Preparation method of human adipose-derived MSCs (mesenchymal stem cells) and application of human adipose-derived mesenchymal stem cell in preparation of medicine for treating diseases
A technology of stem cells and fat, applied in the field of immunology, can solve the problems of uncontrolled disease of patients, large difference in curative effect, serious side effects and other problems, and achieve strong immune regulation, no side effects and long-lasting curative effect Effect
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Embodiment 1
[0041] Example 1 Preparation of human adipose MSCs
[0042] 1. Collection of fat: HIV, HBV, HCV, HTLV, EBV, CMV, Treponema pallidum infection test (donor screening) is carried out on the donor, and the fat is collected under sterile conditions after the test is qualified. Liposuction can be used, and the liposuction sites include: waist, abdomen, buttocks, legs, etc., or surgical methods can be used to collect abdominal or pelvic fat. The amount of fat is above 10ml. After collection, the fat is placed in a preservation solution and stored at a constant temperature of 2-8°C.
[0043] 2. Fat transportation: Put the fat in the preservation solution, store it in the vaccine box at a constant temperature of 2-8°C, and send it to the laboratory within 48 hours.
[0044] 3. Handover of fat: the laboratory receives fat, registers customer information, and encodes it.
[0045] 4. Preparation of fat microblocks: absorb and discard the fat preservation solution, add an equal volume o...
Embodiment 2
[0051] Example 2 Quality Control of Human Fat MSCs
[0052] 1. Identification of human adipose MSCs surface markers by flow cytometry: Take human adipose MSCs and detect cell surface markers by flow cytometry. The results of flow cytometry are as follows
[0053]
[0054] Among them, positive markers CD29, CD73, CD90, CD105, and CD49d were positively expressed, negative markers CD14, CD34, and CD45 were negatively expressed, and Actin negatively expressed excluded fibroblasts, proving that the obtained cells were MSCs. The negative expression of HLA-DR, CD80, and CD86 proves that human adipose MSCs have low immunogenicity and can be used for allogeneic use without matching.
[0055] 2. Adipogenic induction and identification of human adipose MSCs: seed human adipose MSCs in a 24-well plate at a density of 2×104 / cm2, add 0.8ml of low-sugar DMEM culture medium with 15% FBS and 1ng / ml bFGF to each well; When the confluence reaches 80%, change to induction medium (high glucose...
Embodiment 3
[0061] Embodiment 3 Human adipose MSCs administration method
[0062] 1. P2- Six generations of human adipose MSCs, the cell viability after resuscitation is more than 85%, diluted with normal saline or electrolyte injection, put into a transfer bag, marked with the patient's name, and ready for reinfusion.
[0063] 2. Give dexamethasone 5-10 mg or promethazine 20-50 mg half an hour before cell infusion.
[0064] 3. Open the dorsal vein of the hand, connect the transfer bag containing human adipose MSCs, and adjust the infusion speed.
[0065] 4. Pay attention to observe the patient's reaction during the infusion, and treat the symptoms in time. The whole process takes about 30 minutes.
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