Ready-to-use activated human umbilical cord mesenchymal stem cells as well as preparation method and application thereof

Abstract

The invention discloses a ready-to-use activated human umbilical cord mesenchymal stem cell as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out passage on P0-generation cells for three times to obtain P3-generation cells; cryopreserving P3 generation cells, and establishing a cell seed bank; resuscitating the cells in the seed bank for three times of passage to obtain P6-generation cells; and carrying out activation culture on the P6-generation cells, and resuspending the activated P6-generation cells in a cell protective agent to obtain the human umbilical cord mesenchymal stem cell original suspension. The invention also discloses an umbilical cord mesenchymal stem cell cryopreservation preparation. The cryopreservation preparation is obtained by mixing the human umbilical cord mesenchymal stem cell original suspension with a cryopreservation protective agent, sub-packaging and cryopreserving. Experiments prove that the human umbilical cord mesenchymal stem cell cryopreserved preparation prepared by the invention can be stored for a long time, is convenient to store, transport and use clinically, can complete effectiveness and safety index detection within the shelf life, and has the same activity as a fresh preparation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to ready-to-use activated human umbilical cord mesenchymal stem cells and a preparation method and application thereof. Background Art

[0002] Mesenchymal stromal / stem cells (MSCs) are a type of multipotent stem cell derived from the mesoderm and are found in various tissues of the human body, such as bone marrow, umbilical cord blood, peripheral blood, umbilical cord, and placenta. Because of their multiple biological characteristics, such as self-renewal, multidirectional differentiation, and immune regulation, and in particular, because they do not express MHCⅡ and costimulatory molecules CD80 and CD86, they have low immunogenicity and do not cause immune rejection when transplanted between allogeneic donors. MSCs can be used to treat a variety of intractable diseases and have great clinical application prospects. Their possible mechanisms of action are: (1) in vivo expansion and differentiation replacement; (2) affecting surrounding cells through intercellular contact and / or secretion of cytokines or extracellular vesicles; and (3) apoptosis of transplanted MSCs, which, during the process of efferocytosis, induces macrophage differentiation into the suppressive phenotype M2, exerting an immunoregulatory effect.

[0003] Despite its enormous potential for clinical application, several technical challenges remain to be addressed during industrialization. The first is obtaining batch-to-batch stable MSCs. Effectively evaluating clinical research data requires the use of batch-to-batch stable MSCs. MSC isolation and in vitro expansion methods are among the factors influencing batch-to-batch stability, and research and development in these areas is fundamental to achieving batch-to-batch stable MSCs. Secondly, to facilitate clinical use, research into dosage forms and formulations is essential. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing human umbilical cord mesenchymal stem cells.

[0005] The method for preparing human umbilical cord mesenchymal stem cells provided by the present invention comprises the following steps:

[0006] (1) digesting and screening the isolated umbilical cord tissue pieces and then culturing them until the cell confluence reaches 60%-80%, thereby obtaining P0 mesenchymal stem cells;

[0007] (2) subculturing the P0 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P1 generation mesenchymal stem cells;

[0008] (3) subculturing the P1 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P2 generation mesenchymal stem cells;

[0009] (4) subculturing the P2 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P3 generation mesenchymal stem cells;

[0010] (5) freezing the P3 mesenchymal stem cells to obtain a cell seed bank;

[0011] (6) resuscitating the cells from the cell seed bank and performing subculture until the cell confluence reaches 70%-90%, thereby obtaining P4 mesenchymal stem cells;

[0012] (7) subculturing the P4 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P5 generation mesenchymal stem cells;

[0013] (8) subculturing the P5 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P6 generation mesenchymal stem cells;

[0014] (9) activating and culturing the P6 generation mesenchymal stem cells to obtain activated P6 generation mesenchymal stem cells; the activation culture medium does not contain serum and contains ascorbic acid, α-tocopheryl acetate, vitamin A, palmitic acid, reduced glutathione, and 4-hydroxyethylpiperazineethanesulfonic acid;

[0015] (10) Resuspending the activated P6 mesenchymal stem cells in a cell protectant to obtain the human umbilical cord mesenchymal stem cell original suspension.

[0016] In the above method, in step (1), the digestion and screening may include the following steps: cutting the isolated umbilical cord into 3-9 mm 3 The tissue blocks are then digested with collagenase (e.g., digested at 37°C for 30-50 min), filtered after digestion, the filtrate below the filter is collected, added to complete culture medium and mixed to obtain mixed solution 1; the filtrate above the filter is collected and added to a pancreatic enzyme substitute for secondary digestion (e.g., digested at 37°C for 30-50 min), filtered after digestion, the filtrate below the filter is collected, added to complete culture medium and mixed to obtain mixed solution 2; mixed solution 1 and mixed solution 2 are combined, washed with phosphate buffer and filtered, the filtrate below the filter is collected and centrifuged (e.g., centrifuged at 2800-3200g for 10 minutes), the supernatant is discarded, complete culture medium is added to the cell pellet, resuspended, and then inoculated into a cell culture flask for culture to obtain primary cells.

[0017] In the above method, in step (2), the subculture may include the following steps: discarding the supernatant, adding phosphate buffer for washing, and adding pancreatic enzyme substitute (such as 0.01-0.03 mL / cm 2) for digestion; after digestion, phosphate buffer was added for washing; after washing, centrifugation was performed (e.g., 300-500g for 5 minutes), the supernatant was discarded, the cells were resuspended in complete culture medium, passaged at a ratio of 1:3, and cultured after replenishing complete culture medium until the cell confluence reached 70%-90% to obtain P1 mesenchymal stem cells.

[0018] In the above method, in step (3), the subculture may include the following steps: discarding the supernatant, adding phosphate buffer for washing, and adding pancreatic enzyme substitute (such as 0.01-0.03 mL / cm 2 ) for digestion; after digestion, phosphate buffer was added for washing; after washing, centrifugation was performed (e.g., 800-1200g for 5 minutes), the supernatant was discarded, the cells were resuspended in complete culture medium, passaged at a ratio of 1:3, and cultured after replenishing complete culture medium until the cell confluence reached 70%-90% to obtain P2 mesenchymal stem cells.

[0019] In the above method, in step (4), the subculture may include the following steps: discarding the supernatant, adding phosphate buffer for washing, and adding pancreatic enzyme substitute (such as 0.01-0.03 mL / cm 2 ) for digestion; after digestion, phosphate buffer was added for washing; after washing, centrifugation was performed (e.g., 800-1200g for 5 minutes), the supernatant was discarded, the cells were resuspended in complete culture medium, passaged at a ratio of 1:3, and cultured after replenishing complete culture medium until the cell confluence reached 70%-90% to obtain P3 mesenchymal stem cells.

[0020] In the above method, in step (5), the step of resuspending the P3 mesenchymal stem cells in a cryopreservation protection solution to obtain a cell suspension is further included before the cryopreservation.

[0021] In some embodiments, the cryopreservation solution is composed of a serum substitute and dimethyl sulfoxide, and the volume ratio of the serum substitute to the dimethyl sulfoxide is preferably 9:1.

[0022] In some embodiments, the concentration of the cell suspension is (2-4)×10 6 cells / mL, preferably 3.5×10 6 cells / mL.

[0023] In some embodiments, the cryopreservation method may include the following steps: dispensing the cell suspension into cryovials, placing the cryovials into cryotubes, placing the cryotubes into cryoboxes, placing the cryoboxes into a cryorack, and placing them in a -80°C ultra-low temperature freezer for 24-72 hours before transferring them to liquid nitrogen. Specifically, after one month of storage in liquid nitrogen, a sterility test is performed; cells that pass the test are designated as cell seed banks.

[0024] In some embodiments, the step (5) may include the following steps: washing (phosphate buffer washing), digesting (trypsin substitute digestion) and centrifuging (e.g., 800-1200g centrifugation for 5 minutes) the P3 mesenchymal stem cells in sequence, and then resuspending them in a cryopreservation protection solution to obtain a cell suspension; and freezing the cell suspension.

[0025] In the above method, in step (6), the subculture may include the following steps: resuscitating the cell seed bank cells, then adding complete culture medium and mixing them, and then centrifuging (such as centrifugation at 300-500g for 5 minutes), discarding the supernatant, and then resuspending the cells with complete culture medium and inoculating them into a cell culture flask. After replenishing the complete culture medium, culturing is carried out until the cell confluence reaches 70%-90%, and P4 mesenchymal stem cells are obtained.

[0026] In the above method, in step (7), the subculture may include the following steps: discarding the supernatant, adding phosphate buffer for washing, and adding pancreatic enzyme substitute (such as 0.01-0.03 mL / cm 2 ) for digestion; after digestion, phosphate buffer was added for washing; after washing, centrifugation was performed (e.g., 800-1200g for 5 minutes), the supernatant was discarded, the cells were resuspended in complete culture medium, passaged at a ratio of 1:3, and cultured after replenishing complete culture medium until the cell confluence reached 70%-90% to obtain P5 mesenchymal stem cells.

[0027] In the above method, in step (8), the subculture may include the following steps: discarding the supernatant, adding phosphate buffer for washing, and adding pancreatic enzyme substitute (such as 0.01-0.03 mL / cm 2 ) for digestion; after digestion, phosphate buffer was added for washing; after washing, centrifugation was performed (e.g., 800-1200g for 5 minutes), the supernatant was discarded, the cells were resuspended in complete culture medium, passaged at a ratio of 1:3, and cultured after replenishing complete culture medium until the cell confluence reached 70%-90% to obtain P6 mesenchymal stem cells.

[0028] In the above method, in step (9), the activation culture may include the following steps: discarding the supernatant, adding phosphate buffer for washing, and adding activation culture medium (such as 0.2-0.3 mL / cm 2 ) to obtain activated P6 mesenchymal stem cells. The activation culture conditions are preferably 37° C., 5% CO 2 culturing for 16-48 hours.

[0029] Furthermore, the concentration of ascorbic acid in the activation medium may be 45-55 mg / L, 45-50 mg / L, or 50-55 mg / L. In some embodiments, the concentration of ascorbic acid in the activation medium is 45 mg / L, 50 mg / L, or 55 mg / L, preferably 50 mg / L.

[0030] The concentration of the α-tocopheryl acetate in the activation medium may be 0.3-0.5 mg / L, 0.3-0.4 mg / L, or 0.4-0.5 mg / L. In some embodiments, the concentration of the α-tocopheryl acetate in the activation medium is 0.3 mg / L, 0.4 mg / L, or 0.5 mg / L, preferably 0.4 mg / L.

[0031] The concentration of vitamin A in the activation medium may be 1.0-1.5 mg / L or 1.0-1.38 mg / L or 1.38-1.5 mg / L. In some embodiments, the concentration of vitamin A in the activation medium is 1.0 mg / L or 1.38 mg / L or 1.5 mg / L, preferably 1.38 mg / L.

[0032] The concentration of palmitic acid in the activation medium may be 4.5-5.5 mg / L or 4.5-5.13 mg / L or 5.13-5.5 mg / L. In some embodiments, the concentration of palmitic acid in the activation medium is 4.5 mg / L or 5.13 mg / L or 5.5 mg / L.

[0033] The concentration of the reduced glutathione in the activation medium may be 0.5-0.7 mg / L or 0.5-0.62 mg / L or 0.62-0.7 mg / L. In some embodiments, the concentration of the reduced glutathione in the activation medium is 0.5 mg / L or 0.62 mg / L or 0.7 mg / L.

[0034] The concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the activation medium may be 3200-3900 mg / L, or 3200-3580 mg / L, or 3580-3900 mg / L. In some embodiments, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the activation medium is 3200 mg / L, or 3580 mg / L, or 3900 mg / L.

[0035] Furthermore, the activation medium also contains glycine, alanine, glutamine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, vitamin H, choline chloride, calcium pantothenate, folic acid, nicotinamide, vitamin B6, vitamin B2, vitamin B1, vitamin B12, inositol (inactive inositol), calcium chloride, copper sulfate, ferric nitrate, ferric sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, zinc sulfate, D-glucose, hypoxanthine, linoleic acid, lipoic acid, tetramethylethylenediamine, sodium pyruvate, and thymidine.

[0036] The concentration of glycine in the activation medium is 18.75 mg / L.

[0037] The concentration of alanine in the activation medium is 4.45 mg / L.

[0038] The concentration of glutamine in the activation medium was 542 mg / L.

[0039] The concentration of arginine in the activation medium is 147.5 mg / L.

[0040] The concentration of asparagine in the activation medium is 7.5 mg / L.

[0041] The concentration of aspartic acid in the activation medium is 6.65 mg / L.

[0042] The concentration of cysteine ​​in the activation medium is 35.12 mg / L.

[0043] The concentration of cystine in the activation medium is 31.29 mg / L.

[0044] The concentration of glutamic acid in the activation medium is 7.35 mg / L.

[0045] The concentration of the histidine in the activation medium is 31.48 mg / L.

[0046] The concentration of isoleucine in the activation medium is 54.47 mg / L.

[0047] The concentration of leucine in the activation medium is 59.05 mg / L.

[0048] The concentration of lysine in the activation medium is 91.25 mg / L.

[0049] The concentration of methionine in the activation medium is 17.24 mg / L.

[0050] The concentration of phenylalanine in the activation medium is 35.48 mg / L.

[0051] The concentration of proline in the activation medium is 17.25 mg / L.

[0052] The concentration of serine in the activation medium is 26.25 mg / L.

[0053] The concentration of threonine in the activation medium is 53.45 mg / L.

[0054] The concentration of tryptophan in the activation medium was 9.02 mg / L.

[0055] The concentration of tyrosine in the activation medium is 55.79 mg / L.

[0056] The concentration of valine in the activation medium is 52.85 mg / L.

[0057] The concentration of the vitamin H in the activation medium is 0.0035 mg / L.

[0058] The concentration of the choline chloride in the activation medium is 8.98 mg / L.

[0059] The concentration of the calcium pantothenate in the activation medium is 2.24 mg / L.

[0060] The concentration of folic acid in the activation medium is 2.65 mg / L.

[0061] The concentration of the nicotinamide in the activation medium is 2.02 mg / L.

[0062] The concentration of vitamin B6 in the activation medium is 2.031 mg / L.

[0063] The concentration of vitamin B2 in the activation culture medium is 0.219 mg / L.

[0064] The concentration of vitamin B1 in the activation medium is 2.17 mg / L.

[0065] The concentration of vitamin B12 in the activation medium is 0.68 mg / L.

[0066] The concentration of the optically inactive inositol in the activation medium is 12.6 mg / L.

[0067] The concentration of calcium chloride in the activation medium is 116.6 mg / L.

[0068] The concentration of the copper sulfate in the activation medium is 0.0013 mg / L.

[0069] The concentration of the ferric nitrate in the activation medium is 0.05 mg / L.

[0070] The concentration of the ferric sulfate in the activation medium is 0.417 mg / L.

[0071] The concentration of the magnesium chloride in the activation medium is 28.64 mg / L.

[0072] The concentration of the magnesium sulfate in the activation medium is 48.84 mg / L.

[0073] The concentration of potassium chloride in the activation medium is 311.8 mg / L.

[0074] The concentration of the sodium bicarbonate in the activation medium is 4876 mg / L.

[0075] The concentration of sodium chloride in the activation medium is 6999.5 mg / L.

[0076] The concentration of the sodium hydrogen phosphate in the activation medium is 71.02 mg / L.

[0077] The concentration of the sodium dihydrogen phosphate in the activation medium is 62.5 mg / L.

[0078] The concentration of zinc sulfate in the activation medium is 0.432 mg / L.

[0079] The concentration of D-glucose in the activation medium is 3151 mg / L.

[0080] The concentration of the hypoxanthine in the activation medium is 2.39 mg / L.

[0081] The concentration of the linoleic acid in the activation medium is 0.042 mg / L.

[0082] The concentration of the lipoic acid in the activation medium is 0.105 mg / L.

[0083] The concentration of the tetraethylenediamine in the activation medium is 0.081 mg / L.

[0084] The concentration of sodium pyruvate in the activation medium is 165 mg / L.

[0085] The concentration of thymine in the activation medium is 0.365 mg / L.

[0086] Furthermore, the activation medium is composed of a solvent and a solute, the solvent is water, and the solutes and their concentrations are as follows: glycine 18.75 mg / L, alanine 4.45 mg / L, glutamine 542 mg / L, arginine 147.5 mg / L, asparagine 7.5 mg / L, aspartic acid 6.65 mg / L, cysteine ​​35.12 mg / L, cystine 31.29 mg / L, glutamic acid 7.35 mg / L, histidine 31.48mg / L, Isoleucine 54.47mg / L, Leucine 59.05mg / L, Lysine 91.25mg / L, Methionine 17.24mg / L, Phenylalanine 35.48mg / L, Proline 17.25mg / L, Serine 26.25mg / L, Threonine 53.45mg / L, Tryptophan 9.02mg / L, Tyrosine 55.79mg / L, Valine 52.85mg / L, Vitamin H 0.0035mg / L, Choline chloride 8.98mg / L, Calcium pantothenate 2.24mg / L, Folic acid 2.65mg / L, Nicotinamide 2.02mg / L, Vitamin B6 2.031mg / L, Vitamin B2 0.219mg / L, Vitamin B1 2.17mg / L, Vitamin B12 0.68mg / L, inositol 12.6mg / L, ascorbic acid 50mg / L, α-tocopheryl acetate 0.4mg / L, vitamin A 1.38mg / L, calcium chloride 116.6mg / L, copper sulfate 0.0013mg / L, ferric nitrate 0.05mg / L, ferric sulfate 0.417mg / L, magnesium chloride 28.64mg / L, magnesium sulfate 48.84mg / L, potassium chloride 311.8mg / L, sodium bicarbonate 4876mg / L, sodium chloride 6999.5mg / L, disodium hydrogen phosphate 7 1.02 mg / L, sodium dihydrogen phosphate 62.5 mg / L, zinc sulfate 0.432 mg / L, D-glucose 3151 mg / L, hypoxanthine 2.39 mg / L, linoleic acid 0.042 mg / L, palmitic acid 5.13 mg / L, lipoic acid 0.105 mg / L, tetramethylethylenediamine 0.081 mg / L, sodium pyruvate 165 mg / L, thymine 0.365 mg / L, reduced glutathione 0.62 mg / L, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) 3580 mg / L. In the above method, the following steps are further included between step (9) and step (10): discarding the supernatant, adding phosphate buffer for washing, and adding pancreatic enzyme substitute (such as 0.01-0.03 mL / cm 2 ) for digestion; after digestion, add cell protectant and centrifuge (such as 800-1200g centrifugation for 5 minutes).

[0087] In the above method, in step (10), the cell protectant consists of human serum albumin solution and compound electrolyte injection.

[0088] In some embodiments, the final concentration (mass fraction) of the human serum albumin in the cell protectant may be 0.5%-1%, and the final concentration (mass fraction) of the human serum albumin in the cell protectant is preferably 0.5% or 1%.

[0089] In the above method, complete culture medium is used for cell culture in steps (1), (2), (3), (4), (6), (7) and (8).

[0090] In some embodiments, the complete culture medium consists of DMEM / F12 basal medium and serum replacement. The volume ratio of the DMEM / F12 basal medium to the serum replacement is preferably 9:1.

[0091] The human umbilical cord mesenchymal stem cells prepared according to the above method also fall within the protection scope of the present invention.

[0092] Another object of the present invention is to provide a cryopreserved preparation of human umbilical cord mesenchymal stem cells.

[0093] The human umbilical cord mesenchymal stem cell cryopreservation preparation provided by the present invention comprises the above-mentioned human umbilical cord mesenchymal stem cells and a cryoprotectant.

[0094] Furthermore, the cryoprotectant comprises compound electrolyte injection, dimethyl sulfoxide and human albumin solution.

[0095] In some embodiments, the volume ratio of the compound electrolyte injection, the dimethyl sulfoxide, and the human albumin solution is 65:10:25.

[0096] Furthermore, the concentration of the human umbilical cord mesenchymal stem cells in the human umbilical cord mesenchymal stem cell cryopreservation preparation is (3-6)×10 6 cells / mL.

[0097] In some embodiments, the concentration of the human umbilical cord mesenchymal stem cells in the human umbilical cord mesenchymal stem cell cryopreservation preparation is 5×10 6 cells / mL or 6×10 6 cells / mL.

[0098] Another object of the present invention is to provide a method for preparing the above-mentioned human umbilical cord mesenchymal stem cell cryopreservation preparation.

[0099] The preparation method of the human umbilical cord mesenchymal stem cell cryopreservation preparation provided by the present invention comprises the following steps: resuspending the human umbilical cord mesenchymal stem cells with a cryoprotectant to obtain a cell suspension; and cryopreserving the cell suspension to obtain the human umbilical cord mesenchymal stem cell cryopreservation preparation.

[0100] Furthermore, the freezing method includes the following steps: packaging the cell suspension into freezing bags to obtain a packaged preparation; then placing the packaged preparation into a freezing box and freezing it; and after freezing, transferring it to liquid nitrogen for long-term storage.

[0101] In some embodiments, cryopreservation can be performed as follows: cryopreservation boxes are stacked and placed in a cryorack at 10±4°C. Within 30 minutes, the cryorack is transferred to a cryorack holder in a -80°C ultra-low temperature freezer at 10±4°C, where it is placed for 24-72 hours. Finally, the cryorack is transferred to a liquid nitrogen tank. The cryorack holder is preferably 5±2 cm from the bottom of the -80°C ultra-low temperature freezer and 20±5 cm from the four sides of the -80°C ultra-low temperature freezer.

[0102] In some embodiments, freezing can be performed as follows: the cryopreservation box is transferred to a programmed cooling device (Haier; model CJ-L37) in an environment of 10±4°C, and the cooling program is set as follows: starting temperature 4°C → 1°C / min to -8°C → 20°C / min to -40°C → 10°C / min to -15°C → 1°C / min to -20°C → -20°C maintained for 5 minutes → 2°C / min to -40°C → 5°C / min to -80°C → -80°C maintained for 5 minutes. After the program is completed, the cryopreservation box is placed in a freezing rack and then transferred as a whole into a liquid nitrogen tank.

[0103] In some embodiments, freezing can be performed as follows: the cryopreservation box is transferred to a programmed cooling apparatus (Songyang; model CX-34) in an environment of 10±4°C, and the cooling program is set as follows: 10°C / min to 4°C → 1°C / min to -5°C → 25°C / min to -45°C → 10°C / min to -12°C → 1°C / min to -40°C → 10°C / min to -90°C → -90°C maintained for 15 minutes. After the program is completed, the cryopreservation box is placed in a freezing rack and then transferred as a whole into a liquid nitrogen tank.

[0104] It is also an object of the present invention to provide a kit of reagents.

[0105] The complete reagent set provided by the present invention comprises the above-mentioned complete culture medium, the above-mentioned activation culture medium, the above-mentioned cryopreservation protection solution and the above-mentioned cell preservation solution.

[0106] Furthermore, the complete set of reagents also includes the above-mentioned cryoprotectant.

[0107] Furthermore, the kit also includes phosphate buffer (pH 7.40±0.20) and pancreatic enzyme substitute.

[0108] The last object of the present invention is to provide the application in any one of the following M1)-M8):

[0109] M1) Use of the human umbilical cord mesenchymal stem cells in preparing the human umbilical cord mesenchymal stem cell cryopreservation preparation;

[0110] M2) Use of the above-mentioned reagent set in preparing the above-mentioned human umbilical cord mesenchymal stem cells or the above-mentioned human umbilical cord mesenchymal stem cell cryopreservation preparation;

[0111] M3) Use of the human umbilical cord-derived mesenchymal stem cells or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation prepared according to the above method in the preparation of a product for the treatment or adjuvant treatment of ischemic stroke;

[0112] M4) Use of the human umbilical cord-derived mesenchymal stem cells, the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation, or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation prepared according to the above method in the preparation of a product for treating or assisting in the treatment of spinal cord injury;

[0113] M5) Use of the human umbilical cord-derived mesenchymal stem cells, the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation, or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation prepared according to the above method in the preparation of a product for preventing and / or treating graft-versus-host disease;

[0114] M6) Use of the human umbilical cord-derived mesenchymal stem cells, the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation, or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation prepared according to the above method in the preparation of a product for the treatment or adjuvant treatment of liver failure;

[0115] M7) Use of the human umbilical cord-derived mesenchymal stem cells, the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation, or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation prepared according to the above method in the preparation of a product for treating or assisting in the treatment of diabetic nephropathy;

[0116] M8) Use of the above-mentioned human umbilical cord-derived mesenchymal stem cells, or the above-mentioned human umbilical cord-derived mesenchymal stem cell cryopreservation preparation, or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation prepared according to the above-mentioned method in the preparation of a product for the treatment or auxiliary treatment of acute respiratory distress syndrome.

[0117] Any of the above-mentioned ischemic strokes may be permanent ischemic strokes.

[0118] Any of the above-mentioned spinal cord injuries may be subacute spinal cord injury.

[0119] In any of the above applications, the product may be a medicine.

[0120] The present invention provides a method for preparing ready-to-use activated human umbilical cord mesenchymal stem cells, the method comprising the following steps: applying enzymatic technology to digest umbilical cord tissue blocks, and culturing adherently to obtain P0 primary cells; passage the P0 cells three times to obtain P3 cells; harvesting and freezing the P3 cells, and establishing a cell seed bank after passing the test; thawing the frozen seed bank cells, passage three times to obtain P6 cells; culturing the P6 cells using an activation medium to obtain activated P6 cells; harvesting the activated P6 cells to obtain human umbilical cord mesenchymal stem cells, and then resuspending them with a cell protectant to obtain the human umbilical cord mesenchymal stem cell original suspension. The present invention also provides a method for preparing a frozen preparation of umbilical cord mesenchymal stem cells, the method comprising the following steps: mixing the human umbilical cord mesenchymal stem cell original suspension with a cryoprotectant, subpackaging, and freezing to obtain a frozen preparation of human umbilical cord mesenchymal stem cells. Experiments have shown that the human umbilical cord mesenchymal stem cell cryopreservation preparation prepared by the present invention can be preserved for a long time, is convenient for storage, transportation and clinical use, can complete the effectiveness and safety index testing during the shelf life, and the cryopreserved preparation has the same activity as the fresh preparation.

[0121] The beneficial effects of the present invention are as follows:

[0122] 1. The present invention optimizes the components and concentration of the activation medium and uses the optimized activation medium (without animal serum) to activate and culture P6 MSCs, which can not only ensure the stable expansion and efficient activation of MSCs in vitro, but also reduce the residual animal serum and enhance the immunoregulatory function of MSCs (Example 9), greatly improving its clinical efficacy.

[0123] 2. During the preparation stage, cell viability is negatively correlated with operation time, which directly limits the operability time of the preparation. The present invention preserves cells in a cell protectant during the preparation stage, which significantly increases the duration of cell viability (Example 4), thereby extending the preparation time and ensuring product quality.

[0124] 3. During cell cryopreservation, the addition of a cryoprotectant reduces damage to the cells caused by ice crystal formation inside and outside the cells, thereby ensuring cell activity. Literature has reported that the cryopreservation process can affect the immunoregulatory function of mesenchymal stem cells (Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing). However, the umbilical cord mesenchymal stem cell injection prepared by the present invention does not suffer from cryopreservation damage, that is, the immune regulatory ability of the cells is not impaired after cryopreservation, and the immune regulatory ability is no different from that of fresh cells (Example 6), thus ensuring the function of the cells.

[0125] 4. Cryopreservation dosage forms require large-scale freezing of cells. Currently, most cell freezing processes use programmable cooling devices, but they are all scientific research level and cannot be used for large-scale cell freezing. The present invention provides a new method for freezing mesenchymal stem cells (Group D1-1 / Group E1-1 in Example 5), which does not require professional programmable cooling equipment or a complicated cooling process, and the freezing effect can be achieved by cooling in one step. This method is suitable for freezing large quantities of products, is simple to operate, and is easy to transfer. It avoids the impact of long-term transfer operations under extremely low temperature conditions on the quality of stem cell drugs and is suitable for mass industrial production of mesenchymal stem cell drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 This is the morphology of human umbilical cord mesenchymal stem cells from the cell seed bank.

[0127] Figure 2 Figure 1 shows the osteogenic and adipogenic differentiation abilities of seed bank cells. The first row (three images) corresponds to adipogenic differentiation. The second row (three images) corresponds to osteogenic differentiation. The first column (two images) corresponds to KY210015, the second column (two images) corresponds to KY210016, and the third column (two images) corresponds to KY210017.

[0128] Figure 3 The effect of cell protection solution on the viability of human umbilical cord mesenchymal stem cells. ** P<0.01, *** P<0.001, **** P<0.0001.

[0129] Figure 4 The results of the long-term survival test of human umbilical cord mesenchymal stem cell cryopreservation preparations. ** P<0.01, *** P<0.001, ****P < 0.0001. The first two images correspond to KY210015, the second two images correspond to KY210016, and the third two images correspond to KY210017.

[0130] Figure 5 The effect of different cryopreservation methods on the viability of human umbilical cord mesenchymal stem cell cryopreservation preparations. ** P<0.01, *** P<0.001, **** P<0.0001.

[0131] Figure 6 Comparison of the inhibition of IFN-γ secretion by activated PBMCs with cryopreserved and fresh human umbilical cord mesenchymal stem cell preparations.

[0132] Figure 7 This is the LFB staining of the myelin sheath of the corpus callosum in the peri-infarct area of ​​rats with permanent cerebral ischemia after injection of cryopreserved human umbilical cord mesenchymal stem cells (400×).

[0133] Figure 8 These are the results of spinal cord histopathological diagnosis in rats with subacute spinal cord injury after injection of cryopreserved human umbilical cord mesenchymal stem cells.

[0134] Figure 9 The results of PGE2 expression in activated human umbilical cord mesenchymal stem cells. **** P<0.0001.

[0135] Figure 10 Effects of cryopreserved human umbilical cord mesenchymal stem cells on the body weight of mice with acute respiratory distress syndrome. * P<0.05.

[0136] Figure 11 Effects of cryopreserved human umbilical cord mesenchymal stem cells on the expression of serum inflammatory factors in mice with acute respiratory distress syndrome. * P<0.05, *** P<0.001, **** P<0.0001.

[0137] Figure 12 Effects of cryopreserved human umbilical cord mesenchymal stem cells on serum MPO activity in mice with acute respiratory distress syndrome. **** P<0.0001.

[0138] Figure 13 Survival curve of liver failure model mice after injection of cryopreserved human umbilical cord mesenchymal stem cells.

[0139] Figure 14Effects of cryopreserved human umbilical cord mesenchymal stem cells on serum factor expression in liver failure model mice. **** P<0.0001.

[0140] Figure 15 Effects of cryopreserved human umbilical cord mesenchymal stem cells on liver histopathological scores in mice with liver failure model.

[0141] Figure 16 H&E pathological sections of liver tissue in liver failure model mice after injection of cryopreserved human umbilical cord mesenchymal stem cell preparation.

[0142] Figure 17 Effects of cryopreserved human umbilical cord mesenchymal stem cells on urine protein levels in diabetic nephropathy model rats. ** P<0.01, *** P<0.001, **** P<0.0001.

[0143] Figure 18 Effects of cryopreserved human umbilical cord mesenchymal stem cells on serum creatinine levels in diabetic nephropathy model rats. * P<0.05, **** P<0.0001. DETAILED DESCRIPTION

[0144] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0145] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0146] The complete culture medium in the following examples consists of DMEM / F12 basal medium (GIBCO, 12500-096) and serum replacement (Shanghai Yikesai, product number FSP500); the volume ratio of DMEM / F12 basal medium to serum replacement is 9:1.

[0147] The phosphate buffer (pH 7.40±0.20) in the following examples is a product of Youkang Biotechnology (Beijing) Co., Ltd., with the product number BS040.

[0148] The collagenase phosphate buffer in the following examples is a solution obtained by dissolving 3 g of collagenase (Gibco, product number 17101-015) in 1000 mL of phosphate buffer.

[0149] The pancreatic enzyme substitute in the following examples is a product of Thermo Fisher Scientific (China) Co., Ltd., with a product number of 12604-021.

[0150] The serum substitute in the following examples is a product of Shanghai Yikesai Biological Products Co., Ltd., with the product number FSP500.

[0151] The DMSO in the following examples is a product of Origen Biomedical, with the product number CP-70.

[0152] The human albumin solution (human albumin mass fraction is 20%) in the following examples is a product of Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd., with the product number being National Medicine Standard S10940026.

[0153] The compound electrolyte injection in the following examples is a product of Shanghai Better Medical Products Co., Ltd., with the product number being National Medicine Standard H20000475.

[0154] The osteogenic differentiation medium in the following examples is a product of Saiye (Guangzhou) Biotechnology Co., Ltd., with the product number HUXUC-90021.

[0155] The adipogenic differentiation medium (including liquid A and liquid B) in the following examples is a product of Saiye (Guangzhou) Biotechnology Co., Ltd., with the product number HUXUC-90031.

[0156] The Ficoll solution in the following examples is a product of Tianjin Haoyang Huake Biotechnology Co., Ltd., with the product number LTS1077.

[0157] In the following examples, RPMI1640 culture medium is a product of Gibco, with product number 11835.

[0158] The PHA in the following examples is a product of Roche, with product number 11082132001.

[0159] The IL-2 in the following examples is a product of PeproTech, with the product number being 200-02-10VG.

[0160] The cell filter in the following examples is a product of Jiete Biotechnology, with the product number CSS013100.

[0161] Example 1. Preparation of human umbilical cord mesenchymal stem cell suspension

[0162] 1. Isolation of primary cells

[0163] (1) Obtain the umbilical cord of a healthy fetus in vitro that has obtained informed consent and meets the acceptance criteria (no history of genetic diseases, negative test results for infectious pathogens such as hepatitis, syphilis, and AIDS), tie both ends of the umbilical cord, place it in a 100 mL beaker, and wash it 2-4 times with phosphate buffer.

[0164] (2) The umbilical cord that has been processed in step (1) is transferred to a kidney-shaped dish, blood clots are removed, and the umbilical cord is cut into 0.5-1 cm tissue segments using sterile scissors. After washing with phosphate buffer 2-4 times, the umbilical cord is cut into 3-9 mm segments. 3 Organization block.

[0165] (3) Transfer the tissue block obtained in step (2) to a 100 mL plasma bottle, add 30-50 mL of collagenase phosphate buffer, and digest at 37°C for 30-50 min. Filter through a cell strainer, and collect the filtrate above the filter for secondary digestion in step (4). Collect the filtrate below the filter, add 10-20 mL of complete culture medium, and mix well to obtain mixed solution 1.

[0166] (4) Take the filtrate obtained in step (3), add 30-50 mL of trypsin substitute, digest at 37°C for 30-50 min, filter through a cell strainer, collect the filtrate below the strainer, add 10-20 mL of complete culture medium and mix well to obtain mixed solution 2.

[0167] (5) Combine Mixture 1 and Mixture 2, add 80-120 mL of phosphate buffered saline, and filter through a cell strainer. Collect the filtrate below the strainer and centrifuge at 2800-3200 g for 10 minutes. Discard the supernatant, resuspend the cells in 30-40 mL of complete culture medium, and take a sample for counting.

[0168] (6) Take the resuspended cells obtained in step (5) and centrifuge at 10,000-16,000 / cm 2 Inoculate the cell culture flask with complete culture medium (0.2-0.3 mL / cm 2 ), and after labeling, culture in a carbon dioxide incubator at 37°C and 5% CO2. Observe regularly under an inverted microscope and change the medium every 3-4 days to obtain primary cells.

[0169] 2. Establishment of cell seed bank

[0170] (1) When the primary cell fusion degree reaches 60%-80%, P0 mesenchymal stem cells are obtained.

[0171] (2) The P0 mesenchymal stem cells obtained in step (1) were subcultured: the supernatant was discarded, phosphate buffer was added for washing, and pancreatic enzyme substitute (0.01-0.03 mL / cm 2) for digestion. Transfer the cell suspension to a centrifuge tube; add phosphate buffer for washing, and after washing, transfer the cell suspension to a centrifuge tube; take a sample and count; centrifuge the centrifuge tube (300-500g for 5 minutes), discard the supernatant, resuspend the cells in complete medium equilibrated to room temperature, subculture at a ratio of 1:3, and replenish the complete medium (0.2-0.3mL / cm 2 ), and after labeling, cultured in a 37°C, 5% CO2 incubator. When the cell confluence reaches 70%-90%, P1 mesenchymal stem cells are obtained.

[0172] (3) The P1 mesenchymal stem cells obtained in step (2) were subcultured: the supernatant was discarded, phosphate buffer was added for washing, and pancreatic enzyme substitute (0.01-0.03 mL / cm 2 ) for digestion. Transfer the cell suspension to a centrifuge tube; add phosphate buffer for washing, and then transfer the cell suspension to a centrifuge tube; take a sample and count; centrifuge the centrifuge tube (800-1200g for 5 minutes), discard the supernatant, resuspend the cells in complete medium equilibrated to room temperature, subculture at a ratio of 1:3, and replenish the complete medium (0.2-0.3mL / cm 2 ), and after labeling, cultured in a 37°C, 5% CO2 incubator. When the cell confluence reaches 70%-90%, P2 mesenchymal stem cells are obtained.

[0173] (4) The P2 mesenchymal stem cells obtained in step (3) were subcultured: the supernatant was discarded, phosphate buffer was added for washing, and pancreatic enzyme substitute (0.01-0.03 mL / cm 2 ) for digestion. Transfer the cell suspension to a centrifuge tube; add phosphate buffer for washing, and then transfer the cell suspension to a centrifuge tube; take a sample and count; centrifuge the centrifuge tube (800-1200g for 5 minutes), discard the supernatant, resuspend the cells in complete medium equilibrated to room temperature, subculture at a ratio of 1:3, and replenish the complete medium (0.2-0.3mL / cm 2 ), and after labeling, cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 70%-90%, P3 mesenchymal stem cells were obtained.

[0174] (5) The P3 mesenchymal stem cells obtained in step (4) were harvested and frozen: the supernatant was discarded, phosphate buffer was added for washing, and pancreatic enzyme substitute (0.01-0.03 mL / cm 2) for digestion. Transfer the cell suspension to a centrifuge tube; add phosphate buffer for washing, and after washing, transfer the cell suspension to a centrifuge tube; take a sample and count; centrifuge the centrifuge tube (800-1200g for 5 minutes), discard the supernatant, and resuspend the cells in cryopreservation protection solution (serum substitute: DMSO = 9:1) to adjust the cell concentration to 3.5×10 6 Cells / mL, and the cell suspension is divided into cryopreservation tubes. The cryopreservation tubes are placed in a cryopreservation box, placed on a cryopreservation rack according to a certain loading method, placed in a -80°C ultra-low temperature refrigerator for freezing for 24-72 hours, and then transferred to liquid nitrogen or vapor phase liquid nitrogen for long-term storage. After being stored under liquid nitrogen or vapor phase liquid nitrogen conditions for 1 month, a sterility test (including mycoplasma test, intracellular and exogenous viral factor test, tumorigenicity test, etc.) is performed with reference to the current edition of the Chinese Pharmacopoeia. After passing the test, a cell seed bank is obtained, and the identification of the cell seed bank in Example 2 is performed.

[0175] 3. Preparation of Human Umbilical Cord Mesenchymal Stem Cell Suspension

[0176] (1) Transfer the cells of the revived cell seed bank to a centrifuge tube, add complete culture medium, mix well, and centrifuge (300-500g for 5 minutes), discard the supernatant, and resuspend the cells in complete culture medium equilibrated to room temperature at a density of 12,000-18,000 cells / cm 2 The cells were inoculated into cell culture flasks at a density of 100 μg / cm and supplemented with complete culture medium (0.2-0.3 mL / cm 2 ), and after labeling, cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 70%-90%, P4 mesenchymal stem cells were obtained.

[0177] (2) The P4 mesenchymal stem cells obtained in step (1) were subcultured: the supernatant was discarded, phosphate buffer was added for washing, and pancreatic enzyme substitute (0.01-0.03 mL / cm 2 ) for digestion. Transfer the cell suspension to a centrifuge tube; add phosphate buffer for washing, and then transfer the cell suspension to a centrifuge tube; take a sample and count; centrifuge the centrifuge tube (800-1200g for 5 minutes), discard the supernatant, resuspend the cells in complete medium equilibrated to room temperature, subculture at a ratio of 1:3, and replenish the complete medium (0.2-0.3mL / cm 2 ), and after labeling, cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 70%-90%, P5 mesenchymal stem cells were obtained.

[0178] (3) The P5 mesenchymal stem cells obtained in step (2) were subcultured: the supernatant was discarded, phosphate buffer was added for washing, and pancreatic enzyme substitute (0.01-0.03 mL / cm 2) for digestion. Transfer the cell suspension to a centrifuge tube; add phosphate buffer for washing, and then transfer the cell suspension to a centrifuge tube; take a sample and count; centrifuge the centrifuge tube (800-1200g for 5 minutes), discard the supernatant, resuspend the cells in complete medium equilibrated to room temperature, subculture at a ratio of 1:3, and replenish the complete medium (0.2-0.3mL / cm 2 ), and after labeling, cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 70%-90%, P6 mesenchymal stem cells were obtained.

[0179] (4) The P6 mesenchymal stem cells obtained in step (3) were activated and cultured: the supernatant was discarded, and phosphate buffer was added to wash 2-3 times. After washing, activation medium (0.2-0.3 mL / cm2) was added to the cells equilibrated to room temperature. 2 ), and after labeling, cultured in a carbon dioxide incubator at 37° C. and 5% CO 2 for 16-48 hours to obtain activated P6 mesenchymal stem cells.

[0180] The activation medium is composed of solvent and solute. The solvent is water. The solute includes amino acids, vitamins, inorganic salts and other components. The components and concentrations of each type of solute are as follows:

[0181] Amino Acids: Glycine 18.75mg / L, Alanine 4.45mg / L, Glutamine 542mg / L, Arginine 147.5mg / L, Asparagine 7.5mg / L, Aspartic Acid 6.65mg / L, Cysteine ​​35.12mg / L, Cystine 31.29mg / L, Glutamic Acid 7.35mg / L, Histidine 31.48mg / L, Isoleucine 54.47mg / L, Leucine 59.05mg / L, Lysine 91.25mg / L, Methionine 17.24mg / L, Phenylalanine 35.48mg / L, Proline 17.25mg / L, Serine 26.25mg / L, Threonine 53.45mg / L, Tryptophan 9.02mg / L, Tyrosine 55.79mg / L, Valine 52.85mg / L;

[0182] Vitamins: Vitamin H 0.0035mg / L, Choline chloride 8.98mg / L, Calcium pantothenate 2.24mg / L, Folic acid 2.65mg / L, Nicotinamide 2.02mg / L, Vitamin B6 2.031mg / L, Vitamin B2 0.219mg / L, Vitamin B12 2.17mg / L, Vitamin B12 0.68mg / L, Inositol 12.6mg / L, Ascorbic acid 50mg / L, α-Tocopheryl acetate 0.4mg / L, Vitamin A 1.38mg / L;

[0183] Inorganic salts: calcium chloride 116.6 mg / L, copper sulfate 0.0013 mg / L, ferric nitrate 0.05 mg / L, ferric sulfate 0.417 mg / L, magnesium chloride 28.64 mg / L, magnesium sulfate 48.84 mg / L, potassium chloride 311.8 mg / L, sodium bicarbonate 4876 mg / L, sodium chloride 6999.5 mg / L, disodium hydrogen phosphate 71.02 mg / L, sodium dihydrogen phosphate 62.5 mg / L, zinc sulfate 0.432 mg / L;

[0184] Other ingredients: D-glucose 3151 mg / L, hypoxanthine 2.39 mg / L, linoleic acid 0.042 mg / L, palmitic acid 5.13 mg / L, lipoic acid 0.105 mg / L, tetramethylethylenediamine 0.081 mg / L, sodium pyruvate 165 mg / L, thymine 0.365 mg / L, reduced glutathione 0.62 mg / L, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) 3580 mg / L.

[0185] (5) Take the activated P6 mesenchymal stem cells obtained in step (4): discard the supernatant, add phosphate buffer to wash, and add pancreatic enzyme substitute (0.01-0.03mL / cm 2 ) for digestion; transferring the cell suspension to a centrifuge tube, adding a cell protective agent and mixing, and then transferring the cell suspension to a centrifuge tube; sampling and counting; centrifuging the centrifuge tube (800-1200g for 5 minutes) to obtain human umbilical cord mesenchymal stem cells; adding a cell protective agent to the human umbilical cord mesenchymal stem cells and resuspending the cells to obtain a human umbilical cord mesenchymal stem cell original suspension (cell concentration is (5-6)×10 6 The cell protectant is composed of human albumin solution (20%) and compound electrolyte injection, and the volume ratio of human albumin solution (20%) to compound electrolyte injection is 1:19.

[0186] Example 2: Identification of cell seed banks

[0187] 1. Cell morphology identification

[0188] The cells of the revived cell seed bank (different batches of cell seed banks prepared according to the method in step 2 of Example 1, respectively denoted as KY230015, KY230016, and KY230017) were transferred to a centrifuge tube, added with complete medium, mixed and centrifuged (300-500g for 5 minutes), the supernatant was discarded, and the cells were resuspended in complete medium equilibrated to room temperature at a density of 12,000-18,000 cells / cm 2 The cells were inoculated into cell culture flasks at a density of 100 μg / cm and supplemented with complete culture medium (0.2-0.3 mL / cm 2), and after labeling, culture in a 37°C, 5% CO2 incubator. When the cell confluence reaches 70%-90%, observe and photograph under an inverted microscope with a 4× objective lens. Figure 1 This is the cell morphology diagram of the seed bank.

[0189] 2. Surface marker identification

[0190] The cells of the revived cell seed bank (different batches of cell seed banks prepared according to the method in step 2 of Example 1, respectively recorded as KY230015, KY230016, and KY230017) were transferred to a centrifuge tube, added with complete culture medium, mixed and centrifuged (300-500g for 5 minutes), the supernatant was discarded, and the cells were resuspended in phosphate buffer and divided into 11 tubes, 100 μL / tube, and Mouse IgG1-FITC (BD, 555748), CD19-FITC (BD, 555412), CD 31-FITC (BD, 555445), CD34-FITC (BD555821), Mouse IgG1-PE (BD, 555749), CD11b-PE (BD, 555388), CD45-PE (BD, 555483), CD73-PE (BD, 550257), CD90-PE (BD, 555596), CD105-PE (BD, 560839), and HLA-DR-PE (BD, 555812) were added in a 20 μL volume. Cells were resuspended and mixed thoroughly and incubated in the dark for 1 hour. After incubation, cells were washed two to three times with phosphate buffered saline and filtered through a 200-mesh filter. Flow cytometer analysis was performed using a BD FACSCalibur flow cytometer, with 10,000 cells per sample.

[0191] The results are shown in Table 1. The results showed that the positive rates of surface markers CD73, CD90 and CD105 in the seed bank cells were higher than 95%; the positive rates of CD11b, CD19, CD31, CD34, CD45 and HLA-DR were lower than 2%.

[0192] Table 1. Detection results of cell surface markers in seed banks (taking three seed banks as an example)

[0193] KY230015 KY230016 KY230017 CD19 0.59％ 0.37％ 0.89％ CD31 0.74％ 0.62％ 0.79％ CD34 0.63％ 0.35％ 0.94％ CD11b 0.25％ 0.64％ 0.73％ CD45 0.17％ 0.49％ 0.96％ CD73 99.24％ 99.09％ 99.36％ CD90 99.54％ 99.19％ 99.28％ CD105 99.36％ 99.12％ 99.23％ HLA-DR 0.49％ 0.68％ 0.92％

[0194] 3. Multidirectional differentiation ability test

[0195] 1) Osteogenic differentiation

[0196] The cells of the revived cell seed bank (different batches of cell seed banks prepared according to the method in step 2 of Example 1, respectively denoted as KY230015, KY230016, and KY230017) were transferred to a centrifuge tube, and complete medium was added. After mixing, centrifugation was performed (300-500g for 5 minutes), the supernatant was discarded, and the cells were resuspended in complete medium equilibrated to room temperature and cultured at a density of 20,000 cells / cm 2 Cells were seeded at a density of 100 μg / cm2 in a six-well plate, 2 mL of complete culture medium was added, and the plate was labeled and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 70%, 2 mL of osteogenic differentiation medium was replaced. The medium was changed every 3 days, and after 21 days of induction, the cells were stained with Alizarin Red.

[0197] 2) Adipogenic differentiation

[0198] The cells of the revived cell seed bank (different batches of cell seed banks prepared according to the method in step 2 of Example 1, respectively denoted as KY230015, KY230016, and KY230017) were transferred to a centrifuge tube, and complete medium was added. After mixing, centrifugation was performed (300-500g for 5 minutes), the supernatant was discarded, and the cells were resuspended in complete medium equilibrated to room temperature and cultured at a density of 20,000 cells / cm 2 Cells were seeded at a density of 100 μg / ml in a six-well plate, 2 mL of complete culture medium was added, and the plate was labeled and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 100%, the medium was replaced with adipogenic differentiation medium. First, 2 mL of adipogenic differentiation medium A was added. After 3 days of induction, the medium was replaced with 2 mL of adipogenic differentiation medium B. After 1 day of induction, the medium was replaced with 2 mL of adipogenic differentiation medium A. Liquid A and liquid B were used alternately. When sufficient lipid droplets of appropriate size appeared, the cells were stained with Oil Red O.

[0199] The results are as follows Figure 2 The results showed that the seed bank cells prepared by the present invention have good osteogenic and adipogenic differentiation abilities.

[0200] Example 3: Effect of Cytoprotectants on the Viability of Human Umbilical Cord Mesenchymal Stem Cells

[0201] Take the human umbilical cord mesenchymal stem cells obtained in Example 1 (the human umbilical cord mesenchymal stem cells in step 3 (5) of Example 1), add different groups of cell protectants according to Table 2, resuspend the cells, filter, and adjust the cell concentration to 5×10 6 cells / mL, 6×10 6 cells / mL, placed in room temperature (20-30°C) and 2-8°C environment, and the cell viability was detected after 0 hour, 1 hour, 2 hours, 3 hours and 4 hours respectively.

[0202] Cell viability detection method: Take 20 μL of sample and mix it evenly with 20 μL of AO / PI fluorescent dye, pipette 20 μL into the counting plate, and analyze it using a fluorescence counter.

[0203] The results are as follows Figure 3 The results showed that compared with the cell protectant group without human albumin, when the final concentration of human albumin in the cell protectant was 0.2%, although the cell viability was greater than 95% after being placed at room temperature or 2-8°C for 1 hour, the cell viability decreased to about 90% after 4 hours, which was not statistically different from the cell protectant group without human albumin, and the cell viability of subsequent preparations could not be guaranteed; when the final concentration of human albumin in the cell protectant was 0.5% and 1%, the cell viability was greater than 95% after being placed at room temperature or 2-8°C for 4 hours, which was significantly higher than the cell protectant group without human albumin and the cell protectant group with a final concentration of human albumin of 0.2% ( ** P<0.01).

[0204] Table 2. Cytoprotectant formulations and groupings (taking 100 mL as an example)

[0205]

[0206]

[0207] Note: The formula for calculating the final concentration of human albumin is: [0.2 × volume of human albumin solution / (volume of human albumin solution + volume of compound electrolyte injection)] × 100%.

[0208] Example 4: Preparation and long-term stability study of cryopreserved human umbilical cord mesenchymal stem cell preparations

[0209] 1. Preparation of cryopreserved human umbilical cord mesenchymal stem cell preparations

[0210] (1) Take the human umbilical cord mesenchymal stem cells obtained in Example 1 (the human umbilical cord mesenchymal stem cells in step 3 (5) of Example 1), add different groups of cell protectants according to Table 3 to resuspend the cells, filter, sample and count, centrifuge, add cryoprotectant (the volume ratio of compound electrolyte injection, dimethyl sulfoxide and human serum albumin solution is 65:10:25), and adjust the cell concentration to 5×10 6 cells / mL or 6×10 6 cells / mL.

[0211] Table 3. Cytoprotectant formulation and grouping (taking 100 mL as an example)

[0212]

[0213] Note: The formula for calculating the final concentration of human albumin is: [0.2 × volume of human albumin solution / (volume of human albumin solution + volume of compound electrolyte injection)] × 100%.

[0214] (2) The cell suspension obtained in step (1) is dispensed into cryopreservation bags at a rate of 5 mL / bag using an automated dispensing system (described in utility model patent No. ZL202420914542.9, which is available to the public from the applicant). The automated dispensing system includes modules for preparation filling, cutting, heat sealing, and weighing, which can automatically complete the above operations.

[0215] (3) The preparation obtained in step (2) was placed in an aluminum cryobox, placed on a cryorack according to a certain loading method (the loading method is the D1-1 group / E1-1 group in Example 5), placed in a -80°C ultra-low temperature freezer for 24-72 hours, and then directly transferred to liquid nitrogen for long-term storage.

[0216] 2. Long-term viability testing of cryopreserved human umbilical cord mesenchymal stem cell preparations

[0217] The mesenchymal stem cell cryopreservation preparations were removed from liquid nitrogen after 0, 3, 6, 9, 12, 18, 24, and 36 months of cryopreservation, and thawed. The cells in the preparations were evenly mixed, and 20 μL of the sample was mixed evenly with 20 μL of AO / PI fluorescent dye. 20 μL was aspirated and added to a counting plate for analysis using a fluorescence counter.

[0218] The results are as follows Figure 4 The results showed that at a given time point, the final concentration of human serum albumin in the cell protectant would affect the freezing density of 5×10 6 cells / mL and 6×10 6 The cell viability of human umbilical cord mesenchymal stem cell cryopreservation preparations prepared with 1% and 2% human serum albumin was not significantly different when the final concentration of human serum albumin in the cell protectant was 0.5% and 1%, but both were significantly better than the cell protectant group without human serum albumin ( ** P<0.01); when the final concentration of human albumin in the cell protectant was 0.5% and 1%, the viability of the human umbilical cord mesenchymal stem cell cryopreservation preparation was guaranteed to be no less than 85% after three years of storage under liquid nitrogen conditions; when the cell protectant did not contain human albumin, the viability of the human umbilical cord mesenchymal stem cell cryopreservation preparation was only guaranteed to be no less than 80% after three years of storage under liquid nitrogen conditions.

[0219] Example 5: Effects of Different Cryopreservation Methods on Cryopreserved Human Umbilical Cord Mesenchymal Stem Cell Preparations

[0220] The human umbilical cord mesenchymal stem cell cryopreservation preparations D1 and E1 prepared in Example 4 were placed in aluminum cryopreservation boxes and divided into three groups. The cells were cryopreserved according to the following freezing methods for each group:

[0221] Group D1-1 / Group E1-1: Place cryopreservation boxes containing cryopreserved human umbilical cord mesenchymal stem cell preparations in a stacked format into seven cryoracks (17 cm L × 10 cm W × 67 cm H) at 10 ± 4°C. Each cryorack can accommodate 50 boxes, with the remaining space filled with a preparation cryopreservation simulant (a preparation with thermodynamic parameters consistent with the cryopreserved preparation to ensure consistent freezing rates for each batch). Within 30 minutes, transfer the cryoracks to a -80°C ultralow-temperature freezer (made of polypropylene, 80 cm L × 20 cm W × 70 cm H) at 10 ± 4°C. The cryoracks were secured in a -80°C ultralow-temperature freezer for 24–72 hours, maintaining a distance of 5 ± 2 cm from the bottom and 20 ± 5 cm from the four sides. The entire cryorack was then transferred to a liquid nitrogen tank.

[0222] Group D1-2 / Group E1-2: Transfer the cryopreservation boxes containing the cryopreserved human umbilical cord mesenchymal stem cell preparation to a programmed cooling apparatus (Haier; model CJ-L37) at 10±4°C. Based on the internal volume of the programmed cooling apparatus, a maximum of 60 cryopreservation boxes can be placed. Set the cooling program based on the preparation characteristics and manufacturer's recommendations: starting temperature 4°C → 1°C / min to -8°C → 20°C / min to -40°C → 10°C / min to -15°C → 1°C / min to -20°C → -20°C for 5 min → 2°C / min to -40°C → 5°C / min to -80°C → -80°C for 5 min. After the program is completed, place the cryopreservation boxes in a cryorack and transfer the entire box to a liquid nitrogen tank.

[0223] Group D1-3 / Group E1-3: Transfer the cryopreservation boxes containing the cryopreserved human umbilical cord mesenchymal stem cell preparation to a programmed cooling apparatus (Songyang; Model CX-34) at 10±4°C. Based on the internal volume of the programmed cooling apparatus, a maximum of 60 cryopreservation boxes can be placed. Set the cooling program based on the preparation characteristics and manufacturer's recommendations: 10°C / min to 4°C → 1°C / min to -5°C → 25°C / min to -45°C → 10°C / min to -12°C → 1°C / min to -40°C → 10°C / min to -90°C → -90°C for 15 min. After the program is completed, place the cryopreservation boxes in a cryorack and transfer the entire box to a liquid nitrogen tank.

[0224] The three groups of human umbilical cord mesenchymal stem cell cryopreservation preparations were taken out from the liquid nitrogen storage tank after 0 months, 3 months, 6 months, 9 months, and 12 months of cryopreservation, respectively. The cells in the preparations were evenly mixed, and 20 μL of the sample was mixed evenly with 20 μL of AO / PI fluorescent dye. 20 μL was aspirated and added to a counting plate for analysis using a fluorescence counter.

[0225] The results are as follows Figure 5 The results showed that the cell viability of the three groups of human umbilical cord mesenchymal stem cell cryopreservation preparations prepared by different cryopreservation methods was greater than 85%, and there was no statistical difference between the groups (p>0.05).

[0226] Example 6: Comparative Study of the Inhibition of IFN-γ Secretion by Cryopreserved and Fresh Human Umbilical Cord Mesenchymal Stem Cells

[0227] 1. Take the human umbilical cord mesenchymal stem cell cryopreservation preparation E1 prepared in Example 4, and after thawing, add 10,000-12,000 cells / cm 2 The cells were seeded at a density of 100 μg / cm2 in a six-well plate, 2 mL of complete culture medium was added, and the plate was labeled and incubated in a carbon dioxide incubator at 37°C and 5% CO2 for 24 hours. The supernatant was collected and recorded as SD1. The cells of the resuscitated cell seed bank were serially passaged to P6. The P6 cells were cultured at a density of 10,000-12,000 cells / cm2. 2 The cells were inoculated at a density of 100 μg / mL in a six-well plate, 2 mL of complete culture medium was added, and the plate was marked and incubated in a carbon dioxide incubator at 37°C and 5% CO2 for 24 hours. The supernatant was collected and recorded as SX1.

[0228] 2. PBMC cell suspension was separated using Ficoll. The specific steps were as follows: peripheral blood of the donor (healthy person) was collected, phosphate buffer was added at a ratio of 2:1, and diluted whole blood was obtained; Ficoll solution was added to a 50 mL centrifuge tube at a ratio of 1:1, and then the diluted whole blood was slowly added, centrifuged at 1800 rpm for 20 min, and mononuclear cells in the buffy coat were aspirated with a disposable pipette; 20 mL phosphate buffer was added, and the sample was counted and centrifuged (1500 rpm, 10 min), the supernatant was discarded, and the suspension was resuspended in RPMI1640 medium and counted at 1×10 5 cells / well were seeded in 96-well plates.

[0229] 3. 100 μL of SX1 supernatant was added to the fresh preparation (SX) group, and 100 μL of SD1 supernatant was added to the frozen preparation (SD) group. PHA (final concentration 5 μg / mL) and IL-2 (final concentration 100 U / mL) were also added, with a total volume of 200 μL / well. Three replicates were set up for each group. After labeling, the cells were incubated in a 37°C, 5% CO2 incubator for 72 hours, and the supernatant was collected. PBMC monoculture served as a control group.

[0230] 4. Use ELISA kit to detect the expression level of IFN-γ in the supernatant of SX, SD and control groups, and calculate the IFN-γ inhibition rate (%) according to the following formula: IFN-γ inhibition rate (%) = [(IFN-γ expression level in control group - IFN-γ expression level in experimental group) / IFN-γ expression level in control group] × 100%.

[0231] The results are as follows Figure 6 As shown, the results showed that there was no statistical difference in the level of inhibition of IFN-γ secretion by paracrine factors in the cells of the frozen preparation (SD) experimental group and the fresh preparation (SX) experimental group (p>0.05).

[0232] Example 7: Efficacy trial of cryopreserved human umbilical cord mesenchymal stem cell preparations in treating acute ischemic stroke

[0233] 1. Experimental animals and groups

[0234] Forty-eight male SPF SD rats aged 6-8 weeks were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and raised in Tianjin Weihao Pharmaceutical Technology Co., Ltd. The experiment was carried out after the adaptation period.

[0235] 2. Test methods

[0236] A permanent middle cerebral artery occlusion (MCAO) model was established in rats using the suture method. The MCAO model rats were randomly divided into two groups, 16 in each group, and a sham operation group (rats in the sham operation group only underwent a simulated surgical procedure) using the block randomization method. The treatment methods for each group were as follows:

[0237] Experimental group: The MCAO model rats were injected with a cryopreserved mesenchymal stem cell preparation (the cryopreserved mesenchymal stem cell preparation prepared in Group E1 in Example 4) into the tail vein.

[0238] Vehicle control group: an equal volume of cryoprotectant was injected into the tail vein of MCAO model rats.

[0239] Sham operation group: The rats in the sham operation group were injected with an equal volume of compound electrolyte injection into the tail vein.

[0240] The dosing information of each group is shown in Table 4. The details are as follows: the dosing frequency was 3 times, 24 h, 72 h, and 120 h after modeling, and the dosing volume was 1 mL / kg. The dose of the experimental group (single dose) was 1×10 7 The experimental endpoint was 6 days after treatment (7 days after model establishment) for 8 rats in each group and 13 days after treatment (14 days after model establishment) for 8 rats in each group.

[0241] Table 4. Grouping and medication information

[0242] Group Volume / dose Dosage time Dosage method / frequency N Sham operation group 1 mL / kg 24h, 72h, 120h iv / 3 8+8 Vehicle control group 1 mL / kg 24h, 72h, 120h iv / 3 8+8 experimental group <![CDATA[1mL / kg,1×10 7 pieces / kg]]> 24h, 72h, 120h iv / 3 8+8

[0243] The following parameters of rats in each group were measured at different time points: body weight, behavioral score (balance beam test), LFB myelin staining, etc. The scoring criteria for the balance beam test are shown in Table 5.

[0244] Table 5. Scoring criteria for the Beam Balance Test (BBT)

[0245] Standard Content score Stand firmly on the wooden bar without shaking for 2 minutes 1 Stand firmly on the wooden bar, sway left and right without sliding down, for 2 minutes 2 Stand on a wooden plank and slide down to the side without falling for 2 minutes 3 Fall off the wooden plank after standing on it for less than 2 minutes 4 Trying to stand on the wooden planks but falling off within seconds 5 No ability to stand 6

[0246] 3. Test results

[0247] (1) Effects of cryopreserved human umbilical cord mesenchymal stem cells on body weight (g) in rats with permanent cerebral ischemia

[0248] The results are shown in Table 6. From day 1 to 14 after modeling, the body weight of the animals in the vehicle control group decreased significantly, with a statistically significant difference compared to the sham-operated group (P < 0.001). Stem cell therapy was administered three times, starting one day after modeling. Compared to the vehicle control group, the experimental groups showed varying degrees of weight gain, with significant improvement observed from day 6 to 13 of treatment (P < 0.05).

[0249] Table 6. Effects of cryopreserved human umbilical cord mesenchymal stem cells on body weight (g) in rats with permanent cerebral ischemia

[0250] time Quantity (pieces) Sham operation group Vehicle control group experimental group Before modeling 16 251.0±3.4 254.5±6.6 255.5±4.9 Before treatment 16 237.2±3.8 <![CDATA[209.4±12.2 ### ]]> 205.8±7.4 D2 after treatment 16 256.6±4.5 <![CDATA[187.2±9.1 ### ]]> 190.5±15.5 D6 after treatment 16 290.6±7.8 <![CDATA[163.7±15.7 ### ]]> <![CDATA[181.9±22.9 * ]]> D10 after treatment 8 312.3±12.7 <![CDATA[163.3±25.4 ### ]]> <![CDATA[196.8±34.5 * ]]> D13 after treatment 8 333.0±16.6 <![CDATA[161.6±23.4 ### ]]> <![CDATA[205.5±41.1 * ]]>

[0251] Note: Compared with the sham operation group: ### P<0.001; compared with the vehicle control group: * P<0.05.

[0252] (2) Effects of cryopreserved human umbilical cord mesenchymal stem cells on BBT (scores) in rats with permanent cerebral ischemia

[0253] The results are shown in Table 7. The results showed that from 1 to 14 days after modeling, animals in the vehicle control group showed significant impairment in balance, with significant differences compared to the sham-operated group (P < 0.001). With the passage of time after modeling, BBT scores slightly recovered. Stem cell therapy was administered three times, starting 1 day after modeling. Compared to the vehicle control group, the scores in the experimental groups improved to varying degrees from 2 to 13 days after treatment, with significant differences observed from 2 to 13 days (P < 0.05).

[0254] Table 7. Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on BBT (scores) in rats with permanent cerebral ischemia (mean ± SD, n = 8-16)

[0255]

[0256] Note: Compared with the sham operation group: ### P<0.001; compared with the vehicle control group: * P<0.05.

[0257] (3) Effects of cryopreserved human umbilical cord mesenchymal stem cells on infarct size in rats with permanent cerebral ischemia

[0258] The results are shown in Table 8. The results showed that significant infarct areas were observed in the brain tissue of animals in the vehicle control group. Compared with the vehicle control group, the extent of cerebral infarction in the experimental group improved to varying degrees on day 13. The improvement rate was 23.5% on day 6 (P = 0.0650) and 24.4% on day 13 (P < 0.01). The formula for calculating the infarct area (%) on day 6 of treatment was as follows: Infarct area (%) = (Infarct area / Total brain area) × 100%. The formula for calculating the infarct area (%) on day 13 of treatment was as follows: Infarct area (%) = (Area of ​​the healthy hemisphere - Normal brain area on the infarcted side) / (Area of ​​the healthy hemisphere × 2) × 100%.

[0259] Table 8. Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on the extent of cerebral infarction (%) in rats with permanent cerebral ischemia (mean ± SD, n = 8)

[0260]

[0261] Note: Compared with the sham operation group: ### P<0.001; compared with the vehicle control group: ** P<0.01.

[0262] (4) Effects of cryopreserved human umbilical cord mesenchymal stem cells on LFB in the corpus callosum surrounding the infarcted area in rats with permanent cerebral ischemia

[0263] The results are as follows Figure 7The results are shown in Table 9. The results showed that stem cell treatment was given three times starting from 1 day after model establishment. Compared with the vehicle control group, the experimental group could significantly improve the demyelinating disease of the corpus callosum in the peripheral area of ​​cerebral infarction at 6 days and 13 days after treatment (P<0.01-0.001).

[0264] Table 9. Effects of cryopreserved human umbilical cord mesenchymal stem cells on the myelin LFB (% Area) in the corpus callosum surrounding the infarct in rats with permanent cerebral ischemia (mean ± SD, n = 8)

[0265]

[0266]

[0267] Note: Compared with the sham operation group: ### P<0.001; compared with the vehicle control group: ** P<0.01, *** P<0.001.

[0268] Example 8: Effectiveness Test of Cryopreserved Human Umbilical Cord Mesenchymal Stem Cell Preparation in Treating Subacute Spinal Cord Injury

[0269] 1. Experimental animals and groups

[0270] Thirty male 6-8 week old SPF grade SD rats were purchased from Spefoc (Beijing) Biotechnology Co., Ltd. and kept in the Experimental Animal Center of Youji (Tianjin) Pharmaceutical Technology Co., Ltd. The experiment was carried out after the adaptation period.

[0271] 2. Test methods

[0272] A spinal cord injury (SCI) model was established in SD rats using the Allen's method. The rats were randomly divided into two groups (10 rats each) using block randomization, and a sham-operated group (rats in the sham-operated group underwent only a simulated surgical procedure) with 10 rats. Each group was treated as follows: In the experimental group, the SCI model rats were injected with a cryopreserved mesenchymal stem cell preparation (the cryopreserved mesenchymal stem cell preparation prepared in Group E1 in Example 4) via the tail vein.

[0273] Vehicle control group: an equal volume of cryoprotectant was injected into the tail vein of SCI model rats.

[0274] Sham-operated group: The rats in the sham-operated group were injected with an equal volume of 0.9% sodium chloride injection into the tail vein.

[0275] The rats in each group were given the drug twice, 72 hours and 7 days after model establishment, with a volume of 400 μL per rat. The dose (single dose) of the experimental group was 3×10 7The following indicators were tested at different times: spinal cord injury BBB behavioral score and spinal cord tissue pathology diagnosis. The BBB behavioral score criteria for spinal cord injury are as follows:

[0276] 0 points: no visible hindlimb movement.

[0277] 1 point: Slight movement in one or two joints, usually the hip and / or knee.

[0278] 2 points: One joint has wide range of motion or one joint has wide range of motion and the other joint has slight motion.

[0279] 3 points: Both joints have wide range of motion.

[0280] 4 points: All three joints of the hind limbs can move slightly.

[0281] 5 points: Two joints can move slightly, and the third joint can move widely.

[0282] 6 points: Two joints have wide range of motion, and the third joint can move slightly.

[0283] 7 points: All three joints of the hind limbs can move widely.

[0284] 8 points: The paw can touch the ground without bearing weight.

[0285] 9 points: Occasionally the palmar surface of the claw bears weight and moves, or the dorsal surface of the claw bears weight and moves, but there is no palmar surface of the claw supporting movement.

[0286] 10 points: Occasional weight-bearing movement of the palmar surface of the paw; no coordinated movement of the forelimbs and hindlimbs.

[0287] 11 points: The palmar surface can be seen to bear weight more often, but there is no coordinated movement of the front and back limbs.

[0288] 12 points: Weight-bearing movement of the palm can be seen more often, and coordinated movements of the front and back limbs can be seen occasionally.

[0289] 13 points: Common weight-bearing movements of the palm surface, and coordinated movements of the forelimbs and hindlimbs are common.

[0290] 14 points: There are continuous weight-bearing movements of the palm surface and coordinated movements of the forelimbs and hindlimbs; or there are common palm surface movements, continuous coordinated movements of the forelimbs and hindlimbs, and occasional dorsal movements.

[0291] 15 points: Continuous palmar movement and coordinated forelimb and hindlimb movements, with no or occasional forelimb grasping during forward movement; the active paw is parallel to the body at initial contact.

[0292] 16 points: Continuous palmar movement and coordinated forelimb and hindlimb movements are visible in the gait, and claws are often seen during the forward movement of the forelimbs; the active claw is parallel to the body at initial contact and rotates after weight transfer.

[0293] 17 points: Continuous palmar movement and coordinated forelimb and hindlimb movements are visible in the gait, and claws are often seen during the forward movement of the forelimbs; the active claw position is parallel to the body at initial contact and after weight transfer.

[0294] 18 points: Continuous palmar movement and coordinated forelimb and hindlimb movements are visible in the gait, and the forelimbs can continuously grasp the ground during forward movement; the active paw position is parallel to the body at initial contact, and rotates after weight transfer.

[0295] 19 points: Gait shows continuous palmar movement and coordinated forelimb and hindlimb movements, with sustained claw grip during forelimb forward movement; active claw position is parallel to the body at initial contact and after weight transfer; tail is sometimes or always drooping.

[0296] 20 points: Continuous palmar movement, continuous coordinated gait, continuous toe grip, active paw position parallel to the body at initial contact and after weight transfer, trunk instability, tail continuously raised.

[0297] 21 points: Continuous palmar movement, continuous coordinated gait, continuous toe grip, active paw position always parallel to the body during movement, continuous trunk stability, and continuous tail lift.

[0298] 3. Test results

[0299] 1) The effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on the BBB score after spinal cord injury in rats are shown in Table 10. The results showed that the BBB score of rats in the sham-operated group was normal, while the BBB score of rats in the vehicle control group was 0 after model establishment (before drug administration), which was significantly lower than that in the sham-operated group (P < 0.001), indicating successful model establishment. Compared with the vehicle control group, the experimental group significantly improved the BBB score (P < 0.05).

[0300] Table 10. Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on BBB scores after spinal cord injury in rats (x±s, n=10)

[0301]

[0302] Note: Compared with the sham operation group: *** P<0.001; compared with the vehicle control group: * P<0.05.

[0303] 2) Effect of cryopreserved human umbilical cord mesenchymal stem cell preparations on spinal cord histopathology after spinal cord injury in rats

[0304] The results are as follows Figure 8Nissl staining results showed that compared with the vehicle control group, the experimental group effectively improved the size and number of Nissl bodies in neurons. H&E staining results showed that compared with the vehicle control group, the experimental group effectively alleviated the swelling and degeneration of white matter nerve fibers, focal or small patchy necrosis, or the formation of "cavities," and alleviated the disappearance of pyknotic nuclei or degeneration and swelling in the spinal cord gray matter or some neurons.

[0305] Example 9: Secretory Potential of Activated Human Umbilical Cord Mesenchymal Stem Cells

[0306] PGE2 is an important paracrine factor that affects the biological properties and therapeutic effects of MSCs (Ke, Chen; Human umbilical cord mesenchymal stem cells hUC-MSCs exert immunosuppressive activities through a PGE2-dependent mechanism. 10.1016 / j.clim.2010.01.015). To test the biological properties and therapeutic effects of human umbilical cord mesenchymal stem cells prepared by the present invention, the P6 mesenchymal stem cells described in Example 1 were used for the following experiments:

[0307] Activation group: discard the supernatant, add phosphate buffer to the cell culture flask to wash 2-3 times, and then add activation medium (0.2-0.3mL / cm 2 ), and after labeling, cultured in a carbon dioxide incubator at 37°C and 5% CO2 for 24 hours. The supernatant was collected, centrifuged, and the PGE2 content in the supernatant was detected using an ELISA kit.

[0308] Control group: discard the supernatant, add phosphate buffer to the cell culture flask and wash 2-3 times, then add complete culture medium (0.2-0.3mL / cm 2 ), and after labeling, cultured in a carbon dioxide incubator at 37°C and 5% CO2 for 24 hours. The supernatant was collected, centrifuged, and the PGE2 content in the supernatant was detected using an ELISA kit.

[0309] The results are as follows Figure 9 The results showed that compared with the control group, the activated human umbilical cord mesenchymal stem cells in the activated group secreted significantly more PGE2 ( ** P<0.01).

[0310] Example 10: Effectiveness Test of Cryopreserved Human Umbilical Cord Mesenchymal Stem Cell Preparations in Treating Acute Respiratory Distress Syndrome

[0311] 1. Experimental animals and groups

[0312] Forty-two 6-8 week old SPF-grade C57BL / 6J mice, half male and half female, were purchased from Spefox (Beijing) Biotechnology Co., Ltd. and raised in the Experimental Animal Center of Tianjin International Joint Institute of Biomedicine. The experiment was carried out after the adaptation period.

[0313] 2. Test methods

[0314] The ALI / ARDS model was established in C57BL / 6 mice by bronchial instillation of lipopolysaccharide (LPS). The mice were randomly divided into three groups using the block randomization method, with 14 mice in each group. The treatment methods for each group were as follows:

[0315] Experimental group: ALI / ARDS model mice were injected with a cryopreserved mesenchymal stem cell preparation (the cryopreserved mesenchymal stem cell preparation prepared in Group E1 in Example 4) into the tail vein.

[0316] Vehicle control group: An equal volume of cryoprotectant was injected into the tail vein of ALI / ARDS model mice.

[0317] Model group: ALI / ARDS model mice were injected with an equal volume of 0.9% sodium chloride injection via the tail vein.

[0318] The frequency of administration for each group of mice was 2 times (1 time / 3 days), the administration volume was 100ul / mouse, and the dosage of the experimental group (single dose) was 1×10 6 The observation period was 4 days. The efficacy of MSCs in the experimental ALI / ARDS model was examined, including changes in body weight, expression of serum inflammatory factors (IL-1β, IL-10, TNF-α, PGE2, IL-6), and myeloperoxidase (MPO) activity.

[0319] 3. Test results

[0320] 1) Effect of cryopreserved human umbilical cord mesenchymal stem cell preparations on the body weight of ALI / ARDS model mice

[0321] The results are as follows Figure 10 The results are shown in Table 11. The experimental group showed a relatively stable and positively distributed body weight, with no abnormal fluctuations. The model and vehicle control groups showed abnormal fluctuations, with a non-positive distribution and a downward trend, and both were significantly lower than the experimental group (P < 0.05). In summary, the experimental group alleviated the weight loss of LPS-induced ALI / ARDS mice to a certain extent.

[0322] Table 11. Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on the body weight of ALI / ARDS model mice

[0323]

[0324]

[0325] 2) Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on the expression of serum inflammatory factors in ALI / ARDS model mice

[0326] The results are as follows Figure 11 As shown in Table 12. The results showed that human umbilical cord mesenchymal stem cell cryopreservation preparations had a certain protective effect on LPS-induced ALI / ARDS mice. The mechanism may be that human umbilical cord mesenchymal stem cell cryopreservation preparations transplantation downregulated the levels of pro-inflammatory factors (IL-1β, IL-6) and upregulated the production of anti-inflammatory factors (IL-10) and PGE2, thereby changing the lung environment from a pro-inflammatory response to an anti-inflammatory response, and maintaining a balance between inflammatory and anti-inflammatory mediators in the lungs, thereby preventing the further progression of the inflammatory response in the lungs.

[0327] Table 12. Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on the expression of serum inflammatory factors in ALI / ARDS model mice (mean ± SD)

[0328]

[0329] 3) Effect of cryopreserved human umbilical cord mesenchymal stem cell preparations on the expression of MPO activity in ALI / ARDS model mice

[0330] The results are as follows Figure 12 The results are shown in Table 13. The results showed that the MPO activity in the model group and the vehicle control group was significantly increased, while the MPO activity in the experimental group after transplantation of cryopreserved human umbilical cord mesenchymal stem cell preparation was significantly lower than that in the model group and the vehicle control group ( **** P<0.0001). This result indicates that after treatment with cryopreserved human umbilical cord mesenchymal stem cell preparation, MPO activity in the serum of ALI / ARDS mice was significantly reduced, thereby alleviating the degree of lung inflammation in the mice.

[0331] Table 13. Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on serum MPO activity in ALI / ARDS model mice (mean ± SD)

[0332] Group MPO(U / L) Model Group 95.187±14.542 Vehicle control group 78.704±9.369 experimental group 50.588±9.285

[0333] Example 11: Effectiveness Test of Cryopreserved Human Umbilical Cord Mesenchymal Stem Cell Preparations in Treating Liver Failure

[0334] 1. Experimental animals and groups

[0335] Thirty-six 3-week-old SPF-grade NOD / SCID mice, half male and half female, were purchased from Beijing Huafukang Biotechnology Co., Ltd. and raised in the Experimental Animal Center of the Institute of Hematology, Chinese Academy of Medical Sciences. The experiment was carried out after the adaptation period.

[0336] 2. Test methods

[0337] A NOD / SCID mouse liver failure model was established using carbon tetrachloride (CCl4) and randomly divided into three groups using the block randomization method, with 12 mice in each group. The treatment methods for each group were as follows:

[0338] Experimental group: The NOD / SCID model mice were injected with a cryopreserved mesenchymal stem cell preparation (the cryopreserved mesenchymal stem cell preparation prepared in Group E1 in Example 4) into the tail vein.

[0339] Vehicle control group: an equal volume of cryoprotectant was injected into the tail vein of NOD / SCID model mice.

[0340] Model group: NOD / SCID model mice were injected with an equal volume of 0.9% sodium chloride injection via the tail vein.

[0341] The frequency of administration for each group of mice was 3 times (1 time / 2 weeks), the administration volume was 400ul / mouse, and the dosage (single dose) of the experimental group was 4×10 6 The experimental observation period was 2 weeks. The efficacy of MSCs in the experimental liver failure model was evaluated, including changes in body weight, survival rate, clinical indicators (liver function, commissioned by Tianjin Aidikang Medical Testing), and pathomorphological scores (scoring criteria are shown in Table 16).

[0342] 3. Test results

[0343] 1) Effect of cryopreserved human umbilical cord mesenchymal stem cell preparations on body weight in liver failure model mice

[0344] The results are shown in Table 14. The results showed that the body weight of the mice in the experimental group was significantly higher than that before treatment (P<0.05), while the body weight of the model group and the vehicle control group gradually decreased and was lower than that before treatment. The experimental group alleviated the weight loss of mice with liver failure induced by CCL4 to a certain extent.

[0345] Table 14. Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on body weight in liver failure model mice (g, mean ± SD)

[0346]

[0347] 2) Effect of cryopreserved human umbilical cord mesenchymal stem cell preparations on the survival of liver failure model mice

[0348] The results are as follows Figure 14The results are shown in Table 15. The results showed that the animals in the model group began to die on the 7th day of treatment. At the same time, the total number of deaths in the model group and the vehicle control group in the late treatment period was 4 and 3, respectively. All animals in the experimental group survived without abnormal deaths. Combined with the clinical observation experimental results, the experimental group improved the survival rate of mice with liver failure to a certain extent and prolonged their survival time.

[0349] Table 15. Number of surviving mice in liver failure model at different times after injection of cryopreserved human umbilical cord mesenchymal stem cell preparations (n ​​= 1)

[0350] Group Number of animals 0 weeks 2 weeks 4 weeks 6 weeks Model Group 12 12 11 9 8 Vehicle control group 12 12 12 10 9 experimental group 12 12 12 12 12

[0351] 3) Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on serum factor levels in liver failure model mice

[0352] Liver function is an effective indicator for detecting whether the liver is damaged and the degree of damage, mainly including albumin (ALB), aspartate aminotransferase (AST) and total bilirubin (TBL). The test results of liver function indicators are as follows Figure 13 As shown. The results showed that: for ALB values, compared with the model group, the ALB value content of the test group was higher, and the liver albumin synthesis function of the liver failure mice was improved after treatment. For AST values, compared with the model group, the AST content of the test group was significantly reduced. After treatment in the treatment group (high dose), no necrosis of hepatocytes in the liver of the liver failure mice occurred, and no abnormal release of aspartate aminotransferase in the cytoplasm occurred. For TBL values, compared with the model group, the TBL of the test group was relatively low. In summary, after the liver failure model was given drug treatment in the test group, the liver function of the mice was improved and recovered to a certain extent.

[0353] 3) Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on liver histopathological scores and morphology in mice with liver failure

[0354] Table 16. Pathology scoring criteria

[0355] Scoring indicators 3 points 2 points 1 point 0 points Integrity of liver lobule structure severe Moderate Mild / partial whole Hepatocellular edema (water degeneration) severe Moderate Mild / partial none Liver World Edition severe Moderate Mild / partial normal Inflammatory cell infiltration severe Moderate Mild / small amount none Hepatocellular ballooning / / have none

[0356] The results are as follows Figure 15 and Figure 16 The results showed that inflammatory cell infiltration, hepatocyte degeneration, and severe necrosis were observed in the liver tissue of the model group and the vehicle control group. After treatment, the pathological score of the experimental group was significantly lower than that of the model group and the vehicle control group (P<0.0001). The morphology of the liver tissue cells in the experimental group was normal, with the hepatic plates radiating outward in a cord-like pattern centered on the central vein, and the hepatic lobule structure was intact.

[0357] Example 12: Effectiveness Test of Cryopreserved Human Umbilical Cord Mesenchymal Stem Cell Preparations in Treating Graft-Versus-Host Disease

[0358] 1. Experimental animals and groups

[0359] Twenty male 7-8 week old SPF BALB / c mice were purchased from the Institute of Laboratory Animals, Chinese Academy of Medical Sciences and raised at Beijing Zhaoyan New Drug Research Center Co., Ltd. The experiment was carried out after the adaptation period.

[0360] 2. Test methods

[0361] 1×10 bone marrow and spleen cells were isolated from C57BL / 6 mice 7 implanted with 10.0 Gy 60 A mouse aGVHD model was established in BALB / c mice irradiated with Coγ-rays.

[0362] The animals were randomly divided into 3 groups using block randomization, with 10 animals in each group. The treatment methods for each group were as follows:

[0363] Experimental Group 1: A cryopreserved mesenchymal stem cell preparation (the cryopreserved mesenchymal stem cell preparation prepared in Group E1 in Example 4) was injected into the tail vein of aGVHD model mice twice (on the 6th and 12th days after model establishment).

[0364] Experimental Group 2: The aGVHD model mice were injected with cryopreserved mesenchymal stem cells (the cryopreserved mesenchymal stem cells prepared in Group E1 in Example 4) into the tail vein twice (on the 12th and 24th days after model establishment).

[0365] Model group: aGVHD model mice without any treatment.

[0366] The administration volume of experimental group 1 and experimental group 2 was 0.1 ml / 10 g body weight, and the administration dose (single dose) was 1×10 6 The experimental observation period was 28 days. The efficacy of MSCs on aGVHD model mice was observed, including weight changes, survival rate, and clinical index scores (scoring criteria are shown in Table 17).

[0367] Table 17. Clinical index scores after treatment of mouse aGVHD model

[0368]

[0369]

[0370] 3. Test results

[0371] 1) Effect of cryopreserved human umbilical cord mesenchymal stem cell preparations on the body weight of aGVHD model mice

[0372] The results are shown in Table 18. The results showed that the body weight of each group gradually decreased after model establishment, with the largest decrease on D7, followed by a slight recovery, which lasted for about a week before continuing to decrease.

[0373] Table 18. Effect of cryopreserved human umbilical cord mesenchymal stem cell preparations on the body weight of aGVHD model mice (g, mean ± SD)

[0374] Group Number of animals D0 D7 D14 D21 D28 Model Group 10 18.14±1.05 13.60±0.85 16.40 - - Experimental group 1 9 18.33±0.60 12.78±1.12 15.25±0.07 13.05±1.06 - Experimental Group 2 8 18.93±0.82 13.89±0.86 16.09±1.65 12.98±1.15 -

[0375] 2) Effect of cryopreserved human umbilical cord mesenchymal stem cell preparations on the survival of aGVHD model mice

[0376] The results are shown in Table 19. The results showed that animals in the model group began to die after day 3, with 9 animals dead by day 7, 1 surviving by day 14, and all animals dead by day 21. Meanwhile, the total deaths in experimental groups 1 and 2 were 3 and 0, respectively, on day 7 after treatment, 7 and 2, respectively, on day 14, and 7 and 5, respectively, on day 21. All animals had died by day 28. Experimental groups 1 and 2 improved the survival rate and prolonged the survival of aGVHD model mice to a certain extent.

[0377] Table 19. Number of surviving mice in aGVHD model at different times after injection of cryopreserved human umbilical cord mesenchymal stem cells (n = 1)

[0378] Group Number of animals D1 D3 D7 D14 D21 D28 Model Group 10 10 10 1 1 0 0 Experimental group 1 9 9 9 6 2 2 0 Experimental Group 2 8 8 8 8 7 3 0

[0379] 3) Effects of cryopreserved human umbilical cord mesenchymal stem cell preparations on clinical index scores in aGVHD model mice

[0380] The results are shown in Table 20. The results show that the clinical scores of animals in each group gradually increased from model establishment to D7, decreased on D14, confirming that the clinical manifestations were alleviated, but increased again on D21 until death on D28.

[0381] Table 20. Clinical index scores of aGVHD model mice after injection of cryopreserved human umbilical cord mesenchymal stem cell preparation (mean ± SD)

[0382] Group Number of animals D4 D7 D14 D21 D28 Model Group 10 3.30±0.48 7.00±0.00 5.00 - - Experimental group 1 9 3.90±0.32 7.33±0.52 5.00 9.00 - Experimental Group 2 8 4.00±0.00 6.90±0.64 4.43±0.53 9.00 -

[0383] Example 13: Effectiveness Test of Cryopreserved Human Umbilical Cord Mesenchymal Stem Cell Preparations in Treating Diabetic Nephropathy

[0384] 1. Experimental animals and groups

[0385] Twenty-one male SPF SD rats aged 5-7 weeks were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and raised in Tianjin Weijia Pharmaceutical Technology Co., Ltd. The experiment was carried out after the adaptation period.

[0386] 2. Test methods

[0387] Mice with diabetic nephropathy were prepared by a single intraperitoneal injection of 60 mg / kg streptozotocin (STZ) (Sigma-Aldrich, S0130) in sodium citrate buffer (0.1 M, pH 4.5). A control group of mice was prepared by injecting an equal volume of sodium citrate buffer in the same manner.

[0388] The patients were randomly divided into 3 groups using block randomization. The treatment method for each group was as follows:

[0389] Treatment group: The diabetic nephropathy model group mice were injected with the cryopreserved mesenchymal stem cell preparation (the cryopreserved mesenchymal stem cell preparation prepared in Group E1 in Example 4) into the tail vein.

[0390] Model group: The diabetic nephropathy model group mice were injected with an equal volume of cryoprotectant through the tail vein.

[0391] Control group: mice in the control group were injected with an equal volume of cryoprotectant via the tail vein.

[0392] The mice in each group were given the drug three times, which were injected into the tail vein once a week for three consecutive weeks after the diabetic nephropathy model was successfully established (i.e., 24h urine protein was greater than or equal to 30mg). The dosage was based on the body weight of each animal. The treatment group (single dose) was 1×10 7 Urine was collected at different times using metabolic cages for urine protein and serum creatinine testing. Urine protein: Urine was collected weekly in metabolic cages for 24 hours after dosing, and the urine protein content was determined using a urine protein quantification kit (CBB method) (Nanjing Jiancheng Bioengineering Institute, C035-2-1). Serum creatinine: Blood was collected from the jugular vein weekly after dosing, centrifuged at 4°C, 4000 rpm for 10 minutes, and the upper serum was collected. Serum creatinine content was determined using a creatinine (CRE) assay kit (sarcosine oxidase method, microplate method) (Nanjing Jiancheng Bioengineering Institute, C011-2-1).

[0393] 3. Test results

[0394] 1) The results of the effect of cryopreserved human umbilical cord mesenchymal stem cells on urine protein in diabetic nephropathy rats are as follows Figure 17 The results showed that compared with the model group, the urine protein content of the rats in the treatment group decreased significantly 2 weeks after administration (P < 0.05).

[0395] 2) Effect of cryopreserved human umbilical cord mesenchymal stem cell preparation on serum creatinine in rats with diabetic nephropathy

[0396] The results are as follows Figure 18The results showed that compared with the model group, the serum creatinine level of the rats in the treatment group decreased significantly 3 weeks after administration (P<0.05).

[0397] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for preparing human umbilical cord mesenchymal stem cells, comprising the following steps: (1) digesting and screening the isolated umbilical cord tissue pieces and then culturing them until the cell confluence reaches 60%-80%, thereby obtaining P0 mesenchymal stem cells; (2) subculturing the P0 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P1 generation mesenchymal stem cells; (3) subculturing the P1 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P2 generation mesenchymal stem cells; (4) subculturing the P2 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P3 generation mesenchymal stem cells; (5) freezing the P3 mesenchymal stem cells to obtain a cell seed bank; (6) resuscitating the cells from the cell seed bank and performing subculture until the cell confluence reaches 70%-90%, thereby obtaining P4 mesenchymal stem cells; (7) subculturing the P4 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P5 generation mesenchymal stem cells; (8) subculturing the P5 generation mesenchymal stem cells until the cell confluence reaches 70%-90%, thereby obtaining P6 generation mesenchymal stem cells; (9) activating and culturing the P6 generation mesenchymal stem cells to obtain activated P6 generation mesenchymal stem cells; the activation culture medium does not contain serum and contains ascorbic acid, α-tocopheryl acetate, vitamin A, palmitic acid, reduced glutathione, and 4-hydroxyethylpiperazineethanesulfonic acid; (10) Resuspending the activated P6 mesenchymal stem cells in a cell protectant to obtain the human umbilical cord mesenchymal stem cell original suspension.

2. The method according to claim 1, wherein: In the steps (1), (2), (3), (4), (6), (7) and (8), complete culture medium is used for cell culture; Alternatively, the complete culture medium consists of DMEM / F12 basal medium and serum replacement; Alternatively, the volume ratio of the DMEM / F12 basal medium to the serum replacement is 9:

1.

3. The method according to claim 1 or 2, characterized in that: In the step (5), the step of resuspending the P3 mesenchymal stem cells with a cryopreservation protection solution to obtain a cell suspension is also included before the cryopreservation; Alternatively, the cryopreservation protection solution consists of a serum substitute and dimethyl sulfoxide; Or, the volume ratio of the serum substitute to the dimethyl sulfoxide is 9:1; Alternatively, the concentration of the cell suspension is (2-4)×10 6 cells / mL.

4. The method according to any one of claims 1 to 3, characterized in that: In the step (9), the concentration of ascorbic acid in the activation medium is 45-55 mg / L; The concentration of α-tocopheryl acetate in the activation medium is 0.3-0.5 mg / L; The concentration of vitamin A in the activation medium is 1.0-1.5 mg / L; The concentration of palmitic acid in the activation medium is 4.5-5.5 mg / L; The concentration of the reduced glutathione in the activation medium is 0.5-0.7 mg / L; The concentration of the 4-hydroxyethylpiperazineethanesulfonic acid in the activation medium is 3200-3900 mg / L; Alternatively, the activation medium further comprises glycine, alanine, glutamine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, vitamin H, choline chloride, calcium pantothenate, folic acid, nicotinamide, vitamin B6, vitamin B2, vitamin B1, vitamin B12, inositol, calcium chloride, copper sulfate, ferric nitrate, ferric sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, zinc sulfate, D-glucose, hypoxanthine, linoleic acid, lipoic acid, tetramethylethylenediamine, sodium pyruvate, and thymidine; Alternatively, the concentration of glycine in the activation medium is 18.75 mg / L; The concentration of alanine in the activation medium is 4.45 mg / L; The concentration of glutamine in the activation medium is 542 mg / L; The concentration of arginine in the activation medium is 147.5 mg / L; The concentration of asparagine in the activation medium is 7.5 mg / L; The concentration of aspartic acid in the activation medium is 6.65 mg / L; The concentration of cysteine ​​in the activation medium is 35.12 mg / L; The concentration of cystine in the activation medium is 31.29 mg / L; The concentration of glutamic acid in the activation medium is 7.35 mg / L; The concentration of histidine in the activation medium is 31.48 mg / L; The concentration of isoleucine in the activation medium is 54.47 mg / L; The concentration of leucine in the activation medium is 59.05 mg / L; The concentration of lysine in the activation medium is 91.25 mg / L; The concentration of methionine in the activation medium is 17.24 mg / L; The concentration of phenylalanine in the activation medium is 35.48 mg / L; The concentration of proline in the activation medium is 17.25 mg / L; The concentration of serine in the activation medium is 26.25 mg / L; The concentration of threonine in the activation medium is 53.45 mg / L; The concentration of tryptophan in the activation medium is 9.02 mg / L; The concentration of tyrosine in the activation medium is 55.79 mg / L; The concentration of valine in the activation medium is 52.85 mg / L The concentration of vitamin H in the activation medium is 0.0035 mg / L; The concentration of choline chloride in the activation medium is 8.98 mg / L; The concentration of calcium pantothenate in the activation medium is 2.24 mg / L; The concentration of folic acid in the activation medium is 2.65 mg / L; The concentration of nicotinamide in the activation medium is 2.02 mg / L; The concentration of vitamin B6 in the activation medium is 2.031 mg / L; The concentration of vitamin B2 in the activation medium is 0.219 mg / L; The concentration of vitamin B1 in the activation medium is 2.17 mg / L; The concentration of vitamin B12 in the activation medium is 0.68 mg / L; The concentration of the inositol in the activation medium is 12.6 mg / L; The concentration of calcium chloride in the activation medium is 116.6 mg / L; The concentration of copper sulfate in the activation medium is 0.0013 mg / L; The concentration of the ferric nitrate in the activation medium is 0.05 mg / L; The concentration of the ferric sulfate in the activation medium is 0.417 mg / L; The concentration of magnesium chloride in the activation medium is 28.64 mg / L; The concentration of magnesium sulfate in the activation medium is 48.84 mg / L; The concentration of potassium chloride in the activation medium is 311.8 mg / L; The concentration of sodium bicarbonate in the activation medium is 4876 mg / L; The concentration of sodium chloride in the activation medium is 6999.5 mg / L; The concentration of the sodium hydrogen phosphate in the activation medium is 71.02 mg / L; The concentration of sodium dihydrogen phosphate in the activation medium is 62.5 mg / L; The concentration of zinc sulfate in the activation medium is 0.432 mg / L The concentration of D-glucose in the activation medium is 3151 mg / L; The concentration of hypoxanthine in the activation medium is 2.39 mg / L; The concentration of the linoleic acid in the activation medium is 0.042 mg / L; The concentration of the lipoic acid in the activation medium is 0.105 mg / L; The concentration of tetraethylenediamine in the activation medium is 0.081 mg / L; The concentration of sodium pyruvate in the activation medium is 165 mg / L; The concentration of thymine in the activation medium is 0.365 mg / L; In the step (10), the cell protectant is composed of a human albumin solution and a compound electrolyte injection; Alternatively, the final concentration of the human serum albumin in the cell protectant is 0.5%-1%.

5. Human umbilical cord mesenchymal stem cells prepared according to the method of any one of claims 1 to 4. A human umbilical cord mesenchymal stem cell cryopreservation preparation comprising the human umbilical cord mesenchymal stem cells according to claim 5 and a cryoprotectant.

7. The cryopreserved human umbilical cord mesenchymal stem cell preparation according to claim 6, wherein: The cryoprotectant comprises compound electrolyte injection, dimethyl sulfoxide and human albumin solution; Alternatively, the volume ratio of the compound electrolyte injection, the dimethyl sulfoxide, and the human albumin solution is 65:10:25; Alternatively, the concentration of the human umbilical cord mesenchymal stem cells in the human umbilical cord mesenchymal stem cell cryopreservation preparation is (3-6)×10 6 cells / mL.

8. A method for preparing the cryopreserved human umbilical cord mesenchymal stem cell preparation according to claim 6 or 7, comprising the steps of: resuspending the human umbilical cord mesenchymal stem cells according to claim 5 with a cryoprotectant to obtain a cell suspension; and freezing the cell suspension to obtain the cryopreserved human umbilical cord mesenchymal stem cell preparation.

9. A set of reagents comprising the complete culture medium according to any one of claims 1 to 4, the activation culture medium according to any one of claims 1 to 4, the cryoprotective solution according to any one of claims 1 to 4, and the cell preservation solution according to any one of claims 1 to 4.

10. Application in any of the following M1)-M8): M1) Use of the human umbilical cord mesenchymal stem cells according to claim 5 in the preparation of the human umbilical cord mesenchymal stem cell cryopreservation preparation according to claim 6 or 7; M2) Use of the kit of reagents according to claim 9 in the preparation of the human umbilical cord mesenchymal stem cells according to claim 5 or the human umbilical cord mesenchymal stem cell cryopreservation preparation according to claim 6 or 7; M3) Use of the human umbilical cord-derived mesenchymal stem cells according to claim 5, or the human umbilical cord mesenchymal stem cell cryopreservation preparation according to claim 6 or 7, or the human umbilical cord mesenchymal stem cell cryopreservation preparation prepared according to the method of claim 8 in the preparation of a product for the treatment or adjuvant treatment of ischemic stroke; M4) Use of the human umbilical cord-derived mesenchymal stem cells according to claim 5, or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation according to claim 6 or 7, or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation prepared according to the method of claim 8 in the preparation of a product for treating or assisting in the treatment of spinal cord injury; M5) Use of the human umbilical cord-derived mesenchymal stem cells according to claim 5, or the human umbilical cord mesenchymal stem cell cryopreservation preparation according to claim 6 or 7, or the human umbilical cord mesenchymal stem cell cryopreservation preparation prepared according to the method of claim 8 in the preparation of a product for preventing and / or treating graft-versus-host disease; M6) Use of the human umbilical cord-derived mesenchymal stem cells according to claim 5, or the human umbilical cord mesenchymal stem cell cryopreservation preparation according to claim 6 or 7, or the human umbilical cord mesenchymal stem cell cryopreservation preparation prepared according to the method of claim 8 in the preparation of a product for the treatment or auxiliary treatment of liver failure; M7) Use of the human umbilical cord-derived mesenchymal stem cells according to claim 5, or the human umbilical cord mesenchymal stem cell cryopreservation preparation according to claim 6 or 7, or the human umbilical cord mesenchymal stem cell cryopreservation preparation prepared according to the method of claim 8 in the preparation of a product for treating or assisting in the treatment of diabetic nephropathy; M8) Use of the human umbilical cord-derived mesenchymal stem cells described in claim 5, the human umbilical cord mesenchymal stem cell cryopreservation preparation described in claim 6 or 7, or the human umbilical cord mesenchymal stem cell cryopreservation preparation prepared according to the method of claim 8 in the preparation of a product for the treatment or auxiliary treatment of acute respiratory distress syndrome.

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