Adipose derived stem cell large-scale culture method

A technology for large-scale culture and quality stem cells, which is applied in the field of large-scale culture of adipose-derived mesenchymal stem cells, which can solve the problems of restricting the application of adipose-derived stem cells, unable to collect cells at one time, and increase the workload, so as to achieve good cell shape and a large number of harvested cells. , the effect of fast cell proliferation

Active Publication Date: 2015-05-13
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inoculation density is 1:3~1:5 to inoculate cells. In large-scale culture and passage, this will increase the workload, and it is impossible to collect a large number of uniform cells

Method used

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  • Adipose derived stem cell large-scale culture method
  • Adipose derived stem cell large-scale culture method
  • Adipose derived stem cell large-scale culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051]After primary culture and separation of adipose-derived mesenchymal stem cells confluence reaches 80%, use a pipette to absorb the culture medium in the φ10cm cell culture dish, add 10ml PBS to wash twice, add 2ml TrypLE to digest the adherent cells, and observe under the microscope For cell digestion, after the cells are basically no longer attached to the wall, add 3ml of serum-free medium (UltraCULTURE MEDIUM) to stop digestion, transfer the cell suspension to a centrifuge tube, centrifuge at 1000rpm / min for 5min, remove the supernatant, and use 10ml of serum-free The cells were resuspended in the serum medium, and 20 μl of the cell suspension was taken, and the total number of cells and cell activity information were obtained with a Countstar automatic cell counter. Dilute the cell suspension at 1.3×10 4 / cm 2 Inoculate the cell density into a φ15cm cell culture dish (UltraCULTURE MEDIUM), add 15ml of serum-free medium (UltraCULTURE MEDIUM) containing 10ng / ml EGF, m...

Embodiment 2

[0057] Embodiment 2: cell proliferation curve (traditional method, non-enzyme method)

[0058] After the confluence of adipose-derived mesenchymal stem cells separated by primary culture reaches 90%, use a pipette to absorb the culture medium in the φ15cm cell culture dish, add 10ml PBS to wash twice, add 3ml non-enzymatic TrypLE to digest the adherent cells, and mirror Observe the digestion of the cells. After the cells are basically no longer attached to the wall, add 5ml of serum-free medium (UltraCULTURE MEDIUM) to stop the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000rpm / min for 5min, remove the supernatant, and use Resuspend the cells in 15ml of serum-free medium, take 20μl of the cell suspension, and use the Countstar automatic cell counter to obtain the total number of cells and cell activity information. Dilute the cell suspension at 1.3×10 4 / cm 2 Inoculate the cell density into a φ15cm cell culture dish, add 15ml serum-free mediu...

Embodiment 3

[0060] result: figure 2 The results show that the cell doubling time obtained by the enzyme-free method is significantly better than that of the cells passaged by the traditional method. Example 3: Detection of cell surface markers (P1, P6, P15)

[0061] (1) The culture method of Example 1. Cells were cultured, and the cells of the 1st, 6th, and 15th passages were collected respectively, and the cell surface markers between different passages were detected by flow cytometry. Digest and collect cells separately. After counting, take 4×106 cells from each batch and divide into 4 tubes; wash once with staining buffer, centrifuge at 200 g for 5 min; discard the supernatant, blow and mix the cells with staining buffer; add CD45, CD59, CD90, and HLA-DRA antibodies were 10 μl each, and one tube was used as a blank control; at 4°C, avoid light for 15-20 minutes; wash once with staining buffer, and centrifuge at 1500rpm for 5 minutes; discard the supernatant of directly labeled cells...

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Abstract

The invention discloses an adipose derived stem cell (ADSC) large-scale culture method. The method selects a non-enzymatic digestion method (TrypLE) to collect adipose derived stem cells adhering to walls, adopts a minimum TrypLE dosage to maximumly reduce the damage of the cell digestion process to cells, and selects the optimum inoculation density of 1.3*10<4>/cm<2> to culture cells, uses a serum-free medium (UltraCULTURE MEDIUM), and adds appropriate concentration EGF into the medium so as to conduct large-scale culture in a phi 15cm culture dish. Thus, the passage number of ADSC is increased to over 15 and the original dryness can still be maintained, thereby greatly satisfying the requirement of mesenchymal stem cells for scientific research and application.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a large-scale culture method of adipose-derived mesenchymal stem cells. Background technique [0002] Stem cells (stem cells) are different from mature cells because they can maintain the ability of self-renewal and expansion for a long time, and secondly, stem cells can differentiate into various cell lines. These characteristics of stem cells make them a good source of seed cells. Among them, adult pluripotent stem cells (MSCs) derived from mesenchymal cells have the advantage of being easy to obtain and culture in vitro, and have become one of the most feasible cell sources for various clinical cell therapies. Adipose-derived mesenchymal stem cells (ADSCs) are stem cells that exist in adipose tissue and have the potential for multilineage differentiation. In 2001, adipose-derived mesenchymal stem cells were isolated from adipose tissue suspension extracted from human liposuction for ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 王一飞陈海佳葛啸虎卢瑞珊马岩岩王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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