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Animal-derived-component-free passaging method for passaging of stem cells

A technology of animal origin and stem cells, applied in the field of biomedicine, can solve the problems of prolonging the digestion process, affecting the state of stem cell culture, and uneven digestion

Pending Publication Date: 2021-04-20
辽宁省肿瘤医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional trypsin is extracted from pig pancreas, and it is difficult to avoid heterologous protein allergy and pig-derived pathogen infection during application
Recently invented mild digestive enzymes and mild cell dissociation reagents can replace trypsin for passaging digestion. However, due to the lack of trypsin-related components, the digestion process is prolonged and the digestion is uneven, and it is easy to form cell clumps. Serious Affects stem cell culture status

Method used

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  • Animal-derived-component-free passaging method for passaging of stem cells
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  • Animal-derived-component-free passaging method for passaging of stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035]Example 1 Extract the human amniotic membrane, umbilical cord serum, routinely cultured to the third generation, and cell identification was carried out.

[0036]I. Materials and methods.

[0037]1, material.

[0038]DMEM / F12 medium (Hyclone, United States); ElitegroTM-Advanced (GMP Degree); PBS buffer (Biological Industries, Israel); collagenase IV (Thermofisher, USA); MESENCULTTM Osteogenic DiffERENTIATION KIT (HUMAN) (STEMCELL, USA); MESENCULTTMAdipogenic DiffERENTIATION KIT (HUMAN) (STEMCELL, USA);

[0039]MESENCULTTM-ACF CHONDROGENIC DIFFERENTIATION KIT (STEMCELL, USA); Alizarin Red S (Sedae Biology, China); Oil Red O (Fair Biology, China); Alcian Blue (Sedai Biology, China); CD34 FITC direct target antibody (BioLegend , United States); CD73FITC direct target antibody (BioLegend, United States); CD90FITC direct target antibody (Biolegend, US); HLA-DRFITC direct antibody (BioLegend, USA).

[0040]No animal source component digestion: The concentration unit Mg / L is calculated from the...

Embodiment 2

[0053]Type 3 generations of amnunopions, umbilical-cord metamorphosis stem cells in 75cm2Culture in the culture flask, the treatment of 80-90%.

[0054]I. Materials and methods.

[0055]1, material.

[0056]DMEM / F12 medium (Hyclone, United States); ElitegroTM-Advanced (GMP Degree) (Dako, China); PBS buffer (Biological Industries, Israel).

[0057]2, method.

[0058]Culture of Hamscs Cells: Take the third generation of amniotic membranes under the culture of animals source components, umbilical tape, tanneous stem cells, according to 5 × 104 / cm2Turn on 75cm2The bottle was cultured, and the cell cell density was observed after inoculation, and the cell convergence was 80-90%.

Embodiment 3

[0059]Example 3 Apply an animal-free component digestible liquid, digestive termination liquid, a human amniocenger, umbilical-cord metamorphosis stem cells.

[0060]I. Materials and methods.

[0061]1, material.

[0062]PBS buffer (Biological Industries, Israel); mild digestive enzyme (Friend Kang, China); no animal source component digestion; digestive termination; DMEM / F12 medium (Hyclone, United States); ElitegroTM-Advanced (GMPDEGREE) (Dako is China).

[0063]No animal source component digestion: The concentration unit Mg / L is calculated from the following concentrations of components: EDTA-2NA100, recombinant human trypsin 50, potassium hydrogen phosphate 60, potassium chloride 400, sodium bicarbonate 350, Sodium chloride 8000.

[0064]Digestive Termination: Meeting in a concentration unit, is comprised of the following concentrations of components: sodium chloride 8000, dihydrate sodium hydrogen sodium hydrogen phosphate 125, potassium chloride 400, potassium phosphate 60, magnesium sul...

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Abstract

The invention belongs to the technical field of biological medicine, and relates to an animal-derived-component-free passaging method for passaging of stem cells, in particular to preparation of animal-derived-component-free digestive juice and digestive stopping solution for clinical-grade stem cells and an animal-derived-component-free passaging method for passaging of the stem cells. The animal-derived-component-free passaging method for passaging of the clinical-grade stem cells comprises the following steps: extracting human amniotic membrane and umbilical cord mesenchymal stem cells, performing routine culture to a third generation, and carrying out cell identification; inoculating the third-generation human amniotic membrane and umbilical cord mesenchymal stem cells into a 75cm<2> culture bottle for culture until the convergence degree reaches 80-90%; and making the human amniotic membrane and umbilical cord mesenchymal stem cells passage by applying animal-derived-component-free digestive juice and digestive stopping solution. The animal-derived-component-free passaging method for passaging of the stem cells provided by the invention not only meets the animal-derived-component-free standard, but also can effectively digest the stem cells to unicellular morphology, and thereby the stem cells are uniformly inoculated, and the optimal culture state is achieved.

Description

Technical field[0001]The present invention belongs to the field of biomedical technology, and it is directed to a biological source transmission method for passaged stem cells, and more particularly to a clinical stem cell without animal source component digestible liquid, digestive termination liquid, and stem cells without animal source.Background technique[0002]Stem cells are a class of cells having self-renewal and multi-diffiling potential. Studies have shown that stem cells can induce a variety of functional cells constituting human body under certain conditions, and is the secret cells that form a variety of organisms and organs of the human body. It is the secret of human organ regeneration and repair.[0003]An animal-free component medium is a type of medium that extracts commonly used in the production of clinical stem cell production, which is capable of avoiding dissimilar proteins allergic and xenografts in the production process without contacting any components contain...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 康悦赵峰
Owner 辽宁省肿瘤医院
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