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Adipose stem cell separation and culture method

A technology of adipose stem cells and culture methods, which is applied in the direction of animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of increasing the cost of collagenase, to promote rapid proliferation, maintain cell stemness and biological characteristics, The effect of high separation efficiency

Active Publication Date: 2019-06-04
济南新生命干细胞有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other methods such as tissue block attachment method, adsorption column method, direct centrifugation method and mechanical shaking method, this method has the advantage of high stem cell yield, but the use of collagenase also increases the cost

Method used

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  • Adipose stem cell separation and culture method
  • Adipose stem cell separation and culture method
  • Adipose stem cell separation and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Obtaining a new collagenase and its expression, purification and activity identification

[0039] 1. Obtaining collagenase

[0040] According to the collagenase gene sequence published by NCBI, a pair of primers (as shown in Seq ID No.7 and Seq ID No.8) were designed, and Bacillus cereus (Bacillus cereus) strain ACCC10263 (purchased from China Agricultural Microbiological Culture Collection Management Center) genomic DNA as a template for PCR amplification, electrophoresis and recovery of about 3.0kb bands, sequenced to obtain the nucleotide sequence Seq ID No.1 of the collagenase-encoding gene, and the amino acid sequence of collagenase encoded by it It is Seq ID No.2.

[0041] 2. Construction of engineering bacteria

[0042] The collagenase coding gene shown in Seq ID No.1 was connected to the multiple cloning site of the expression vector pET28a to obtain the recombinant expression vector Z1. The recombinant expression vector Z1 was transformed into Esc...

Embodiment 2

[0071] Embodiment 2, the acquisition of mutants and their expression, purification and activity identification

[0072] 1. Acquisition of Mutants

[0073] According to the functional region of collagenase, design some collagenase mutants that have 2-3 amino acid replacements in the specific position of amino acid sequence Seq ID No.2 with the collagenase obtained in Example 1, wherein four mutants (A, B, The mutation site information of C and D) is as follows:

[0074] Mutant A replaces amino acid d(Asp) with m(Met) at position 254 of the collagenase Seq ID No.2 sequence, and replaces amino acid r(Arg) with g(Gly) at position 255; the mutation The amino acid sequence of body A is shown in Seq ID No.3, and the nucleotide sequence of its encoding gene is shown in Seq ID No.4;

[0075] Mutant B replaces amino acid k (Lys) with l (Leu) at position 382 of the collagenase Seq ID No.2 sequence, and replaces amino acid e (Glu) with p (Pro) at position 383;

[0076] Mutant C replace...

Embodiment 3

[0089] Embodiment 3, the application of mutant A and D in the separation and cultivation of adipose stem cells as collagenase

[0090] 1. Influence of the amount (concentration) of mutants A and D on the separation effect of adipose stem cells

[0091]Using the mutants A and D prepared in Example 2 as collagenases, the adipose stem cells were separated according to the following steps, wherein the digestion time was the same, the amount (concentration) of collagenase added was different, and commercialized type I collagen Enzyme (invitrogen, Cat. No. 17100-017) as control (CK1):

[0092] (1) Negative pressure liposuction collects human adipose tissue, divides it into 100mL portions, sterilizes the surface of each container containing adipose tissue, and carefully transfers it to a biological safety cabinet. After standing still, the swelling fluid and adipose tissue Layer, and remove the lower liquid and upper layer of grease;

[0093] (2) Add an equal volume of normal salin...

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Abstract

The invention discloses an adipose stem cell separation and culture method. The adipose stem cell separation and culture method comprises the following steps: S1, collecting adipose tissues; S2, adding collagenase into the adipose tissues collected in the step S1, thereby digesting the adipose tissues so as to obtain adipose stem cells; S3, enriching the adipose stem cells obtained in the step S2;and S4, performing purification and subculture on the adipose stem cells enriched in the step S3. In the step S2, the amino acid sequence of the collagenase is as shown in Seq ID No. 3; volume percentage content of the adipose tissues in the digestive system is 40-60%; content of the collagenase in the digestive system is 0.20-0.30 g / L; and the digestion time is 15-30 minutes. The adipose stem cell separation and culture method disclosed by the invention has the advantages of being low in collagenase dosage and high in adipose stem cell separation efficiency; and thus, the method is of greatvalues to scientific research as well as large-scale isolation and culture of adipose stem cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to methods for separating and culturing fat stem cells. Background technique [0002] Due to congenital deformity, trauma, tumor resection and other causes of soft tissue defects and the increasing demand for plastic surgery, soft tissue filling is a common technique used in plastic surgery. As an ideal soft tissue filling material, autologous fat is better in biocompatibility than artificial tissue substitutes, allogeneic and heterogeneous materials, without immune rejection, and has the advantages of easy material acquisition, simple operation, and good filling shape. However, the low postoperative fat survival rate (25%-80%) of autologous fat transplantation greatly limits its wide application in clinic. In recent years, with the gradual understanding of the survival mechanism after fat transplantation, people use adipose-derived stem cells to help transplanted fat have a higher sur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N9/54
Inventor 宋珂慧胡振生刘振中庄秀萍郭伟李明
Owner 济南新生命干细胞有限公司
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