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Method for culturing hepatic cells on a large scale

A technology of emulsifier and base-based chitosan, which is applied in biochemical equipment and methods, tissue culture, immobilized enzymes, etc., can solve the problems of poor porous microcarriers, achieve high cell seeding efficiency, increase contact opportunities, Little damage to liver cells

Active Publication Date: 2012-10-10
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the culture of hepatocytes, the existing porous microcarriers are not ideal enough, so it is of great significance to seek an ideal porous microcarrier with hepatocyte specificity for the current large-scale culture of hepatocytes in vitro

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  • Method for culturing hepatic cells on a large scale
  • Method for culturing hepatic cells on a large scale
  • Method for culturing hepatic cells on a large scale

Examples

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preparation example Construction

[0039] Preparation and Application of Macroporous Microcarrier

[0040] 1. Preparation of Macroporous Microcarriers

[0041] 1) Preparation of silk fibroin: boil raw silk in 5g / L sodium carbonate solution for 1 hour, then wash with a large amount of deionized water or distilled water to remove sericin, and dry at 60-70°C to obtain silk protein. Dissolve an appropriate amount of silk fibroin in a ternary solution of calcium chloride / water / ethanol (molar ratio 1:8:2) at 80±2°C, dialyze in deionized water or distilled water at room temperature for 3 days to remove the salt and ethanol, and filtered to remove insoluble impurities to prepare an aqueous solution of silk fibroin. Stir the silk fibroin aqueous solution at a low speed at 50±2°C, evaporate part of the water, and obtain a silk fibroin solution with a concentration of about 7-10w / v%, and set aside;

[0042] 2) Preparation of galactosylated chitosan (GC): take 2.2g chitosan, dissolve in 1.0%-2.5% acetic acid aqueous s...

Embodiment 1

[0055] Embodiment 1. Human liver cell line CL-1 cells were cultivated with silk fibroin / galactosylated chitosan under static / microgravity rotary culture;

[0056] 1. Configuration of complete medium: add 15ml of fetal bovine serum, 3.5mmol of HEPES, 10,000U of penicillin, and 10,000U of streptomycin for every 100ml of DMEN (high glucose type) medium

[0057] 2. Pretreatment of silk fibroin / galactosylated chitosan macroporous microcarriers: place 0.1g silk fibroin / galactosylated chitosan macroporous microcarriers in a 50ml plastic centrifuge tube, and use 0.1mol / L, After soaking overnight in phosphate buffered saline (PBS) with a pH of 7.0 and calcium and magnesium ions, suck out the PBS, and then wash with PBS for 3 times. Soak with the complete medium prepared in 1) for more than 10 hours, and set aside;

[0058] 3. Human hepatocytes (CL-1) cultured on silk fibroin / galactosylated chitosan macroporous microcarriers under static / microgravity rotation conditions

[0059]1. Mic...

Embodiment 2

[0064] Embodiment 2, Microcarrier cultivation of human liver cell line CL-1 cells under microgravity rotary culture (silk fibroin / galactosylated chitosan macroporous microcarrier SF / GC, solid microcarrier: cytodex 3);

[0065] 1. Configuration of complete medium: add 15ml of fetal bovine serum, 3.5mmol of HEPES, 10,000U of penicillin, and 10,000U of streptomycin for every 100ml of DMEN (high glucose type) medium

[0066] 2. Microcarrier pretreatment:

[0067] 1. Pretreatment of silk fibroin / galactosylated chitosan macroporous microcarriers: put 0.2g silk fibroin / galactosylated chitosan macroporous microcarriers in a 50ml plastic centrifuge tube, and use 0.1mol / L, After soaking overnight in phosphate buffered saline (PBS) with a pH of 7.0 and calcium and magnesium ions, suck out the PBS, and then wash with PBS for 3 times. Soak with the complete medium prepared in 1) for more than 10 hours, and set aside;

[0068] 2. Pretreatment of solid microcarriers (cytodex 3): put 0.25g ...

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Abstract

The inevention discloses a novel silk protein-galactosylation chitosan macroporous carrier with the specificity of hepatic cells, a preparation method thereof, and a method for culturing the hepatic cells under the condition of microgravity rotary culture using same. Compared with the normal solid frame material, the novel microcarrier has larger surface area / volume and has a sinusoid structure which is extremely similar to a hepatic sinus structure in human body, thereby being better convenient for the hepatic cells to be stuck on the frame material, the mutual contact among cells, the transmission of oxygen gas and nutrient component, and the discharge of metabolites, and further heightening the culture density of the hepatic cells and the function of the hepatic cells.

Description

technical field [0001] The invention relates to a method for large-scale culture of liver cells, in particular to a method for large-scale culture of liver cells by using a novel hepatocyte-specific macroporous microcarrier. Background technique [0002] The bioartificial liver began to develop gradually in the late 1980s. Although it has experienced more than 20 years of development and made some progress, it has not been widely and effectively used in clinical practice. It has become a complete substitute for liver transplantation to treat various acute and chronic liver functions. The best method for exhaustion, and how to realize the large-scale culture of hepatocytes in vitro with strong functions is the bottleneck problem that strongly restricts the development of bioartificial liver. [0003] Hepatocytes are anchorage-dependent cells, and their effective function must depend on the attachment to supports such as scaffold materials. In addition to the basic requiremen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08L89/00C08L5/08C08J9/28C08J3/24C12N5/08C12N11/00
Inventor 周焕城张志高毅龚独辉蒋泽生刘勇
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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