phosphate buffers or the addition of histidine. In the case of the phosphate buffer, of course, its ionic strength must be included in the total ionic strength. Particularly cell-compatible are phosphate buffers consisting of a mixture of KH₂P0₄ and KJrTPO. or KH-.P0. and Na-HPO., whereby the range for the buffer concentration, also for the histidine buffer, is advantageously between 1 and 10 mM.

The results of fusion are positively influenced by the exchange of physiological cations for bioccmpatible cations which at least partially compensate for the negative surface charges of the πiembranes. In particular, biocompatible cations exhibiting a low charge density and an electrostatically weakly bound hydrate shell contribute toward improved bringing together of the two cells in that the hydrate structure at the surface of the cellular membranes is less heavily pronounded and more weakly bound, thus causing a reduced steric hindrance as the cells approach each other.

Particularly preferred media contain oligo- and/or polycationic oligo- and/or polypep ides which, due to the fact that they are multiple-charged, compensate for a greater proportion of the negative surface charge of the membranes, yet still exhibit an electrostatically weakly bound hydrate shell. Particularly preferred are oligo- and/or polypeptides which have an isoelectric point above pH = 7 since, in the case of a physiological pH value, they carry an excess positive charge. It is favorable to use proteins, particularly cytochrome and/or histones as polypeptides. It is also advantageous to use oligo- and/or polylysine as bioccmpatible cations.

Problems may occur with products of electrolysis as a result of the alternating voltage which is applied for the dielectrophoretiσ collection and orientation of the cells, even if an alternating field of relatively high frequency (for example in the MHz range) is employed. Therefore, to trap products of electrolysis, it is advisable to add further non-electrolytes to the solution. Catalase is particularly suitable in small quantities for decomposing H₂0₂. it is advantageous to add radical scavengers to the medium individually or in combination, particularly glutathione, albumin, cysteine, $-mercaptoethanol or tocopherol, whereby the individual concentrations are approximately 1 mM and 1 mg/ml for catalase, cysteine and albumin.

The radical scavengers are particularly practical if the calcium and magnesium salts are used in the form of chlorides. Some of the problems of electrolysis can be eliminated from the outset through the use of the corresponding acetates.

The healing of the penetrations in the cellular membranes and the complete rounding off of the cells is performed to conclude the fusion process usually at a temperature above room temperature, for example 37°C, in order to accelerate these processes.

In order to produce viable cells in a modified fusion process such that the survival rate of the fused cells is improved, the cells, after being fused, are transferred into an aftertreatment medium containing as is components:

-NaCl with a concentration between 5 mM and 140 mM,

-KC1 with a concentration between 5 mM and 140 mM,

-phosphate buffer with a phosphate concentration between 5 mM and 30 mM,

-Mg chloride or acetate with a concentration between 0.05 mM and 2 mM and

-Ca chloride or acetate with a concentration between 0.05 mM and 2 mM

whereby the overall osmolarity of the components guarantees an isotonic property of the solution.

The cells remain for approximately 30 minutes in this aftertreatment solution which is preferably at a temperature of 37°C. The healing of the membrane penetrations is then largely completed and the selection system of the cellular membranes is fully restored, with the result that the cells react less sensitively to ambient influences. Subsequently, the cells are cultivated in known manner.

It is advantageous to use a medium for the aftertreatment of the fused cells in which the sum of the individual concentrations of NaCl and KC1 exhibits approximately the value of 145 mM whereby the KC1 concentration is between 5 mM and 70 mM. Particularly preferred in this connection is the KC1 concentration range between 20 mM and 60 mM. It is practical to adjust the ratio of concentration of calcium and magnesium ions in the medium for the aftertreatment of fused cells in the range between 1:2 and 1:10. Particularly preferred is a ratio of concentration of the calcium and magnesium ions in the range between 1:4 and 1:6.

Although increasing the frequency of the electric alternating field for the collection and orientation of the cells can drastically restrain the above-described problems of electrolysis phenomena, this is itself limited by the fact that a higher frequencies of the electric alternating field there is a reduction of the shielding effect of the cellular membranes for the cell interior with the result that the alternating field acts on the cell interior where it may cause changes which are detrimental to the viability of the cell.

In order to apply a medium, of the type described above, before and after the application of the filed pulses, an electric alternating field with a frequency below 100 kHz is used. In the frequency range below 100 kHz (frequencies down into the 1 Hz range are possible) the shielding of the cellular membrane for the cell interior is virtually complete. The products of electrolysis which occur in increased quantities at this relatively low frequency of the electric alternating field are trapped and decomposed by the radical scavengers which are added to the medium.

It is particularly advantageous to select the frequency of the electric alternating field such that it is below the Maxwell-Wagner rotation frequency and outside other rotation frequency ranges of the cells which are to be fused.

Consequently, initially, the cells are brought in a particularly non-damaging manner to the contact distance required for fusion - furthermore, thanks to the now possible lengthening of the residence time of the cells in the electric field it is possible to obtain better orientation of the cells - and, secondly, there are none of the rotational or spinning motions of the cells which occur in low frequency ranges and which may partially cancel out the orienting effect of dielectrophoresis on the cells.

For each cell type there are one or more frequency ranges in which a maximum rotational motion of the cell in suspension medium is caused (Biochimica et Biophysica Acta 694, 227 - 277 (1982), p. 239 ff) . The position of these frequency ranges is mainly dependent on the type of cell, the size of cell and on the conductivity of the suspension medium.

It is particularly advantageous to perform the fusion process at low alternating field frequencies and, after the actual fusion process, to transfer the fused cells into one of the above-described media for the aftertreatment of the fused cells and to allow the healing processes to take place in this medium. This assures, initially, extremely non-damaging treatment of the cells during fusion and also guarantees that the newly fused cells, which, due to the increased permeability of their cellular membranes, react particularly sensitively to the ambient conditions and encounter particularly favorable conditions for completing the healing process of the membranes while retaining their capability of division. A particularly pronounced increase in the yield of viable, fused cells is obtained particularly in the case of very sensitive systems, such as the hybridoma cells. Yeast cells react less sensitively to the ambient conditions. These and further aspects and advantages of the invention are explained in greater detail below with reference to the following non-limiting examples:

Example 1

FUSION OF SP 2/0 MYELOMA CELLS WITH MURINE LYMPHOCYTES.

A cell suspension of myeloma cells (SP 2/0) and lymphocyte cells in the ratio of 1:10 and a total suspension

7 density of 1.1 x 10 cells/ml are transferred into a fusion - chamber in a medium of the composition described further below.

In the fusion chamber the electrodes (platinum wires with, a diameter of 0.2 mm) are disposed at a distance of 0.2 mm from each other. The fusion chamber is controlled to a temperature of

20°C during fusion.

The myeloma cells show a lager diameter than the lymphocyte cells. The amplitude of the field pulses which create penetrations in the membranes must be selected according to the Laplace equation (Biochimica et Biophysica Acta 694, 227 to 277; equation (5)) in accordance with the requirements of the smaller lymphocyte cell. In the large cell, the myeloma cell, therefore, there are also penetrations of pores outside the region of the contact zone with the lymphocyte cell. This is partly a reason for the greater sensitivity of these newly fused cells (the so-called hybridoma cells) to the ambient influences. In order to prevent the fusion of two or more myeloma cells and to control fusion in the direction of the hybridoma cells, the lymphocyte cells are present in a 10-fold excess. This ratio guarantees that, basically, every myeloma cell has contact with a lymphocyte cell.

The medium according to the invention, in which the cells are suspended during fusion, contains 0.28 M inositol property of the fusion medium. Consequently, the electrolyte part can be kept very small so that the conductivity of the solution likewise remains very low. The low conductivity of the solution has the advantage that, during the use of the electric alternating field for dielectrophoresis, there is only very slight heating of the solution, as a result of which problems with thermal convection currents in the fusion chamber, which would adversely affect the bonding of the cell chains, are prevented.

A physiological pH value (approximately 7) of the fusion medium is assured with the aid of a phosphate buffer (concentration 1 mM; KH₂0₄/K₂HP0₄) .

An optimum ionic strength of the solution is obtained with the additional components of 0.1 mM calcium acetate and 0.5 mM magnesium acetate. The electrical fusion conditions are:

Sinusoidal alternating field for 30 s with a field strength of 250 Van and a frequency of 1.5 MHz.

Three direct voltage pulses with a field strength of 3.5 kVcrn^"" and 20us duration are applied at intervals of 1 s. Subsequently, a sinusoidal alternating field is again applied to the electrodes for 30 s with a frequency of 1.5 MHz and a field strength of 250 Vcm^" . Directly after this, the cells are kept in the fusion medium for about 10 minutes at 37°C.

Subsequently, the fused cells - which at this point in time exhibit increased permeability of the cellular membrane - are, until the membrane penetrations have completely healed and until rounding-off, transferred into an aftertreatment medium with a temperature of 37°C and are left there for approximately 30 minutes.

The aftertreatment medium is tailored to the fusion medium and consists basically of an aqueous solution of the following components:

80 mM NaCl

60 mM KC1 8 mM Na₂HOP₄ 1.5 mM KH₂P0₄ 0.5 mM Mg acetate 0.1 mM Ca acetate

With the aid of the fusion medium according to the invention as well as the aftertreatment of the fused cells according to the invention, it has been possible to increase the yield of a few individual viable cells (prior art, e.g. FEBS Letters 137, 11 to 13 (1982)) to 140 viable, i.e division-capable, hybridoma cells. The good reproducibility of the yields of division-capable cells is to be particularly emphasized in this connection. Hitherto it was only possible to fuse cells with good yields, but without being able to obtain satisfactory and reproducible results as regards their survival rate.

Example 2:

FUSION OF SP 2/0 MYELOMA CELLS WITH MURINE LYMPHOCYTES.

Conditions as in example 1, but the concentration of inositol in the fusion medium is replaced by a 0.28 M concentration of glucosamine.

Thus, this medium also posseses the properties of a medium according to the invention for the production of viable, fused cells. With 128 viable hybridoma cells, the result of fusion is similarly satisfactory to that of the solution containing inositol.

Example 3:

FUSION OF 2/0 MYELOMA CELLS WITH MURINE LYMPHOCYTES.

Conditions as in example 1, but in this case components were additionally added to the fusion medium to allow the low-frequency collection and orientation of the cells:

1 mM glutathione 1 mg/ml high-purity albumin 0.1 mg/ml catalase (purified by means of dialysis)

-16-

The frequency of the electric alternating field was able to be lowered to 20 kHz and the field strength of the alternating ffiieelldd ttoo 118800 VVccmm^"1.. AA ffuurrtthh*er possible frequency of the electric alternating field is 800 Hz.

As compared to the process with a collection frequency of 1.5 MHz (example 1), the collection frequency of 20 kHz made it possible to achieve a further drastic increase in the yield to 280 viable hybridoma cells.

In the following examples, individiual aspects of the invention are once again illustrated with reference to the example of the fusion of yeast cells of the strains Saccharomyces cerevisiae AH 22 pADH and Saccharomyces cerevisiae AH215. In each case, a comparison is made to the prior art which is described in the publication FEMS Microbiol, Letters 2A_^ (1984), 81 to 85. Since the fused yeast cells react with low sensitivity to ambient influences, an aftertreatment in a separate medium is not necessary for the production of viable, fused cells, and, therefore, the effect of the individual measures in the optimization of the actual fusion process and of the corresponding medium can be illustrated with reference to examples 4 to 8.

Example 4:

PRODUCTION OF YEAST CELL HYBRIDS.

q

The mixture ratio of the two cell types was 1.5 x 10

Q cells/ml and 2 x 10 cells/ml. Fusion was performed in so-called helix chambers (see FEMS Microbiol. Letters 2A_, 81 to 85 (1984)) at a temperature between 20 and 25°C.

The electrical field conditions of fusion were:

Sinusoidal alternating field with a field strength of between 250 and 275 V cm^" and a frequency of 2 MHz. The alternating field was applied both before and after the application of two square-wave field pulses (field strength 10 kV cm^" , pulse duration 10μ s, interval 0.5 s).

After the actual fusion process the cells remained for a further 10 minutes in the fusion chamber at 20 to 25°C. whereafter the temperature was raised for 20 minutes to 30°C in order to accelerate the healing of the membranes and the rounding-off of the cells.

Subsequently, the cells were cultivated in known manner. The yield of division-capable hybrids can be deduced from the number of colonies which grew.

The composition of the aqueous solutions in which the cells were suspended for experiments 1 to 4 can be seen from Table 1.

Table 1:

Control

Experiment 1 2 3 4

CaCl₂ 0.1 mM 0.1 mM 0 0.1 mM

MgCl₂ 0.5 mM 0.5 mM 0 0.5 mM

Inositol 0.28 M 0.28 M 0 0.28 M

Sorbitol 0.92 M 0.92 M 1.2 M 0.92 M

No. of division- capable hybrids 672 536 42

Relative yield in % 100 79.8 6.3

Experiment 3 corresponds in its fusion conditions to the prior art (FEMS Microbiol. Letters 24, 81 to 85 (1984)). With 42 hybrids, the yield likewise corresponds to the prior art (50 to 60 hybrids).

In control experiment 4 the cell suspension was not exposed to an electric field.

Experiments 1 and 2 show the enormous, reproducible increase in the yields of viable hybrids with the partial replacement of sorbitol by inositol according to the invention as well as with calcium and magnesium concentrations according to . the invention.

Example 5:

PRODUCTION OF YEAST CELL HYBRIDS.

The conditions and the treatment of the fused cells are the same as those in example 4. The compositions of the media for the production of viable, fused cells can be taken from Table 2.

Table 2:

Control

Experiment 1 2 3 4

Ca acetate 0.1 mM 0 0 0.1 mM

Mg acetate 0.5 mM 0 0 0.5 mM

CaCl₂ 0 0.1 mM 0 0

MgCl₂ 0 0.5 mM 0 0

Inositol 0.28 M 0.28 M 0 0.28 M

Sorbitol 0.92 M 0.92 M 1.2 M 0.92 M

No. of division- capable hybrids 990 584 119 2

Relative yield in % 100 59 12 0.2

Experiment 3 corresponds once again in its fusion conditions to the prior art and was performed under identical conditions of experiment 3 in example 4. From this it becomes clear what low level of reproducibility is obtainable as regards the yields of division-capable hybrids if fusion is performed accordirg to the prior art (pure sorbitol solution, no calcium or magnesium ions) .

In the control experiment 4, the cell suspension was once again not exposed to an electric field. The two hybrids which were produced stem from spontaneous, i.e. non-induced, cell fusion. Experiment 2 corresponds directly to experiments 1 and 2 in example 4; once again, reference must be made to the good reproductibility of the yields.

Example 1 shows that the exchange of the chlorides for the acetates provides a further clear increase in the yield of viable, fused cells. This means that, even with the high alternating field frequency of 2 MHz used here, there are problems of electrolysis if chlorides are used.

If experiment 1 is conducted in the same manner, but with the difference that the 0.92 M sorbitol and 0.28 M inositol in the solution are replaced by 1.2 M glucosamine, then, with 870 hybrids, the result is a very good yield within the reproducibility.

Example 6:

PRODUCTION OF YEAST CELL HYBRIDS

The conditions and the treatment of the fused cells were selected as in example 4.

The compositions of the media for the production of viable, fused cells can be seen from Table 3.

Table 3:

Control

Experiment 1 2 3 4

Catalase 0.094 mg/ml 0.092 mg/ml 0 0.092 mg/ml

Glutathione 0.1 mM 0 0 0 βπmercaptoethanol 0 1.95 mM 0 1.95 mM

BSA* 1 mg/ml 1 mg/ml 0 1 mg/ml

Inositol 0.28 M 0.28 M 0.28 M 0.28 M

Sorbitol 0.92 M 0.92 M 0.92 M 0.92 M

Ca acetate 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Mg acetate- 0.5 mM 0.5 mM 0.5 mM 0.5 mM

No. of division- capable hybrids 1429 1272 793 3

Relative yield in % 100 89 55.5 0.2

* BSA = Bovine Serum Albumin

.experiment 3 corresponds to the fusion conditions of experiment 1 in example 5. Once again, the very good reproducibility of the yields when fusion is performed in media according to the invention becomes clear.

In control experiment 4 the cell suspension was once again not exposed to an electric field. The three hybrids produced stem from spontaneous, i.e non-induced, cell fusion.

As shown by experiments 1 and 2, it is possible through the addition of radical scavengers to trap and decompose products of electrolysis, thereby achieving a further increase in the yield of viable, fused cells. This increase proves again that, with a 2 MHz frequency of the alternating field, there are problems with electrolysis during fusion. Overall, therefore, an increase in the yield of yeast cell hybrids by a factor of approximately 30 can reproducibly be realized with the fusion media according to the invention.

Example 7:

PRODUCTION OF YEAST CELL HYBRIDS

The solution used for the suspension of the cells during fusion is identical to the composition given in Table 3 (example 6) under experiment 1. The field pulses are likewise applied as in the preceding examples.

Only the alternating field conditions were varied (see Table 4). Thanks to the radical scavengers which were added, it is possible to greatly reduce the frequency of the alternating field, thus preventing the electric field from acting on the interior of the cell. The products of electrolysis which occur in increased quantities are trapped by the ccmponents catalase, glutathione and albumin, so that there is no additional damage to the cells. Conversely, the collection and orientation of the cells is performed under non-damaging <onditions by means of dielectrophoresis (in addition to the frequency of the alternating field it is also possible to reduce the field strength), which results, in experiments 2 and 3, in a further increase in the yield of viable hybrids as compared to high-frequency fusion technology (2 MHz).

In control experiment 4 the cell suspension was once again not exposed to an electric field. -23-

Table 4

Control

Experiment 1 2 3 4

Frequency 2 MHz 10 kHz 20 kHz

Amplitude 5 V 4-4.5 V 4-4.5 V

No. of division- capable hybrids 1355 1720 1610 1

Relative yield in % 79 100 94

Example 8:

PPODUCTION OF YEAST CELL HYBRIDS

On the basis of fusion experiment 1 in example 5, this example demonstrates in experiments 2 and 3 the effect of the addition of bioccmpatible cations which act on the hydrate structure in the region of the cellular membrane, in this case taking the example of cytochrcme.

As in the preceding examples, the cell suspension in control experiment 4 is once again not exposed to an electric field.

A slight increase in the yield of viable hybrids can be registered due to the addition of cytochrcme. Cytochrome results, above all, in improved membrane contact between the cells, thus raising the probability of fusion. The influence of cytochrcme on the capability of the yeast cell hybrids to divide appears to be less significant, with the result that, overall, the already very good yields from experiment 1 in example 5 are in the main further stabilized. -24-

Table 5:

Control

Experiment 1 2 3 4

Ca acetate 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Mg acetate 0.5 mM 0.5 mM 0.5 mM 0.5 mM

Cytochrome 0 1 mM 5 mM 0

Sorbitol + Inositol 1.2 M 1.2 M 1.2 M 1.2 M

No. of 910 1090 1120 0 division- capable hybrids

Relative yield in % 81 97 100 0

While the foregoing invention has been described with reference to its preferred embodiments, various modifications and alterations will occur to those skilled in the art. All such variations and modifications are intended to fall within the scope of the appended claims.

Claims

What is claimed is:

1. Medium for the production of viable, fused cells by means of field-induced electrical fusion whereby the medium exhibits a pH value compatible for the cells to be fused and comprises a largely isotonic aqueous solution of non-electrolytes and electrolytes and the ratio of conetentration of non-electrolytes to electrolytes in the solution is at least 10:1, the non-electrolyte part being composed essentially of at least one multiply substituted derivative of one or more of the basic chemical building blocks — cyclohexane, tetraphdrofuran and tetrahydropyran — with the cyclohexane derivatives exhibiting at least two hydroxyl groups and the tetrahydrofuran as well as the tetrahydropyran derivatives exhibiting at least one hydroxyl group and one amino group as substituents on the ring and/or on an aliphatic side chain whereby the isotonic property of the solution is constantly guaranteed by the overall osmolarity of the electrolyte and non-electrolyte parts.

2. Medium as defined in claim 1 wherein the non-electrolyte part comprises a cyclohexane derivative which carries an amino group on the ring.

3. Medium as defined in claim 1 wherein the medium comprises a cyclohexane derivative with six hydroxyl groups on the ring.

4. Medium as defined in claim 3 wherein inositol is present in the solution as the cyclohexane derivative. -26-

5. Medium as defined in claim 1 wherein the tetrahydrofuran and/or tetrahydropyran derivatives exhibit an amino group on the ring.

6. Medium as defined in claim 1 wherein the tetrahydrofuran derivatives exhibit a furanoside structure.

7. Medium as defined in claim 1 wherein the tetrahydrofuran derivatives exhibit a furanoside structure.

8. Medium as defined in claim 1 wherein the tetrahydropyran derivatives are amino sugars.

9. Medium as defined in claim 8 wherein glucosamine is present in the solution as amino sugar.

10. Medium as defined in claim 8 wherein galactosamine is present in the solution as amino sugar.

11. Medium as defined in claim 1 wherein the electrolyte part is composed mainly of calcium and magnesium salts.

12. Medium as defined in claim 1 wherein the electrolyte part is composed of calcium and magnesium salts as well as of a phosphate bufrer with the buffer concentration being greater than or equal to the total concentration of the magnesium and calcium ions.

13. Medium as defined in claim 1 wherein the ratio of concentration of the calcium to the magnesium ions is in the range between 1:2 and 1:10.

14. Medium as defined in claim 11 wherein the calcium and magnesium salts are chlorides.

15. Medium as defined in claim 11 wherein the calcium and magnesium salts are acetates.

16. Medium as defined in claim 11 wherein the calcium concentration is in the range between 0.05 mM and 0.2 mM and the magnesium concentration is between 0.05 mM and 1 mM.

17. Medium as defined in claim 16 wherein the magnesium concentration is adjusted in the range between 0.05 and 0.6 mM.

18. Medium as defined in claim 12 wherein the phosphate buffer is composed of a mixture of KH K>₄ and K₂HP0₄.

19. Medium as defined in claim 12 wherein the phosphate buffer concentration is between 1 mM and 10 mM.

20. Medium as defined in claim 1 wherein the pH value of the solution is adjusted by a histidine buffer.

21. Medium as defined in claim 20 wherein the concentration of histidine is between 1 mM and 10 mM.

22. Medium as defined in claim 1 wherein said medium has a pH value of approximately 7.

23. Medium as defined in claim 1 wherein the electrolyte part comprises bioccmpatible cations which replace physiological cations on negatively charged membrane surfaces and change the water structure in the region of the cellular membrane.

24. Medium as defined in claim 23 wherein the bioccmpatible cations are oligo- and/or polycationic oligo- and/or polypeptides, respectively.

25. Medium as defined in claim 24 wherein cytochrome and/or histones are contained as polypeptides.

26. Medium as defined in claim 1 wherein said medium contains, as a further non-electrolyte, pure catalase in small quantities for the decomposition of H 0₂.

27. Medium as defined in claim 1 wherein said medium comprises radical scavengers as^" additional non-electrolytes.

28. Medium as defined in claim 27 wherein glutathione and/or albumin are contained as radical scavengers.

29. Medium as defined in claim 28 wherein glutathione •ind/or albumin are contained with a concentration of approximately 1 mM and/or with a concentration of approximately 1 rig/ml, respectively.

30. Medium as defined in claim 27 wherein cysteine, tocopherol and g-mercaptoethanol are added to the medium individually or in combination as additional radical scavengers.

31. A process for utilizing a medium designed for the production of viable, fused cells by means of field-induced electrical fusion, said medium comprising a largely isotonic aqueous solution of non-electrolytes with electrolytes and the ratio of concentration of non-electrolytes to electrolytes in the solution being at least 10:1, the non-electrolyte part being composed essentially of at least one multiply substituted derivative of one or more of the basic chemical building blocks — cyclohexane, tetraphdrofuran and tetrahydropyran — with the cyclohexane derivatives exhibiting at least two hydroxyl groups and the tetrahydrofuran as well as the tetrahydropyran derivatives exhibiting at least one hydroxyl group and one amino group as substituents on the ring and/or on an aliphatic side chain whereby the isotonic property of the solution is constantly guaranteed by the overall osmolarity of the electrolyte and non-electrolyte parts, the process comprising the steps of: after the cells are fused, transfering the cells into an aftertreatment medium containing as its components -NaCl with a concentration between 5 mM and 140 mM, -KC1 with a concentration between 5 mM and 140 mM, -phosphate buffer with a phosphate concentration between 5 mM and

-Mg chloride or acetate with a concentration between 0.05 mM and 2 mM and

-Ca chloride or acetate with a concentration between 0.05 mM and 2 mM whereby the overall osmolarity of the components guarantees an isotonic property of the solution.

32. Process as defined in claim 31 wherein in the medium for the aftertreatment of the fused cells the sum of the individual concentrations of NaCl and KCl exhibits approximately the value of 145 mM whereby the KCl concentration is between 5 mM and 70 M.

33. Process as defined in claim 32 wherein the KCl concentration is in the range between 20 mM and 60 mM.

34. Process as defined in claim 31 wherein the ratio of concentration of calcium and magnesium ions in the medium for the aftertreatment of fused cells is in the range between 1:2 and 1:10.

35. Process as defined in claim 34 wherein the ratio of concentration of calcium and magnesium ions is in^* the range between 1:4 and 1:6.

36. A process for utilizing a medium designed for the production of viable, fused cells by means of field-induced electrical fusion said medium comprising a largely isotonic aqueous solution of non-electrolytes and electrolytes with the ratio of concentration of non-electrolytes to electrolytes in the solution being at least 10:1, the non-electrolyte part being composed essentially of at least one multiply substituted derivative of one or more of the basic chemical building blocks — cyclohexane derivatives exhibiting at least two hydroxyl groups and the tetrahydrofuran as well as the tetrahydropyran derivatives exhibiting at least one hydroxyl group and one amino group as substituents on the ring and/or on an aliphatic side chain whereby the isotonic property of the solution is constantly guaranteed by the overall osmolarity of the electrolyte and non-electrolyte parts, said medium containing, as a further non-electrolyte, pure catalase in small quantities for the decomposition of H₂0.-, th^e process comprising the steps of: applying an electrins alternating field for the orientation and collection of cells in the production of viable, fused cells, said electric alternating field having a frequency below 100 kHz.

37. Process as defined in claim 36 wherein the frequency of the electric alternating field is below the Maxwell-Wagner rotation frequency and outside other rotation frequency ranges of the cells which are to be fused.

38. Process as defined in claim 36 wherein, after being fused, the cells are transferred into an aftertreatment medium containing as its components

-NaCl with a concentration between 5 mM and 140 mM,

-KCl with a concentration between 5 mM and 140 mM,

-phosphate buffer with a phosphate concentration between 5 mM and 30 mM,

-Mg chloride or acetate with a concentration between 0.05 mM and 2 mM, and

-Ca chloride or acetate with a concentration between 0.05 mM and mM

39. Process as defined in claim 38 wherein in the medium for the aftertreatment of the fused cells the sum of the individual concentrations of NaCl and KCl exhibits approximately the value of 145 mM whereby the KCl concentration is between 5 mM and 70 mM.

40. Process as defined in claim 39 wherein the KCl concentration is in the range between 20 mM and 60 mM.

41. Process as defined in claim 38 wherein the ratio of concentration of calcium and magnesium ions in the medium for the aftertreatment of fused cells is in the range between 1:2 and 1:10.

42. Process as defined in claim 41 wherein the ratio of concentration of calcium and magnesium ions falls in the range between 1:4 and 1:6.