Mesenchymal stem cell storage solution as well as preparation and use methods thereof

Abstract

The invention relates to the technical field of mesenchymal stem cells, in particular to mesenchymal stem cell storage liquid as well as a preparation method and a use method thereof. The stock solution comprises propylene glycol, corn glycopeptide and a basic culture medium, the basic culture medium is a DMEM / F12 culture medium, and supplemental factors are added into the basic culture medium; in the process, propylene glycol serves as a protective agent and can permeate into the cells, mechanical damage of ice crystals to the cells during cryopreservation is avoided, meanwhile, corn glycopeptide can be combined with water in the storage liquid and can protect the cells outside the cells, and besides, the storage liquid can be used for storing the cells. The corn glycopeptide and the basal culture medium can also provide nutrient substances normally required before cell freezing and after cell recovery, the supplementary factors can regulate and control the activity of the cells and ensure good activity of the cells, in addition, the permeability of cell membranes can be effectively improved by applying an electromagnetic field in the cryopreservation process and cooperating with calcium ions and the like in the basal culture medium, and the cryopreservation effect of the cells is improved. And better permeation of propylene glycol is facilitated, so that the cryopreservation effect is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cells, and in particular to a mesenchymal stem cell storage solution and a preparation and use method thereof. Background Art

[0002] Mesenchymal stem cells are an important member of the stem cell family. They originate from the early developmental mesoderm and are pluripotent stem cells. Mesenchymal stem cells were originally discovered in the bone marrow. They are increasingly attracting attention because of their multidirectional differentiation potential, hematopoietic support, promotion of stem cell engraftment, immune regulation and self-replication.

[0003] Under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into a variety of tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelium, etc. They still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathology. The existing cell culture method is used to culture and proliferate mesenchymal stem cells, but in actual research and application, it is often necessary to store the cultured and proliferated stem cells to facilitate research and application; this process is often achieved by cell cryopreservation.

[0004] Storage fluid, also known as freezing fluid, is needed during cell storage. Storage fluid can protect cells during cell freezing to prevent them from being mechanically damaged by ice crystals, thereby ensuring the activity of cells after recovery. There are many existing freezing fluids, but most of them have complex components that are inconsistent with the normal growth environment of cells. At the same time, in order to improve the permeability of small molecules such as dimethyl sulfoxide and glycerol, and to protect cells extracellularly with sucrose and glucose, a large number of complex components are often added to reduce the damage to cells during freezing. However, too many components have a serious impact on the activity of cells, which makes the cells often less active after recovery, causing inconvenience to practical applications. Summary of the invention

[0005] The purpose of the present invention is to provide a mesenchymal stem cell storage solution and a preparation and use method thereof, so as to solve the problem that the cell activity is seriously affected when the existing cells are revived after being frozen.

[0006] To achieve the above object, the present invention adopts the following technical scheme: a mesenchymal stem cell storage solution, comprising the following components by mass: 5-10 parts of propylene glycol, 10-20 parts of corn glycopeptide and 40-60 parts of basal culture medium.

[0007] Furthermore, the corn glycopeptide is a product of covalent bonding of low molecular weight corn peptide obtained by protease hydrolysis of zein and D-glucosamine.

[0008] Furthermore, the basic culture medium is DMEM / F12 culture medium; DMEM / F12 serum-free culture medium is a mixture of DMEM culture medium and F12 culture medium in a volume ratio of 1:1, and the DMEM culture medium is a DMEM low-glucose culture medium.

[0009] Furthermore, supplementary factors are added to the basal culture medium, including leukemia inhibitory factor, insulin and platelet-derived growth factor, the concentration of leukemia inhibitory factor is 10-50 ng / mL, the concentration of insulin is 10-20 μg / mL, and the concentration of platelet-derived growth factor is 20-30 μg / mL.

[0010] The method for preparing a mesenchymal stem cell storage solution comprises the following steps:

[0011] S1. Sterilize the clean bench and required consumables with ultraviolet light;

[0012] S2, taking DMEM / F12 medium, adding various supplementary factors thereto according to the concentration, to obtain a basal medium after the supplementary factors are added;

[0013] S3, adding propylene glycol to the basal culture medium obtained in S1 and stirring to mix, and then adding corn glycopeptide and stirring to dissolve, to obtain a stock solution;

[0014] S4. Filter the stock solution obtained in S3 to obtain the storage solution, and refrigerate it for later use.

[0015] Furthermore, the ultraviolet lamp sterilization time is 20 to 40 minutes, filtration is performed using a 0.22 μm organic filter membrane, and the refrigeration temperature is 4°C.

[0016] The method for using the mesenchymal stem cell storage solution comprises the following steps:

[0017] S1, cells in the logarithmic growth phase after incubation and centrifugation, adding storage solution to adjust the cell density;

[0018] S2, treating the conditioned cells with a pulsed electromagnetic field;

[0019] S3. The cells treated with S2 are divided into cryopreservation tubes for cryopreservation.

[0020] Furthermore, in the S1, after adding the storage solution, the cell density is adjusted to 3-8×10 6 Pieces / mL.

[0021] Furthermore, in the S2, the frequency of the pulse electromagnetic field is 50-100 Hz, the intensity is 1-10 mT, and the treatment time is 10-30 min.

[0022] Furthermore, in the S3, the freezing procedure is: cooling to 4°C at a cooling rate of -1°C / min and maintaining for 10 minutes; then when the temperature is greater than -25°C, the cooling rate is -1°C / min; when the temperature is less than or equal to -25°C, the cooling rate is -5°C / min; when the temperature is lower than -80°C, it can be directly put into liquid nitrogen.

[0023] Beneficial effects of the present invention:

[0024] 1. The storage solution of the present invention does not contain dimethyl sulfoxide, has low biological toxicity, and will not cause serious toxicity to cells after cell recovery, which is beneficial to ensure the activity of cells;

[0025] 2. The present invention uses corn glycopeptide to protect cells outside the cell. Corn glycopeptide can bind water, which helps to reduce the mechanical damage of ice crystals. While protecting the cells, it can also provide the necessary nutrients for the cells before and after freezing and recovery, and also plays a positive role in ensuring cell activity;

[0026] 3. The present invention uses a pulsed electromagnetic field to treat cells before freezing. During the process, the pulsed electromagnetic field improves the permeability of the cell membrane. At the same time, combined with calcium ions in the basal culture medium, the permeability of the cell membrane can be further improved, thereby facilitating the penetration of propylene glycol, thereby enhancing the protective effect of propylene glycol on the cell during the freezing process, and also playing a positive and beneficial role in ensuring cell activity. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0028] Example 1

[0029] Preparation of stock solution:

[0030] In a sterilized clean bench, DMEM low-glucose medium was taken and mixed evenly with F12 medium at a ratio of 1:1 to obtain a basal medium, and leukemia inhibitory factor, insulin and platelet-derived growth factor were added to the basal medium to make the concentration of leukemia inhibitory factor 30 ng / mL, the concentration of insulin 15 μg / mL, and the concentration of platelet-derived growth factor 25 μg / mL.

[0031] Take 50 parts of the basic culture medium obtained above by weight, add 8 parts of propylene glycol thereto, stir and mix, then add 15 parts of corn glycopeptide thereto, stir and dissolve thoroughly to obtain a storage solution, filter the storage solution through a 0.22 μm organic filter membrane, and then seal it in a refrigerator at 4°C for later use.

[0032] In this embodiment, the preparation method of the product of low molecular weight corn peptide covalently bound to D-glucosamine obtained by protease hydrolysis of corn glycopeptide zein is prior art and will not be described in detail.

[0033] Cryopreservation of Mesenchymal Stem Cells:

[0034] Take umbilical cord mesenchymal stem cells in the logarithmic growth phase, remove the old culture medium, rinse with PBS buffer, add 37℃ trypsin for digestion, add trypsin inhibitor to terminate digestion, centrifuge, remove the supernatant, resuspend in the prepared storage solution, and blow evenly to adjust the cell density in the storage solution to 3-8×10 6 Pieces / mL.

[0035] The conditioned cells were treated with a pulsed electromagnetic field with a frequency of 50 Hz, an intensity of 5 mT and a treatment time of 20 min.

[0036] The cells after the above treatment were divided into cryopreservation tubes, and 1.5 mL was added to each tube; the cells were cryopreserved using a cooling device, and the cryopreservation program was set as follows: the temperature was cooled to 4°C at a cooling rate of -1°C / min and maintained for 10 minutes; then when the temperature was greater than -25°C, the cooling rate was -1°C / min; when the temperature was less than or equal to -25°C, the cooling rate was -5°C / min; when the temperature was lower than -80°C, the cells could be directly put into liquid nitrogen.

[0037] Resuscitation of mesenchymal stem cells:

[0038] Take the cells that have been frozen for three months and immerse them directly in a circulating water system at 37°C. Shake them continuously during the process to melt the cells as quickly as possible.

[0039] After the cells thawed, take out the cryopreservation tube, transfer the cell suspension to a centrifuge tube, add 20 mL of complete medium (DMEM medium + 10% fetal bovine serum + 1% double antibody), centrifuge, remove the supernatant, add complete medium to resuspend the cells, and adjust the cell density to 8000 cells / cm 2 Inoculate into culture medium and continue culturing at 37°C.

[0040] The cell activity was detected 24 hours after the start of culture, and the results are shown in the following table:

[0041] Table 1 Cell activity after resuscitation in Example 1

[0042]

[0043] It can be seen that the cell storage solution of the present invention has good activity after cell recovery, and the cell activity can reach more than 98%, which is convenient for application and promotion.

[0044] Example 2

[0045] In this comparative example, leukemia inhibitory factor, insulin and platelet-derived growth factor were not added to the basal culture medium, and other steps were the same as those in Example 1.

[0046] After the final recovery, the cell activity was tested at 24h, and the results are shown in the following table:

[0047] Table 2 Cell activity after recovery in Example 2

[0048]

[0049] It can be seen that although cells with good activity can be obtained in this example, they are obviously inferior to those in Example 1.

[0050] Comparative Example 1

[0051] The corn glycopeptide in Example 1 was replaced by sucrose and glucose, and the ratio of sucrose to glucose was 1:1. Others were the same as in Example 1.

[0052] After the final recovery, the cell activity was tested at 24h, and the results are shown in the following table:

[0053] Table 3 Comparative Example 1 Cell Activity after Recovery

[0054]

[0055]

[0056] It can be seen that the effect of using sucrose and glucose is obviously inferior to the corn glycopeptide of the present invention. On the one hand, not only is the activity significantly lower than Example 1 of the present invention, but on the other hand, the cell activity fluctuates greatly and is not stable, indicating that the stability is poor in actual operation and is easily affected by the external environment.

[0057] Comparative Example 2

[0058] The corn glycopeptide in Example 1 was replaced by corn peptide, and the rest was the same as Example 1.

[0059] After the final recovery, the cell activity was tested at 24h, and the results are shown in the following table:

[0060] Table 4 Comparative Example 2 Cell Activity after Recovery

[0061]

[0062] It can be seen that the effect of using corn peptide is obviously inferior to the corn glycopeptide of the present invention.

[0063] Comparative Example 3

[0064] The corn glycopeptide in Example 1 was replaced with corn peptide + glucose, and the ratio of corn peptide to glucose was 1:1. Others were the same as in Example 1.

[0065] After the final recovery, the cell activity was tested at 24h, and the results are shown in the following table:

[0066] Table 5 Comparative Example 3 Cell Activity after Recovery

[0067]

[0068] It can be seen that the effect of using corn peptide + glucose is better than that of comparative example 2, but obviously not as good as that of corn glycopeptide in Example 1 of the present invention.

[0069] Comparative Example 4

[0070] In this comparative example, the basal culture medium is completely DMEM serum-free culture medium, and the rest is the same as in Example 1.

[0071] After the final recovery, the cell activity was tested at 24h, and the results are shown in the following table:

[0072] Table 6 Comparative Example 4 Cell Activity after Recovery

[0073]

[0074] It can be seen that when DMEM medium is used exclusively, although other conditions remain unchanged, the final cell activity is significantly reduced, which is probably due to the excessively high calcium ion concentration in the DMEM medium.

[0075] Comparative Example 5

[0076] In this comparative example, no pulsed electromagnetic field treatment was performed during the cell freezing stage, and the rest was the same as in Example 1.

[0077] After the final recovery, the cell activity was tested at 24h, and the results are shown in the following table:

[0078] Table 7 Comparative Example 5 Cell Activity after Recovery

[0079]

[0080] It can be seen that when the pulsed electromagnetic field is not applied for treatment, the final cell activity is also significantly lower than that in Example 1, which shows that the pulsed electromagnetic field plays a positive and beneficial role in the penetration of propylene glycol.

[0081] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. A mesenchymal stem cell storage solution, characterized in that: The invention comprises the following components by weight: 5 to 10 parts of propylene glycol, 10 to 20 parts of corn glycopeptide and 40 to 60 parts of basic culture medium.

2. The mesenchymal stem cell storage solution according to claim 1, characterized in that: The corn glycopeptide is a product of covalent bonding of low-molecular-weight corn peptide obtained by protease hydrolysis of zein and D-glucosamine.

3. The mesenchymal stem cell storage solution according to claim 1, characterized in that: The basic culture medium is DMEM / F12 culture medium; DMEM / F12 serum-free culture medium is prepared by mixing DMEM culture medium and F12 culture medium in a volume ratio of 1:1, and the DMEM culture medium is a DMEM low-glucose culture medium.

4. The mesenchymal stem cell storage solution according to claim 3, characterized in that: The basic culture medium is added with supplementary factors, which include leukemia inhibitory factor, insulin and platelet-derived growth factor. The concentration of leukemia inhibitory factor is 10-50 ng / mL, the concentration of insulin is 10-20 μg / mL, and the concentration of platelet-derived growth factor is 20-30 μg / mL.

5. The method for preparing the mesenchymal stem cell storage solution according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Sterilize the clean bench and required consumables with ultraviolet light; S2, taking DMEM / F12 medium, adding various supplementary factors thereto according to the concentration, to obtain a basal medium after the supplementary factors are added; S3, adding propylene glycol to the basal culture medium obtained in S1 and stirring to mix, and then adding corn glycopeptide and stirring to dissolve, to obtain a stock solution; S4. Filter the stock solution obtained in S3 to obtain the storage solution, and refrigerate it for later use.

6. The method for preparing the mesenchymal stem cell storage solution according to claim 5, characterized in that: The ultraviolet lamp sterilization time is 20 to 40 minutes, 0.22 μm organic filter membrane is used for filtration, and the refrigeration temperature is 4°C.

7. The method for using the mesenchymal stem cell storage solution according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, cells in the logarithmic growth phase after incubation and centrifugation, adding storage solution to adjust the cell density; S2, treating the conditioned cells with a pulsed electromagnetic field; S3. The cells treated with S2 are divided into cryopreservation tubes for cryopreservation.

8. The method for using the mesenchymal stem cell storage solution according to claim 7, characterized in that: In the S1, after adding the storage solution, the cell density was adjusted to 3-8×10 6 Pieces / mL.

9. The method for using the mesenchymal stem cell storage solution according to claim 7, characterized in that: In the S2, the frequency of the pulse electromagnetic field is 50-100 Hz, the intensity is 1-10 mT, and the treatment time is 10-30 min.

10. The method for using the mesenchymal stem cell storage solution according to claim 7, characterized in that: In the S3, the freezing procedure is: cooling to 4°C at a cooling rate of -1°C / min and maintaining for 10 minutes; then when the temperature is greater than -25°C, the cooling rate is -1°C / min; when the temperature is less than or equal to -25°C, the cooling rate is -5°C / min; when the temperature is lower than -80°C, it can be directly put into liquid nitrogen.

Images