Ch.4 Adipose in Tissue Engineering
Cell Sources & Tissue Procurement Cell line: homogeneous, same stage of differentiation Primary Cell: in vivo condition 과유사 (varying stage of development) 위치 (epididymal 과 subcutaneous fat) 에따라 growth 와 differentiation capacity 가다름 Ideal source of autologous cell: abundant, expendable, readily obtainable Cell line Derivation Comments 3T3-L1 Mouse embryo (17-19일) Dexamethasone, methylisobutylxanthine, IGF-1 : 분화촉진 3T3-F442A Mouse embryo (17-19 일 ) Insuline - lipid 축적 TA1 Ob17 Mouse embryo 의mesenchymal stem cell Obese의 Epididymal ( 정소 ) fat Eariler preadipocyte stage Later preadipocyte stage
Processing Liposuctioned Tissue Novel Device: 100-1500ml (Filtration, Cleansing, Dissociation) 1 PBS세척 2 Collagenase (300 U/ml) 처리 (37, 30-45min) 3 Medium/10%FBS로희석및 Stopcock Valve를통해용액배출 4 5min후상분리 -upper: Mature adipocyte -lower: Preadipocyte(stromal-vascular cell) Isolation of Stromal-Vascular Cells 1 Lower Layer를 centrifugation(400g,10min) 2 Pellets을적혈구lysing buffer를넣은뒤 centrifugation(400g,10min) 3 DMEM & Ham s F12/10%FBS배지에 Pipetting후 100μm nylon mesh에 filtering 40.2-1 10 5 cells/cm 2 로flask에분주 (DMEM & Ham s F12 /10%FBS, bfgf 10ng/ml ) 5 Confluent하면 subculture
Differentiation of lipo-derived Preadipocytes Adipogenic induction 1 Confluent해진세포를 PBS로 2회세척 2 Serum-free medium으로교체 (DMEM & Ham s F12,15mM NaHCO3, 15mM HEPES, 33uM biotin, 17uM pantothenate, 10μg /ml transferrin, 100nM hydrocortisone, 66nM insulin, 0.2nM T3) ** Isobutylmethylxanthine(IBMX) 는 0.25mM의농도로초기 4-5일에만첨가 3 1-3주간배양
Mature adiopcyte Cell counting: Hoescht( 회크스트 ) staining33258 후형광현미경으로 counting Mature adipocyte 는배지에떠있기때문에일반적인배양방법으로배양하기어려움 따라서 Ceiling Culture(Sugihara et al) 방법이개발 Mature adipocyte 는 dedifferentiation 이되어세포질내의지방이제거되고 fibroblast-like cell 형태로되어야증식할수있음 배양과정초기의 fibroblast-like cell 형태는 adipocyte 보다 contamination 된세포들임 Ceiling Culture 1 플라스크에배지를완전히채운후 48 시간동안뒤집어 incubation 2 배지와미부착세포를모두꺼내어 150 μm로 filtering 하여죽은세포의 aggregate 를제거한후다시플라스크에접종후 48 시간동안뒤집어 incubation 3 위과정을반복하여순수한 adipocyte 를얻을때까지반복 4 순수한 adipocyte 를얻으면 10 일간 ceiling culture 후플라스크를뒤집고배지를정상적인양만큼넣은후 2-3 일간격으로교체 5 2-3 주간배양을하면 dedifferentiation 이되어세포질내의지방이제거되고 fibroblast-like cell 형태로증식
Breast Reconstruction 외과적수술 ( 유방절제술 ) 후또는미용적인목적으로이용 Breast는인체의 image와여성다움을나타내기때문에 mastectomy는여성에게정신적인영향을끼침현재 flap과 free flap이많이이용됨 Flap: Transverse Rectus Abdominis Musculocutaneous (TRAM, 복부의근피부부위 ) - 단점 : 치료기간이길고수술위험이크고수술부위에상처가크게생김
Breast Cancer(U.S) 200,000 환자수 150,000 100,000 50,000 0 1992 1998 2000 년도 발생연령 : 35-50year(49%), 51-64year(36%) 발생빈도 : 110case per 100,000 women 81year 이상의고령일경우는 11%
Fat Cell Adipose tissue 는신체에있어풍부하고가장큰조직이며쉽게증식할수있고외형의변화없이적출가능 자가지방이식 : 이식한지방의부피가 40-60% 감소 ( 충분한혈관이없어영양분과산소공급의부족으로세포가괴사 ) 지방흡입술에의한세포적출 ; Mechanical force 에의해세포의 90% 가손상, 나머지 10% 도 cysts( 과립 ) 나이식후부분적으로괴사 Mature adipocyte 는세포배양이힘들며따라서최근에는 preadipocytes 를이용하여배양 Preadipocyte: morphology가 fibroblast와비슷하며분화하는동안 lipid축적 비만과식이요법에관한연구에이용되나아직까지도세포의부착, 이동과주위환경에대한영향에관한연구는부족 환자의연령과폐경기등에따라지방세포의생물학적영향이다름 암환자들은방사선치료및약물치료를받았기때문에치료결과가확실하지않음
Scaffold PLGA에 preadipocyte를배양하여 adipose tissue을재건 (Patrick et al, 1999) 구조적으로는조직이비슷하게재건되었으나 too rigid하고환자가 uncomfortable함 최근에는 Non-biodegradable polymer (flurotex monofilamentexpended polytetrafluoroethylene) 에여러종류의 ECM을 coating 하여세포를배양 Injectable materials: alginate, hyaluronic acid gel HYAFF11(hyaluronic acid modified by estrificsion) 으로제조된 sponge와 Nonwoven mesh 그리고 collagen sponge에 human preadipocyte를접종한후 nude mice에이식 (3주, 8주후조직검사 ) 이중에서 HYAFF11 sponge의 preadipocyte가증식과분화가잘이루어짐 (Dennis von Heimburg et al, Plastic & Reconstructive surgery, 108: 2001)
(C. W. Patrick, The Anatomical Record, 2001) A: PLGA 에 rat preadipocyte 를배양하여재건한 adipose tissue B: Rat 의 epididymal fat pad
Microenviroment Growth factor, po2, ph, ECM, support cell PLGA microcarrier bead에 insulin, bfgf, IGF-1을 loading 하여 preadipocyte를 adipocyte 로분화시킴 Glucocorticoid(+), Arachidonic acid Retinoic acid(+/-)