Serum-free cartilage cell culture solution

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

Active Publication Date: 2015-05-20
SHAANXI RUISHENG BIOTECH
View PDF8 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Serum-free cartilage cell culture solution
  • Serum-free cartilage cell culture solution
  • Serum-free cartilage cell culture solution

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 number were calculated. After washing with PBS, culture medium A was used respectively (culture medium A was a mixed culture medium of DMEM and F12 according to the 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 Pieces / cm 2 Inoculated into culture flasks, placed in an incubator at 37°C, 5% CO 2 Culture under conditions; after 48~72h, half of the medium was changed, and the culture was continued, and then the medium was changed every 48h. 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 medium used in this example is: the basal medium is a mixed medium of DMEM and...

Embodiment 2

[0029] The obtained human cartilage tissue was digested overnight by enzymatic digestion, and the primary chondrocytes were collected by centrifugation. After that, follow 0.5x10 4 / cm 2 Cell density was seeded into cell culture flasks at 37°C, 5% CO 2 The cells were cultured in a conditioned incubator; when the cells reached 60%-80% confluence, they were passaged by trypsin digestion. 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 it was passed to the 3rd and 6th generations.

[0030] The serum-free medium used in this example is: the basal medium is a mixed medium of DMEM and F12 in a volume ratio of 1:1; 6g / L of dextran is added for stirring and dissolved, and then 0.5mL / L of lipid concentration is added respectively. agent, 75 mg / L sodium pyruvate, 0.1 μM T3, 0.05 mg / L hydrocortisone, 0.5 μg / L dexamethasone, 1 μg / L sodium selenite, 25 μM β-mercapto...

Embodiment 3

[0033] The obtained human cartilage tissue was digested by enzymatic digestion overnight, and primary chondrocytes were collected by centrifugation. 4 / cm 2 Cell density was seeded into cell culture flasks at 37°C, 5% CO 2 The cells were cultured in a conditioned incubator; when the cells reached 60%-80% confluence, they were passaged by trypsin digestion, and the serum-free medium of this example was used for culture.

[0034] The serum-free medium used in this example is: the basal medium is DMEM as the basal medium; 15g / L of dextran is added with stirring to dissolve, and then 0.1mL / L of lipid concentrate and 55mg / L of pyruvate are added respectively. Sodium, 0.5μM T3, 0.1mg / L Hydrocortisone, 5μg / L Dexamethasone, 2μg / L Sodium Selenite, 25μM β-mercaptoethanol, 100mg / L V C , 50 μg / mL glutamine, 25 μg / L bFGF, 10 μg / L EGF, 10 μg / L TGF-β, 5 μg / L PDGF, 5 μg / L BMP, 1 mg / L transferrin, 100 μg / L IGF, 125 μg / L GH, 20 nM progesterone, 75 μM butanediamine, 1% non-essential amino ac...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

A serum-free cartilage cell culture solution is obtained by adding exogenous additives to a basic culture solution. The basic culture solution is a DMEM culture solution or an F12 culture solution or a DMEM culture solution-F12 culture solution mixed culture solution, and the exogenous additives are nonessential amino acids, glucan, vitamin C, glutamine, sodium pyruvate, insulin or insulin-like growth factor, transferrin, growth hormone, triiodothyronine, hydrocortisone, dexamethasone, sodium selenite, beta-mercaptoethanol, lipid concentrate, progesterone, succinimide, a basic fibroblast growth factor, an epidermal growth factor, a transforming growth factor, a bone morphogenetic protein and a platelet derived growth factor respectively. The serum-free cartilage cell solution can avoid potential risks due to the use of serum, improves the extracellular matrix secreting ability of cartilage cells, makes the growth propagation state approach the growth propagation state of the cells in a serum-containing culture solution, and allows the dedifferentiation rate and the matrix secreting ability of low density inoculation cartilage cells to be obviously better than those of the cells in the serum-containing culture solution respectively.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N5/077
Inventor 田智泉
Owner SHAANXI RUISHENG BIOTECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap