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Method for preparing composite growth factor of natural cells

A compound growth factor and cell technology, applied in the field of biological tissue culture, can solve the problems that the biological activity of the product is difficult to achieve the activity of natural products, the physiological effect of protein is difficult to achieve, and the difference in natural substances and quantities, so as to achieve increased granulation tissue formation and fibroblast formation. Accelerated cell maturation and increased capillary effects

Inactive Publication Date: 2007-05-09
西安组织工程工程技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preparation of such factors mainly relies on genetic engineering. The disadvantage is that genetic engineering can only prepare a single type of factor, and the biological activity of the product is difficult to achieve the activity of natural products.
In addition, current relevant literature reports basically culture cells on culture flasks or some kind of matrix, and obtain supernatant by two-dimensional culture. Cells cultured in this way are different from the growth mode in vivo, so its The synthesized and secreted protein is difficult to achieve the ideal physiological effect, and there are qualitative and quantitative differences from natural products

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] The embodiment of the present invention is illustrated by taking the supernatant obtained from air-liquid surface culture of skin tissue.

[0014] 1. Under sterile conditions, mix collagen, hyaluronic acid, and chondroitin sulfate in a weight ratio of 9:1:1, and prepare a 6% solution by weight with 0.1% acetic acid solution (can be 1-10% Select within the range), then add fetal bovine serum and 10mg / ml DMEM medium according to 10% of its volume, adjust the pH value to 7.4, add the 5-40 generations of human fibroblast suspension cultured in vitro, so that the final concentration is 10 5 cells / ml in CO 2 After curing in an incubator at 37° C. for 30 minutes, add DMEM culture solution containing 10% fetal bovine serum for cultivation, and change the medium every day.

[0015] 2. After culturing for 3 days, press 10 4 cells / cm 2 The 5-40 generation human epidermal cells cultured in vitro were planted, continued to be cultured, and the medium was changed every day, and t...

Embodiment 2

[0019] The embodiment of the present invention is exemplified by the cultivation of fibroblasts and epidermal cells in a bioreactor.

[0020] 1. In the rotary culture device (a kind of airtight cylindrical culture bottle, the culture system that the culture bottle axially rotates at a speed of 20 to 40 revolutions / min by external force is a prior art device), adding 10% fetal bovine Serum DMEM culture fluid and human fibroblasts cultured in vitro are added to microcarriers (60-250 μm in diameter) at a ratio of 5 mg to 40 mg / ml for rotary culture. Fibroblasts are attached to microcarriers to grow and proliferate to form Fibroblast clusters with microcarriers as the core;

[0021] 2. After culturing for 5 days, add epidermal cells cultured in vitro to the culture device, and use epidermal cell culture medium to culture for 3 days. Epidermal cells wrap fibroblast clusters to form multiple small skin tissues;

[0022] 3. Start to collect the supernatant, and use a 0.22μm filter m...

Embodiment 3

[0025] The embodiment of the present invention is exemplified by the cultivation of bone marrow mesenchymal stem cells and extracellular matrix.

[0026] 1. under aseptic conditions, extracellular matrix (being the mixture of various types of collagen, same as example 1) and 0.1% acetic acid solution are prepared into 1% solution by weight, add 1 / 10 volume of fetal bovine serum , 10mg / ml DMEM culture solution, adjust the pH value to 7.2, add the bone marrow mesenchymal stem cell suspension cultured in vitro for 5 to 40 generations, so that the final concentration is 10 5 cells / ml in CO at 37°C 2 After solidifying in the incubator for 30 minutes, add the culture medium for cultivation, and change the medium every day.

[0027] 2. Start to collect the supernatant every day, filter the harvested supernatant at 4°C with a 0.22 μm filter membrane, then concentrate it to 1 / 10 of the liquid volume with a hollow fiber column system, and then store the concentrated sample at 40°C Add...

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Abstract

This invention discloses a method for extracting natural composite growth factor. The method comprises: constructing artificial tissue / organ in vitro through tissue-engineering method, collecting the supernatant during tissue / organ culture, separating and purifying to obtain natural composite growth factor. The method can obtain large quantities of amino acids or proteins including growth factor and nutrition factor, which, after separation and purification, can be used in cell culture, cosmetics, foods and drugs.

Description

technical field [0001] The invention belongs to the technical field of biological tissue culture, and in particular relates to a preparation method of a natural cell compound growth factor. Background technique [0002] Cells cultured in vitro can autocrine and paracrine amino acids, polypeptides or proteins and other cytokines, including growth factors, nutritional factors, etc. These factors have biological regulation effects on various cell physiological functions and metabolic activities, and their content in the body is very small, but High biological activity. These factors can play a role in combination with specific receptors present on target cells, and their main biological effects are: chemotaxis induces inflammatory cells to stimulate target cell proliferation and differentiation, promotes target cell synthesis and secretion of extracellular matrix (such as collagen etc.); can regulate the formation, development and metabolic functions of T cells, B cells, histi...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N5/06A61K38/17A61K8/64A23L1/305A23L33/17C12N5/071
Inventor 张勇杰金岩
Owner 西安组织工程工程技术研究中心
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