Environment for cryopreservation of human and animal cells

 

(57) Abstract:

The invention relates to biotechnology and medicine, specifically to the media for cryopreservation of animal and human cells. The medium contains fetal calf serum, dimethylsulfoxide, medium RPMI-1640, antibiotics, acetone, D-glucuronic acid, lecithin, cholesterol, linoleic acid in a predetermined ratio of the components. The invention provides enhancement of the survival of the cells by increasing their resistance to cryopreservation and reducing the toxic effects of cryoprotectants. table 2.

The invention relates to medicine, specifically to the media for reconservation of human and animal cells.

Known medium for cryopreservation of human and animal cells, containing salt, cryoprotector, pyruvate, lactate, glucose, antibiotics, sulfonamides, bovine serum albumin, phenol red, HEPES buffer, for example modified environment M 1 and M 2 [1]. However, these environments do not contain amino acids, vitamins, whey, which is necessary for adaptation and protection of cells during the procedure of cryopreservation. This leads to unwanted damage and death of cells during cryopreservation at Samurais salt basis, the cryoprotectant, pyruvate, lactate, glucose, antibiotics and sulfonamides, also contains amino acids, vitamins and serum, for example the medium RPMI-1640 with the addition of 10-20% fetal calf serum, 5-10% DMSO, 100 IU/ml penicillin sodium salt and 100 µg/MD streptomycin sulfate [6]. This environment due to the presence of amino acids, vitamins and serum promotes the protection and adaptation of cells to cryopreservation conditions. However, it does not contain the stabilizer of cell membranes and colloidal structures, which increase the resistance of cells to freeze. There are no chemicals that reduce the toxic effect of cryoprotectants (dimethyl sulfoxide) on the cells.

The new technical problem - increasing the survival rate of cells by increasing the resistance of cells to cryopreservation and reducing the toxic effects of cryoprotectants.

The set task is solved by the application of a new environment containing medium RPMI-1640, fetal calf serum, dimethylsulfoxide, antibiotics and sulfonamides, and it additionally contains ndimethylacetamide, D-glucuronic acid, lecithin, cholesterol, and linoleic acid in the following ratio of components,%:

Embryo is a lot 0,01-0,03

Lecithin 0.05 to 0.15

Cholesterol 0,05-0,15

Linoleic acid 0,025-0,075

The sulfonamides and antibiotics 0.1 to 1.5

Medium RPMI-1640 100

The difference between the supplied invention from the known analogues is that the environment further comprises acetone, D-glucuronic acid, lecithin, cholesterol, linoleic acid in the following ratio of components,%:

Fetal calf serum 10-20

Dimethyl sulfoxide 5-15

Acetamid 10-30

D-glucuronic acid 0,01-0,03

Lecithin 0.05 to 0.15

Cholesterol 0,05-0,15

Linoleic acid 0,025-0,075

The sulfonamides and antibiotics 0.1 to 1.5

Medium RPMI-1640 100

A comparison of the proposed technical solutions with other shows that the proposed composition of the ingredients of the medium for cryopreservation of animal and human cells, allowing to increase the survival rate of the cells during cryopreservation, the description of which is not found in the literature.

Given the above, one should consider the claimed solution corresponding to the criterion of “significant differences”. The use of this environment in the experiment on the cells of animals and humans have shown that it is “industrially applicable” PI:

- preparation of liposomes using ultrasound according to the standard method of 1 part (by weight) of cholesterol, 5 parts of linoleic acid, 10 parts of lecithin [4];

- mixing liposomes with medium RPMI-1640, fetal calf serum, D-glucuronic acid, penicillin and streptomycin until dissolved substances;

- add (immediately before use) in the environment of dimethyl sulfoxide and cooling it to room temperature.

The resulting environment contains, vol.%:

Fetal calf serum 10-20

Dimethyl sulfoxide 5-15

Acetamid 10-30

D-glucaro new acid 0,01-0,03

Lecithin 0.05 to 0.15

Cholesterol 0,05-0,15

Linoleic acid 0,025-0,075

The sulfonamides and antibiotics 0.1 to 1.5

Medium RPMI-1640 100

The concentration of the components in the proposed environment for cryopreservation of human and animal cells selected on the basis of data interpretation of experimental studies.

Due to the fact that the main damaging effect on the cells during cryopreservation provides cryoprotector dimethyl sulfoxide was resolved, that the comparison environment (prototype) concentration of cryoprotectant and fetal calf seemai environment. Therefore, the concentration of these components in the prototype environment, respectively, were within the limits, vol%: dimethysulfoxide 5-15, fetal calf serum 10-20.

Antibiotics and sulfonamides used in conventional doses (penicillin sodium salt 100 IU/ml, streptomycin sulfate, 100 μg/ml) was estimated to be 0.1 to 1.5 vol.%.

Example 1

Cardiomyocytes were isolated from the hearts of newborn rats breed “Wistar” with the help of enzymes [8]. The viability (survival), cellularity and morphology of the cells was determined by obsidionalis methods using Trypanosoma blue, hemocytometer and staining with azure II-eosin, respectively [3, 5]. Brought the cell concentration to 1107/ml of medium of the following composition: medium RPMI-1640 - 80%, fetal calf serum at 20%). To 1 ml of cell suspension was added dropwise environment for cryopreservation containing, vol.%:

Fetal calf serum - 10

The sulfoxide 5

Acetamid 10

D-glukhareva acid of 0.01

Lecithin 0,05

Cholesterol 0,05

Linoleic acid 0,025

Penicillin sodium salt 100 IU/ml

Streptomycin sulfate, 100 μg/ml

Medium RPMI-1640 100

Material paramesh the flax was cooled to-40C with a speed of 1-2C/min And then transferred to liquid nitrogen (-S) and kept for 1 and 5 years [1]. After thawing was evaluated by the number of surviving cells, cellularity and morphology of kariolou, as described above.

The control used the prototype environment, containing, vol.%:

Medium RPMI-1640 85

Fetal calf serum 10

The sulfoxide 5

The results are presented in table 1. The analysis showed that the number of viable cells (survival) after thawing in the environment through 1 year experience 54,72,2, prototype 42,50,9, P<0.05) and 5 years (experience 53,11,3, prototype 40,31,2, Pu<0,05) was significantly higher than that of the prototype.

Example 2

Cardiomyocytes were isolated from the hearts of newborn rats breed “Wistar” with the help of enzymes [8]. The viability (survival), cellularity and morphology of the cells was determined by conventional methods using Trypanosoma blue, hemocytometer and staining with azure II-eosin, respectively [3, 5]. Brought the cell concentration to 1107/ml of medium of the following composition: medium RPMI-1640 - 80%, fetal calf serum at 20%). To 1 ml of cell suspension was added dropwise environment for cryopreservation, containing, vol.%:

Fetal calf serum 15ptx2">Cholesterol 0,01

Linoleic acid is 0.05

Penicillin sodium salt 100 IU/ml

Streptomycin sulfate, 100 μg/ml

Medium RPMI-1640 100

The material was mixed and placed in containers for cryopreservation of 1.5 ml and froze for a two-step scheme. Originally was cooled to-40C with a speed of 1-2C/min And then transferred into liquid nitrogen (-S) and kept for 1 and 5 years [1]. After thawing was evaluated by the number of surviving cells, cellularity and morphology of kariolou, as described above. The control used the prototype environment, containing, vol.%:

Medium RPMI-1640 75

Fetal calf serum 15

The sulfoxide 10

Penicillin sodium salt 100 IU/ml

Streptomycin sulfate, 100 μg/ml

The results are presented in table 1. The analysis showed that the number of viable cells (survival) after thawing in the environment through 1 year experience 67,13,3, prototype 48,11,7, P<0.05) and 5 years (experience 60,21,9, prototype 45,52,2, Pu<0,05) was significantly higher than that of the prototype.

Example 3

Cardiomyocytes were isolated from the hearts of newborn rats breed “Wistar” with the help of enzymes [8]. The viability (survival), cellularity and morph the key Azur-II eosin, respectively [3, 5]. Brought the cell concentration to 1107ml of medium of the following composition: medium RPMI-1640 - 80%, fetal calf serum at 20%). To 1 ml of cell suspension was added dropwise environment for cryopreservation, containing, vol.%:

Fetal calf serum 20

Dimethyl sulfoxide 15

Acetamid 30

D-glucuronic acid 0,03

Lecithin 0,15

Cholesterol 0,015

Linoleic acid 0,075

Penicillin sodium salt 100 IU/ml

Streptomycin sulfate, 100 μg/ml

Medium RPMI-1640 100

The material was mixed and placed in containers for cryopreservation of 1.5 ml and froze for a two-step scheme. Originally was cooled to-40C with a speed of 1-2C/min And then transferred into liquid nitrogen (-S) and kept for 1 and 5 years [1]. After thawing was evaluated by the number of surviving cells, cellularity and morphology of kariolou as described above. In the control using the prototype environment, containing, vol.%:

Medium RPMI-1640 75

Fetal calf serum 20

Dimethyl sulfoxide 15

Penicillin sodium salt 100 IU/ml

Streptomycin sulfate, 100 μg/ml

The results are presented in table 1. The analysis showed that kolichestvo 49,92,4, Pu<0.05) and 5 years (experience 53,72,4, prototype 42,14,4, Pu<0,05) was significantly higher than that of the prototype.

Example 4

10 ml of Peripheral blood stabilized with heparin (100 U/ml) was carefully layered on 5 ml of ficollhypaque with a density of 1.077 g/cm3. Centrifuged at 3000 rpm for 15-20 minutes Interphase layer containing monocytes and lymphocytes were collected and washed from ficollhypaque fresh portion of RPMI-1640 medium with 10% fetal calf serum. Cellularity of viable mononuclear brought up to 1107/ ml and [2]. 1 Piece suspension kariolou was mixed with the medium for cryopreservation of the following composition,%:

Fetal calf serum 15

The sulfoxide 10

Acetamid 20

D-glucuronic acid 0,02

Lecithin 0,1

Cholesterol 0,01

Linoleic acid is 0.05

Penicillin sodium salt 100 IU/ml

Streptomycin sulfate, 100 μg/ml

Medium RPMI-1640 100

And frozen in liquid nitrogen as described above. The control used the environment of the prototype, consisting,%:

Medium RPMI-1640 75

Fetal calf serum 15

The sulfoxide 10

Penicillin sodium salt 100 IU/ml

osobnosti) using Trypanosoma blue, with subsequent morphological analysis. The results are presented and in table 1. The analysis showed that the number of viable cells (survival) after thawing in the environment through 1 year experience 85,72,28, prototype 761,3, P<0,05) was significantly higher than that of the prototype.

Justification of the choice of ingredients

The damage and loss of cells during preservation is mainly due to the intracellular formation of ice crystals, which break the cell membrane, as well as the toxic effects of cryoprotectants (dimethyl sulfoxide) [5].

Diethylsulfoxide efficiency is now considered one of the best reprotection among the known analogues (glycerol, methanol, sucrose, propylene glycol, polyethylene glycol and others). To reduce its toxic action on Wednesday introduced acetamide, which is based on the ability of some amides to reduce the chemical activity of solutions of dimethyl sulfoxide [5].

To increase the stability of cell membranes to cryopreservation in it add: d-glucuronosyl acid, lecithin, cholesterol, and linoleic acid.

D-glucuronosyl acid included in the composition of glycosaminoglycans, which form nutrilett lipid, and lecithin - protein layers of the cell membranes. These substances have the protective, plastic and antioxidant functions that suffer during cryopreservation [5, 7, 9].

Thus, the application of the proposed environment for cryopreservation of animal and human cells can increase cell survival by increasing their resistance to cryopreservation and reduce the toxic effects of cryoprotectants (dimethyl sulfoxide).

Bibliography

1. Developmental biology of mammals. Methods/ M NAAT. - M.: Mir, 1990.

2. Goldberg E. D., Digi A. M., Shakhov B. N. Methods of tissue culture in Hematology, Tomsk, Tomsk state University, 1992.

3. Immunological methods/ G. Primal. - M.: Medicine, 1987.

4. Immunological research methods/ I. Berkovitsa, of B. Pernis. - M.: Mir, 1988.

5. Culture of animal cells. Methods/ R. Fresno. - M.: Mir. - 1989.

6. Lymphocytes: Methods/J. Klaus. - M.: Mir. - 1990, S. 239-242.

7. Serov centuries, Schechter A. B. connective tissue. - M.: Medicine. - 1981.

8. Physiology and pathology of the heart/ N.Sperelakis. - M.: Medicine. - B2, so 2. - 1990.

9. Yushkov, B., Popov, K., Severin M. C., Hawks A. P. Glycoproteins and haematopoiesis. - Yekaterinburg. - 1994.

Fetal calf serum 10-20

Dimethyl sulfoxide 5-15

Acetamid 10-30

D-glucuronic acid 0,01-0,03

Lecithin 0.05 to 0.15

Cholesterol 0,05-0,15

Linoleic acid 0,025-0,075

Streptomycin 100 µg/ml

Penicillin 100 IU/ml

Medium RPMI-1640 To 100

 

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