Improvements in and relating to preservation of cells

The subject of the present invention is a method of conserving at low temperature cells from animal tissues and usable as culture media.

Viable cells, obtained from tissues of various animals, find many important uses. For example, cells from animal organs, for example kidney cells, provide culture media for viruses, particularly in the manufacture of vaccines.

An object of the invention is to provide a method for the storage at low temperature of cells originating from animal tissues, making it possible to keep these cells in a viable state for long periods and in a state such that they can maintain growth. of a virus, for the manufacture of a vaccine.

The expression "tissue", as used in this presentation, denotes an agglomeration of cells, with their intercellular substance, forming one of the components of the structure of an animal. This definition includes tissues in the form of organs and bone marrow, but excludes body fluids, blood and cells present in the animal as isolated cells, for example erythorcytes and leukocytes.

The method according to the invention, in which cells of animal tissue are brought into contact with a protective additive to reduce the deterioration of the cells during freezing and thawing, is characterized (1) in that the cell combination additive until the combination has solidified and the latent heat of fusion has been eliminated, (2) in that the solidified cells are continued to cool to -50 ° C at a speed not exceeding about 3 ° C / min, and (3) in that the cells are continued to cool frozen and keeps frozen cells below -75 ° C.

The method according to the invention is generally applicable to the conservation of cells from any animal, preferably mammals. For example, bone marrow and kidney, liver, heart, spleen and lung tissue from birds, reptiles, fish, rabbits, monkeys, cats, dogs, rodents, horses and⁰ man, can be preserved by the method according to the invention.

The cells to be preserved by the method according to the invention can be obtained in known manner from donor animals. Several ways of obtaining different types of cells and cellular agglomerations are described in detail in the examples.

The method according to the invention is applicable to the conservation of tissues in the form of agglomerations¹¹ relatively small, for example in the form of bone marrow or finely chopped tissue, as well as in the preservation of tissue in the form of large cellular agglomerations, including whole organs, for example kidneys and lungs. The pro⁵ assigned according to the invention is also applicable to the conservation of cells initially present in the animal as part of a tissue cell agglomeration, but which have been separated into isolated cells before freezing.

The cells to be preserved are brought into contact with a protective additive which reduces the deterioration of these cells. cells during the freezing and thawing stages. The cells can be brought into contact with the protective additive in any suitable manner, for example by perfusion of a whole organ with a liquid medium containing a protective additive, in addition during cell agglomerations or cells isolated in a medium. liquid containing a protective additive, or by pouring onto the cellular agglomerations a liquid medium containing a protective additive. When an entire organ is to be preserved by the method according to the invention, it suffices to perfuse the organ with the liquid medium containing the protective additive, then to freeze the organ without further treatment. When bone marrow, chopped tissue or isolated cells from a tissue are to be preserved according to the invention, it is preferable to prepare a suspension of the cell agglomerations or isolated cells in a medium containing a protective additive, then to freeze the entire suspension.

Among the protective additives which can be used in the present process, mention may be made of the following compounds: glycerol; ethylene glycol; aldoses and ketoses such as xylose, perythrosis, parabinosis, rhibose, glucose, fructose and galactose; polysaccharides such as maliosis, sucrose, lactose and raffinose; dextran; polyvinylpyrrolidone; serum albumin; dimethyl sulfoxide; mannitol; and Mash. It is also possible to use mixtures of two or more of these protective additives.

Some of these protective additives, such as glycerol and monosaccharides, cross the membrane of individual cells. Other protective additives, notably polysaccharides and polyvinylpyrrolidone, do not penetrate through the cell membrane. The chemical and physiological properties of these various “intracellular” and “extracellular” protective additives are well known.

In the process according to the invention, it is preferable to use the protective additive in a liquid medium additionally containing nutrients for the cells. These nutrients can include, for example, amino acids, saccharides, inorganic salts, proteins, vitamins and autologous, homologous or heterologous animal serum. Examples of perfusion medium and / or suspension which can be used in the process according to the invention are given below. All percentages are given by volume, unless otherwise indicated.

(AT)

10% egg yolk 20% serum 20% glycerol 50% MEM from Eagle

Eagle's minimum essential medium (MEM) is a commercially available synthetic nutrient medium for the cultivation of mammalian cells, either in single layers or in suspensions. It is a complex mixture containing amino acids, vitamins, inorganic salts and glucose.

(B)

10% egg yolk 20% serum 20% glycerol 50 o / o TC-199

"TC-199" is a nutritious medium which is found on the market and which is a little richer than the

SAME.

(C)% egg yolk

20¹% serum

10% dimethyl sulfoxide

O

II (CHsSCHs)

60% of MEM of Eagle (D)

10% egg yolk 20% serum 10% dimethyl sulfoxide 60% TC-199 (E)

20% dimethyl sulfoxide serum ° / o

70% of MEM of Eagle (F)% of "Centrolex F"

20% serum

10¹% of dimethyl sulfoxide

60% of Eagle's MEM

"Centrolex F" is a nutritive medium which is commercially available and which comprises an aqueous dispersion at 1% by weight of a crude fraction of soy phospholipids. This phospholipid fraction essentially consists of a 1: 1: 1 mixture of lecithin, cephalin and phosphoinositide.

(G)

2.5% by weight of glucose 0.6% by weight of methylcellulose (viscosity 15 centipoises)% of serum 16.9% of water% of dimethylsulfoxide 50% of MEM of Eagle

Methylcellulose improves the yield of the infusion by increasing the viscosity of the perfusion medium and also preserves the integrity of the cell membrane during trypsinization. The serum of media A to G above can be autologous serum, homologous serum or heterologous serum.

To freeze cells, they can be cooled to solidification temperature and further cooled to remove latent heat of fusion (i.e., cool until the phase changes (liquid state in the solid state is finished) in the usual way, for example by immersion in a coolant, such as liquid nitrogen, or by passing a cold gas over the material to be frozen. There are commercially available devices for freezing biological substances by these methods. The terms "solidification" and "liquid-solid phase change", as used in this paper, apply to both (a) the liquid in which tissue cells or agglomerations of cells are suspended and (b ) to the liquid present inside the solid walls of isolated cells or inside the solid structure of tissues and organs.

It is important that, after the end of the liquid-solid phase change, the solidified tissue cells are cooled to a temperature of -50 ° C at a rate not exceeding 3 ° C / min. It has been found that faster cooling in this temperature range adversely affects cell viability. The temperature of -50 ° C corresponds approximately to the temperature at which all the freezing water present in the system has been transformed into a solid. Some of the water in biological systems, for example water bound by proteins or other hydrophilic compounds, is not freezable. The exact temperature at which all freezable water solidifies varies to some extent depending on the particular system under consideration, but in general all freezable water solidifies at a temperature of -50 ° C.

When the temperature of -50 ° C has been reached, the frozen cells can be further cooled to any desired speed to a temperature below -75 ° C. Any refrigerant capable of lowering the temperature to below -75 ° C, can be used, for example solid carbon dioxide, mixtures of solid carbon dioxide and a solvent, helium, neon, argon and nitrogen. A storage temperature of -75 ° C is sufficient for short storage times. For prolonged storage, a temperature below -130 ° C is preferable. At liquid nitrogen temperature (-170 ° C to -190 ° C), viable tissue cells can be stored almost indefinitely. Nitrogen is the preferred refrigerant because it can be obtained in large quantities, it is biologically inert and non-toxic, and it allows the implementation of extremely low temperatures. Containers and apparatuses suitable for freezing tissue cells at limited speed, as well as apparatuses for storage at temperatures of -75 to -196 ° C of frozen tissue cells, are commercially available.

When it is desired to use the tissue cells preserved by the method according to the invention, the frozen cells are thawed quickly by proceeding in the usual manner, preferably by means of a hot water bath, generally maintained at 37 ° C. .

In a preferred implementation of the method according to the invention, the cells of tissue to be preserved are cooled so that they pass through the phase change as quickly as possible, and in any case in less than 8 to 10 minutes. Preferably, the cellular agglomerations are cooled so that they pass the phase change in 2 min or less. Cooling during the phase change can be done most quickly and efficiently on organs or on small suspensions of tissue cells. However, it is possible to get the phase change through relatively large tissue cell suspensions in less than 10 min using a container with a large surface / volume ratio and a low temperature refrigerant, such as liquid nitrogen. . The improvement in the viability of frozen cells, obtained by rapidly passing through the phase change, is demonstrated by Examples 5 and 6 below.

In another preferred implementation of the method according to the invention, the organs of animals which must be frozen whole or chopped are perfused under pressure before freezing. Any of the mi6 4 (39593 infusion sites described above can be used. While the ordinary infusion, that is to say the infusion without backpressure, has the effect of impregnating the organ with the protective additive and bringing the cells into contact with the latter, the infusion under sufficient backpressure to causing slight turgor and / or distension of the organ improves the viability of the cells. Back pressure is usually achieved during the infusion by the pressure of the infusion fluid in the intact venous systems. If necessary, back pressure can be achieved by partial constriction of the organ's blood vessels during the infusion.

The method according to the invention can be applied to the conservation of isolated cells which have been obtained from agglomerations of tissue cells by trypsinization, or the agglomerations of cells can be preserved by the method according to the invention and subsequently disintegrated into cells isolated, if desired.

To compare the viability of frozen and thawed cells with that of non-frozen control cells, one can compare the cell multiplication rates in culture medium over a period of up to 2 weeks. When the culture medium is seeded with cells from an unfrozen control, in a concentration of 5 × 10⁵ cells per ml, and the incubated medium is incubated at 37 ° C. in an atmosphere comprising 5% carbon dioxide and 95% air, the concentration of the cells decreases slightly during the first days, then increases until a maximum concentration of approximately 1 to 2 X 10® cells per ml. The non-frozen control cells reach a concentration of 1 to 2 × 10® cells per ml. After 7 to 9 days of culture. The viability of frozen and thawed cells can therefore be assessed according to the number of culture days necessary to reach the concentration of 1 to 2 X 10® cells per ml. When the frozen and thawed cells reach this concentration in no more than 3 days longer than the non-frozen controls, the viability of the cells can be considered satisfactory. Similarly, the relative viability of two or more frozen and thawed cell samples can be compared by the time it takes for cultured cells to reach a concentration of 1 to 2 X 10® cells per ml.

A second method of assessing cell viability is the technique of incorporating glycine. This method is based on the ability of conserved cells to synthesize proteins. This technique consists of measuring the capture and incorporation of a labeled acid using a radioactive isotope, i.e. measuring the cell's ability to admit the amino acid, and then to incorporate into the protein fraction of the cell. The amino acid used is glycine (aminoacetic acid) labeled with C¹⁴. This test shows whether the cell is capable of actively absorbing glycine, and also whether glycine, when it is present inside the cell, can be used for protein synthesis. To determine the incorporation of glycine, cells, which have been subjected to the freeze-thaw process, are incubated with glycine-2-C¹⁴ at 37 ° C, and samples are taken at variable intervals. The protein fraction of the cells, which is insoluble in acids, is precipitated with cold 10% trichloroacetic acid. The precipitate is washed with cold 5% ifcriohloroacetic acid, then washed several times with 95% ethanol, and finally with absolute ethanol. Next, the acid precipitated protein fraction is spread on blanks (metal discs), dried and counted the radioactivity in a gas current meter.

A third method of evaluating cell viability is based on Warburg's current manometric technique for measuring respiration ("Manometric Techniques" by WC Umbreït, RH Burris and JF Stauffer, Burges Publishing Company 1957), with the use of '' 10 glucose solution^-3 molar as a medium. In this test, the oxygen consumed by the respiration of the cells is measured by means of a special manometer. The amount of oxygen consumed is measured over a period of time, and the ability of the cells to breathe, compared to that of control cells, indicates how viable the cells are.

Example 1

Two samples were used in this example, one being subjected to the freezing, preservation and thawing operations of the method according to the invention and the other serving as an unfrozen control.

- Preparation of the control sample

One rabbit was sacrificed and the renal circulation was isolated as described above. The kidneys were perfused at a rate of 1 ml / min with a total of 50 ml of an infusion fluid of the following composition (by volume):

10% egg yolk 20% glycerol 20% rabbit serum 50% Eagle MEM

After the end of the infusion, the kidneys were removed and the cortical tissue was chopped. The chopped tissue was trypsinized four times with p, 25% trypsin solution, the cells obtained in the first place being left to remove the cytoxine. . Finally, the trypsinized cells were washed twice with GKN containing 10% rabbit serum and resuspended in 25 ml of culture medium. We prepared 50 identical tubes, containing 5 X10⁵ cells per ml, and incubated at 37 ° C.

- Preparation of the test sample

The kidneys were perfused and the cortical tissue minced as for the control sample. The chopped tissue was suspended in 10 ml of the above-described infusion fluid in a cylindrical aluminum container, and the suspension was cooled in a speed-controlled freezer at a speed of about 1 ° C / min to that the temperature reaches -50 ° C. Then the container was placed in the vapor phase of a liquid nitrogen refrigerator and kept at a temperature of -170 ° C for four days. After removing it from the refrigerator, the container was heated by immersion in a water bath at 37 ° C. Then, the thawed mince was trypsinized using a 0.1% trypsin solution, proceeding exactly the same as for the control sample. Identical tubes were prepared containing 1.1 X 10® cells per ml and incubated at 37 ° C.

- Results:

Multiplication curves making it possible to compare the multiplication in the sample subjected to the freezing and thawing process with that in the control sample were established by conventional methods. These multiplication curves showed that the test sample multiplied at about the same speed as the control sample and reached approximately the same maximum concentration of cells.

Example 2

Six preparations of monkey kidney cells, designated samples 1-6, were obtained by the methods described in detail above. The perfusion medium used to obtain samples 1 to 6 has the following composition:

% egg yolk 20% calf serum 10% dimethyl sulfoxide 60 ® / o from MEM from Eagle

Sample 1 - The kidney was perfused in situ, excised, frozen whole to a temperature of -47 ° C at a rate of 0.8 ° C / min, stored in vapor of liquid nitrogen (about -170 ° C) overnight, quickly thawed in a 37 ° C bath, chopped and trypsinized. Trypsinization was carried out using 0.19% trypsin in a buffered solution, at room temperature, for two successive 1 hour periods.

Sample 2 - Same procedure as for sample 1, except that the trypsinization was carried out using 0.2 ° / o of trypsin in a solution buffered at ordinary temperature, for an initial period of 10 min, then for three successive periods of 40 min.

Sample 3 - The kidney was perfused in situ, excised, minced, frozen to a temperature of -42 ° C at a speed of about 1.3 ° C / min, stored in vapor of liquid nitrogen ( about -170 ° C) overnight, quickly thawed in a 37 ° C bath and trypsinized, trypsinization being carried out as in sample 1.

Sample 4 - Same procedure as for sample 3, except that trypsinization was carried out as for sample 2.

Sample 5 - The kidney was perfused in situ, excised, minced and trypsinized as for sample 1. This sample served as a perfused control, not frozen.

Sample 6 - The kidney was excised, chopped and trypsinized as for sample 1. This sample served as an uninfused and unfrozen control.

A count of viable cells of each of these six preparations was carried out after trypsinization, by vital staining techniques. The bottle and tube cultures were then brought to a constant concentration (1.5 × 10⁵ cells per ml for test tubes and 7.5 X 10⁴ cells per ml for bottles) and observed periodically. The 7^e day of "9593

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GG tube groups of each of these six preparations were prepared for the determination of virus sensitivity by comparison of the titers. (Susceptibility to virus is the ability of cell culture to support the multiplication of a particular type of virus. The titer is □ equal to the negative logarithm of the dilution factor corresponding to the lowest concentration at which the culture transmits the virus, ie if the virus preparation can be diluted to a concentration of 10^-7 times the original value and multiplication 10 can still occur, the title is 7.)

Also on the 7th^e day, tube and bottle cultures were seeded with a virus (polio, adenovirus and measles virus), collected after ₁₃a period of time estimated as giving the optimum yield of virus, and titrated with respect to a standard culture, to obtain the comparative yields of virus.

In addition to the evaluation of cell cultures ²⁰as for susceptibility to the virus and to transmission, unseeded cultures were transferred on the 7th day from the culture medium to a maintenance medium, in order to evaluate the long-term conservation of the cell cultures prepared from tissue of kidney preserved. (An environment²³maintenance medium is a nutritious medium capable of maintaining cell metabolism but not of maintaining multiplication). Long-term observation of all kidney cell cultures intended for virus transmission is a normal precaution, taken to ensure³⁰No latent virus is present in the cells. _M !□

Table I brings together qualitative observations · &lt;on the cell cultures in the culture medium and in the maintenance medium, as well as the virus titers, 3 p pq indicating the sensitivity to the virus.

Notes:

(1) Based on Trypan blue coloring. All 40 suspensions gave 85 to 90% viability.

The differences in the number of viable cells are due to the differences in kidney size, the differences in the degree of trypsinization and the formation of cell fragments as a result of deterioration from freezing and thawing.

(2) Development corresponds to the formation of confluent cell layers. Quality refers to the appearance of individual cells (^{+ + + +} indicates the optimum). The term "clean" means absence of⁵⁰granules. The quality of cell multiplication was essentially the same in the tubes and in the bottles.

(3) These titles allow a comparison between the 53 test samples and the control samples.

(4) These observations relate to the appearance of the cultures which were transferred from the culture medium to the maintenance medium on 7^e day. ₆₀ (5) The term "weak", as used in this table, means that the cell sheet is not contiguous.

Table II gives the comparative yields of the 6 cultures in bottles for each of the six preparations.

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TABLE Π

Virus titers in bottle cultures

4M 593

<td rowspan="2">.Sample No.</td><td colspan="2">, Polyo</td><td colspan="2">Adeno-virus</td><td colspan="2">Measles</td>

<td>473 g</td><td>830 g</td><td>473 g</td><td>830 g</td><td>473 g</td><td>830 g</td>

<td> 1</td><td> 7,17</td><td> —</td><td> 2,50</td><td> __</td><td> 2,50</td><td> _</td>

<td> 2 ·</td><td> 6,50</td><td> —.</td><td> —</td><td> —</td><td> —</td><td> —</td>

<td> 3</td><td> 7,17</td><td> 7,50</td><td> 3,38</td><td> —</td><td> 2,63</td><td> —</td>

<td> 4</td><td> 7,17</td><td> —</td><td> 3,16</td><td> —</td><td> 2,83</td><td> —,</td>

<td> 5</td><td> —</td><td> 7,63</td><td> —.</td><td> 3,83</td><td> —</td><td> 3,5</td>

<td> 6</td><td> 7,50</td><td> 7,63</td><td> 4,16</td><td> —</td><td> 2,83</td><td> —</td>

This example shows that the method according to the invention makes it possible to obtain agglomerations of preserved tissue cells, containing a high proportion of viable cells and capable of supporting the multiplication of viruses.

Example 3

The results presented in this example illustrate the importance of cooling the agglomerations of frozen cells between the end of the phase change and -50 ° C. at a speed not greater than 3 ° C./min.

In this example, we infused right away. rabbit kidneys in situ using an infusion medium of the following composition:

20% rabbit serum% dimethyl sulfoxide 70% MEM from Eagle

The kidneys were chopped, the mince was suspended in the infusion medium, the suspension was cooled from the point of. freezing until, and including the phase change in a period of 2 min, then it was cooled to a temperature of -50 ° C at the rates indicated in Table III. Then, the samples were quickly cooled to liquid nitrogen temperature, quickly thawed in a 37 ° C water bath, trypsinized and cultured by conventional methods. The multiplication rates corresponding to the various cooling rates are presented in Table III below. In this table, each cell concentration reading is the average of four culture tubes.

TABLE ΠΙ

Concentration of viable cells (cells / ml)

<td rowspan="2">Culture time (days)</td><td colspan="4">Freezing speed ('C / min)</td>

<td> 0,96</td><td> 3,1</td><td> 4,1</td><td> 8,0</td>

<td> 0</td><td>5.4X10® -</td><td>6.2 X 10®</td><td>5.0 X 10®</td><td>5.2 X 10®</td>

<td> 4</td><td> ——</td><td>7.3 X 10</td><td> —</td><td> —</td>

<td> 6</td><td>1,5X10®</td><td>2.5 X 10®</td><td>4.3 X 10⁴</td><td> —</td>

<td> 8</td><td>. 8,8X10®</td><td>1,0X10®</td><td>8.7 X 10⁴</td><td></td>

<td> - 9</td><td>'1.0 X 10⁶</td><td> ——</td><td> —</td><td> ——-</td>

<td> 10</td><td> —</td><td> - -</td><td>2.9 X 10®</td><td>2.2 X 10⁴</td>

<td> 12</td><td> --</td><td> —</td><td>3.8 X 10®</td><td> --</td>

<td> 13</td><td> —</td><td> —</td><td> ——</td><td>4.1 X 10⁴</td>

With the freezing rates of 0.96 and 3.1 ° C / min, the concentration of cells reached approximately 1 X 10⁶ cells per ml in 8 to 9 days, while at the freezing speeds of 4.1 and 8.0 ° C / min; the cells have not yet reached this concentration after 12 to 13 days. Culture cells from unfrozen control kidneys reached a concentration of approximately 1 X 10^e cells per ml in 7 days.

Example 4

This example shows that the cooling rate before the change from liquid to solid phase is not of decisive importance.

In this example, three samples of rabbit bone marrow were suspended in media of the following composition (in percent by volume):

70% Hanks medium 15% dimethyl sulfoxide 15% rabbit serum

The samples were cooled to freezing at various rates, then cooled to the temperature of liquid nitrogen and warmed to room temperature under substantially the same conditions. The viability of frozen and thawed cells was compared with that of control samples which had not been cooled.

The results are collated in Table IV.

TABLE IV

TABLE V

<td>Cooling rate (from room temperature to freezing point)</td><td>Viability in ° / o of that of the control (cell respiration method)</td>

<td>l ° C / min</td><td> 74</td>

<td>3-4 ° C / min</td><td> 69</td>

<td>8 ° C / min</td><td> 72</td>

Example 5

This example shows that the time during which the cells pass through the change from liquid to solid phase must be less than 8-10 min, and preferably as short as possible.

Three samples of rabbit bone marrow (group A) were suspended in media of the following composition (in percent by volume):

° / o Hanks medium 15% dimethyl sulfoxide 15% rabbit serum and three other samples of rabbit bone marrow (group B) were suspended in media of the following composition (percentage by volume):

% Hanks medium 15% glycerol 15% rabbit serum

Each group of samples was cooled to the freezing point at a rate of 1 to 2 ° C / min, passed through the phase change in varying amounts of time, and then cooled to -25 ° C to 1 ° C / min, they were rapidly cooled to the temperature of liquid nitrogen, they were thawed in a water bath at 37 ° C and the viability was compared with that of 'non-frozen control samples. The results are collated in Table V. The cell viability of samples from group A was measured by the glycine-C incorporation method.¹⁴ and the cell viability of the group B samples was measured by the cellular respiration method. These two viability determinations have been shown to give results which are comparable to each other and to those of a determination on an irradiated animal. The viabilities are slightly lower than in the previous example, due to the inhibitory effect of the additive present. The results based on two measurement methods and two additives show that the quickest crossing of the heat of fusion is greater than the slower crossings for optimum viability.

<td>Duration of phase change (min)</td><td colspan="2">Viability, in ° / o of that of the witness Group A Group B</td>

<td> 1-2</td><td> 52</td><td> 45</td>

<td> 8-10</td><td> 35</td><td> 33</td>

<td> 15-25</td><td> 2</td><td> 3</td>

Example 6

This example also illustrates the importance of crossing the heat of fusion as quickly as possible when cooling agglomerations of tissue cells. In this example, rabbit kidneys were obtained by the general methods described above. These rabbit kidneys were perfused with an infusion medium containing:

2.5% by weight of glucose

0.6% by weight of methylcellulose (viscosity 15 centipoises)% of rabbit serum 16.9% of water% of dimethylsulfoxide 50% of MEM of Eagle

Before use, the perfusion medium was stirred for 1 hour in an atmosphere composed of 95% by volume of oxygen and 5% by volume of carbon dioxide at a pressure of one atmosphere.

Then the kidneys were chopped and hung in the infusion medium. Four samples of rabbit kidney cells in suspension were cooled to the melting temperature at a rate of 1-2 ° C / min, they were made to pass through the phase change in various periods of time, they were cooled to -50 ° C at a rate of 1 ° C / min, cooled rapidly to the temperature of liquid nitrogen, thawed in a water bath at 37 ° C, cultured, and the multiplication rate was compared with that of non-frozen control samples. The culture media were seeded with cells in a concentration of 5 × 10⁵ cells per ml. The unfrozen control reached a concentration of 1 X 10 "cells per ml in 7 days. The viable cell concentrations measured over time in cultures from frozen rabbit kidney cells are collated in Table VI.

TABLE VI

Concentration of viable cells (cells / ml)

<td rowspan="2">Cultivation time (days)</td><td colspan="4">Duration of phase change (min)</td>

<td> 2</td><td> 4</td><td> 8</td><td> 16</td>

<td> 0</td><td>5.4 X 105</td><td>5.0 X IO "</td><td>5.0 X 1 () 5</td><td>5.2 X 105</td>

<td> 4</td><td>1.1 X 105</td><td>6.6 X 10⁴</td><td> ,,, .</td><td> —</td>

<td> 5</td><td>2.5 X 105</td><td> _</td><td> _</td><td>8.1 X 10⁴</td>

<td> 6</td><td>5.7 X IO⁵</td><td> _</td><td>7.6 X 10⁴</td><td> —</td>

<td> 7</td><td>1.1 X 10 "</td><td>8.7 X 10⁴</td><td> —</td><td>4.7 X 10⁵</td>

<td> 8</td><td>1.3 'X 10'</td><td>3.9 X 10⁵</td><td>3.2 X 10 "</td><td>1.0 X 105</td>

<td> 9</td><td>1.4 X 10 "</td><td>4.0 X 10⁵</td><td> _</td><td> —</td>

<td> 10</td><td> _</td><td> _</td><td>1.6 X 10 "</td><td>1.2 X 105</td>

<td> 11</td><td> —</td><td>1.5 X 10 "</td><td>1.8 X 10 "</td><td>4.8 X 10⁵</td>

<td> 12</td><td> —</td><td>1.7 X 10 "</td><td>1.4 X 10 "</td><td>1.1 X 10 "</td>

<td> 13</td><td> -'</td><td> —</td><td>1.5 X 10 "</td><td>9.8 XW⁵</td>

439593 15

The results presented in Table VI show that when the cells are cooled so as to cross the period of fusion heat in 2 min, the multiplication rate is approximately equal to that of the non-frozen controls. When the duration of crossing of the heat of fusion period was 4 to 8 min, multiplication to a concentration of 1 X 10⁶ cells per ml was delayed until 10^e about a day. When the cells cross the period of heat of fusion in 16 min, the rate of multiplication is notably slowed down.

A comparison of Examples 5 and 6 shows that the importance of cooling the cells so that they quickly pass the period of heat of fusion is demonstrated equally well by three different criteria of viability; i.e. incorporation of glycine-C¹⁴, cellular respiration and the multiplication time of cells in culture by 5 × 10⁵ per ml at 1 X 1Q⁸ cells per ml.

Example 7

This example illustrates the importance of maintaining back pressure during organ perfusion. Two whole rabbit kidneys were obtained by the methods described above and were perfused in situ with an infusion medium of the following composition:

2.5% by weight of glucose 0.6% by weight of methylcellulose (viscosity 15 centipoises)% of rabbit serum 16.9% of water% of dimethylsulfoxide 50% of MEM of Eagle

Before use, the perfusion medium was stirred for 1 hour in an atmosphere composed of 95% by volume of oxygen and 5% by volume of carbon dioxide at a pressure of one atmosphere.

Both kidneys were perfused completely, but in one kidney a vein was cut so that there was essentially no back pressure during the infusion. The two whole kidneys were placed in aluminum containers and cooled to -50 ° C at an average speed of 1 ° C / min, quickly frozen to the temperature of liquid nitrogen , they were thawed in a 37 ° C water bath, chopped, trypsinized and cultured to an initial cell concentration of 5 X 10⁵ cells per ml. The cells from the kidney perfused with back pressure reached a concentration of 1.4 X 10⁸ cells per ml in 9 days. The cells of the second kidney, perfused without back pressure, multiplied poorly and reached a maximum concentration of only 4 X 10⁴ cells per ml.

Example 8

This example illustrates the application of the method according to the invention to the conservation of rat lungs.

A rat lung perfused in situ with a medium of the following composition:

2.5% by weight of glucose 0.6 ° / o by weight of methylcellulose (viscosity 15 centipoise)% of fetal calf serum

16.9% water% dimethyl sulfoxide% MEM from Eagle

Before use, the perfusion medium was stirred for 1 hour in an atmosphere composed of% by volume of oxygen and 5% by volume of carbon dioxide at a pressure of one atmosphere.

We chopped the perfused lung, hung it in perfusion medium, cooled it to the freezing point, made it go through the phase change in about 6 min, we still got it cooled to -50 ° C to 1-2 ° C / min, continued to cool to liquid nitrogen temperature, then thawed by immersion in 37 ° C water bath ° C. The lung tissue was disintegrated into isolated cells by treatment with a buffered medium containing trypsin and collagenase, by the methods described above. Frozen and thawed cells, cultured at an initial concentration of 5 X 10⁵ cells per ml, have reached a concentration of approximately 1 X 10⁸ cells per ml in 10 days. A control cell culture, originating from a rat lung subjected to identical treatment, excluding freezing and thawing operations, passed from an initial concentration of 5 × 10⁵cells per ml at a concentration of 1 X 10⁸ cells per ml in about 9 days.

Example 9

This example illustrates a preferred method of trypsinization, especially suitable for the trypsinization of tissues, in particular chopped tissues, which have been frozen by the method according to the invention.

Trypsinization solution

The trypsinization solution used in this example was prepared as follows:

A 10% by weight trypsin solution was formed by adding trypsin powder to an aqueous solution containing the following materials:

Methylcellulose (15 cps.) 6.0 g / 1

NaCl .............. 8.0 g / 1

KOI .............. 0.4 g / 1

N / A₂HPO₄ .......... 0.06 g / 1

KH₂PO₄ ............ 0.06 g / 1

Glucose ............ 50.0 g / 1

The mixture thus obtained was stirred by means of a magnetic stirrer for 10 min at 4 ° C., then it was centrifuged at 1000 rpm at 4 ° C. The clarified liquid was decanted and diluted with 50 vices of an aqueous solution containing the following materials:

NaCl .............. 8.0 g / 1

KC1 .............. 0.04 g / 1

Na, HPO ............ 0.06 g / 1

KH₂PO₄ ............ 0.06 g / 1

NaHCOg ............ 0.5 g / 1

The final diluted solution was filtered to sterilize it, and the filtrate (containing 0.2% by weight of trypsin) was used as the trypsinization solution, as described below.

- Cell culture media

The culture media used in this example and in the following example contain from 2 to 40% by volume of calf serum, 0.5% by weight of lactalbumin hydrolyzate, 200 micromoles of glutamine and from 60 to 98% in MEM volume (defined above).

-— Trypsinization method

The following trpsini'sation method was used in this example:

A tube of albumin, containing chopped tissue frozen for 3.5 min, is immersed in a bath maintained at 37 ° C. with periodic inversion of the tube to ensure correct heating and thawing of the tissue. The thawed chopped tissues are decanted in a fluted trypsinization flask and a part (for example 200 ml) of the trypsinization solution described above is introduced into the flask. Next, the fabric is trypsinized by shaking the contents of the flask for about 1 hour, using a magnetic stirrer at the start of shaking, the contents of the flask being at 4 ° C and warming up to approximately ordinary temperature at the end of the hour. The stirring is sufficient to cause good agitation of the tissue-solution mixture, but not so strong as to cause foaming of the trypsinization solution. The non-trypsinized tissues are allowed to settle and the liquid layer, containing the trypsinized cells, is decanted through a gauze. If the trypsinization is incomplete, another portion (for example 200 ml) of the trypsinization solution is introduced into the flask containing the non-trypsinized tissues and the trypsinization is repeated (for example for 0.5 to 1.0 hour) until that trypsinization is complete. The contents of the flask are allowed to settle and the liquid layer, containing the trypsinized cells, is decanted through a gauze. The, or the filtrate portions thus obtained are centrifuged at 600 rpm for 15 min. The clarified liquid is decanted from the centrifuge tubes and the cells, which are deposited during centrifugation, are resuspended in 10 to 20 ml of the culture medium described above, containing 10% by volume of calf serum and 90%. by volume of MEM. The resuspended cells are again centrifuged at 600 rpm, the clarified liquid is decanted and the cells, which have settled, are resuspended in 10 to 20 ml of culture medium. The quantity and the viability of these latter resuspended cells are determined by the above-described vital staining method. The resuspended cells are stored at 4 ° C. until use (for example, as described in Example 10).

- Results:

The following table collects the results obtained when minced monkey kidney tissue and minced rabbit kidney go tissue, which had been previously frozen by the process according to the invention, were thawed and trypsinized by the above-described method. For comparison, the table contains the results obtained when monkey kidney tissue and₂₅ Chopped and unfrozen rabbits were treated similarly.

Trypsinization results

<td>Animal</td><td>Treatment prior</td><td>Tissue</td><td>Time</td><td>Solution</td><td>° / o of viability</td><td>β / o of trypsinization</td><td>Cell yield (2)</td>

<td>Monkey Cynomologus</td><td>Not frozen</td><td>Cortex</td><td>1.5 hrs</td><td>200 ml</td><td> 92 (1)</td><td> 64,5 (1)</td><td>6.7 X 107 (1)</td>

<td>Monkey Cynomologus</td><td>DMSO + O frozen₂(3)</td><td>Cortex</td><td>1 h. 0.5 h</td><td>200 ml 200 ml</td><td> 76,5 (1)</td><td> * 50-80 (1)</td><td>6.9 X 10⁷cells per kidney</td>

<td>Monkey Rhesus</td><td>DMSO + O frozen₂(3)</td><td>Cortex</td><td>1 h 0.5 h</td><td>200 ml 200 ml</td><td> 75</td><td> &lt;50</td><td>4.1 XW⁷cells per kidney</td>

<td>Rabbit</td><td>Not frozen</td><td>Cortex</td><td>1 hr</td><td>100 ml</td><td> 89</td><td> —</td><td>5.4 X 10⁷</td>

<td>Rabbit</td><td>DMSO + O frozen₂(3)</td><td>Cortex</td><td>1 hr</td><td>100 ml</td><td> 87,5(1)</td><td> 73,5 (1)</td><td>7.9 X 10⁷ (T)</td>

<td>Rabbit</td><td>Not frozen</td><td>full</td><td>1 hr</td><td>100 ml</td><td> 88</td><td> —</td><td>6.7 X 10⁷</td>

<td>Rabbit</td><td>DMSO + O frozen₂(3)</td><td>full</td><td>1 hr</td><td>100 ml</td><td> 88 (1)</td><td> 76 (1)</td><td>9.2 X 10⁷ (1)</td>

(1) Average of two tests.

(2) Yield in cells per gram of frozen tissue, unless otherwise noted.

(3) Tissue chopped and frozen by the method according to the invention after treatment with a solution at 10% by weight of dimethyl sulfoxide saturated with oxygen and additionally containing serum, MEM and methylcellulose.

* Estimate.

It turned out that the conditions of agitation in the above-described trypsinization method are critical. Excellent results (as evidenced by the high percentage of viability of trypsinized cells) have been obtained when the stirring has been carried out for a period of time not exceeding 1 to 4 hours at 30 ° C and reaching 12 to 5 p.m. at 4 ° C. Duration

-optimum agitation is inversely proportional to the temperature of the tissue-solution mixture. When the conditions for this stirring have been varied considerably (for example when the stirring has been carried out for hours at room temperature), a

439 593 very marked reduction in cell viability. It is also very desirable to agitate the tissue-trypsinization solution mixture at a speed close to. stirring speed at which foam is formed, but below this speed.

Example 10

This example illustrates the improvement in cell multiplication achieved when the preferred method of trypsinization described in Example 9 is combined with the preferred method of cell culture described below.

- Cell culture method

Portions of the cell suspension obtained in Example 9 are diluted to a concentration of 5 × 10⁵ cells per ml, then introduced into normal 12.5 cm X 15 mm test tubes and into normal 237 ml pharmaceutical bottles. 1 ml of the diluted suspension is introduced into the tubes and 10 ml into the bottles. Comparative tests were carried out in the tubes and in the bottles to determine whether the configuration of the container had any effect on the multiplication of cells. No effect due to the configuration of the container was observed.

A current of a gaseous mixture, consisting of 5% by volume of carbon dioxide and 95% by volume of air, was passed in contact with the surface of the cell suspensions for a short period of time, for example 1 to 4 s. This gas mixture was borrowed from pressurized tanks (the gauge pressure in these tanks was 14 to 155 kg / cm²). Between the tanks and the surface of the suspensions, this mixture passed through a filter (16.5 cm in length and 2.5 cm in diameter), then a small diameter tube (25 mm in length and 1.59 mm in diameter), and had a considerable speed - at the time of contact with the surface of the suspensions. Then, the tubes and bottles were sealed and incubated at 37 ° C. In the case of tubes, the original culture medium was replaced with fresh culture medium - every 48 hours. This was not necessary in the bottles, since the medium which they contain is not appreciably exhausted during the cell culture period. . - · .....

- Rhesus monkey fabric

Three suspensions of rhesus monkey kidney tissue cells, which had been trypsinized as described in Example 9, were cultured by the culture method described above in culture media containing 10, 20 and 40% by weight of serum of calf, respectively. Three other suspensions of rhesus monkey kidney tissue cells were prepared in the same manner, except that the surfaces of the suspensions were not contacted with the air-carbon dioxide mixture. After 24 hours, the cell suspensions which had been treated with the air-carbon dioxide mixture exhibited more adhesions to the internal walls of the glass vessels and better proliferation than the other cell suspensions (not treated with gas). In addition, the cell suspensions which had been treated with the air-carbon dioxide mixture had a pH of 7.1 to 7.3, while the cell suspensions which had not been so treated had a pH from 7.6 'to 7.8. After 6-7 days of incubation, completely confluent coatings-suspensions of cells which had been treated with the air-carbon dioxide mixture were observed on the walls of the container. No progress in multiplication and coating formation was observed with cell suspensions which had not been treated with the air-carbon dioxide mixture. After 8 days, the counting of the cells in the tube containing the suspension containing 10 ° / o by volume of serum and which had been treated with the air-carbon dioxide mixture gave 1.3 X 10^e cells per ml.

- Cynomologus monkey fabric

The two frozen cynomologus monkey kidney tissue suspensions, prepared as described in Example 9, were combined and cultured - in tubes and in bottles, in culture media containing serum in a concentration of 2%, 5 % and 10% by volume. The tubes and bottles were as described above. These samples were subjected to the above-described cell culture method. Three other samples of the combined suspensions were treated in the same way, except that they were not brought into contact with the air-carbon dioxide mixture. After 24 hours of incubation, cell adhesions to the internal surfaces of the container were observed in all the containers, but there were more cells adhering to the containers when the cell suspensions had been brought into contact with the air-carbon dioxide mixture. After 6 days of incubation, a confluent coating was observed on the internal surfaces of all the containers, but greater cell proliferation was observed when the cell suspensions had been treated with the air-carbon dioxide mixture. The quantities of cells in the tubes were as follows:; /.

<td>Concentration of serum ("/ o in flight)</td><td>Concentration of cells treated with air-CO »</td><td>Centration of cells. not treated by air-COo</td>

<td> ' 2</td><td>1.1 X 10 "</td><td>1.7 X 1Q⁴</td>

<td> 5</td><td>1.1 X 10 "</td><td>2.6 X 10⁴</td>

<td> 10</td><td>1.3 X 10 "</td><td>2.2 X 10⁴ ’</td>

These results show how advantageous it is to bring the cell suspensions into contact with a stream of an air-CO mixture.₂, having culture. Apparently carbon dioxide is absorbed by the suspensions and delays an undesirable rise in the pH of the suspensions above about 7.3 during cell proliferation. In general, it is preferable to bring the cell suspensions into contact with streams of air-CO mixtures.₂ containing from 2 to 20% by volume of CO₂ and from 80 to 98% by volume of air. On the other hand, good results can be obtained by using standard buffers to prevent the pH from rising above 7.3 during cell culture. It is preferable that the pH is not less than 7.0.