Method for preparing a cell-derived extracellular matrix membrane

A cell and decellularization technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of difficult tissue collection, uncontrolled biodegradation of amniotic membrane, low biocompatibility, etc.

Inactive Publication Date: 2009-10-21
REGENPRIME
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  • Abstract
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Problems solved by technology

[0004] Although autologous chondrocyte transplantation (ACI) is a clinically proven cell transplant therapy (Brittberg, M. et al., New England Journal of Medicine 331:889, 1994), there is a problem: since the site of cartilage damage should The periosteum is sutured after the periosteum is collected, and the periosteum may proliferate excessively and cause pain in the affected part after the operation
The main component of the amniotic membrane is type I collagen, and therefore, it has the disadvantage of low biocompatibility for chondrocytes compared to ECM membranes
In addition, amnion may remain in the body long after transplantation due to uncontrolled biodegradation of the amniotic membrane
In addition, it is difficult to collect tissue with the consent of the donor

Method used

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  • Method for preparing a cell-derived extracellular matrix membrane
  • Method for preparing a cell-derived extracellular matrix membrane
  • Method for preparing a cell-derived extracellular matrix membrane

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[0034] One object of the present invention relates to a method for preparing an ECM membrane for tissue engineering through high-density culture and an ECM membrane prepared by the same method. In particular, the present invention relates to a method for preparing a chondrocyte-derived ECM membrane, the method comprising the following steps: (a) isolating chondrocytes from animal-derived cartilage, and then culturing them; (b) obtaining Chondrocyte / ECM membrane; and (c) ECM membrane obtained by removing cells from the chondrocyte / ECM membrane, and drying the resulting membrane, and ECM membrane prepared by the same method.

[0035] In the present invention, the above method may include an additional step (d): re-culturing to obtain a thick ECM membrane with high compressive force by replanting chondrocytes on the obtained ECM membrane.

[0036]In one embodiment of the present invention, by controlling the thickness of chondrocytes, chondrocytes are used to prepare ECM membrane...

Embodiment 1

[0060] Example 1: Isolation of porcine chondrocytes

[0061] 2-week-old pigs that were not infected with cholera, other viruses, or infectious diseases were sacrificed by excessive paralysis in accordance with animal care laws. In a sterile environment, remove the cartilage from the posterior knee joint. The removed cartilage was then cut into small pieces on a clean work surface and treated with 0.1% collagenase for 12 hours. After filtering the cells through a 0.4 μm filter, the chondrocytes were isolated by centrifugation.

Embodiment 2

[0062] Example 2: Preparation of chondrocyte-derived ECM membranes

[0063] The chondrocytes isolated in Example 1 were separated at 0.7 × 10 5 The cells were planted on 6-well culture dishes at a concentration of per square centimeter, and cultured in medium (DMEM+20% FBS+1% penicillin-streptomycin+5 μg / mL ascorbic acid) for 3 weeks. The medium was changed every three days. After 3 weeks, the ECM film was washed three times with PBS and detached from the 6-well dish. The prepared ECM film presents a translucent film type ( figure 1 ).

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Abstract

The present invention relates to a method for preparing a cell-derived extracellular matrix membrane, more particularly, to a method for preparing a chondrocyte-derived ECM membrane, the method comprising the steps of forming a suitable thickness of ECM membrane by culturing chondrocytes derived from animal cartilage at a high concentration in vitro, and drying it after decellularization process. The cell-derived ECM membrane scaffold according to the present invention is composed of extracellular matrix secreted by chondrocytes so that the membrane has excellent biocompatibility as well as an immune-previlage effect specific to cartilage. Since the membrane also has a suitable compressive strength, it can be used to replace periosteum for cartilage regeneration or artificial collagen membrane and used as dura mater transplant material, a natural ECM membrane for treating skin loss, materials for cell transplantation and a growth factor delivery vehicle.

Description

technical field [0001] The invention relates to a method for preparing a cell-derived extracellular matrix (ECM) membrane, especially a method for preparing a chondrocyte-derived ECM membrane, which comprises the following steps: cultivating chondrocytes derived from animal cartilage to form cartilage Cells / ECM membrane, removal of chondrocytes from the membrane, fabrication of an ECM membrane of appropriate thickness, and drying for sterilization. Background technique [0002] Articular chondrocytes are special mesoderm-derived cells and are found only in cartilage. Cartilage is a nonvascular tissue whose physical properties vary with the extracellular matrix produced by chondrocytes. Once the cartilage is damaged, its self-rebuilding ability will be very limited, and it will eventually lead to osteoarthritis, which greatly affects the quality of life of the patient. [0003] For damaged cartilage, typical treatments include bone marrow stimulation to induce bone marrow-d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08A61L27/60C12N5/077
CPCC12N2533/90A61L27/3633A61L27/3817A61L27/3683A61L27/60C12N5/0655A61P19/04A61P19/08A61P43/00C12N5/0652
Inventor 朴索拉闵炳显崔秉显
Owner REGENPRIME
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