High-efficiency Extraction Kit for Placenta Stem Cells

Abstract

The present invention belongs to the field of stem cell technology and relates to a kit for efficient extraction of placental stem cells. This kit includes a placental preservation solution, a placental washing solution I, a placental washing solution II, a tissue decomposition solution I, a tissue decomposition solution II, a digestion termination solution, a cell separation solution, and a cell cryopreservation solution. By using the kit of the present invention, a large number of placental stem cells can be extracted in a relatively short time, and it is convenient to use.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cells and relates to a kit for efficiently extracting placental stem cells. Background Art

[0002] Placental stem cells refer to a type of mixed stem cells present in placental tissue, including various stem cells such as hematopoietic stem cells and mesenchymal stem cells.

[0003] Placental hematopoietic stem cells provide a new source for hematopoietic stem cell transplantation. Hematopoietic stem cells are the originators of red blood cells, white blood cells, platelets, etc. They are highly undifferentiated cells and are the original cells of all blood cells and immune cells. Hematopoietic stem cell transplantation can treat more than a dozen life-threatening diseases such as malignant blood diseases, some malignant tumors, and some genetic diseases. The number of hematopoietic stem cells in the placenta is abundant, 5-10 times that of cord blood, breaking through the bottleneck of the use of hematopoietic stem cells from cord blood for a body weight of 30 kg, and can provide sufficient amounts of autologous stem cells to reconstruct the hematopoietic system of an adult.

[0004] Clinical application research on placental hematopoietic stem cells has been widely carried out at present. The diseases treated include: malignant or non-malignant blood diseases such as severe aplastic anemia, myelodysplastic syndrome, leukemia, etc., and non-blood system diseases such as type 2 diabetes, idiopathic pulmonary fibrosis, ankylosing spondylitis, ulcerative colitis, etc.

[0005] Placental mesenchymal stem cells also provide a new source for mesenchymal stem cells. Mesenchymal stem cells are a type of stem cells with multi-directional differentiation potential. They originate from the mesoderm during embryonic development and have the potential to differentiate into multiple mesodermal cell lines, including osteoblasts, adipocytes, chondrocytes, nerve cells, liver cells, epidermal cells, etc. Mesenchymal stem cells have a wide source and no ethical restrictions, are easy to isolate and expand in vitro, and still maintain multi-directional differentiation potential after multiple passages of cell division. So far, no serious side effects have been found in clinical trial studies on mesenchymal stem cells. They are an ideal seed cell for cell therapy and regenerative medicine. Placental mesenchymal stem cells also have the following advantages: First, they have low immunogenicity and immunosuppressive effects, which enables the mesenchymal stem cells used for treatment to be derived not only from autologous but also from allogeneic sources; second, they have a strong immunomodulatory effect, enabling the transplanted mesenchymal stem cells to regulate the immune state of the patient while repairing damaged tissues; third, they have the characteristic of directional differentiation into specific cells, avoiding the risk of tumorigenicity of totipotent stem cells such as embryonic stem cells.

[0006] In recent years, mesenchymal stem cells have been widely used in experimental and clinical research, and a large number of studies have revealed their application value in the diagnosis and treatment of systemic diseases such as cardiovascular, nervous, locomotor, digestive, autoimmune, blood, urinary, ophthalmic, and orthopedic systems.

[0007] In summary, placental stem cells have broad clinical application prospects. Therefore, it has become an urgent task to develop a simple, rapid, efficient, and safe method for obtaining a large number of placental stem cells. However, there is currently no kit for extracting placental stem cells in the existing technology, which has slowed down the progress of related scientific research work. Summary of the Invention

[0008] The purpose of the present invention is to make up for the deficiencies in the existing technology and provide a kit for extracting and obtaining a large number of placental stem cells that is convenient to use and low in cost.

[0009] To achieve the above object, the technical solution adopted by the present invention is: a kit for highly efficient extraction of placental stem cells, including a placental preservation solution, a placental washing solution I, a placental washing solution II, a tissue decomposition solution I, a tissue decomposition solution II, a digestion termination solution, a cell separation solution, and a cell cryopreservation solution.

[0010] The amounts of each component in the kit are as follows:

[0011] 1 bottle of placental preservation solution (500 ml / bottle);

[0012] 2 bottles of placental washing solution I (500 ml / bottle);

[0013] 2 bottles of placental washing solution II (500 ml / bottle);

[0014] 1 bottle of tissue decomposition solution I (500 ml / bottle);

[0015] 1 bottle of tissue decomposition solution II (300 ml / bottle);

[0016] 1 bottle of digestion termination solution (20 ml / bottle);

[0017] 1 bottle of cell separation solution (100 ml / bottle);

[0018] 1 bottle of cell cryopreservation solution (50 ml / bottle).

[0019] The placental preservation solution is a DMEM, DMEM / F12, MEM, or RPMI-1640 medium containing 100 - 200 μg / ml of penicillin, 100 - 200 μg / ml of streptomycin, 2.5 - 5 μg / ml of amphotericin, 0.5 - 1.5 mg / ml of heparin sodium injection, 5 - 10 mg / ml of human albumin, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C.

[0020] The placenta washing solution I is normal saline, PBS, D-Hanks or HBSS containing 100 - 200 μg / ml of penicillin, 100 - 200 μg / ml of streptomycin, 2.5 - 5 μg / ml of amphotericin, 0.5 - 1.5 mg / ml of heparin sodium injection, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C.

[0021] The placenta washing solution II is normal saline, PBS, D-Hanks or HBSS containing 100 - 200 μg / ml of penicillin, 100 - 200 μg / ml of streptomycin, 2.5 - 5 μg / ml of amphotericin, 0.5 - 1.5 mg / ml of heparin sodium injection, 5 - 10 mg / ml of human albumin, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C.

[0022] The tissue digestion solution I is DMEM, DMEM / F12, MEM or RPMI-1640 medium containing 0.5 - 2.5 mg / ml of type IV collagenase, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C.

[0023] The tissue digestion solution II is DMEM, DMEM / F12, MEM or RPMI-1640 medium containing 0.5 - 2.5 mg / ml of trypsin, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C.

[0024] The digestion termination solution is 0.2 g / ml of human albumin.

[0025] The cell separation solution is 60 mg / ml of hydroxyethyl starch.

[0026] The cell cryopreservation solution is KnockoutTM SR, Keratinocyte-SFM medium, TheraPEAK™ MSCGM-CD™ medium, DMEM + 10% FBS or DMEM / F12 + 10% FBS containing 5 - 10 ng / ml of ophiopogonin D, 5 - 10 ng / ml of cassiaside C, and 100 - 200 mg / ml of DMSO.

[0027] The present invention provides a kit for efficiently extracting placental stem cells. In the present invention, the function of the placental preservation solution is to ensure the activity of the placental specimen during transportation after collection, and to eliminate and prevent bacteria and fungi. The functions of placental washing solution I and placental washing solution II are to wash away blood cells from the placental tissue, while eliminating and preventing bacteria and fungi, and at the same time ensuring the activity of placental stem cells. The functions of tissue digestion solution I and tissue digestion solution II are to digest the placental tissue step by step, efficiently extract placental stem cells, while ensuring the activity of placental stem cells, and finally obtain a high-activity single-cell suspension. The function of the digestion termination solution is to terminate the digestion effect of the tissue digestion solution and ensure the activity of placental stem cells. The function of the cell separation solution is to remove red blood cells and obtain mononuclear cells, while ensuring the activity of placental stem cells. The function of the cell cryopreservation solution is to cryopreserve the obtained placental stem cells and ensure the activity of the stem cells after resuscitation.

[0028] The kit for efficiently extracting placental stem cells of the present invention can efficiently and rapidly obtain a large number of placental stem cells, and has the advantages of convenient use and low cost. It will play an important role in stem cell clinical trials and related research, and has extremely broad application prospects. Specific embodiments

[0029] In the following embodiments, the methods used are all conventional methods unless otherwise specified, and the required reagent consumables, experimental instruments, etc. can all be obtained through commercial channels.

[0030] Example 1 Assembly and application of the kit for efficiently extracting placental stem cells

[0031] I. Assembly of the kit for efficiently extracting placental stem cells

[0032] 1. Solution preparation

[0033] In the assembly of the kit for efficiently extracting placental stem cells of the present invention, all components required are commercially available products and can be obtained through commercial channels: penicillin, brand Amresco, catalog number 0242; streptomycin, brand Amresco, catalog number 0382; amphotericin, brand Amresco, catalog number E437; human albumin, brand GRIFOLS, specification 20%: 50 ml; heparin sodium injection, Chengdu Haitong Pharmaceutical Co., Ltd., specification 2 ml: 5000 units; palmatine hydrochloride, brand aladdin, catalog number P408401 - 1 ml; chloroquine phosphate, brand sigma, catalog number C6628 - 25G; hydroxysafflor yellow A, Shanghai Yansheng Biochemical Reagent Co., Ltd., 20 mg; type IV collagenase, brand sigma, catalog number C5138; ophiopogonin D, brand MCE, catalog number HY-N0515; cassiaside C, Wuhan Tianzhi Biotechnology Co., Ltd., catalog number CFN95120; DMSO, brand sigma, catalog number D2650.

[0034] The highly efficient extraction kit for placental stem cells of the present invention comprises the following reagents:

[0035] (1) Placenta preservation solution, preparation method: 100 mg of penicillin, 100 mg of streptomycin, 2.5 mg of amphotericin, 2.5 g of human albumin, 0.5 g of heparin sodium injection, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 5 μg of ophiopogonin D, and 5 μg of cassiaside C are dissolved in 500 ml of DMEM / F12 medium.

[0036] (2) Placenta washing solution I, preparation method: 200 mg of penicillin, 200 mg of streptomycin, 5 mg of amphotericin, 1 g of heparin sodium injection, 4 μg of palmatine hydrochloride, 4 μg of chloroquine phosphate, 4 μg of hydroxysafflor yellow A, 10 μg of ophiopogonin D, and 10 μg of cassiaside C are dissolved in 1000 ml of physiological saline.

[0037] (3) Placenta washing solution II, preparation method: 200 mg of penicillin, 200 mg of streptomycin, 5 mg of amphotericin, 5 g of human albumin, 1 g of heparin sodium injection, 4 μg of palmatine hydrochloride, 4 μg of chloroquine phosphate, 4 μg of hydroxysafflor yellow A, 10 μg of ophiopogonin D, and 10 μg of cassiaside C are dissolved in 1000 ml of physiological saline.

[0038] (4) Tissue digestion solution I, preparation method: 1 g of type IV collagenase, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 5 μg of ophiopogonin D, and 5 μg of cassiaside C are dissolved in 500 ml of DMEM / F12 medium, and then filtered and sterilized with a 0.22 μm filter.

[0039] (5) Tissue digestion solution II, preparation method: 1.25 g of trypsin, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 5 μg of ophiopogonin D, and 5 μg of cassiaside C are dissolved in 500 ml of DMEM / F12 medium, and then filtered and sterilized with a 0.22 μm filter.

[0040] (6) Digestion termination solution, preparation method: commercially available human albumin with a concentration of 0.2 g / ml (brand: GRIFOLS, specification 20%: 50 ml), and it can be aseptically subpackaged.

[0041] (7) Cell separation solution, preparation method: commercially available 60 mg / ml hydroxyethyl starch (manufacturer: Fresenius Kabi (China) Pharmaceutical Co., Ltd., specification 500 ml: 30 g of hydroxyethyl starch 130 / 0.4 and 4.5 g of sodium chloride), and it can be aseptically subpackaged.

[0042] (8)Cell cryopreservation solution, preparation method: Slowly add 10 μg of ophiopogonin D, 10 μg of cassiaside C, and 200 ml of DMSO to 800 ml of Keratinocyte-SFM (brand Gibco, catalog number: 17005042) medium, gently mix well, and filter and sterilize with a 0.22-μm filter.

[0043] 2. Kit assembly

[0044] The amount of each component in each kit is as follows: 1 bottle of placental preservation solution (500 ml / bottle), 2 bottles of placental washing solution I (500 ml / bottle), 2 bottles of placental washing solution II (500 ml / bottle), 1 bottle of tissue decomposition solution I (500 ml / bottle), 1 bottle of tissue decomposition solution II (300 ml / bottle), 1 bottle of digestion termination solution (20 ml / bottle), 1 bottle of cell separation solution (100 ml / bottle), and 1 bottle of cell cryopreservation solution (50 ml / bottle). Dispense each component in the kit according to the above reagent amounts, and package to obtain a kit for extracting placental stem cells.

[0045] All components are stored at -20°C, with a shelf life of 3 years. Before use, thaw in a 4°C refrigerator or a 37°C water bath, and store at 4°C after thawing. Use up within three months.

[0046] The kit specification is 1 time / box and can be used to extract one placenta.

[0047] II. Extraction of placental stem cells

[0048] Now use the kit in step one to extract placental stem cells. The specific extraction method includes the following steps:

[0049] 1. Placenta transportation: Collect a complete placenta, add an appropriate amount of placental preservation solution to submerge the placenta, and store it in a vaccine box at a constant temperature of 2-8°C and send it to the laboratory within 24 hours.

[0050] 2. Wash the placenta: Aspirate the placental preservation solution, add an appropriate volume of placental washing solution I, shake it several times repeatedly, aspirate the placental washing solution I, and do the same above. Then add an appropriate volume of placental washing solution I and shake and wash 2-3 times until the blood stains on the placenta surface are washed clean. Remove the amnion and decidua on the placenta surface, add an appropriate volume of placental washing solution II, cut the placental tissue into pieces, and wash the blood in the tissue blocks clean. Collect the placental washing solution II, centrifuge at 1800 rpm at room temperature for 10 minutes, discard the supernatant, resuspend the cell pellet with 50 ml of placental washing solution II, add the cell separation solution according to the ratio of placental washing solution II: cell separation solution = 4:1, mix well, let it stand at room temperature for 30 minutes and then layer, and collect the upper layer liquid.

[0051] 3. Digest the placenta: Collect the minced placental tissue blocks, add tissue digestion solution I with the same volume as the placental tissue blocks, and digest in a 37°C water bath shaker for 15 - 20 min. Then add tissue digestion solution II with 1 / 4 volume of the placental tissue blocks and digest in a 37°C water bath shaker for 15 - 20 min. Filter the placental tissue blocks after the two digestions through a 200-mesh analytical sieve to collect the filtrate, add 10 ml of digestion termination solution, mix well, and centrifuge at 1800 rpm at room temperature for 10 min. Discard the supernatant, resuspend the cell pellet with an appropriate amount of placental washing solution II, add cell separation solution according to the ratio of placental washing solution II:cell separation solution = 4:1, mix well, and centrifuge at 300 rpm at room temperature for 10 min to collect the upper layer liquid.

[0052] 4. Harvest the cells: Combine the upper layer liquids collected in steps 2 and 3, and measure the number of mononuclear cells in a hematology analyzer to be 7.32×10 11 cells.

[0053] 5. Cryopreserve the cells: Centrifuge the obtained cell suspension at 1800 rpm at room temperature for 10 min, discard the supernatant, slowly add cell cryopreservation solution, gently mix, then label the basic information of the cells on the cryopreservation bag, cool to -80°C in a programmable freezer, and transfer to liquid nitrogen for long-term storage. Thaw it when needed for use.

[0054] It can be seen that with the kit of the present invention, a large number of placental stem cells can be obtained from a complete placenta within 2.5 hours, which is 7.32×10 11 cells, indicating that the kit of the present invention can obtain a large number of placental stem cells in a relatively short time.

[0055] Example 2 Assembly and Application of the High-Efficiency Placental Stem Cell Extraction Kit

[0056] I. Assembly of the High-Efficiency Placental Stem Cell Extraction Kit

[0057] 1. Solution preparation

[0058] (1) Placenta preservation solution, preparation method: Dissolve 50 mg of penicillin, 50 mg of streptomycin, 2.5 mg of amphotericin, 2.5 g of human albumin, 0.5 g of heparin sodium injection, 1 μg of palmatine hydrochloride, 1 μg of chloroquine phosphate, 1 μg of hydroxysafflor yellow A, 5 μg of ophiopogonin D, and 5 μg of cassiaside C in 500 ml of DMEM / F12 medium.

[0059] (2) Placenta washing solution I, preparation method: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 5 mg of amphotericin, 1 g of heparin sodium injection, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 5 μg of ophiopogonin D, and 5 μg of cassiaside C in 1000 ml of physiological saline.

[0060] (3) Placenta washing solution II, preparation method: 200 mg of penicillin, 200 mg of streptomycin, 5 mg of amphotericin, 10 g of human albumin, 1 g of heparin sodium injection, 4 μg of palmatine hydrochloride, 4 μg of chloroquine phosphate, 4 μg of hydroxysafflor yellow A, 10 μg of ophiopogon saponin D, 10 μg of cassiaside C, dissolved in 1000 ml of physiological saline.

[0061] (4) Tissue digestion solution I, preparation method: 250 mg of type IV collagenase, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 5 μg of ophiopogon saponin D, 5 μg of cassiaside C, dissolved in 500 ml of DMEM / F12 medium, and filtered through a 0.22 μm filter for sterilization.

[0062] (5) Tissue digestion solution II, preparation method: 250 mg of trypsin, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 5 μg of ophiopogon saponin D, 5 μg of cassiaside C, dissolved in 500 ml of DMEM / F12 medium, and filtered through a 0.22 μm filter for sterilization.

[0063] (6) Digestion termination solution, preparation method: commercially available human albumin with a concentration of 0.2 g / ml, aseptically aliquoted.

[0064] (7) Cell separation solution, preparation method: commercially available 60 mg / ml hydroxyethyl starch, aseptically aliquoted.

[0065] (8) Cell cryopreservation solution, preparation method: 5 μg of ophiopogon saponin D, 5 μg of cassiaside C, 100 ml of DMSO were slowly added to 900 ml of Knockout TM SR (brand invitrogen, catalog number 10828 - 028), gently mixed, and filtered through a 0.22 μm filter for sterilization.

[0066] 2. Kit assembly

[0067] The amount of each component in each kit is as follows: 1 bottle of placenta preservation solution (500 ml / bottle), 2 bottles of placenta washing solution I (500 ml / bottle), 2 bottles of placenta washing solution II (500 ml / bottle), 1 bottle of tissue digestion solution I (500 ml / bottle), 1 bottle of tissue digestion solution II (300 ml / bottle), 1 bottle of digestion termination solution (20 ml / bottle), 1 bottle of cell separation solution (100 ml / bottle), 1 bottle of cell cryopreservation solution (50 ml / bottle). Aliquot each component in the kit according to the above reagent amounts, and package to obtain a kit for extracting placental stem cells.

[0068] All components are stored at -20°C, with a validity period of 3 years. Before use, thaw them in a 4°C refrigerator or a 37°C water bath, and store them at 4°C after thawing. Use them up within three months.

[0069] The kit specification is 1 time / box and can be used to extract one placenta.

[0070] II. Extraction of placental stem cells

[0071] Using the kit in Step 1, extract placental stem cells with reference to the method in Example 1, and count the obtained placental stem cells in the same method. As a result, for a complete placenta, 9.11×10 11 placental stem cells can be obtained after 2.5 hours of separation, indicating that the kit of the present invention can obtain a large number of placental stem cells in a short time.

[0072] Example 3 Assembly and Application of a Kit for Efficient Extraction of Placental Stem Cells

[0073] I. Assembly of a Kit for Efficient Extraction of Placental Stem Cells

[0074] 1. Solution preparation

[0075] (1) Placenta preservation solution, preparation method: Dissolve 75 mg of penicillin, 75 mg of streptomycin, 1.25 mg of amphotericin, 2.5 g of human albumin, 0.5 g of heparin sodium injection, 1.5 μg of palmatine hydrochloride, 1.5 μg of chloroquine phosphate, 1.5 μg of hydroxysafflor yellow A, 7 μg of ophiopogonin D, and 7 μg of cassiaside C in 500 ml of DMEM medium.

[0076] (2) Placenta washing solution I, preparation method: Dissolve 75 mg of penicillin, 75 mg of streptomycin, 1.25 mg of amphotericin, 1 g of heparin sodium injection, 1.5 μg of palmatine hydrochloride, 1.5 μg of chloroquine phosphate, 1.5 μg of hydroxysafflor yellow A, 7 μg of ophiopogonin D, and 7 μg of cassiaside C in 1000 ml of physiological saline.

[0077] (3) Placenta washing solution II, preparation method: Dissolve 75 mg of penicillin, 75 mg of streptomycin, 1.25 mg of amphotericin, 10 g of human albumin, 1 g of heparin sodium injection, 1.5 μg of palmatine hydrochloride, 1.5 μg of chloroquine phosphate, 1.5 μg of hydroxysafflor yellow A, 7 μg of ophiopogonin D, and 7 μg of cassiaside C in 1000 ml of physiological saline.

[0078] (4)Tissue digestion solution Ⅰ, preparation method: 1 g of type Ⅳ collagenase, 1.5 μg of palmatine hydrochloride, 1.5 μg of chloroquine phosphate, 1.5 μg of hydroxysafflor yellow A, 7 μg of ophiopogonin D, 7 μg of cassiaside C, dissolve in 500 ml of DMEM medium, and filter and sterilize with a 0.22 μm filter.

[0079] (5)Tissue digestion solution Ⅱ, preparation method: 1 g of trypsin, 1.5 μg of palmatine hydrochloride, 1.5 μg of chloroquine phosphate, 1.5 μg of hydroxysafflor yellow A, 7 μg of ophiopogonin D, 7 μg of cassiaside C, dissolve in 500 ml of DMEM / F12 medium, and filter and sterilize with a 0.22 μm filter.

[0080] (6)Digestion termination solution, preparation method: commercially available human albumin with a concentration of 0.2 g / ml, and aseptically dispense.

[0081] (7)Cell separation solution, preparation method: commercially available 60 mg / ml hydroxyethyl starch, and aseptically dispense.

[0082] (8)Cell cryopreservation solution, preparation method: 8 μg of ophiopogonin D, 8 μg of cassiaside C, slowly add 150 ml of DMSO to 850 ml of DMEM + 10% FBS, gently mix, and filter and sterilize with a 0.22 μm filter.

[0083] 2. Kit assembly

[0084] The amount of each component in each kit is: 1 bottle of placental preservation solution (500 ml / bottle), 2 bottles of placental washing solution Ⅰ (500 ml / bottle), 2 bottles of placental washing solution Ⅱ (500 ml / bottle), 1 bottle of tissue digestion solution Ⅰ (500 ml / bottle), 1 bottle of tissue digestion solution Ⅱ (300 ml / bottle), 1 bottle of digestion termination solution (20 ml / bottle), 1 bottle of cell separation solution (100 ml / bottle), 1 bottle of cell cryopreservation solution (50 ml / bottle). Dispense each component in the kit according to the above reagent amounts, and package to obtain a kit for extracting placental stem cells.

[0085] All components are stored at -20°C, with a validity period of 3 years. Thaw in a 4°C refrigerator or a 37°C water bath before use, and store at 4°C after thawing. Use up within three months.

[0086] The kit specification is 1 time / box, and can be used to extract one placenta.

[0087] II. Extraction of placental stem cells

[0088] Using the kit in Step 1, extract placental stem cells according to the method in Example 1, and count the obtained placental stem cells in the same way. As a result, from a complete placenta, 8.69×10 11 placental stem cells can be obtained after 2.5 hours of separation, indicating that the kit of the present invention can obtain a large number of placental stem cells in a relatively short time.

[0089] Example 4 Assembly and Application of a Kit for Efficient Extraction of Placental Stem Cells

[0090] I. Assembly of the Kit for Efficient Extraction of Placental Stem Cells

[0091] 1. Solution Preparation

[0092] (1) Placenta preservation solution. Preparation method: Dissolve 75 mg of penicillin, 50 mg of streptomycin, 2.5 mg of amphotericin, 2.5 g of human albumin, 250 mg of heparin sodium injection, 1 μg of palmatine hydrochloride, 1 μg of chloroquine phosphate, 1 μg of hydroxysafflor yellow A, 2.5 μg of ophiopogonin D, and 2.5 μg of cassiaside C in 500 ml of MEM medium.

[0093] (2) Placenta washing solution I. Preparation method: Dissolve 100 mg of penicillin, 200 mg of streptomycin, 2.5 mg of amphotericin, 1.5 g of heparin sodium injection, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 5 μg of ophiopogonin D, and 5 μg of cassiaside C in 1000 ml of physiological saline.

[0094] (3) Placenta washing solution II. Preparation method: Dissolve 200 mg of penicillin, 200 mg of streptomycin, 5 mg of amphotericin, 5 g of human albumin, 1.5 g of heparin sodium injection, 4 μg of palmatine hydrochloride, 4 μg of chloroquine phosphate, 4 μg of hydroxysafflor yellow A, 7 μg of ophiopogonin D, and 10 μg of cassiaside C in 1000 ml of physiological saline.

[0095] (4) Tissue digestion solution I. Preparation method: Dissolve 750 mg of type IV collagenase, 1 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 2 μg of ophiopogonin D, and 2 μg of cassiaside C in 500 ml of DMEM / F12 medium, and filter and sterilize it with a 0.22 μm filter.

[0096] (5) Tissue digestion solution II. Preparation method: Dissolve 750 mg of trypsin, 1 μg of palmatine hydrochloride, 1 μg of chloroquine phosphate, 1 μg of hydroxysafflor yellow A, 2.5 μg of ophiopogonin D, and 2.5 μg of cassiaside C in 500 ml of MEM medium, and filter and sterilize it with a 0.22 μm filter.

[0097] (6) Digestion termination solution, preparation method: commercially available human albumin with a concentration of 0.2 g / ml, just aseptically subpackage it.

[0098] (7) Cell separation solution, preparation method: commercially available hydroxyethyl starch with a concentration of 60 mg / ml, just aseptically subpackage it.

[0099] (8) Cell cryopreservation solution, preparation method: Slowly add 10 μg of ophiopogonin D, 10 μg of cassiaside C, and 100 ml of DMSO to 900 ml of Knockout TM SR, gently mix well, and filter and sterilize with a 0.22 μm filter.

[0100] 2. Kit assembly

[0101] The amount of each component in each kit is as follows: 1 bottle of placental preservation solution (500 ml / bottle), 2 bottles of placental washing solution I (500 ml / bottle), 2 bottles of placental washing solution II (500 ml / bottle), 1 bottle of tissue decomposition solution I (500 ml / bottle), 1 bottle of tissue decomposition solution II (300 ml / bottle), 1 bottle of digestion termination solution (20 ml / bottle), 1 bottle of cell separation solution (100 ml / bottle), and 1 bottle of cell cryopreservation solution (50 ml / bottle). Subpackage each component in the kit according to the above reagent amounts, and package to obtain a kit for extracting placental stem cells.

[0102] All components are stored at -20°C, with a validity period of 3 years. Before use, thaw in a 4°C refrigerator or a 37°C water bath, and store at 4°C after thawing. Use up within three months.

[0103] The kit specification is 1 time / box, and it can be used to extract one placenta.

[0104] II. Extraction of placental stem cells

[0105] Use the kit in step one to extract placental stem cells with reference to the method in Example 1, and count the obtained placental stem cells with the same method. As a result, for a complete placenta, 6.96×10 11 placental stem cells can be obtained after 2.5 hours of separation, indicating that the kit of the present invention can obtain a large number of placental stem cells in a relatively short time.

[0106] Example 5 Assembly and application of a kit for highly efficient extraction of placental stem cells

[0107] I. Assembly of a kit for highly efficient extraction of placental stem cells

[0108] 1. Solution preparation

[0109] (1)Placenta preservation solution, preparation method: 100 mg of penicillin, 100 mg of streptomycin, 2.5 mg of amphotericin, 2.5 g of human albumin, 0.5 g of heparin sodium injection, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 1 μg of hydroxysafflor yellow A, 5 μg of ophiopogon saponin D, and 5 μg of cassiaside C are dissolved in 500 ml of DMEM / F12 medium.

[0110] (2)Placenta washing solution Ⅰ, preparation method: 100 mg of penicillin, 100 mg of streptomycin, 5 mg of amphotericin, 1 g of heparin sodium injection, 2 μg of palmatine hydrochloride, 4 μg of chloroquine phosphate, 4 μg of hydroxysafflor yellow A, 5 μg of ophiopogon saponin D, and 5 μg of cassiaside C are dissolved in 1000 ml of physiological saline.

[0111] (3)Placenta washing solution Ⅱ, preparation method: 200 mg of penicillin, 200 mg of streptomycin, 5 mg of amphotericin, 10 g of human albumin, 1 g of heparin sodium injection, 4 μg of palmatine hydrochloride, 4 μg of chloroquine phosphate, 4 μg of hydroxysafflor yellow A, 8 μg of ophiopogon saponin D, and 8 μg of cassiaside C are dissolved in 1000 ml of physiological saline.

[0112] (4)Tissue decomposition solution Ⅰ, preparation method: 1 g of type Ⅳ collagenase, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 2.5 μg of ophiopogon saponin D, and 2.5 μg of cassiaside C are dissolved in 500 ml of DMEM / F12 medium, and then filtered and sterilized with a 0.22 μm filter.

[0113] (5)Tissue decomposition solution Ⅱ, preparation method: 1 g of trypsin, 2 μg of palmatine hydrochloride, 2 μg of chloroquine phosphate, 2 μg of hydroxysafflor yellow A, 5 μg of ophiopogon saponin D, and 5 μg of cassiaside C are dissolved in 500 ml of DMEM / F12 medium, and then filtered and sterilized with a 0.22 μm filter.

[0114] (6)Digestion termination solution, preparation method: commercially available human albumin with a concentration of 0.2 g / ml is aseptically dispensed.

[0115] (7)Cell separation solution, preparation method: commercially available 60 mg / ml hydroxyethyl starch is aseptically dispensed.

[0116] (8)Cell cryopreservation solution, preparation method: 9 μg of ophiopogon saponin D, 9 μg of cassiaside C, and 200 ml of DMSO are slowly added to 800 ml of Knockout TM SR, gently mixed, and then filtered and sterilized with a 0.22 μm filter.

[0117] 2. Kit assembly

[0118] The amounts of each component in each box are as follows: 1 bottle of placental preservation solution (500 ml / bottle), 2 bottles of placental washing solution I (500 ml / bottle), 2 bottles of placental washing solution II (500 ml / bottle), 1 bottle of tissue decomposition solution I (500 ml / bottle), 1 bottle of tissue decomposition solution II (300 ml / bottle), 1 bottle of digestion termination solution (20 ml / bottle), 1 bottle of cell separation solution (100 ml / bottle), and 1 bottle of cell cryopreservation solution (50 ml / bottle). The components in the kit are sub-packaged according to the above reagent dosages, and the kit for extracting placental stem cells is obtained through packaging.

[0119] All components are stored at -20°C with a validity period of 3 years. Before use, they are thawed in a 4°C refrigerator or in a 37°C water bath, and then stored at 4°C and used up within three months.

[0120] The kit specification is 1 time / box and can be used to extract one placenta.

[0121] II. Extraction of Placental Stem Cells

[0122] Using the kit in Step 1, extract placental stem cells with reference to the method in Example 1, and count the obtained placental stem cells using the same method. As a result, for a complete placenta, 8.61×10 11 placental stem cells can be obtained after 2.5 hours of separation, indicating that the kit of the present invention can obtain a large number of placental stem cells in a relatively short time.

Claims

1. Placenta stem cell high-efficiency extraction kit, characterized in that, It includes placenta preservation solution, placenta washing solution I, placenta washing solution II, tissue decomposition solution I, tissue decomposition solution II, digestion termination solution, cell separation solution, and cell cryopreservation solution; The placenta preservation solution is a DMEM, DMEM / F12, MEM, or RPMI-1640 medium containing 100 - 200 μg / ml of penicillin, 100 - 200 μg / ml of streptomycin, 2.5 - 5 μg / ml of amphotericin, 0.5 - 1.5 mg / ml of heparin sodium injection, 5 - 10 mg / ml of human albumin, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C; The placenta washing solution I is a normal saline, PBS, D-Hanks, or HBSS containing 100 - 200 μg / ml of penicillin, 100 - 200 μg / ml of streptomycin, 2.5 - 5 μg / ml of amphotericin, 0.5 - 1.5 mg / ml of heparin sodium injection, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C; The placenta washing solution II is a normal saline, PBS, D-Hanks, or HBSS containing 100 - 200 μg / ml of penicillin, 100 - 200 μg / ml of streptomycin, 2.5 - 5 μg / ml of amphotericin, 0.5 - 1.5 mg / ml of heparin sodium injection, 5 - 10 mg / ml of human albumin, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C; The tissue decomposition solution I is a DMEM, DMEM / F12, MEM, or RPMI-1640 medium containing 0.5 - 2.5 mg / ml of type IV collagenase, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C; The tissue decomposition solution II is a DMEM, DMEM / F12, MEM, or RPMI-1640 medium containing 0.5 - 2.5 mg / ml of trypsin, 2 - 4 ng / ml of palmatine hydrochloride, 2 - 4 ng / ml of chloroquine phosphate, 2 - 4 ng / ml of hydroxysafflor yellow A, 5 - 10 ng / ml of ophiopogonin D, and 5 - 10 ng / ml of cassiaside C; The digestion termination solution is 0.2 g / ml of human albumin; The cell separation solution is 60 mg / ml of hydroxyethyl starch; The cell cryopreservation solution is KnockoutTM SR, Keratinocyte-SFM medium, TheraPEAK™ MSCGM-CD™ medium, DMEM + 10% FBS or DMEM / F12 + 10% FBS containing 5 - 10 ng / ml of ophiopogonin D, 5 - 10 ng / ml of cassiaside C, and 100 - 200 mg / ml of DMSO.

2. The highly efficient extraction kit for placental stem cells according to claim 1, wherein The amounts of each component in the kit are: 1 bottle of placental preservation solution, 500 ml / bottle; 2 bottles of placental washing solution I, 500 ml / bottle; 2 bottles of placental washing solution II, 500 ml / bottle; 1 bottle of tissue digestion solution I, 500 ml / bottle; 1 bottle of tissue digestion solution II, 300 ml / bottle; 1 bottle of digestion termination solution, 20 ml / bottle; 1 bottle of cell separation solution, 100 ml / bottle; 1 bottle of cell cryopreservation solution, 50 ml / bottle.