Stem cell cryopreservation liquid and stem cell cryopreservation method

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a stem cell cryopreservation solution and a stem cell cryopreservation method. The stem cell freezing solution comprises human serum albumin, trehalose, a DMEM / F12 culture medium and propylene glycol. The stem cell cryopreservation liquid does not contain animal-derived substances and a conventional cell cryopreservation protective agent dimethyl sulfoxide (DMSO), deep hypothermia storage of the stem cells can be achieved, and the form and characteristics of the stem cells after resuscitation are well maintained.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a stem cell cryopreservation solution and a stem cell cryopreservation method. Background Technology

[0002] In the field of stem cell research and application, the effective preservation of stem cells is crucial. Stem cell cryopreservation, as a key method for long-term preservation of stem cells, requires significant optimization in its technology to maintain the biological characteristics and functional integrity of stem cells.

[0003] Currently, vitrification cryopreservation is the mainstream method for cryopreservation in the industry, and its core lies in inhibiting the formation of ice crystals. However, in practice, inhibiting ice crystal formation is extremely difficult. Even minor disturbances can easily induce ice crystal formation, and once formed, the ice crystals will grow explosively in an irregular and sharp form, causing irreversible damage to stem cells and greatly reducing the stem cell recovery rate.

[0004] To inhibit ice crystal formation, traditional cryopreservation techniques often add high concentrations of dimethyl sulfoxide (DMSO). However, DMSO has strong cytotoxicity and epigenotoxicity. Related studies have shown that using cryopreservation solutions containing 10% DMSO significantly impairs the cellular characteristics and function of mesenchymal stem cells. Furthermore, when used as an excipient in cell therapy drugs, it may cause gastrointestinal side effects. Based on this, drug regulatory agencies in several countries, including the China Food and Drug Administration and the US FDA, have called for the search for alternatives to DMSO.

[0005] Meanwhile, existing cell cryopreservation solutions also present numerous problems. For example, commonly used cell cryopreservation solutions contain 10% DMSO, 20%-90% fetal bovine serum (FBS), and the remaining incomplete culture medium. Among these, FBS is an animal-derived substance with a complex composition, posing potential risks in clinical applications and increasing the likelihood of contamination.

[0006] Furthermore, Japan and Western countries have long monopolized the global cryopreservation market due to their advantages in traditional vitrification cryopreservation technology. They essentially control the cryopreservation solutions used in assisted reproduction, cell therapy, and umbilical cord blood hematopoietic stem cell cryopreservation, which to some extent restricts the development of related industries in China.

[0007] Therefore, developing a safe, efficient, and cost-effective stem cell cryopreservation solution and method that can effectively reduce ice crystal damage, has simple composition, no animal-derived risks, and is cost-controllable has always been a research topic for researchers in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a stem cell cryopreservation solution and a stem cell cryopreservation method to solve the problems of toxicity risks or high costs associated with existing stem cell cryopreservation solutions. The technical problems to be solved by this invention are not limited to the described technical topics; those skilled in the art will clearly understand other technical topics not mentioned herein through the following description.

[0009] To achieve the above objectives, the present invention provides a stem cell cryopreservation solution comprising: human serum albumin, trehalose, DMEM / F12 culture medium, and propylene glycol.

[0010] Preferably, the volume fraction of human serum albumin is 8-12%, the mass concentration of trehalose is 3-5%, the volume fraction of DMEM / F12 medium is 68-77%, and the volume fraction of propylene glycol is 15-20%.

[0011] Human serum albumin must meet injectable grade standards to avoid immune reactions. Its main functions are: regulating osmotic pressure, maintaining cell colloid osmotic pressure, preventing cell dehydration or swelling, and acting as a stabilizer to prevent cell adhesion.

[0012] Trehalose can form a non-specific protective membrane, reducing cell damage from low temperatures; it stabilizes cell membrane protein structure, protects cell viability, and can further stabilize cell structure by forming a glassy matrix. When used in combination with hydroxyethyl starch, human serum albumin, etc., it synergistically and significantly improves cell viability.

[0013] DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F-12, rich in amino acids, vitamins, and inorganic salts, which enables cells to rapidly regain activity and normal growth and proliferation after thawing. Its inorganic salts maintain osmotic pressure balance inside and outside the cell, preventing morphological changes or rupture due to abnormal osmotic pressure and ensuring cell integrity. Furthermore, DMEM / F12 medium provides a suitable liquid environment for cells, allowing them to disperse evenly and reducing mechanical damage to cells from ice crystals during freezing.

[0014] Propylene glycol (purity ≥99.9%) can penetrate the cell membrane and enter the cell before the cell freezing suspension solidifies, protecting the cell from damage caused by high concentrations of electrolytes. It can bind with water molecules, reducing the formation of intracellular ice crystals and mitigating the mechanical damage caused by ice crystals. Furthermore, propylene glycol can prevent cell dehydration and protein denaturation due to solution effects, helping to maintain normal cell morphology and function and reducing the impact of freezing on cell structure and metabolism.

[0015] A method for cryopreserving stem cells, wherein the above-mentioned stem cell cryopreservation solution is used to cryopreserve cells.

[0016] Preferably, the cells are suspended in the above-mentioned stem cell cryopreservation solution to obtain a cell suspension, which is then pre-cooled.

[0017] Preferably, the cell density in the cell suspension is 5 × 10⁻⁶. 6 -1×10 7 cells / mL.

[0018] Preferably, the temperature for cryopreservation of the stem cells is ≤-150℃.

[0019] Preferably, the stem cells need to be pre-cooled before cryopreservation, and the pre-cooling meets at least one of the conditions in (1)-(2):

[0020] (1) First, treat the product at 2℃-8℃ for more than 30 minutes in the program cooling box, and then treat it at -80℃ for more than 4 hours.

[0021] (2) Treat at -80℃ for more than 4 hours.

[0022] Compared with existing technologies, the present invention has the following advantages: the stem cell cryopreservation solution has a simple composition, is non-cytotoxic, and the configuration of the components can stabilize the cell structure and reduce the mechanical damage to the cells caused by ice crystals during cryopreservation.

[0023] The stem cell preservation solution obtained by this invention can achieve deep cryopreservation of stem cells, and the vitality and characteristics of stem cells are well maintained after revival. Attached Figure Description

[0024] Figure 1 Cell recovery and counting results after direct cryopreservation, programmed temperature cryopreservation, and direct cryopreservation in the control group (CTRL).

[0025] Figure 2 Microscopic images of direct cryopreservation, programmed temperature freezing, and the control group (CTRL) after direct cryopreservation followed by thawing and inoculation for 72 hours.

[0026] Figure 3 The flow cytometry graph is a result of direct cryopreservation followed by thawing and inoculation for 72 hours.

[0027] Figure 4 The flow cytometry graph shows the results of 72 hours of inoculation after the cryopreserved material was thawed and inoculated following a controlled cooling process. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Example 1

[0031] Stem cell cryopreservation solution: Add human serum albumin, trehalose, and propylene glycol to DMEM / F12 culture medium, mix well, and then filter through a 0.22μm filter membrane to obtain the stem cell cryopreservation solution.

[0032] The final concentrations of each component in the stem cell cryopreservation solution are as follows: 10% by volume of human serum albumin, 40 mM of trehalose, 15% by volume of propylene glycol, with the remainder supplemented by DMEM / F12 medium.

[0033] Stem cell cryopreservation method: Stem cells were uniformly dispersed in the stem cell cryopreservation solution obtained in this embodiment to achieve a stem cell concentration of 5 × 10⁻⁶. 6 -1×10 7 cells / mL.

[0034] Comparative Example 1

[0035] Stem cell cryopreservation solution: A commercially available finished cryopreservation solution (containing DMSO).

[0036] Stem cell transport: Stem cells were uniformly dispersed in the comparative example stem cell cryopreservation solution to achieve a stem cell concentration of 5 × 10⁻⁶. 6 -1×10 7 cells / mL.

[0037] Take cultured P4 generation umbilical cord mesenchymal stem cells into a 50mL centrifuge tube, measure the volume to 45mL, mix well, and then take 20μL of cells and mix with 20μL of LAOPI staining solution to count the cells. The cell viability is 94.61%. Divide the cell suspension into two tubes according to the required number of cells in the example and control example, and centrifuge for later use.

[0038] After centrifugation, the cells were resuspended in 6 mL of the stem cell cryopreservation solution obtained in Example 1, and aliquoted into cryovials, 1 mL per tube, for a total of 6 tubes. Three tubes were for direct cryopreservation (numbered 1-3), and the other three tubes were for programmed cooling cryopreservation (numbered 4-6). The cells were also resuspended in 3 mL of commercially available stem cell cryopreservation solution, and aliquoted into cryovials, 1 mL per tube, for a total of 3 tubes, which constituted the control group (CTRL) (numbered 7-9).

[0039] Cells in tubes 1-3 and 7-9 were placed directly in a -80°C freezer, while cells in tubes 4-6 were placed in a programmed cooling box and treated at 2°C-8°C for 30 minutes. After pre-cooling overnight in a -80°C freezer, the cells were transferred to liquid nitrogen for storage.

[0040] One month later, cells in tubes 1-9 were resuscitated. Each tube of cells was resuspended in 5 mL of pre-cooled 0.9% sodium chloride injection solution, mixed thoroughly, and then 20 μL of cells were mixed with 20 μL of LAOPI staining solution for cell counting. The results are shown in Table 1. Figure 1 As shown.

[0041] Table 1. Cell recovery and counting results after direct cryopreservation, programmed cooling cryopreservation, and direct cryopreservation (control group).

[0042]

[0043] From Table 1 and Figure 1 The average survival rates of cells frozen directly and frozen under programmed temperature were 85.57% and 94.44%, respectively; the average survival rate of cells frozen directly in the control group (CTRL) was 90.26%.

[0044] Through Table 1 and Table Figure 1 It can be found that the average cell survival rate after cryopreservation of umbilical cord mesenchymal stem cells by direct cryopreservation or cryopreservation using a programmed cooling box is ≥85%, which meets the ideal survival rate expectation. Moreover, the average survival rate of umbilical cord mesenchymal stem cells after cryopreservation using the programmed cooling box is 94.44%, which is higher than the average survival rate of 90.26% of the control group (CTRL).

[0045] The applicant further cultured cells obtained through direct cryopreservation and programmed cooling after thawing and viability testing. Both groups of cells were seeded in 100mm culture dishes and cultured at 37°C with 5% CO2 for 72 hours, followed by microscopic observation. The results are as follows... Figure 2 and Figure 3 As shown, the cells are dense, uniform in shape, and spindle-shaped, consistent with the morphological characteristics of mesenchymal stem cells.

[0046] The two groups of cells were then digested and harvested after inoculation and culture. After washing the cells with PBS, they were divided into four tubes for antibody staining: tube 1 was a positive isotype control, tube 2 a negative isotype control, tube 3 a positive antibody, and tube 4 a negative antibody. After mixing the antibodies with the cells, the tubes were incubated at 4°C for 30 min. Each tube was then washed with PBS, and flow cytometry was used to detect nine cell surface markers: positive markers CD90+, CD73+, CD105+, and CD29+; and negative markers CD45-, CD34-, CD14-, CD79a-, and HLA-DR-. The results are as follows: Figure 4 As shown, all positive indicators are greater than 95%, and all negative indicators are less than 2%.

[0047] pass Figure 2 , Figure 3 , Figure 4It can be observed that when umbilical cord mesenchymal stem cells are cryopreserved using this cryopreservation solution, the cell morphology and characteristics are well maintained after thawing.

[0048] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A stem cell cryopreservation solution, characterized in that, Including human serum albumin, trehalose, DMEM / F12 medium, and propylene glycol.

2. The stem cell cryopreservation solution according to claim 1, characterized in that, The volume fraction of human serum albumin was 8-12%, the mass concentration of trehalose was 3-5%, the volume fraction of DMEM / F12 medium was 68-77%, and the volume fraction of propylene glycol was 15-20%.

3. The stem cell cryopreservation solution according to claim 1, characterized in that, Human serum albumin is a clinical-grade reagent with an initial concentration of 20%.

4. The stem cell cryopreservation solution according to claim 1, characterized in that, Propylene glycol is biological grade propylene glycol (purity ≥99.9%).

5. A method for cryopreserving stem cells, characterized in that, Stem cells are cryopreserved using the cryopreservation solution described in any one of claims 1-4.

6. A method for cryopreserving stem cells according to claim 5, characterized in that, The cell cryopreservation density is 5 × 10⁻⁶. 6 -1×10 7 cells / mL.

7. A method for cryopreserving stem cells according to claim 5, characterized in that, The freezing temperature is ≤-150℃.

8. A method for cryopreserving stem cells according to claim 5, characterized in that, Pre-cooling is required before freezing.

9. The stem cell cryopreservation method according to claim 8, characterized in that, The precooling satisfies at least one of the conditions in (1)-(2): (1) The pre-cooling treatment is as follows: first, it is treated at 2℃-8℃ for more than 30 minutes in a programmed cooling box, and then treated at -80℃ for more than 4 hours. (2) The pre-cooling treatment is: treatment at -80℃ for more than 4 hours.

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