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Serum-free media for chondrocytes and methods of use thereof

a chondrocyte and serum-free technology, applied in the field of cell and tissue culture, can solve the problems of phenotypical instability, difficult standardization, and serious physical debilitation, and achieve the effect of safe, effective and inexpensive culture medium

Inactive Publication Date: 2007-11-29
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides a safe, effective, and inexpensive method for culturing articular chondrocytes using chemically defined culture media and without the need for serum. The method allows for the attachment and proliferation of chondrocytes under serum-free conditions, and can also be used to prime chondrocytes prior to implantation. The defined cell culture media used in the invention promote cell attachment, proliferation, and redifferentiation capacity, and can be used as a redifferentiation-sustaining medium for chondrocytes embedded in a matrix intended for implantation. The invention also provides a basal medium that can be used in combination with other supplements such as PDGF and lipid components to further enhance the growth and redifferentiation of chondrocytes.

Problems solved by technology

Damage of cartilage produced by trauma or disease, e.g., rheumatoid and osteoarthritis, can lead to serious physical debilitation.
However, during tissue culture, these cells become phenotypically unstable, adopt a fibroblastic morphology, and then cease to produce type II collagen and proteoglycans characteristic of hyaline-like articular cartilage.
However, even though serum is widely used for mammalian cell culture, there are several problems associated with its use (Freshney (1994) Serum-free media.
In Culture of Animal Cells, John Wiley & Sons, New York, 91-99): 1) serum contains many unidentified or non-quantified components and therefore is not “defined;” 2) the composition of serum varies from lot to lot, making standardization difficult for experimentation or other uses of cell culture; 3) many of the serum components affect cell attachment, proliferation, and differentiation making it difficult to control these parameters; 4) some components of serum are inhibitory to the proliferation of specific cell types and to some degree may counteract its proliferative effect, resulting in sub-optimal growth; and 5) serum may contain viruses and other pathogens which may affect the outcome of experiments or provide a potential health hazard if the cultured cells are intended for implantation in humans.
However, attachment and proliferation of cells in the known media are often not optimal.
Additionally, the amounts of starting cell material available for autologous chondrocyte implantation are generally limited.
Attempts to culture articular chondrocytes at subconfluent densities in DM have been only partially successful.

Method used

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  • Serum-free media for chondrocytes and methods of use thereof
  • Serum-free media for chondrocytes and methods of use thereof
  • Serum-free media for chondrocytes and methods of use thereof

Examples

Experimental program
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Effect test

example 1

[0052] Human articular cartilage biopsy samples from donors of 16-51 years of age were trimmed of extraneous material, minced and subjected to enzymatic digestion using 0.25% protease from Bascilus Thermopropolipycus for 1-2 hrs followed by an overnight digestion in 0.1% collagenase / DMEM at 37° C. Isolated articular chondrocytes were washed twice in DMEM containing 10% human serum albumin (DMEM / 10% HSA). The isolated primary human articular chondrocytes (HAC) were seeded at 5,000-6,000 cells / cm2 in T75 flasks using the following separate media conditions: [0053] 1) DMEM / 10% FBS (DMEM supplemented with 10% fetal bovine serum and 100 μg / ml gentamycin); [0054] 2) cDRF (as defined in Table 3); [0055] 3) cDRF / P (cDRF supplemented with 10 ng / ml PDGF); [0056] 4) cDRF / L (cDRF supplemented with 5 μl / ml CDLM (as defined in Table 4)); and [0057] 5) cDRF / P / L (cDRF supplemented with 10 ng / ml PDGF and 5 μl / ml CDLM)

[0058] Two flasks were used per each condition. At the end of each passage, nearly...

example 2

[0059] Hyaline cartilage biopsy samples collected from multiple donors were used to compare cell yields as a function of the passage number for chondrocytes cultured in DMEM / 10% FBS or in a completely defined serum-free medium according to this invention. Samples were collected and treated as described in Example 1. Isolated chondrocytes were washed twice in DMEM containing 10% human serum albumin (DMEM / 10% HSA). The isolated primary human articular chodrocytes (HAC) were seeded at 6,000 cells / cm2 in T75 flasks using the following media conditions: [0060] 1) DMEM / 10% FBS (DMEM supplemented with 10% fetal bovine serum and 100 μg / ml gentamycin); and [0061] 2) cDRF / P / L (cDRF supplemented with 10 ng / ml PDGF and 5 μl / ml CDLM)

[0062] At the end of each passage nearly confluent cells were harvested by trypsinization, counted, washed in DMEM / 10% HSA and reseeded at 6,000 cells / cm2 in respective media. A comparison of cell yields at the end of each passage for chondrocytes propagated in DMEM...

example 3

[0063] In this experiment, human articular chondrocytes from three donors, ages 16, 22, and 55, were isolated and treated as described in Example 1. Chondrocytes were seeded at 6,000 cells / cm2 in T75 flasks and grown in DMEM / 10% FBS until near confluence. The cells were then harvested by trypsinization, washed in seeding media, and immediately frozen in 10% DMSO / 40% HSA / 50% DMEM. For the second passage, ampules of frozen cells were thawed out, rinsed in DMEM / 10% HSA and reseeded at 3,000-4,000 cells / cm2 in the following media: 1) DMEM / 10% FBS; 2) cDRF; 3) cDRF / P; and 4) cDRF / P / L (see Example 1 for the description of the media). Two flasks were used per each set of media conditions. At the end of each passage nearly confluent cells were harvested by trypsinization, washed in DMEM / 10% HSA and reseeded in the corresponding media. At the end of the third passage, cells were harvested and counted. Growth index expressed as a number of doublings per day at the end of a seven-day period wa...

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Abstract

The present invention provides defined serum-free cell culture media useful in culturing fibroblasts, especially articular chondrocytes, that avoids problems inherent in the use of serum-containing media. The defined media comprise platelet-derived growth factor (PDGF), and chemically defined lipids, or combinations of these compounds. In another aspect, the present invention also provides tissue culture methods that comprise incubating chondrocytes in the defined serum free media. The methods enhance attachment and proliferative expansion of chondrocytes seeded at low density while maintaining their redifferentiation potential.

Description

[0001] This is a continuation of U.S. application Ser. No. 10 / 350,816, filed Jan. 24, 2003, which is incorporated herein by reference. U.S. application Ser. No. 10 / 350,816 claims the benefit of U.S. Provisional Application No. 60 / 389,078, filed Jun. 14, 2002, and of U.S. Provisional Application No. 60 / 351,949, filed Jan. 25, 2002.FIELD OF THE INVENTION [0002] The present invention relates to the field of cell and tissue culture. More specifically, the invention relates to methods and compositions for ex vivo propagation of cells capable of forming cartilaginous tissue intended for treatment or repair of cartilage defects. BACKGROUND OF THE INVENTION [0003] Articular cartilage is composed of chondrocytes encased within the complex extracellular matrix produced by these cells. The unique biochemical composition of this matrix provides for the smooth, nearly frictionless motion of articulating surfaces of the knee joint. With age, tensile properties of human articular cartilage change ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/06C12N5/02C12N5/077
CPCC12N5/0655C12N2500/25C12N2500/36C12N2500/38C12N2501/58C12N2501/115C12N2501/135C12N2501/39C12N2500/99C12N2500/90
Inventor BROWN, LIESBETH MARIA
Owner GENZYME CORP
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