CN117981747A

CN117981747A - Cell freezing solution and application thereof in cell preparation

Info

Publication number: CN117981747A
Application number: CN202410406620.9A
Authority: CN
Inventors: 高爽; 常建桥; 安文强; 杜星芳; 刘俊江; 冯志华; 李欢欢; 崔丞丰; 赵伦
Original assignee: Beijing Weige Stem Cell Technology Co ltd
Current assignee: Beijing Weige Stem Cell Technology Co ltd
Priority date: 2024-04-07
Filing date: 2024-04-07
Publication date: 2024-05-07

Abstract

The present disclosure provides a gel-forming cell cryopreservation solution comprising dimethyl sulfoxide, a soluble calcium salt, and thrombin. When the cell cryopreservation liquid is used for cryopreserving cells, the cells can maintain activity under the low-temperature condition. The cell suspension recovered by using the cell freezing solution is directly mixed with fibrinogen, so that in-situ gel forming can be realized, a cell preparation wrapped by gel materials can be obtained, and the loss of cells caused by liquid loss is reduced. Meanwhile, the gel can maintain the activity of cells in the gel, so that better efficacy exertion is realized.

Description

Cell freezing solution and application thereof in cell preparation

Technical Field

The invention relates to the technical field of biology, in particular to a cell freezing solution and application thereof in a cell preparation.

Background

Cell therapy refers to a therapeutic method for treating a disease or improving physiological functions by implantation of autologous or allogenic cells. Compared with small molecular drugs, large molecular drugs and protein drugs, the biggest characteristic of cell therapy is that living cells are used as drugs, so that the cell therapy has higher requirements on preservation of cell activity. With rapid development of biotechnology and in-depth knowledge of cellular properties, cell therapy has rapidly progressed and is widely used in clinical practice, particularly in the fields of autoimmune diseases, degenerative diseases, cardiovascular diseases, tumors, and the like.

Cell therapy drugs that have been applied clinically can be classified into fresh cell preparations and frozen cell preparations in terms of preservation conditions of the cell drugs. Wherein, the fresh cell preparation refers to clinical application after cells in the culture and expansion process are subjected to digestion and washing steps and resuspended by using physiological simulation solution and buffer solution. The frozen cell preparation is prepared by suspending the cultured and amplified cells in frozen solution, and preserving at low temperature (generally below-80deg.C) to stop enzyme activity in the cells completely and stop cell metabolism activity, thereby realizing long-term preservation of cells. Before use, the frozen cells can be placed in an environment with the temperature not higher than 37 ℃ for thawing recovery, and then clinical application is carried out. In theory, the fresh cell preparation is directly used without the process of cryopreservation and resuscitation, and has the characteristics of high cell activity rate and better cell function. Accordingly, fresh cell preparations have extremely high requirements on the temperature and humidity of the preservation environment, and most of them require special preservation solutions to provide balanced conditions and nutrient systems for the cells. Even under such conditions, the preservation time of the cells is not more than 72 hours in many cases. The geographical range of applicability of fresh cell preparations is therefore limited. On the other hand, the time required for the projects such as sterile detection, mycoplasma detection, virus detection and the like in the cell medicine release test is longer and is 14-180 days, and the validity period of the fresh cell preparation is far exceeded. These test items therefore require a risk release before the result is not achieved, increasing the risk to the patient to some extent.

Cell cryopreservation formulations can solve this problem to some extent. When the cells are frozen, cryoprotectants such as glycerol or dimethyl sulfoxide are used, and can improve the permeability of cell membranes to water, so that the water in the cells is not easy to form sharp ice crystals at low temperature, and the damage of the sharp ice crystals to the cells is reduced. The cells can be stored for several years or even decades under cryopreservation conditions, during which time there is sufficient time for adequate stability studies of the cryopreservation system. In addition, the freezing condition can be maintained by liquid nitrogen or dry ice, and the frozen state can be transported to a place far from the preparation place without additional power input maintenance. Therefore, the frozen cell preparation has larger application space in the geographic range. Of course, cell cryopreservation formulations present advantages as well as a number of attendant problems. Firstly, the frozen stock solution needs to be added with a permeable or non-permeable cryoprotectant, and the substances often have certain cytotoxicity or tissue toxicity, so that when the frozen stock preparation is clinically applied, the frozen stock preparation needs to be diluted or washed to a certain extent to reduce the toxicity of the cryoprotectant. Secondly, the cell activity of frozen cells is mostly lower than that of fresh cells, so that the frozen solution needs to be subjected to detailed formula research to ensure that the frozen solution can maintain the effects of cell viability, apoptosis and the like in the aspect of freezing.

Cell drugs can be morphologically divided into cell suspensions and non-cell suspensions. The cell suspension is prepared by re-suspending cells in physiological simulation solution or buffer solution. Non-cellular suspensions are understood to mean tissue engineering methods, in which cells are combined with a material or, by means of a defined culture, form cell aggregates, after which the cells are applied. For example, the cartilage repair techniques ACI and MACI approved by the united states drug administration are autologous chondrocyte suspensions, injected into cartilage defect sites, and tightly covered by periosteum or collagen membrane to prevent loss of autologous chondrocytes, thereby achieving in-situ cartilage repair. But at present, after chondrocytes are combined with a scaffold material by a certain tissue engineering method, such as CartiOne ™, the chondrocytes are directly transplanted to a cartilage defect part, so that the loss of the chondrocytes can be directly avoided. At present, except for immune cells used for treating tumor diseases, the administration is mostly carried out through intravenous infusion. Stem cells and terminally differentiated somatic cells are used in clinical applications for the treatment of autoimmune diseases, degenerative diseases, and cardiovascular diseases. Part of the application scenes more require that cells stay locally and function for a longer period of time. In this case, bonding with the material is a possible option.

The form of a material will have a direct impact on its function, properties and application. The materials reported in the prior literature and clinical studies are porous materials, fibrous materials, gel materials and the like. The gel material has certain fluidity, can better adapt to the shapes of different application parts, and has good application prospect. It has also been reported that cytosolic drugs bound to gel materials can remain inside tissues for longer periods of time, thereby exerting greater efficacy.

Disclosure of Invention

The present disclosure provides a cell cryopreservation solution capable of forming a gel, which can maintain the activity of cells under low temperature conditions when the cells are cryopreserved. The cell freezing solution contains thrombin, and the principle that fibrin and thrombin form gel is utilized, so that the cell suspension is directly mixed with fibrinogen after recovery, in-situ gel forming can be realized, a cell preparation wrapped by gel materials can be obtained, and the loss of cells along with the loss of liquid is reduced. Meanwhile, the gel can maintain the activity of cells in the gel, so that better efficacy exertion is realized.

An aspect of the present disclosure provides a cell cryopreservation solution comprising dimethyl sulfoxide, a soluble calcium salt, and thrombin.

In the present disclosure, non-cytotoxic thrombin is added to a cell cryopreservation solution as an impermeable cryoprotectant to reduce the concentration of dimethyl sulfoxide with cell and tissue toxicity. Meanwhile, the DMSO is not removed by the cell cryopreservation liquid in a cleaning mode, so that the convenience of using the cell cryopreservation preparation is improved.

In some embodiments, the concentration of dimethyl sulfoxide in the cell cryopreservation solution is 0.01% (v/v) to 10% (v/v), e.g., can be 0.01% (v/v), 0.05% (v/v), 0.1 (v/v), 0.5 (v/v), 1 (v/v), 2 (v/v), 4 (v/v), 6 (v/v), 8 (v/v), 10 (v/v), or any value therebetween. In some preferred embodiments, the concentration of dimethyl sulfoxide in the cell cryopreservation solution is 0.01% (v/v) to 5% (v/v), more preferably 0.01% (v/v) to 3% (v/v).

In some embodiments, the concentration of thrombin in the cell cryopreservation solution is 1-100 IU/mL, e.g., 1 IU/mL、2 IU/mL、5 IU/mL、10 IU/mL、20 IU/mL、30 IU/mL、40 IU/mL、50 IU/mL、60 IU/mL、70 IU/mL、80 IU/mL、90 IU/mL、100 IU/mL or any value therebetween. In some preferred embodiments, the concentration of thrombin in the cell cryopreservation solution is 2-50 IU/mL, more preferably 5-25 IU/mL.

In some embodiments, the concentration of the soluble calcium salt in the cell cryopreservation solution is 1-60 mM, which may be, for example, 1mM, 5mM, 10mM, 15mM, 20mM, 30mM, 40 mM, 50mM, 60 mM or any value therebetween. In some preferred embodiments, the concentration of the soluble calcium salt in the cell cryopreservation solution is 1-20 mM.

In some embodiments, the thrombin source may be human fibrinogen, thrombin of other animal species, such as bovine thrombin, rabbit thrombin, murine thrombin, sheep thrombin, porcine thrombin, and thrombin of other species origin, or a mixture of two or more thrombin.

In some embodiments, the soluble calcium salt comprises one or more of calcium chloride, calcium bromide, calcium nitrate, calcium chlorate, calcium perchlorate, calcium bicarbonate, and calcium dihydrogen phosphate. Preferably, the soluble calcium salt is calcium chloride.

In some embodiments, the cell cryopreservation solution further comprises at least one of a permeable cryopreservation protectant, a non-permeable cryopreservation protectant, a buffer solution, and a physiological solution.

In some embodiments, the buffer solution comprises a phosphate buffer solution, a HEPES buffer solution, or Hank's buffer solution.

In some embodiments, the physiological solution comprises physiological saline or dextrose injection.

In some embodiments, the permeable cryopreservation protective agent comprises propylene glycol or ethanol.

In some embodiments, the impermeable cryopreservation protective agent comprises albumin, trehalose, dextran, polyethylene glycol, dextran, sucrose, hydroxyethyl starch.

In some embodiments, the cell cryopreservation solution comprises dimethyl sulfoxide, calcium chloride, thrombin, glucose, dextran, glycerol, and albumin.

In some embodiments, the cell cryopreservation solution comprises 0.01-10% (v/v) dimethyl sulfoxide, 1-60 mmol/L calcium chloride, 1-100 IU/mL thrombin, 0.01-0.5 mmol/L glucose, 1-10% (w/v) dextran, 0.01-5% (v/v) propylene glycol, and 1-10% (w/v) albumin.

In some embodiments, the cell cryopreservation solution comprises 0.01-3% (v/v) dimethyl sulfoxide, 1-20 mmol/L calcium chloride, 2-50 IU/mL thrombin, 0.1-0.3 mmol/L glucose, 2-8% (w/v) dextran, 0.5-2% (v/v) propylene glycol, and 2-8% (w/v) albumin.

In some embodiments, the cell cryopreservation solution further comprises a phosphate buffer solution. In some embodiments, the phosphate buffer solution comprises 120-180 (e.g., 120, 140, 160, 180) mmol/L sodium chloride, 0.5-5 (e.g., 0.5, 1, 1.5, 2, 2.5, 2.68, 3, 3.5, 4, 4.5, 5) mmol/L potassium chloride, 0.5-5 (e.g., 0.5, 1, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5) mmol/L potassium dihydrogen phosphate, and 1-20 (e.g., 1, 5, 10, 15, 20) mmol/L disodium hydrogen phosphate. In some embodiments, the phosphate buffer solution further comprises 0.05-5 (e.g., 0.05, 0.1, 0.5, 1, 2, 3, 4, 5) mmol/L magnesium ions.

Another aspect of the present disclosure provides the use of the cell cryopreservation solution for cryopreserving cells or for forming a gel material.

In some embodiments, the cell density in the cell cryopreservation solution is 1×10 ⁷/mL~5×10⁷/mL, e.g., 1×10 ⁷/mL、2×10⁷/mL、3×10⁷/mL、4×10⁷/mL、5×10⁷/mL or any value in between. Preferably, the cell density in the cell cryopreservation solution is 1X 10 ⁷/mL~3×10⁷/mL.

In some embodiments, the gel material is encapsulated with cells.

In some embodiments, the cells comprise stem cells.

In some embodiments, the cells comprise mesenchymal stem cells.

In some embodiments, the cells comprise adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, or umbilical cord mesenchymal stem cells.

Yet another aspect of the present disclosure provides a composition for forming a gel, the composition comprising the cell cryopreservation solution and a fibrinogen solution.

In some embodiments, the fibrinogen solution comprises fibrinogen and a solvent.

In some embodiments, the fibrinogen concentration in the fibrinogen solution is 5-100 mg/mL, e.g., 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, or any value therebetween. Preferably, the fibrinogen concentration in the fibrinogen solution is 10-75 mg/mL, more preferably 30-60 mg/mL.

In some embodiments, the mixing volume ratio of the cell cryopreservation solution to the fibrinogen solution is 1: (0.5-2), for example, 1:0.5, 1:0.75, 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, or any value therebetween. Preferably, the mixing volume ratio of the cell freezing solution to the fibrinogen solution is 1: (0.8-1.2).

In some embodiments, the solvent comprises physiological saline, water for injection, or phosphate buffered solution.

In some embodiments, the fibrinogen source may be human fibrinogen, or fibrinogen of other animal species, such as bovine fibrinogen, rabbit fibrinogen, murine fibrinogen, sheep fibrinogen, pig fibrinogen, or fibrinogen of other species, or a mixture of two or more fibrinogen.

In some embodiments, the fibrinogen and thrombin species may be the same or different.

In yet another aspect of the present disclosure, a cell preparation prepared from the composition is provided.

In some embodiments, the cell preparation is in the form of a gel.

In some embodiments, the cells in the cell preparation are encapsulated by a gel. Preferably, more than 90% of the cells in the cell preparation are encapsulated by the gel.

In some embodiments, the cells comprise stem cells.

In some embodiments, the cells comprise mesenchymal stem cells.

Yet another aspect of the present disclosure provides a method for preparing the cell preparation, comprising mixing the cell cryopreservation solution in which cells are frozen with the fibrinogen solution after thawing, to form a gel.

In some embodiments, the disclosure also provides the use of the cell cryopreservation solution, the composition, the cell preparation, or the cell preparation obtained by the preparation method in the preparation of a medicament for cell therapy.

In some embodiments, the cell therapy drug is used to treat autoimmune diseases, degenerative diseases, cardiovascular diseases, tumors.

The technical scheme of the invention has the following beneficial effects:

1. the cell freezing solution can provide low-temperature preservation for cells for more than 6 months.

2. The cell frozen by the cell frozen stock solution can form a stable gel structure with fibrinogen solution after being recovered.

3. After the cells frozen by the cell frozen stock solution are recovered, a stable gel structure can be formed with the fibrinogen solution, and the cells in the formed gel can still keep activity.

4. After the cells frozen by the cell frozen stock solution are recovered, a stable gel structure can be formed with the fibrinogen solution, and the gel structure can wrap most cells (> 90%).

5. The cell frozen by the cell frozen stock solution can form a stable gel structure with fibrinogen solution after being recovered. The processes of cell cryopreservation, cell resuscitation and gel structure formation can not cause massive cell death and massive cell apoptosis.

6. The cell frozen by the cell frozen stock solution can form a stable gel structure with the fibrinogen solution after being recovered, and the cell can survive stably in the gel.

Drawings

Figure 1 shows a statistical plot of toxicity of DMSO at different concentrations for adipose-derived mesenchymal stem cells of three different donors after 24 hours of continuous culture.

Figure 2 shows a statistical plot of toxicity of DMSO at different concentrations for adipose-derived mesenchymal stem cells of three different donors after 72 hours of continuous culture.

FIG. 3 shows a statistical plot of the time required for gel formation of cell cryopreservation solutions of varying concentrations of thrombin, calcium chloride, DMSO content with varying concentrations of fibrinogen solution.

Figure 4 shows a statistical plot of cell viability after 3 months and 6 months of cryopreservation of adipose-derived mesenchymal stem cells from three different donors in the cryopreservation solution of the present invention.

Figure 5 shows a photograph of the macroscopic morphology of the gel formed by the cell cryopreservation solution.

FIG. 6 shows fluorescence microscopy images after thawing of cells after cryopreservation in a cryopreservation solution, after gel formation with fibrinogen solution, after dead staining of cells in the gel.

Figure 7 shows a statistical plot of the proportion of cells contained in the gel after resuscitating after cryopreservation in a cryopreservation solution, forming a gel with fibrinogen solution.

FIG. 8 shows a statistical plot of cell viability in gels after recovery of cells after cryopreservation in cryopreservation solution, and after formation of gels with fibrinogen solution.

Figure 9 shows a statistical plot of the proportion of non-apoptotic cells in the gel after resuscitating cells after cryopreservation in a cryopreservation solution, forming a gel with fibrinogen solution.

FIG. 10 shows fluorescence microscopy pictures after thawing of cells after cryopreservation in a cryopreservation solution, after gel formation with fibrinogen solution, and further incubation for 24 hours, after dead staining of cells in the gel.

Detailed Description

The present invention will be described in further detail with reference to the following examples and the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Among them, thrombin, calcium chloride and fibrinogen were purchased from Shanghai Laiyes blood products Co.

Phosphate buffer (pH 7.2-7.4) was purchased from Beijing Soy Bao technology Co., ltd., P1020.

Dextran was purchased from Cord pharmaceutical Condition, national drug standard H22021777.

Albumin was purchased from aladine, B265993.

Nattokinase purchased from Sian Tian Guangdong Biotechnology Co., ltd., T1025.

Cell basal medium was purchased from sammer femto technology (china), C12571500BT.

Fetal bovine serum was purchased from sammer feishier technology (china), C0235.

The cell complete medium is cell basal medium+10% fetal bovine serum.

Example 1

The cell cryopreservation solution is used for low-temperature preservation of cells and comprises a cryopreservation protective agent, a buffer solution or a physiological solution, calcium chloride, thrombin and the like. When the cell cryopreservation liquid is used for cryopreserving cells, the cells can maintain activity under the low-temperature condition. The cell suspension is directly mixed with fibrinogen after recovery, so that in-situ gel formation can be realized. Wherein the fibrinogen solution is composed of fibrinogen and corresponding solvent.

The use process comprises two steps of freezing and gelling.

The freezing step is to mix the cells with the cell freezing solution, then put the mixture into a freezing tube, and cool the cells to below-80 ℃ for preservation by a program cooling box or a program cooling instrument.

The gel forming step is to mix the frozen cells with fibrinogen solution in a ratio of 1:1 to form gel after the frozen cells are melted rapidly.

1. Dimethyl sulfoxide (DMSO) concentration exploration test

DMSO can enter the cell through the cell membrane, and can avoid the formation of larger ice crystals in the cell to puncture the cell in the freezing process, so that the cell-free preparation is a common permeable freezing protective agent. But it has some cytotoxicity. When it becomes part of the cell product, it is necessary to control the concentration or to remove it by washing. The cell cryopreservation liquid does not remove DMSO in a cleaning mode, so that convenience in use of the cell cryopreservation preparation is ensured. Therefore, the cell can continue to grow in the gel containing DMSO after the frozen stock solution forms the gel by exploring the tolerable DMSO concentration range of the cell through the test, thereby exerting the efficacy.

Three different donor adipose-derived mesenchymal stem cells were seeded in 96-well plates, respectively, with 4000 cells per well. After incubation for 24 hours. All wells inoculated with cells were equally divided into 8 groups of 6 duplicate wells per sample. Cell complete media containing different concentrations of DMSO (0.1%, 0.2%, 0.5%, 0.8%, 1.0%, 2.0%, 5.0%, v/v) were added to wells of each group as experimental groups, and medium groups without DMSO were used as control groups. After further incubation for 24 hours and 72 hours, each group of cells was assessed for activity using the CCK-8 kit.

The specific method is that a culture medium is used for diluting the CCK-8 reagent by 10 times to prepare working solution. The medium in each well was then aspirated, 100uL of this working solution was added and incubated with cells for 100 min at 37 ℃. Wells without cells in the wells were designated as blank and incubated under the same conditions. And finally, measuring the absorbance of each hole at 450 nm by using an enzyme-labeling instrument, wherein the absorbance of an experimental group is marked as an A measured value, the absorbance of a control group is marked as an A control value, and the absorbance of a blank group is marked as an A blank value. The cell viability calculation method comprises the following steps: cell viability (%) = (a measured value-a blank value)/(a control value-a blank value) ×100%.

The results of evaluating the cell activities of adipose-derived mesenchymal stem cells of three different donors are shown in fig. 1-2. When the DMSO content in the environment is more than 2%, the cell activity is obviously reduced. When the DMSO content in the environment is 2%, the cell activity is maintained to be more than 90% after 24 hours, but the cell activity is obviously reduced to be less than 50% after 72 hours. When the DMSO content in the environment is less than or equal to 2%, the effect on the cell activity is not great. From this test result, it was determined that the final gel product formed had a DMSO concentration in the range of 0.01% to 5%, a sub-optimal range of 0.01% to 2.5%, and an optimal range of 0.01% to 1.5%. According to the reverse thrust of the gel forming process, the concentration range of DMSO in the frozen stock solution is 0.01-10%, the suboptimal range is 0.01-5%, and the optimal range is 0.01-3%.

2. Thrombin, calcium chloride, fibrinogen concentration discovery assay

The concentration of thrombin, calcium chloride, DMSO and fibrinogen in the fibrinogen solution in the cell cryopreservation solution have an important effect on the rate of gel formation, so that it is necessary to explore their appropriate concentrations by experimentation, as well as the concentrations in the corresponding cryopreservation solution and fibrinogen.

Frozen stock solutions and fibrinogen solutions were prepared according to the orthogonal test designs of table 1 below.

Wherein the frozen stock solution is prepared by dissolving phosphate buffer solution (pH 7.2-7.4), and other components are added into glucose (0.15 mmol/L), dextran (5%, w/v), glycerol (1%, v/v) and albumin (5%, w/v).

The fibrinogen solution dissolves fibrinogen using physiological saline.

Adipose-derived mesenchymal stem cells of three different donors were resuspended in a cryopreservation solution, the cell density was adjusted to 2×10 ⁶/mL, and then mixed with an equal volume of fibrinogen solution to form a gel. Continuous measurements of absorbance at 350 nm were performed with an microplate reader during gel formation, automatically scanned every 10 seconds. The absorbance curve was analyzed and when the slope of the curve was 0, the gel was completely formed, and the time for complete gel formation was recorded. As a result, as shown in FIG. 3, the gel forming time was varied from 5 seconds to 150 seconds for each of the above groups. The effect levels of fibrinogen concentration in the thrombin, calcium chloride, DMSO and fibrinogen solutions on gel formation time were also calculated using SUMIF functions, respectively. Among the factors studied, the calcium chloride concentration has the greatest effect on the gel time, followed by thrombin concentration, DMSO concentration, fibrinogen concentration in that order.

According to the test result, the concentration of the thrombin in the cell freezing solution is in the range of 1-100 IU/mL, the suboptimal range is 2-50 IU/mL, and the optimal range is 5-25 IU/mL. The concentration of calcium chloride in the gel in the cell freezing solution is in the range of 1-60 mM, the suboptimal range is 1-20 mM, and the optimal range is 2-10 mM.

From this test result, it can be obtained that the fibrinogen concentration in the fibrinogen solution is in the range of 5-100 mg/mL, the suboptimal range is 10-75 mg/mL, and the optimal range is 30-60 mg/mL.

Example 2

The basic solvent of the cell cryopreservation solution prepared in this example is water, and the composition formula is shown in table 2 below:

and (3) placing the prepared cell frozen stock solution in a refrigerator at the temperature of 2-8 ℃ for precooling and preserving.

The adipose-derived mesenchymal stem cells of three different donors were cultured using a cell complete medium under culture conditions of 37 ℃,5% CO ₂ concentration and complete humidity, and the cell confluency was about 85% as observed by microscope. Cells were then collected by digestion with recombinant trypsin (TryplE) and cell viability and cell density were calculated by acridine orange/propidium iodide (AO/PI) staining.

According to the cell counting result, taking 1.2×10 ⁸ of living cells, centrifugally collecting, removing supernatant, slowly adding 6 mL of prepared cell freezing solution into cell sediment, uniformly mixing to uniformly disperse the cells in the cell freezing solution, wherein the cell density is 2×10 ⁷/mL.

The cell suspension was aliquoted into 6 cryopreservation tubes, 1mL per tube.

The frozen tube was placed in a program cooling box (Corning, coolCell) and then the program cooling box was placed in a-80 ℃ low temperature refrigerator. And taking the cell cryopreservation tube out of the program cooling box after 24 hours, and storing in a low-temperature refrigerator at the temperature of-80 ℃.

Sampling at 3 months and 6 months respectively, taking out the freezing tube from the refrigerator, placing the freezing tube into a water bath kettle at 37 ℃ for rapid melting, and then carrying out cell density and cell activity detection analysis. The results obtained are shown in FIG. 4. When the cell density of frozen storage is 2X 10 ⁷ cells/mL, the cell viability of the adipose-derived mesenchymal stem cells of three different donors is above 90% after 6 months of frozen storage.

Example 3

The basic solvent of the cell cryopreservation solution prepared in this example is water, and the composition formula is shown in table 3 below:

Adipose-derived mesenchymal stem cells were cultured in the same manner as in example 2, and the cell confluency was about 85% as observed by a microscope. Cells were then collected by digestion with recombinant trypsin (TryplE) and cell viability and cell density were calculated by acridine orange/propidium iodide (AO/PI) staining.

According to the cell counting result, taking the living cells 6×10 ⁷, centrifugally collecting, removing the supernatant, slowly adding 3 mL prepared cell freezing solution into the cell sediment, uniformly mixing to uniformly disperse the cells in the cell freezing solution, wherein the cell density is 2×10 ⁷/mL.

The cell suspension was aliquoted into 3 cryopreservation tubes, 1mL per tube.

And placing the freezing tube in a program cooling box, and then placing the program cooling box in a low-temperature refrigerator at-80 ℃. And taking the cell cryopreservation tube out of the program cooling box after 24 hours, and storing in a low-temperature refrigerator at the temperature of-80 ℃.

Sampling at 6 months, taking out the frozen storage tube from the refrigerator, and rapidly melting in a water bath kettle at 37 ℃.

A fibrinogen solution was prepared as shown in table 4 below, with water as the solvent:

1 mL of the formulated fibrinogen solution was aspirated into a 2 mL syringe.

The thawed 1 mL cell suspension was aspirated into another 2 mL syringe.

Two syringes were placed on a plastic holder of a twin mixer and connected to a Y-extension tube, while pushing out the two components to form a gel completely free of flow in about 5 minutes. The macroscopic morphology of the gel is shown in figure 5.

Example 4

Cell cryopreservation solutions were prepared under the conditions of example 3. Adipose mesenchymal stem cells were cryopreserved according to a cryopreservation density of 2X 10 ⁷/mL. Sampling and resuscitating after 6 months of freezing.

A fibrinogen solution was prepared according to the conditions of example 3.

The resuscitated cell suspension and fibrinogen solution were removed using a twin mixer as described in example 3 to provide a gel containing cells.

Cell-containing gels were visualized by staining using Calcein/propidium iodide (Calcein-AM/PI) cell activity and toxicity detection kit (Beyotime, C2015M), where Calcein-AM was able to permeate cell membranes and hydrolyze with cell endogenous esterases to produce the polar molecule Calcein (Calcein) with strong negative charge that is unable to permeate cell membranes, and thus is retained in the cells, where Calcein emits strong green fluorescence. PI dyes do not cross the cell membrane of living cells, but bind to DNA within dead cells where the cell membrane integrity is compromised, exhibiting red fluorescence. The use of Calcein-AM/PI dye allows the cell-encapsulated gel to appear green on fluorescent microscopy and red on dead cells. The staining results are shown in fig. 6. The results showed that the vast majority of cells were living cells within the gel (stained green) and only a very small number of cells were dead cells (stained red).

Example 5

Cell cryopreservation solutions were prepared under the conditions of example 3. Adipose-derived mesenchymal stem cells were frozen at a frozen density of 2X 10 ⁷/mL and 3X 10 ⁷/mL, respectively. Sampling and resuscitating after 6 months of freezing.

A fibrinogen solution was prepared according to the conditions of example 3.

Nattokinase was dissolved in phosphate buffer solution at a concentration of 50 FU/mL.

0.2 And 2mL nattokinase solution is added into the gel, and the gel is incubated for 30 minutes at 37 ℃, so that the gel can be observed to be completely degraded by the nattokinase and is in a cell suspension state.

The resuscitated cell suspension without gel formation was also recorded as control 1.

And simultaneously adding an equivalent amount of nattokinase solution into the cell suspension subjected to recovery without gel formation for treating for the same time, and verifying the damage of the nattokinase to cells. Is designated as control group 2.

The cells were collected by centrifugation, and the results of counting the cells are shown in FIG. 7. Recovery (%) = (number of cells after sol/number of cells added) ×100. The recovery rate of control 1 was 100%, indicating that the counting method was correct. The number of recovered cells in control group 2 was 99.8%, indicating no significant damage to cells by the nattokinase solution. Cell recovery was 100.7% and 96.1% for the 2X 10 ⁷/mL cell-gel group and 3X 10 ⁷/mL cell-gel group, respectively. The cell freezing solution capable of forming gel can form effective package on most cells after forming gel.

Example 6

Cell cryopreservation solutions were prepared under the conditions of example 2. Adipose-derived mesenchymal stem cells were frozen at a frozen density of 2X 10 ⁷/mL and 3X 10 ⁷/mL, respectively. Sampling and resuscitating after 6 months of freezing.

A fibrinogen solution was prepared according to the conditions of example 2.

The resuscitated cell suspension and fibrinogen solution were removed using a twin mixer as described in example 2 to provide a gel containing cells.

The resulting cell gel was subjected to lysis treatment using a nattokinase solution under the conditions of example 4.

Cells were collected by centrifugation and cell viability was measured using AOPI. The results are shown in FIG. 8. The number of living cells in each group was >95%. The processes of cell cryopreservation, cell resuscitation and gel formation do not lead to significant cell death.

The centrifugally collected cells were stained using an Annexin-V-PI apoptosis staining kit and the staining results were detected by flow cytometry. The non-apoptotic cells are not stained by Annexin-V and PI, the non-Annexin-V stained by PI is necrotic cells or mechanically damaged cells, the non-PI stained by Annexin-V stained is early apoptotic cells, and the Annexin-V and PI are late apoptotic cells simultaneously. The results are shown in FIG. 9.

From the results, it can be seen that >92% of the cells are in the non-apoptotic state. That is, it is demonstrated that the processes of cell cryopreservation, cell resuscitation and gel formation using the cell cryopreservation solution do not cause significant damage to cells to cause apoptosis.

Example 7

The basic solvent of the cell cryopreservation solution prepared in this example is water, and the composition formula is shown in table 5 below:

According to the cell counting result, taking living cells 3×10 ⁷, centrifugally collecting, removing supernatant, slowly adding 3 mL prepared cell freezing solution into cell sediment, uniformly mixing to uniformly disperse the cells in the cell freezing solution, wherein the cell density is 1×10 ⁷/mL.

The cell suspension was aliquoted into 3 cryopreservation tubes, 1mL per tube.

A fibrinogen solution was prepared as shown in table 6 below, with water as the solvent:

1 mL of the formulated fibrinogen solution was aspirated into a 2 mL syringe.

The thawed 1 mL cell suspension was aspirated into another 2 mL syringe.

Two syringes were placed on a plastic holder of a twin mixer and connected to a Y-extension tube, while pushing out the two components to form a gel completely free of flow in about 3 minutes.

The gel was placed in a 24-well culture plate, a cell complete medium was added, and after culturing for 24 hours, the gel containing cells was stained with Calcein/propidium iodide (Calcein-AM/PI) cell activity and toxicity detection kit, and the photograph of the gel was shown under a fluorescence microscope as shown in fig. 10. It can be seen that the vast majority of cells in the gel are living cells (stained green) and only a small number of cells are dead cells (stained red). This result demonstrates that the use of the cell cryopreservation solution to cryopreserve cells and the gel formed during use can maintain the activity of the cells in a simulated physiological environment.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A cell freezing solution comprises dimethyl sulfoxide, soluble calcium salt and thrombin.

2. The cell freezing solution according to claim 1, wherein in the cell freezing solution,

The concentration of the dimethyl sulfoxide is 0.01% (v/v) to 10% (v/v); and/or

The concentration of thrombin is 1-100 IU/mL; and/or

The concentration of the soluble calcium salt is 1-60 mM; and/or

The soluble calcium salt comprises one or more of calcium chloride, calcium bromide, calcium nitrate, calcium chlorate, calcium perchlorate, calcium bicarbonate and calcium dihydrogen phosphate; and/or

The cell cryopreservation solution also comprises at least one of a permeable cryopreservation protective agent, an impermeable cryopreservation protective agent, a buffer solution and a physiological solution.

3. The cell cryopreservation solution according to claim 2, wherein the buffer solution or physiological solution comprises a phosphate buffer solution, a HEPES buffer solution or Hank's buffer solution, physiological saline or dextrose injection; and/or

The permeable cryopreservation protective agent comprises propylene glycol or ethanol; and/or

The impermeable cryopreservation protective agent comprises albumin, trehalose, dextran, polyethylene glycol, dextran, sucrose and hydroxyethyl starch; and/or

The cell freezing solution comprises 0.01-10% (v/v) dimethyl sulfoxide, 1-60 mmol/L calcium chloride, 1-100 IU/mL thrombin, 0.01-0.5 mmol/L glucose, 1-10% (w/v) dextran, 0.01-5% (v/v) propylene glycol and 1-10% (w/v) albumin.

4. Use of a cell cryopreservation solution according to any one of claims 1-3 for cryopreserving cells or for forming a gel material, said gel material encapsulating cells.

5. The use according to claim 4, wherein the cell density in the cell cryopreservation solution is 1 x 10 ⁷/mL~5×10⁷/mL; and/or the cells comprise stem cells.

6. A composition for forming a gel, the composition comprising the cell cryopreservation solution of any one of claims 1-3 and a fibrinogen solution.

7. The composition of claim 6, wherein the fibrinogen solution comprises fibrinogen and a solvent; and/or

The concentration of the fibrinogen in the fibrinogen solution is 5-100 mg/mL; and/or

The solvent comprises normal saline, water for injection or phosphate buffer solution; and/or

The mixing volume ratio of the cell freezing solution to the fibrinogen solution is 1: (0.5-2).

8. A cell preparation prepared from the composition of claim 6 or 7, said cell preparation being in the form of a gel.

9. The method for producing a cell preparation according to claim 8, comprising thawing the cell frozen solution in which cells are frozen, and mixing the thawed solution with the fibrinogen solution to form a gel.

10. Use of a cell cryopreservation solution according to any one of claims 1-3, a composition according to claim 6 or 7 or a cell preparation according to claim 8 or a cell preparation obtained by a method according to claim 9 for the preparation of a medicament for cell therapy.