Method of cryoconservation of multipotent mesenchymal stromal cells

FIELD: medicine.

SUBSTANCE: method of cryoconservation of multipotent mesenchymal stromal cells (MMSC) includes preparation of culture mixture in culture medium, mixing cell culture in culture medium with cryoprotector, step-by-step controlled cooling and freezing mixture of culture medium with cryoprotector to storage temperature and further storage of frozen mixture at low temperatures. As cryoprotector for mixture saturation used is xenon gas, introduced till saturation is achieved into prepared mixtute of cell culture MMSC in DMEM medium, which is in open cryo test tubes by blowing culture medium with xenon at 0°C with further cooling and formation of primary monolith of culture medium with cryoprotector, directed later at cooling to storage temperature.

EFFECT: invention ensures preservation of cells in cryopreservation.

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The invention relates to medicine, more specifically to Cryobiology, in particular to methods and products for cryopreservation of cell suspensions person used as therapeutic drugs.

Today there are many ways (protocols) cryopreservation of cell suspensions of human rights. All known methods and techniques differ from each other only by differences in the concentration of the added liquid chriopractic, the use of different composition closewith solutions and the rate of freezing of cell suspensions. The main characteristic features of this type closewith compounds are liquid, easily diffuse through the cell membrane and is able to bind molecules outside - and intracellular water, with a shift of the freezing point of the solution and prevents the growth of ice crystals inside and outside of cells.

It is known that the commonly used liquid cryoprotectants: DMSO, polietilenglikol, glycerin (A.S. No. 1412039), propylene glycol, polyvinylpyrrolidone, described in patents (EA 009073 B1, EN 2283119, US 20050026133 A1) does not sufficiently provide the assigned functions, in particular to protect cell cultures from formed during freezing crystalline structures of ice, which, ultimately, break the cell membrane or displace the latter in hyperosmotic Cana is s, where cells are exposed to strong mechanical deformation and the effects of high solute concentrations, which, ultimately, is the cause denaturation of proteins membrane elements, cells, reducing the viability of cell cultures during cryopreservation.

As the closest analogue of the claimed invention can be seen EN 2303631 C1, publ. 27.07.2007, revealing the way cryopreservation of MSC, with the cryoprotectant DMSO (5-10%), lack of which is the high toxicity of DMSO and its pungent smell, which can cause allergies in working with it staff, which also limits the use of DMSO as cryoprotectant cell suspensions.

It is also known and applied under high pressure in the chamber a mixture of inert gases xenon, krypton, argon for cryopreservation of animal organs and tissues in the process of low temperature preservation (see "Method of cryopreservation of organs and tissues IN SITU" RF Patent №2268590 A01N 1/02. Publ. 21.01.2006 bull. No. 3).

In the experiments, in particular, used a mixture gas of Kr, Ar, Xe in the ratio of 2.5; 47,5; 5,0% vol. under the pressure of 1.5 ATM for cryopreservation of organs and tissues of the rat in situ. The chamber was placed a rat weighing about 300 g, were intensively cooled it under running cold water to 0°C with simultaneous saturation of the mixture of inert gas under pressure is receiving 1.5 ATM for full saturation of the gas mixture tissues of the body, then replace the water in this mixture. Then the rat was frozen to -43°C and then reducing the pressure of the gas medium, cooled to the temperature of liquid nitrogen and kept for 6 hours. After defrosting of the rats were removed heart and transplanted it to the recipient. All functions of the rat heart donor was fully preserved when transferring the rat recipient.

Also close to the proposed method can be considered a method of conservation of the kidneys by a combination of cooling at a temperature of(+2)-(+4)°and hyperbaria argon at a pressure of 1.5-2 ATM (see Shumakov V.I. and other "Conservation bodies" M, Medicine, 1975, s-128). The disadvantage of this method is the relatively low reliability and long duration of doing conservation, which can be explained insufficiently reduced cellular metabolism due to the use of temperatures above 0°C.

However, the use of such complex multiphase and multicomponent methods using mixtures of noble gases for cryopreservation of cell cultures of human and these technologies from available sources is not known.

Thus, used and known liquid cryoprotectants at a known concentration and known mixtures of inert gases, injected under high pressure for cryopreservation of organs and tissues have limited critisicim action and require the I for the implementation of complex equipment (pressure chambers, the cryochamber) and large expenditures of time and money, do not allow to eliminate possible side effects, the occurrence of which can be crucial when working with stem cells that inhibits the introduction of cellular technology in medical practice.

Object of the invention is the finding of the cryoprotectant and the development of the method of cryopreservation with minimal damaging effect on cells and cell suspension, no toxic effects after defrost and does not require the use of special unique techniques and equipment while reducing the time and cost to the cryopreservation multipotent mesenchymal stromal cells (MMSC) person that enhance the quality and viability of cell cultures.

The task is solved by the fact that as a cryoprotectant during cryopreservation mixture MSC from the culture medium using chemically pure gas xenon - Xe composition of 99.999%, and cryopreservation is carried out by blowing gas Heh before saturation of the mixture of cell culture (suspension MSC) in DMEM (Sigma), placed in open plastic cryovials, and blowing Heh carried out at atmospheric pressure in an ice bath at 0°C for 10-20 min, then cryoprobes volume of 2 ml with a mixture MSC saturated with xenon closed and placed in cryocontainers that plush is up in the freezer of the type for Sanyo controlled freezing to 70-85°C with a cooling rate of 1°C per minute and before the formation of the monolith mixture of culture medium with cryoprotectant Heh and then for long-term storage in the repository liquid nitrogen for later use.

The main component is cryoconserved proposed method gas xenon is produced by the domestic industry, i.e. the sentence "industrially applicable".

Xenon gas is colorless and odorless; (symbol Heh Xenonum) from the group of noble gases; the density of 5.85 kg/m3. Apply Xe gas discharge lamps, as well as in research and medical purposes (see Polytechnical dictionary. Ed 3-E. M., "Soviet encyclopedia", 1989, s).

Currently, gas xenon, due to its low toxicity, are widely used in anesthesiology as drugs and for the treatment of several diseases (see Boers E.N., Potapov V.N., Makeev GN. / Xenon in anesthesiology. - Moscow: "Pulse", 2000. - 389 S.).

For laboratory testing and research on the effectiveness of the proposed method of cryopreservation xenon MSC in culture medium DMEM - multipotential mesenchymal stromal cells (MSC) were isolated from subcutaneous fat 4 healthy men aged 27-42, and cultivated in the laboratory in accordance with the regulations.

After reaching 70% of confluency cells were mobilized by treatment with 0.25% solution of trypsin in EDTA (Invitrogen), transplanted to new mattresses larger area (4 mattress each at the rate of 1,000-3,000 cells/cm2 . Counting cells produced on the Hematology analyzer Cobas Micros FROM.

Further, upon reaching confluency 75-80%, cultures were randomly divided into groups for laboratory experience (table 1).

For cryopreservation MMSC was removed from the mattress with a solution of trypsin-versene by the standard method and, after centrifugation, resuspendable and counted on the Hematology analyzer.

Table 1
The scheme of experiment for testing the proposed method of cryopreservation MSK
Group numberCryoconservedA number of cultures
1Control4
2DMSO 5%4
3DMSO 10%4
4Xenon 100%4

For processing a mixture of cell cultures xenon (group 4) open the bottles (plastic cryoprobes) volume of 2 ml of the firm "Cryo.S with the culture of cells in the environment of DEM blew Heh within 10-20 minutes while gradually lowering the temperature to 0°C in an ice bath, then the bottles were corked, was placed in a container for controlled cooling Nalgene Cryo, and then in the refrigerator Sanyo MDF - 192 at -84°C for 1 hour. In subsequent samples (samples) were kept in pairs LNs at a temperature of minus 196°C for 48 hours and 6 months. This scheme ensures a uniform temperature in the range 0-84°C. the Intensity of cooling is 1°C/min

For processing cells DMSO (groups 2 and 3) preparing a cell culture containing twice the number of cells in half the volume of medium and the solution is pre-sterilized filtered DMSO in the culture medium so that, when mixed to obtain the standard number of cells in a medium containing 5 or 10% DMSO.

Cryoprobes volume of 2 ml with cultures treated with DMSO, was placed in the container, Nalgene Cryo and placed in the refrigerator Sanyo MDF - 192 at minus 84°C for one hour, then the samples were kept in pairs LN2at a temperature of minus 196°C. the Samples were divided into groups and were kept 48 hours and 6 months. respectively.

Defrost all specimens were prepared by placing cryoprobes in a water bath at 37°C. After defrosting assessed the viability of cell cultures options (groups), assessment of their count of clonogenic activity of radioisotope method. After thawing the samples was investigated the possibility of spontaneous d is ferentiate MSC in four Orthodox directions (adipogen, osteogenic, myogenic and chondrogen).

To remove DMSO, the cells after thawing before sowing on culture medium was repeatedly washed by centrifugation. Order to mitigate the possible effects of this procedure on the studied parameters, the samples in which the cryoprotectant used xenon introduced by the proposed method were processed by the same Protocol.

Viability after cryopreservation and thawing of cells was determined by the percentage of stained (0.4% solution Trypanosoma blue) in the camera Goryaeva, simultaneously assessed the proliferative activity of cells by incorporation into macromolecules labeled selective precursor of DNA synthesis (214C - thymidine Amersham Pharmacia Biotech). Radionuclide with an activity of 37 KBq/ml of the medium was brought to 75 cm2mattresses simultaneously with h6cells. The experiment was interrupted after three days of culturing, the cells were removed from the mattress by the standard method and was calculated on the Hematology analyzer. Counting the radioactivity produced in alcohol-toluene scintillator on a liquid scintillation counter Beta-2 (efficiency accounts for carbon - 98%). The results were expressed in units of becquerels 106cells.

Count of clonogenic activity was examined after fixation of cells, staining and counting of cells, as is compared to the number of colonies, consisting of not less than 20 cells, seeded and expressed in percentage relative to the control.

Prepotential thawed cells was studied by creating conditions for differentiation MSC in different directions.

For differentiation in adipokines direction into the culture medium was made by a combination of isobutylmethylxanthine, dexamethasone, insulin and indomethacin.

For differentiation in chondrogen direction into the culture medium was made by a combination of insulin, transforming growth factor and ascorbate.

For differentiation in cardiomyogenesis direction into the culture medium was made recombinant IL3 and IL6.

To stimulate differentiation in osteogenic direction into the culture medium was made dihydrovitamin D3, ascorbic acid and glycerol.

Statistical data processing was performed using software package Statistica 3.04. Results were considered significant p≤0.05.

The results of these studies are presented in table 2. During the research it was confirmed that the use as a cryoprotectant DMSO does not ensures complete safety of the cells from the damaging effects of temperature changes during cryopreservation and subsequent thawing (table 2) and leads to a decrease in their count of clonogenic activity.

Thus, the share of money neratinib cell forms after thawing when using DMSO was significantly higher than in the original cultures with increasing concentrations of DMSO with 5 to 10% showed a tendency to increase the percentage of degenerative forms.

However, in a variant of the experience when used as a cryoprotectant xenon gas composition 99,999% under the proposed method, the number of degenerative cell forms did not differ from the initial benchmarks, showing the high efficiency of the proposed method and the results of the study of synthetic parameters of cells with preservation of which as a protector used xenon headlights, also did not differ from control and fully kept all their functions (table 2), while the count of clonogenic activity in % was 10-20% higher than in the variants with DMSO.

Table 2
Characteristics MSC at different times after cryopreservation
IndicatorsControlCryoconserved
DMSO 5% 48 hDMSO 5% 6 months.DMSO 10% 48 hDMSO 10% 6 months.Heh 48 hHeh 6 months.
The share of degenerative cell forms in % of total2,6±1,94,2±1,16,6±0,9*8,4±1,2*7,8±1,3*3,1±1,42,9±1,7
The inclusion of the 214C - thymidine into DNA (Bq/106cells)71,1±1,462,4±1,2*56,6±1,3*65,5±1,8*60,7±2,9*70,6±2,9to 70.7±1,1
Count of clonogenic activity, %10089,76±1,69*79,60±1,82*91,12±1,5385,37±1,07*99,20±2,0799,43±1,54
* - significant difference from control (p≤0,05)

The results, obtained the studies of the synthetic activity of cells after cryopreservation, confirm microscopy data. So using DMSO to cryoprotective accompanied by a significant decrease in synthetic activity on average by 14%. The results are consistent with literature data and can be explained by the displacement of the cells formed in the environment of large ice crystals in hyperosmolar channels (figure 1), where cells are exposed to mechanical deformation and osmotic shock.

In figure 1 cryoprotector DMSO 10%, storage 48 hours. On the picture can be clearly seen deformed cells MMSC, replaced by ice crystals in hyperosmolar channels.

In figure 2 cryoprotector gas Heh 48 hours. Ice crystals are not visually determined. Deformation of cells no. The structure of the sample is almost uniform.

Cryoprotector xenon, is introduced under the proposed method, forming clusters of water prevents the formation of ice crystals within cells and in the extracellular environment (figure 2), showing a unique non-obvious effect uniform freezing of the object.

Apparently, in this case, the damaging effects of freezing and thawing significantly lower than when using the cryoprotectant DMSO (options 2 and 3), suggesting that gas xenon, input on the proposed method in a mixture MSC in DMEM (Sigma) for 10-20 min, more efficient the n as a cryoprotectant, than the standard method using DMSO.

However, many cryoprotectants, including DMSO, have the potential to cause unwanted differentiation of stem cells, which reduces their prepotential, unipotential. The latter is an important point, because raises the question of the feasibility of cryopreservation poly potent cells using DMSO.

In order to clarify the question about the presence of such effects in xenon was additionally studied the ability of cryopreserved using xenon cells in DMEM as cryoprotectant MSC to differentiate in 4 directions - androgena, adipokines, osteogenic, cardiomyogenesis.

It should be noted that in none of the cases was not observed in any of the growth and differentiation of cryopreserved cells compared to intact.

As in the patent and scientific literature is not aware of such technical and technological solutions, similar to the present method, the proposal fully complies with the criteria of the invention of "novelty."

The novelty of the proposed method cryopreservation MSC is also the use of a known drug (xenon gas)is introduced through the blow-off to saturation without pressure in the mixture (suspension) MSC in the rede DMEM (Sigma) for a new purpose as a cryoprotectant at a low temperature freezing with the new non-obvious effect of increasing quality MMSC after thawing while reducing labor costs for implementation fashion and high quality cryopreservation, which is confirmed by table 2 and figure 2.

Studies on the use of xenon gas as an effective cryoprotectant multipotent mesenchymal stromal cells showed significant qualitative advantages over the standard used for this purpose DMSO (5% and 10%).

Cryoprotector xenon composition 99,999%, input on the proposed method, unlike DMSO, does not cause unwanted differentiation MMSC, and also provides a higher quality safety MMSC during cryoprotective and can be recommended as a promising cryoprotector for multipotent mesenchymal stromal cells used in medical practice.

1. Method of cryopreservation multipotent mesenchymal stromal cells (MSC), including the preparation of the mixture of culture in culture medium, mixed culture of cells in culture medium with cryoprotectant, speed controlled cooling and freezing of the mixture of culture medium with cryoprotectant to a temperature of storage and subsequent storage of the frozen mixture at low temperatures, characterized in that as a cryoprotectant for saturation of the mixture using gas xenon, entered before saturation in the prepared mixture of cell culture MMS is in DMEM, in the public cryovials by blowing culture medium xenon at 0°C. with further cooling and the formation of primary monolith culture medium with cryoprotectant, which is then channelled to the freezing temperatures of storage.

2. Method of cryopreservation multipotent mesenchymal stromal cells according to claim 1, characterized in that the saturation of the mixture as a cryoprotectant use chemically pure xenon gas composition 99.999%and as the basis of a mixture of culture medium MMSC use DMEM (Sigma), placed in cryoprobes volume of 2 ml, and the purge xenon and primary cooling of the mixture is carried out in 10-20 min at atmospheric pressure in an ice bath, followed by closure and dive saturated gas cryovials in the freezer for controlled freezing step up (-70)-(-85°C when the intensity of cooling of 1°C in minute and then for cryogenic storage in the storage of liquid nitrogen.

3. Method of cryopreservation multipotent mesenchymal stromal cells according to claim 1, characterized in that for freezing up (-70)-(-85°C use freezer type Sonyo, and full of freezing and cryo-storage of cryovials frozen culture mixture using a cryo-Depositary with liquid nitrogen.



 

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EFFECT: invention makes it possible to increase HSC sterility during freezing, integrity and their viability.

FIELD: medicine.

SUBSTANCE: invention relates to preservation of corpses. Application of, at least, one compound of formula (I): R-(OCH2)n-OR' in which R and R' are similar or different, represent linear or branched alkyl radical, including from 1 to 5 carbon atoms, and n represents index, which has value, including from 1 to 8, and/or formula where R2, R3, R4 and R5 represent, independently, linear or branched alkyl radical, which includes from 1 to 8 atoms, or, or R2 and R5 and/or R3 and R4 together and with two oxygen atoms, with which they are bound, form saturated or unsaturated 5- or 6-member heterocycle, possibly substituted with one or several groups selected from OH, CH2OH or linear or branched alkyl radical, which includes from 1 to 8 carbon atoms; and R1 represents group CH-R6-CH, where R6 forms bond or represents linear or branched alkylene radical, which includes from 1 to 5 carbon atoms, or saturated or unsaturated carbocycle, which includes from 3 to 8 carbon atoms; or R1 represents saturated or unsaturated carbocycle, which includes from 3 to 8 carbon atoms; for preservation of human or animal body and/or for dead body embalming. Preservation is performed by introduction into corpse of composition, which contains said compounds and glycerol.

EFFECT: invention makes it possible to reduce preservation process toxicity.

11 cl, 6 ex, 1 tbl

FIELD: agriculture.

SUBSTANCE: method involves aerosol treatment of the hair and skin surface of whole cut velvet antlers with solution of a cultural liquid with metabolism products of Bacillus subtil is or Bacillus licheniformis probiotic bacteria or a nisin solution, its specific activity being no less than 1000 IU/mg, in an amount of 1.0-3.00 mcg/ml or solution of a mixture of nisin, its specific activity being no less than 1000 IU/mg, in an amount of 0.1-100 mcg/ml and lysozyme in an amount of 0.1-200 mcg/ml. Treated whole velvet antlers are frozen till temperature is no more than 20°C. Then the velvet antlers are dried till moisture content is no more than 10% (weight).

EFFECT: prolonged storage life of velvet antlers.

11 cl, 1 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.

EFFECT: enhanced effectiveness in producing living descendants.

39 cl, 5 dwg, 1 tbl

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