WO2014112675A1 - Natural cryopreservative agent for bone marrow-derived stem cells and cryopreservation method for bone marrow-derived stem cells using same - Google Patents
Natural cryopreservative agent for bone marrow-derived stem cells and cryopreservation method for bone marrow-derived stem cells using same Download PDFInfo
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- WO2014112675A1 WO2014112675A1 PCT/KR2013/000563 KR2013000563W WO2014112675A1 WO 2014112675 A1 WO2014112675 A1 WO 2014112675A1 KR 2013000563 W KR2013000563 W KR 2013000563W WO 2014112675 A1 WO2014112675 A1 WO 2014112675A1
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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
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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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
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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
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- the present invention relates to a natural cryopreservative used for cryopreservation of bone marrow-derived stem cells, and more particularly to cryopreservatives configured to protect cells and increase cell viability during cryopreservation of bone marrow-derived stem cells.
- BMSCs Bone marrow mesenchymal stem cells
- MSCs mesenchymal stem cells
- tissue regeneration has been used as cell therapy because it has no carcinogenic properties for autologous transplantation.
- MSCs derived from bone marrow and adipose tissue were used to treat bone and cartilage. These cells have also been reported to be able to treat a wide range of diseases, including spinal cord injury and myocardial infarction.
- cryopreservation of MSCs has not been developed for proper use in various clinical conditions, and large numbers of autologous cells, especially for cell therapies requiring repeated transplantation, have been developed. As it needs to be secured, clinically applicable cryopreservation standards are needed.
- Cryopreservation is a process that freezes and stores below -80 ° C to maintain biological functions in liquid nitrogen (-196 ° C) for various uses of tissues and cells. Enable long term storage. Cryopreservation is increasing in importance in recent years due to the development of cell and tissue transplantation, genetic technology, tissue engineering, in vitro fertilization, and preservation of biological data. Therefore, if long-term cryopreservation of stem cells is possible, high viability and differentiation into tissue-specific cells can be diversified in clinical applications, and autologous transplantation can prevent transplant rejection as well as homogeneous cell therapy. It will be important to
- Cryopreservatives improve the survival rate of cells after thawing by reducing the physical and chemical cell damage that occurs during freezing and thawing.
- Cryopreservatives fall into two categories. Intracellular drugs are DMSO, glycerol, and ethylene glycol, which penetrate into the cell to prevent ice crystal formation and membrane rupture. Extracellular mixtures are sucrose, trehalose, dextrose and polyvinylpyrrolidone that cannot penetrate the cell membrane and serve to reduce the high osmotic effect during the freezing process.
- the most widely used cryopreservative is DMSO, first described by Lovelock et al. In 1959. It is a hygroscopic polar compound and is known to be toxic at room temperature.
- DMSO stabilizes cellular proteins, phospholipid bilayers and plasma membranes at high concentrations of 10% (v / v) and is known to be associated with neurotoxicity in the brain (Y Liu, X Xu, X Ma et.al., 2010). ).
- thawed cells Injecting thawed cells into patients without removal of DMSO causes many side effects and toxic reactions. Examples of side effects include sedation, headache, nausea, vomiting, high blood pressure, adycardia, hypotension, and anaphylactic shock. Toxicity is known to be correlated with the amount of DMSO. DMSO should be removed or the concentration reduced. While removal of DMSO prior to infusion is most reasonable for patients, it may be costly, time consuming, or loss of cells in the removal process, so if cryopreservation reduces DMSO concentrations, there is no need to remove cryopreservatives. Thawed cells can also be injected directly.
- FBS Fetal Bovine Serum
- FBS Fetal Bovine Serum
- cryopreservatives commonly used are 10% DMSO (v / v) and up to 90% FBS (v / v), which are widely used in various types of stem cells, including human bone marrow-derived stem cells
- DMSO v / v
- FBS v / v
- cryopreservatives containing approved materials must be used for the clinical application of cryopreserved cells, and cryopreservatives need to be removed from animal-derived biological materials or components of infectious diseases. .
- Disaccharides such as sucrose and trehalose are widely used as non-toxic natural cryopreservatives and act as cell membrane stabilizers when dehydration occurs during freezing.
- trehalose is commonly found in organisms such as yeasts and nematodes that maintain life even in frozen or dehydrated plants, and these characteristics are being studied as alternative cryopreservatives.
- the usefulness of trehalose as a cryopreservative interacts with lipid membranes, stabilizes unstable proteins during freeze-thaw, inherent chemical safety, and also enables stable glasses formation during cryopreservation.
- cryopreservatives Another cause of loss of cell viability during or after freezing is the oxidation of oxygen free radicals.
- Peroxide radicals cause oxidative damage such as lipid peroxidation, protein oxidation, and DNA damage in cells, and have a protective effect by adding catalase. Therefore, the addition of antioxidants such as catalase to cryopreservatives can be expected to have cell regeneration after thawing bone marrow-derived mesenchymal stem cells. Despite improved cryopreservation, cell viability may be reduced within 24 hours after thawing.
- the mechanism of cell death during apoptosis by cryopreservation is known to be initiated by caspase activity. Protease inhibition of caspase strains in frozen injury inhibits apoptosis in various experimental environments, and zVAD-fmk is known as a caspase inhibitor.
- Korean Patent Publication No. 10-2012-0117209 discloses a cryopreservation medium composition of human-derived mononuclear cells containing DMSO, hydroxyethyl starch, autologous plasma and animal-free serum medium.
- Patent No. 10-0908481 discloses a mesenchymal stem cell culture medium including umbilical cord blood serum, insulin, hydrocortisone, EFG and LIF.
- Another object of the present invention is to provide an effective cryopreservation method for reducing physical and chemical cell damage in cryopreservation process.
- the object of the present invention is to prepare a cryopreservative with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk; Cryopreserving the bone marrow-derived mesenchymal stem cells in the cryopreservative for three weeks; Measuring and evaluating cell viability and proliferation after thawing; Measuring expression of specific surface markers and stem cell markers according to the conditions; It was achieved by identifying and evaluating the differentiation capacity after myocyte differentiation induction by the above conditions.
- the present invention has the effect of providing a cryopreservative to protect the cells in cryopreservation of bone marrow-derived mesenchymal stem cells, it is possible to maintain the long-term cryopreservation of the stem cells and to maintain a high survival rate and to differentiate into tissue-specific cells.
- FIG. 1 is a graph showing the cell viability after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,
- Figure 2 is a graph showing the cell growth curve after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,
- FIG. 3 is a diagram showing the results of flow cytometry after thawing of BMSCs cryopreserved with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,
- A is solution 1 of Table 1
- B is Solution 4
- C shows solution 7.
- FIG. 4 is a diagram showing RT-PCR results after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,
- FIG. 5 shows the differentiation capacity of BMSCs cryopreserved with a solution of trehalose, catalase and zVAD-fmk mixed with 5% (4th solution) DMSO or 2.5% (7th solution) DMSO, followed by differentiation into myocytes.
- A MyoD and desmin protein expression in Western blot of myocytes and
- B MyoD and desmin protein expression in immunofluorescence staining of myocytes.
- the experimental results were expressed as mean ⁇ standard error, and the difference between the means was obtained by t test calculator.
- the statistics were analyzed using GraphPad software, and statistical significance was set at p ⁇ 0.01 and p ⁇ 0.05.
- Cryopreservation of bone marrow-derived stem cells was tested in cryopreservatives with different combinations of ingredients, as shown in Table 1. 100% viability). All groups were allowed to contain 1 ⁇ 10 6 cells in 1 mL of the final freezing medium and frozen using a controlled-rate freezer (Cryo, Rockville, MD, USA). Each experimental group was stored in a liquid nitrogen tank for three weeks.
- the thawing of bone marrow-derived mesenchymal stem cells was rapidly thawed in a water tank set at 37 ° C., and the thawed cells were cultured for two passages until the required cell number was reached.
- Example 2 Survival analysis of bone marrow-derived mesenchymal stem cells
- Example 3 Analysis of proliferation capacity of bone marrow-derived mesenchymal stem cells
- the growth rate was similar in the 5% DMSO group containing 30% FBS, and the growth rate was higher in the group containing 5% DMSO, trehalose, catalase and zVAD-fmk.
- PTT population doubling time
- the control group was 47.6 hours, and the 5% DMSO group added with trehalose and catalase and the group added with catalase inhibitors were 45.4 and 45.7 hours, respectively (FIG. 2).
- Example 2 the cryopreserved bone marrow-derived mesenchymal stem cells were thawed under various cryopreservative conditions, and the expression of specific surface markers was analyzed by flow cytometry.
- FACS antibodies were analyzed by flow cytometry using PE-conjugated monoclonal antibodies control, CD44, CD45, CD73, CD90, CD105, SSEA4, HLA ABC and HLA DR (FACSCalibur, BD biosciences, San Jose, CA, USA) to analyze the expression.
- CD44 hyaluronan receptor
- CD73 marker of SH-3 and SH-4
- CD90 marker of Thy-1
- CD105 encodedoglin
- CD45 marker of hematopoietic lineage
- SSEA-4 stage-specific embryonic antigen
- HLA-ABC Class I major histocompatibility antigens
- HLA-DR MHC Class II
- trehalose, catalase and zVAD-fmk were added to 5% and 2.5% DMSO, showing similar results for the expression of specific surface markers under all cryopreserved conditions. Therefore, even after cryopreservation, human bone marrow-derived mesenchymal stem cells were confirmed to have characteristics (FIG. 3).
- RT-PCR Reverse transcription-polymerase chain reaction
- Each bone marrow-derived mesenchymal stem cells stored in the cryopreservative of the present invention were thawed and dispensed into cell culture plates.
- 10% horse-serum Gibco / BRL, Carlsbad, CA, USA
- 0.5% chick embryo extract Gibco / BRL, Carlsbad, CA, USA
- 1% penicillin / streptomycin A low-glucose formulation (DMEM) differentiation medium (Hyclone, Logan, UT, USA) was added and the medium was changed every 2-3 days to induce differentiation for 7 days. Thereafter, Western blotting was performed to compare the differentiation ability to myocytes.
- DMEM low-glucose formulation
- Differentiation-induced bone marrow-derived mesenchymal stem cells were washed with PBS after removal of medium and RIPA lysis buffer (15 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.5, 2 mM) Protein was extracted by adding EDTA pH 8.0, 1 ⁇ protease inhibitor cocktail). After quantification, the extracted protein was isolated from 10% SDS-poly acrylamide gel, and polyclonal anti-human MyoD (1: 1000, BD Pharmingen, San Diego, CA) was used as the primary antibody and secondary antibody was used as secondary antibody.
- RIPA lysis buffer 15 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.5, 2 mM Protein was extracted by adding EDTA pH 8.0, 1 ⁇ protease inhibitor cocktail). After quantification, the extracted protein was
- immunofluorescence staining was performed after Western blotting in order to compare the differentiation potential into myocytes.
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Abstract
The present invention relates to a cryopreservative agent for bone marrow-derived stem cells, and the present invention is achieved by the steps of: preparing a cryopreservative agent of various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk; cryopreserving for three weeks a bone marrow-derived mesenchymal stem cell introduced into the cryopreservative agent; after thawing, measuring and evaluating the cell viability and cell proliferation; and measuring the expression of a specific surface marker and stem cell marker according to the conditions, and by confirming and evaluating the differentiation ability after inducing the myocyte differentiation according to the conditions, where the present invention has an effect of providing a cryopreservative agent which protects cells when subjecting bone marrow-derived mesenchymal stem cells to cryopreservation, allows a long-term cryopreservation of stem cells and exhibits excellent effects such as maintaining high viability and achieving differentiation into tissue-specific cells.
Description
본 발명은 골수유래 줄기세포의 냉동보존에 사용되는 천연 냉동보존제에 관한 것이며 더욱 상세하게는 골수유래 줄기세포의 냉동보존시 세포를 보호하고 세포 생존율을 높이기 위해 구성한 냉동보존제에 관한 것이다.The present invention relates to a natural cryopreservative used for cryopreservation of bone marrow-derived stem cells, and more particularly to cryopreservatives configured to protect cells and increase cell viability during cryopreservation of bone marrow-derived stem cells.
골수유래 중간엽 줄기세포(bone marrow mesenchymal stem cells, BMSCs)는 자가 재생이 가능하고 배양환경에 따라 골세포, 연골세포, 지방세포, 근육모세포와 신경세포로도 분화가 가능함이 입증됨으로써 세포치료와 조직공학에서 응용되고 있다.Bone marrow mesenchymal stem cells (BMSCs) are self-renewal and have been proven to be able to differentiate into bone cells, chondrocytes, adipocytes, myoblasts and neurons, depending on the culture environment. It is applied in tissue engineering.
세포치료(cell therapy)는 많은 종류의 질병 치료에 적용되며 조직재생에서 중간엽 줄기세포(mesenchymal stem cells, MSCs)의 이용은 자가조직 이식에 발암성이 없는 특성으로 세포치료제로서 사용되고 있다. 동물실험에서는 골수와 지방조직에서 유래한 MSCs를 이용하여 뼈와 연골 치료에 효과가 있었다. 또한 이러한 세포들은 척수 손상 및 심근경색 등 질병의 다양한 범위에서 치료가 가능한 것으로 발표되었다. 그러나 MSCs의 세포 및 동물실험에서의 재생연구와 발전에도 불구하고 여러 임상 조건에 적절하게 사용하기 위한 MSCs의 냉동보존법이 개발되지 않고 있으며 특히 반복적인 이식이 필요한 세포치료를 위해서는 많은 수의 자가조직 세포 확보가 필요하므로 임상적으로 적용 가능한 냉동보존 표준법이 필요한 상황이다.Cell therapy is applied to the treatment of many diseases and the use of mesenchymal stem cells (MSCs) in tissue regeneration has been used as cell therapy because it has no carcinogenic properties for autologous transplantation. In animal experiments, MSCs derived from bone marrow and adipose tissue were used to treat bone and cartilage. These cells have also been reported to be able to treat a wide range of diseases, including spinal cord injury and myocardial infarction. However, despite the regeneration research and development in cell and animal experiments of MSCs, cryopreservation of MSCs has not been developed for proper use in various clinical conditions, and large numbers of autologous cells, especially for cell therapies requiring repeated transplantation, have been developed. As it needs to be secured, clinically applicable cryopreservation standards are needed.
냉동보존법(cryopreservation)은 조직과 세포를 다양하게 이용하기 위해 -80℃이하로 냉동하고 저장하여 일반적으로 액체질소(-196℃)에서 생물학적인 기능을 유지하는 과정으로 세포배양, 이식, 장거리 운송 및 장기저장을 가능하게 한다. 최근 의료와 생명공학 산업에서 세포 및 조직 이식, 유전기술, 조직공학, 체외수정, 생물자료의 보존 등이 발전함으로써 냉동보존은 그 중요성이 증가되고 있다. 따라서 줄기세포의 장기간 냉동보존이 가능하고 높은 생존율 유지 및 조직 특이적 세포로 분화가 가능하다면 임상 적용에의 활용이 다양화 될 수 있고 동종의 세포치료뿐만 아니라 이식거부 면역을 방지할 수 있는 자가이식에 중요하게 적용될 것이다. Cryopreservation is a process that freezes and stores below -80 ° C to maintain biological functions in liquid nitrogen (-196 ° C) for various uses of tissues and cells. Enable long term storage. Cryopreservation is increasing in importance in recent years due to the development of cell and tissue transplantation, genetic technology, tissue engineering, in vitro fertilization, and preservation of biological data. Therefore, if long-term cryopreservation of stem cells is possible, high viability and differentiation into tissue-specific cells can be diversified in clinical applications, and autologous transplantation can prevent transplant rejection as well as homogeneous cell therapy. It will be important to
냉동보존제는 냉동보존시 동결과 해동과정 동안 발생하는 물리적, 화학적인 세포손상을 줄여줌으로써 해동 후 세포의 생존율을 향상시킨다. 냉동보존제는 두 가지로 구분된다. 세포내 약물은 얼음결정 형성과 막파열을 방지하기 위해 세포 안쪽으로 침투하는 역할을 하는 DMSO, glycerol, ethylene glycol이다. 세포밖 혼합물은 세포막에 침투할 수 없고 냉동과정 중에 고삼투압 효과를 줄이는 역할을 하는 sucrose, trehalose, dextrose, polyvinylpyrrolidone이다. 가장 널리 사용되는 냉동보존제는 1959년에 Lovelock 등에 의해 처음 설명된 DMSO이며 흡습성의 극지 화합물로 실온에서는 독성이 존재한다고 알려져 있다. DMSO는 10% (v/v)의 높은 농도에서 세포 단백질, 인지질의 이중층과 원형질막을 안정화하며 뇌의 신경독성과 관련이 있는 것으로 알려져 있다(Y Liu,X Xu,X Ma et.al.,2010).Cryopreservatives improve the survival rate of cells after thawing by reducing the physical and chemical cell damage that occurs during freezing and thawing. Cryopreservatives fall into two categories. Intracellular drugs are DMSO, glycerol, and ethylene glycol, which penetrate into the cell to prevent ice crystal formation and membrane rupture. Extracellular mixtures are sucrose, trehalose, dextrose and polyvinylpyrrolidone that cannot penetrate the cell membrane and serve to reduce the high osmotic effect during the freezing process. The most widely used cryopreservative is DMSO, first described by Lovelock et al. In 1959. It is a hygroscopic polar compound and is known to be toxic at room temperature. DMSO stabilizes cellular proteins, phospholipid bilayers and plasma membranes at high concentrations of 10% (v / v) and is known to be associated with neurotoxicity in the brain (Y Liu, X Xu, X Ma et.al., 2010). ).
냉동 후에 해동된 세포를 DMSO의 제거 없이 환자에게 주입하게 되면 많은 부작용과 독성반응을 일으킨다. 부작용의 예는 진정작용, 두통, 메쓰꺼움, 구토, 고혈압, 서맥(adycardia), 저혈압, 과민증 쇼크(anaphylactic shock) 등이 있으며 이러한 독성은 DMSO의 양과 상관관계가 있다고 알려져 있어 독성을 줄이기 위해서는 냉동보존제에서 DMSO를 제거하거나 농도를 줄여야 한다. 환자에게 주입 전에 DMSO를 제거하는 방법이 가장 합리적인 반면에 제거과정에서 비용이 많이 들거나 시간이 걸리거나 세포가 손실되는 결과를 가져올 수도 있으므로 세포냉동 보존시 DMSO 농도를 줄이게 된다면 냉동보존제를 제거할 필요 없이 해동된 세포를 직접 주입할 수도 있다.Injecting thawed cells into patients without removal of DMSO causes many side effects and toxic reactions. Examples of side effects include sedation, headache, nausea, vomiting, high blood pressure, adycardia, hypotension, and anaphylactic shock. Toxicity is known to be correlated with the amount of DMSO. DMSO should be removed or the concentration reduced. While removal of DMSO prior to infusion is most reasonable for patients, it may be costly, time consuming, or loss of cells in the removal process, so if cryopreservation reduces DMSO concentrations, there is no need to remove cryopreservatives. Thawed cells can also be injected directly.
한편, FBS(Fetal Bovine Serum,소태아혈청)는 생체막을 안정화하고 냉동보존제로서 삼투압을 조정하기 때문에 냉동보존하는 동안에 세포를 보호하는 것으로 알려져 있다. 그러나 FBS는 인체의 구성성분이 아니므로 치료의 목적을 위한 세포의 보존에 이용하기에는 바람직하지 못하며 바이러스 전염, 프리온 질환, 초기의 이종개체 면역반응을 일으키는 단백질을 이환시킬 우려가 있다. FBS 대신에 자가조직의 혈청 사용은 윤리와 안전문제를 해결할 수 있으나 생산에 많은 시간이 소요되고 양이 매우 제한되어 있다.Meanwhile, FBS (Fetal Bovine Serum) is known to protect cells during cryopreservation because it stabilizes biological membranes and adjusts osmotic pressure as cryopreservative. However, since FBS is not a component of the human body, it is not preferable to use for preservation of cells for therapeutic purposes, and there is a risk of infecting proteins causing viral transmission, prion disease, and early heterogeneous immune responses. The use of autologous serum instead of FBS can solve ethics and safety issues but is time consuming and very limited in production.
보통 일반적으로 사용하는 냉동보존제의 공식화된 구성은 10% 의 DMSO(v/v), 최대 90%의 FBS(v/v)이고 이것은 인간의 골수유래 줄기세포를 포함한 다양한 종류의 줄기세포에 널리 사용되고 있으나 DMSO, FBS 모두 다수의 단점이 있으므로 냉동보존된 세포의 임상적용을 위해서는 승인된 재료가 포함된 냉동보존제를 사용해야 하며 동물유래 생물학적인 재료나 전염성 질병의 구성성분을 제거한 냉동보존제가 필요한 실정이다..Commonly formulated formulations of cryopreservatives commonly used are 10% DMSO (v / v) and up to 90% FBS (v / v), which are widely used in various types of stem cells, including human bone marrow-derived stem cells However, since both DMSO and FBS have a number of disadvantages, cryopreservatives containing approved materials must be used for the clinical application of cryopreserved cells, and cryopreservatives need to be removed from animal-derived biological materials or components of infectious diseases. .
Sucrose와 trehalose와 같은 이당류는 독성이 없는 천연 냉동보존제로 널리 사용되고 있으며 이는 냉동시 탈수 현상이 일어날 때 세포막 안정제로 작용한다. 일반적으로 trehalose는 냉동이나 탈수에서도 생명을 유지하는 효모나 선충류 같은 생물에서 흔하게 발견되었으며 이러한 특성 때문에 대체 냉동보존제로서 연구가 이루어지고 있다. 냉동보존제로서 trehalose의 유용성은 지질막과 상호 보호작용을 하고 냉동-해동 과정에서 불안정한 단백질을 안정화시키며 고유의 화학적 안전과 나아가 냉동보존 동안 stable glasses 형성 등이 가능하게 한다. Disaccharides such as sucrose and trehalose are widely used as non-toxic natural cryopreservatives and act as cell membrane stabilizers when dehydration occurs during freezing. In general, trehalose is commonly found in organisms such as yeasts and nematodes that maintain life even in frozen or dehydrated plants, and these characteristics are being studied as alternative cryopreservatives. The usefulness of trehalose as a cryopreservative interacts with lipid membranes, stabilizes unstable proteins during freeze-thaw, inherent chemical safety, and also enables stable glasses formation during cryopreservation.
냉동과정 중이나 냉동 후의 세포 생존율의 손실을 가져오는 다른 원인으로는 산소유리기 형성에 의한 산화 작용이 있다. 과산화수소(peroxide radicals)는 세포에 지질과산화 작용, 단백질 산화, DNA 손상과 같은 산화력 손상을 발생시키는데 catalase 첨가에 의해 세포 보호 효과가 있다. 따라서 냉동보존제에 catalase와 같은 항산화제의 첨가는 골수유래 중간엽 줄기세포의 해동 후 세포재생 효과가 있을 것으로 기대해 볼 수 있다. 냉동보존법을 개선함에도 불구하고 해동과정 후 24시간 이내에 세포 생존율 감소 결과가 나타날 수 있다. 냉동보존에 의한 세포자멸사 중에 세포사의 메커니즘은 caspase 활성에 의해 시작되는 것으로 알려져 있다. 냉동손상에서 caspase 계통의 protease 억제는 다양한 실험환경에서 세포자멸사를 저해하며 caspase 억제제로는 zVAD-fmk 등이 알려져 있다. Another cause of loss of cell viability during or after freezing is the oxidation of oxygen free radicals. Peroxide radicals cause oxidative damage such as lipid peroxidation, protein oxidation, and DNA damage in cells, and have a protective effect by adding catalase. Therefore, the addition of antioxidants such as catalase to cryopreservatives can be expected to have cell regeneration after thawing bone marrow-derived mesenchymal stem cells. Despite improved cryopreservation, cell viability may be reduced within 24 hours after thawing. The mechanism of cell death during apoptosis by cryopreservation is known to be initiated by caspase activity. Protease inhibition of caspase strains in frozen injury inhibits apoptosis in various experimental environments, and zVAD-fmk is known as a caspase inhibitor.
줄기세포의 동결보존방법에 대한 국내특허로는 공개특허 제10-2012-0117209호에서 PBS(phosphate buffered saline) 용액 중 수크로스, 에틸렌글리콜 및 폴리비닐피롤리돈을 포함하는 줄기세포 동결보존액이 개시된 바 있다. 국내 등록특허 제10-1101308호에는 DMSO, 하이드록시에틸 스타치, 자가혈장 및 무동물혈청 배지 를 함유한 인간유래 단핵세포의 냉동보존용 배지 조성물을 개시하였다. 중간엽 줄기세포 배지에 대한 국내특허로는 등록특허 제10-0908481호에서 제대혈 혈청, 인슐린, 하이드로코르티존, EFG 및 LIF를 포함하는 중간엽 줄기세포 배양배지를 개시하였다.As a domestic patent for a cryopreservation method of stem cells, a stem cell cryopreservation solution containing sucrose, ethylene glycol and polyvinylpyrrolidone in PBS (phosphate buffered saline) solution is disclosed in Korean Patent Publication No. 10-2012-0117209. There is a bar. Korean Patent No. 10-1101308 discloses a cryopreservation medium composition of human-derived mononuclear cells containing DMSO, hydroxyethyl starch, autologous plasma and animal-free serum medium. As a domestic patent for mesenchymal stem cell medium, Patent No. 10-0908481 discloses a mesenchymal stem cell culture medium including umbilical cord blood serum, insulin, hydrocortisone, EFG and LIF.
따라서, 본 발명의 목적은 골수유래 중간엽 줄기세포의 냉동보존에서 세포를 보호하고 세포 생존율을 향상시킬 수 있는 천연 냉동보존제를 제공하는 데 있다.Accordingly, it is an object of the present invention to provide a natural cryopreservative that can protect cells and improve cell viability in cryopreservation of bone marrow-derived mesenchymal stem cells.
본 발명의 다른 목적은 냉동보존 과정에서 물리적, 화학적 세포손상을 줄이는 효과적인 냉동보존방법을 제공하는 데 있다.Another object of the present invention is to provide an effective cryopreservation method for reducing physical and chemical cell damage in cryopreservation process.
본 발명의 상기 목적은 DMSO, FBS, 트레할로스, 카탈라제 및 zVAD-fmk의 다양한 조성으로 냉동보존제을 제조하는 단계와; 상기 냉동보존제에 넣은 골수유래 중간엽 줄기세포를 3주간 냉동보존하는 단계와; 해동 후 세포생존율 및 증식능력을 측정하고 평가하는 단계와; 상기 조건별로 특정표면 마커 및 줄기세포 마커의 발현을 측정하는 단계와; 상기 조건별로 근세포 분화유도 후 분화능을 확인하고 평가함으로써 달성하였다.The object of the present invention is to prepare a cryopreservative with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk; Cryopreserving the bone marrow-derived mesenchymal stem cells in the cryopreservative for three weeks; Measuring and evaluating cell viability and proliferation after thawing; Measuring expression of specific surface markers and stem cell markers according to the conditions; It was achieved by identifying and evaluating the differentiation capacity after myocyte differentiation induction by the above conditions.
본 발명은 골수유래 중간엽 줄기세포의 냉동보존에서 세포를 보호하는 냉동보존제를 제공하는 효과가 있으며 줄기세포의 장기간 냉동보존이 가능하고 높은 생존율 유지 및 조직 특이적 세포로 분화 가능한 뛰어난 효과가 있다.The present invention has the effect of providing a cryopreservative to protect the cells in cryopreservation of bone marrow-derived mesenchymal stem cells, it is possible to maintain the long-term cryopreservation of the stem cells and to maintain a high survival rate and to differentiate into tissue-specific cells.
도 1은 DMSO, FBS, 트레할로스, 카탈라제 및 zVAD-fmk의 다양한 조성으로 냉동보존된 BMSCs의 해동 후 세포생존율을 나타낸 그래프이고,1 is a graph showing the cell viability after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,
도 2는 DMSO, FBS, 트레할로스, 카탈라제 및 zVAD-fmk의 다양한 조성으로 냉동보존된 BMSCs의 해동 후 세포 성장곡선을 나타낸 그래프이고,Figure 2 is a graph showing the cell growth curve after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,
도 3은 DMSO, FBS, 트레할로스, 카탈라제 및 zVAD-fmk의 다양한 조성으로 냉동보존된 BMSCs의 해동 후 유세포 분석 결과를 나타낸 도 이고, (A)는 [표 1]의 1번 용액, (B)는 4번 용액, (C)는 7번 용액을 나타내었다.3 is a diagram showing the results of flow cytometry after thawing of BMSCs cryopreserved with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk, (A) is solution 1 of Table 1, (B) is Solution 4, (C) shows solution 7.
도 4는 DMSO, FBS, 트레할로스, 카탈라제 및 zVAD-fmk의 다양한 조성으로 냉동보존된 BMSCs의 해동 후 RT-PCR 결과를 나타낸 도 이고,4 is a diagram showing RT-PCR results after thawing of cryopreserved BMSCs with various compositions of DMSO, FBS, trehalose, catalase and zVAD-fmk,
도 5는 트레할로스, 카탈라제 및 zVAD-fmk을 5%(4번 용액) DMSO 또는 2.5%(7번 용액) DMSO와 혼합한 용액으로 냉동보존된 BMSCs의 해동한 다음 근세포로 분화유도한 후 분석한 분화능을 나타낸 도 이고, (A) 근세포의 웨스턴 블럿에서 MyoD 및 desmin 단백질 발현을 나타내고 (B) 근세포의 면역형광염색에서 MyoD 및 desmin 단백질 발현을 나타내었다.FIG. 5 shows the differentiation capacity of BMSCs cryopreserved with a solution of trehalose, catalase and zVAD-fmk mixed with 5% (4th solution) DMSO or 2.5% (7th solution) DMSO, followed by differentiation into myocytes. (A) MyoD and desmin protein expression in Western blot of myocytes and (B) MyoD and desmin protein expression in immunofluorescence staining of myocytes.
이하, 실시예를 들어 구체적인 내용을 상세하게 설명한다.Hereinafter, specific examples will be described in detail with reference to Examples.
재료준비Material Preparation
1-1: 골수 유래 중간엽 줄기세포 준비1-1: Preparation of Bone Marrow-derived Mesenchymal Stem Cells
경북대학교 치과대학 구강병리학교실에서 공급받은 인간 골수 유래 중간엽 줄기세포를 실험 재료로 사용하였다.Human bone marrow-derived mesenchymal stem cells supplied from the Department of Oral Pathology, College of Dentistry, Kyungpook National University, Korea, were used as experimental materials.
1-2: 냉동보존제 준비1-2: Cryopreservative Preparation
모든 냉동바이알은 세포 첨가 후에 최종 배지가 1 mL가 되도록 제조하였다. DMSO는 10%, 5%, 2.5% (v/v)의 비율로 첨가하였으며, 트레할로스(trehalose) (Sigma-Aldrich, St. Louis, MO, USA), 카탈라제(catalase)(Sigma-Aldrich, St. Louis, MO, USA), zVAD-fmk (R&D systems, Minneapolis, MN, USA)를 각각 60 mmol/L, 100 μg/mL 및 30 μM의 농도로 세포 동결바이알에 첨가하였다(표 1).All frozen vials were prepared so that the final medium was 1 mL after cell addition. DMSO was added at a rate of 10%, 5%, 2.5% (v / v), trehalose (Sigma-Aldrich, St. Louis, MO, USA), catalase (Sigma-Aldrich, St. Louis, MO, USA), zVAD-fmk (R & D systems, Minneapolis, MN, USA) were added to the cell freeze vials at concentrations of 60 mmol / L, 100 μg / mL and 30 μM, respectively (Table 1).
통계분석Statistical analysis
실험결과는 평균±표준오차로 나타내었으며, 평균의 차이는 t test calculator를 이용하였다. 통계는 GraphPad software 를 사용하여 분석하였으며, 통계적 유의성은 p<0.01, p<0.05 인 경우로 하였다.The experimental results were expressed as mean ± standard error, and the difference between the means was obtained by t test calculator. The statistics were analyzed using GraphPad software, and statistical significance was set at p <0.01 and p <0.05.
실시예 1: 골수유래 중간엽 줄기세포의 냉동보존 및 해동Example 1 Cryopreservation and Thawing of Bone Marrow-derived Mesenchymal Stem Cells
골수유래 줄기세포의 냉동보존은 [표 1]과 같이 상이한 성분의 조합을 가진 냉동보존제에 넣고 실험하였으며, 10% (v/v) DMSO와 30% FBS 용액이 든 Solution 1번을 표준 보조 용액(100% 생존도)으로 사용하였다. 모든 군에서 최종 냉동배지 1 mL에 1×106 의 세포가 함유되도록 하였고 속도제어 냉동기(controlled-rate freezer)(Cryo, Rockville, MD, USA)를 사용하여 냉동시켰다. 각 실험군은 3주간 액체질소 탱크에서 보관하였다. Cryopreservation of bone marrow-derived stem cells was tested in cryopreservatives with different combinations of ingredients, as shown in Table 1. 100% viability). All groups were allowed to contain 1 × 10 6 cells in 1 mL of the final freezing medium and frozen using a controlled-rate freezer (Cryo, Rockville, MD, USA). Each experimental group was stored in a liquid nitrogen tank for three weeks.
골수유래 중간엽 줄기세포의 해동은 37℃로 설정된 수조에서 신속하게 세포를 해동하였고 해동한 세포를 배양하여 필요한 세포수에 도달할 때까지 2계대 동안 배양하여 실험에 사용하였다.The thawing of bone marrow-derived mesenchymal stem cells was rapidly thawed in a water tank set at 37 ° C., and the thawed cells were cultured for two passages until the required cell number was reached.
실시예 2: 골수유래 중간엽 줄기세포의 생존율 분석Example 2: Survival analysis of bone marrow-derived mesenchymal stem cells
상기 실시예 1에 따라 실험한 결과 얻은 7건의 냉동보존제에 저장되었던 골수유래 중간엽 줄기세포를 해동 후 생존율을 확인하기 위해 1일째, 2일째, 3일째 각각 cck-8 assay를 실시하였다. 세포의 생존율은 Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, USA)을 이용하여 제조사의 방법으로 실험하였다. In order to confirm the survival rate after thawing the bone marrow-derived mesenchymal stem cells stored in the seven cryopreservatives obtained as a result of the experiment according to Example 1, cck-8 assay was performed on the 1st, 2nd and 3rd days, respectively. Cell viability was tested by the manufacturer's method using Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, USA).
실험 결과 대조군(10% DMSO+30% FBS)과 비교하였을 때 5% DMSO에 트레할로스와 카탈라제를 첨가한 군에서 대조군에 비해 1일째, 2일째에 통계적으로 유의한 차이를 보이는(p<0.05) 생존율을 보였으며 5% DMSO에 트레할로스, 카탈라제 및 zVAD-fmk를 함께 넣은 군에서도 통계적으로 유의한 차이를 보이며(p<0.01) 1일째, 2일째, 3일째 모두 가장 높은 생존율을 나타내었다. 반면 2.5% DMSO를 넣은 군에서는 대조군과 비교하였을 때 비슷하거나 낮은 생존율을 나타내었다(도 1).Experimental results showed that 5% DMSO added trehalose and catalase to the control group (10% DMSO + 30% FBS) showed statistically significant difference on the 1st and 2nd day compared to the control group (p <0.05). In addition, trehalose, catalase and zVAD-fmk in 5% DMSO group showed statistically significant difference (p <0.01), and the highest survival rates were seen in 1st, 2nd and 3rd day. On the other hand, in the group containing 2.5% DMSO showed a similar or lower survival rate compared to the control (Fig. 1).
실시예 3: 골수유래 중간엽 줄기세포의 증식능력 분석Example 3: Analysis of proliferation capacity of bone marrow-derived mesenchymal stem cells
3주 냉동저장 후의 골수유래 중간엽 줄기세포의 성장곡선 실험은 손 등의 방법으로 실험하였다.Growth curve experiments of bone marrow-derived mesenchymal stem cells after 3 weeks of freezing were performed by the hand method.
해동된 골수유래 중간엽 줄기세포를 8일간 배양하여 각각의 군에서 증식 능력을 분석한 결과, 전반적으로 2일째부터 증가하기 시작하여 6일째까지 서서히 증식하다가 7일째부터 급격히 증가하는 것을 확인할 수 있었다. As a result of culturing thawed bone marrow-derived mesenchymal stem cells for 8 days and analyzing the proliferative capacity in each group, it was confirmed that the overall growth started from day 2 and gradually proliferated until day 6 and then increased rapidly from day 7.
대조군과 비교하였을 때 30% FBS를 포함한 5% DMSO 군에서는 비슷한 성장속도를 보였고 5% DMSO와 trehalose, catalase를 첨가한 군과 zVAD-fmk를 포함한 군에서는 성장속도가 더 높음을 확인하였다. 각각의 PDT(population doubling time)를 계산하였을 때 대조군은 47.6시간이었고 트레할로스와 카탈라제를 첨가한 5% DMSO 군과 카탈라제 억제제를 추가로 첨가한 군에서 각각 45.4, 45.7 시간이었다(도 2). Compared with the control group, the growth rate was similar in the 5% DMSO group containing 30% FBS, and the growth rate was higher in the group containing 5% DMSO, trehalose, catalase and zVAD-fmk. When the population doubling time (PDT) was calculated, the control group was 47.6 hours, and the 5% DMSO group added with trehalose and catalase and the group added with catalase inhibitors were 45.4 and 45.7 hours, respectively (FIG. 2).
실시예 4 : 배양조건에 따른 특정표면 마커의 발현 분석Example 4 Analysis of Expression of Specific Surface Markers According to Culture Conditions
상기 실시예 2에 따라 다양한 냉동보존제 조건으로 냉동보존된 골수유래 중간엽 줄기세포를 해동하여 특정 표면 표지자의 발현을 유세포 분석법(Flow cytometry analysis)으로 분석하였다. 세포특이적 표면항원 발현을 분석하기 위해 FACS 항체는 PE-conjugated monoclonal antibodies인 대조군, CD44, CD45, CD73, CD90, CD105, SSEA4, HLA ABC 및 HLA DR을 사용하여 유세포분석(FACSCalibur,BD biosciences,San Jose,CA,USA)으로 발현유무를 분석하였다.According to Example 2, the cryopreserved bone marrow-derived mesenchymal stem cells were thawed under various cryopreservative conditions, and the expression of specific surface markers was analyzed by flow cytometry. To analyze cell specific surface antigen expression, FACS antibodies were analyzed by flow cytometry using PE-conjugated monoclonal antibodies control, CD44, CD45, CD73, CD90, CD105, SSEA4, HLA ABC and HLA DR (FACSCalibur, BD biosciences, San Jose, CA, USA) to analyze the expression.
실험 결과 CD44(hyaluronan receptor), CD73(SH-3와 SH-4의 marker), CD90(Thy-1의 marker), CD105(endoglin)가 발현되었고, CD45 (hematopoietic lineage의 marker), SSEA-4(stage-specific embryonic antigen)는 발현되지 않았다. 그리고 Class Ⅰ major histocompatibility (MHC) antigens (HLA-ABC)가 발현된 반면에 MHC Class Ⅱ (HLA-DR)은 발현되지 않았다. 대조군과 마찬가지로 5%와 2.5% DMSO에 트레할로스, 카탈라제 및 zVAD-fmk를 첨가하여 냉동보존된 모든 조건에서 특정 표면 마커의 발현에 있어 유사한 결과를 나타내었다. 따라서 냉동보존 후에도 사람의 골수유래 중간엽 줄기세포의 특성을 가지고 있음을 확인할 수 있었다(도 3).As a result, CD44 (hyaluronan receptor), CD73 (marker of SH-3 and SH-4), CD90 (marker of Thy-1), CD105 (endoglin) were expressed, and CD45 (marker of hematopoietic lineage), SSEA-4 ( stage-specific embryonic antigen) was not expressed. Class I major histocompatibility (MHC) antigens (HLA-ABC) were expressed, whereas MHC Class II (HLA-DR) was not expressed. As with the control, trehalose, catalase and zVAD-fmk were added to 5% and 2.5% DMSO, showing similar results for the expression of specific surface markers under all cryopreserved conditions. Therefore, even after cryopreservation, human bone marrow-derived mesenchymal stem cells were confirmed to have characteristics (FIG. 3).
실시예 5: 줄기세포 마커의 발현 분석Example 5: Expression Analysis of Stem Cell Markers
본 발명의 냉동보존제를 넣어 3주간 냉동보존한 골수유래 중간엽 줄기세포에서 냉동보존 후 해동하였을 때에도 줄기세포 마커를 발현하는지 확인하기 위해 RT-PCR(Reverse transcription-polymerase chain reaction)을 시행하였다. PCR에 사용된 프라이머는 Moon 등의 논문을 참고하였다(표 2). 모든 군에서 줄기세포 marker인 GATA-4, SCF, Vimentin, FGF-5, CK-18, Oct-4의 발현이 뚜렷이 확인되었으며 각 조건간에 차이는 나타나지 않았다(도 4).Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine whether stem cell markers were expressed even when thawed after cryopreservation in bone marrow-derived mesenchymal stem cells cryopreserved for three weeks. Primers used for PCR refer to Moon et al. (Table 2). In all groups, expression of the stem cell markers GATA-4, SCF, Vimentin, FGF-5, CK-18, and Oct-4 was clearly observed, and there was no difference between the conditions (FIG. 4).
실시예 6: 근세포로의 분화능 분석Example 6: Analysis of Differentiation Capacity into Myocytes
본 발명 냉동보존제에 저장되었던 각 골수유래 중간엽 줄기세포를 해동하여 세포배양 플레이트에 분주하였다. 24시간 후 근세포로 분화를 유도하기 위해 10% horse- serum(Gibco/BRL,Carlsbad,CA,USA), 0.5% chick embryo extract(Gibco/BRL, Carlsbad,CA,USA), 1% 페니실린/스트렙토마이신(Hyclone,Logan,UT,USA)이 첨가된 DMEM(low-glucose formulation) 분화배지로 교체하였고 2-3일에 한번씩 배지를 갈아주며 7일간 분화를 유도하였다. 그 후 근세포로의 분화능을 비교하기 위해 웨스턴 블랏팅 하기와 같이 시행하였다.Each bone marrow-derived mesenchymal stem cells stored in the cryopreservative of the present invention were thawed and dispensed into cell culture plates. To induce differentiation into myocytes after 24 hours, 10% horse-serum (Gibco / BRL, Carlsbad, CA, USA), 0.5% chick embryo extract (Gibco / BRL, Carlsbad, CA, USA), 1% penicillin / streptomycin A low-glucose formulation (DMEM) differentiation medium (Hyclone, Logan, UT, USA) was added and the medium was changed every 2-3 days to induce differentiation for 7 days. Thereafter, Western blotting was performed to compare the differentiation ability to myocytes.
분화 유도된 골수유래 중간엽 줄기세포는 배지를 제거하고 PBS로 세척한 후 RIPA lysis buffer(15mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50mM Tris-HCl pH 7.5, 2mM EDTA pH 8.0, 1×protease inhibitor cocktail)를 첨가하여 단백질을 추출하였다. 추출한 단백질은 정량 후 일정량 단백질을 10% SDS-poly acrylamide겔에서 분리하였으며 1차 항체로는 polyclonal anti-human MyoD(1:1000,BD Pharmingen,San Diego,CA)을 사용하였으며 2차 항체로는 secondary peroxidase-conjugated antibody(1:1000,Amersham Bioscience,Uppsala,Sweden)를 사용하여 SuperSignal West Pico 형광발색시약(Therm scientific,Inc.,Bremen, Germany)을 이용하여 X-ray 필름에 감광시켜 특정 단백질 발현을 확인하였다.Differentiation-induced bone marrow-derived mesenchymal stem cells were washed with PBS after removal of medium and RIPA lysis buffer (15 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.5, 2 mM) Protein was extracted by adding EDTA pH 8.0, 1 × protease inhibitor cocktail). After quantification, the extracted protein was isolated from 10% SDS-poly acrylamide gel, and polyclonal anti-human MyoD (1: 1000, BD Pharmingen, San Diego, CA) was used as the primary antibody and secondary antibody was used as secondary antibody. Using a peroxidase-conjugated antibody (1: 1000, Amersham Bioscience, Uppsala, Sweden), photosensitize X-ray films using SuperSignal West Pico fluorescence reagents (Therm scientific, Inc., Bremen, Germany) to express specific protein expression. Confirmed.
웨스턴 블랏팅 결과 모든 군에서 근세포 마커인 MyoD와 desmin이 발현되는 것으로 확인되었다. 그 중에서도 5% DMSO에 트레할로스와 카탈라제 및 zNAD-fmk를 첨가한 군이 2.5% DMSO에 같은 조성인 군에서보다 MyoD와 desmin의 발현이 더 증가한 것을 확인할 수 있었다(도 5 (A)).Western blotting revealed that myoD and desmin, myocyte markers, were expressed in all groups. Among them, the addition of trehalose, catalase, and zNAD-fmk in 5% DMSO increased the expression of MyoD and desmin in 2.5% DMSO than in the same composition (FIG. 5 (A)).
또, 근세포로의 분화능을 비교하기 위해 웨스턴 블랏팅 후 면역형광염색을 하기와 같이 시행하였다.In addition, immunofluorescence staining was performed after Western blotting in order to compare the differentiation potential into myocytes.
각 solution 별로 4-chamber slide (BD Biosciences, Bedford, MA, USA)에 세포를 seeding 하여 7일간 분화유도된 골수유래 중간엽 줄기세포를 desmin (BD Pharmingen, San Diego, CA)과 MyoD (BD Pharmingen, San Diego, CA) 를 각각 1:400과 1:200으로 PBS로 희석하여 37℃ 에서 1시간 동안 반응시킨 후, 2차 항체로 0.5 mg/ml의 fluorescein isothiocyanate (FITC) conjugated goat anti-mouse (Santa cruz Biotechnology, Santa Cruz, CA, USA)를 1:800, 1:400의 비율로 실온에서 1시간 정도 반응시켰으며 핵 염색은 DAPI(PI, Sigma, USA)를 사용하였으며, 형광현미경(Nikon ECLIPSE Ti-S, Tokyo, Japan)으로 관찰하였다.Each solution was seeded on 4-chamber slides (BD Biosciences, Bedford, MA, USA) to differentiate derivatized bone marrow-derived mesenchymal stem cells for 7 days from desmin (BD Pharmingen, San Diego, CA) and MyoD (BD Pharmingen, San Diego, CA) was diluted with PBS at 1: 400 and 1: 200, respectively, and reacted at 37 ° C. for 1 hour, followed by 0.5 mg / ml of fluorescein isothiocyanate (FITC) conjugated goat anti-mouse (Santa). cruz Biotechnology, Santa Cruz, CA, USA) was reacted for 1 hour at room temperature at a ratio of 1: 800, 1: 400, and nuclear staining was performed using DAPI (PI, Sigma, USA), and a fluorescence microscope (Nikon ECLIPSE Ti). -S, Tokyo, Japan).
면역형광염색 결과 대조군과 같이 5%와 2.5% DMSO에 트레할로스, 카탈라제 및 zVAD-fmk를 넣은 군이 모두 근세포 마커인 MyoD와 desmin이 발현됨을 확인할 수 있었다(도 5 (B)). 따라서 냉동보존 후 해동된 골수유래 중간엽 줄기세포는 분화배지에서 배양시 근세포로 분화가능함을 알 수 있었다.As a result of immunofluorescence staining, it was confirmed that the groups in which trehalose, catalase, and zVAD-fmk were added in 5% and 2.5% DMSO, as in the control group, expressed myoD and desmin, myocyte markers (FIG. 5 (B)). Therefore, it can be seen that bone marrow-derived mesenchymal stem cells thawed after cryopreservation can differentiate into myocytes when cultured in differentiation medium.
Claims (2)
- 냉동보존제 전체부피 1mL에 대하여 1×106 개의 세포를 함유하고 5%(v/v) DMSO, 60㎍의 트레할로스(trehalose), 100㎍의 카탈라아제(catalase), 0.03㎍의 zVAD-fmk로 구성되는 것이 특징인 골수유래 줄기세포의 냉동보존제.Contain 1 × 10 6 cells per 1 mL of total volume of cryopreservative and consist of 5% (v / v) DMSO, 60 μg trehalose, 100 μg catalase, 0.03 μg zVAD-fmk Cryopreservative of bone marrow-derived stem cells characterized in that.
- 제1항 기재의 골수유래 줄기세포의 냉동보존제를 냉동바이알에 넣은 후 속도제어 동결기를 사용하여 동결하고 액체질소 탱크에 보관하는 것을 특징으로 하는 골수유래 줄기세포의 냉동보존방법.A cryopreservation method of bone marrow-derived stem cells according to claim 1, wherein the cryopreservative of the bone marrow-derived stem cells is placed in a frozen vial and frozen using a speed-controlled freezer and stored in a liquid nitrogen tank.
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