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A production process for preparing "ten billion" fat-derived regenerative cells

A technology for regenerative cells and fat sources, which is applied in the field of regenerative medicine, can solve the problems of high cost, less harvested culture medium, and less harvested cells, and achieve the effects of reducing total cost, increasing added value, and improving culture efficiency

Active Publication Date: 2020-05-12
SHENZHEN ALPHA BIOTECHNOLOGY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Approximately ~1 billion (1x10 9 ), the “100 million” level culture of adipose-derived regenerative cells has always been a technical bottleneck in this field. The problems of the existing technology are highlighted in the high cost, the small amount of harvested cells, and the small amount of harvested conditioned medium that can generate added value

Method used

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  • A production process for preparing "ten billion" fat-derived regenerative cells
  • A production process for preparing "ten billion" fat-derived regenerative cells
  • A production process for preparing "ten billion" fat-derived regenerative cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Flake carrier and preparation.

[0052] Weigh 50-150 grams of the sheet-shaped carrier that is reused for the first or second time, wash with ultrapure water, and control to dry.

[0053] Put it in a drying oven at 60°C for 24 hours to dry completely.

[0054] Each 150 grams of the sheet-shaped carrier is added to a cell culture vessel with a working volume of 3.75 liters in a German Eppendorf bioreactor, and the upper and lower parts are fixed with mesh plates to form a fixed packed bed with a volume of about 3 liters.

[0055] Pour pure water into the outer isolation interlayer of the reaction vessel, and the amount of water is half of the total interlayer volume. Add 3.75 liters of 0.01M phosphate buffer to the reaction vessel. All other parts are installed, fixed and sealed according to the manufacturer's instructions.

[0056] Move the entire fixed-bed reaction vessel into an autoclave at 121°C and autoclave for 30 minutes.

[0057] After cooling to room temperature, connec...

Embodiment 2

[0060] 150 grams of the second reused sheet carrier constitutes a 3 liter fixed packed bed.

[0061] Calculate the surface area provided by the sheet carrier: provide 1200cm per gram of sheet carrier 2 Surface area, the 150g sheet carrier used has 1.8x10 5 cm 2 The surface area.

[0062] The cell culture medium is DMEM / F12 basic medium and 10% fetal bovine serum.

[0063] Calculation of the first cell seeding volume on the first day: cell seeding density per unit area 3x10 3 Cells / cm 2 , 150g sheet carrier has 1.8x10 5 cm 2 Surface area, a total of 5.4x10 inoculated 8 Cells, calculated as a packed bed with a volume of 3 liters, the cell seeding density per unit volume is 1.8x10 5 Cells / ml.

[0064] After inoculation, the magnetic stirrer speed was set to 25 rpm, stirred for 5 minutes, stopped for 25 minutes, and repeated 4 times, that is, after 2 hours, the magnetic stirrer speed was set to continuous 25 rpm for 24 hours, and the pH of the system was maintained in the range of 7.2-7.4....

Embodiment 3

[0074] Fifty grams of flake carriers constitute a fixed packed bed of 1 liter.

[0075] Calculate the surface area provided by the sheet carrier: 1200cm per gram of sheet carrier 2 Surface area, the 50g sheet carrier used has 6x10 4 cm 2 The surface area.

[0076] The cell culture medium is DMEM / F12 basic medium and 10% fetal bovine serum.

[0077] Calculation of the first cell seeding volume on the first day: cell seeding density per unit area 6x10 3 Cells / cm 2 , 50g sheet carrier has 6x10 4 cm 2 Surface area, inoculated 3.6x10 8 Cells, calculated as a packed bed with a volume of 1 liter, the cell seeding density per unit volume is 3.6x10 5 Cells / ml.

[0078] After inoculation, the magnetic stirrer speed was set to 25 rpm, stirred for 5 minutes, stopped for 25 minutes, and repeated 4 times, that is, after 2 hours, the magnetic stirrer speed was set to continuous 25 rpm for 24 hours, and the pH of the system was maintained in the range of 7.2-7.4.

[0079] The second cell inoculation on...

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Abstract

The invention belongs to the technical field of regenerative medicine and biology, and particularly relates to a multiplication culture method for ten-billion-level adipose-derived regenerative cells.Through a cell batch inoculation process, adipose-derived regenerative cells are inoculated and attached to a fixed packed bed arranged through a sheet carrier in a cell culture vessel, and in combination with a fed-batch perfusion process, the billion level of the prior art is increased to the ten billion level for cell multiplication. In the method, the use of disposable cell culture vessels and a large number of collagen microcarriers is abandoned, so that the fixed cost with the highest ratio in the costs of goods sold is greatly reduced, which is a significant progress of the method. Thecell culture vessel adopted in the method and a cell culture vessel adopted in the closest prior art are the updated iterative model with the same volume of products of the same company, so that themethod adopts relatively parallel test conditions and has comparability with the prior art.

Description

Technical field [0001] The invention belongs to the technical field of regenerative medicine, and specifically relates to a large-scale expansion and culture process for preparing "tens of billions" grade fat-derived regenerative cells, and in particular to an adherent culture using a fixed packed bed combined with cell "batch inoculation" And "fed-batch perfusion" process, industrially amplify fat-derived regenerative cells. Background technique [0002] Adult stem cells have the ability to self-replicate and differentiate into a variety of cells, and can repair damaged and aging tissues in the human body. Therefore, they have very important prospects for clinical applications in regenerative medicine, especially the rapid development of mesenchymal stem cells and antibody-targeted drugs. , So that many diseases that could not be treated are expected to be overcome. With the transformation of research results into clinical applications, the market demand for cells and cell prod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12M3/04C12M1/36
CPCC12M25/18C12M41/48C12N5/0667C12N2533/30
Inventor 周莹
Owner SHENZHEN ALPHA BIOTECHNOLOGY INC
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