CN106244528A - Chondrocyte serum-free medium and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of in-vitro tissue cell separation, in particular to a chondrocyte serum-free culture medium which comprises a basic culture medium, a serum substitute and a growth factor, solves the problems caused by the serum culture medium in the prior art, has good adherence and higher proliferation speed in the growth process of chondrocytes, is beneficial to obtaining more cell amount, can maintain the characteristics of the chondrocytes while proliferating the chondrocytes, and has no obvious dedifferentiation phenomenon.

Description

A kind of chondrocyte serum-free medium and preparation method thereof

Technical field

The present invention relates to technical field of biomedical materials, be specifically related to a kind of chondrocyte serum-free medium and system thereof Preparation Method.

Background technology

The vesselless tissue that cartilage is made up of chondrocyte and extracellular matrix, chondrocyte is unique in articular cartilage Cell type, its major function be by secretion II Collagen Type VI (type II collagen) and proteoglycan (aggrecan, Etc. Acan) function of articular cartilage is kept.Under normal physiological conditions, synthesis and the catabolism of chondrocyte maintain dynamically Balance；And chondrocyte relies primarily on motion and the extruding absorption nutrient substance in joint, so self-regeneration energy after cartilage injury Force rate is more weak, and the reparation after its damage is the most all one of difficult problem of medical circle.Clinical practice and skill through more than ten years Art develops, and font chondrocyte cell transplantation technology has become clinical treatment cartilage injury's most efficient method.

Most animals cell relies on serum the most to some extent could growing multiplication in vitro.Conventional chondrocyte training Support is to carry out under the DMEM culture medium condition containing the serum of 10%.Serum can provide the nutrient needed for cell growth Matter, simultaneously containing all kinds of somatomedin in serum, can promote the propagation of cell.But serum composition is complicated, cellular products is being entered Row can be disturbed by serum composition when analyzing or purify, and the difficult quality of different batches serum keeps consistent, it addition, Possibly together with a certain amount of cytotoxic substance and inhibiting substances in serum, cell is dedifferented effect, affect some cell merit The expression of energy.

Under the condition of culture having serum, along with passage number increases, chondrocyte is easy to dedifferente generation fiber finer Born of the same parents' state, it is impossible to the cartilage specificity typeⅡ Collagen of secretion chondrocyte secretion and Dan Baiduotang proteoglycan PG, loses normal cartilage thin Born of the same parents' phenotype.And serum-free medium can solve some problems that serum brings well, such as Publication No. CN 104630138 The patent of invention of A discloses a kind of serum-free cultured chondrocytes liquid, but, due to chondrocyte, to belong to adherent growth class thin Born of the same parents, lack in above-mentioned patent kinds of ingredients and promote adherent associated protein and protease inhibitor, easily cause cell growth adherent not The problem such as firm, additionally, there are and delay chondrocyte to dedifferente inefficient problem, and chondrocyte In vitro culture easily occurs point Change, cause collagen protein to be expressed and change.

Summary of the invention

The present invention is directed to problems of the prior art, it is provided that a kind of chondrocyte serum-free medium, not only solve The problem that blood serum medium of the prior art brings, and the most adherent and value-added speed during chondrocyte growth Faster, be conducive to obtaining more cell concentration, the characteristic of chondrocyte can be kept simultaneously while chondrocyte proliferation, do not go out Now obvious dedifferen tiation.

Another object of the present invention is to provide the preparation method of a kind of chondrocyte serum-free medium.

The present invention is achieved through the following technical solutions this purpose:

A kind of chondrocyte serum-free medium, is made up of basal medium, serum substitute and somatomedin, its In:

Described basal medium is DMEM culture medium, F12 culture medium or the mixture of the two；

Described serum substitute includes: glucosan 5-20g/L, lipid concentrating agents 0.1-1ml/L, Sodium Pyruvate 50- 100mg/L, triiodothyronine 0.01-2uM, hydrocortisone 0.05-5mg/L, dexamethasone 0.1-10ug/L, Monohydrated selenium dioxide Sodium 1-5ug/L, beta-mercaptoethanol 25-50uM, vitamin C 10-100mg/L, glutamine 50-500ug/ml, transferrins 1- 20mg/L, insulin or insulin like growth factor 5-100ug/L, growth hormone 1 GH1 0-200ug/L, progesterone 5-20nM, butanediamine 20-100uM, choline chloride 2.5-3.2mg/L, laminin，LN 5-30ug/L, STI 2-10mg/L；

L-arginine 250.0-320.0mg/L, L-ASPARTIC ACID 12.00-25.00mg/L, Pidolidone 15.0- 25.0mg/L, glycine 8.0-12.0mg/L, L-Histidine 13.0-16.0mg/L, L-hydroxyproline 18.0-22.0mg/L, L- Isoleucine 35.0-55.0mg/L, L-Leu 40.0-60.0mg/L, METHIONINE 12.0-18.0mg/L, L-phenylpropyl alcohol ammonia Acid 12.0-18.0mg/L, L-PROLINE 18.0-22.0mg/L, Serine 25.0-32.0mg/L, L-threonine 18.0- 22.0mg/L, L-Trp 2.0-6.0mg/L, TYR 20.19-25.89mg/L, Valine 18.0-22.0mg/L；

Described somatomedin includes: basic fibroblast growth factor 5-60ug/L, epidermal growth factor 0.5-30ug/L, turn Change somatomedin 1-25ug/L, bone morphogenetic protein(BMP) 1-20ug/L, platelet derived growth factor 1-18ug/L, bHLH transcription factor 0.2-5ug/L, transcription factor E2F-1 0.01-2ug/L.

As preferably, chondrocyte serum-free medium, by basal medium, serum substitute and somatomedin group Become, wherein:

Described basal medium is DMEM culture medium, F12 culture medium or the mixture of the two；

Described serum substitute includes: glucosan 10g/L, lipid concentrating agents 0.5ml/L, Sodium Pyruvate 80mg/L, triiodo Desiodothyroxine 0.2uM, hydrocortisone 2mg/L, dexamethasone 0.2ug/L, sodium selenite 2ug/L, beta-mercaptoethanol 25uM, Vitamin C 50mg/L, glutamine 200ug/mL, transferrins 5mg/L, insulin or insulin like growth factor 40ug/L, Growth hormone 20ug/L, progesterone 10nM, butanediamine 50uM, choline chloride 2.5mg/L, LN1 2ug/L, soybean protein Enzyme inhibitor 6mg/L；

L-arginine 300mg/L, L-ASPARTIC ACID 20mg/L, Pidolidone 20mg/L, glycine 10mg/L, L-Histidine 15mg/L, L-hydroxyproline 20mg/L, ILE 40mg/L, L-Leu 60mg/L, METHIONINE 15mg/L, L-benzene Alanine 15mg/L, L-PROLINE 20mg/L, Serine 30mg/L, L-threonine 20mg/L, L-Trp 5mg/L, L-cheese Propylhomoserin 22mg/L, Valine 20mg/L；

Basic fibroblast growth factor 20ug/L, epidermal growth factor 5ug/L, TGF-1 0ug/L, bone formation Albumen 5ug/L, platelet derived growth factor 18ug/L, bHLH transcription factor 0.2ug/L, transcription factor E2F-1 0.05ug/ L。

The preparation method of a kind of chondrocyte serum-free medium, comprises the steps:

Choose basal medium, DMEM culture medium, F12 culture medium or the mixture of the two；

Under room temperature gnotobasis, in basal medium, add serum substitute and relevant growth factors；

0.22um membrane filtration is degerming, obtains chondrocyte serum-free medium.

Relative to prior art, the invention have the benefit that

The chondrocyte serum-free medium of the present invention, is made up of basal medium, serum substitute and somatomedin, Not only solve the problem that blood serum medium of the prior art brings, and during chondrocyte growth the most adherent also Value-added speed faster, is conducive to obtaining more cell concentration, can keep chondrocyte while chondrocyte proliferation simultaneously , there is not obvious dedifferen tiation in characteristic.

Accompanying drawing explanation

Fig. 1 is the cellular morphology figure of comparative example 1.

Fig. 2 is the cellular morphology figure of comparative example 2.

Fig. 3 is the cellular morphology figure of embodiment 1.

Fig. 4 is the cellular morphology figure of embodiment 2.

Fig. 5 is the cellular morphology figure of embodiment 3.

Fig. 6 is that 5 groups of cells cultivate growth curve figure.

Wherein, group 1 expression comparative example 1, group 2 expression comparative example 2, group 3 expression embodiment 1, group 4 expression embodiment 2, organize 5 Represent embodiment 3.

Detailed description of the invention

Describe the present invention below in conjunction with drawings and the specific embodiments.

Embodiment 1.

A kind of chondrocyte serum-free medium, is made up of basal medium, serum substitute and somatomedin, its In:

Described basal medium is DMEM culture medium；

Described serum substitute includes: glucosan 5g/L, lipid concentrating agents 0.1ml/L, Sodium Pyruvate 50mg/L, triiodo first Shape gland propylhomoserin 0.01uM, hydrocortisone 0.05mg/L, dexamethasone 0.1ug/L, sodium selenite 1ug/L, beta-mercaptoethanol 25uM, vitamin C 10mg/L, glutamine 50ug/ml, transferrins 1mg/L, insulin or insulin like growth factor 5ug/ L, growth hormone 1 GH1 0ug/L, progesterone 5nM, butanediamine 20uM, choline chloride 2.5mg/L, laminin，LN 5ug/L, soybean protein Enzyme inhibitor 2mg/L；

L-arginine 250.0mg/L, L-ASPARTIC ACID 12.00mg/L, Pidolidone 15.0mg/L, glycine 8.0mg/L, L-Histidine 13.0mg/L, L-hydroxyproline 18.0mg/L, ILE 35.0mg/L, L-Leu 40.0mg/L, L-first Methyllanthionine 12.0mg/L, L-phenylalanine 12.0mg/L, L-PROLINE 18.0mg/L, Serine 25.0mg/L, L-threonine 18.0mg/L, L-Trp 2.0mg/L, TYR 20.19mg/L, Valine 18.0mg/L；

Described somatomedin includes: basic fibroblast growth factor 5ug/L, epidermal growth factor 0.5ug/L, conversion growth Factor 1ug/L, bone morphogenetic protein(BMP) 1ug/L, platelet derived growth factor 1ug/L, bHLH transcription factor 0.2ug/L, transcribe because of Sub-E2F-1 0.01ug/L.

Embodiment 2.

Chondrocyte serum-free medium, is made up of basal medium, serum substitute and somatomedin, wherein:

Described basal medium is F12 culture medium；

Described serum substitute includes: glucosan 10g/L, lipid concentrating agents 0.5ml/L, Sodium Pyruvate 80mg/L, triiodo Desiodothyroxine 0.2uM, hydrocortisone 2mg/L, dexamethasone 0.2ug/L, sodium selenite 2ug/L, beta-mercaptoethanol 25uM, Vitamin C 50mg/L, glutamine 200ug/mL, transferrins 5mg/L, insulin or insulin like growth factor 40ug/L, Growth hormone 20ug/L, progesterone 10nM, butanediamine 50uM, choline chloride 2.5mg/L, LN1 2ug/L, soybean protein Enzyme inhibitor 6mg/L；

L-arginine 300mg/L, L-ASPARTIC ACID 20mg/L, Pidolidone 20mg/L, glycine 10mg/L, L-Histidine 15mg/L, L-hydroxyproline 20mg/L, ILE 40mg/L, L-Leu 60mg/L, METHIONINE 15mg/L, L-benzene Alanine 15mg/L, L-PROLINE 20mg/L, Serine 30mg/L, L-threonine 20mg/L, L-Trp 5mg/L, L-cheese Propylhomoserin 22mg/L, Valine 20mg/L；

Basic fibroblast growth factor 20ug/L, epidermal growth factor 5ug/L, TGF-1 0ug/L, bone formation Albumen 5ug/L, platelet derived growth factor 18ug/L, bHLH transcription factor 0.2ug/L, transcription factor E2F-1 0.05ug/L

Embodiment 3.

A kind of chondrocyte serum-free medium, is made up of basal medium, serum substitute and somatomedin, its In:

Described basal medium be DMEM culture medium with F12 culture medium according to the ratio of 1:1 mix mixture；

Described serum substitute includes: glucosan 20g/L, lipid concentrating agents 1ml/L, Sodium Pyruvate 100mg/L, triiodo first Shape gland propylhomoserin 2uM, hydrocortisone 5mg/L, dexamethasone 10ug/L, sodium selenite 5ug/L, beta-mercaptoethanol 50uM, dimension are raw Element C100mg/L, glutamine 500ug/ml, transferrins 20mg/L, insulin or type-1 insulin like growth factor 00ug/L, life Long hormone 200ug/L, progesterone 20nM, butanediamine 100uM, choline chloride 3.2mg/L, laminin，LN 30ug/L, soybean protein Enzyme inhibitor 10mg/L；

L-arginine 320.0mg/L, L-ASPARTIC ACID 25.00mg/L, Pidolidone 25.0mg/L, glycine 12.0mg/ L, L-Histidine 16.0mg/L, L-hydroxyproline 22.0mg/L, ILE 55.0mg/L, L-Leu 60.0mg/L, L- Methionine 18.0mg/L, L-phenylalanine 18.0mg/L, L-PROLINE 22.0mg/L, Serine 32.0mg/L, L-revive ammonia Acid 22.0mg/L, L-Trp 6.0mg/L, TYR 25.89mg/L, Valine 122.0mg/L；

Described somatomedin includes: basic fibroblast growth factor 60ug/L, epidermal growth factor 30ug/L, conversion growth Factor 25ug/L, bone morphogenetic protein2 0ug/L, platelet derived growth factor 18ug/L, bHLH transcription factor 5ug/L, transcribe because of Sub-E2F-1 2ug/L.

Comparative example 1.

Add the DMEM culture medium of 10% hyclone.

Comparative example 2.

Serum-free cultured chondrocytes liquid disclosed in the patent of invention of Publication No. CN 104630138 A.

Contrast on effect checking test:

1, take rabbit cartilaginous tissue respectively some, through PBS 2-3 time, shred to 1mm³, add II Collagenase Type, in 37 DEG C shaking table digestion 2-4h；

2, filter, the centrifugal lower floor's chondrocyte of collecting of filtrate, counting；

3, according to count results, 1 × 10 is taken⁷Cell concentration, is divided into 5 groups and is utilized respectively embodiment 1～3, and contrast The culture medium of example 1,2 preparation carries out culture experiment.

4, the cell-seeding-density of above 5 groups of tests is 2 × 10⁵/ mL, carries out when cell grows to 80% passing on training Supporting, cultivate and counted respectively to 6 days and 10 days, count results is as shown in table 1, and cellular morphology is as shown in Fig. 1～5, according to cytometer Count the cell proliferation curve of result drafting as shown in Figure 6:

15 groups of culture experiment cell counts of table

Group	Inoculating cell amount (105)	Cell concentration (10 after 6 days5)	Cell concentration (10 after 10 days5)
				Comparative example 1	2	3.86	10.21
Comparative example 2	2	4.28	11.85
				Embodiment 1	2	6.88	17.29
Embodiment 2	2	6.89	18.35
				Embodiment 3	2	7.12	17.89

Interpretation of result: from the data of table 1 and Fig. 1～6, cell concentration and growth rate that embodiment 1～3 obtains are bright Aobvious it is better than comparative example 1,2, and cellular morphology is clear-cut, be passaged to P9 generation still in " paving stone " shape, and the cell of comparative example 1 For cell, fibroblast shape occurs in P5；There is fibroblast shape in P7 in the cell of comparative example 2, and the cartilage of the present invention is described Cell non-serum culture medium is optimal in terms of maintaining chondrocyte characteristic, and growth rate is also the most optimum.

Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (3)

1. a chondrocyte serum-free medium, is made up of basal medium, serum substitute and somatomedin, wherein:

Described basal medium is DMEM culture medium, F12 culture medium or the mixture of the two；

Described serum substitute includes: glucosan 5-20g/L, lipid concentrating agents 0.1-1ml/L, Sodium Pyruvate 50-100mg/L, Triiodothyronine 0.01-2uM, hydrocortisone 0.05-5mg/L, dexamethasone 0.1-10ug/L, sodium selenite 1-5ug/ L, beta-mercaptoethanol 25-50uM, vitamin C 10-100mg/L, glutamine 50-500ug/ml, transferrins 1-20mg/L, pancreas Island element or insulin like growth factor 5-100ug/L, growth hormone 1 GH1 0-200ug/L, progesterone 5-20nM, butanediamine 20-100uM, Choline chloride 2.5-3.2mg/L, laminin，LN 5-30ug/L, STI 2-10mg/L；

L-arginine 250.0-320.0mg/L, L-ASPARTIC ACID 12.00-25.00mg/L, Pidolidone 15.0-25.0mg/L, Glycine 8.0-12.0mg/L, L-Histidine 13.0-16.0mg/L, L-hydroxyproline 18.0-22.0mg/L, ILE 35.0-55.0mg/L, L-Leu 40.0-60.0mg/L, METHIONINE 12.0-18.0mg/L, L-phenylalanine 12.0- 18.0mg/L, L-PROLINE 18.0-22.0mg/L, Serine 25.0-32.0mg/L, L-threonine 18.0-22.0mg/L, L- Tryptophan 2.0-6.0mg/L, TYR 20.19-25.89mg/L, Valine 18.0-22.0mg/L；

Described somatomedin includes: basic fibroblast growth factor 5-60ug/L, epidermal growth factor 0.5-30ug/L, conversion life Long factor 1-25ug/L, bone morphogenetic protein(BMP) 1-20ug/L, platelet derived growth factor 1-18ug/L, bHLH transcription factor 0.2- 5ug/L, transcription factor E2F-1 0.01-2ug/L.

Chondrocyte serum-free medium the most according to claim 1, by basal medium, serum substitute and growth The factor forms, wherein:

Described basal medium is DMEM culture medium, F12 culture medium or the mixture of the two；

Described serum substitute includes: glucosan 10g/L, lipid concentrating agents 0.5ml/L, Sodium Pyruvate 80mg/L, triiodo first shape Gland propylhomoserin 0.2uM, hydrocortisone 2mg/L, dexamethasone 0.2ug/L, sodium selenite 2ug/L, beta-mercaptoethanol 25uM, dimension are raw Element C50mg/L, glutamine 200ug/m L, transferrins 5mg/L, insulin or insulin like growth factor 40ug/L, growth Hormone 20ug/L, progesterone 10nM, butanediamine 50uM, choline chloride 2.5mg/L, LN1 2ug/L, soybean protein enzyme press down Preparation 6mg/L；

L-arginine 300mg/L, L-ASPARTIC ACID 20mg/L, Pidolidone 20mg/L, glycine 10mg/L, L-Histidine 15mg/L, L-hydroxyproline 20mg/L, ILE 40mg/L, L-Leu 60mg/L, METHIONINE 15mg/L, L-benzene Alanine 15mg/L, L-PROLINE 20mg/L, Serine 30mg/L, L-threonine 20mg/L, L-Trp 5mg/L, L-cheese Propylhomoserin 22mg/L, Valine 20mg/L；

Basic fibroblast growth factor 20ug/L, epidermal growth factor 5ug/L, TGF-1 0ug/L, bone morphogenetic protein(BMP) 5ug/L, platelet derived growth factor 18ug/L, bHLH transcription factor 0.2ug/L, transcription factor E2F-1 0.05ug/L.

3. the preparation method of the chondrocyte serum-free medium described in claim 1 or 2, comprises the steps:

Choose basal medium, DMEM culture medium, F12 culture medium or the mixture of the two；

Under room temperature gnotobasis, in basal medium, add serum substitute and relevant growth factors；

0.22um membrane filtration is degerming, obtains chondrocyte serum-free medium.