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Construction method of human adipose derived stem cell bank

A mesenchymal stem cell and construction method technology, applied in the field of mesenchymal stem cell preparation, can solve the problems of low cell purity, complicated operation process, tedious removal of red blood cells, etc., and achieve simplified process flow, simple steps, and easier control of process stability Effect

Inactive Publication Date: 2017-05-31
四川华皓生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a method for constructing a human adipose-derived mesenchymal stem cell bank, which can effectively solve the problems in the prior art of complex operation process, low cell purity and cumbersome removal of red blood cells

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  • Construction method of human adipose derived stem cell bank
  • Construction method of human adipose derived stem cell bank
  • Construction method of human adipose derived stem cell bank

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Isolation of adipose-derived mesenchymal stem cells

[0034] (1) Sign an informed consent form with the donor, collect 30mL female (25 years old) abdominal adipose tissue, and collect 10mL donor blood samples at the same time and test the donor's blood type, HLA-ABC / DR matching, infectious diseases (two and a half hepatitis B, Hepatitis C, cytomegalovirus, Epstein-Barr virus, syphilis, HIV), ensure that its various indicators are normal, and then cut the donor’s fat tissue into 1-5mm 3 Size and divide them into 3 parts, respectively, put them in a 50mL centrifuge tube, add 30mL sterile saline, centrifuge at 20℃, 4000r / min for 15min, the purpose is to deoil adipose tissue and remove red blood cells in adipose tissue ;

[0035] (2) After centrifugation, remove the upper oil layer, and then transfer the adipose tissue in the middle layer to a new centrifuge tube, and wash 1-2 times with normal saline;

[0036] (3) Collect the adipose tissue mass in step (2), weigh 2g a...

Embodiment 2

[0038] Example 2 Recovery, subculture and cryopreservation of adipose tissue

[0039] (1) Resuscitate the adipose tissue frozen for 2 months, 6 months, and 12 months in a 37℃ water bath;

[0040] (2) Take out the adipose tissue and add it to a 50mL centrifuge tube, add 30mL of sterile physiological saline, and centrifuge at 4°C and 1200r / min for 3min;

[0041] (3) Collect the adipose tissue mass in step (2), weigh 2g and inoculate it in a T175 culture flask (Corning), add 12mL complete medium, and then place it at 37°C, 5% CO 2 Cultivate in a humid incubator, and change the medium every 7 days; see the growth of P0 generation cells after resuscitation and culture of adipose tissue frozen for 2 months, 6 months, and 12 months Figure 1-2 , 1-3, 1-4;

[0042] (4) When the cell fusion degree is 70%, trypLE TM Select (Gibco) digested cells, centrifuged after digestion, centrifuged at 1200r / min for 3min, and collected the cells according to 1×10 4 Cells / cm 2 Inoculate it in a T175 culture...

Embodiment 3

[0045] Example 3 Identification of Biological Characteristics of Adipose Mesenchymal Stem Cells

[0046] 1. Identification of multi-directional differentiation potential

[0047] P3 generation adipose-derived mesenchymal stem cells were seeded in a six-well plate, and each well was seeded with 1×10 5 Add 2mL complete medium to each well, set 3 replicates for each sample, change the medium every two days, and add osteogenic, adipogenic, and chondrogenic induction medium (Cyagen ) 2mL each, with the addition of complete medium as a blank control, change the induction medium every 2-3 days, after 2-3 weeks of continuous cultivation, remove the induction medium and wash it with PBS once, add 2mL paraformaldehyde solution (4% , V / v) Fix for 30min, then remove the paraformaldehyde solution and wash twice with PBS, stain with staining solution, the staining time for osteogenic induction and chondrogenesis induction is 2-3min; the staining time for lipogenic induction is 30min, then remove...

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Abstract

The invention provides a construction method of a human adipose derived stem cell bank. The construction method comprises the following steps: (1) collecting adipose tissues, adding a saline solution and centrifuging; (2) removing an oil layer and washing the adipose tissues; (3) inoculating the washed adipose tissues into a complete culture medium and culturing under the conditions that the temperature is 37 DEG C and the concentration of CO2 is 5 percent; (4) replacing the culture medium for one time every 5 to 8 days; when the cell confluence is 70 percent to 80 percent, digesting cells with TrypLETM Select; (5) carrying out subculture; (6) detecting the quality; (7) cryopreserving; (8) establishing the bank. According to the construction method provided by the invention, oil and red blood cells are removed by adopting a centrifugal method and collagenase is not used for digesting; the operation time can be shortened and a process is simple and feasible; a lot of the red blood cells are not generated, and the red blood cells do not need to be removed by adopting a red blood cell lysis solution or low-molecular-weight heparin calcium; potential risks on human adipose derived stem cells are not caused; meanwhile, the cost is saved.

Description

Technical field [0001] The invention belongs to the technical field of preparation of mesenchymal stem cells, and specifically relates to a method for constructing a human adipose-derived mesenchymal stem cell bank. Background technique [0002] Adipose tissue exists in large quantities in the human body. It is a kind of good tissue engineering material that is easy to obtain. It has good cosmetic medical treatment effects for soft tissue defect filling, breast enhancement, and wrinkle removal. However, the survival rate of adipose tissue transplantation is low and it is easily absorbed. , Fat liquefaction and necrosis have greatly restricted the application of adipose tissue. Studies have shown that adipose-derived mesenchymal stem cells can significantly promote the survival rate of adipose tissue transplantation. [0003] Adipose-derived mesenchymal stem cells (AD-MSCs) are a type of adult stem cells that exist in adipose tissue and have the potential for self-renewal and multi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 李俊李升岐黄海森徐能飞石玥唐梦涛
Owner 四川华皓生物科技有限公司
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