Preparation method of novel medical cell repairing agent

A repairing agent and cell technology, applied in the field of preparation of new medical cell repairing agents, can solve the problems of rejection, not a skin substitute, allergies, etc., and achieve the effect of small rejection reaction

Active Publication Date: 2018-10-23
北京壹典壹生生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, synthetic skin has been used to treat skin damage, but it is not a perfect skin substitute, and there are also a series of problems: the currently used synthetic skin seed cells are autologous or allogeneic epidermal cells
[0004] The invention patent with application number 200810136415.6 discloses a preparation method of human umbilical cord mesenchymal stem cell wound varnish using methylcellulose as the cell matrix, but the invention contains the antibiotic gentamicin, which is not suitable for people allergic to antibiotics; The use of human umbilical cord mesenchymal stem cells has no matching type, and there may be rejection reactions, and the rapid preparation of the smear contains animal-derived components of fetal bovine serum, which may cause allergies and have animal-derived safety hazards

Method used

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  • Preparation method of novel medical cell repairing agent
  • Preparation method of novel medical cell repairing agent
  • Preparation method of novel medical cell repairing agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Preparation of human amniotic mesenchymal stem cells

[0036] Take aseptically collected amniotic membrane tissue, and wash it in pre-cooled PBS in a biological safety cabinet. If the placenta is accompanied by chorion, the chorion and amnion need to be separated first, the amnion is taken out, and the chorion is discarded; Add an equal volume of 2U / ml neutral protease to digest for 30min, then add a 1:1 mixture of 5.5mg / ml V-type collagenase and 2U / ml hyaluronidase twice and a half volume of amniotic membrane to digest for 3.5h; the collection and digestion are complete The cell suspension was added to the balance solution without calcium and magnesium ions, washed several times, counted the mononuclear cells after dilution with glacial acetic acid, and obtained the total number of cells; adjusted the cell concentration to 1.0×10 5cells / ml, add serum-free mesenchymal stem cell medium, culture in an incubator at 37°C with a concentration of 5% CO2 and saturated humid...

Embodiment 2

[0043] 1. Preparation of human amniotic mesenchymal stem cells

[0044] Take aseptically collected amniotic membrane tissue, and wash it in pre-cooled PBS in a biological safety cabinet. If the placenta is accompanied by chorion, the chorion and amnion need to be separated first, the amnion is taken out, and the chorion is discarded; Add an equal volume of 2U / ml neutral protease to digest for 30min, then add a 1:1 mixture of 5.5mg / ml V-type collagenase and 2U / ml hyaluronidase twice and a half volume of amniotic membrane to digest for 3.5h; the collection and digestion are complete The cell suspension was added to the balance solution without calcium and magnesium ions, washed several times, counted the mononuclear cells after dilution with glacial acetic acid, and obtained the total number of cells; adjusted the cell concentration to 1.0×10 5 cells / ml, add serum-free mesenchymal stem cell medium, culture in an incubator at 37°C with a concentration of 5% CO2 and saturated humi...

Embodiment 3

[0050] Embodiment 3 application effect detection

[0051] 1. Biological characteristics of human amniotic mesenchymal stem cells prepared in Example 1:

[0052] a. Identification of molecular surface markers by flow cytometry:

[0053] The third-generation amniotic mesenchymal stem cells in good growth state were taken for flow cytometric analysis to see whether they expressed CD34, CD44, CD45, CD73, CD90, CD105, CD326, HLA-DR and other surface molecules.

[0054] b. Cell immunofluorescence staining experiment:

[0055] Take the third-generation amniotic mesenchymal stem cells in a good growth state, adjust the cell concentration, and make cell slides. After the cells are full, fix them with 4% paraformaldehyde, do immunofluorescence staining, and observe under a fluorescent microscope to detect whether the cells contain Vimentin.

[0056] c. In vitro multi-lineage differentiation experiment:

[0057] After subculture, human amniotic mesenchymal stem cells were cultured in...

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Abstract

The invention relates to medicine for restoring wound, in particular to a preparation method of a novel medical cell repairing agent. The preparation method has the concrete preparation steps of separation purification and culture of human amniotic mesenchymal stem cells, preparation of a sodium alginate complexing agent and preparation of a cell restoring agent. The invention builds the preparation method of human amniotic mesenchymal stem cells and a corresponding stem cell bank, and makes the preparation for large-scale production and preparation of the human amniotic mesenchymal stem cellrepairing agent and the application of the repairing agent.

Description

Technical field: [0001] The invention relates to a medicament for repairing wounds, in particular to a preparation method of a novel medical cell repairing agent. Background technique: [0002] Skin wound repair is a complex biological process, which initiates a series of complex biological events, including inflammation, tissue regeneration and tissue remodeling. Although many studies have applied different approaches to skin wound repair, low healing quality and non-renewable appendages are still key issues in the skin wound healing process. Currently, skin grafting is the most effective way to repair extensive skin damage. In recent years, synthetic skin has been used to treat skin damage, but it is not a perfect skin substitute, and there are also a series of problems: the currently used synthetic skin seed cells are autologous or allogeneic epidermal cells. The number of autologous epidermal cells is insufficient, while allogeneic epidermal cells have a rejection reac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/28A61K38/18A61P17/02C12N5/0775A61K31/734A61K31/728A61K31/43A61K31/7036
CPCA61K9/0014A61K31/43A61K31/7036A61K31/728A61K31/734A61K35/28A61K38/1808A61K38/1825A61K38/1866A61P17/02C12N5/0668C12N2509/00A61K2300/00
Inventor 马步鹏李栋宋芸娟
Owner 北京壹典壹生生物技术有限公司
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