Adipose-derived 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 adipose mesenchymal stem cells. The kit includes a fat preservation solution, a fat washing solution, a fat decomposition solution, a cell washing solution, a cell culture solution, a cell digestion solution, and a cell freezing solution. The kit of the present invention can efficiently prepare adipose mesenchymal stem cells, obtain a large number of adipose 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 adipose mesenchymal stem cells. Background Art

[0002] Adipose tissue is abundant in the human body and is easy to obtain with minimal trauma. It has broad application prospects in tissue engineering, organ repair, gene therapy, etc. Therefore, adipose-Derived mesenchymalstem cells (AD-MSCs) have become a hot topic in the field of stem cells.

[0003] Adipose-derived 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.

[0004] In January 2012, the Korean FDA approved the launch of the autologous adipose stem cell drug Cuepistem, which is used to treat complicated Crohn's disease complicated by anal fistula. In October of the same year, the US FDA approved the clinical trial of Cytori Therapeutics' adipose stem cells for patients with heart failure. Adipose mesenchymal stem cells have broad prospects for clinical application.

[0005] Adipose-derived mesenchymal stem cells have extremely broad prospects for clinical application. Therefore, it is imperative to develop a simple, rapid, efficient, and safe method to obtain a large number of adipose-derived mesenchymal stem cells. However, there is currently no kit for the efficient preparation of adipose-derived mesenchymal stem cells in the existing technology, which has slowed down the progress of related scientific research. Summary of the invention

[0006] 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 adipose-derived mesenchymal stem cell (AD-MSC) efficient preparation kit for preparing a large number of adipose-derived mesenchymal stem cells (AD-MSCs) in a short period of time.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a highly efficient preparation kit for adipose-derived mesenchymal stem cells, comprising adipose preservation solution, adipose washing solution, adipose decomposition solution, cell washing solution, cell culture solution, cell digestion solution and cell freezing solution.

[0008] The fat preservation solution is DMEM, DMEM / F12, MEM or RPMI-1640 culture medium containing 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin, 5-10ug / ml of aminoethanol, 2-8ug / ml of GLP-1, 5-10ug / ml of forsythiaside B and 4-8ng / ml of eugenol.

[0009] The fat washing solution is physiological saline, PBS, D-Hanks or HBSS containing 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin, 5-10ug / ml of aminoethanol, 2-8ug / ml of GLP-1, 5-10ug / ml of forsythiaside B and 4-8ng / ml of eugenol.

[0010] The lipolysis solution is DMEM, DMEM / F12, MEM or RPMI-1640 culture medium containing collagenase I 1-2 mg / ml, deoxyribonuclease I 0.1-0.2 mg / ml, aminoethanol 5-10ug / ml, GLP-1 2-8ug / ml, forsythiaside B 5-10ug / ml, and eugenol 4-8ng / ml.

[0011] The cell washing solution is PBS, D-Hanks or HBSS containing 5-10ug / ml of aminoethanol, 2-8ug / ml of GLP-1, 5-10ug / ml of forsythiaside B, and 4-8ng / ml of eugenol.

[0012] The cell culture medium is Keratinocyte-SFM medium, TheraPEAK™ MSCGM-CD™ medium, DMEM+10% FBS or DMEM / F12+10% FBS supplemented with 5-10 ng / ml of aminoethanol, 2-8 ng / ml of GLP-1, 5-10 ng / ml of forsythiaside B, and 4-8 ng / ml of eugenol.

[0013] The cell digestion solution is PBS, D-Hanks or HBSS solution containing Trypsin-EDTA 1.25-2.5 mg / ml, deoxyribonuclease I 0.1-0.2 mg / ml, aminoethanol 5-10 ug / ml, GLP-1 2-8 ug / ml, forsythiaside B 5-10 ug / ml, and eugenol 4-8 ng / ml.

[0014] The cell freezing medium is Keratinocyte-SFM culture medium, TheraPEAK™ MSCGM-CD™ culture medium, DMEM+10% FBS or DMEM / F12+10% FBS containing DMSO 100-200 mg / ml, chloroquine phosphate 40-100 ug / ml, and palmatine hydrochloride 5-10 ng / ml.

[0015] Preferably, the fat preservation solution is a DMEM culture medium containing 200ug / ml penicillin, 200ug / ml streptomycin, 8ug / ml aminoethanol, 5ug / ml GLP-1, 8ug / ml forsythiaside B, and 6ng / ml eugenol.

[0016] Preferably, the fat washing solution is PBS containing 200ug / ml penicillin, 200ug / ml streptomycin, 8ug / ml aminoethanol, 5ug / ml GLP-1, 8ug / ml forsythiaside B, and 6ng / ml eugenol.

[0017] Preferably, the lipolysis solution is a DMEM culture medium containing 2 mg / ml collagenase I, 0.15 mg / ml deoxyribonuclease I, 8 ug / ml aminoethanol, 5 ug / ml GLP-1, 8 ug / ml forsythiaside B, and 6 ng / ml eugenol.

[0018] Preferably, the cell washing solution is PBS containing 8ug / ml of aminoethanol, 5ug / ml of GLP-1, 8ug / ml of forsythiaside B, and 6ng / ml of eugenol.

[0019] Preferably, the cell culture medium is Keratinocyte-SFM medium to which aminoethanol 8 ng / ml, GLP-1 5 ng / ml, forsythiaside B 8 ng / ml, and eugenol 6 ng / ml are additionally added.

[0020] Preferably, the cell digestion solution is PBS containing Trypsin-EDTA 2mg / ml, deoxyribonuclease I 0.15mg / ml, aminoethanol 8ug / ml, GLP-1 5ug / ml, forsythiaside B 8ug / ml, and eugenol 6ng / ml.

[0021] The cell freezing solution is a Keratinocyte-SFM culture medium containing 200 mg / ml DMSO, 70 ug / ml chloroquine phosphate, and 8 ng / ml palmatine hydrochloride.

[0022] The present invention provides a kit for efficiently preparing adipose mesenchymal stem cells. In the kit, the function of the fat preservation solution is to ensure the activity of adipose mesenchymal stem cells during the transportation of the fat specimen after collection, and to eliminate and prevent bacteria, fungi, and viruses. The function of the fat washing solution is to wash and remove blood cells from adipose tissue, ensure the activity of adipose mesenchymal stem cells, and eliminate and prevent bacteria and fungi. The function of the fat decomposition solution is to efficiently and quickly digest adipose tissue without damaging the activity of adipose 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 adipose mesenchymal stem cells. The function of the cell culture solution is to quickly and efficiently amplify adipose 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 adipose mesenchymal stem cells. The function of the cell freezing solution is to efficiently freeze the obtained adipose mesenchymal stem cells for use.

[0023] The adipose-derived mesenchymal stem cell efficient preparation kit of the present invention can obtain a large number of adipose-derived mesenchymal stem cells in a relatively short time, and is convenient for clinical use. It is easy to use and low in cost, and will play an important role in stem cell clinical trials and related research, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention shows a kit for efficiently preparing adipose-derived mesenchymal stem cells (top view). The following are marked in the figure: 1, cell washing solution area; 2, cell washing solution; 3, cell culture solution area; 4, cell culture solution; 5, cell freezing solution area; 6, cell freezing solution; 7, cell digestion solution area; 8, cell digestion solution; 9, fat preservation solution; 10, fat decomposition solution; 11, fat washing solution.

[0025] Figure 2 Adipose-derived mesenchymal stem cells (p0, 100×) prepared by the present invention are shown.

[0026] Figure 3 Adipose mesenchymal stem cells (P1, 100×) prepared by the present invention are shown. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1 Assembly of a kit for efficiently preparing adipose-derived mesenchymal stem cells

[0029] In the preparation and assembly of the adipose mesenchymal stem cell efficient preparation kit of the present invention, the required components are all commercially available products and can be purchased through commercial channels: penicillin, brand Amresco, product number 0242; streptomycin, brand Amresco, product number 0382; aminoethanol, brand Sigma, product number 471453; GLP-1, brand sigma, product number: 87805-34-3; forsythiaside B, brand Sigma, product number XG-R11596; eugenol, brand Sigma, product number XG-R11057; collagenase I, brand sigma, product number C0130; deoxyribonuclease I, brand sigma, product number C4263; DMSO, brand sigma, product number D2650; chloroquine phosphate, brand sigma, product number C6628-25G; palmatine hydrochloride, brand aladdin, product number P408401-1ml.

[0030] 1. Solution preparation

[0031] The adipose-derived mesenchymal stem cell efficient preparation kit of the present invention comprises the following reagents:

[0032] (1) Preparation of fat preservation solution: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 10 mg of aminoethanol, 8 mg of GLP-1, 10 mg of forsythiaside B, and 8 ug of eugenol in 1000 ml of DMEM culture medium and filter with a 0.22 um filter for sterilization.

[0033] (2) Preparation of fat washing solution: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 10 mg of aminoethanol, 8 mg of GLP-1, 10 mg of forsythiaside B, and 8 ug of eugenol in 1000 ml of normal saline and filter through a 0.22 um filter for sterilization.

[0034] (3) Preparation of lipolysis solution: Dissolve 2 g of collagenase I, 0.2 g of deoxyribonuclease I, 10 mg of aminoethanol, 8 mg of GLP-1, 10 mg of forsythiaside B, and 8 ug of eugenol in 1000 ml of DMEM culture medium and filter through a 0.22 um filter for sterilization.

[0035] (4) Preparation of cell washing solution: PBS, formula: 8.50g NaCl, Na2HPO4·12H 2 O 3.58 g, NaH 2 PO 4 ·2H 2O 0.39g, add double distilled water to 1000ml, adjust pH to between 7.2-7.4, and sterilize by high pressure. Dissolve aminoethanol 10mg, GLP-1 8mg, forsythiaside B 10mg, and eugenol 8ug in the above-prepared 1000ml PBS medium, and filter and sterilize with a 0.22um filter.

[0036] (5) Preparation of cell culture medium: Dissolve 10ug of aminoethanol, 8ug of GLP-1, 10ug of forsythiaside B, and 8ug of eugenol in 1000ml of Keratinocyte-SFM culture medium and filter through a 0.22um filter for sterilization.

[0037] (6) Preparation method of cell digestion solution: Dissolve 2.5 g of Trypsin (trypsin), 197.28 mg of EDTA-2Na, 200 mg of deoxyribonuclease I, 10 mg of aminoethanol, 8 mg of GLP-1, 10 mg of forsythiaside B, and 8 ug of eugenol in 1000 ml of PBS prepared above (PBS preparation method is the same as above), and filter through a 0.22 um filter for sterilization.

[0038] (7) Preparation of cell freezing solution: Slowly add 200 ml DMSO to 800 ml Keratinocyte-SFM medium and mix gently. Dissolve 100 mg chloroquine phosphate and 10 ug bamipine hydrochloride in 1000 ml of the above solution and filter through a 0.22 μm filter for sterilization.

[0039] 2. Packaging of each component

[0040] The prepared fat preservation solution, fat washing solution, fat 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 fat preservation solution (50ml / bottle), 1 bottle of fat washing solution (100ml / bottle), 1 bottle of fat decomposition solution (50ml / bottle), 6 bottles of cell washing solution (500ml / bottle), 20 bottles of cell culture solution (500ml / bottle), 2 bottles of cell digestion solution (500ml / bottle), 2 bottles of cell freezing solution (500ml / bottle). The components in the kit are packaged according to the above reagent amounts to obtain a high-efficiency preparation kit for adipose mesenchymal stem cells. 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.

[0041] Example 2 Assembly of a kit for efficiently preparing adipose-derived mesenchymal stem cells

[0042] 1. Solution preparation

[0043] The adipose-derived mesenchymal stem cell efficient preparation kit of the present invention comprises the following reagents:

[0044] (1) Preparation of fat preservation solution: Dissolve 100 mg of penicillin, 100 mg of streptomycin, 5 mg of aminoethanol, 2 mg of GLP-1, 5 mg of forsythiaside B, and 4 ug of eugenol in 1000 ml of MEM culture medium and filter with a 0.22 um filter for sterilization.

[0045] (2) Preparation of fat washing solution: Dissolve 100 mg of penicillin, 100 mg of streptomycin, 5 mg of aminoethanol, 2 mg of GLP-1, 5 mg of forsythiaside B, and 4 ug of eugenol in 1000 ml of normal saline and filter through a 0.22 um filter for sterilization.

[0046] (3) Preparation of lipolysis solution: Dissolve 1g of collagenase I, 100mg of deoxyribonuclease I, 5mg of aminoethanol, 2mg of GLP-1, 5mg of forsythiaside B, and 4ug of eugenol in 1000ml of MEM culture medium and filter with a 0.22um filter for sterilization.

[0047] (4) Preparation of cell washing solution: PBS, formula: 8.50g NaCl, Na2HPO4·12H 2 O 3.58 g, NaH 2 PO 4 ·2H 2 O 0.39g, add double distilled water to 1000ml, adjust pH to between 7.2-7.4, and sterilize by high pressure. Dissolve 5mg aminoethanol, 2mg GLP-1, 5mg forsythiaside B, and 4ug eugenol in the above-prepared 1000ml PBS medium, and filter and sterilize with a 0.22um filter.

[0048] (5) Preparation of cell culture medium: Dissolve 5ug of aminoethanol, 2ug of GLP-1, 5ug of forsythiaside B, and 4ug of eugenol in 1000ml of Keratinocyte-SFM culture medium and filter through a 0.22um filter for sterilization.

[0049] (6) Preparation method of cell digestion solution: Dissolve 2.5 g of Trypsin (trypsin), 197.28 mg of EDTA-2Na, 100 mg of deoxyribonuclease I, 5 mg of aminoethanol, 2 mg of GLP-1, 5 mg of forsythiaside B, and 4 ug of eugenol in 1000 ml of PBS prepared above (PBS preparation method is the same as above), and filter through a 0.22 um filter for sterilization.

[0050] (7) Preparation of cell freezing solution: Slowly add 100 ml DMSO to 900 ml Keratinocyte-SFM medium and mix gently. Dissolve 40 mg chloroquine phosphate and 5 ug bamipine hydrochloride in the above prepared 1000 ml solution and filter with a 0.22 μm filter for sterilization.

[0051] 2. Packaging of each component

[0052] The prepared fat preservation solution, fat washing solution, fat 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 fat preservation solution (50ml / bottle), 1 bottle of fat washing solution (100ml / bottle), 1 bottle of fat decomposition solution (50ml / bottle), 6 bottles of cell washing solution (500ml / bottle), 20 bottles of cell culture solution (500ml / bottle), 2 bottles of cell digestion solution (500ml / bottle), 2 bottles of cell freezing solution (500ml / bottle). The components in the kit are packaged according to the above reagent amounts to obtain a high-efficiency preparation kit for adipose mesenchymal stem cells. 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.

[0053] Example 3 Assembly of a kit for efficiently preparing adipose-derived mesenchymal stem cells

[0054] 1. Solution preparation

[0055] The adipose-derived mesenchymal stem cell efficient preparation kit of the present invention comprises the following reagents:

[0056] (1) Preparation of fat preservation solution: Dissolve 150 mg of penicillin, 150 mg of streptomycin, 10 mg of aminoethanol, 8 mg of GLP-1, 10 mg of forsythiaside B, and 8 ug of eugenol in 1000 ml of RPMI-1640 culture medium and filter with a 0.22 um filter for sterilization.

[0057] (2) Preparation of fat washing solution: Dissolve 150 mg of penicillin, 150 mg of streptomycin, 10 mg of aminoethanol, 8 mg of GLP-1, 10 mg of forsythiaside B, and 8 ug of eugenol in 1000 ml of normal saline and filter through a 0.22 um filter for sterilization.

[0058] (3) Preparation of lipolysis solution: Dissolve 1.5 g collagenase I, 0.15 g deoxyribonuclease I, 9 mg aminoethanol, 7 mg GLP-1, 8 mg forsythiaside B, and 4 ug eugenol in 1000 ml DMEM / F12 culture medium and filter through a 0.22 um filter for sterilization.

[0059] (4) Preparation of cell washing solution: PBS, formula: 8.50g NaCl, Na2HPO4·12H 2 O 3.58 g, NaH 2 PO 4 ·2H 2 O 0.39g, add double distilled water to 1000ml, adjust pH to between 7.2-7.4, and sterilize by high pressure. Dissolve aminoethanol 10mg, GLP-1 8mg, forsythiaside B 10mg, and eugenol 8ug in the above-prepared 1000ml PBS medium, and filter and sterilize with a 0.22um filter.

[0060] (5) Preparation of cell culture medium: Dissolve 10ug of aminoethanol, 8ug of GLP-1, 10ug of forsythiaside B, and 8ug of eugenol in 1000ml of TheraPEAK™ MSCGM-CD™ culture medium and sterilize by filtering with a 0.22um filter.

[0061] (6) Preparation method of cell digestion solution: Dissolve 2.5 g of Trypsin (trypsin), 197.28 mg of EDTA-2Na, 200 mg of deoxyribonuclease I, 10 mg of aminoethanol, 8 mg of GLP-1, 10 mg of forsythiaside B, and 8 ug of eugenol in 1000 ml of PBS prepared above (PBS preparation method is the same as above), and filter through a 0.22 um filter for sterilization.

[0062] (7) Preparation of cell freezing solution: Slowly add 100 ml DMSO to 900 ml TheraPEAK™ MSCGM-CD™ medium and mix gently. Dissolve 70 mg chloroquine phosphate and 8 ug bamipine hydrochloride in the above prepared 1000 ml solution and filter with a 0.22 um filter for sterilization.

[0063] 2. Packaging of each component

[0064] The prepared fat preservation solution, fat washing solution, fat 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 fat preservation solution (50ml / bottle), 1 bottle of fat washing solution (100ml / bottle), 1 bottle of fat decomposition solution (50ml / bottle), 6 bottles of cell washing solution (500ml / bottle), 20 bottles of cell culture solution (500ml / bottle), 2 bottles of cell digestion solution (500ml / bottle), 2 bottles of cell freezing solution (500ml / bottle). The components in the kit are packaged according to the above reagent amounts to obtain a high-efficiency preparation kit for adipose mesenchymal stem cells. 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.

[0065] Example 4 Assembly of a kit for efficiently preparing adipose-derived mesenchymal stem cells

[0066] 1. Solution preparation

[0067] The adipose-derived mesenchymal stem cell efficient preparation kit of the present invention comprises the following reagents:

[0068] (1) Preparation of fat preservation solution: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 8 mg of aminoethanol, 5 mg of GLP-1, 8 mg of forsythiaside B, and 6 ug of eugenol in 1000 ml of DMEM culture medium and filter with a 0.22 um filter for sterilization.

[0069] (2) Preparation of fat washing solution: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 8 mg of aminoethanol, 5 mg of GLP-1, 8 mg of forsythiaside B, and 6 ug of eugenol in 1000 ml of normal saline and filter through a 0.22 um filter for sterilization.

[0070] (3) Preparation of lipolysis solution: Dissolve 2 g of collagenase I, 150 mg of deoxyribonuclease I, 8 mg of aminoethanol, 5 mg of GLP-1, 8 mg of forsythiaside B, and 6 ug of eugenol in 1000 ml of DMEM culture medium and filter through a 0.22 um filter for sterilization.

[0071] (4) Preparation of cell washing solution: PBS, formula: 8.50g NaCl, Na2HPO4·12H 2 O 3.58 g, NaH 2 PO 4 ·2H 2 O 0.39g, add double distilled water to 1000ml, adjust pH to between 7.2-7.4, and sterilize by high pressure. Dissolve aminoethanol 8mg, GLP-1 5mg, forsythiaside B 8mg, and eugenol 6ug in the above-prepared 1000ml PBS medium, and filter and sterilize with a 0.22um filter.

[0072] (5) Preparation of cell culture medium: Dissolve 8ug of aminoethanol, 5ug of GLP-1, 8ug of forsythiaside B, and 6ug of eugenol in 1000ml of Keratinocyte-SFM culture medium and filter through a 0.22um filter for sterilization.

[0073] (6) Preparation method of cell digestion solution: Dissolve 2g of Trypsin (trypsin), 197.28mg of EDTA-2Na, 150mg of deoxyribonuclease I, 8mg of aminoethanol, 5mg of GLP-1, 8mg of forsythiaside B, and 6ug of eugenol in 1000ml of PBS prepared above (PBS preparation method is the same as above), and filter through a 0.22um filter for sterilization.

[0074] (7) Preparation of cell freezing solution: Slowly add 200 ml DMSO to 800 ml Keratinocyte-SFM medium and mix gently. Dissolve 70 mg chloroquine phosphate and 8 ug bamipine hydrochloride in 1000 ml of the above solution and filter through a 0.22 μm filter for sterilization.

[0075] 2. Packaging of each component

[0076] The prepared fat preservation solution, fat washing solution, fat 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 fat preservation solution (50ml / bottle), 1 bottle of fat washing solution (100ml / bottle), 1 bottle of fat decomposition solution (50ml / bottle), 6 bottles of cell washing solution (500ml / bottle), 20 bottles of cell culture solution (500ml / bottle), 2 bottles of cell digestion solution (500ml / bottle), 2 bottles of cell freezing solution (500ml / bottle). The components in the kit are packaged according to the above reagent amounts to obtain a high-efficiency preparation kit for adipose mesenchymal stem cells. 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.

[0077] Example 5 Preparation of adipose-derived mesenchymal stem cells

[0078] Adipose-derived mesenchymal stem cells were prepared using the kit in Example 4. Taking 20 ml of fat as an example, all manipulation steps were completed on a clean bench. The specific preparation method included the following steps:

[0079] 1. Fat transportation: Collect 20 ml of fat, add an equal volume of fat preservation solution, store at a constant temperature of 2-8℃ in a vaccine box, and send to the laboratory within 3 days.

[0080] 2. Fat washing: Aspirate the fat preservation solution, add an equal volume of fat washing solution, shake repeatedly several times, let the fat and fat washing solution stand naturally, aspirate the fat washing solution, add an equal volume of fat washing solution and shake and wash 2-3 times as above, until the lower layer of fat washing solution is clear, aspirate the fat washing solution to obtain fat.

[0081] 3. Digest fat: Add an equal volume of fat decomposition solution (preheated to 37°C) to the obtained fat and place it in a constant temperature shaking incubator at 37°C, 200rpm, and digest for 30-40 minutes until the fat becomes crumbly.

[0082] 4. Separation of SVF (stromal vascular fraction): The digested adipose tissue was centrifuged at 700 g for 10 min, and the upper layer of fat and the lower layer of collagenase solution were carefully removed from top to bottom with a pipette (note that a small amount of solution should be left above the SVF precipitate to avoid disturbing the precipitated cells), and the cell washing solution was added and mixed, and filtered through a 100 um filter. 1 ml of the suspension was aspirated and counted, which was 1.56 × 10 8 The cells were centrifuged at 400 g for 8 min, and the supernatant was discarded to obtain SVF.

[0083] 5. Cell seeding: According to the number of SVF cells, 1.0×10 6 Add 20 ml of cell culture medium at a density of cells / ml, inoculate the cell culture bottle, 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.

[0084] 6. Cell passaging: Primary culture: When the cells reach 60% to 80% fusion (such as attached Figure 2 The old cell culture medium was discarded, cell washing solution was added, and the mixture was allowed to stand for 1 min. The cell washing solution was discarded, and cell digestion solution was added. The digestion time was 1-2 min. Then 2-3 ml of cell culture solution was added to stop the digestion. The bottom of the bottle was repeatedly blown until most of the cells fell off. The cells were transferred into a 50 ml centrifuge tube and the cell count was 3.23 × 10 9 The cells were centrifuged at 400 g for 8 min, and the supernatant was discarded to obtain 3.23 × 10 9 P0 adipose-derived mesenchymal stem cells. 5 / ml of cell culture medium was added, inoculated into the culture bottle, and placed in a carbon dioxide incubator for continued culture, which was counted as the P1 generation. After about 3 days, when the P1 generation cells reached 80%-90% fusion (if attached Figure 3 As shown above, the cells were harvested and counted to obtain 2.48×10 10 The P1 adipose-derived mesenchymal stem cells were subcultured as above (once every 3-5 days), and so on, until the P3 generation was harvested, and the cell count was 1.19×10 12 Adipose-derived mesenchymal stem cells.

[0085] 7. Cell identification: P3 generation AD-MSCs were taken and cell surface markers were detected by flow cytometry. The results are shown in Table 1.

[0086] Table 1 Detection of surface markers of AD-MSCs

[0087] AD-MSCs surface markers CD29 CD73 CD90 CD49d CD34 CD45 Expression rate (%) 99.11 98.87 98.56 98.73 1.35 0.47

[0088] Among them, the expression of positive markers CD29, CD73, CD90, and CD49d 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).

[0089] 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.

[0090] It can be seen that using the kit of the present invention, a large amount of 1.19×10 12 This indicates that the kit of the present invention can obtain a large number of adipose-derived mesenchymal stem cells in a relatively short period of time.

[0091] Example 6 Preparation of Adipose-Derived Mesenchymal Stem Cells

[0092] The kit of the present invention assembled in Example 3 was used to prepare adipose-derived mesenchymal stem cells. The method in Example 5 was referred to to prepare adipose-derived mesenchymal stem cells. The obtained mesenchymal stem cells were counted by the same method. A large number of 1.36×10 12 This indicates that the kit of the present invention can obtain a large number of adipose-derived mesenchymal stem cells in a relatively short period of time.

[0093] Example 7 Preparation of Adipose-Derived Mesenchymal Stem Cells

[0094] The kit of the present invention assembled in Example 2 was used to prepare adipose-derived mesenchymal stem cells. The method in Example 5 was referred to to prepare adipose-derived mesenchymal stem cells. The obtained mesenchymal stem cells were counted by the same method. The result was that 1.27×10 were obtained from only 20 ml of fat within 15 days. 12 This indicates that the kit of the present invention can obtain a large number of adipose-derived mesenchymal stem cells in a relatively short time.

Claims

1. A kit for efficiently preparing adipose-derived mesenchymal stem cells, characterized in that: Including fat preservation solution, fat washing solution, fat decomposition solution, cell washing solution, cell culture solution, cell digestion solution and cell freezing solution; The fat preservation solution is DMEM, DMEM / F12, MEM or RPMI-1640 culture medium containing 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin, 5-10ug / ml of aminoethanol, 2-8ug / ml of GLP-1, 5-10ug / ml of forsythiaside B, and 4-8ng / ml of eugenol; The fat washing solution is physiological saline, PBS, D-Hanks or HBSS containing 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin, 5-10ug / ml of aminoethanol, 2-8ug / ml of GLP-1, 5-10ug / ml of forsythiaside B, and 4-8ng / ml of eugenol; The lipolysis solution is DMEM, DMEM / F12, MEM or RPMI-1640 culture medium containing collagenase I 1-2 mg / ml, deoxyribonuclease I 0.1-0.2 mg / ml, aminoethanol 5-10ug / ml, GLP-1 2-8ug / ml, forsythiaside B 5-10ug / ml, and eugenol 4-8ng / ml; The cell washing solution is PBS, D-Hanks or HBSS containing 5-10ug / ml aminoethanol, 2-8ug / ml GLP-1, 5-10ug / ml forsythiaside B, and 4-8ng / ml eugenol; The cell culture medium is Keratinocyte-SFM medium, TheraPEAK™ MSCGM-CD™ medium, DMEM+10% FBS or DMEM / F12+10% FBS supplemented with 5-10 ng / ml aminoethanol, 2-8 ng / ml GLP-1, 5-10 ng / ml forsythiaside B, and 4-8 ng / ml eugenol; The cell digestion solution is PBS, D-Hanks or HBSS solution containing Trypsin-EDTA 1.25-2.5 mg / ml, deoxyribonuclease I 0.1-0.2 mg / ml, aminoethanol 5-10 ug / ml, GLP-1 2-8 ug / ml, forsythiaside B 5-10 ug / ml, and eugenol 4-8 ng / ml; The cell freezing medium is Keratinocyte-SFM culture medium, TheraPEAK™ MSCGM-CD™ culture medium, DMEM+10% FBS or DMEM / F12+10% FBS containing DMSO 100-200 mg / ml, chloroquine phosphate 40-100 ug / ml, and palmatine hydrochloride 5-10 ng / ml.

Images