Fat SVF cell clinical high-efficiency preparation and cryopreservation method, and applications thereof

A fat and cell technology, applied in the field of the preparation of fat SVF cells, can solve the problems of low cell viability, low SVF purity, and large amount of collagenase used, so as to improve digestion ability, strong cell proliferation activity, and prevent excessive cell proliferation. effects of digestion

Active Publication Date: 2017-12-15
SHANGHAI LIFE SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a high-efficiency preparation and cryopreservation method of adipose SVF cells and its application. The preparation method can solve the problems of the large amount of collagenase used in the prior art, the low cell viability, and the low purity of the extracted SVF. problem, it can reduce the amount of collagenase used and improve cell viability and SVF purity to ensure the application effect in clinical treatment

Method used

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  • Fat SVF cell clinical high-efficiency preparation and cryopreservation method, and applications thereof
  • Fat SVF cell clinical high-efficiency preparation and cryopreservation method, and applications thereof
  • Fat SVF cell clinical high-efficiency preparation and cryopreservation method, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] (1) Sample screening

[0071] Medical history collection and testing of fat donors: including basic information of the donor, past medical history, family history, etc.

[0072] Past history and family history should collect detailed information about genetic diseases (single gene and polygenic diseases, including cardiovascular diseases and tumors, etc.).

[0073] Inspection and screening prove that the infection of specific unmanned viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.) is free of Treponema pallidum infection. If necessary, the donor's ABO blood type, HLA-I and II types need to be collected Data for retrospective inquiry.

[0074] (2) A method for clinically efficient preparation of fat SVF cells

[0075] Preparation of experimental materials:

[0076] The preparation of the preservation solution: It is composed of 0.9% normal saline + 100ug / mL penicillin + 100ug / mL streptomycin.

[0077] Preparation of washing solution: 500ml of normal saline, add 5ml of 20% h...

Embodiment 2

[0110] (1) Cryopreservation method of human adipose SVF cells

[0111] A method for freezing human adipose SVF cells, the freezing method includes:

[0112] Step S1': Use clinical grade DMSO (Dimethyl Sulfoxide, Article No. WAK-DMSO-10, Manufacturer Cryosure) and Serum Replacement (KnockOut™ Serum Replacement, KSR, Article No. 10828-028, Manufacturer Gibco) in accordance with 1:9 Mix by volume to form a freezing solution;

[0113] Step S2': slowly add the above cryopreservation solution to the centrifuge tube containing human fat SVF cells at a ratio of 1:1, pipette to mix the cells, mix well, and add the mixed cell suspension to the 2mL cryopreservation tube. Add 1.5mL to each tube, tighten the lid after adding, seal with the sealing film, mark the sample number, name, and freeze storage time, and put the marked cryotube into the freezer box;

[0114] Step S3': Place the sample in the cryopreservation box in the program cooling device to perform program cooling. The program cooling ...

Embodiment 3

[0121] A pharmaceutical preparation comprising: fat SVF cells, normal saline and albumin; wherein the mass ratio of normal saline to albumin is 98:2.

[0122] The human adipose SVF cells are prepared by the clinical-grade high-efficiency preparation method of human adipose SVF cells described in Example 1. The concentration of fat SVF cells is 0.5*10 7 / mL ~1.5*10 8 / mL.

[0123] The drug is used as a preparation for the treatment of osteoarthritis. Its dosage is in accordance with the clinical treatment dosage, and the dosage is 1*10 in a single time. 8 / 2mL ~2*10 8 / 2mL.

[0124] In summary, the clinical-grade efficient preparation and cryopreservation method of human adipose SVF cells of the present invention and its use. The washing solution and digestion solution used in the preparation method make the prepared adipose SVF cells have high cell viability and purity. Improved.

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Abstract

The invention discloses a fat SVF cell clinical high-efficiency preparation and cryopreservation method, and applications thereof. The fat SVF cell clinical high-efficiency preparation and cryopreservation method comprises following steps: S1, fat samples are collected under aseptic conditions, and are preserved in a preserving fluid, and a washing liquid is added for pre-treatment of the fat samples; S2, after pre-treatment, the fat samples are subjected to centrifugalization so as to obtain yellow fat tissue, the fat tissue is added into a preheated digestive juice, an obtained mixture is sealed, under constant temperature conditions, the fat tissue and the digestive juice are mixed to be uniform, digestion is carried out until no large tissue block is left or even large tissue blocks are left, a large amount of fat is gathered, and then centrifugation is carried out, wherein the volume ratio of the fat tissue to the preheated digestive juice is controlled to be 1:1; and S3, after centrifugation, upper layer fat is removed, the washing liquid is added for resuspending and uniform mixing of an obtained residue, filtering is carried out, the washing liquid is added again, centrifugation is carried out, and an obtained supernatant is removed so as to obtain fat SVF cells. According to the fat SVF cell clinical high-efficiency preparation and cryopreservation method, the washing liquid and the digestive juice are used for increase the cell number, the cell living rate, and the purity of the obtained fat SVF cells, and the effect of the extracted SVF cells in clinical treatment is ensured.

Description

Technical field [0001] The invention relates to a preparation method of adipose SVF cells, in particular to a clinical-grade efficient preparation and cryopreservation method of adipose SVF cells and uses thereof. Background technique [0002] Adult stem cells are a group of stem cells with multiple differentiation potentials that exist in various tissues and organs of the human body recently discovered. Studies have found that human Adipose-derived Stem cells (hASCs) as seed cells have many advantages: (1) easy to obtain materials (only by vacuum liposuction); (2) have many advantages Differentiation potential in different directions; (3) adipose-derived mesenchymal stem cells have low immunogenicity; (4) have an immunosuppressive regulatory effect. [0003] Adipose SVF (Stromal Vascular Fraction) cells are stromal cell groups separated and enriched from adipose tissue. Mature adipocytes have been removed. They contain a variety of cellular components, including adipose stem cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/071C12N5/077A01N1/02A61K35/35A61P29/00A61P19/02A61P19/08
CPCA01N1/02A61K35/35C12N5/0653C12N5/0656C12N5/0667C12N5/069C12N2509/00
Inventor 李文荣
Owner SHANGHAI LIFE SCI & TECH CO LTD
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