KR20220134422A - Method for Cryopreservation and thawing of cartilage tissue - Google Patents
Method for Cryopreservation and thawing of cartilage tissue Download PDFInfo
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- KR20220134422A KR20220134422A KR1020210140356A KR20210140356A KR20220134422A KR 20220134422 A KR20220134422 A KR 20220134422A KR 1020210140356 A KR1020210140356 A KR 1020210140356A KR 20210140356 A KR20210140356 A KR 20210140356A KR 20220134422 A KR20220134422 A KR 20220134422A
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- South Korea
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- cryopreservation
- cartilage tissue
- solution
- tissue
- thawing
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- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
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- A—HUMAN NECESSITIES
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- A01N1/02—Preservation of living parts
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- A—HUMAN NECESSITIES
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- A01N1/02—Preservation of living parts
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Abstract
Description
본 발명은 연골조직의 동결 보존 및 해동방법에 대한 것으로, 동종 연골 이식재로 활용할 연골조직을 조직 내 세포 생존율 유지하면서 동결 보존하고 동결보존된 연골조직을 해동하는 방법에 관한 것이다.The present invention relates to a method for cryopreserving and thawing cartilage tissue, and to a method for cryopreserving cartilage tissue to be used as an allogeneic cartilage graft material while maintaining cell viability in the tissue and thawing the cryopreserved cartilage tissue.
연골 손상은 관절의 퇴행성 변화, 관절의 무리한 사용 또는 외상에 의한 손상에 의하여 발생될 수 있으며 연골의 제한된 재생 능력 때문에 쉽게 골관절염으로 이어질 수 있다.Cartilage damage can be caused by degenerative changes in the joint, excessive use of the joint, or damage caused by trauma, and can easily lead to osteoarthritis due to the limited regenerative capacity of cartilage.
연골 손상이 광범위하게 진행되어 넓은 범위의 관절 연골 결손을 치유하기 위한 대표적인 치료방법 중 하나로 동종 골연골 이식 수술이 사용되어지고 있다. 동종 골연골 이식 수술은 기존에 많이 시행되고 있는 미세골절술 및 연골세포 자가 이식수술보다 넓은 범위의 연골 결손에 대해 우월한 치료 효과를 나타낸다. 하지만 동종 골연골 이식재는 공여자의 특성에 따라 품질에 차이가 있으며 부적절한 골연골 이식재를 이식할 경우 숙주 조직과의 융합 능력이 떨어지고 골조직 괴사에 의한 이식물의 붕괴가 보고되고 있다. 성공적인 동종 골연골 이식을 위해서는 이식조직 내 세포 생존율이 중요한 요인으로써 작용할 수 있다. 골연골 이식 수술 후 10년 이상 장기 추적 관찰 연구결과에 따르면 골연골 이식재의 세포 생존율이 높을수록 유의적인 임상적 치료 효능이 관찰되는 것으로 보고되고 있다. 하지만 종래의 동결 보존 및 해동방법은 이식조직의 세포 생존율을 크게 고려하고 있지 않아 동종 골연골의 이식 성공률이 낮은 문제가 있다.As cartilage damage progresses widely, allogeneic osteochondral transplantation is being used as one of the representative treatment methods for healing a wide range of articular cartilage defects. Allogeneic osteochondral transplantation has a superior therapeutic effect on a wide range of cartilage defects compared to microfracture and chondrocyte autologous transplantation, which are frequently performed. However, the quality of allogeneic osteochondral graft materials differs depending on the characteristics of the donor, and when improperly transplanted osteochondral graft materials are transplanted, the ability to fuse with the host tissue decreases and the graft collapses due to bone tissue necrosis. For successful allogeneic osteochondral transplantation, cell viability in the transplanted tissue can act as an important factor. According to the results of long-term follow-up studies for more than 10 years after osteochondral transplantation surgery, it is reported that the higher the cell viability of the osteochondral graft material, the more significant clinical therapeutic efficacy is observed. However, the conventional cryopreservation and thawing method does not take into account the cell viability of the grafted tissue, so there is a problem that the success rate of transplantation of allogeneic osteochondral is low.
한편, 연골조직은 다른 조직에 비하여 상대적으로 면역거부 반응이 낮아 이식된 부위에서 장기간 연골조직 및 세포의 생존이 유지되기 때문에 골연골 조직 이식에 비하여 비교적 우월한 임상적 치유 결과를 보인다. On the other hand, cartilage tissue exhibits comparatively superior clinical healing results compared to osteochondral tissue transplantation because cartilage tissue has a relatively low immune rejection response compared to other tissues, and long-term survival of cartilage tissue and cells is maintained at the transplanted site.
종래 연골조직 보관 방법은 일반적으로 냉장보관 4℃ 및 냉동보관 -20℃ 이하로 분류된다. 냉장보관의 경우 최대 6개월까지 연골조직 내 세포생존율을 보존할 수 있기 때문에 6개월 이상 장기 보존을 위해서는 연골조직의 냉동보관 기술이 요구된다. 연골조직의 동결보관은 냉장보관에 비하여 상대적으로 장기간 보관에 용이하지만, 동결과정에 사용되는 동결 보존액, 급격한 온도 변화 (동결, 해동) 및 해동액의 처치와 같은 물리화학적 변화로 인하여 세포 및 세포외기질에 손상을 줄 수 있기 때문에 연골조직의 세포 생존율을 유지하면서 장시간 연골조직을 보존할 수 있는 동결 보존 및 해동방법이 필요한 실정이다.Conventional cartilage tissue storage methods are generally classified into refrigerated storage at 4°C and frozen storage at -20°C or lower. In the case of refrigeration, cell viability in cartilage tissue can be preserved for up to six months, so for long-term preservation of more than six months, cryopreservation technology of cartilage tissue is required. Freezing storage of cartilage tissue is relatively easier for long-term storage compared to refrigeration, but due to physicochemical changes such as cryopreservation solution used in the freezing process, rapid temperature change (freezing, thawing), and treatment of thawing solution, cellular and extracellular Since it can damage the matrix, there is a need for a cryopreservation and thawing method that can preserve the cartilage tissue for a long time while maintaining the cell viability of the cartilage tissue.
본 발명은 동종 골연골 이식술의 성공률을 높이기 위해 동결보관 후에도 연골조직의 세포생존율을 유지시킬 수 있고, 동결보존액 내 다이메틸 설폭사이드(Dimethyl Sulfoxide, DMSO)의 잔류량을 분석할 수 있는 연골조직의 동결보존 및 해동방법을 제공함에 있다.In order to increase the success rate of allogeneic osteochondral transplantation, the present invention can maintain the cell viability of cartilage tissue even after cryopreservation, and freeze the cartilage tissue that can analyze the residual amount of dimethyl sulfoxide (DMSO) in the cryopreservation solution. To provide a method of preservation and thawing.
상기와 같은 목적을 달성하기 위해, 본 발명의 연골조직의 동결보존 및 해동방법은 이식용 연골조직의 세포생존율을 유지하면서 보존하기 위해서, 개체로부터 분리된 연골조직에 동결보존액을 처리하는 단계; 동결보존액이 처리된 연골조직을 동결 보존시키는 단계; 및 상기 동결 보존된 연골조직을 30~40℃ 수조에서 해동시킨 후, 인산완충액으로 세척하여 동결보존액을 제거하는 단계를 포함하며, 상기 동결보존액은 5~10 v/v% 다이메틸 설폭사이드(Dimethyl Sulfoxide, DMSO)를 포함하는 무혈청 동결보존액인 것을 특징으로 하는 연골조직 동결보존 및 해동방법을 제공한다.In order to achieve the above object, the cryopreservation and thawing method of cartilage tissue of the present invention comprises the steps of: treating the cryopreservation solution to the cartilage tissue separated from the subject in order to preserve the cell viability of the cartilage tissue for transplantation; Cryopreserving the cryopreservation solution-treated cartilage tissue; and thawing the cryopreserved cartilage tissue in a water bath at 30-40° C. and washing with a phosphate buffer to remove the cryopreservation solution, wherein the cryopreservation solution is 5-10 v/v% dimethyl sulfoxide (Dimethyl). Sulfoxide, DMSO) provides a method for cryopreservation and thawing of cartilage tissue, characterized in that it is a serum-free cryopreservation solution.
본 발명은 동종 골연골 이식술에 활용할 연골조직의 세포 생존율을 높일 수 있는 연골조직의 동결보존 및 해동 방법을 제공하며, 특히 본 발명에 따른 연골조직의 동결보존 및 해동 방법은 동결보존 후 연골조직 내 세포 생존율을 유지시킴으로써 동종 연골 이식술 시 광범위 연골 결손의 재생에 임상적 치료 효능을 극대화 시킬 수 있을 뿐 아니라, 동결보존액 내 다이메틸 설폭사이드(Dimethyl Sulfoxide, DMSO)의 잔류량을 분석할 수 있는 장점을 제공한다.The present invention provides a cryopreservation and thawing method of cartilage tissue that can increase the cell viability of cartilage tissue to be used for allogeneic osteochondral transplantation. In particular, the cryopreservation and thawing method of cartilage tissue according to the present invention provides By maintaining the cell viability, it is possible to maximize the clinical therapeutic efficacy in the regeneration of extensive cartilage defects during allogeneic cartilage transplantation, as well as to analyze the residual amount of dimethyl sulfoxide (DMSO) in cryopreservation solution. do.
도 1은 본 발명의 동종 연골조직의 동결보존 및 해동 과정을 도식화 한 모식도이다.
도 2는 동결보존액의 종류에 따른 연골조직 내 세포생존율 및 세포 증식율을 각각 트리판 블루 염색 분석(Trypan Blue staining assay)과 WST-1 분석을 통해 정량적으로 평가한 결과이다.
도 3은 연골조직에 DMSO가 포함된 무혈청 동결보존액을 서로 다른 시간으로 각각 처치한 후 마이크로-CT 분석을 통하여 각 시간대에 연골조직 내 동결보존액 삼투 과정을 평가한 결과이다.
도 4는 통제속도냉각장치(Controlled Rate Freezers; CRF)의 온도 하강 조절에 따라 동결 과정 중 연골조직 내의 온도 변화 그래프에 따라 최적화 된 동결방법을 평가한 결과이다.
도 5는 연골조직의 냉동 보관 온도에 따른 연골조직 내 세포생존율 및 세포 증식율을 각각 트리판 블루 염색 분석(Trypan Blue staining assay)과 WST-1 분석을 통해 정량적으로 평가한 결과이다.
도 6은 동결보존 된 연골조직의 잔여 동결보존액을 제거하기 위하여 PBS 용액으로 조직을 세척할 때 필요되는 최소의 세척 시간과 회수를 마이크로-CT 분석을 통하여 최적화 시킨 결과이다.
도 7은 본 발명에 따른 동결보존 방법으로 연골조직을 동결보존을 진행하고 조직의 동결보존 전후 세포생존율 및 세포증식율을 각각 Live & Dead 분석 및 트리판 블루 염색 분석(Trypan Blue staining assay)과 WST-1 분석을 통해 정량적으로 비교한 결과이다.
도 8은 본 발명에 따른 동결보존 방법으로 연골조직을 동결보존 진행하고 조직의 동결보존 전후 생화학적 방법 및 조직학적 염색 방법으로 정량적 및 이미지 방면으로 비교한 결과이다.
도 9는 본 발명에 따른 동결보존 방법으로 연골조직을 동결보존 진행하고 조직의 동결보존 전후 세포의 세포외기질을 생산하는 능력을 평가하기 위해 면역조직화학적 분석(collagen type II)와 사프라닌-O 염색 실험을 통하여 비교한 결과이다.1 is a schematic diagram schematically illustrating the cryopreservation and thawing process of allogeneic cartilage tissue of the present invention.
2 is a result of quantitative evaluation of cell viability and cell proliferation in cartilage tissue according to the type of cryopreservation solution through Trypan Blue staining assay and WST-1 analysis, respectively.
3 is a result of evaluating the osmotic process of the cryopreservation solution in the cartilage tissue at each time through micro-CT analysis after treating the cartilage tissue with a serum-free cryopreservation solution containing DMSO at different times.
4 is a result of evaluating the freezing method optimized according to the temperature change graph in the cartilage tissue during the freezing process according to the temperature drop control of the Controlled Rate Freezers (CRF).
5 is a result of quantitative evaluation of the cell viability and cell proliferation rate in the cartilage tissue according to the cryopreservation temperature of the cartilage tissue through Trypan Blue staining assay and WST-1 analysis, respectively.
6 is a result of optimizing the minimum washing time and recovery required when washing the tissue with PBS solution through micro-CT analysis in order to remove the cryopreservation solution remaining in the cryopreserved cartilage tissue.
7 is a cryopreservation method according to the present invention to proceed with cryopreservation of cartilage tissue, and the cell viability and cell proliferation rate before and after cryopreservation of the tissue, respectively, Live & Dead analysis and Trypan Blue staining assay (Trypan Blue staining assay) and WST- 1 This is the result of quantitative comparison through analysis.
8 shows the results of quantitative and image comparison of cartilage tissue cryopreservation using the cryopreservation method according to the present invention, and biochemical method and histological staining method before and after cryopreservation of the tissue.
9 shows immunohistochemical analysis (collagen type II) and safranin- This is the result of comparison through the O staining experiment.
이하에서 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 광범위 관절 연골 결손을 치유하기 위한 동종 연골 이식재의 동결보존 및 해동방법에 관한 것으로, 이식재의 품질을 개선하고 최종적으로 연골 치유 및 재생 효과를 높일 수 있다. 현재 동종 연골조직의 6개월 이상 장기 보관에는 동결보존 시스템이 필수적으로 요구되어지나, 조직의 동결과정에서 각종 위해요소(동결과정에서의 조직 내 얼음생성 및 잠열 방출, 보존액의 독성)로 인하여 조직의 세포 및 세포외기질의 파괴가 야기되고, 이에 따라 동결보존 후 조직의 품질이 저하되는 한계점이 있다. The present invention relates to a method for cryopreserving and thawing an allogeneic cartilage graft material for healing a wide range of articular cartilage defects, which can improve the quality of the graft material and finally increase the cartilage healing and regeneration effect. Currently, a cryopreservation system is essential for long-term storage of allogeneic cartilage tissue for more than 6 months. There is a limitation in that the destruction of cells and extracellular matrix is caused, and thus the quality of the tissue is deteriorated after cryopreservation.
따라서, 본 발명은 연골조직의 세포 생존율을 유지할 수 있도록 연골조직에 최적화된 동결보존액의 처치, 동결온도의 설정 및 해동 조건을 제어하는 연골조직의 동결보존 및 해동방법에 관한 것이다.Accordingly, the present invention relates to a cryopreservation and thawing method of cartilage tissue that controls the treatment of cryopreservation solution optimized for cartilage tissue, setting of freezing temperature and thawing conditions so as to maintain cell viability of cartilage tissue.
본 발명은 개체로부터 분리된 연골조직에 동결보존액을 처리하는 단계; 동결보존액이 처리된 연골조직을 동결 보존시키는 단계; 및 상기 동결 보존된 연골조직을 30~40℃ 수조에서 해동시킨 후, 인산완충액으로 세척하여 동결보존액을 제거하는 단계를 포함하는 연골조직 동결보존 및 해동방법을 제공한다.The present invention comprises the steps of treating the cryopreservation solution to the cartilage tissue separated from the subject; Cryopreserving the cryopreservation solution-treated cartilage tissue; And after thawing the cryopreserved cartilage tissue in a 30-40 ° C water bath, it provides a method for cryopreserving and thawing cartilage tissue comprising the step of washing with a phosphate buffer solution to remove the cryopreservation solution.
상기 동결보존액을 처리하는 단계는 동결보존액을 4℃에서 3-5시간 동안 처리하는 것이 바람직하며, 상기 범위를 벗어나면 세포생존율과 세포증식율이 감소하는 문제가 야기될 수 있다.In the step of treating the cryopreservation solution, it is preferable to treat the cryopreservation solution at 4° C. for 3-5 hours, and out of the above range may cause a problem in that the cell viability and cell proliferation rate decrease.
상기 동결보존액으로는 투과성 동결 보존액 또는 비투과성 동결 보존액일 수 있다. 투과성 동결 보호제로는 글리세롤 (glycerol), 에틸렌글라이콜 (Ethylene glycol, EG) 또는 다이메틸 설폭사이드 (Dimethyl Sulfoxide, DMSO)를 포함하는 보존액일 수 있다. 비투과성 동결 보존액으로는 글루코스 (glucose), 덱스트란 (Dextran), 폴리비닐피롤리돈 (polyvinylpyrolidone)을 포함하는 동결 보존액일 수 있다. 종래 투과성 동결보호제로 많이 사용되는 DMSO 또는 글리세롤과, 비투과성 동결보호제로 많이 사용되는 덱스트란, 글루코스의 성능을 비교한 결과, 도 2와 같이 세포생존율과 세포증식율 측면에서 DMSO가 포함된 동결보존액의 성능이 가장 우수함을 확인하였다.The cryopreservation solution may be a permeable cryopreservation solution or an impermeable cryopreservation solution. The permeable cryoprotectant may be a preservative containing glycerol, ethylene glycol (EG), or dimethyl sulfoxide (DMSO). The impermeable cryopreservation solution may be a cryopreservation solution containing glucose, dextran, and polyvinylpyrrolidone. As a result of comparing the performance of DMSO or glycerol, which are commonly used as permeable cryoprotectants, and dextran, and glucose, which are often used as impermeable cryoprotectants, as shown in FIG. 2, in terms of cell viability and cell proliferation, the cryopreservation solution containing DMSO was It was confirmed that the performance was the best.
상기 동결 보존시키는 단계는 통제속도냉각장치(Controlled Rate Freezers; CRF)를 통해 동결보존액을 처리한 연골조직이 빙점에 도달하였을 때 냉각 속도를 15-20℃/min으로 냉각시키고 챔버 온도를 영하 40-80℃까지 하강함으로써 조직 내 각 부위에서 과냉각 또는 잠열 방출 현상을 제지하고 균일한 속도로 냉각되게 하는 방법으로 연골조직의 동결 온도 및 유지시간을 조절할 수 있다. In the cryopreservation step, when the cartilage tissue treated with the cryopreservation solution through a Controlled Rate Freezers (CRF) reaches the freezing point, the cooling rate is cooled to 15-20°C/min and the chamber temperature is lowered to minus 40- By lowering to 80°C, it is possible to control the freezing temperature and retention time of cartilage tissue by stopping the supercooling or latent heat release phenomenon in each part of the tissue and cooling it at a uniform rate.
상기 연골조직의 보관 온도는 -80℃ 내지 -180 ℃의 범위에서 조절할 수 있고, 이러한 범위를 벗어나면 연골 조직의 품질 및 연골세포 생존율이 감소되는 문제가 야기될 수 있다.The storage temperature of the cartilage tissue can be adjusted in the range of -80 ℃ to -180 ℃, out of this range may cause a problem that the quality of the cartilage tissue and the chondrocyte viability is reduced.
상기 동결보존액을 제거하는 단계는 흔들리는 장비 내에서 인산완충액으로 3-4회 세척하는 것이 바람직하다. In the step of removing the cryopreservation solution, it is preferable to wash 3-4 times with a phosphate buffer solution in a shaking device.
상기 동결보존액을 제거하는 단계 이후, 마이크로-CT 분석을 통해 동결보존액 내 다이메틸 설폭사이드(Dimethyl Sulfoxide, DMSO)의 잔류량을 분석하는 단계를 더 포함할 수 있다.After removing the cryopreservation solution, the method may further include analyzing the residual amount of dimethyl sulfoxide (DMSO) in the cryopreservation solution through micro-CT analysis.
특히, 본 발명에 따르면, 상기 DMSO의 잔류량 분석은 DMSO 용액에 함유하고 있는 황 원자의 밀도로 인하여 마이크로-CT 분석을 통하여 실시간 관찰이 가능하여 연골조직 내 동결보존액의 포집과 DMSO의 잔류량을 분석할 수 있다.In particular, according to the present invention, the analysis of the residual amount of DMSO can be observed in real time through micro-CT analysis due to the density of sulfur atoms contained in the DMSO solution, so that the collection of cryopreservation solution in cartilage tissue and the residual amount of DMSO can be analyzed. can
도 6과 같이, 인산완충액(PBS) 교체 회수 1회에서 4회까지 마이크로-CT 값은 하강하는 것을 확인할 수 있고, 4회 이후 마이크로-CT 값은 큰 변화가 없는 것을 확인할 수 있다. 따라서, 본 발명에 따른 동결보존 및 해동방법 중 PBS 용액으로 특히 4회 세척하였을 때 조직 내의 동결보존액은 제거되었음을 마이크로-CT 분석을 통해 확인할 수 있었다.As shown in FIG. 6 , it can be seen that the micro-CT value is decreased from 1 to 4 times of replacement of the phosphate buffer (PBS), and it can be confirmed that the micro-CT value does not change significantly after 4 times. Therefore, it could be confirmed through micro-CT analysis that the cryopreservation solution in the tissue was removed when the cryopreservation and thawing method according to the present invention was washed 4 times with PBS solution.
이하, 본 발명의 이해를 돕기 위하여 실시예를 통해 본 발명의 동결보존 및 해동방법에 대해 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. Hereinafter, the cryopreservation and thawing method of the present invention will be described in detail by way of Examples to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples.
<실시예 1> 연골조직의 동결 보존액 최적화<Example 1> Optimization of cryopreservation solution of cartilage tissue
1. 연골조직용 동결보존액의 선정1. Selection of cryopreservation solution for cartilage tissue
연골조직의 동결보존에 적합한 동결보존액을 선정하기 위하여 다양한 동결보존액을 처치하여 동결한 후 다시 해동하여 조직 내 세포생존율 및 증식율을 분석하였다. 동결보존액의 후보 그룹으로는 투과성 동결보호제로 많이 사용되고 있는 DMSO (CryoStor® CS10), 글리세롤 (Glycerol) 그리고 비투과성 동결보호제로 많이 사용되고 있는 덱스트란 (dextran), 글루코스 (glucose)를 선정하여 비교하였다. 상기 동결보존액 처치 후 -180℃에서 4주간 동결보존한 후 37℃ 수조에서 해동 후 인산완충액(PBS)로 세척하여 조직내 동결보존액을 제거하였다. 해동된 연골조직은 즉시 미세절편기를 이용하여 연골조직 절편으로 제작하고, LIVE & DEAD 시약으로 염색한 후 confocal 현미경으로 이미지를 촬영하였다. In order to select a cryopreservation solution suitable for cryopreservation of cartilage tissue, various cryopreservation solutions were treated, frozen, and then thawed again to analyze the cell viability and proliferation rate in the tissue. As a candidate group for cryopreservation, DMSO (CryoStor ® CS10), which is widely used as a permeable cryoprotectant, and glycerol, and dextran and glucose, which are often used as an impermeable cryoprotectant, were selected and compared. After treatment with the cryopreservation solution, cryopreservation was performed at -180° C. for 4 weeks, thawed in a water bath at 37° C., and washed with phosphate buffer (PBS) to remove the cryopreservation solution in the tissue. The thawed cartilage tissue was immediately prepared into cartilage tissue sections using a microsection, stained with LIVE & DEAD reagent, and then images were taken with a confocal microscope.
조직 내 세포 생존율을 정량적으로 분석하기 위하여 연골조직을 수술칼로 잘게 다진 후 0.1% 콜라게나이제 II를 함유한 DMEM (Dulbecco's Modified Eagle's Medium, GENDEPOT) 배지에서 16시간 노출시킨 후 분리한 연골세포를 Trypan Blue Staining을 통하여 세포생존율을 측정하였다. In order to quantitatively analyze the cell viability in the tissue, the cartilage tissue was minced with a surgical knife and exposed to DMEM (Dulbecco's Modified Eagle's Medium, GENDEPOT) medium containing 0.1% collagenase II for 16 hours. Then, the isolated chondrocytes were treated with Trypan Blue. Cell viability was measured through staining.
또한 조직 내 세포의 증식률을 분석하기 위하여 상기 방법으로 분리한 연골 세포를 배양하고 WST-1 (Water Soluble Tetrazolium-1) 분석을 통하여 14일까지 세포 증식률을 관찰하였다.In addition, in order to analyze the proliferation rate of cells in the tissue, the chondrocytes isolated by the above method were cultured and the cell proliferation rate was observed until 14 days through WST-1 (Water Soluble Tetrazolium-1) analysis.
트리판 블루 염색 결과, 도 2a와 같이 DMSO 동결보존액 그룹의 세포 생존율은 71.3%로, 나머지 세 그룹 (Glycerol: 64.2%, Dextran: 58.8%, Glucose: 58.4%)에 비하여 유의적으로 높은 세포 생존율을 나타냈다. 연골조직으로부터 분리된 연골 세포를 2주간 배양하여 증식률을 WST-1로 분석하였을 때, 도 2b와 같이 동결보존하지 않은 정상대조군 (control) 그룹과 DMSO 동결보존액에서 보존한 후 조직에서 분리된 세포의 증식률에 유의적인 차이가 관찰되지 않았다. 하지만 글리세롤, 덱스트란 및 글루코스 그룹은 배양 14일째 정상대조군 (control) 그룹에 비하여 유의적으로 세포의 증식률이 감소하였다.As a result of trypan blue staining, as shown in Figure 2a, the cell viability of the DMSO cryopreservation group was 71.3%, which was significantly higher than that of the other three groups (Glycerol: 64.2%, Dextran: 58.8%, Glucose: 58.4%). showed When the chondrocytes isolated from the cartilage tissue were cultured for 2 weeks and the proliferation rate was analyzed with WST-1, as shown in FIG. 2b, the cells separated from the tissue after preservation in the cryopreservation solution and the normal control group that were not cryopreserved in DMSO. No significant difference was observed in the proliferation rate. However, the glycerol, dextran and glucose group significantly decreased the cell proliferation rate compared to the normal control group on the 14th day of culture.
2. 연골조직 동결보존을 위한 DMSO 동결보존액의 처리 시간 선정2. Selection of processing time of DMSO cryopreservation solution for cryopreservation of cartilage tissue
연골조직 동결보존 후 조직 내 세포 생존율이 가장 유의적으로 높았던 DMSO 동결보존액의 적정 처리 시간을 최적화하기 위하여, 4 ℃에서 각각 2시간, 4시간, 6시간, 8시간, 10시간 동결보존액 처리에 따른 연골조직을 마이크로-CT 분석을 진행하였다. After cryopreservation of cartilage tissue, in order to optimize the appropriate processing time of the DMSO cryopreservation solution, which had the highest intra-tissue cell viability, 2 hours, 4 hours, 6 hours, 8 hours, and 10 hours cryopreservation solution The cartilage tissue was subjected to micro-CT analysis.
동결보존액에 함유하고 있는 다이메틸 설폭사이드(Dimethyl Sulfoxide, DMSO)는 황원자를 포함하고 있고 이는 높은 전자 밀도를 표현하고 있다. 따라서 마이크로-CT 분석을 통하여 조직 내에 동결보존액 삼투 현상을 평가하였다.Dimethyl sulfoxide (DMSO) contained in the cryopreservation solution contains a sulfur atom, which expresses a high electron density. Therefore, the cryopreservation solution osmosis in the tissue was evaluated through micro-CT analysis.
그 결과, 도 3과 같이 4시간까지 처리 시간이 증가할수록 마이크로-CT 값은 증가하는 추세를 보였고 4시간 이후 마이크로-CT 값은 변하지 않는 것을 관찰하였다. 이는 동결보존액을 연골조직에 처리하였을 때 4시간 후 포화 상태에 도달한다는 것을 알 수 있으므로 동결보존액 처치 시간을 4시간으로 설정하였다.As a result, as shown in FIG. 3 , as the treatment time increased up to 4 hours, the micro-CT value showed a tendency to increase, and it was observed that the micro-CT value did not change after 4 hours. It can be seen that the cryopreservation solution reaches the saturation state after 4 hours when the cartilage tissue is treated, so the cryopreservation solution treatment time was set to 4 hours.
<실시예 2> 통제속도냉각장치(Controlled Rate Freezers; CRF)를 통한 동결 온도 설정 <Example 2> Freezing temperature setting through Controlled Rate Freezers (CRF)
CRF를 이용한 연골조직의 동결 온도 조건을 최적화하기 위하여, 기존에 세포 및 타 조직에 대한 CRF 동결 조건을 참고하여 표 1과 같은 3가지 조건을 선정한 후 연골조직에서 평가하였다. 연골조직의 세포 생존율을 유지하면서 동결보존하기 위해서는 앞서 설명한 것과 같이 각종 위해요소(동결과정에서의 조직 내 얼음생성 및 잠열 방출, 보존액의 독성)로 인하여 조직의 세포 및 세포외기질의 파괴가 야기되고, 이에 따라 동결보존 후 조직의 품질이 저하되는 문제를 극복하기 위해 장기간 동결보존하기 위한 액체 질소 등을 활용하는 냉동장치로 보내기 전에 적정한 온도까지 도달시키는 과정이 중요하므로 이를 위해 CRF를 활용하였다.In order to optimize the freezing temperature conditions of the cartilage tissue using CRF, three conditions as shown in Table 1 were selected with reference to the existing CRF freezing conditions for cells and other tissues, and then evaluated in the cartilage tissue. For cryopreservation while maintaining cell viability of cartilage tissue, as described above, destruction of tissue cells and extracellular matrix is caused due to various hazardous factors (ice formation and release of latent heat in the tissue during the freezing process, toxicity of the preservative solution), Accordingly, in order to overcome the problem of deterioration of tissue quality after cryopreservation, it is important to reach an appropriate temperature before sending it to a freezer using liquid nitrogen for long-term cryopreservation, so CRF was used for this purpose.
[표 1][Table 1]
CRF 온도 설정에 사용된 동결보존액의 조건은 상기 실시예 1에서 사용한 DMSO 용액을 이용하여 동결 보존하였으며 기기 동결 과정에서 동결 그래프를 관찰하였다. The conditions of the cryopreservation solution used to set the CRF temperature were cryopreserved using the DMSO solution used in Example 1, and the freezing graph was observed during the device freezing process.
그 결과, 도 4와 같이 Protocol B는 동결보존액을 처리한 조직의 빙점에 도달하였을 때 냉각 속도를 15-20℃/min으로 냉각시키고 챔버 온도를 영하 40-80℃까지 하강함으로써 연골 조직 내 각 부위에서 과냉각 또는 잠열 방출 현상을 억제하고 균일한 속도로 냉각되도록 최적화된 조건이다. 이는 다른 조건에 비하여 연골조직 및 동결보존액의 빙점에 도달했을 때 본 동결보존기의 우점으로 급속한 냉각을 진행하는 과정에서 샘플의 온도가 안정적으로 하강하는 것을 관찰할 수 있고 Protocol A 및 Protocol C와 비교하였을 때 상대적으로 적은 과냉각 및 잠열방출 현상을 관찰할 수 있었다.As a result, as shown in FIG. 4, Protocol B cooled the cooling rate to 15-20 ° C/min when the cryopreservation solution-treated tissue reached the freezing point and lowered the chamber temperature to -40-80 ° C. It is an optimized condition to suppress supercooling or latent heat release and to cool at a uniform rate. Compared to other conditions, when the freezing point of the cartilage tissue and cryopreservation solution is reached, it is the advantage of this cryopreservation machine. In this case, relatively little supercooling and latent heat release could be observed.
<실시예 3> 연골조직의 동결 보관 온도 최적화 <Example 3> Optimization of freezing storage temperature of cartilage tissue
상기 CRF 조건의 조직 동결 후 연골조직의 보관 온도를 최적화하기 위하여, 각각 -80℃와 -180℃에서 연골조직을 4주간 보관한 후 해동하여 상기와 같은 방법으로 세포 생존율 및 세포 증식율을 평가하였다.In order to optimize the storage temperature of the cartilage tissue after tissue freezing under the CRF conditions, the cartilage tissue was stored at -80°C and -180°C for 4 weeks and then thawed to evaluate the cell viability and cell proliferation rate in the same manner as above.
그 결과, 도 5와 같이 -180℃ 보관 그룹이 -80℃ 보관 그룹에 비하여 높은 세포 생존율을 보였으나, 통계적 유의성은 관찰되지 않았고, 세포 증식율 또한 유의적인 차이를 보이지 않았다. As a result, as shown in FIG. 5 , the -180°C storage group showed a higher cell viability than the -80°C storage group, but no statistical significance was observed, and the cell proliferation rate also did not show a significant difference.
<실시예 4> 연골조직의 해동 조건 최적화 <Example 4> Optimization of thawing conditions of cartilage tissue
상기 방법으로 동결된 연골조직의 해동 조건을 최적화하기 위하여, 37℃ 수조에서 동결된 조직을 해동한 후 PBS 용액으로 세척하여 동결보존액을 제거하였고 이 과정에서 마이크로-CT 분석을 통하여 동결보존액의 제거 정도를 평가하였다.In order to optimize the thawing conditions of the cartilage tissue frozen by the above method, the frozen tissue was thawed in a water bath at 37° C. and washed with PBS solution to remove the cryopreservation solution. In this process, the degree of removal of the cryopreservation solution through micro-CT analysis was evaluated.
동결된 조직을 해동한 후 PBS 용액으로 흔들리는 장비에서 조직을 세척하였고 5분 간격으로 용액을 교체하였다. 마이크로-CT 분석 결과, 도 6과 같이 용액 교체 회수 1회에서 4회까지 마이크로-CT 값은 하강하는 것을 확인할 수 있고, 4회 이후 마이크로-CT 값은 큰 변화가 없는 것을 확인할 수 있다. 따라서 PBS 용액으로 4회 세척하였을 때 조직 내의 동결보존액은 제거되었음을 알 수 있다.After thawing the frozen tissue, the tissue was washed in a shaking device with PBS solution, and the solution was replaced every 5 minutes. As a result of the micro-CT analysis, it can be confirmed that the micro-CT value decreases from the first to the fourth solution replacement times as shown in FIG. 6 , and it can be confirmed that the micro-CT value does not change significantly after the fourth time. Therefore, it can be seen that the cryopreservation solution in the tissue was removed when washing 4 times with PBS solution.
<실시예 5> 연골조직 동결보존 전후 비교 <Example 5> Comparison before and after cryopreservation of cartilage tissue
동결보존액을 처리한 연골 조직이 빙점에 도달하였을 때 통제속도냉각장치를 통하여 냉각 속도를 15-20℃/min으로 냉각시키고 챔버 온도를 영하 40-80℃까지 하강함으로써 조직 내 각 부위에서 과냉각 또는 잠열 방출 현상을 제지하고 균일한 속도로 냉각되는 최적화 된 조건으로 연골조직의 동결보존을 진행하고 연골조직의 동결보존 전후의 품질을 다음과 같은 방법으로 분석하였다.When the cartilage tissue treated with the cryopreservation solution reaches the freezing point, the cooling rate is cooled to 15-20℃/min through the controlled-rate cooling device and the chamber temperature is lowered to -40-80℃ to supercool or latent heat in each part of the tissue. The cryopreservation of the cartilage tissue was carried out under the optimized conditions of restraining the release phenomenon and cooling at a uniform rate, and the quality of the cartilage tissue before and after cryopreservation was analyzed in the following way.
도 7A와 같이 동결보존 전후의 연골조직을 1mm 두께로 짜른 후 LIVE & DEAD 키트 염색용액(5 μL 칼세인 AM(성분 A) 및 20 μL 에티듐 호모다이머-1(성분 B)을 10 mL DPBS에 추가하여 염색 용액을 만든다)에 1시간 처리하였다. 이렇게 처리한 후 조직을 형광현미경 관찰 분석을 통하여 이미지 사진으로 세포 생존율을 확인하였고 이미지 J 분석을 통하여 정량적으로 조직의 세포 생존율을 측정하였다. 세포의 이미지 J 계수는 동결보존 전후에 연골조직에서 71.3%의 세포 생존율을 관찰할 수 있다. As shown in Figure 7A, after squeezing the cartilage tissue before and after cryopreservation to a thickness of 1 mm, the LIVE & DEAD kit staining solution (5 μL calcein AM (component A) and 20 μL ethidium homodimer-1 (component B) was added to 10 mL DPBS. added to make a dyeing solution) for 1 hour. After this treatment, the cell viability of the tissue was confirmed by image photography through fluorescence microscopy analysis, and the cell viability of the tissue was quantitatively measured through image J analysis. Image J count of cells can observe 71.3% cell viability in cartilage tissue before and after cryopreservation.
또, 도 7B와 같이 동결보존 전후의 연골 조직을 블레이드로 1mm3 크기로 균일하게 자른 후 0.15% 콜라게네이즈(Collagenase, Type II)를 16시간 처리한 후 0.25μm 필터로 필터링 한 후 트리판 블루(Trypan blue) 염색을 통하여 세포 생존율을 측정하였다. 그 결과, 세포의 수동 계수는 동결보존 전후에 연골조직에서 71.9%의 세포 생존율을 관찰할 수 있다. In addition, as shown in Figure 7B, the cartilage tissue before and after cryopreservation was uniformly cut to a size of 1 mm 3 with a blade, treated with 0.15% collagenase (Type II) for 16 hours, filtered with a 0.25 μm filter, and then trypan blue Cell viability was measured through (Trypan blue) staining. As a result, manual counting of cells can observe a cell viability of 71.9% in cartilage tissue before and after cryopreservation.
그리고, 도 7C는 위에서 콜라게네이즈로 획득한 세포를 96well 에 1x104 밀도로 분주한 후 0일, 7일, 14일 시간대에 High Sensitive Water Soluble Tetrazolium Salt(WST) 용액을 1시간 처리하여 살아있는 세포의 Dehydrogenase와 반응함으로써 흡광도 값을 통하여 세포 증식 능력을 측정하였다. WST-1 분석 결과, 동결보존에 따른 연골 조직 내 세포증식율의 감소는 관찰되지 않았으며, 세포 증식 능력이 유지되는 것을 관찰할 수 있었다.And, Figure 7C shows the cells obtained with collagenase from above were dispensed in 96 wells at 1x10 4 density, and then treated with High Sensitive Water Soluble Tetrazolium Salt (WST) solution for 1 hour on
동결된 조직을 해동한 후 PBS 용액으로 흔들리는 장비에서 조직을 세척하였고 5분 간격으로 용액을 교체하였다. 마이크로-CT 분석 결과, 도 6과 같이 용액 교체 회수 1회에서 4회까지 마이크로-CT 값은 하강하는 것을 확인할 수 있고, 4회 이후 마이크로-CT 값은 큰 변화가 없는 것을 확인할 수 있다. 따라서 PBS 용액으로 4회 세척하였을 때 조직 내의 동결보존액은 제거되었음을 알 수 있다.After thawing the frozen tissue, the tissue was washed in a shaking device with PBS solution, and the solution was replaced every 5 minutes. As a result of the micro-CT analysis, it can be confirmed that the micro-CT value decreases from the first to the fourth solution replacement times as shown in FIG. 6 , and it can be confirmed that the micro-CT value does not change significantly after the fourth time. Therefore, it can be seen that the cryopreservation solution in the tissue was removed when washing 4 times with PBS solution.
도 8은 연골조직의 동결보존 전후 글리코사미노글리칸(Glycosaminoglycan; GAG) 함량의 변화에 대해 생화학적 분석 및 조직병리학적 분석을 진행하였다. 생화학적 방법으로는 동결보존 전후의 연골조직을 수술용 블레이드로 1mm 두께로 자른후 동결건조기에서 24시간 동결건조를 진행하였다. 다음으로 동결건조 된 연골 조직을 무게를 측정한 후 파파인(Papain, Sigma) 용액에 1mg/ml 비율로 혼합한 후 60℃ 오븐에서 24시간 용해시켰다. 마지막으로 위의 용액을 Glycosaminoglycan Assay Blyscan™(1,9-디메틸메틸렌 블루) 키트를 사용하여 단위 중량 당 연골 조직내 GAG 함량으로 분석하였다. 8 shows biochemical and histopathological analysis of changes in glycosaminoglycan (GAG) content before and after cryopreservation of cartilage tissue. As a biochemical method, cartilage tissue before and after cryopreservation was cut to a thickness of 1 mm with a surgical blade and then freeze-dried in a freeze dryer for 24 hours. Next, the lyophilized cartilage tissue was weighed and mixed with papain (Papain, Sigma) solution at a ratio of 1 mg/ml and dissolved in an oven at 60° C. for 24 hours. Finally, the above solution was analyzed as the GAG content in cartilage tissue per unit weight using the Glycosaminoglycan Assay Blyscan™ (1,9-dimethylmethylene blue) kit.
조직학적 방법으로는 동결보존 전후의 연골조직을 4% 포르말린에 7일 동안 처리한 후 5% 질산 용액을 24시간 처리하여 석회질을 제거하였다. 다음으로 조직 프로세싱 및 임베딩을 통하여 조직 블락을 제조하였고 마이크로톰(Microtome) 기기를 사용하여 4μm 두께로 조직 절편을 제조하였다. 마지막으로 GAG 특이적 염색 방법인 사프라닌-O(Safranin-O) 염색을 진행하여 조직내의 GAG 발현의 정도를 이미지로 확인하였다. 분석결과, 동결보존 전후 연골조직의 GAG 함량 및 발현 분포에는 유의미한 차이가 없는 것을 관찰할 수 있었다.As a histological method, cartilage tissue before and after cryopreservation was treated in 4% formalin for 7 days and then treated with 5% nitric acid solution for 24 hours to remove calcification. Next, tissue blocks were prepared through tissue processing and embedding, and tissue sections were prepared to a thickness of 4 μm using a Microtome instrument. Finally, Safranin-O (Safranin-O) staining, a GAG-specific staining method, was performed to confirm the level of GAG expression in the tissue as an image. As a result of the analysis, it was observed that there was no significant difference in the GAG content and expression distribution of cartilage tissue before and after cryopreservation.
도 9는 상기에 기술한 방법으로 연골조직 동결보존 전후 조직에서 연골 세포를 분리한 후 2주 동안 세포를 증식시킨 후 그룹 당 500,000개의 세포를 50 ml 튜브에 넣고 400g에서 20분동안 원심분리하여 세포 펠렛 (Cell pellet)을 제작하였다. 연골 분화 배지는 100 nM 덱사메타손, 50 μg/ml 아스코르브산-2 인산염, 40 ㎍/ml L-프롤린, 10 μl/ml ITS 보충제, 1.25 mg/ml 소 혈청 알부민, 100 ㎍/ml 피루브산 나트륨염, 10 ng/ml 전환성장인자-베타 (Sigma-Aldrich, MO, USA)로 보충된 DMEM으로 구성되었다.Figure 9 is a method described above after isolating chondrocytes from the tissue before and after cryopreservation of cartilage tissue and proliferating the cells for 2 weeks. Then, 500,000 cells per group were placed in a 50 ml tube and centrifuged at 400 g for 20 minutes. A cell pellet was prepared. Chondrogenic differentiation medium was 100 nM dexamethasone, 50 μg/ml ascorbic acid-2 phosphate, 40 μg/ml L-proline, 10 μl/ml ITS supplement, 1.25 mg/ml bovine serum albumin, 100 μg/ml sodium pyruvate, 10 It consisted of DMEM supplemented with ng/ml transforming growth factor-beta (Sigma-Aldrich, MO, USA).
연골 분화 유도 3주 후, 샘플을 4% 포르말린으로 고정하고 파라핀 블락을 제조하였다. 4 μm 두께의 절편을 준비하고 제 2형 콜라겐(Type 2 collagen, 1/100; Abcam, Cambridge, UK; catalog No. ab34712)에 대한 면역조직화학분석을 진행하여 조직내 제 2형 콜라겐 분포를 측정하였고 Safranin O(Sigma-Aldrich, MO, USA)로 염색하여 황산화된 글리코사미노글리칸의 분포를 관찰하였다. Three weeks after induction of chondrogenesis, samples were fixed with 4% formalin and paraffin blocks were prepared. A 4 μm-thick section was prepared and immunohistochemical analysis was performed on
그 결과, 동결보존 후 연골 조직으로부터 분리된 연골세포 펠렛은 sGAG 와 제 2형 콜라겐이 균질하고 풍부하게 분포하고 있는 것을 관찰할 수 있었다. 이러한 결과를 통해, 연골 조직의 동결보존 이후에도 조직 내 연골세포가 생물학적으로 정상적인 기능을 유지하고 있을 뿐만 아니라 분화능력 특성 또한 유지되어 있음을 확인 할 수 있었다.As a result, it was observed that sGAG and type II collagen were homogeneously and abundantly distributed in the chondrocyte pellet isolated from cartilage tissue after cryopreservation. Through these results, even after cryopreservation of cartilage tissue, it could be confirmed that the chondrocytes in the tissue not only maintained a biologically normal function, but also the differentiation ability characteristics were maintained.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited by this. It is clear. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.
Claims (6)
동결보존액이 처리된 연골조직을 동결 보존시키는 단계; 및
상기 동결 보존된 연골조직을 30~40℃ 수조에서 해동시킨 후, 인산완충액으로 세척하여 동결보존액을 제거하는 단계를 포함하며,
상기 동결보존액은 5~10 v/v% 다이메틸 설폭사이드(Dimethyl Sulfoxide, DMSO)를 포함하는 무혈청 동결보존액인 것을 특징으로 하는 연골조직 동결보존 및 해동방법.treating the cryopreservation solution on the cartilage tissue separated from the subject;
Cryopreserving the cryopreservation solution-treated cartilage tissue; and
After thawing the cryopreserved cartilage tissue in a water bath at 30-40 ° C, washing with phosphate buffer to remove the cryopreservation solution,
The cryopreservation solution is a method for cryopreservation and thawing of cartilage tissue, characterized in that it is a serum-free cryopreservation solution containing 5-10 v/v% dimethyl sulfoxide (DMSO).
상기 동결보존액을 처리하는 단계는 동결보존액을 4℃에서 3-5시간 동안 처리하는 것을 특징으로 하는 연골조직 동결보존 및 해동방법.The method according to claim 1,
In the step of treating the cryopreservation solution, the cryopreservation and thawing method of cartilage tissue, characterized in that the cryopreservation solution is treated at 4° C. for 3-5 hours.
상기 동결 보존시키는 단계는 통제속도냉각장치(Controlled Rate Freezers; CRF)를 통해 동결보존액을 처리한 연골조직이 빙점에 도달하였을 때 냉각 속도를 15-20℃/min으로 냉각시키고 챔버 온도를 영하 40-80℃까지 하강함으로써 조직 내 각 부위에서 과냉각 또는 잠열 방출 현상을 제지하고 균일한 속도로 냉각되게 하는 것을 을 특징으로 하는 연골조직 동결보존 및 해동방법.The method according to claim 1,
In the cryopreservation step, when the cartilage tissue treated with the cryopreservation solution through a Controlled Rate Freezers (CRF) reaches the freezing point, the cooling rate is cooled to 15-20°C/min and the chamber temperature is lowered to minus 40- A method for cryopreservation and thawing of cartilage tissue, characterized in that by lowering to 80°C, supercooling or latent heat release from each part of the tissue is prevented and cooled at a uniform rate.
상기 동결보존액을 제거하는 단계는 흔들리는 장비 내에서 인산완충액으로 3-4회 세척하는 것을 특징으로 하는 연골조직 동결보존 및 해동방법.The method according to claim 1,
The step of removing the cryopreservation solution is a method of cryopreservation and thawing of cartilage tissue, characterized in that it is washed 3-4 times with a phosphate buffer solution in a shaking device.
상기 동결보존액을 제거하는 단계 이후, 마이크로-CT 분석을 통해 동결보존액 내 다이메틸 설폭사이드(Dimethyl Sulfoxide, DMSO)의 잔류량을 분석하는 단계를 더 포함하는 것을 특징으로 하는 연골조직 동결보존 및 해동방법.The method according to claim 1,
Cartilage tissue cryopreservation and thawing method, characterized in that after the step of removing the cryopreservation solution, further comprising the step of analyzing the residual amount of dimethyl sulfoxide (DMSO) in the cryopreservation solution through micro-CT analysis.
상기 DMSO의 잔류량 분석은 DMSO 용액에 함유하고 있는 황 원자의 밀도로 인하여 마이크로-CT 분석을 통하여 실시간 관찰이 가능하여 연골조직 내 동결보존액의 포집과 DMSO의 잔류량을 분석하는 것을 특징으로 하는 연골조직 동결보존 및 해동방법.6. The method of claim 5,
The analysis of the residual amount of DMSO can be observed in real time through micro-CT analysis due to the density of sulfur atoms contained in the DMSO solution. Preservation and thawing methods.
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