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Digestive juice for obtaining highly active chondrocytes

A chondrocyte and digestive juice technology, applied in the direction of bone/connective tissue cells, animal cells, cell dissociation methods, etc., can solve the problems of inability to achieve chondrocyte digestion and separation, denaturation of type II collagenase, and reduced digestion, etc. Good protection, improve digestion rate, prevent damage

Inactive Publication Date: 2019-11-05
杨慧慧
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most common method for isolating chondrocytes is to obtain them through type II collagenase digestion, but this method has many shortcomings, mainly including: (1) Although type II collagenase is a mild digestive enzyme, it still exists The damage to cells, especially when the digestion is excessive, the damage to cells is very obvious; (2) Type Ⅱ collagenase is susceptible to denaturation due to the influence of temperature, pH, other proteases, etc., so type Ⅱ collagenase is used In the process of chondrocytes, it is very easy to lose activity, reduce the digestion effect, and seriously lead to the inability to realize the digestion and separation of chondrocytes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The present invention provides a digestive solution capable of obtaining highly active chondrocytes, based on complete medium, and the digestive liquid capable of obtaining highly active chondrocytes contains the following components:

[0027] Type Ⅱ collagenase 0.15g / L

[0028] Type IV Collagenase 0.0.25g / L

[0029] Human albumin 12.5g / L

[0030] Mesna 5g / L.

[0031] Wherein, the base liquid is DMEM medium with 8v / v% serum.

[0032] Among them, the DMEM medium contains calcium chloride, ferric nitrate, potassium chloride, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate, succinic acid, succinic acid, L-arginine hydrochloride, L-cyst amino acid, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, calcium D-pantothenate, choline bitartrate, folic acid, inositol, niacinamide, riboflavin, thiamine hydrochloride, hydrochloric acid ...

Embodiment 2

[0043] The present invention provides a digestive solution capable of obtaining highly active chondrocytes, based on complete medium, and the digestive liquid capable of obtaining highly active chondrocytes contains the following components:

[0044] Type Ⅱ collagenase 0.1g / L

[0045] Type IV Collagenase 0.5g / L

[0046] Human albumin 20g / L

[0047] Shi Leizuosheng 2g / L.

[0048] Wherein, the base liquid is DMEM medium with 8v / v% serum.

[0049] Among them, the DMEM medium contains calcium chloride, ferric nitrate, potassium chloride, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate, succinic acid, succinic acid, L-arginine hydrochloride, L-cyst amino acid, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, calcium D-pantothenate, choline bitartrate, folic acid, inositol, niacinamide, riboflavin, thiamine hydrochloride, hydrochloric a...

Embodiment 3

[0060] The present invention provides a digestive solution capable of obtaining highly active chondrocytes, based on complete medium, and the digestive liquid capable of obtaining highly active chondrocytes contains the following components:

[0061] Type Ⅱ collagenase 0.2g / L

[0062] Type IV Collagenase 0.05g / L

[0063] Human serum albumin 5g / L

[0064] Amifostine 8g / L.

[0065] Wherein, the base liquid is DMEM medium with 8v / v% serum.

[0066] Among them, the DMEM medium contains calcium chloride, ferric nitrate, potassium chloride, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate, succinic acid, succinic acid, L-arginine hydrochloride, L-cyst amino acid, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, calcium D-pantothenate, choline bitartrate, folic acid, inositol, niacinamide, riboflavin, thiamine hydrochloride, hydrochloric ...

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Abstract

The invention relates to the technical field of in vitro tissue cell separation, and provides a digestive juice capable of obtaining highly active chondrocytes. By combining type II collagenase and type IV collagenase, digestion rate of the digestive juice is improved. By adding human albumin in the digestive juice, the decomposition of type II collagenase and type IV collagenase by other exogenous proteases is buffered to ensure that type II collagenase is not damaged and has high activity; by adding a cell protective agent in the digestive juice, chondrocytes are greatly protected and are effectively prevented from being damaged during the digestive process, which facilitate to obtain more cartilage primary cells with higher activity.

Description

technical field [0001] The invention belongs to the technical field of in vitro tissue cell separation, and in particular relates to a digestive fluid capable of obtaining highly active chondrocytes. Background technique [0002] Cartilage is avascular tissue composed of chondrocytes and extracellular matrix. Chondrocytes are the only cell type in articular cartilage. Their main function is to maintain articular cartilage by secreting type II collagen and proteoglycan (aggrecan, Acan). function. Under normal physiological conditions, the synthesis and catabolism of chondrocytes maintain a dynamic balance; while chondrocytes mainly rely on joint movement and extrusion to absorb nutrients, so the self-repair ability of cartilage damage is relatively weak, and its repair after damage has always been It is one of the problems in the medical field. In recent years, with the emergence of tissue engineering technology and new biomaterials, autologous chondrocyte transplantation h...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2509/00
Inventor 杨慧慧
Owner 杨慧慧
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