Umbilical cord mesenchymal stem cell efficient preparation kit

Abstract

The present invention belongs to the field of stem cell technology and relates to a kit for efficiently preparing umbilical cord mesenchymal stem cells. The kit comprises umbilical cord preservation solution, umbilical cord washing solution, umbilical cord decomposition solution, cell washing solution, cell culture solution, cell digestion solution, and cell freezing solution. The kit of the present invention can efficiently prepare umbilical cord mesenchymal stem cells, obtain a large number of umbilical cord mesenchymal stem cells in a relatively short time, and is convenient for clinical use.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cells and relates to a kit for efficiently preparing umbilical cord mesenchymal stem cells. Background Art

[0002] Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) refer to a type of multipotent stem cells that exist in the umbilical cord tissue of newborns and have the potential for self-renewal and multidirectional differentiation. They can differentiate into a variety of tissue cells such as bone, cartilage, fat, heart, liver, kidney, lung, pancreas, nerve, muscle, etc., covering almost all tissue cells in the human body.

[0003] UC-MSCs can be obtained by tissue block adherence method and collagenase digestion method. The primary culture time of tissue block adherence method is about two to three weeks, which is a long culture time, while the collagenase digestion method can quickly obtain a large number of mesenchymal stem cells that grow on the wall. This method is simple and easy to operate, can better maintain cell vitality, and greatly shortens the primary culture time. The present invention adopts the collagenase digestion method.

[0004] UC-MSCs have the following advantages: 1. The umbilical cord is rich in MSCs; 2. The umbilical cord MSCs are more primitive, with stronger proliferation and differentiation abilities; 3. UC-MSCs can be expanded multiple times to provide a large number of seed cells for clinical treatment, sufficient to meet treatment needs; 4. Low immunogenicity; 5. It has a wide range of clinical applications and great medical prospects: it has certain therapeutic effects on liver fibrosis, cartilage damage, myocardial infarction, diabetes, cirrhosis, lupus erythematosus, Parkinson's syndrome, Alzheimer's disease, spinal cord injury and many other diseases. With the advancement of science and technology, it can be used for the repair and reconstruction of tissues and organs.

[0005] Umbilical cord mesenchymal stem cells have the following functions and uses: 1. Repair damaged or diseased tissues and organs, and are used to treat cartilage damage, cardiovascular and cerebrovascular diseases, liver disease, bone and muscle degenerative diseases, brain and spinal cord nerve damage, and Alzheimer's disease; 2. They have strong immunomodulatory effects and can be used to treat autoimmune diseases such as lupus erythematosus and scleroderma, reduce immune rejection reactions after cell or organ transplantation, and improve the success rate of cell or organ transplantation; 3. Promote hematopoietic recovery function. Compared with single hematopoietic stem cell transplantation, co-transplantation of mesenchymal stem cells and hematopoietic stem cells can significantly improve the therapeutic effect of diseases such as leukemia and refractory anemia.

[0006] Umbilical cord mesenchymal stem cells have extremely broad prospects for clinical application. Therefore, it is urgent to develop a simple, rapid, high-tech and safe method to obtain a large number of umbilical cord mesenchymal stem cells. However, there is no kit for the efficient preparation of umbilical cord mesenchymal stem cells in the existing technology, which makes the relevant scientific research work progress slowly. Summary of the invention

[0007] The purpose of the present invention is to make up for the deficiencies in the prior art and to provide an easy-to-use, low-cost umbilical cord mesenchymal stem cell efficient preparation kit for preparing a large number of umbilical cord mesenchymal stem cells (UC-MSCs) in a short period of time.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is: an umbilical cord mesenchymal stem cell efficient preparation kit, including umbilical cord preservation solution, umbilical cord washing solution, umbilical cord decomposition solution, cell washing solution, cell culture solution, cell digestion solution and cell freezing solution.

[0009] The umbilical cord preservation solution is DMEM, DMEM / F12, MEM or RPMI-1640 culture medium containing 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin, 2-8ug / ml of resveratrol, 2-8ug / ml of icariin, 5-10ng / ml of rapamycin, 2-8ng / ml of trametinib and 2-8ng / ml of ethanolamine.

[0010] The umbilical cord washing solution is physiological saline, PBS, D-Hanks or HBSS containing 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin, 2-8ug / ml of resveratrol, 2-8ug / ml of icariin, 5-10ng / ml of rapamycin, 2-8ng / ml of trametinib and 2-8ng / ml of ethanolamine.

[0011] The umbilical cord decomposition solution is DMEM, DMEM / F12, MEM or RPMI-1640 culture medium containing collagenase II 1-2 mg / ml, deoxyribonuclease I 0.1-0.2 mg / ml, resveratrol 2-8 ug / ml, icariin 2-8 ug / ml, rapamycin 5-10 ng / ml, trametinib 2-8 ng / ml, and ethanolamine 2-8 ng / ml.

[0012] The cell washing solution is PBS, D-Hanks or HBSS containing 2-8ug / ml of resveratrol, 2-8ug / ml of icariin, 5-10ng / ml of rapamycin, 2-8ng / ml of trametinib and 2-8ng / ml of ethanolamine.

[0013] The cell culture medium is Keratinocyte-SFM, a serum-free complete culture medium for human mesenchymal stem cells, DMEM+10% FBS or DMEM / F12+10% FBS, to which resveratrol 2-8 ng / ml, icariin 2-8 ng / ml, and metformin 2-8 ng / ml are additionally added.

[0014] The cell digestion solution is PBS, D-Hanks or HBSS solution containing 1.25-2.5 mg / ml Trypsin-EDTA and 0.1-0.2 mg / ml deoxyribonuclease I.

[0015] The cell freezing medium is Keratinocyte-SFM containing 60-200 mg / ml DMSO, 2-8 ng / ml ethanolamine, 2-8 ng / ml chloroplatinic acid hexahydrate, a serum-free complete medium for human mesenchymal stem cells, DMEM+10% FBS or DMEM / F12+10% FBS.

[0016] Preferably, the umbilical cord preservation solution is a DMEM culture medium containing 200ug / ml penicillin, 200ug / ml streptomycin, 6ug / ml resveratrol, 6ug / ml icariin, 8ng / ml rapamycin, 6ng / ml trametinib, and 6ng / ml ethanolamine.

[0017] Preferably, the umbilical cord washing solution is PBS containing 200ug / ml penicillin, 200ug / ml streptomycin, 6ug / ml resveratrol, 6ug / ml icariin, 8ng / ml rapamycin, 6ng / ml trametinib, and 6ng / ml ethanolamine.

[0018] Preferably, the umbilical cord decomposition solution is a DMEM culture medium containing 2 mg / ml collagenase II, 0.2 mg / ml deoxyribonuclease I, 6 ug / ml resveratrol, 6 ug / ml icariin, 8 ng / ml rapamycin, 6 ng / ml trametinib, and 6 ng / ml ethanolamine.

[0019] Preferably, the cell washing solution is PBS containing 6 ug / ml of resveratrol, 6 ug / ml of icariin, 8 ng / ml of rapamycin, 6 ng / ml of trametinib, and 6 ng / ml of ethanolamine.

[0020] Preferably, the cell culture medium is Keratinocyte-SFM to which 6 ng / ml of resveratrol, 6 ng / ml of icariin and 6 ng / ml of metformin are additionally added.

[0021] Preferably, the cell digestion solution is a PBS solution containing 2 mg / ml Trypsin-EDTA and 0.1 mg / ml deoxyribonuclease I.

[0022] Preferably, the cell freezing solution is Keratinocyte-SFM containing 100 mg / ml DMSO, 6 ng / ml ethanolamine, and 6 ng / ml chloroplatinic acid hexahydrate.

[0023] The present invention provides a kit for efficiently preparing umbilical cord mesenchymal stem cells. In the kit, the function of the umbilical cord preservation solution is to ensure the activity of the umbilical cord mesenchymal stem cells during the transportation of the umbilical cord specimen after collection, and to eliminate and prevent bacteria and fungi. The function of the umbilical cord washing solution is to wash and remove the blood cells of the umbilical cord tissue, ensure the activity of the umbilical cord mesenchymal stem cells, and eliminate and prevent bacteria and fungi. The function of the umbilical cord decomposition solution is to efficiently and quickly digest the umbilical cord tissue without damaging the activity of the umbilical cord mesenchymal stem cells, and obtain a single cell suspension. The function of the cell washing solution is to wash the obtained stem cells without damaging the activity of the umbilical cord mesenchymal stem cells. The function of the cell culture solution is to quickly and efficiently amplify the umbilical cord mesenchymal stem cells. The function of the cell digestion solution is to digest the adherent mesenchymal stem cells during cell passage, while not damaging the activity of the umbilical cord mesenchymal stem cells. The function of the cell freezing solution is to efficiently freeze the obtained umbilical cord mesenchymal stem cells for standby use.

[0024] The umbilical cord mesenchymal stem cell efficient preparation kit of the present invention can obtain a large number of umbilical cord mesenchymal stem cells (the number of cells can reach 10 11 It is easy to use and low-cost, and will play an important role in stem cell clinical trials and related research, with a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention shows an umbilical cord mesenchymal stem cell high-efficiency preparation kit (top view). The following are marked in the figure: 1 cell washing solution area; 2 cell washing solution; 3 cell culture solution; 4 cell culture solution area; 5 cell freezing solution; 6 cell freezing solution area; 7 cell digestion solution area; 8 cell digestion solution; 9 umbilical cord preservation solution; 10 umbilical cord washing solution; 11 umbilical cord decomposition solution.

[0026] Figure 2 The umbilical cord mesenchymal stem cells (p0, 40×) prepared by the present invention are shown.

[0027] Figure 3 The umbilical cord mesenchymal stem cells (P3, 200×) prepared by the present invention are shown.

[0028] Figure 4 The flow cytometry results of umbilical cord mesenchymal stem cells prepared by the present invention are shown. DETAILED DESCRIPTION

[0029] The methods used in the following examples are conventional methods unless otherwise specified, and the required reagents, consumables and experimental instruments can be purchased through commercial channels.

[0030] Example 1 Assembly of a kit for efficiently preparing umbilical cord mesenchymal stem cells

[0031] In the preparation and assembly of the umbilical cord mesenchymal stem cell efficient preparation kit of the present invention, all the required components are commercially available products and can be purchased through commercial channels: penicillin, brand Amresco, item number 0242; streptomycin, brand Amresco, item number 0382; resveratrol, brand Sigma, item number R5010; icariin, Shanghai Microcrystal Biotechnology, item number 489-32-7; rapamycin, brand TargetMol , product number T1537; trametinib, brand Meilun Bio, product number MB5401; ethanolamine, brand Sigma, product number 8008490100; collagenase II, brand sigma, product number C6885; deoxyribonuclease I, brand sigma, product number C4263; metformin, brand, Sigma, product number PHR1084-500MG; DMSO, brand sigma, product number D2650; chloroplatinic acid hexahydrate, brand Sigma, product number 206083.

[0032] 1. Solution preparation

[0033] The kit for efficiently preparing umbilical cord mesenchymal stem cells of the present invention comprises the following reagents:

[0034] (1) Preparation of umbilical cord preservation solution: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 8 mg of resveratrol, 8 mg of icariin, 10 ug of rapamycin, 8 ug of trametinib, and 8 ug of ethanolamine in 1000 ml of DMEM culture medium and filter through a 0.22 um filter for sterilization.

[0035] (2) Preparation of umbilical cord washing solution: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 8 mg of resveratrol, 8 mg of icariin, 10 ug of rapamycin, 8 ug of trametinib, and 8 ug of ethanolamine in 1000 ml of normal saline and filter through a 0.22 um filter for sterilization.

[0036] (3) Preparation of umbilical cord decomposition solution: Dissolve 2 g collagenase II, 0.2 g deoxyribonuclease I, 8 mg resveratrol, 8 mg icariin, 10 ug rapamycin, 8 ug trametinib, and 8 ug ethanolamine in 1000 ml DMEM culture medium and filter through a 0.22 um filter for sterilization.

[0037] (4) Preparation of cell washing solution: PBS, the formula is NaCl 8.50g, Na2HPO4·12H2O 3.58g, NaH2PO4·2H2O 0.39g, add double distilled water to 1000ml, adjust the pH to between 7.2-7.4, and sterilize by high pressure. Resveratrol 8mg, icariin 8mg, rapamycin 10ug, trametinib 8ug, ethanolamine 8ug are dissolved in the above-prepared 1000ml PBS medium, and sterilized by filtering with a 0.22um filter.

[0038] (5) Preparation of cell culture medium: Mix 450 ml of serum-free basal medium (Lonza brand, item number 00190620) and 50 ml of additives (Lonza brand, item number 00192125). Dissolve 8 ug of resveratrol, 8 ug of icariin, and 8 ug of metformin in the above-prepared 1000 ml serum-free basal medium and filter with a 0.22 um filter for sterilization.

[0039] (6) Preparation of cell digestion solution: Dissolve 2.5 g of Trypsin and 197.28 mg of EDTA-2Na in 1000 ml of PBS (PBS preparation method is the same as above), mix well, dissolve 200 mg of deoxyribonuclease I in 1000 ml of the above preparation, and filter through a 0.22 um filter for sterilization.

[0040] (7) Preparation of cell freezing solution: Slowly add 60 ml of the prepared cell culture medium (i.e., serum-free growth medium for mesenchymal stem cells) to 940 ml of the prepared cell culture medium, and gently mix. Dissolve 8 ug of ethanolamine and 8 ug of chloroplatinic acid hexahydrate in 1000 ml of the prepared medium, and sterilize by filtering with a 0.22 um filter.

[0041] 2. Packaging of each component

[0042] The prepared umbilical cord preservation solution, umbilical cord washing solution, umbilical cord decomposition solution, cell washing solution, cell culture solution, cell digestion solution and cell freezing solution are respectively loaded into the kit. The amount of each component in each box is: 1 bottle of umbilical cord preservation solution (50ml / bottle), 1 bottle of umbilical cord washing solution (50ml / bottle), 1 bottle of umbilical cord decomposition solution (50ml / bottle), 10 bottles of cell washing solution (500ml / bottle), 20 bottles of cell culture solution (500ml / bottle), 2 bottles of cell digestion solution (500ml / bottle), 1 bottle of cell freezing solution (500ml / bottle). The components in the kit are packaged according to the above reagent amounts to obtain an umbilical cord mesenchymal stem cell efficient preparation kit. The kit is stored at -20℃, with a validity period of 3 years. It should be thawed in a 4℃ refrigerator or a 37℃ water bath before use, stored at 4℃ after thawing, and used up within three months.

[0043] Example 2 Assembly of a kit for efficiently preparing umbilical cord mesenchymal stem cells

[0044] 1. Solution preparation

[0045] The kit for efficiently preparing umbilical cord mesenchymal stem cells of the present invention comprises the following reagents:

[0046] (1) Preparation of umbilical cord preservation solution: Dissolve 100 mg of penicillin, 100 mg of streptomycin, 2 mg of resveratrol, 2 mg of icariin, 5 ug of rapamycin, 2 ug of trametinib, and 2 ug of ethanolamine in 1000 ml of MEM culture medium and filter with a 0.22 um filter for sterilization.

[0047] (2) Preparation of umbilical cord washing solution: Dissolve 100 mg of penicillin, 100 mg of streptomycin, 2 mg of resveratrol, 2 mg of icariin, 5 μg of rapamycin, 2 μg of trametinib, and 2 μg of ethanolamine in 1000 ml of normal saline and filter through a 0.22 μm filter for sterilization.

[0048] (3) Preparation of umbilical cord dissociation solution: Dissolve 1 g collagenase II, 0.21 g deoxyribonuclease I, 2 mg resveratrol, 2 mg icariin, 5 μg rapamycin, 2 μg trametinib and 2 μg ethanolamine in 1000 ml DMEM medium and filter through a 0.22 μm filter for sterilization.

[0049] (4) Preparation of cell washing solution: Dissolve 2 mg of resveratrol, 2 mg of icariin, 5 ug of rapamycin, 2 ug of trametinib, and 2 ug of ethanolamine in 1000 ml of PBS culture medium and filter through a 0.22 um filter for sterilization.

[0050] (5) Preparation of cell culture medium: Mix 450 ml of serum-free basal medium (Lonza brand, item number 00190620) and 50 ml of additives (Lonza brand, item number 00192125). Dissolve 2 ug of resveratrol, 2 ug of icariin, and 3 ug of metformin in the above-prepared 1000 ml serum-free basal medium and filter with a 0.22 um filter for sterilization.

[0051] (6) Preparation of cell digestion solution: Dissolve 2.5 g of Trypsin and 197.28 mg of EDTA-2Na in 1000 ml of PBS and mix well. Dissolve 200 mg of deoxyribonuclease I in 1000 ml of the above solution and filter through a 0.22 um filter for sterilization.

[0052] (7) Preparation of cell freezing solution: 100 ml is slowly added to 900 ml of the prepared cell culture medium, i.e., serum-free growth medium for mesenchymal stem cells, and gently mixed. 2 ug of ethanolamine and 2 ug of chloroplatinic acid hexahydrate are dissolved in 1000 ml of the prepared solution, and sterilized by filtering with a 0.22 um filter.

[0053] 2. Packaging of each component

[0054] The prepared umbilical cord preservation solution, umbilical cord washing solution, umbilical cord decomposition solution, cell washing solution, cell culture solution, cell digestion solution and cell freezing solution are respectively loaded into the kit. The amount of each component in each box is: 1 bottle of umbilical cord preservation solution (50ml / bottle), 1 bottle of umbilical cord washing solution (50ml / bottle), 1 bottle of umbilical cord decomposition solution (50ml / bottle), 10 bottles of cell washing solution (500ml / bottle), 20 bottles of cell culture solution (500ml / bottle), 2 bottles of cell digestion solution (500ml / bottle), 1 bottle of cell freezing solution (500ml / bottle). The components in the kit are packaged according to the above reagent amounts to obtain an umbilical cord mesenchymal stem cell efficient preparation kit. The kit is stored at -20℃, with a validity period of 3 years. It should be thawed in a 4℃ refrigerator or a 37℃ water bath before use, stored at 4℃ after thawing, and used up within three months.

[0055] Example 3 Assembly of a kit for efficiently preparing umbilical cord mesenchymal stem cells

[0056] 1. Solution preparation

[0057] The kit for efficiently preparing umbilical cord mesenchymal stem cells of the present invention comprises the following reagents:

[0058] (1) Preparation of umbilical cord preservation solution: Dissolve 150 mg of penicillin, 150 mg of streptomycin, 2 mg of resveratrol, 2 mg of icariin, 5 ug of rapamycin, 2 ug of trametinib, and 2 ug of ethanolamine in 1000 ml of DMEM / F12 culture medium and filter through a 0.22 um filter for sterilization.

[0059] (2) Preparation of umbilical cord washing solution: Dissolve 150 mg of penicillin, 150 mg of streptomycin, 2 mg of resveratrol, 2 mg of icariin, 5 μg of rapamycin, 2 μg of trametinib, and 2 μg of ethanolamine in 1000 ml of normal saline and filter through a 0.22 μm filter for sterilization.

[0060] (3) Preparation of umbilical cord dissociation solution: Dissolve 1 g collagenase II, 0.21 g deoxyribonuclease I, 2 mg resveratrol, 2 mg icariin, 5 μg rapamycin, 5 μg trametinib and 5 μg ethanolamine in 1000 ml DMEM / F12 culture medium and filter through a 0.22 μm filter for sterilization.

[0061] (4) Preparation of cell washing solution: Dissolve 2 mg of resveratrol, 2 mg of icariin, 5 ug of rapamycin, 5 ug of trametinib, and 5 ug of ethanolamine in 1000 ml of PBS culture medium and filter through a 0.22 um filter for sterilization.

[0062] (5) Preparation of cell culture medium: Dissolve 5ug of resveratrol, 5ug of icariin, and 5ug of metformin in 1000ml of pre-prepared DMEM / F12+10% FBS, and filter through a 0.22um filter for sterilization.

[0063] (6) Preparation of cell digestion solution: Dissolve 2.5 g of Trypsin and 197.28 mg of EDTA-2Na in 1000 ml of PBS and mix well. Dissolve 150 mg of deoxyribonuclease I in 1000 ml of the above solution and filter through a 0.22 um filter for sterilization.

[0064] (7) Preparation of cell freezing solution: 200 ml is slowly added to 800 ml of the prepared cell culture medium, i.e., serum-free growth medium for mesenchymal stem cells, and gently mixed. 5 ug of ethanolamine and 5 ug of chloroplatinic acid hexahydrate are dissolved in 1000 ml of the prepared solution, and sterilized by filtering with a 0.22 um filter.

[0065] 2. Packaging of each component

[0066] The prepared umbilical cord preservation solution, umbilical cord washing solution, umbilical cord decomposition solution, cell washing solution, cell culture solution, cell digestion solution and cell freezing solution are respectively loaded into the kit. The amount of each component in each box is: 1 bottle of umbilical cord preservation solution (50ml / bottle), 1 bottle of umbilical cord washing solution (50ml / bottle), 1 bottle of umbilical cord decomposition solution (50ml / bottle), 10 bottles of cell washing solution (500ml / bottle), 20 bottles of cell culture solution (500ml / bottle), 2 bottles of cell digestion solution (500ml / bottle), 1 bottle of cell freezing solution (500ml / bottle). The components in the kit are packaged according to the above reagent amounts to obtain an umbilical cord mesenchymal stem cell efficient preparation kit. The kit is stored at -20℃, with a validity period of 3 years. It should be thawed in a 4℃ refrigerator or a 37℃ water bath before use, stored at 4℃ after thawing, and used up within three months.

[0067] Example 4 Assembly of a kit for efficiently preparing umbilical cord mesenchymal stem cells

[0068] 1. Solution preparation

[0069] The kit for efficiently preparing umbilical cord mesenchymal stem cells of the present invention comprises the following reagents:

[0070] (1) Preparation of umbilical cord preservation solution: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 6 mg of resveratrol, 6 mg of icariin, 8 ug of rapamycin, 6 ug of trametinib, and 6 ug of ethanolamine in 1000 ml of DMEM culture medium and filter through a 0.22 um filter for sterilization.

[0071] (2) Preparation of umbilical cord washing solution: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 6 mg of resveratrol, 6 mg of icariin, 8 ug of rapamycin, 6 ug of trametinib, and 6 ug of ethanolamine in 1000 ml of PBS and filter through a 0.22 um filter for sterilization.

[0072] (3) Preparation of umbilical cord dissociation solution: Dissolve 2 g collagenase II, 0.2 g deoxyribonuclease I, 6 mg resveratrol, 6 mg icariin, 8 ug rapamycin, 8 ug trametinib, and 6 ug ethanolamine in 1000 ml DMEM culture medium and filter through a 0.22 um filter for sterilization.

[0073] (4) Preparation of cell washing solution: PBS, the formula is NaCl 8.50g, Na2HPO4·12H2O 3.58g, NaH2PO4·2H2O 0.39g, add double distilled water to 1000ml, adjust the pH to between 7.2-7.4, and sterilize by high pressure. Dissolve 6mg of resveratrol, 6mg of icariin, 8ug of rapamycin, 6ug of trametinib, and 6ug of ethanolamine in the above-prepared 1000ml PBS medium and filter and sterilize with a 0.22um filter.

[0074] (5) Preparation of cell culture medium: Dissolve 6ug of resveratrol, 6ug of icariin, and 6ug of metformin in 1000ml of Keratinocyte-SFM (Gibco, catalog number 17005042) prepared above, and filter through a 0.22um filter for sterilization.

[0075] (6) Preparation of cell digestion solution: Dissolve 2 g of Trypsin and 197.28 mg of EDTA-2Na in 1000 ml of PBS and mix well. Dissolve 100 mg of deoxyribonuclease I in 1000 ml of the above solution and filter through a 0.22 um filter for sterilization.

[0076] (7) Preparation of cell freezing solution: Slowly add 100 ml of the solution to 900 ml of Keratinocyte-SFM and mix gently. Dissolve 5 ug of ethanolamine and 5 ug of chloroplatinic acid hexahydrate in 1000 ml of the solution and sterilize by filtering with a 0.22 um filter.

[0077] 2. Packaging of each component

[0078] The prepared umbilical cord preservation solution, umbilical cord washing solution, umbilical cord decomposition solution, cell washing solution, cell culture solution, cell digestion solution and cell freezing solution are respectively loaded into the kit. The amount of each component in each box is: 1 bottle of umbilical cord preservation solution (50ml / bottle), 1 bottle of umbilical cord washing solution (50ml / bottle), 1 bottle of umbilical cord decomposition solution (50ml / bottle), 10 bottles of cell washing solution (500ml / bottle), 20 bottles of cell culture solution (500ml / bottle), 2 bottles of cell digestion solution (500ml / bottle), 1 bottle of cell freezing solution (500ml / bottle). The components in the kit are packaged according to the above reagent amounts to obtain an umbilical cord mesenchymal stem cell efficient preparation kit. The kit is stored at -20℃, with a validity period of 3 years. It should be thawed in a 4℃ refrigerator or a 37℃ water bath before use, stored at 4℃ after thawing, and used up within three months.

[0079] Example 5 Preparation of umbilical cord mesenchymal stem cells

[0080] Umbilical cord mesenchymal stem cells are prepared using the kit in Example 4. The specific preparation method includes the following steps. Taking a 5 cm umbilical cord as an example, the operation is as follows:

[0081] 1. Transportation of umbilical cord specimens: Collect 5 cm of umbilical cord, add an equal volume of umbilical cord preservation solution, store at a constant temperature of 2-8°C in a vaccine box, and send to the laboratory within 48 hours.

[0082] 2. Wash the umbilical cord: Aspirate the umbilical cord preservation solution, add an equal volume of umbilical cord washing solution, shake repeatedly several times, aspirate the supernatant, and wait until the umbilical cord washing solution becomes clear.

[0083] 3. Preparation and culture of umbilical cord mesenchymal stem cells:

[0084] 1) Place the umbilical cord in a 10 cm culture dish, cut it into several sections, and remove the three blood vessels in the middle;

[0085] 2) The white connective tissue between the amnion and the blood vessels is Walton's jelly. Use long-handled toothed forceps to tear it off and place it in a sterile dish.

[0086] 3) Use sterile long-handled surgical scissors to cut the Walton's jelly into 1 mm thick pieces in a centrifuge tube. 3 The shearing time for the homogenate block is 15-20min.

[0087] 4) Add an equal volume of umbilical cord dissociation solution (preheated to 37°C) to the tissue homogenate block, place in a constant temperature shaking incubator at 37°C, 200rpm, and digest for 30-45 minutes (take out and shake gently every 15 minutes and put back) until the umbilical cord tissue homogenate block becomes fluffy.

[0088] 4. Collect cells: Add 5 times the volume of cell washing solution to the tissue homogenate digested with umbilical cord decomposition solution at a volume ratio of 1:5, filter through a sterile 200-mesh filter to remove the residue, and take 1 ml of the suspension to count the cells, which is 8.1×10 7 The remaining liquid was centrifuged at 400 g for 8 min, the supernatant was discarded, and the lower layer of cells was obtained.

[0089] 5. Cell planting: According to the cell count, 1.0×10 6 Add 50 ml of cell culture medium at a density of cells / ml, inoculate the T175 culture flask, and place it in a carbon dioxide incubator. The culture conditions are: 37±0.5℃, carbon dioxide volume fraction of (5±0.2)%, and the medium is changed every 2-3 days.

[0090] 6. Cell passaging: After about 3-5 days, observe the cells under an inverted microscope. The results are shown in the attached Figure 2 When the primary culture cells reached 70% to 80% fusion, the old cell culture medium was discarded, and cell washing solution was added. The cells were allowed to stand for about 1 min, and the cell washing solution was discarded. Cell digestion solution was added. The digestion time was 1.5-2 min, and then 2-3 ml of cell culture solution was added to stop digestion. The bottom of the bottle was repeatedly blown until most of the cells fell off, and the cells were transferred to a 50 ml centrifuge tube. The cell count was 3.5 × 10 8 , centrifuged at 400 g for 8 min, discarded the supernatant, and obtained 3.5 × 10 8 P0 umbilical cord mesenchymal stem cells. 5 Add cell culture medium to T175 culture flasks and place them in a carbon dioxide incubator for culture. This is called P1. When the P1 cells reach 80%-90% fusion after about 3 days, harvest the cells as above and count them to obtain 1.8×10 9 The P1 umbilical cord mesenchymal stem cells were subcultured as above (once every 3 days) until the P3 generation was harvested. The cells were observed under an inverted microscope. The results are shown in the attached Figure 3 The cells were harvested and counted to 2.3 × 10 11 Umbilical cord mesenchymal stem cells.

[0091] 7. Cell identification: Take P3 generation UC-MSCs and detect cell surface markers by flow cytometry. The flow cytometry results are attached. Figure 4 The flow cytometry results are shown in Table 1.

[0092] Table 1 Detection of surface markers of UC-MSCs

[0093] UC-MSCs surface markers CD29 CD73 CD90 CD105 CD34 CD45 Expression rate (%) 97.331 99.21 99.01 98.42 1.05 0.43

[0094] Among them, the expression of positive markers CD29, CD73, CD90, and CD105 was greater than 95%, and the expression of negative markers CD34 and CD45 was less than 2%, proving that the cells were mesenchymal stem cells (MSCs).

[0095] 8. Cell cryopreservation: Centrifuge the P3 cell suspension obtained above at 400g for 8 minutes, discard the supernatant, slowly add the cell freezing solution, gently mix, and then mark the cell name, generation and freezing date on the cryopreservation tube; put it in a cryopreservation box, freeze it at -80℃ overnight, and transfer it to liquid nitrogen for long-term storage the next day, and revive it when needed.

[0096] The kit of the present invention can obtain a large number of umbilical cord mesenchymal stem cells (2.3×10 11 ), indicating that the kit of the present invention can obtain a large number of umbilical cord mesenchymal stem cells in a relatively short time.

[0097] Example 6 Preparation of umbilical cord mesenchymal stem cells

[0098] The kit of the present invention assembled in Example 3 was used to prepare umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells were prepared with reference to the method in Example 5, and the obtained mesenchymal stem cells were counted by the same method. As a result, 2.1×10 umbilical cord cells were amplified within 12 days. 11 umbilical cord mesenchymal stem cells, indicating that the kit of the present invention can obtain a large number of umbilical cord mesenchymal stem cells in a relatively short time.

[0099] Example 7 Preparation of umbilical cord mesenchymal stem cells

[0100] The kit of the present invention assembled in Example 2 was used to prepare umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells were prepared with reference to the method in Example 5, and the obtained mesenchymal stem cells were counted by the same method. The result was that 2.2×10 umbilical cords were obtained within 12 days. 11 umbilical cord mesenchymal stem cells, indicating that the kit of the present invention can obtain a large number of umbilical cord mesenchymal stem cells in a relatively short time.

Claims

1. A kit for efficiently preparing umbilical cord mesenchymal stem cells, characterized in that: Including umbilical cord preservation solution, umbilical cord washing solution, umbilical cord decomposition solution, cell washing solution, cell culture solution, cell digestion solution and cell freezing solution; The umbilical cord preservation solution is DMEM, DMEM / F12, MEM or RPMI-1640 culture medium containing 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin, 2-8ug / ml of resveratrol, 2-8ug / ml of icariin, 5-10ng / ml of rapamycin, 2-8ng / ml of trametinib and 2-8ng / ml of ethanolamine; The umbilical cord washing solution is physiological saline, PBS, D-Hanks or HBSS containing 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin, 2-8ug / ml of resveratrol, 2-8ug / ml of icariin, 5-10ng / ml of rapamycin, 2-8ng / ml of trametinib and 2-8ng / ml of ethanolamine; The umbilical cord decomposition solution is DMEM, DMEM / F12, MEM or RPMI-1640 culture medium containing collagenase II 1-2 mg / ml, deoxyribonuclease I 0.1-0.2 mg / ml, resveratrol 2-8 ug / ml, icariin 2-8 ug / ml, rapamycin 5-10 ng / ml, trametinib 2-8 ng / ml, and ethanolamine 2-8 ng / ml; The cell washing solution is PBS, D-Hanks or HBSS containing 2-8ug / ml of resveratrol, 2-8ug / ml of icariin, 5-10ng / ml of rapamycin, 2-8ng / ml of trametinib and 2-8ng / ml of ethanolamine; The cell culture medium is Keratinocyte-SFM, serum-free complete medium for human mesenchymal stem cells, DMEM+10% FBS or DMEM / F12+10% FBS supplemented with 2-8 ng / ml resveratrol, 2-8 ng / ml icariin and 2-8 ng / ml metformin; The cell digestion solution is PBS, D-Hanks or HBSS solution containing 1.25-2.5 mg / ml Trypsin-EDTA and 0.1-0.2 mg / ml deoxyribonuclease I; The cell freezing medium is Keratinocyte-SFM containing 60-200 mg / ml DMSO, 2-8 ng / ml ethanolamine, 2-8 ng / ml chloroplatinic acid hexahydrate, a serum-free complete medium for human mesenchymal stem cells, DMEM+10% FBS or DMEM / F12+10% FBS.

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