WO2020186870A1 - Cryoprotectant and application thereof to umbilical cord mesenchymal stem cell cryopreservation - Google Patents

Abstract

The present invention relates to the technical field of stem cells, and in particular, to a cryoprotectant and an application thereof to umbilical cord mesenchymal stem cell cryopreservation. The composition provided by the present invention can significantly increase the activity of umbilical cord mesenchymal stem cells under cryopreservation conditions, and a stem cell formulation prepared from the composition has a good osteogenic differentiation and chondrogenic differentiation potential, accordingly can have a good cartilage injury repair function, and thus achieves the effect of treating osteoarthritis.

Description

Cryopreservation solution and its application in cryopreservation of umbilical cord mesenchymal stem cells

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on March 21, 2019, the application number is 201910216830.0, and the invention title is "Cryopreservation Solution and Its Application in the Cryopreservation of Umbilical Cord Mesenchymal Stem Cells". The entire content is incorporated into this application by reference.

Technical field

The present invention relates to the technical field of stem cells, in particular to cryopreservation liquid and its application in cryopreservation of umbilical cord mesenchymal stem cells.

Background technique

Osteoarthritis (OA) is a chronic bone and joint disease that seriously endangers human health. It has the largest number of joint diseases, exceeding the sum of other arthritis such as rheumatoid arthritis and ankylosing spondylitis. At the pathological level, osteoarthritis is manifested by the destruction of articular cartilage and the abnormal proliferation of osteophytes, resulting in clinical manifestations such as joint pain, restricted mobility and joint deformities. At the same time, osteoarthritis is the most important cause of the decline in the young and middle-aged labor force, causing a significant economic burden.

Osteoarthritis is characterized by severe, localized cartilage destruction, deep into the bone. Articular cartilage is composed of cartilage cells and extracellular matrix. Since there are no blood vessels, lymphatic vessels and nerve tissues in the cartilage, and cartilage cells have limited proliferation ability and cannot migrate, their self-repair ability is very weak after articular cartilage injury. With aging, a variety of cells in the joints such as chondrocytes, synovial cells, and mesenchymal stem cells undergo cell senescence and functional degradation. Therefore, transplantation of biologically active chondrocytes into the joint can supplement apoptotic cells or resist cell senescence, and cell transplantation can provide a new idea for the treatment of osteoarthritis.

Cell therapy for osteoarthritis has developed rapidly in recent years and has received widespread attention. Umbilical cord-derived mesenchymal stem cells for the treatment of osteoarthritis not only have the advantages of easy expansion and differentiation of mesenchymal stem cells from other sources, anti-inflammatory and recruitment effects, but also large amounts of acquisition, no ethical problems, no pain in the process of obtaining materials, and high proliferation Characteristics, pluripotent differentiation characteristics, low immunogenicity, non-tumorigenicity and other characteristics, are currently the most commonly used seed cells for clinical cell therapy. However, how to improve the viability of umbilical cord mesenchymal stem cells and how to improve the recovery ability of umbilical cord mesenchymal stem cells after cryopreservation are problems that need to be solved urgently.

Summary of the invention

In view of this, the technical problem to be solved by the present invention is to provide cryopreservation solution and its application in cryopreservation of umbilical cord mesenchymal stem cells. The freezing solution can maintain the viability of mesenchymal stem cells during the freezing period.

The cryopreservation liquid provided by the present invention is composed of the following volume parts:

In some embodiments, the cryopreservation liquid of the present invention is composed of the following volume parts of liquid;

In the present invention, the mass fraction of human albumin in the human albumin injection is 20%; the mass fraction of glucose in the glucose injection is 5%; the hydroxyethyl starch in the hydroxyethyl starch injection The mass fraction of starch is 6%.

The composition provided by the present invention is used in a cell cryopreservation solution and can significantly improve the survival rate of cells. After 6 months of freezing, the cell survival rate can still reach more than 95%.

Application of the cryopreservation solution of the present invention in the cryopreservation of umbilical cord mesenchymal stem cells.

The present invention also provides a method for cryopreserving umbilical cord mesenchymal stem cells, which resuspends the umbilical cord mesenchymal stem cells with the cryopreservation solution of the present invention.

In the present invention, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

In the present invention, the resuspension until the cell density is 1×10 ⁶ to 1×10 ⁸ cells/ml.

In some embodiments, the resuspended to a cell density of 5×10 ⁷ cells/mL.

In the present invention, the freezing temperature is -196°C.

In the present invention, after the freezing, the step of resuscitation is also included. The resuscitation conditions are 37°C-42°C.

Application of the cryopreservation liquid of the present invention in the preparation of stem cell preparations.

The present invention also provides a stem cell preparation, which comprises: umbilical cord mesenchymal stem cells and the cryopreservation solution of the present invention.

In the stem cell preparation of the present invention, the density of the umbilical cord mesenchymal stem cells is 1×10 ⁶ to 1×10 ⁸ cells/ml.

In some embodiments, in the stem cell preparation, the density of the umbilical cord mesenchymal stem cells is 5×10 ⁷ cells/mL.

In some embodiments, the stem cell preparation includes: 5×10 ⁷ cells/mL umbilical cord mesenchymal stem cells and cryopreservation fluid; the cryopreservation fluid is composed of the following volumes of liquid;

The stem cell preparation of the present invention is used in the preparation of medicines for treating osteoarthritis.

Studies have shown that the stem cell preparation of the present invention still has a good potential for osteogenic differentiation and cartilage differentiation after cryopreservation. Therefore, it has the effects of treating osteoarthritis and repairing cartilage damage.

The present invention also provides a method for treating osteoarthritis, which is administering the stem cell preparation of the present invention.

The cryopreservation solution provided by the present invention is composed of DMSO, human albumin injection, glucose injection and hydroxyethyl starch injection, which can maintain the activity of umbilical cord mesenchymal stem cells, and the recovered cells still have good The potential of osteogenic differentiation and cartilage differentiation, which can have a good effect of repairing cartilage damage, thereby playing the effect of treating osteoarthritis.

The composition provided by the present invention can significantly improve the activity of umbilical cord mesenchymal stem cells under cryopreservation conditions, and the stem cell preparations prepared therefrom have good osteogenic differentiation and chondrogenic differentiation potential, and thus can have It has a good effect of repairing cartilage damage and thus has the effect of curing osteoarthritis.

Description of the drawings

Figure 1 shows the cell morphology after resuscitation and culture;

Figure 2 shows osteogenic differentiation of cells;

Figure 3 shows the differentiation of cells into cartilage.

detailed description

The present invention provides a cryopreservation solution and its application in the cryopreservation of umbilical cord mesenchymal stem cells. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invent technology.

The test materials used in the present invention are all common commercially available products, which are all available in the market.

Dimethyl sulfoxide (DMSO), liquid at room temperature.

The mass fraction of albumin in the human albumin injection is 20%. That is, every 100 mL of human albumin injection contains 20 g of human albumin.

The mass fraction of glucose in the glucose injection is 5%. That is, every 100mL glucose injection contains 5g glucose.

The mass fraction of hydroxyethyl starch in the hydroxyethyl starch injection is 6%. That is, every 100 mL of hydroxyethyl starch injection contains 6 g of hydroxyethyl starch and 0.9 g of sodium chloride.

The preparation of the umbilical cord mesenchymal stem cells includes:

1. Place the umbilical cord tissue in DMEM/F12 basal medium containing 1% double antibody, soak and transport to the laboratory, wash with normal saline and 75% alcohol to remove residual blood;

2. Cut the umbilical cord tissue into 2-3cm segments, remove the tissue outer membrane and arteries and veins, tear the remaining tissue into strips and cut them to 30-50mm ³ , select the number of inoculation culture dishes according to the length of the umbilical cord, each 3-5mL Inoculate a 15cm dish with the length of the umbilical cord. Use tweezers to take the corresponding tissue block and place it on the culture dish. After the tissue block is fixed on the culture dish, add 10-15ml DMEM/F12 complete medium containing 5-20ng/ml final concentration of EGF , Gently shake the petri dish to make the medium evenly distributed in the petri dish and cultivate in the incubator;

3. Change the medium: When it is observed that there are at least 5 cells that have migrated out of each dish, use a pipette to remove the culture medium and tissue blocks in the dish, and use a pipette to suck up 15-20mL of new DMEM/F12 completely Add the culture medium to a 15 cm petri dish, and shake the petri dish crosswise to make the culture medium evenly distributed in the petri dish. Transfer to a carbon dioxide incubator with 5% CO ₂ , 37°C and a saturated humidity of 95% to continue the cultivation;

4. Passaging: ①When the cell confluence reaches 80%-85%, put the cells in a clean workbench, use a pipette to remove the culture medium in the dish, add 15-20mL of physiological saline to each dish and wash it twice. Add 2-3mL 0.25% trypsin solution to each petri dish, and shake the petri dish from side to side to make the trypsin infiltrate the entire bottom of the dish and incubate for 1-2 min. Observe under an inverted microscope, 80% cells shrink , When it becomes round and floats, quickly move it to the clean workbench, use a pipette to add 5-10ml DMEM/F12 complete medium to terminate the digestion, gently pipette, transfer the cell suspension to the centrifuge tube and sample for counting, after the centrifuge tube is balanced Place in a centrifuge and centrifuge at 500-800g for 5-10 minutes.

②Pour out the supernatant after centrifugation, adjust the cell seeding density to 1-5×10 ⁴ cell/mL with DMEM/F12 complete medium solution according to the cell count results, and use a pipette to draw 15-20 mL of The cell suspension was inoculated into a 15 cm petri dish. Shake the petri dish crosswise to distribute the cell suspension evenly in the petri dish.

The following examples further illustrate the present invention:

Example

1. Take the P3-P5 generation hUC-MSCs, when the cell confluence reaches 80-85%, wash the cells twice with saline, add 2-3ml 0.25% trypsin for 1-2min, add 5-10ml complete medium Stop the digestion, and centrifuge the obtained cell suspension at 500-800g for 5-10min, discard the supernatant;

2. Resuspend the cells in physiological saline, sample and count, centrifuge at 500g-800g for 5-10min, discard the supernatant;

3. Resuspend the cells in the cryopreservation solution shown in Table 1 to a density of 1×10 ⁶ ～1×10 ⁸ cells/ml.

Table 1 Each group plan

Respectively freeze the cells using the protocol described in Table 1, repeat three times for each group, and recover the cells 1 week, 1 month, 3 months, 6 months and 1 year after freezing to detect their viability and compare the results As shown in Table 2;

Table 2 Cell viability

To 1 week 1 month 3 months 6 months 12 months Group 1 98.16% 98.54% 96.47% 96.88% 93.55% Group 2 99.89% 99.15% 97.21% 98.6% 96.22% Group 3 97.15% 97.64% 93.97% 95.11% 91.76% Group 4 99.63% 95.77% 95.26% 89.8% 83.97% Group 5 98.34% 94.56% 95.42% 92.45% 88.4%

The results showed that the cell viability of groups 1 to 3 was higher than that of groups 4 to 5, and there was a significant difference after statistical analysis, p<0.05. Among them, group 2 had the highest cell viability, which was better than the other groups, p<0.05.

4. After the cells were cryopreserved for one week, the cells of group 2 were resuscitated and cultured, and their cell morphology (Figures 1-2) and proliferation speed were recorded by taking pictures.

Table 3 Group 2 cell proliferation rate after resuscitation

Using the 3d count result, substitute the formula DT=t×[lg2/(lgNt-lgNo)] to calculate the doubling time. The calculation method of the cell doubling time of other groups is the same as that of group 2. The results are shown in Table 4.

Table 4 Cell doubling time

Group Doubling time Group 1 35.63h Group 2 28.62h

Group 3 31.5h Group 4 37.17h Group 5 34.37h

The results showed that the cell doubling time of group 2 was significantly better than that of the other groups. After statistical analysis, there was a significant difference, p<0.05.

5. Detection of differentiation effect,

After the cells were cryopreserved for one week, the recovery group 2 was tested for bone differentiation and chondrogenic differentiation

5.1 Osteogenic differentiation

hUC-MSCs were plated, after the cell fusion reached more than 80%, the original medium was discarded, and the osteoinduction medium was added (10% FBS, 5μg/ml insulin, 0.1μM dexamethasone, 0.2mM vitamin C and 10mM β-glycerophosphate). The medium was changed once in 3 days, and the differentiation was 15 days.

After the mineralized nodules are formed, stain with Alizarin Red. Wash with normal saline 3 times, fix with 10% neutral formaldehyde for 20 min, then wash 2 times with normal saline, soak with Alizarin Red dye solution, wash 2 times with normal saline, observe and take pictures under an inverted microscope, showing mineralized nodules.

The cells after cryopreservation and resuscitation were tested for osteogenic differentiation. The induction medium was as described above. After 22 days of induction, it was stained with Alizarin Red. The osteogenic differentiation results of each example and comparative example are shown in Figure 2.

The results showed that hUC-MSCs stained positively after induction, and the cells were stained red, suggesting that hUC-MSCs have the potential to induce differentiation in vitro. Compared with the control group, Example 2 has a higher degree of staining, indicating that the differentiation is better.

5.2 Chondrogenic differentiation

hUC-MSCs were plated, after the cell fusion reached more than 80%, the original medium was discarded and added to chondrogenic induction medium (10% FBS, 10ng/ml transforming growth factor, 50mg/L (left-handed) vitamin C was added to IMDM, 0.1nmol/L dexamethasone, 50mg/mL ITS, 1mmol/L sodium pyruvate, 5.35μg/mL linoleic acid, 1.25ng/mL bovine serum albumin). The medium was changed once in 3 days, and the differentiation was 30 days.

After the cells form a spherical shape, stain with Alcian Blue, wash 3 times with normal saline, fix with 10% neutral formaldehyde for 20 minutes, then wash 2 times with normal saline, counterstain with Mai's hematoxylin for 5 minutes, wash 2 times with normal saline, observe under an inverted microscope Take pictures.

The cryopreserved cells were tested for chondrogenic differentiation. The induction medium was as described above. After 30 days of induction, it was stained with alcian blue. The chondrogenic differentiation results of each example and comparative example are shown in Figure 3.

The results showed that hUC-MSCs stained positively after chondrogenesis induction, and bright blue proteoglycan particles appeared in the cytoplasm of the cells, indicating that hUC-MSCs had the potential to differentiate into chondrocytes in vitro. Compared with the control group, Example 2 has a higher degree of staining, indicating that the differentiation is better.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be considered This is the protection scope of the present invention.

Claims (10)

A kind of cryopreservation liquid, which is composed of the following volume parts:

The cryopreservation liquid according to claim 1, characterized in that it is composed of the following volume parts of liquid;

The cryopreservation liquid according to claim 1 or 2, characterized in that: The mass fraction of human albumin in the human albumin injection is 20%; The mass fraction of glucose in the glucose injection is 5%; The mass fraction of hydroxyethyl starch in the hydroxyethyl starch injection is 6%. The use of the cryopreservation solution of any one of claims 1 to 3 in the cryopreservation of umbilical cord mesenchymal stem cells. A method for cryopreservation of umbilical cord mesenchymal stem cells, characterized in that the umbilical cord mesenchymal stem cells are resuspended with the cryopreservation solution of any one of claims 1 to 3. The cryopreservation method according to claim 5, wherein the resuspended to a cell density of 5×10 ⁷ cells/mL. Use of the cryopreservation liquid of any one of claims 1 to 3 in the preparation of stem cell preparations. A stem cell preparation comprising: umbilical cord mesenchymal stem cells and the cryopreservation liquid according to any one of claims 1 to 3. The stem cell preparation according to claim 8, wherein the density of the umbilical cord mesenchymal stem cells is 5×10 ⁷ cells/mL. The use of the stem cell preparation according to any one of claims 7 to 9 in the preparation of a medicine for treating osteoarthritis.

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