A serum-free chondrocyte culture medium

A technology of chondrocytes and culture medium, which is applied in the field of biomedical materials, can solve the problems of difficult clinical application of chondrocytes, accelerated dedifferentiation rate of chondrocytes, and limited amount of cartilage tissue, so as to maintain extracellular matrix secretion ability, good extracellular Matrix secretion ability, effect of promoting cell function

Active Publication Date: 2017-11-14
SHAANXI RUISHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Chinese patents 200580037602.3, 200710096843.6, 200510030198.9, 200910265976.0, and 200610024459.0 disclose serum-free culture fluids that can be used for cell culture. Part of the plant protein is added during the preparation process. These ingredients have not yet met the standards for cell culture, which increases the difficulty of preparation; at the same time, The invention is not aimed at specific cells, and its culture effect on chondrocytes has not been reported, but the research results show that the use of a certain serum-free culture medium is particularly beneficial for the in vitro isolation, culture, expansion and matrix secretion of chondrocytes, and can maintain the cells Phenotype
[0008] The serum-free culture medium for fibroblast culture invented by Chinese patent 201310003115.1 is a specific culture medium for fibroblasts. The culture medium is not aimed at the characteristics of chondrocyte culture. Adjusting the addition of cytokines will cause chondrocytes to degenerate. The rate of differentiation is accelerated and the chondrocyte phenotype cannot be maintained
[0009] Other serum-free culture media used for cell culture also have the following problems: the addition of serum albumin and various adhesion factors, etc., resulting in higher cost of culture media
In addition, due to the limited amount of cartilage tissue available during clinical and preclinical studies
In clinical practice, the quantity of chondrocyte seed cells is relatively large. In the early stage of chondrocyte culture, low-density monolayer culture is used. The first three generations of cells proliferate rapidly, but dedifferentiate quickly. After passage, the proliferation is slow, and most of the cell phenotypes are lost. Unable to maintain normal chondrocyte phenotype, thus causing great difficulties to the normal function of chondrocytes and their clinical application

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  • A serum-free chondrocyte culture medium
  • A serum-free chondrocyte culture medium
  • A serum-free chondrocyte culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The obtained human cartilage tissue was obtained by enzymatic digestion to obtain primary chondrocytes, and the cell viability and quantity were calculated. After being washed with PBS, culture medium A (culture medium A was a mixed culture medium of DMEM and F12 at a volume ratio of 1:1, Then add 10% FBS) and the serum-free chondrocyte culture medium prepared in this example to resuspend, press 2×10 4 piece / cm 2 Inoculate into a culture bottle and place in an incubator at 37°C, 5% CO 2 Cultivate under the same conditions; change the medium after 48~72h, continue to cultivate, and change the medium every 48h thereafter. The adherence rate of primary human chondrocytes in serum-free group and serum group can reach about 60% within 48 hours, and subculture can be carried out when the confluence rate of chondrocytes reaches about 50%.

[0026] The serum-free culture medium used in this example is: the basic culture medium is a mixed culture medium of DMEM and F12 accordi...

Embodiment 2

[0029] The obtained human cartilage tissue was digested overnight by enzyme digestion, the primary chondrocytes were collected by centrifugation, and suspended in culture medium A (culture medium A was a mixed culture medium of DMEM and F12 at a volume ratio of 1:1, and then added 10% FBS) After that, according to 0.5x10 4 / cm 2 Cell density seeded into cell culture flasks, placed at 37°C, 5% CO 2 Conditioned incubator for culture; when the cells reached 60%~80% confluence, the trypsin digestion method was used for subculture. The first group was cultured with culture medium A, and the second group was cultured with the serum-free culture medium of this example. Observation was carried out when passing to the 3rd and 6th generations.

[0030] The serum-free culture medium used in this example is: the basic culture medium is a mixed culture medium of DMEM and F12 according to the volume ratio of 1:1; add 6g / L dextran and stir to dissolve, and then add 0.5mL / L lipid concentrat...

Embodiment 3

[0033] The obtained human cartilage tissue was digested overnight by enzymatic digestion, and the primary chondrocytes were collected by centrifugation. After being suspended in culture medium B (culture medium B was DMEM basic culture medium, and 10% FBS was added), the 0.5x10 4 / cm 2 Cell density seeded into cell culture flasks, placed at 37°C, 5% CO 2 Culture in a conditioned incubator; when the cells reach 60% to 80% confluence, trypsin digestion is used for passage, and the serum-free culture medium of this example is used for culture.

[0034] The serum-free culture medium used in this example is: the basic culture medium is DMEM as the basic culture medium; add 15g / L dextran and stir to dissolve, then add 0.1mL / L lipid concentrate and 55mg / L pyruvic acid respectively Sodium, 0.5 μM T3, 0.1 mg / L hydrocortisone, 5 μg / L dexamethasone, 2 μg / L sodium selenite, 25 μM β-mercaptoethanol, 100 mg / L V C , 50 μg / mL glutamine, 25 μg / L bFGF, 10 μg / L EGF, 10 μg / L TGF-β, 5 μg / L PDGF,...

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Abstract

A serum-free chondrocyte culture medium, in which exogenous additives are added to the basic culture medium. The basal culture fluid is DMEM culture fluid or F12 culture fluid or the mixed culture fluid of DMEM culture fluid and F12 culture fluid, and the exogenous additives are non-essential amino acids, dextran, vitamin C, glutamine, sodium pyruvate, insulin or Insulin-like growth factor, transferrin, growth hormone, triiodothyronine, hydrocortisone, dexamethasone, sodium selenite, beta-mercaptoethanol, lipid concentrate, progesterone, butylenediamine, base fibroblast growth factor, epidermal growth factor, transforming growth factor, bone morphogenic protein, platelet-derived growth factor. The culture medium does not add albumin, which can avoid the potential hidden dangers caused by the use of serum, improve the ability of chondrocytes to secrete extracellular matrix, and is close to the growth and proliferation state in the serum-containing culture medium. The improvement of matrix secretion ability is obviously better than that of serum-containing culture medium.

Description

technical field [0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a serum-free chondrocyte culture solution. Background technique [0002] Articular cartilage defects caused by trauma or bone disease are very common in clinic. Because cartilage has no blood supply and nerves, the content of chondrocytes is low and they are terminally differentiated cells, so the regeneration ability of cartilage is very limited. After more than ten years of clinical application and technical development, autologous chondrocyte implantation (ACI) technology has become the most effective method for clinical treatment of cartilage injuries, and the curative effect rate of short, medium and long-term follow-up results is 75-95%. However, due to the limited in vitro expansion ability of chondrocytes, how to obtain enough seed cells (ie, chondrocytes) that maintain the cell phenotype has become a bottleneck restricting the clinical applicati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
Inventor 田智泉
Owner SHAANXI RUISHENG BIOTECH
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