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A 3D method for culturing autologous adipose-derived MSCs-derived neural progenitor cells

A technology of neural precursor cells and adipocytes, applied in the field of cell culture, can solve the problems of small culture capacity, limit the industrial transformation of nerve tissue repair medicine, complex operation, etc., meet the requirements of purity and preparation, promote MSCs adhesion, Guaranteed effect of single cell adhesion

Active Publication Date: 2020-04-10
北京再生生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Neurosphere culture is to inoculate MSCs into hydrophobic culture containers to form multi-cell aggregated cell spheres. It belongs to the semi-suspension culture system. Its culture capacity is small, the operation is complicated, and it is difficult to pass and expand, resulting in low cell yield, which limits the Industrial transformation of NPCs nerve tissue repair medicine

Method used

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  • A 3D method for culturing autologous adipose-derived MSCs-derived neural progenitor cells
  • A 3D method for culturing autologous adipose-derived MSCs-derived neural progenitor cells
  • A 3D method for culturing autologous adipose-derived MSCs-derived neural progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Preparation of Collagen 3D Scaffold

[0058] Collagen sponge (article number: 1050030, manufacturer: Aiweiting) was trimmed into circular sheets with a diameter of 8 cm and a thickness of 0.3 cm, and each 5 sheets were stacked together as a culture unit to contain 10 ng / recombinant human laminin, Soak in 100ng / mL recombinant human vitronectin in DMEM / F12, incubate overnight at 37°C; soak in saline, rinse 3 times, drain, place in a sterile airtight container, store at 4°C for no more than 30 days, and set aside.

Embodiment 2

[0059] Example 2: Culture of P1 generation adipose MSCs

[0060] 2.1 Fat collection

[0061] Donor physical examination before collection, should have no tumor history, no virus infection, no mycoplasma infection. Fat collection is collected in professional medical collection institutions. Abdominal subcutaneous liposuction 50mL. Immediately after collection, place them in a 125mL ice-bath sterile bottle (product number 2019-0250, manufacturer Nalgene), which contains 20mL of preservation solution, which is DMEM / F12 containing 50ug / mL gentamicin sulfate.

[0062] 2.2 Isolation of fat cells

[0063] Remove small blood vessels and connective tissue, chop them into fluid form, and rarely see particles; resuspend in 2 times the volume of medical saline containing 50ug / mL gentamicin sulfate, centrifuge at 500g for 10 minutes, and take the fat layer and sediment layer; Repeat washing 2 times, collect fat layer and precipitate layer, digest with double volume of digestion solutio...

Embodiment 3

[0066] Example 3: Collagen 3D scaffold pretreatment

[0067] Stack five collagen 3D scaffolds with a diameter of 8 cm, place them in a 15 cm tissue culture dish (product number 168381, manufacturer NUNC), add 30 mL of MSCs medium, and place at 37 °C, 5% CO 2 Cultivate overnight in the incubator; take out the collagen 3D scaffold, drain the medium, transfer to a new 15cm tissue culture dish, and set aside.

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Abstract

The invention discloses a method for 3D culture of autologous fat MSCs (mesenchymal stem cells) derived neural precursor cells. The method includes the steps of: S1. preparing a collagen 3D scaffold; S2. collecting adipose tissue and separating adipose cells, using an MSCs medium for culture to harvest P0 generation MSCs, resuspending the P0 generation MSCs with the MSCs medium, and performing culture to harvest P1 generation MSCs; S3. pretreating the collagen 3D scaffold; S4. using the MSCs medium to resuspend the P1 generation MSCs, conducting inoculation into a tissue culture dish containing the collagen 3D scaffold, and using an NPCs (neural precursor cells) medium to conduct further culture for 6 days, thus harvesting NPCs. The method provided by the invention employs laminin and vitronectin to coat collagen sponge so as to construct the collagen 3D scaffold, can promote MSCs adhesion and inhibit neuronal differentiation, then uses the MSCs medium for pretreatment of the collagen 3D scaffold so as to prevent agglomeration of MSCs during adhesion, and maximumly guarantees single cell adhesion. According to the invention, abdominal adipose tissue is collected and separated, MSCs is amplified, and the collagen 3D scaffold is employed to culture NPCs, and the obtained NPCs can meet the purity and preparation demands of autologous fat MSCs derived NPCs.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for 3D culturing neural precursor cells derived from autologous adipose MSCs. Background technique [0002] Neural stem cells and neural progenitor cells are the precursor cells of nerve cells such as neurons and glial cells, and there is no accurate method to distinguish between them. Neural precursor cells (neural precursor cells, NPCs) play an important role in the development of the nervous system and the repair of nerve damage. The difficulty in repairing adult nerve damage is related to the lack or silence of NPCs. Previous studies have shown that transplantation of allogeneic NPCs can alleviate Parkinson's disease, multiple sclerosis, glioma and other diseases, can promote neurodegenerative diseases, trauma and other axonal injury remyelination and repair, and relieve clinical symptoms. Immune rejection, low chimerism rate, and the medical prospect of autolo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079
CPCC12N5/0618C12N2500/32C12N2500/38C12N2501/11C12N2501/115C12N2501/392C12N2501/50C12N2506/1384C12N2513/00C12N2533/50C12N2533/52C12N2533/54
Inventor 张洪钿苑春慧
Owner 北京再生生物科技研究院有限公司