KR20020004744A - Method for separating chondrocytes, media for culturing the separated chondrocytes, and chondrocytes cultured in the media - Google Patents

Abstract

PURPOSE: An isolating method of a cartilage cell, a medium for cultivating the cartilage cell and a cultivated cartilage cell are provided, thereby selectively producing the healthy cartilage cell and improving its cultivation rate in the medium. CONSTITUTION: The isolating method of a cartilage cell comprises the steps of: preparing a cartilage tissue; isolating a cartilage cell from the tissue; preparing stack gradient; loading the cartilage cell suspension to the stack gradient and centrifuging it; and recovering the cartilage cell from each fraction of the stack gradient, in which the stack gradient is prepared by: producing an isotonic percoll solution by mixing percoll and dilution; concentrating the isotonic percoll and producing 10%, 20%, 30%, 40%, 50% and 60% of isotonic percoll solution by using the dilution; and inserting each concentration percoll solution into each tube. The medium for cultivating the cartilage cell contains insulin, transferrin, selenium, T3 hormone, TGF-beta, IGF, FGF, PDGF, KGF, EGF, HGF or the combination thereof.

Description

Chondrocyte separation method, medium for culturing chondrocytes isolated according to the above method and chondrocytes cultured in the medium {METHOD FOR SEPARATING CHONDROCYTES

The present invention relates to a method for separating chondrocytes from cartilage, a medium for culturing chondrocytes separated by the method, and a chondrocyte cultured in the medium, and more specifically, to the cell transplantation of the missing cartilage and cartilage The present invention relates to a method for isolating only chondrocytes with active metabolic activity so that the cartilage substrate is secreted so as to be suitable for in vitro production, a medium through which the isolated chondrocytes can grow better, and a chondrocyte cultured in the medium.

Cartilage inherently has various types, such as joint or hyaline cartilage, elastic cartilage and fibrocartilage, and especially articular cartilage is found on the articular surface of the bone, for example the joint, and causes the characteristic smooth slide movement of the movable joint. Articular cartilage is firmly attached to the bone supporting the joint, and depending on the type of joint and the position within the joint, the thickness is 5-7 mm or less in humans, and it is a nerve, avascular, and non-negative. In adults, cartilage provides nutrients to chondrocytes in cartilage tissue through a synovial membrane, through a diffuse system through the dense matrix of cartilage.

However, as mechanical and structural changes in cartilage composition occur with aging, cartilage and bones in adults have limited recovery capacity, and damage to cartilage caused by rheumatoid disease and / or osteoarthritis or trauma is severe pain. And can cause physical deformities and movement disorders.

As a treatment for treating such cartilage damage or defect, various methods have been studied. The method of culturing cartilage cells on a biodegradable matrix in vitro to make a cartilaginous structure and transplanting it to a damaged cartilage site or damaged cartilage. And a method of directly injecting a cellular preparation including chondrocytes in a specific way.

Therefore, the more cartilage substrates used to effectively treat the above-described cartilage damage or deficiency, the more active the secretion of the cartilage substrate, the better the results can be obtained. However, in the past, rather than selecting only the most preferable chondrocytes, the chondrocytes isolated from the collected chondrocytes were centrifuged and cultured as they are, because of the morphological and biochemical characteristics of the chondrocytes described below. .

First, take the articular cartilage as an example, look at the morphological and biochemical properties of cartilage structure. Chondrocytes are the only cellular component of articular cartilage, and are composed of heterogeneous cells, depending on morphological differences such as cell density, array direction, properties, composition, protein distribution, and collagen bundles. In addition, the structure and components vary depending on the depth from the joint surface, and it is known to be composed of four layers such as the top layer, the middle layer, the deep layer, and the lime layer, and also the joint surface in terms of the biochemical properties, components, and composition of the substrate molecules. And vary from depth to depth, which is due to differences in the metabolic activity of the cells.

In addition, the characteristics of the cells in each layer of articular cartilage can be seen that the celestial cartilage cells are flat, long round cells arranged in parallel to the joint plane, and have a higher amount of collagen and a smaller amount than other layers. It has been reported to contain protein sugars. However, when the cells of the celestial layer are isolated and cultured, the cells of the celestial layer are known to synthesize small amounts of collagen and protein, while they break down protein sugar faster than cells in the deep layer.

In contrast, deep cartilage cells are rounded ellipsoids that form columns and contain large molecules of collagen and high protein sugar, which are known to migrate towards the tidemark of the limestone layer. .

The results of the studies on the chondrocytes published to date as described above are all histological observations or results obtained by separating four layers of articular cartilage with the naked eye and then cutting the cells from each layer.

Of course, histological classification of articular cartilage provides basic knowledge about the heterogeneous cell populations of chondrocytes in tissues, but it is difficult to distinguish anatomically between layers of tissues, and because cartilage cells with different characteristics coexist in the same layer. In order to purely separate chondrocytes in the chondrocytes composed of heterotypic cell groups, and to separate only chondrocytes with the most active cartilage substrate secretion, a more objective and systematic test method by the difference of the cells themselves is required in addition to the tissue dissection method.

In order to solve the above problems, the present invention through the more objective and systematic separation method by the difference between the chondrocytes themselves, chondrocytes having the same morphological characteristics and biochemical characteristics, particularly target activity is active cartilage substrate It is an object of the present invention to provide a method for isolating chondrocytes that are active in secretion, a medium for culturing the isolated chondrocytes and chondrocytes cultured in the medium.

Figure 1a is an electron micrograph of A layer chondrocytes immediately after separation according to the chondrocyte separation method according to the present invention.

Figure 1b is an electron micrograph of D-layer chondrocytes immediately after separation according to the chondrocyte separation method according to the present invention.

Figure 2a is an optical micrograph after the monolayer culture of the separated A-layer chondrocytes for 4 days.

Figure 2b is an optical micrograph after the monolayer culture of the separated B-layer chondrocytes for 4 days.

Figure 2c is an optical micrograph after the monolayer culture of the isolated C-layer chondrocytes for 4 days.

Figure 2d is an optical micrograph after the monolayer culture of the isolated D-layer chondrocytes for 4 days.

Figure 3a is an electron micrograph of layer A chondrocytes after monolayer culture for 4 days

Figure 3b is an electron micrograph of layer B chondrocytes after monolayer culture for 4 days

4 is a graph showing the extent of proliferation of chondrocytes recovered from each layer.

Figure 5 is a photograph comparing the expression level of type 2 collagen of isolated chondrocytes by Western blot analysis.

Figure 6 is a graph showing the protein synthesis of chondrocytes of each layer separated by DMB analysis.

The present invention comprises the steps of preparing cartilage tissue; Separating the prepared cartilage tissue into respective cells; Preparing a stack gradient to separate the separated chondrocytes into homozygous cell groups; Centrifuging by loading the separated chondrocyte suspension on the prepared stack gradient; And it provides a chondrocyte separation method comprising the step of recovering the centrifuged chondrocytes for each fragment (fraction) of the stack gradient.

The present invention further comprises the step of culturing the chondrocytes recovered for each fragment in the step of recovering the centrifuged chondrocytes for each fragment in the chondrocyte separation method and determining the fragments with the most active cartilage substrate secretion. It provides a method for separating cartilage cells.

The present invention further provides a medium for culturing chondrocytes separated by the chondrocyte separation method and determined as the fragment having the most active cartilage substrate secretion, and the chondrocytes cultured in the medium.

In order to better describe the inherent features of the present invention and how to perform it, examples are described below. However, the present invention should not be considered to be limited by the detailed description of the embodiments.

Example 1

In the present invention, homogeneous chondrocytes were isolated from rabbit articular cartilage using the method of Percoll density gradients centrifugation, and the morphological characteristics of the isolated chondrocytes were observed under an electron microscope, followed by culturing each cell. Their growth and phenotypic characteristics were observed.

1) Isolation of Chondrocytes

Cartilage tissue was aseptically collected from both knee joints (about 250 g). Cartilage tissue isolated from the knee joint was chopped and washed several times with PBS (phosphate buffered saline) containing 200 units / ml penicillin and 200 ㎍ / ml streptomycin. The chopped tissue was washed twice with PBS again and treated with 0.1% collagenase for 4 hours at 37 ° C. for 4 hours to separate single chondrocytes, followed by centrifugation, and then 0.02% trypsin to prevent aggregation of cells. 10 minutes at 37 ℃, and washed with PBS.

2) Preparation of Percoll Density Gradient Stack

First, an isotonic Percoll solution was prepared by mixing Percoll stock solution with 10 × PBS solution. Thereafter, 60% concentrated solution was prepared and diluted with PBS to prepare Percoll solution of 10%, 20%, 30%, 40%, 50%, 60%. A percol density gradient stack was prepared by loading 2 ml of each solution into a 15 ml polypropylene tube.

3) Percoll density gradient centrifugation

After the cell suspension prepared in 1) was loaded on the top layer of the percol density gradient stack prepared in 2), centrifugation was performed at 400 g and 20 ° C. for 20 minutes.

4) Identification of Isolated Chondrocytes

After centrifugation, cartilage cells of each fraction were recovered from the top using a Paster pipette. As a result, cells separated between the cell suspension and between 10% (A fraction), between 10% and 20% (B fraction), between 20% and 30% (C fraction) and between 40% and 50% (D fraction) Could get Cartilage cells in each layer were washed and diluted with 10% fetal bovine serum, DMEM (dulbecco minimum essential medium) containing penicillin and streptomycin.

The recovery rate of the separated cells was 60% or more cells recovered in the B layer. The survival rate of the cells in each of the recovered layers was measured by trypan blue exclusion method, and showed 60% or more survival rate for each layer.

5) Culture of Isolated Chondrocytes

Cell culture was added to the isolated chondrocytes at a cell density of cell number / ml and then cultured at 37 ° C. and 95% O ₂ /5% CO ₂ . In particular, 10% fetal calf serum or 10% autologous serum is added to the medium, and additionally, insulin, transferrin, selenium or a mixture thereof, or transforming growth factor-β (TGF-β), insulin-like Insuline-like growth factor (IGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), epidermal growth factor (Keratocyte growth factor), epidermal growth factor (Epidermal growth factor; EGF), hepatocyte growth factor (HGF) or a mixture thereof may be cultured in a medium further comprising.

The badge was changed twice a week.

Experimental Example

In order to determine the morphological characteristics, biochemical characteristics, proliferation rate and phenotype of chondrocytes isolated and cultured chondrocytes separated by Example 1 described above, the following experiment was performed, and the results were described.

1) electron microscopy and its results

Isolated chondrocytes were fixed with a mixture of glutaraldehyde and paraformaldehyde immediately after separation and after incubation for a period of time. After fixation, it was dehydrated with ethanol and stained with uranyl acetate. The stained chondrocytes were soaked in Polarbed 812 (Polaron, Watford, U.K.), ultra-stained incision and stained and observed by TEM (Zeiss EM 902A).

As observed in the electron microscope, as shown in FIG. 1A, the cells of the layer A were generally composed of large cells. These cells had a large distribution of lipids in the cytoplasm and most of them had a small nucleus. On the other hand, as shown in Figure 1b cells of the D layer was composed of cells generally smaller than the cells of the A layer. The cells had a very large nucleus and well-developed Golgi apparatus.

In addition, after culturing the cells separated by each layer for 4 days in a monolayer culture and observed with an optical microscope, as shown in Figures 2a, 2b, 2c and 2d, cells of layer A are generally C, D layer Cells in the D layer were observed to grow in a small, non-flat form, whereas the cells in the D-layer grew larger and broader than the cells of the.

In addition, as a result of observing the morphology of the cells of each layer by electron microscopy by fixing the cells cultured for 4 days, as shown in Figures 3a and 3b, the cells of the layer A still had a small nucleus and cells of the layer C They had a large nucleus. However, the cells in each layer showed no significant difference as seen in the observations immediately after separation.

2) Cell Proliferation Measurement Experiment

To measure cell proliferation, chondrocytes were cultured in a 24well culture vessel at a concentration of 1 × 10 ⁴ cells / 0.5ml. Chondrocytes cultured in monolayers for a certain period of time were separated with 0.05% trypsin / EDTA, centrifuged at 1700 rpm for 15 minutes, and the cells were collected again to count the cells directly with a hemocytometer (FIG. 4).

In direct counting, cells in layer A and cells in layer C showed a similar degree of proliferation, and cells recovered in layer B showed the fastest growth. On the other hand, cells recovered in the D layer hardly proliferated.

3) Protein expression experiment by Western blotting analysis

Chondrocytes in monolayer cultures were scraped from the culture vessel for each period, followed by EBC buffer (40 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40, 2 g / ml aprotinine, 2 g / ml pepstatin, 2 g). protein was isolated by treatment with / ml leupeptin, 100 g / ml PMSF). After electrophoresis at 100 volts for 3 hours using 8% polyacrylamide gel, 10 volts using transfer buffer (12.5 mM Tris, 0.1 M glycine, pH 8.3) ) Was transferred to nitrocellulose membrane overnight. Block with membrane blocking solution (5% Carnation nonfat dry milk, in TNE), and then add antibody solution (human collagen type II) to the blocking solution to a concentration of 1 μg / ml overnight. The reaction was carried out at 4 ℃.

As a result of observing the expression level of type II collagen protein using Western blotting, as shown in FIG. 5, the expression of type 2 collagen, which is one of the phenotypes of chondrocytes of articular cartilage Was strong in cells of layer A and B, but not in cells of layer C and D. In addition, type 1 collagen was not expressed in all layers of cells.

4) Measurement of proteoglycan secretion of chondrocytes

Normally, very low concentrations of protein sugar are present in the matrix of chondrocytes. DMB assay was used to determine the difference between the synthesis of these proteins in the cells isolated by Percoll density gradient centrifugation.

In more detail, the dimethyl methylene blue (DMB) assay for measuring the production of protein, the extracellular matrix of chondrocytes, was attached to a 24-well culture vessel at a constant concentration of cells. After culturing for a period of time, the culture solution was taken and the concentrations of the protein sugar produced and discharged into the medium were measured and compared. 20 µl of the culture medium in which the cells were cultured was taken, mixed with 150 µl of DMB solution, and measured using an ESISA at a wavelength of 525 nm within 10 minutes. The standard curve is obtained by mixing 20 μl of DMEM containing 0, 0.11, 0.22, 0.44, 0.88, 1.75, 3.5, 7, 14 μg of chondroitin sulfate with 150 μl of DMB solution. It was based on what was measured.

As shown in Figure 6, chondrocytes recovered in the A and B layers were synthesized more protein than the chondrocytes in layer C (Fig. 6: Cells of fraction D have difficulty in measuring DMB due to difficulty in recovery Not displayed).

Example 2

The cartilage tissues of young rabbits were isolated, washed several times with PBS, and then chopped. The cartilage cells were treated with 0.1% hyaluronidase, 0.2% trypsin, and 0.2% collagenase. The chondrocytes were cultured in DMEM (Dulbecco's Modified Eagle Medium: 4500mg / L D-glucose, L-glutamine, pyridoxine hydrochloride, 110mg / L sodium pyruvate, without sodium bicarbonate) containing 10% FBS as a control. In the medium (Medium A), which further added 10 ng / ml of IGF-I to the medium (Medium A), the medium further added 5 ng / ml of TGF-β (Medium B_), and the medium further added basic FGF (Medium C). The chondrocytes were cultured.

Alginate was used to three-dimensionally culture monolayered cells for 4 weeks under each condition.

1. Experiment Method

The proliferation rate of chondrocytes was combined with 1) direct cell count and 2) ³ H-Thymidine Incorporation. After the medium exchange, ³ H-Thymidine Incorporation method was treated with [ ³ H] -Thymidine (0.5 μCi / well) every 72 hours for 0, 6, 12, 24, 36, and 48 hours. And liquid scintillation analyzer to measure radioactivity.

The chondrocyte phenotype was mainly measured by the expression of type II collagen by 1) immunofluorescence staining and 2) Western blot analysis. 3) The amount of synthesized proteoglycan was measured using DMB (dimethyl methylene blue). Absorbance was measured at a wavelength of 525 nm.

2. Results

end. Chondrocyte Morphology

The control group is a typical chondrocyte-shaped multipolar type, the chondrocytes of medium A are 1.5-2 times larger, and the round shape, the chondrocytes of medium B are similar to the control group, but the cells tend to gather together. Chondrocytes were smaller and fusiform than chondrocytes grown in other media.

2. Growth patterns of monolayer cultured chondrocytes

The growth rate was obtained by dividing the number of cells after the incubation by the initial seeding number, and the growth patterns of chondrocytes in the control medium and the medium A and the medium B were similar, whereas the cartilage cells of the medium C showed a high growth rate of about 12 times.

DNA synthesis rate was measured by [ ³ H] -Thymidine uptake method, and the control group and chondrocytes of medium A and medium B showed similar tendency showing maximum value at 12 hours after medium exchange. The maximum value at 12 hours was maintained up to 24 hours, and the value was twice as high.

3. Results of Chondrocyte Phenotyping

As a result of Western blotting analysis, in the chondrocytes of the control group and the medium A, collagen type 2 was expressed well from the beginning of monolayer culture to about 2 weeks, and gradually decreased from 3 weeks of culture. Chondrocytes of medium B and medium C showed very low expression, and only a very small amount was expressed from 1 week of culture. When cultured monolayered cells with alginate bead for 4 weeks, all groups were re-expressed. Immunohistochemical staining resulted in the longest expression of type 2 collagen in the chondrocytes of medium A. The amount of proteoglycan synthesized and secreted by these mediums was the highest in medium B, and a relatively high amount was synthesized in medium A.

The present inventors obtained four cartilage cell groups in rabbit knee joints using Percoll density gradient centrifugation, and observed morphological characteristics, phenotypic characteristics and glycoprotein synthesis in each cell group. As a result of preparing the percol density gradient stack according to the method of the present inventors and centrifuging, sometimes four or more chondrocyte layers were separated, but almost all of them could be separated into four layers. In addition, the separated cells were able to recover more than 70% and the cells showed a survival rate of more than 60%. The survival rate of the cells was low because the cells were separated one by one during the separation process and centrifuged at room temperature for a long time.

Electron microscopy (TEM) was used to examine the morphological differences of the cells in each layer. Freshly isolated cells from the tissues were clearly morphologically different. Didn't look

As a result of observing the monolayer culture for 4 days with an optical microscope, as shown in Figure 4, the cells of the A layer was generally grown in a larger and wider flat form than the cells of the C layer, D layer. In addition, cells in layers A, B, and C showed rapid proliferation. However, even after D-layer culture, D-layer cells grew in small, unflattened form and showed little growth.

In the cells of A and B layers, the expression of collagen type II, one of the chondrocyte phenotypes, was strongly expressed, and a large amount of protein was also synthesized. No expression of type 2 collagen was observed in the cells of layer C and D. The cells of layer D were difficult to recover and very difficult to measure the degree of synthesis of protein due to the low proliferation rate. This was synthesized.

In addition, the present inventors experimented with the effects of growth factors on the proliferation of chondrocytes and the change of the chondrocyte phenotype by adding FGF, IGF-I and TGF-β, respectively, among the growth factors in the culture medium of articular chondrocytes of rabbits. As a result, the most effective growth factor for the proliferation of chondrocytes was basic FGF, which can be a secondary chondro-progenitor cell in three-dimensional culture, and IGF increases collagen expression and TGF-β. Has been found to increase the synthesis of proteoglycans.

Recently, cell transplantation has been introduced as a method for treating cartilage defects, and further, in vitro production of cartilage using tissue engineering techniques has been attempted. Either way, healthy cartilage cells with active metabolic activity and cartilage matrix secretion are essential. Selection of chondrocytes by the chondrocyte separation method according to the present invention can be used as a useful method for selectively obtaining healthy chondrocytes, and the chondrocyte culture rate when the chondrocytes are separated from the medium according to the present invention. Since the remarkably improved, a plurality of chondrocytes cultured in the medium can be more easily provided for cell transplantation or in vitro production of cartilage.

Claims (7)

Preparing cartilage tissue; Separating the prepared cartilage tissue into respective cells; Preparing a stack gradient to separate the separated chondrocytes into isoforms; Centrifuging by loading the separated chondrocyte suspension on the prepared stack gradient; And Cartilage cell separation method comprising the step of recovering the centrifuged chondrocytes for each fragment (fraction) of the stack gradient. The method of claim 1, further comprising the step of culturing the collected chondrocytes for each fragment in the step of recovering the centrifuged chondrocytes for each fragment, and then determining the fragment having the most active cartilage matrix secretion. . The method of claim 1 or 2, wherein preparing the stack gradient Mixing percoll stock solution and dilution solution to prepare Isotonic Percoll solution; Preparing a percol solution of 10%, 20%, 30%, 40%, 50%, 60% by diluting the prepared percol solution with 60% concentrated solution and diluting with the dilution solution, respectively; Chondrocyte separation method comprising the step of making a stack by putting a predetermined amount of percol solution of each concentration in a tube (tube). The method according to claim 1 or 2, wherein each fragment of the step of recovering the centrifuged chondrocytes is between the suspension and 10% (A fraction), between 10% and 20% (B fraction), 20% and 30% Chondrocyte separation method between (C fraction) and between 40% and 50% (D fraction). As a medium for culturing chondrocytes isolated according to the method of claim 1 or 4, insulin, transferrin, selenium, T3 hormone, TGF-β, IGF, FGF, PDGF, KGF, EGF, HGF or a combination thereof A medium for culturing isolated chondrocytes further comprising. Cultured chondrocytes characterized in that the chondrocytes isolated according to the method of claim 1 or 4 are cultured in the medium according to claim 5. The cultured chondrocytes according to claim 6, wherein the isolated chondrocytes are A-layer or B-layer chondrocytes.