CN111019887A - Culture method for preventing dedifferentiation of chondrocytes - Google Patents

Culture method for preventing dedifferentiation of chondrocytes Download PDF

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CN111019887A
CN111019887A CN201911095362.2A CN201911095362A CN111019887A CN 111019887 A CN111019887 A CN 111019887A CN 201911095362 A CN201911095362 A CN 201911095362A CN 111019887 A CN111019887 A CN 111019887A
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chondrocytes
culture medium
growth factor
dedifferentiation
culture method
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陈锦阳
李静静
刘军权
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Zhejiang Healthfuture Biomedical Technology Co ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
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Abstract

The invention discloses a cultivation method for preventing dedifferentiation of chondrocytes, which comprises the following steps: s1, digesting human articular cartilage tissues by using collagenase; s2, diluting the cells with PBS, and centrifuging to collect the cells; s3, centrifugally washing with PBS to obtain primary human chondrocytes; s4, culturing primary human chondrocytes in a culture medium for 5-7 generations, wherein the culture medium comprises the following components: 10-15% of serum, amino acids and growth factors. The culture method for preventing the dedifferentiation of the chondrocytes can effectively reverse the dedifferentiated chondrocytes, so that the capability of normal chondrocytes is well maintained, and an experimental foundation is laid for the application of the culture method to autologous chondrocyte transplantation.

Description

Culture method for preventing dedifferentiation of chondrocytes
Technical Field
The invention relates to the technical field of biology, in particular to a cultivation method for preventing dedifferentiation of chondrocytes.
Background
Articular cartilage is mainly avascular tissue composed of chondrocytes and extracellular matrix, and chondrocytes are the only cell type in articular cartilage, and have the main function of maintaining the function of articular cartilage through secretion of type II collagen, proteoglycan and the like. Cartilage cells mainly absorb nutrients by means of movement and extrusion of joints, and most importantly, cartilage tissues lack blood vessels, lymph and a nervous system, so that the self-repair capacity of the cartilage after damage is weak, and repair and transplantation after the damage are always one of the problems in the medical field. Through more than ten years of clinical application and technical development, autologous chondrocyte transplantation technology has become the most effective method for clinically treating cartilage injury, but clinical research has limited tissue material availability for patients, and because chondrocytes are terminally differentiated cells, have limited in vitro proliferation capacity and are easy to dedifferentiate, the culture and amplification of chondrocytes in vitro become the key to be solved at present.
According to the culture method in the prior art, with the increase of the number of passages, chondrocytes are easy to dedifferentiate to present a fibrocyte state, cartilage specific type II collagen and proteoglycan secreted by the chondrocytes cannot be secreted, and the normal chondrocyte state is lost.
Disclosure of Invention
The present invention provides a culture method for preventing the dedifferentiation of chondrocytes, in order to solve the above-mentioned technical problems.
The invention is realized according to the following technical scheme.
A culture method for preventing dedifferentiation of chondrocytes, comprising the steps of:
s1, digesting human articular cartilage tissues by using collagenase;
s2, diluting the cells with PBS, and centrifuging to collect the cells;
s3, centrifugally washing with PBS to obtain primary human chondrocytes;
s4, culturing primary human chondrocytes in a culture medium for 5-7 generations, wherein the culture medium comprises the following components: 10-15% of serum, amino acids and growth factors.
Further, the culture medium comprises the following components in parts by weight: 10-15% serum; the amino acids comprise L-arginine 150.0-200.0mg/L, L-aspartic acid 8.0-10.0mg/L, L-glutamic acid 6.0-12.0mg/L, glycine 15.0-18.0mg/L, L-histidine 18.0-20.0mg/L, L-hydroxyproline 12.0-16.0mg/L, L-isoleucine 20.0-30.0mg/L, L-leucine 25.0-35.0mg/L, L-methionine 20.0-25.0mg/L, L-phenylalanine 6.0-10.0mg/L, L-proline 16.0-20.0mg/L, L-serine 35.0-40.0mg/L, L-threonine 7.0-15.0mg/L, L-tryptophan 8.0-10.0mg/L, L, L-tyrosine 15.0-20.0mg/L, L-valine 10.0-16.0 mg/L; the growth factors include: basic fibroblast growth factor 25-45ug/L, epidermal growth factor 5-25ug/L, bone morphogenetic protein 10-25ug/L, platelet derived growth factor 15-35ug/L, interleukin-60.5-2.5 ug/L; the balance is F12 basic culture medium.
Further, the culture medium comprises the following components in parts by weight: 12-15% serum; the amino acids include L-arginine 160.0-180.0mg/L, L-aspartic acid 8.0-10.0mg/L, L-glutamic acid 8.0-10.0mg/L, glycine 15.0-17.0mg/L, L-histidine 18.0-20.0mg/L, L-hydroxyproline 14.0-16.0mg/L, L-isoleucine 22.0-28.0mg/L, L-leucine 28.0-32.0mg/L, L-methionine 22.0-24.0mg/L, L-phenylalanine 7.0-9.0mg/L, L-proline 17.0-19.0mg/L, L-serine 37.0-39.0mg/L, L-threonine 10.0-15.0mg/L, L-tryptophan 8.0-10.0mg/L, L, L-tyrosine 16.0-18.0mg/L, L-valine 12.0-16.0 mg/L; the growth factors include: 30-40ug/L of basic fibroblast growth factor, 10-20ug/L of epidermal growth factor, 15-20ug/L of bone morphogenetic protein, 20-30ug/L of platelet-derived growth factor and-61.0-2.0 ug/L of interleukin; the balance is F12 basic culture medium.
Further, the culture medium comprises the following components in parts by weight: 15% serum; the amino acid comprises 170.0mg/L, L mg of L-arginine, 9.0mg/L, L mg of aspartic acid, 9.0mg/L of glutamic acid, 16.0mg/L, L mg of glycine, 19.0mg/L, L mg of histidine, 15.0mg/L, L mg of hydroxyproline, 25.0mg/L, L mg of isoleucine, 30.0mg/L, L mg of leucine, 23.0mg/L, L mg of phenylalanine, 8.0mg/L, L mg of proline, 18.0mg/L, L mg of serine, 38.0mg/L, L mg of threonine, 12.0mg/L, L mg of tryptophan, 9.0mg/L, L of tyrosine, 17.0mg/L, L and 14.0mg/L of valine; the growth factors include: 35ug/L of basic fibroblast growth factor, 15ug/L of epidermal growth factor, 18ug/L of bone morphogenetic protein, 25ug/L of platelet-derived growth factor and 61.5ug/L of interleukin; the balance is F12 basic culture medium.
Further, the collagenase in step S1 is 0.5-2% collagenase type II.
Further, the collagenase in step S1 is 1.5% type II collagenase.
The present invention obtains the following advantageous effects.
The culture method for preventing the dedifferentiation of the chondrocytes can effectively prevent the dedifferentiation of the chondrocytes, so that the capability of normal chondrocytes is well maintained, and an experimental foundation is laid for the application of the culture method to autologous chondrocyte transplantation.
Detailed Description
The present invention will be further illustrated with reference to the following examples.
Example 1
A culture method for preventing dedifferentiation of chondrocytes, comprising the steps of:
s1, digesting human articular cartilage tissues by using 1.5% type II collagenase;
s2, diluting the cells with PBS, and centrifuging to collect the cells;
s3, centrifugally washing with PBS to obtain primary human chondrocytes;
s4, culturing primary human chondrocytes in a culture medium for 5-7 generations, wherein the culture medium comprises the following components: the culture medium comprises the following components in parts by weight: 10% serum; the amino acid comprises 150.0mg/L, L mg of L-arginine, 8.0mg/L, L mg of aspartic acid, 6.0mg/L of glutamic acid, 15.0mg/L, L mg of glycine, 18.0mg/L, L mg of histidine, 12.0mg/L, L mg of hydroxyproline, 20.0mg/L, L mg of isoleucine, 25.0mg/L, L mg of leucine, 20.0mg/L, L mg of phenylalanine, 6.0mg/L, L mg of proline, 16.0mg/L, L mg of serine, 35.0mg/L, L mg of threonine, 7.0mg/L, L mg of tryptophan, 8.0mg/L, L of tyrosine, 15.0mg/L, L and 10.0mg/L of valine; the growth factors include: 25ug/L of basic fibroblast growth factor, 5ug/L of epidermal growth factor, 10ug/L of bone morphogenetic protein, 15ug/L of platelet-derived growth factor and 60.5ug/L of interleukin; the balance is F12 basic culture medium.
Example 2
A culture method for preventing dedifferentiation of chondrocytes, comprising the steps of:
s1, digesting human articular cartilage tissues by using 1.5% type II collagenase;
s2, diluting the cells with PBS, and centrifuging to collect the cells;
s3, centrifugally washing with PBS to obtain primary human chondrocytes;
s4, culturing primary human chondrocytes in a culture medium for 5-7 generations, wherein the culture medium comprises the following components: the culture medium comprises the following components in parts by weight: 15% serum; the amino acid comprises 200.0mg/L, L mg of L-arginine, 10.0mg/L, L mg of aspartic acid, 12.0mg/L of glutamic acid, 18.0mg/L, L mg of glycine, 20.0mg/L, L mg of histidine, 16.0mg/L, L mg of hydroxyproline, 30.0mg/L, L mg of isoleucine, 35.0mg/L, L mg of leucine, 25.0mg/L, L mg of phenylalanine, 10.0mg/L, L mg of proline, 20.0mg/L, L mg of serine, 40.0mg/L, L mg of threonine, 15.0mg/L, L mg of tryptophan, 10.0mg/L, L of tyrosine, 20.0mg/L, L and 16.0mg/L of valine; the growth factors include: basic fibroblast growth factor 45ug/L, epidermal growth factor 25ug/L, bone morphogenetic protein 25ug/L, platelet derived growth factor 35ug/L, interleukin-62.5 ug/L; the balance is F12 basic culture medium.
Example 3
A culture method for preventing dedifferentiation of chondrocytes, comprising the steps of:
s1, digesting human articular cartilage tissues by using 1.5% type II collagenase;
s2, diluting the cells with PBS, and centrifuging to collect the cells;
s3, centrifugally washing with PBS to obtain primary human chondrocytes;
s4, culturing primary human chondrocytes in a culture medium for 5-7 generations, wherein the culture medium comprises the following components: the culture medium comprises the following components in parts by weight: 15% serum; the amino acid comprises 170.0mg/L, L mg of L-arginine, 9.0mg/L, L mg of aspartic acid, 9.0mg/L of glutamic acid, 16.0mg/L, L mg of glycine, 19.0mg/L, L mg of histidine, 15.0mg/L, L mg of hydroxyproline, 25.0mg/L, L mg of isoleucine, 30.0mg/L, L mg of leucine, 23.0mg/L, L mg of phenylalanine, 8.0mg/L, L mg of proline, 18.0mg/L, L mg of serine, 38.0mg/L, L mg of threonine, 12.0mg/L, L mg of tryptophan, 9.0mg/L, L of tyrosine, 17.0mg/L, L and 14.0mg/L of valine; the growth factors include: 35ug/L of basic fibroblast growth factor, 15ug/L of epidermal growth factor, 18ug/L of bone morphogenetic protein, 25ug/L of platelet-derived growth factor and 61.5ug/L of interleukin; the balance is F12 basic culture medium.
Example 4
A culture method for preventing dedifferentiation of chondrocytes, comprising the steps of:
s1, digesting human articular cartilage tissues by using 1.5% type II collagenase;
s2, diluting the cells with PBS, and centrifuging to collect the cells;
s3, centrifugally washing with PBS to obtain primary human chondrocytes;
s4, culturing primary human chondrocytes in a culture medium for 5-7 generations, wherein the culture medium comprises the following components: the culture medium comprises the following components in parts by weight: 14% serum; the amino acid comprises 160.0mg/L, L mg of L-arginine, 8.0mg/L, L mg of aspartic acid, 10.0mg/L of glutamic acid, 15.0mg/L, L mg of glycine, 20.0mg/L, L of histidine, 16.0mg/L, L-isoleucine, 26.0mg/L, L-leucine, 29.0mg/L, L-methionine, 22.0mg/L, L-phenylalanine, 9.0mg/L, L-proline, 17.0mg/L, L-serine, 39.0mg/L, L-threonine, 14.0mg/L, L-tryptophan, 10.0mg/L, L-tyrosine, 18.0mg/L, L-valine, 13.0 mg/L; the growth factors include: 36ug/L of basic fibroblast growth factor, 12ug/L of epidermal growth factor, 16ug/L of bone morphogenetic protein, 28ug/L of platelet-derived growth factor and 61.7ug/L of interleukin; the balance is F12 basic culture medium.
Example 5 examples 1-4 cultured chondrocyte count results
Figure RE-GDA0002391995610000041
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (6)

1. A culture method for preventing dedifferentiation of chondrocytes, comprising: the method comprises the following steps:
s1, digesting human articular cartilage tissues by using collagenase;
s2, diluting the cells with PBS, and centrifuging to collect the cells;
s3, centrifugally washing with PBS to obtain primary human chondrocytes;
s4, culturing primary human chondrocytes in a culture medium for 5-7 generations, wherein the culture medium comprises the following components: 10-15% of serum, amino acids and growth factors.
2. A culture method for preventing dedifferentiation of chondrocytes according to claim 1, wherein: the culture medium comprises the following components in parts by weight: 10-15% serum; the amino acids comprise L-arginine 150.0-200.0mg/L, L-aspartic acid 8.0-10.0mg/L, L-glutamic acid 6.0-12.0mg/L, glycine 15.0-18.0mg/L, L-histidine 18.0-20.0mg/L, L-hydroxyproline 12.0-16.0mg/L, L-isoleucine 20.0-30.0mg/L, L-leucine 25.0-35.0mg/L, L-methionine 20.0-25.0mg/L, L-phenylalanine 6.0-10.0mg/L, L-proline 16.0-20.0mg/L, L-serine 35.0-40.0mg/L, L-threonine 7.0-15.0mg/L, L-tryptophan 8.0-10.0mg/L, L, L-tyrosine 15.0-20.0mg/L, L-valine 10.0-16.0 mg/L; the growth factors include: basic fibroblast growth factor 25-45ug/L, epidermal growth factor 5-25ug/L, bone morphogenetic protein 10-25ug/L, platelet derived growth factor 15-35ug/L, interleukin-60.5-2.5 ug/L; the balance is F12 basic culture medium.
3. A culture method for preventing dedifferentiation of chondrocytes according to claim 1, wherein: the culture medium comprises the following components in parts by weight: 12-15% serum; the amino acids include L-arginine 160.0-180.0mg/L, L-aspartic acid 8.0-10.0mg/L, L-glutamic acid 8.0-10.0mg/L, glycine 15.0-17.0mg/L, L-histidine 18.0-20.0mg/L, L-hydroxyproline 14.0-16.0mg/L, L-isoleucine 22.0-28.0mg/L, L-leucine 28.0-32.0mg/L, L-methionine 22.0-24.0mg/L, L-phenylalanine 7.0-9.0mg/L, L-proline 17.0-19.0mg/L, L-serine 37.0-39.0mg/L, L-threonine 10.0-15.0mg/L, L-tryptophan 8.0-10.0mg/L, L, L-tyrosine 16.0-18.0mg/L, L-valine 12.0-16.0 mg/L; the growth factors include: 30-40ug/L of basic fibroblast growth factor, 10-20ug/L of epidermal growth factor, 15-20ug/L of bone morphogenetic protein, 20-30ug/L of platelet-derived growth factor and-61.0-2.0 ug/L of interleukin; the balance is F12 basic culture medium.
4. A culture method for preventing dedifferentiation of chondrocytes according to claim 1, wherein: the culture medium comprises the following components in parts by weight: 15% serum; the amino acid comprises 170.0mg/L, L mg of L-arginine, 9.0mg/L, L mg of aspartic acid, 9.0mg/L of glutamic acid, 16.0mg/L, L mg of glycine, 19.0mg/L, L mg of histidine, 15.0mg/L, L mg of hydroxyproline, 25.0mg/L, L mg of isoleucine, 30.0mg/L, L mg of leucine, 23.0mg/L, L mg of phenylalanine, 8.0mg/L, L mg of proline, 18.0mg/L, L mg of serine, 38.0mg/L, L mg of threonine, 12.0mg/L, L mg of tryptophan, 9.0mg/L, L of tyrosine, 17.0mg/L, L and 14.0mg/L of valine; the growth factors include: 35ug/L of basic fibroblast growth factor, 15ug/L of epidermal growth factor, 18ug/L of bone morphogenetic protein, 25ug/L of platelet-derived growth factor and 61.5ug/L of interleukin; the balance is F12 basic culture medium.
5. A culture method for preventing dedifferentiation of chondrocytes according to claim 1, wherein: the collagenase in step S1 is 0.5-2% collagenase type II.
6. A culture method for preventing dedifferentiation of chondrocytes according to claim 1, wherein: the collagenase in step S1 is 1.5% collagenase type II.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101490245A (en) * 2006-06-20 2009-07-22 建新公司 Serum-free media and their uses for chondrocyte expansion
CN104480066A (en) * 2014-12-30 2015-04-01 陕西瑞盛生物科技有限公司 Chondrocyte culture medium and chondrocyte culture method
CN104630138A (en) * 2013-11-06 2015-05-20 陕西瑞盛生物科技有限公司 Serum-free cartilage cell culture solution
CN106244528A (en) * 2016-09-28 2016-12-21 广州赛莱拉干细胞科技股份有限公司 Chondrocyte serum-free medium and preparation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101490245A (en) * 2006-06-20 2009-07-22 建新公司 Serum-free media and their uses for chondrocyte expansion
CN104630138A (en) * 2013-11-06 2015-05-20 陕西瑞盛生物科技有限公司 Serum-free cartilage cell culture solution
CN104480066A (en) * 2014-12-30 2015-04-01 陕西瑞盛生物科技有限公司 Chondrocyte culture medium and chondrocyte culture method
CN106244528A (en) * 2016-09-28 2016-12-21 广州赛莱拉干细胞科技股份有限公司 Chondrocyte serum-free medium and preparation method thereof

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Title
KARIN BENZ等: "Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 *
张辉等: "碱性成纤维细胞生长因子对国产多孔钽-软骨细胞复合物软骨细胞表型维持及去分化的影响", 《第二军医大学学报》 *

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Application publication date: 20200417