Cryopreservation composition for stem cells and preparation method thereof

Abstract

The invention relates to the technical field of stem cell cryopreservation, in particular to a cryopreservation composition for stem cells and a preparation method of the cryopreservation composition. Comprising the following steps: dissolving thioether modified trehalose in an acetic acid buffer solution, adding ferric nitrate and a hydrogen peroxide solution, and reacting for a period of time to obtain sulfoxide modified trehalose. Adding sulfoxide modified trehalose and phosphatidylcholine into the precooled basic culture medium, and stirring until the materials are completely dissolved, so as to obtain a basic solution; and adding the antioxidant, the bacteriostatic agent, the platelet lysis buffer and ferric nitrate into the basic solution, stirring in a dark place, and then filtering and sterilizing to obtain the cryopreservation composition for the stem cells. According to the prepared cryopreservation composition for the stem cells, through the synergistic interaction between the sulfoxide modified trehalose and the phosphatidylcholine, and no DMSO or animal serum is added, the high efficiency, safety and clinical suitability of cell cryopreservation are achieved.

Description

Technical Field

[0001] The present application relates to the technical field of stem cell cryopreservation, and in particular to a cryopreservation composition for stem cells and a preparation method thereof. Background Art

[0002] Stem cells are a type of cell with the ability to self-renew and multidirectionally differentiate. They can generate identical stem cells through division (self-replication) and, depending on the microenvironment and induction signals, can differentiate into different types of mature cells (such as neurons, cardiomyocytes, and hepatocytes). They are currently used in various biomedical fields, including regenerative medicine, gene therapy, and immunotherapy.

[0003] Traditional stem cell cryopreservation solutions use DMSO and animal serum to improve stem cell survival rates. However, DMSO is cytotoxic and animal serum increases the risk of cell viral infection. Therefore, it is necessary to invent a stem cell cryopreservation composition that uses as little or no DMSO and animal serum as possible. Summary of the Invention

[0004] In response to the above problems, the purpose of the present application is to provide a stem cell cryopreservation composition that does not require the addition of DMSO and animal serum and can improve the survival rate of stem cells and inhibit stem cell differentiation.

[0005] To achieve the above objectives, the present application provides a method for preparing a composition for cryopreservation of stem cells, comprising the following steps:

[0006] S1. Dissolve thioether-modified trehalose in an acetate buffer solution, then add ferric nitrate and hydrogen peroxide solution, and react for a period of time to obtain sulfoxide-modified trehalose;

[0007] In the above process, ferric nitrate is used as a catalyst, and hydrogen peroxide is used as an oxidant to react with thioether-modified trehalose to generate sulfoxide-modified trehalose.

[0008] S2. Add sulfoxide-modified trehalose and phosphatidylcholine to the pre-cooled basal medium and stir until completely dissolved to obtain the basal solution;

[0009] S3. Add antioxidant, antibacterial agent, platelet lysate and ferric nitrate to the base solution, stir in the dark, and then filter and sterilize to obtain a cryopreservation composition for stem cells.

[0010] Furthermore, the final concentration of the sulfoxide-modified trehalose in the cryopreservation composition is 6-10% W / V.

[0011] Preferably, the final concentration of the sulfoxide-modified trehalose in the freezing composition is 7-9% W / V.

[0012] Furthermore, the final concentration of the phosphatidylcholine in the cryopreservation composition is 1.5-2.5% W / V.

[0013] Furthermore, the basal culture medium is DMEM / F12 or RPMI-1640.

[0014] Furthermore, the concentration of the platelet lysate is 15-30% V / V.

[0015] Furthermore, the antioxidant is vitamin E or glutathione, wherein the concentration of vitamin E in the cryopreservation composition is 0.2-0.8 mM or the concentration of glutathione is 0.2-0.8 mM.

[0016] Furthermore, the antibacterial agent is ε-polylysine, and its concentration in the frozen composition is 0.02-0.1% W / V.

[0017] Furthermore, the filter membrane used for the filtration and sterilization is less than 0.25 μm.

[0018] Furthermore, the concentration of the ferric nitrate in the cryopreservation composition is 10-50 μM.

[0019] The present application also provides a cryopreservation composition for stem cells obtained by the above-mentioned preparation method.

[0020] In summary, this application has the following beneficial effects:

[0021] The cryopreservation composition for stem cells prepared in the present application utilizes the synergistic effect between sulfoxide-modified trehalose and phosphatidylcholine to inhibit the differentiation of stem cells during the cryopreservation process and increase the survival rate of stem cells after recovery; phosphatidylcholine is the main component of the phospholipid bilayer of the cell membrane, and can reduce the low-temperature-induced membrane phase transition and maintain the fluidity of the membrane by filling the gaps in the space of the phospholipid tails; sulfoxide-modified trehalose has good biocompatibility, can enter the cell to balance the osmotic pressure inside and outside the cell, and can also inhibit the formation of ice crystals in the cell and reduce ice crystal penetration damage. Moreover, the hydroxyl groups of phosphatidylcholine can form hydrogen bonds with the sulfoxide groups of sulfoxide-modified trehalose, destroying the orderly arrangement of water molecules and reducing the formation of ice crystals; phosphatidylcholine stabilizes the cell membrane skeleton, and sulfoxide-modified trehalose regulates osmotic pressure and reduces ice crystal formation. The dual effects form a hard and soft protective layer, which greatly improves the cell survival rate. The cryopreservation composition for stem cells prepared in the present application achieves high efficiency, safety and clinical adaptability of cell cryopreservation through the synergistic enhancement between sulfoxide-modified trehalose and phosphatidylcholine without adding DMSO and animal serum. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] The raw materials involved in the specific embodiments of the present application are of pharmaceutical grade. In addition, thioether-modified trehalose is selected from Shanghai Jizhi Biochemical Technology Co., Ltd. JZ-THIO-001, and phosphatidylcholine is selected from Sigma-Aldrich Cat# P5638.

[0024] Example 1

[0025] A method for preparing a cryopreservation composition for stem cells comprises the following steps:

[0026] S1. Dissolve 0.05 mol of thioether-modified trehalose in 50 mL of acetate buffer solution, then add 0.5 g of ferric nitrate and 1.5 mL of 30% w / w hydrogen peroxide solution, and react at 30°C for 5 h to obtain sulfoxide-modified trehalose.

[0027] S2. Take 7 g of sulfoxide-modified trehalose and slowly add it to 50 mL of 4°C pre-cooled DMEM / F12 basal medium and stir magnetically (300 rpm) until completely dissolved; take 1.5 g of phosphatidylcholine powder and dissolve it in 10 mL of 4°C pre-cooled anhydrous ethanol, sonicate for 10 min (40 kHz), then slowly add it dropwise to the DMEM / F12 basal medium containing sulfoxide-modified trehalose and stir magnetically (200 rpm) for 30 min to obtain the basal solution;

[0028] S3. Take 5 mL of 100 mM vitamin E, 0.05 g of ε-polylysine, 20 mL of platelet lysate, and 0.5 mg of ferric nitrate and add them to the basal solution. Stir (100 rpm) for 20 min in the dark at 4°C. Add DMEM / F12 basal medium to 100 mL, then filter and sterilize with a 0.22 μm filter membrane to obtain a cryopreservation composition for stem cells.

[0029] Example 2

[0030] A method for preparing a cryopreservation composition for stem cells comprises the following steps:

[0031] S1. Dissolve 0.05 mol of thioether-modified trehalose in 50 mL of acetate buffer solution, then add 0.5 g of ferric nitrate and 1.5 mL of 30% w / w hydrogen peroxide solution, and react at 30°C for 5 h to obtain sulfoxide-modified trehalose.

[0032] S2. Take 8 g of sulfoxide-modified trehalose and slowly add it to 50 mL of 4°C pre-cooled DMEM / F12 basal medium and stir magnetically (300 rpm) until completely dissolved; take 2 g of phosphatidylcholine powder and dissolve it in 10 mL of 4°C pre-cooled anhydrous ethanol, sonicate for 10 min (40 kHz), and then slowly add it dropwise to the DMEM / F12 basal medium containing sulfoxide-modified trehalose and stir magnetically (200 rpm) for 30 min to obtain the basal solution;

[0033] S3. Take 5 mL of 100 mM vitamin E, 0.05 g of ε-polylysine, 20 mL of platelet lysate, and 0.5 mg of ferric nitrate and add them to the basal solution. Stir (100 rpm) for 20 min in the dark at 4°C. Add DMEM / F12 basal medium to 100 mL, then filter and sterilize with a 0.22 μm filter membrane to obtain a cryopreservation composition for stem cells.

[0034] Example 3

[0035] A method for preparing a cryopreservation composition for stem cells comprises the following steps:

[0036] S1. Dissolve 0.05 mol of thioether-modified trehalose in 50 mL of acetate buffer solution, then add 0.5 g of ferric nitrate and 1.5 mL of 30% w / w hydrogen peroxide solution, and react at 30°C for 5 h to obtain sulfoxide-modified trehalose.

[0037] S2. Take 9 g of sulfoxide-modified trehalose and slowly add it to 50 mL of 4°C pre-cooled DMEM / F12 basal medium and stir magnetically (300 rpm) until completely dissolved; take 2.5 g of phosphatidylcholine powder and dissolve it in 10 mL of 4°C pre-cooled anhydrous ethanol, sonicate for 10 min (40 kHz), and then slowly add it dropwise to the DMEM / F12 basal medium containing sulfoxide-modified trehalose and stir magnetically (200 rpm) for 30 min to obtain the basal solution;

[0038] S3. Take 5 mL of 100 mM vitamin E, 0.05 g of ε-polylysine, 20 mL of platelet lysate, and 0.5 mg of ferric nitrate and add them to the basal solution. Stir (100 rpm) for 20 min in the dark at 4°C. Add DMEM / F12 basal medium to 100 mL, then filter and sterilize with a 0.22 μm filter membrane to obtain a cryopreservation composition for stem cells.

[0039] Comparative Example 1

[0040] The difference between this comparative example and Example 2 is that sulfoxide-modified trehalose is used instead of phosphatidylcholine.

[0041] Comparative Example 2

[0042] The difference between this comparative example and Example 2 is that phosphatidylcholine is used instead of sulfoxide to modify trehalose.

[0043] Comparative Example 3

[0044] The difference between this control example and Example 2 is that 10% DMSO is used instead of phosphatidylcholine-free and sulfoxide-modified trehalose.

[0045] Comparative Example 4

[0046] The difference between this comparative example and Example 2 is that ferric nitrate is not added in step S1.

[0047] Performance Testing

[0048] Functional tests were conducted on Examples 1-3 and Control Examples 1-4. The experimental stem cells selected were adipose-derived mesenchymal stem cells. The experiments were divided into 7 groups, each group corresponding to the cryopreservation compositions prepared in Examples 1-3 and Control Examples 1-4. Three parallel tests were performed in each group, and the average value was taken. Adipose-derived mesenchymal stem cells were divided into 1×10 3 cells / mL were suspended in each freezing composition and cryopreserved. After 6 months of cryopreservation, cell viability (using trypan blue staining) and cell proliferation capacity (using a cell counter to measure cell proliferation times within 3 days) were measured.

[0049] Table 1

[0050] Survival rate (%) proliferation multiples Example 1 93.6 7.2 Example 2 95.3 8.5 Example 3 92.4 6.7 Comparative Example 1 89.7 5.1 Comparative Example 2 82.9 4.2 Comparative Example 3 91.7 6.1 Comparative Example 4 90.3 5.6

[0051] As can be seen from Table 1, compared with other control examples, Examples 1-3 of the present application have a higher stem cell survival rate and a greater stem cell proliferation multiple within the same period of time. Examples 1-3 of the present application, especially Example 2, have better stem cell survival rate and proliferation capacity than other examples. That is, the present application, especially Example 2, provides a cryopreservation composition for stem cells and a preparation method thereof that improves the cryopreservation survival rate and proliferation capacity of stem cells. In summary, the best embodiment of the present application is Example 2.

[0052] The above content is merely an example and explanation of the concept of the present application. Technicians in this technical field may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present application.

Claims

1. A method for preparing a cryopreservation composition for stem cells, characterized in that: The following steps are involved: S1. Dissolve thioether-modified trehalose in acetate buffer solution, add ferric nitrate and hydrogen peroxide solution, and react for a period of time to obtain sulfoxide-modified trehalose; S2. Add sulfoxide-modified trehalose and phosphatidylcholine to the pre-cooled basal medium and stir until completely dissolved to obtain a basal solution; S3. Add antioxidant, antibacterial agent, platelet lysate and ferric nitrate to the base solution, stir in the dark, and then filter and sterilize to obtain a cryopreservation composition for stem cells.

2. The method for preparing a cryopreservation composition for stem cells according to claim 1, characterized in that: The final concentration of the sulfoxide-modified trehalose in the freezing composition is 6-10% W / V.

3. The method for preparing a cryopreservation composition for stem cells according to claim 1, characterized in that: The final concentration of the sulfoxide-modified trehalose in the freezing composition is 7-9% W / V.

4. The method for preparing a cryopreservation composition for stem cells according to claim 1, characterized in that: The final concentration of the phosphatidylcholine in the freezing composition is 1.5-2.5% W / V.

5. The method for preparing a composition for freezing stem cells according to claim 1, characterized in that: The basic culture medium is DMEM / F12 or RPMI-1640.

6. The method for preparing a composition for freezing stem cells according to claim 1, characterized in that: The platelet lysate concentration is 15-30% V / V.

7. The method for preparing a composition for cryopreservation of stem cells according to claim 1, characterized in that: The antioxidant is vitamin E or glutathione, wherein the concentration of the antioxidant in the cryopreservation composition is 0.2-0.8 mM.

8. The method for preparing a composition for cryopreservation of stem cells according to claim 1, characterized in that: The antibacterial agent is ε-polylysine, and its concentration in the freezing composition is 0.02-0.1% W / V.

9. The method for preparing a composition for cryopreservation of stem cells according to claim 1, characterized in that: The concentration of the ferric nitrate in the freezing composition is 10-50 μM.

10. A cryopreservation composition obtained by the method for preparing a cryopreservation composition for stem cells according to any one of claims 1 to 9.