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

Inactive Publication Date: 2015-02-18
张炳强
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] At present, the main treatment methods for autoimmune diseases include hormone therapy and immunosuppressants. On the one hand, the side effects are very serious (gastrointestinal tract reactions, blood system damage, urinary system reactions, affecting fertility, etc.), and on the other hand, the curative effects are different for different patients. Larger, many patients are still unable to control their condition or cannot tolerate the severe side effects of the above treatments, so it is urgent to explore new treatments for autoimmune diseases

Method used

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  • 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
  • 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
  • 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

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a preparation method of human adipose-derived MSCs (mesenchymal stem cells) and an application of the human adipose-derived MSCs in preparation of medicine for treating diseases. The preparation method of the human adipose-derived MSCs comprises steps as follows: adipose tissue collection, adipose tissue transportation, adipose tissue connection, adipose tissue microblock preparation, SVF (stromal vascular fraction) extraction, primary culture, subculturing, cell cryopreservation, cell bank establishment and cell recovery. The human adipose-derived MSCs have a powerful immunity regulation function and can be applied to preparation of medicine for treating autoimmune diseases, and compared with conventional medicine, the human adipose-derived MSCs have the advantages as follows: 1, the treatment is convenient, the curative effect is lasting, the variety and the number of medicine for treatment and toxic and side effects caused by medicine treatment can be reduced, even the medicine such as immunosuppressant and the like for treatment is out of service, the death rate and the disability rate are reduced, and life quality is improved; 2, the safety is good, and toxic and side effects are avoided; 3, the source of adipose tissue is wide, the collection is convenient, fundamental research of the human adipose-derived MSCs is solid, the preparation technology is mature, the popularization degree is high, and the technology is controllable; and 4, the preparation cost of the human adipose-derived MSCs is lower than that of other biological preparation and can be accepted by most patients.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a preparation method of human adipose mesenchymal stem cells and its application in the preparation of disease medicines. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are a kind of adult stem cells with multi-directional differentiation potential and low immunogenicity. They are adhesive, spindle-shaped, and fibroblast-like in shape. The germ layer has the potential to differentiate into a variety of mesoderm cell lineages, including bone cells, adipocytes, chondrocytes, fibroblasts, tenocytes, etc. MSCs are widely found in human bone marrow, fat, periodontal ligament, neonatal umbilical cord, placenta, amniotic membrane, amniotic fluid and other tissues. They come from a wide range of sources and have no ethical restrictions. They are easy to isolate and expand in vitro. It still maintains multi-directional differentiation potential, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/35A61P37/02A61P3/10A61P29/00A61P19/02
Inventor 张炳强
Owner 张炳强
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