KR101037002B1 - The Specific Culture Media for Chondrocyte Cells and Methods of Uses Thereof - Google Patents

Abstract

According to the present invention, cartilage cells can be clinically implanted into femoral articular cartilage (lateral, medial, trochlear) defects and osteochondral defects of the talmur with clinical symptoms in the knee (knee) or ankle joint. It relates to a chondrocyte specific culture method for the early culture of cells.

In particular, in vitro culturing chondrocytes isolated from cartilage tissue harvested for transplantation in vitro, the proliferation rate of chondrocytes is increased, compared to culturing chondrocytes using conventionally available media. In addition to shortening the time, the number of transplantable cells can be obtained quickly.

Chondrocytes, chondrocyte specific culture medium.

Description

The specific culture media for chondrocyte cells and methods of uses thereof

The present invention is specific for culturing chondrocytes showing a higher proliferation rate than conventional culture medium in the process of separating chondrocytes from cartilage tissue collected from the damaged joint cartilage region of human and culturing in vitro as much as the number of clinically transplantable cells. It relates to a chondrocyte specific culture method for the early culture of chondrocytes that can secure a large number of cells in a faster time by culturing using a medium.

Human articular cartilage tissue has a feature that once damaged, it does not normally regenerate in vivo. As such, joint cartilage damage is accompanied by severe pain that limits daily life, and when chronicized, it causes fatal degenerative arthritis.

In recent years, with the rapid development of tissue engineering, a technique for separating and culturing chondrocytes by taking part of the normal cartilage tissue of the patient himself to treat articular cartilage damage was first studied at the Hospital for Joint Disease in New York. The technology has been developed at the University of Gothenburg and Sahlgrenska University Hospital in Sweden, and many industries around the world produce chondrocyte treatments as pharmaceuticals in GMP facilities.

The treatment of damaged articular cartilage is performed by extracting some of the cartilage of the joint which is not used from the patients with damaged knee cartilage, and amplifying the chondrocytes as needed for transplantation in the culture medium in which the proper nutrients are supplied to the chondrocytes in the cell culture period. Injecting or implanting a cartilage defect in a patient regenerates damaged articular cartilage of the patient.

However, chondrocytes are adherent cells, and their proliferation rate is very slow and the intrinsic characteristics of chondrocytes change according to the environmental conditions around the cells during culturing in monolayer culture. A special culture technique and culture medium are necessary to separate cells from the tissues and to proliferate to 12 to 60 million or more chondrocytes, which are sufficient cells for transplantation.

Mammalian cell culture medium consists of about 50 kinds of components, which are largely used for the biosynthesis of cells, the parts used for biological energy metabolism, as a catalyst for various metabolism or physiological phenomena in cells. It can be divided into parts used to control. In other words, the medium used for cell culture is composed of isotonic solution components, buffer components, nutrients including amino acids, vitamins, and inorganic salts as energy sources, and various other supplements. In addition, by supplying about 5 to 20% of serum according to the characteristics of the cell, it supplies various hormones, growth factors, fats, and vitamins suitable for cell proliferation, inhibits protease activity, and acts as a buffer for pH control. Promotes the growth and activity of mammalian cells. The components constituting the medium for culturing mammalian cells vary depending on the type of medium in terms of concentration as well as its composition. The medium types are simpler and commonly used in cell culture, such as Minimal essential medium (MEM), Rosewel Park Memorial Institute (RPMI) -1640, and Dulbecco's modification of Eagle's medium (DMEM), and Iscove's modification of DMEM (IMDM). ), And Ham's F-12 and Connaught Medical Research Laboratories (CMRL) -1666.

The growth curve of mammalian cells usually has a Lag phase of 2 to 3 days and the concentration of viable cells decreases rapidly from the end of the delay phase due to the accumulation of ammonium glutamine metabolite and lactic acid glucose. . Glucose and glutamine are the main carbon and energy sources for mammalian cells. When glucose is metabolized by mammalian cells, glycolysis becomes pyruvate. In addition, a pentose sugar is produced by the pentose phosphate pathway to synthesize nucleic acids. Pyruvic acid produced during glycolysis is decomposed into carbon dioxide and water by the TCA cycle, into lactic acid, and also fatty acid. One of the carbon sources used for mammalian cell metabolism is glutamine, a part of which becomes glutamate, and glutamate enters the TCA cycle, creating a carbon skeleton for other amino acid synthesis. Important excretion of mammalian cells is lactic acid and ammonia, and the release of alanine is also important. Lactate and ammonia are toxic to cells because they change the pH of cells and lysosomes.

As such, since the physiological mechanism and nutritional requirements are different depending on the type of cells cultured, the type of medium used for cell culture should also vary according to characteristics. Therefore, in order to cultivate human-derived cells, growth conditions according to the type and state of the cells and the composition of the required medium will be different. Therefore, the present invention is to develop a chondrocyte culture medium suitable for the characteristics of human chondrocytes.

The present invention was devised to improve the limitations of the physiological and nutritional limitations of the cells isolated from articular cartilage tissue of patients of various ages and health conditions of the human body, and clinically the rejection response to transplantation is almost In order to secure the number of cells required for clinical transplantation in the shortest time possible than the performance of the medium, which is sold and used for commercially available cartilage cells, the patient's own chondrocytes are not only improved in survival rate, proliferation rate, but also in differentiation and intrinsic in cartilage cells. The purpose is to form a specific culture medium composition in the chondrocytes of humans to maintain the characteristics, and to shorten the culture time using this medium.

The present invention creates DR mixed with Rosewel Park Memorial Institute (RPMI) -1640 and Dulbecco's modification of Eagle's medium (DMEM) in a 1: 1 ratio, and compares the composition of the DR with DMEM and RPMI-1640 medium in each medium. Overlapped components select high concentrations while maintaining the same concentration in one of the mediums, while the same source component is selected from the Advanced Basic Media (ABM) -Chondrocyte (ABM-C) medium. Creating a step; And then culturing chondrocytes using the ABM-C medium, culturing short-term chondrocytes using ABM-C medium, or culturing three-dimensional chondrocytes using ABM-C medium. It provides a chondrocyte specific culture method for chondrocyte early culture, characterized in that consisting of a step of.

In order to prepare autologous chondrocytes using DMEM medium by separating chondrocytes from chondrocytes, the present invention is to culture culture period and monolayer continuous culture for 4-5 weeks up to 3rd stage, By using ABM-C medium, the doubling time of chondrocytes can be drastically shortened, monolayer continuous culture to secondary culture, and autologous chondrocytes can be prepared. It can be transplanted to a patient, and by using a short incubation period, the amount of raw materials consumed during the manufacturing process can be reduced, thereby reducing the manufacturing cost and supplying inexpensive chondrocyte therapy.

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According to the present invention, cartilage cells can be clinically implanted into femoral articular cartilage (lateral, medial, trochlear) defects and osteochondral defects of the talmur with clinical symptoms in the knee (knee) or ankle joint. It provides a chondrocyte specific culture medium capable of premature cell culture.

For the above invention, first, a medium composition ABM-Chondrocyte was prepared in which DMEM and RPMI-1640, which are already commercially available media, were mixed at a ratio of 1: 1. (Table 1).

Table 1. Composition of Commercialized Media and Composition of Advanced Basic Media (ABM) -Chondrocyte (ABM-C)

The composition of the basic medium for chondrocyte culture (Advanced Basic Media, ABM) was started in DMEM and RPMI-1640 medium composition. Create a DR with 1: 1 ratio of DMEM and RPMI-1640, and compare the composition of the DR with DMEM and RPMI-1640 medium, and select the high concentration of the overlapping components in each medium. They maintained the same concentration, and the same source components were selected from one of the two to complete the ABM-C medium composition (Table 1). Chondrocytes are classified as somatic cells in which cell proliferation and synthesis are not active. Was DMEM. As shown in Table 1, DMEM high-glucose medium is a medium in which essential amino acid is added twice as much as RPMI1640 medium and contains amino acid 15, inorganic salt 6, vitamin 9 and four other ingredients. Doing. ABM-C medium added asparagine anhydrous, aspartic acid, glutamic acid, hydroxy-proline, proline, which were not found in DMEM medium, and added arginine free base in addition to arginine monohydrochloride.

Insulin은 배양 중인 연골세포의 DNA 합성에 대한 비교적 적은 촉진 효과가 있는 것으로 알려져 있으나 (Prins 등, 1982) 여러 가지 성장 인자와 상승 작용을 하여 세포 증식에 적지 않은 도움을 주며 (Adolphe 등 1984), Swarm rat chondrosarchoma에서 5～10ng/mL의 W1이 proteoglycan 합성과 증식에 적은 효과가 있다(Stevens 등, 1981)고 보고하였다. ABM-C내 insulin의 연골세포 증식을 비교하기 위하여 insulin이 첨가된 CI (DMEM) 배지에서 배양된 passage 2의 연골세포를 75㎠의 세포배양용기에 5,000cell/㎠되게 접종하여 각각 ABM-C과 CI (DMEM-high glucose) 배지 내 insulin을 첨가한 배지와 첨가되지 않은 배지의 연골세포 증식을 비교하였다. Insulin이 첨가되지 않은 CI배지에서 배양된 연골세포와 비교하여 insulin이 첨가된 CI배지는 117.4％, W1이 첨가되지 않은 ABM-C 배지는 483.36％, insulin이 첨가된 ABM-C 배지는 591.7％의 증식률을 보였으며, insulin이 첨가된 ABM-C 배지는 insulin이 첨가되지 않은 ABM-C 배지에서 배양된 연골세포보다 122.43％ 증식률을 보였다. 이는 insulin의 의한 연골세포의 증식률 보다 ABM-C 배지에 의해 연골세포의 증식률이 높아짐을 알 수 있었다. 또한 Doubling time을 비교해 보면, insulin이 첨가된 배지는 첨가되지 않은 배지보다 각각 0.2～0.3일이 빠른 것을 보였으며 insulin이 첨가된 CI배지보다 insulin이 첨가되지 않은 ABM-C 의 doubling time이 약 1 day 빠른 것으로 나타나 insulin이 첨가되지 않은 ABM-C 배지를 이용하여 연골세포치료제 배양도 가능할 것으로 생각되었다.
표 3. ABM-C 배지내 FBS의 농도에 따른 연골세포 증식 비교

To determine the cell proliferation rate of the prepared ABM-C medium, 10% fetal calf serum was added to ABM-C, DMEM high-glucose (GIBCO) and Advanced DMEM high-glucose (GIBCO) medium. Cm 2, 5000 cells / cm 2, and 10000 cells / cm 2) were cultured for 11 days to determine the proliferation rate of chondrocytes by measuring the number of cells every day, thereby increasing the proliferation rate of the ABM-C medium of the present invention (FIG. 2). Proline and Hydroxy-proline are collagen protein (Pro-Hyp-Gly) components that promote the growth of chondrocytes by adding these amino acids, and vitamins d-biotin, aminobenzoic acid and vitamine B12 are added to the Enhanced proliferation. Ascorbic acid stabilizes the structure of collagen fibers by forming hydrogen bonds through hydroxylation of proline residues. Ascorbic acid-2-phosphate was added to DMEM medium and added to ABM-C medium.
Chondrocyte culture through monolayer culture is affected by the concentration of serum (FBS) used. The effect of FBS concentration on ABM-C medium showed that the proliferation of chondrocytes decreased after 6 days of ABM-C containing 2.5% and 5.0% of FBS in doubling time. As a result, the doubling time of ABM-CBM containing 2.5% FBS and CI was the same 3.2days, ABM-CBM medium containing 5% FBS showed 2.7 days, but the proliferation of chondrocytes from day 7 It was confirmed that the fall, ABM-C medium was very dependent on the FBS concentration, the cell proliferation was low at a concentration of 5% or less.
Table 2. Comparison of chondrocyte proliferation according to the concentration of FBS in ABM-C medium

Insulin is known to have a relatively small promoting effect on the DNA synthesis of chondrocytes in culture (Prins et al., 1982) but synergistically with various growth factors (Adolphe et al., 1984), Swarm In rat chondrosarchoma, 5-10 ng / mL of W1 has little effect on proteoglycan synthesis and proliferation (Stevens et al., 1981). To compare chondrocyte proliferation of insulin in ABM-C, the chondrocytes of passage 2 cultured in insulin-added CI (DMEM) medium were inoculated at 5,000 cells / ㎠ in a 75 cm 2 cell culture vessel, respectively. Chondrocyte proliferation was compared between the medium with insulin and the medium without insulin in CI (DMEM-high glucose) medium. Compared to chondrocytes cultured in CI medium without insulin, 117.4% for insulin-containing CI medium, 483.36% for ABM-C medium without W1 and 591.7% for ABM-C medium without insulin The growth rate was increased, and the insulin-added ABM-C medium showed 122.43% growth rate compared to the chondrocytes cultured in the insulin-free ABM-C medium. It was found that the proliferation rate of chondrocytes by ABM-C medium was higher than the proliferation rate of chondrocytes by insulin. Comparing the doubling time, the media with insulin showed 0.2 ~ 0.3 days earlier than the media without insulin, and the doubling time of ABM-C without insulin was about 1 day than the CI medium with insulin. It was considered to be fast, and it was thought that culturing chondrocytes using ABM-C medium without insulin was possible.
Table 3. Comparison of chondrocyte proliferation according to the concentration of FBS in ABM-C medium

In the present invention, when the chondrocytes isolated from the cartilage tissue collected from a patient with a damaged joint are cultured in vitro, the proliferation rate of the chondrocytes is higher than that of the conventionally commercialized medium, so that it is easy to secure the number of cells necessary for clinical transplantation. It is characteristic.

The chondrocyte specific culture medium of the present invention is for culturing chondrocytes isolated from damaged chondrocytes, and may be added with one or more components as necessary, to prevent contamination of microorganisms, including fetal calf, horse or human serum. Antibiotics and antifungal agents can be added and used, preferably 10% FBS.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments, but the present invention is not limited thereto.

Example 1 Chondrocyte Culture Using ABM Medium

<Example 1-1> Chondrocytes Isolation

The collected cartilage tissues were transferred to a 50 mL centrifuge tube, and 25 mL of HBSS (Hanks' Balanced Salt Solution) containing 10 ug / mL Gentamycin was added and shaken several times. Then, the cartilage tissues were washed by removing HBSS. This process was repeated six times. The washed cartilage tissue was transferred to petridish, and bone tissue was removed from the collected cartilage tissue and only the cartilage tissue was cut out. The cartilage tissue contained in Petridish was cut to a size of about 1-2 mm, then transferred to a 15 mL centrifuge tube, shaken several times with HBSS, and centrifuged to remove supernatant. This washing process was repeated three times. Precipitated cartilage tissue was suspended in HBSS solution containing 0.1% collagenase, and then placed in a 25 cm 2 cell culture vessel and allowed to stand at 37 ° C. for 16-18 hours.

<Example 1-2> Primary culture

The chondrocytes isolated from the cartilage tissues that were left in the 0.1% collagenase solution were filtered through a 40 μm nylon network, transferred to a new 15 mL centrifuge tube, centrifuged, the supernatant was removed, and 20% FBS (Fetal Bovine Serum) was added. After floating with DMEM or ABM containing medium, 5ug / mL Gentamycin was added, divided into 1-3 25 cm 2 cell culture vessels, and cultured in a 37 ° C. and 8% carbon dioxide incubator. Every 2-3 days, the culture medium was exchanged with 5 mL of DMEM medium or ABM medium containing 20% FBS for each culture medium and cultured for 9-11 days.

Aspirator aspirates the culture from the culture vessel, dispenses 5 mL HBSS per culture vessel, and shakes the culture vessel gently for 50 times. The washed HBSS is inhaled using an aspirator, and the washing process is repeated two more times. 1 mL of 0.25% trypsin solution was added to each culture vessel and allowed to stand at 37 ° C for 1-2 minutes to release the chondrocytes from the bottom of the culture vessel and transfer them to a 15 mL centrifuge tube. It was. 10 mL of DMEM or ABM medium containing 10% FBS was placed in a 15 mL centrifuge tube, the cells were suspended, 200 μL of cell suspension was collected and suspended in a 15 mL centrifuge tube containing 9.8 mL ISOTON solution. Measured. Chondrocytes collected in the primary culture were divided into five 75 cm 2 cell culture vessels at a density of 5,000 cells / cm 2 and cultured in an incubator at 37 ° C. and 8% carbon dioxide.

The number of chondrocytes primary cultured using the ABM medium showed twice the proliferation rate as that of the chondrocytes cultured in DMEM medium (Table 4).

Table 4. Comparison of Total Cells Harvested After Primary Culture by Media

<Example 1-3> Secondary Culture

After the first subculture, the culture medium was incubated for 5-7 days with 15 mL of DMEM medium or ABM medium containing 10% FBS in culture vessels every 2-3 days. Subcultures were performed when 90-100% confluence cell growth was observed in the culture vessel.

Aspirator aspirates the culture from the culture vessel, dispenses 5 mL HBSS per culture vessel, and shakes the culture vessel gently for 50 times. The washed HBSS is inhaled using an aspirator, and the washing process is repeated two more times. 2 mL of 0.25% trypsin solution was added to each culture vessel and allowed to stand at 37 ° C for 1-2 minutes to release chondrocytes from the bottom of the culture vessel and transfer to a 15 mL centrifuge tube. The supernatant was centrifuged using an aspirator. It was. 10 mL of DMEM or ABM medium containing 10% FBS was placed in a 15 mL centrifuge tube, the cells were suspended, 200 μL of cell suspension was collected and suspended in a 15 mL centrifuge tube containing 9.8 mL ISOTON solution. Measured. The chondrocytes collected in the primary culture were divided into six 175 cm 2 cell culture vessels at a density of 5,000 cells / cm 2 and cultured in an incubator at 37 ° C. and 8% carbon dioxide concentration.

The number of chondrocytes secondarily cultured using the ABM medium showed a growth rate of about 1.9 times that of the chondrocytes cultured in DMEM medium (Table 5).

Table 5. Comparison of Total Cells Harvested After Secondary Culture by Media

<Example 1-4> Tertiary culture

After the second subculture, the chondrocytes were cultured for 9-11 days with the chondrocytes exchanged with 20 mL of DMEM medium or ABM medium containing 10% FBS every 3-4 days. Subcultures were performed when 90-100% confluence cell growth was observed in the culture vessel.

Aspirator aspirates the culture from the culture vessel, dispenses 5 mL HBSS per culture vessel, and shakes the culture vessel gently for 50 times. The washed HBSS is inhaled using an aspirator, and the washing process is repeated two more times. 4 mL of 0.25% trypsin solution was added to each culture vessel and allowed to stand at 37 ° C for 1-2 minutes to release the chondrocytes from the bottom of the culture vessel and transfer them to a 15 mL centrifuge tube. It was. 10 mL of DMEM or ABM medium containing 10% FBS was placed in a 15 mL centrifuge tube, the cells were suspended, 200 μL of cell suspension was collected and suspended in a 15 mL centrifuge tube containing 9.8 mL ISOTON solution. Measured.

Chondrocytes cultured 3 times using ABM medium showed almost the same proliferation rate as DMEM medium (Table 6).

Table 6. Comparison of Total Cells Harvested After Tertiary Culture by Media

Example 1 is the result of culturing using a culture vessel defined for each culture (Fig. 3). The present invention was to calculate the theoretical yield based on the primary culture results of Example 1-2 to develop a medium that is easy to secure the number of cells that can be transplanted in a short time (Table 7). As shown in Table 7, in the case of chondrocytes cultured using ABM, the number of theoretically harvested cells after the second culture can secure 3.5 times as many cells as CI cultured using DMEM medium.

Table 7. Comparison of theoretical yields for each medium based on primary culture results

<Example 1-5> Confirmation of changes in the characteristics of chondrocytes according to the culture medium

In order to confirm the change in the characteristics of chondrocytes cultured in ABM added with FBS, it was compared with the chondrocytes cultured in DMEM medium containing FBS. As a result of confirming the gene expression of the molecular biological level of the cells cultured by the culture step for each culture medium, as shown in Figure 4 there was no significant difference from the chondrocytes cultured in DMEM.

Table 8. RT-PCR Reaction Conditions

Table 9. PCR Primer Sequences for Each Gene

Table 10. PCR reaction conditions for each gene

일반적으로 혈청이 첨가된 배지에서 단층배양으로 연속적으로 배양하면 passage 4～5 이후 연골세포의 특성을 잃어버리는 dedifferentiation이 일어난다 (Benya and Shaffer, 1982; Schulze-Tanzil 등, 2002). 각각 배지로 배양된 연골세포의 passage별 세포특성을 분자생물학적으로 gene의 발현율을 GAPDH와 비교한 결과 (도면 5), 검체 모두 계대배양 수가 증가됨에 따라 거의 gene의 발현도가 떨어지는 경향을 나타내며 hypertrophic marker인 Collagen type X, activated functional marker인 collagen type Ⅱ와 aggrecan, dedifferentiated marker인 collagen type Ⅰ 역시 두 배지 모두에서 감소하는 경향을 보였다. . 이와 마찬가지로 ABM-C 배지에서 연속적인 단층배양에서도 passage 3에서 collagen type Ⅱ의 합성 능력을 상실하였으나 (도면 5) ABM-C을 이용한 연골세포의 배양은 passage 2에서 CI배지에서 배양한 연골세포보다 activated functional chondrocyte의 표지인 aggrecan의 발현이 높게 나타났다. 이를 바탕으로 하여 ABM-CBM 배지를 이용하여 연골세포를 연속적으로 배양하여 연골세포치료제를 제조하기 위해서는 passage2에서 최종제품을 생산하는 short-term culture 법을 확립이 필요하였다.

In general, continuous culturing in monolayer culture in serum-mediated media results in dedifferentiation that results in the loss of chondrocyte characteristics after passages 4-5 (Benya and Shaffer, 1982; Schulze-Tanzil et al., 2002). As a result of comparing the expression rate of genes with GAPDH in the cellular characteristics of chondrocytes cultured in each medium in the passage (Fig. 5), the expression level of genes was almost decreased as the number of subcultures increased. Collagen type X, collagen type II, an activated functional marker, and aggrecan, and collagen type I, a dedifferentiated marker, also decreased in both media. . Likewise, in continuous monolayer culture in ABM-C medium, collagen type II synthesis ability was lost in passage 3 (Fig. 5). The chondrocyte culture using ABM-C was more activated than chondrocytes cultured in CI medium at passage 2. Expression of aggrecan, a marker of functional chondrocytes, was high. Based on this, it was necessary to establish a short-term culture method for producing final products in passage 2 in order to manufacture chondrocytes by continuously culturing chondrocytes using ABM-CBM medium.

DMEM high-glucose를 이용한 연골세포배양방법과 동일하게 1차 배양은 25㎠의 세포용기 3개 접종하여 배양을 실시한 후, 2차 배양은 75㎠의 세포배양용기 5개, 3차 배양은 175㎠의 세포배양용기 6개에서 각각 배양하여 수확되는 세포수를 측정하였다(표 12). 총 배양일은 CI의 경우, 33일 소요된 반면, ABM-CBM은 26일 이였다. 그러나 W1이 첨가되지 않은 ABM-CBM 배지는 P3에서 수확된 연골세포가 3.93×10⁷ cell로 4 vial 기준의 연골세포치료제를 제조할 수 없는 반면, W1이 함유된 CI배지 모두 P3에서 수확된 연골세포만 최소 4.8×10⁷cells 이상 (4 vials 기준으로, ＞ 1.2×10⁷cells/vial)이 수확되었다 (표 12, 도면 6b).
표 12. DMEM 배지를 이용한 연골세포배양법과 동일한 ABM-C의 연골세포 증식비교

ABM-CBM을 이용하여 연골세포를 short-term culture 방법의 효율성을 CI (DMEM) 배지를 이용하여 연골세포를 비교 배양하였다 (표 13, 도면 1). 두 배지 모두 1차 배양은 25㎠의 세포용기 3개 접종하여 배양을 실시한 후, 1차 배양에서 배양된 연골세포들 중 50％를 동결보관하고 나머지 50％의 연골세포를 최소 4.8×10⁷cells 이상의 연골세포를 수확하기 위하여 ABM-C 배지는 2차 배양을 CI (DMEM) 배지는 2차 및 3차을 실시하였다. ABM-C 배지를 이용한 short-term 연골세포 배양은 2차 배양에서 5.26×10⁷cells를 얻을 수 있었으나, CI (DMEM) 배지를 이용한 short-term 연골세포 배양은 2차 배양에서 4.8×10⁷cells 이하인 1.34×10⁷cells의 세포를 얻어 3차 배양을 실시하였다. 이와 같이, W1이 첨가되지 않은 ABM-C 배지를 이용하여 연골세포를 배양하여 CI (DMEM) 배지 보다 17일의 배양기간을 단축하였다 (도면 1).
표 13. ABM-C 배지를 이용한 short-term culture법

[실시예 3] ABM-C 배지를 이용한 3차원 연골세포배양
ABM-C 배지를 이용하여 연골세포를 collagen sponge에 접종하여 TGF-b1과 BMP-2와 같은 세포성장인자의 유무에 따른 3차원 배양 가능여부를 확인하기 위하여 pore size가 50～100um되는 10mm×10mm×2mm 크기의 collagen sponge에 연골세포를 1.0×10⁵ cells를 접종한 후, 표 14와 같은 배양 조건으로 20일 동안 배양하였다. Collagen sponge내 연골세포의 생존 및 배양여부를 확인하기 위하여 20일 동안 배양된 collagen sponge를 H＆E 염색하여 현미경으로 관찰한 결과, ABM-C 배지를 이용하여 collagen sponge 내 연골세포의 3차원 배양가능성도 확인하였다 (도면 7).
표 14. collagen sponge내 연골세포의 배양조건

Example 2 Short-term Chondrocyte Culture Using ABM-C Medium
Insulin-free ABM-C medium also showed rapid growth of CI medium supplemented with insulin, and compared with the current chondrocyte culture method and short-term culture method using ABM-CBM without insulin. To compare the proliferation effect of W1 in chondrocyte culture medium prepared in 25, 75, and 175 cm 2 cell culture vessels, the cartilage tissue was cultured by the primary culture, secondary culture, and tertiary culture. After chondrocytes were collected from collagen cells, the cartilage cells were isolated and inoculated with three 25 cm 2 cell containers, followed by primary culture. Secondary cultures had 75 cm 2 cell culture vessels and tertiary cultures had 175 cm 2 cell cultures. The number of cells harvested by culturing each in a container was measured. In order to compare the proliferation of chondrocytes in the cell culture vessel, a fixed point was placed on the bottom of the culture vessel, and the number of cells was observed and measured every day. Was prepared (Fig. 6a, Table 11). Using this, the doubling time of chondrocytes was calculated (Table 11). The doubling time at P1, P2, and P3 stages of CI medium was 2.4, 4.8, 2.3 days, and the doubling time of ABM-CBM was 1.5, 2.0, 3.8 days. As the culture stage increased, the doubling time increased.
Table 11. Comparison of Chondrocyte Proliferation Using ABM-C Medium Without Insulin

In the same manner as in the chondrocyte culture method using DMEM high-glucose, the primary culture was inoculated with three 25 cm 2 cell containers, and the secondary culture was 75 cm 2 cell culture containers, and the tertiary culture was 175 cm 2. The number of cells harvested by culturing each of six cell culture vessels was measured (Table 12). Total culture days took 33 days for CI, whereas ABM-CBM was 26 days. However, in ABM-CBM medium without W1, the chondrocytes harvested from P3 were 3.93 × 10 ⁷ cells, which could not be used to manufacture cartilage cell therapy based on 4 vial, whereas all of the CI medium containing W1 was harvested from P3. Only cells were harvested at least 4.8 × 10 ⁷ cells or more (> 1.2 × 10 ⁷ cells / vial, based on 4 vials) (Table 12, Figure 6b).
Table 12. Comparison of chondrocyte proliferation of ABM-C identical to chondrocyte culture using DMEM medium

Chondrocytes were cultured using ABM-CBM and cultured chondrocytes using CI (DMEM) medium for the efficiency of the short-term culture method (Table 13, Figure 1). In both cultures, the primary culture was inoculated with three 25 cm 2 cell containers, followed by cryopreservation of 50% of the chondrocytes cultured in the primary culture and at least 4.8 × 10 ⁷ cells for the remaining 50% of chondrocytes. In order to harvest the above chondrocytes, ABM-C medium was subjected to secondary culture and CI (DMEM) medium was subjected to secondary and tertiary. Short-term chondrocyte culture using ABM-C medium yielded 5.26 × 10 ⁷ cells in the secondary culture, while short-term chondrocyte culture using CI (DMEM) medium was 4.8 × 10 ⁷ cells in the secondary culture. Cells of 1.34 × 10 ⁷ cells were obtained and subjected to tertiary culture. As such, chondrocytes were cultured using ABM-C medium to which W1 was not added, which shortened the culture period of 17 days from CI (DMEM) medium (FIG. 1).
Table 13. Short-term culture using ABM-C medium

Example 3 Three-Dimensional Chondrocyte Culture Using ABM-C Medium
Inoculate chondrocytes into collagen sponge using ABM-C medium to determine whether three-dimensional culture is possible depending on the presence of cell growth factors such as TGF-b1 and BMP-2. Chondrocytes were inoculated into 1.0 × 10 ⁵ cells in a collagen sponge having a size of 2 mm, and then cultured for 20 days under the culture conditions shown in Table 14. In order to confirm the survival and cultivation of chondrocytes in the collagen sponge, H & E staining of the collagen sponges cultured for 20 days was observed under a microscope, and the 3D cultureability of the chondrocytes in the collagen sponge was also confirmed using ABM-C medium. (Fig. 7).
Table 14. Culture conditions of chondrocytes in collagen sponge

Technical idea of the chondrocyte specific culture medium and culture method for the early culture of chondrocytes of the present invention can actually repeat the same results, in particular, by implementing the present invention can promote technology development and contribute to industrial development It is well worth protecting.

1 is a graph analyzing the chondrocyte treatment culture using the short-term culture method and the present DMEM medium according to the present invention.

Figure 2 is a graph analyzing chondrocyte proliferation of the cell culture medium according to the inoculation number of chondrocytes according to the present invention, Figure 2a is 2,500 cells / ㎠, Figure 2b is 5,000 cells / ㎠, Figure 2c is 10,000 cells / ㎠ Inoculate the proliferation curve of chondrocytes.

Figure 3 is a graph analyzing the proliferation of chondrocytes in the cell culture vessel for each culture step according to the present invention.

Figure 4 is a photograph of the results of analyzing the molecular biological characteristics of chondrocytes for each culture step according to the present invention.

Figure 5 is a graph of the expression rate of the gene showing the molecular biological characteristics of chondrocytes for each culture step according to the present invention.
6 is a graph showing the growth curves of chondrocytes in the cell culture vessels of each culture step for each medium according to the present invention.
Figure 7 is a H & E staining photograph of the chondrocyte culture day 20 in collagen sponge according to the present invention.

Claims (9)

Create a mixed DR of Rosewel Park Memorial Institute (RPMI) -1640 and Dulbecco's modification of Eagle's medium (DMEM) in a 1: 1 ratio, and compare the composition of the DR with DMEM and RPMI-1640 medium again to duplicate the components in each medium. They select high concentrations and at the same time maintain the same concentrations in one of the mediums and select one of the same sources to form the Advanced Basic Media (ABM) -Chondrocyte (ABM-C) medium. step; And Then, any one of the steps of culturing chondrocytes using the ABM-C medium, culturing short-term chondrocytes using ABM-C medium, or culturing three-dimensional chondrocytes using ABM-C medium. Chondrocyte specific culture method for early culturing chondrocytes, characterized in that consisting of a step. According to claim 1, In the step of culturing chondrocytes using the ABM-C medium, Chondrocyte separation step and the first, secondary, tertiary culture step and the step of confirming the characteristics change of chondrocytes according to the culture medium; chondrocyte specific culture method for the early chondrocyte culture characterized in that the sequence is made sequentially . According to claim 2, The chondrocyte separation step, Transferring the collected cartilage tissue to a 50 mL centrifuge tube, adding 25 mL of HBSS (Hanks' Balanced Salt Solution) containing 10 ug / mL Gentamycin, shaking it several dozen times, and then washing the collected cartilage tissue by removing HBSS; Repeating the above procedure six times and transferring the washed cartilage tissue to petridish, removing bone tissue from the collected cartilage tissue and cutting only the cartilage tissue; Subsequently, the cartilage tissue contained in Petridish was cut to a size of 1-2 mm, transferred to a 15 mL centrifuge tube, placed in HBSS, shaken several times, and centrifuged to remove supernatant; Repeating the flushing process three times and floating the precipitated cartilage tissue in HBSS solution containing 0.1% collagenase, and then placed in a 25cm2 cell culture vessel and allowed to stand for 16-18 hours at 37 ℃. Chondrocyte specific culture method for early culturing chondrocytes. According to claim 2, The primary culture step, The chondrocytes isolated from the cartilage tissues that were left in the 0.1% collagenase solution were filtered through a 40 μm nylon network, transferred to a new 15 mL centrifuge tube, centrifuged, the supernatant was removed, and 20% FBS (Fetal Bovine Serum) was added. Suspended in DMEM or ABM medium containing; Thereafter, 5ug / mL of Gentamycin was added to divide 25 cm 2 -cell culture vessels 1-3 and incubated in a 37 ° C., 8% carbon dioxide incubator; Subsequently, the culture medium was replaced with 5 mL of DMEM medium or ABM medium containing 20% FBS every 2-3 days, followed by culturing for 9-11 days; chondrocyte specific for chondrocyte culture. Cultivation method. According to claim 2, The secondary culture step, After primary subculture, incubate the chondrocytes in culture with 15 mL of DMEM medium or ABM medium containing 10% FBS every 2-3 days and culture for 5-7 days. A chondrocyte-specific culture method for chondrocyte early culture, characterized in that passage is performed when confluence cell growth is observed. According to claim 2, The third culture step, After the second subculture, the culture medium was replaced with DMEM medium or ABM medium containing 10% FBS or 20 mL of ABM medium every 3-4 days in cultured cartilage cells for 9-11 days. A chondrocyte-specific culture method for chondrocyte early culture, characterized in that passage is performed when confluence cell growth is observed. According to claim 2, Checking the characteristic change of chondrocytes according to the culture medium, In order to confirm the change in the characteristics of chondrocytes cultured in ABM added with FBS, it was compared with the chondrocytes cultured in DMEM medium with FBS, and confirmed the gene expression of the molecular biological level of the cells cultured by the culture step for each culture medium. Cartilage cell-specific culture method for the early culture of chondrocytes characterized in that. According to claim 1, The step of culturing the short-term chondrocytes using the ABM-C medium, Performing cartilage cell culture by exchanging medium to compare the proliferative effect of W1 in chondrocyte culture medium prepared in 25, 75, and 175 cm 2 cell culture containers by primary, secondary and tertiary culture methods of cartilage tissue; After that, cartilage tissues were collected using collagenase to separate chondrocytes and inoculated with three 25 cm 2 cell vessels, followed by primary culture. Secondary cultures had 75 cm 2 cell culture vessels and tertiary cultures had 175 cm 2 cells. Measuring the number of cells harvested by culturing each in a culture vessel; In order to compare the proliferation of chondrocytes in the cell culture vessel, a fixed point was placed on the bottom of the culture vessel to observe and measure the number of cells every day. Creating a graph; Cartilage cell-specific culture method for early culturing chondrocytes characterized in that it contains. According to claim 1, Cultivating three-dimensional chondrocytes using the ABM-C medium, Inoculate chondrocytes into collagen sponge using ABM-C medium to determine whether three-dimensional culture is possible depending on the presence of cell growth factors such as TGF-b1 and BMP-2. Inoculating the chondrocytes into 1.0 × 10 ⁵ cells in a collagen sponge having a size of 2 mm and culturing them; And Culturing the chondrocytes in the collagen sponge in three dimensions using ABM-C medium as a result of H & E staining and microscopic observation of the collagen sponges cultured for 20 days to confirm the survival and culture of the chondrocytes in the collagen sponge; Cartilage cell-specific culture method for early culturing chondrocytes characterized in that it comprises.

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