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1 Korean Society for Biotechnology and Bioengineering Journal 31(2): (2016) ISSN / eissn 연구논문 연골세포와중간엽줄기세포의 3 차원 Co-culture 를통한연골화향상 황슬기, 차현명, 임진혁, 이지희, 심혜은, 김동일 * Enhanced Chondrogenesis by Three-dimensional Co-culture of Chondrocytes and Mesenchymal Stem Cells Seul-Gee Hwang, Hyun-Myoung Cha, Jin-Hyuk Lim, Ji-Hee Lee, Hye-Eun Shim, and Dong-Il Kim* Received: 24 February 2016 / Revised: 1 June 2016 / Accepted: 15 June The Korean Society for Biotechnology and Bioengineering Abstract: Two-dimensional cultivation is typically used for cell growth, but the method reduces the characteristics of chondrocytes and stem cells, and limits culture area. Therefore, development of three-dimensional culture method is needed to mimic in vivo environment, improve quality of cells and scale-up efficiently. Improving proliferation and chondrogenesis is available by co-culture of chondrocytes and mesenchymal stem cells (MSCs) that leads to interaction between two kinds of cells. However, the co-culture has problems that permeability of sphere diminishes as aggregate size increased and ratio of two kinds of cells composing each spheres is different. In this work, co-cultivation method using controlled sphere composed of chondrocytes and MSCs was established and enhanced chondrogenesis. Periosteum-derived progenitor cells (PDPCs) that are appropriate for cell therapy source of articular cartilage were used as MSCs. Controlled spheres were formed in the hanging-drop plates and shifted for being induced chondrogenesis in 35-mm non-adhesive culture dishes at a rotation rate of 60 rpm. After inducing chondrogenesis, gene expressions related with chondrogenesis were found to be improved and it was apparent that the utilization of controlled spheres promoted chondrogenesis. As a result, available numbers of cells per unit area were increased 인하대학교공과대학생물공학과 Department of Biological Engineering, Inha University, Incheon , Korea Tel: , Fax: kimdi@inha.ac.kr and chondrogenic differentiation ability was improved compared to typical two-dimensional culture. This approach shows the potential in cartilage regeneration as it can provide sufficient numbers of chondrocytes. Keywords: Chondrocytes, Co-cultures, Mesenchymal stem cells, Periosteum-derived progenitor cells 1. INTRODUCTION 줄기세포는자신과같은능력의세포를만드는자기복제능력과특정한세포로분화되는능력을가지는세포로조직이나장기를재생할수있고, 장기이식후각장기의특성에맞게분화할수있어재생의학에적합하다 [1]. 중간엽줄기세포 (mesenchymal stem cells) 란부착성성질을가지는성체줄기세포의한종류로뼈, 연골, 지방과같은근골격계세포들로분화할수있는다분화능 (multipotency) 을가진줄기세포이다. 중간엽줄기세포는체외배양이용이하고증식능력이뛰어나며원하는조직으로의분화가가능하여퇴행성관절염과척수손상, 심근경색, 당뇨질환등의치료에이용되고있다 [2]. 세포치료제로줄기세포를이용하기위해서는충분한양의세포를얻을수있는배양방법이중요하게요구된다. 중간엽줄기세포의경우부착하여생존하는특성때문에평평한바닥이있는플라스크에서 2 차원으로부착식배양 (adhesion culture) 을한다. 이러한배양방법은줄기세포의고유성질인분화와복제능력을감소시키고부착식으로계대배

2 연골세포와중간엽줄기세포의 3 차원 Co-culture 를통한연골화향상 121 양을할경우에는세포손실과오염이발생할수있다 [3]. 또한, 세포치료제로줄기세포를이용하기위해서는 cells 이상의세포가요구되어배양면적이제한된 2차원배양방법으로대량의세포를얻는것은경제성이낮다. 따라서최근연구에서는 2차원배양방법의공간효율성및줄기세포성질이감소하는문제를보완하기위하여 3차원부유식배양 (suspension culture) 을하는방법이개발되고있다. 현재개발된많은 3차원배양방법중배아줄기세포의응집 (aggregation) 의유도로 embryonic body를형성시키는방법이중간엽줄기세포배양에도적용되어구형의 sphere를형성시키는 3 차원배양방법이개발되었다 [4]. 세포의 aggregation을유도하여 3차원배양하면세포사이의신호전달이활발하게이루어지며 2차원배양방법보다생체내환경을더유사하게재현할수있기때문에세포의치료적잠재성을높일수있다 [5]. 3차원배양방법과함께세포치료제분야에서줄기세포를이용함에있어세포의증식과특성을증대시키기위해공배양 (co-culture) 방법이적용되었다. Co-culture는둘또는그이상의다른종류의세포를같은환경에서배양하여세포간상호작용을통해단일배양 (monoculture) 보다세포의증식이나특성이증대될수있다 [6]. 따라서세포의성질을극대화하기위하여치료목적에맞게 co-culture를이용한다양한시도가있었다 [7]. 그예로연골세포와중간엽줄기세포를 coculture 하면중간엽줄기세포에서분비되는 fibroblast growth factor-1 (FGF-1) 이줄기세포와연골세포의증식에긍정적인영향을미친다 [8]. 이와같은 co-culture 방법은중간엽줄기세포가분비하는 thrombospondin-2가연골의재생능력을증대시켜연골로의분화도증진시킨다고알려져있다 [9]. 본연구에서사용된골막유래전구세포 (periosteum-derived progenitor cells, PDPCs) 는중간엽줄기세포표지자 (cluster of differentiation, CD) 인 CD9, CD90, CD166에양성을나타내고, 조혈모세포에서발현되는표지자인 CD45에음성을나타내는 PDPCs는높은증식력을가지고장기간배양후에도중간엽줄기세포의특이적인성질을유지할수있다. 또한, 윤리적문제가없고세포이식후에도면역거부반응이없다는장점이있다 [10]. 이러한특성을가진 PDPCs의연골화를향상시켜세포치료제에필요한대량의세포를얻는것을목적으로연골세포와 co-culture하여분화능을증진시키고경제적으로세포를배양할수있는 3차원배양방법을적용하였다. 연골세포와 PDPCs를 3차원으로배양하기위하여 hanging-drop 방법으로두세포가함께포함된구형의 sphere를형성시키고, 배양중물질전달능력의감소를방지하기위해 rotation platform을이용하여 3차원 co-culture를하였다. 연골세포만단일로배양한경우와비교하여줄기세포와연골세포를함께 co-culture를하였을때증식률이높았고, 분화능이증진되어연골화가촉진된것을확인하였다. 이를통하여세포치료제로사용가능한대량의연골세포를효율적으로얻을수있는방법을제시하였다. 2. MATERIALS AND METHODS 2.1. 연골세포의분리및배양연골세포의분리를위한연골조직은정형외과의인공관절대체수술후획득한조직을사용하였다. 연골조직을 phosphate buffered saline (PBS; Invitrogen, CA, USA) 으로 3 회세척한다음 Dulbecco's modified Eagle's medium (DMEM; Invitrogen) 으로옮겼다. 세포외기질을제거하기위해 0.2% collagenase type II (Worthington biochemical, NJ, USA) 를첨가하고, 24 시간반응후상등액을취하여 1,200 rpm 에서 3 분간원심분리를하였다. 상등액을제거하고침전물을 PBS 로 3 회세척하여연골조직으로부터연골세포를분리하였다. 분리된연골세포가 80% 증식하였을때계대배양을하였고, 0.05% trypsin-edta (Invitrogen) 를사용하여세포를회수하였다 골막유래전구세포의분리및배양정형외과의수술을통해얻은골막조직을 24 시간이내에초대배양하여골막유래세포 (periosteum-derived cells, PDCs) 를분리하였다. 골막조직을 PBS 로세척한후, 작은절편으로자르고조직의형성층을 24-well plate (SPL Life science, Seoul, Korea) 에부착시키고, 10% fetal bovine serum (FBS; Invitrogen) 과 1% penicillin/streptomycin (P/S; Invitrogen) 이첨가된 DMEM 을넣어주었다. 5% CO 2 가공급되는 37 o C, CO 2 incubator (Sanyo Electric, Osaka, Japan) 에서 7 일동안배양하여조직으로부터세포를분리하였다. 충분한양의 PDCs 를회수하여 FACSVantage (Becton Dickinson, NJ, USA) 로 PDPCs 만선택적으로분리하였다 [10]. 분리된 PDPCs 는 T- flask (SPL Life science) 에 10% FBS 와 1% P/S 가포함된 DMEM 을사용하여배양하였다 연골세포와골막유래전구세포의 3 차원 co-culture 분리된연골세포와 PDPCs 의 aggregation 을유도하여 sphere 를형성시킨후 rotation platform 을이용하여배양하였다. 연골세포와 PDPCs 를 3:7 의비율로혼합하여총 cells/well 의농도로 Perfecta3D 96-well hanging drop plate (3D Biomatrix, MI, USA) 에접종하였다. 배지는 10% FBS 와 1% P/S 가포함된 DMEM 을사용하고, 3 일간배양한후에는세포부착유도인자가없는 35-mm culture dish (SPL Life science) 로세포를옮겼다. 3 차원배양을하기위해서 35-mm culture dish 에서배양을할경우에는 Cha et al. 의연구결과를바탕으로하였다 cells/ml 의농도로 2 ml 의 StemPro Chondrogenesis Differentiation Kit (Invitrogen) 를첨가한후 orbital shaker (Vision Scientific, Daejeon, Korea) 에서 60 rpm 으로 5% CO 2 가공급되는 37 o C, CO 2 incubator 에서배양하였다 WST-1 assay WST-1 assay는기질인 high sensitive water soluble tetrazolium salt가살아있는세포의 dehydrogenase와반응하여수용성 formazan을생성하는미토콘드리아의능력을이용한세포증식

3 122 Korean Society for Biotechnology and Bioengineering Journal 31(2): (2016) 측정법이다. 새로운배지로교환한후에 EZ-Cytox (itsbio, Seoul, Korea) 를첨가하였다. 빛을차단하고 37 o C, 5% CO 2 가공급되는 CO 2 incubator 에서 1 시간반응시켰다. 형성된수용성 formazan 을 96-well plate (Nunc, NY, USA) 에옮기고 Biotrack II plate reader (GE Healthcare, NJ, USA) 를이용하여 450 nm 에서흡광도를측정하였다 역전사중합효소연쇄반응 (RT-PCR) 연골세포와줄기세포의특이유전자를확인하기위해배양한세포를회수하여 RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) 로 total RNA 를추출하였다. 추출한 RNA 의농도를측정한후 Maxime RT Premix Kit (intron, Seongnam, Korea) 에첨가하고 T100 Thermal Cycler (Bio-Rad, CA, USA) 를이용하여 45 o C 에서 60 분, 95 o C 에서 5 분동안반응시켜 cdna 를합성하였다. PCR 은합성된 cdna 와관련유전자의 primer 를 AccuPower PCR Premix (Bioneer, Daejeon, Korea) 에넣고 T100 Thermal Cycler 를이용하여 primer 의합성조건에맞추어반응시켰고, 1.5% agarose gel 에서전기영동을수행하여관련유전자를확인하였다. 정량적 RT-PCR 은합성된 cdna 에 primer 와 iqtm SYBR Green Supermix (Bio-Rad, CA, USA) 를넣고 Rotor-Gene Q (Qiagen, Hilden, Germany) 를사용하여각 primer 의합성조건에맞추어 40 회반복수행하였고, melting curve 를확인하기위해 o C 까지 0.2 o C 마다형광값을확인하였다. 사용한 primer 의정보와조건은 Table 1 과같다 Glycosaminoglycan assay Glycosaminoglycan (GAG) 를정량분석하기위하여배양된세포를 PBS 로세척하고 papain extraction reagent 를첨가한후에 60 o C 에서 3 시간동안반응시켰다. 그후 10,000 g 에서 10 분동안원심분리하여상등액을회수하고, Blyscan Assay Kit (Biocolor, County Antrim, UK) 를사용하여 GAG 를염색하였다. 염색된 GAG 를 12,000 rpm 에서 10 분간원심분리하여얻은침전물에 dissociation reagent 를첨가한후 96-well plate 에옮겨 Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific, MA, USA) 를이용하여 656 nm 에서흡광도 를측정하였다. 3. RESULTS AND DISCUSSION 3.1. 연골세포와골막유래전구세포의 3 차원 co-culture 방법확립 3 차원 co-culture 방법으로연골세포와 PDPCs 를배양하기위하여세포간의 aggregation 을유도하여 sphere 를형성시켰다. Co-culture 에서는두세포의비율이 sphere 의형성에중요한역할을하여한개의 sphere 를형성시킬때 hanging-drop 방법으로연골세포와 PDPCs 의비율조절을하였다. 연골세포의비율이높은환경에서는 sphere 형성이이뤄지지않았다 (Fig. 1(a)-1(c)). 하지만, PDPCs 의비율이높아질수록 aggregation 의유도가증진되어구형에가까운 sphere 가형성되었다 (Fig. 1(d)-1(f)). 연골세포와 PDPCs 의비율이 3:7 일경우에구형의 sphere 형태가가장잘유지되는것을확인하였다 (Fig. 1(d)). 중간엽줄기세포에서 sphere 를형성시켜배양하는방법은세포를서로접촉시켜세포가접촉할곳을잃고부유상태가되어사멸하는 anoikis mechanism 을극복하는것으로밝혀져왔다 [7]. 이러한줄기세포의특성때문에 co-culture 환경에서 PDPCs 의비율이높을수록 sphere 가잘형성된것으로사료된다 Co-culture 환경에서세포증식확인 Hanging-drop 방법으로 aggregation 이유도된연골세포와 PDPCs 의 co-culture 환경에서세포의증식을확인하였다. 연골세포만을배양한단일배양과연골세포와 PDPCs 를 3:7 의비율로혼합한 co-culture 의증식률을비교한결과, co-culture 조건에서단일배양보다더높은증식률을나타내었다 (Fig. 2). 연골세포와줄기세포의 co-culture 를진행하면줄기세포의사이토카인 (cytokine) 생성을통해연골세포의증식을자극하는것으로알려져있다 [12]. 또한, 3 차원배양에서줄기세포와 co-culture 를통해구형의 sphere 형성으로세포의접촉을증가시켜 anoikis mechanism 이유도되지않고세포사멸을억제하여증식률을높일수있다. 따라서본연구에서도 Table 1. Oligonucleotide primers for the detection of target mrnas mrna Sense / antisense sequences Size (bp) Annealing temperature ( o C) GAPDH 5'-GCTCTCCAGAACATCATCCCTGCC-3' 5'-CGTTGTCATACCAGGAAATGAGCTT-3' Nanog 5'-CAGAAAAACAACTGGCCGAA-3' 5'-GGCCTGATTGTTCCAGGATT-3' Oct4 5'-AAGCGATCAAGCAGCGACTA-3' 5'-CCTCAGTTTGAATGCATGGG-3' Sox9 5'-GCCAGGTGCTCAAAGGCTA-3' 5'-TCTCGTTCAGAAGTCTCCAGAG-3' Aggrecan 5'-CTGCTTCCGAGGCATTTCAG-3' 5'-CTTGGGTCACGATCCACTCC-3' GAPDH: glyceraldehyde-3-phosphate dehydrogenase

연골세포와중간엽줄기세포의 3 차원 Co-culture 를통한연골화향상 123 Fig. 2. Determination of proliferation in monocultures and co-cultures with three-dimensional condition.

Comparison of sphere formation in the ratios of chondrocytes:pdpcs as follows: (a) 10:0, (b) 8:2, (c) 7:3, (d) 3:7, (e) 2:8 and (f) 0:10.

4 연골세포와중간엽줄기세포의 3 차원 Co-culture 를통한연골화향상 123 Fig. 2. Determination of proliferation in monocultures and co-cultures with three-dimensional condition. The co-cultures of chondrocytes and PDPCs showed a higher growth rate. The criterion for significant differences is: * for p<0.05. 있기때문에 3 차원 co-culture 를이용하여대량의연골세포를얻는과정에서경제성과효율성을높일수있었다. Fig. 1. Comparison of sphere formation in the ratios of chondrocytes:pdpcs as follows: (a) 10:0, (b) 8:2, (c) 7:3, (d) 3:7, (e) 2:8 and (f) 0:10. When the ratio of chondrocytes and PDPCs was 3:7, the spheres were suitable for three-dimensional co-cultures. 줄기세포를이용한 co-culture 가세포의증식에긍정적인영향을주는것을확인하였다. 형성된 sphere 를 rotation platform 을이용하여대량으로분화배양을할경우에도 agglomerate 가형성되지않고구형의형태를유지하였다 (Fig. 3). 이러한결과는줄기세포가연골로분화되는과정에서 aggregation 발생의원인이되는 E-cadherin 의발현이감소하고, 세포가구형으로변화하는특징을가지고있기때문이다 [13]. 또한, 기존의 2 차원배양방법과단위면적당배양가능한세포의수를비교하면 3 차원배양에서 10 배더많은세포를배양할수 차원배양에서연골세포로의분화확인 3 차원 co-culture 환경에서연골세포로분화를유도한후, 연골세포와줄기세포의특이적인유전자를이용하여분화가이루어졌는지유전자수준에서확인하였다. 분화배양 2, 4, 6 일차에 RT-PCR 을통하여연골세포와관련된유전자인 Sox9 과 Aggrecan 발현을가시적으로관찰하였다. 각각의유전자는중간엽줄기세포인 PDPCs 만단일배양했을때와비교하여 co-culture 조건에서더높은수준으로발현되었다 (Fig. 4). 줄기세포를단일배양한경우에는 Aggrecan 유전자의발현을분화 6 일차에는확인할수없었다. 이는줄기세포비율이높은조건에서 aggregation 을유도하면 Fig. 1 에서확인한것처럼 sphere 의크기가증가하여내부에위치한세포까지물질전달이이루어지기힘들기때문에세포의상태에영향을주거나탈분화가일어난것으로사료된다. 추가적으로, 분화배양 6 일차에연골세포와줄기세포관련유전자발현율을정량하였다. Co-culture 조건에서연골세포유전자인 Sox9 과 Aggrecan 은상대적으로많이발현이되었고, 줄기세포유전자인 Nanog 와 Oct4 는거의발현이되지않았다. 단일배양으 Fig. 3. Sphere formation during chondrogenic differentiation of chondrocytes and PDPCs at (a) day 3, (b) 6 and (c) 9. Homogeneous size of sphere was maintained in the co-culture condition.

124 Korean Society for Biotechnology and Bioengineering Journal 31(2): 120-125 (2016) Fig. 4. mrna expressions of chondrogenesis marker genes after inducing chondrogenesis.

Evaluation of expression level of chondrocyte genes (Sox9 and Aggrecan) and stemness genes (Nanog and Oct4) at day 6.

4의결과와같이연골세포관련유전자는발현율이낮았으며 Nanog와 Oct4 유전자는높게발현되어줄기세포의성질이유지된것을확인하였다 (Fig. 5). 줄기세포와연골세포를함께배양하면줄기세포에의해서분비되는 thrombospondin-2가연골의재생능력을증대시켜연골화를증진시킬수있으며 FGF-1 분비도촉진되어연골세포의증식에도움을줄수있다.

5 124 Korean Society for Biotechnology and Bioengineering Journal 31(2): (2016) Fig. 4. mrna expressions of chondrogenesis marker genes after inducing chondrogenesis. Sox9 and Aggrecan genes were highly detected in co-culture of chondrocytes and PDPCs. Fig. 5. Evaluation of expression level of chondrocyte genes (Sox9 and Aggrecan) and stemness genes (Nanog and Oct4) at day 6. Cocultures showed a significantly enhanced chondrogenesis and had low-level expression of stemness genes. 로 PDCPs를연골세포로분화시키면 Fig. 4의결과와같이연골세포관련유전자는발현율이낮았으며 Nanog와 Oct4 유전자는높게발현되어줄기세포의성질이유지된것을확인하였다 (Fig. 5). 줄기세포와연골세포를함께배양하면줄기세포에의해서분비되는 thrombospondin-2가연골의재생능력을증대시켜연골화를증진시킬수있으며 FGF-1 분비도촉진되어연골세포의증식에도움을줄수있다. 이를바탕으로, 연골세포와 PDPCs의 co-culture 환경조성이연골세포로의분화효율을높일수있음을증명하였다. 연골세포로의분화를단백질수준에서증명하기위하여연골세포의특이단백질인 GAG의발현량을정량분석하였다. 줄기세포와연골세포를함께배양하였을때 GAG의발현량이꾸준히증가하였으며분화배양 12일차에는단일배양에비해서 GAG의발현이 6.2배증가하였다. 단일배양조건에 Fig. 6. Quantification of total GAG at day 0, 6, 12 after inducing 서는 GAG의발현이낮았을뿐만아니라 6일이후에는발현 chondrogenesis. At day 12, co-cultures of chondrocytes and PDPCs showed increased quantification of GAG, whereas amount of GAG 량이현저히감소하였다 (Fig. 6). 연골세포의경우배양환경 was decreased in PDPCs monocultures. The criteria for significant 에따라탈분화가일어나세포의성질이감소할수있기때문 differences are: * for p<0.05 and *** for p< 에세포를배양하는방법이중요하다 [14]. 따라서연골세포의탈분화과정없이연골세포로분화효율을증진시킨 3차원 co-culture 방법을이용하여대량의연골세포를효율적으로얻을수있을것이다.

6 연골세포와중간엽줄기세포의 3 차원 Co-culture 를통한연골화향상 CONCLUSION 본연구에서는퇴행성관절염치료에사용되는연골세포를대량으로얻는것을목적으로 3 차원 co-culture 방법을확립하였다. 연골세포와줄기세포의 aggregation 을유도하여 sphere 형성을위한최적의비율을선정하였고, 형성된 sphere 의증식률을확인하였다. 연골세포와 PDPCs 를 3:7 비율로 hangingdrop 방법을이용하여단일의 sphere 를형성시킨후, rotation platform 에서분화배양할경우에도 sphere 형태가계속유지되었다. 또한, 두세포의 co-culture 를통해서단일배양으로연골세포를배양했을때문제점인느린증식률을극복하였다. Co-culture 조건에서연골세포로분화를유도한결과, 연골세포의특이유전자및단백질의발현이높게나타난것을확인하였다. 이처럼, PDPCs 와연골세포의 co-culture 를통해서연골세포로분화율을높이고, 3 차원배양방법으로단위면적당배양가능한세포수를증가시켜단기간에대량의연골세포를얻을수있었다. 미립운반체 (microcarrier) 또는지지체 (scaffold) 없이 sphere 형성을유도하여개발된 3 차원 co-culture 방법은경제성이매우높아상업적으로큰장점을가지고있기때문에세포치료제생산공정에활용될수있을것으로기대된다. Acknowledgements 본연구는정부 ( 미래창조과학부 ) 의재원으로한국연구재단바이오의료기술개발사업 (No. NRF-2013M3A9B ) 으로수행되었으며이에감사드립니다. REFERENCES 1. Fernandes, T. G., C. A. V. Rodrigues, M. M. Diogo, and J. M. S. Cabral (2014) Stem cell bioprocessing for regenerative medicine. J. Chem. Technol. Biotechnol. 89: Fernndez Vallone, V. B., M. A. Romaniuk, H. Choi, V. Labovsky, J. Otaegui, and N. A. Chasseing (2013) Mesenchymal stem cells and their use in therapy: What has been achieved? Differentiation 85: Breslin, S. and L. O Driscoll (2013) Three-dimensional cell culture: the missing link in drug discovery. Drug Discov. Today 18: Page, H., P. Flood, and E. G. Reynaud (2013) Three-dimensional tissue cultures: current trends and beyond. Cell Tissue Res. 352: Bartosh, T. J., J. H. Ylostalo, A. Mohammadipoor, N. Bazhanov, K. Coble, K. Claypool, R. H. Lee, H. Choi, and D. J. Prockop (2010) Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc. Natl. Acad. Sci. USA 107: Leijten, J. C., N. Georgi, L. Wu, C. A. van Blitterswijk, and M. Karperien (2012) Cell sources for articular cartilage repair strategies: shifting from monocultures to cocultures. Tissue Eng. Part B Rev. 19: Frith, J. E., B. Thomson, and P. G. Genever (2009) Dynamic threedimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng. Part C Methods 16: Wu, L., X. Cai, S. Zhang, M. Karperien, and Y. Lin (2013) Regeneration of articular cartilage by adipose tissue derived mesenchymal stem cells: perspectives from stem cell biology and molecular medicine. J. Cell. Physiol. 228: Jeong, S. Y., D. H. Kim, J. Ha, H. J. Jin, S. Kwon, J. W. Chang, S. J. Choi, W. Oh, Y. S. Yang, and G. Kim (2013) Thrombospondin2 secreted by human umbilical cord bloodderived mesenchymal stem cells promotes chondrogenic differentiation. Stem Cells 31: Choi, Y. S., S. E. Noh, S. M. Lim, C. W. Lee, C. S. Kim, M. W. Im, M. H. Lee, and D. I. Kim (2008) Multipotency and growth characteristic of periosteum-derived progenitor cells for chondrogenic, osteogenic, and adipogenic differentiation. Biotechnol. Lett. 30: Cha, H. M., S. M. Kim, Y. S. Choi, and D. I. Kim (2015) Scaffoldfree three-dimensional culture systems for mass production of periosteum-derived progenitor cells. J. Biosci. Bioeng. 120: Acharya, C., A. Adesida, P. Zajac, M. Mumme, J. Riesle, I. Martin, and A. Barbero (2012) Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation. J. Cell. Physiol. 227: Kinney, M. A., T. A. Hookway, Y. Wang, and T. C. McDevitt (2014) Engineering three-dimensional stem cell morphogenesis for the development of tissue models and scalable regenerative therapeutics. Ann. Biomed. Eng. 42: Meretoja, V. V., R. L. Dahlin, S. Wright, F. K. Kasper, and A. G. Mikos (2014) Articular chondrocyte redifferentiation in 3D co-cultures with mesenchymal stem cells. Tissue Eng. Part C Methods 20:

Product Description Apoptosis 혹은 necrosis등에의하여죽거나손상된세포에서방출되는 Lactate dehydrogenase(ldh) 의양을고감도로측정함으로써 cytotoxicity/cytolysis를간단하게측정할수있는 kit 입니다. Cytot

EZ-LDH Cell Cytotoxicity Assay Kit Cat. No. DG-LDH500 DG-LDH1000 FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. 1 ( 주 ) 대일랩서비스부설두젠생명공학연구소 Product Description Apoptosis 혹은 necrosis등에의하여죽거나손상된세포에서방출되는