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계대 및 배양액이 슈반세포의 증식과 표현형에 미치는 영향
오아영 ( A Young Oh ),김순희 ( Soon Hee Kim ),정수현 ( Su Hyun Jung ),홍현혜 ( Hyun Hye Hong ),전나리 ( John M. Rhee ),이종문,신형식 ( Hyung Sik Shin ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.3
Schwann cell(SCs) has been widely accepted to play indispensable roles during neural development and regeneration but obtaining and culture of SCs is difficulty. SCs have been characterized depending on sequential passages and medium. We measured proliferation and phenotypical stability of SCs using three medium(growth medium, purification medium, high density culture medium). Morphological changes were observed by an optical microscope and cell proliferation was counted using hemacytometer. The effect of sequential passage and medium on the phenotypical stability of SCs was assessed RT-PCR and staining for NF and S-100 as SCs markers. In result, proliferation of SCs showed no difference between three groups up to passage 3. NF and S-100 markers were high expressed in up to passage 3 in PM and HDCM. We concluded that SCs of passage 1, 2 and 3 cultured in HDCM will be useful to cell therapy using SCs or spinal cord regeneration using SCs hybrid scaffold.
3D 형태의 PLGA 지지체에서 뷰틸레이트하이드록시애니솔의 방출거동 및 골수간엽줄기세포의 신경세포화
오아영 ( A Young Oh ),김순희 ( Soon Hee Kim ),모종현 ( Jong Hyun Moh ),김근아 ( Geun Ah Kim ),이종문 ( John M. Rhee ),이해방 ( Hai Bang Lee ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.2
Bone marrow stromal cells(BMSCs) can be differentiated into chondrocytes, osteocytes, neuronal cells under the controlling of culture condition. We fabricated butylated hydorxyanisole(BHA) loaded poly(L-lactide-coglycolide) (PLGA) scaffolds(BHA/PLGA) for neurogenesis of BMSCs and to observe the release behavior of BHA. BHA/PLGA scaffold were manufactured as the concentrations of 200, 400, and 600 μM of BHA by solvent casting/ salt leaching method. We isolated and cultured BMSCs from the tibias of adult female Fischer rat, we seeded BMSCs into the inner core of the hybrid scaffold. Cellular viability and proliferation were assayed by 3-(4,5-dimethylthiazol- 2yl)-2,5-diphenyl-2H- tetrazolium bromide(MTT) test. Morphology of celluar adhesion were confirmed by scanning electron microscope SEM). The effect of BHA/PLGA scaffold on the neural differentiation of rat BMSCs were assessed RT-PCR. According to results, aspect of release of BHA in BHA/PLGA matrix showed that the initial burst of BHA was lower in scaffold than film and released continuously during the desired period in delivery system. BHA 600 μM loaded scaffold revealed low cell viability by MTT assay results. Mophology of BMSCs changed like a neural cell in 200 and 400 μM BHA/PLGA by SEM observation. In addition, 200 and 400 μM BHA/ PLGA scaffold showed higher expression of neural markers. We conclude that neural differentiation of BMSCs in BHA/PLGA scaffold could be possible for long period.
케라틴/PLGA 지지체가 골수간엽줄기세포의 신경분화에 미치는 영향
오아영 ( A Young Oh ),김순희 ( Soon Hee Kim ),김초민 ( Cho Min Kim ),정수현 ( Su Hyun Jung ),전나리 ( Na Ri Jeon ),홍현혜 ( Hyun Hye Hong ),( Sang Jin Lee ),( Mark Van Dyke ),( James J Yoo ),이동원 ( Dong Won Lee ),이종문 ( 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.1
Bone marrow stromal cells(BMSCs) exhibit multiple traits of a stem cell population, and they can expand many times in vitro and be induced to differentiate into multiple cell types. Keratin is the major structural fibrous protein providing outer covering such as wool, hair, and nail. We think keratins are useful as natural biomaterial. We examined the effect of Keratin/PLGA scaffold on the neural differentiation of rat BMSCs. We developed the keratin loaded poly(L-lactide-co-glycolide)(PLGA) scaffolds. Keratin/PLGA 0, 20 and 50 wt% scaffolds were prepared by solvent casting/salt leaching method. BMSCs were harvested from the femurs of adult female Fischer rat. These cells were seeded in prepared Keratin/PLGA scaffold and cultured in Medium 50 ml DMEM, 2 %DMSO, 200 ?M BHA, 25 ?M valproic acid, 10 ?M forskolin, 1 ?M hydro-cotisone, 5?g/ml insulin for 5 day. The effect of Keratin/PLGA scaffold on the neural differentiation of rat BMSCs were assessed in culture using the MTT assay and RT-PCR was conducted to confirm mRNA expression of NSE and Nf for neural marker. We confirmed that effect of Keratin/PLGA scaffold on the neural differentiation of rat BMSCs. According to our results, 20 wt% keratin/ PLGA scaffold have good cell compatibility and the expression of neural markers was higher. In conclusion, Keratin/ PLGA scaffold, on which neural differentiation of rat BMSCs was possible.
케라틴을 함유한 PLGA 지지체가 슈반세포의 증식 및 특성 유지에 미치는 영향
오아영 ( A Young Oh ),김순희 ( Soon Hee Kim ),정수현 ( Su Hyun Jung ),홍현혜 ( Hyun Hye Hong ),전나리 ( Na Ri Jeon ),( Sang Jin Lee ),( Mark Van Dyke ),( James J. Yoo ),이종문 ( John M. Rhee ),강길선 ( Gilson Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.4
Keratin is the major structural fibrous protein such as wool, hair, and nail and is useful as natural protein. Schwann cells(SCs) can stimulate tissue sparing and enhance outgrowth of both intact and lesion axons. We were prepared keratin/PLGA(0, 20 and 50 wt%) scaffolds by solvent casting/salt leaching method. Cellular viability and proliferation were assayed by MTT test. Morphology of cellular adhesion were confirmed by scanning electron microscope(SEM). SCs specific protein was assessed staining by s-100 for SCs marker and RT-PCR was conducted to confirm mRNA expression of NF, P-75 and s-100 for SCs marker. In MTT assay results, cell viability in scaffolds impregnated 20 wt% of keratin were higher than other scaffolds. SEM exhibited normal spindle-shaped morphology on 20 wt% of keratin. SC specific mRNA expression and protein could not be observed in scaffold containing 50 wt% of keratin. These results suggest that keratin provide suitable surface to neural cells and proper content affect on culture condition to improve cell adhesion and proliferation.
유화동결건조법으로 제조된 HA/PLGA 지지체를 이용한 연골 재생
정수현 ( Su Hyun Jung ),장지욱 ( Ji Wook Jang ),김순희 ( Soon Hee Kim ),홍현혜 ( Hyun Hye Hong ),오아영 ( A Young Oh ),이종문 ( John M. Rhee ),강영선 ( Young Sun Kang ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.4
Adult articular cartilage tissue has poor capability of self-repair. Therefore, a variety of tissue engineering approaches are motivated by the clinical need for articular repair. PLGA and hyaluronic acid(HA) has been widely used as biocompatible scaffolds materials to regenerate tissue. HA loaded PLGA scaffolds were prepared by a emulsion freeze-drying method. The chondrocytes were seeded on the HA-PLGA scaffolds and measured by MTT assay. Morphological observation, histology, biological assay for collagen and sGAG, and PCR were performed. In MTT assay result, scaffolds containing HA were higher cell viability then only PLGA scaffolds. Although collagen, sGAG, mRNA and collagen type II were greater than HA-PLGA scaffolds and collagen type I was less than HA/ PLGA scaffolds. When we cultured cartilage cell of rabbit in vitro, we observed better to keep the characteristic of cartilage cell in the HA-PLGA scaffolds than that PLGA scaffolds. This study suggests that HA/PLGA scaffold may serve as a potential cell delivery vehicle and a structural basis for in vitro tissue engineered articular cartilage.
PLGA/DBP 필름에서 DBP 함량에 따른 슈반씨세포와 후각초성세포의 부착과 성장에 미치는 영향
김수미 ( Su Mi Kim ),김순희 ( Soon Hee Kim ),김초민 ( Cho Min Kim ),오아영 ( A Young Oh ),김문석 ( Moon Suk Kim ),강길선 ( Gilson Khang ),이종문 ( John M. Rhee ),이해방 ( Hai Bang Lee ) 한국조직공학·재생의학회 2007 조직공학과 재생의학 Vol.4 No.2
Poly(lactic-co-glycolic acid)(PLGA) is a biodegradable synthetic polymer with acceptable mechanical strength and well-controlled degradation rate. Also, it can be easily fabricated into many shapes. Demineralized bone particle(DBP) contains powerful bioactive molecules. In this study, we fabricated natural/synthetic biomaterial hybrid films using 0, 10, 20, 40 and 80 % of DBP. Schwann cells(SCs) and olfactory ensheathing cells(OECs) were seeded on PLGA/DBP film and confirmed the effects of adhesion and proliferation on SCs and OECs according to content of DBP. PLGA/DBP composite films are easily fabricated and provided bioactive environment that enhance the cell adhesion and proliferation. In this study, we confirmed that specific 10~20% DBP in PLGA/DBP films was superior to adhesion and proliferation of SCs and OECs and content of DBP such as 40% and 80% could not help cell growth and proliferation. These results suggest that DBP, natural material, provide suitable surface to neural cells and proper content affect on culture condition to improve cell adhesion and proliferation. In addition, these information would contribute to design of 3-dimentional PLGA/DBP scaffold for tissue engineered spinal cord regeneration.
김수미 ( Su Mi Kim ),김순희 ( Soon Hee Kim ),박상욱 ( Sang Wook Park ),오아영 ( A Young Oh ),김근아 ( Geun Ah Kim,),이종문 ( John M. Rhee ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.1
Olfactory ensheathing cells(OECs) transplantation is a promising orpotential therapy for spinal cord injury(SCI). Bone marrow stromal cells(BMSCs) contain mesenchymal stem cells and progenitor cells. BMSCs can differentiate into various cell types, including muscle and brain cells. In this study, we examined the feasibility of using BMSCs as a source of stem cells for the differentiation into OECs by co-culture approach. Only OECs and BMSCs were cultured as control group. The experimental group designed that OECs were transferred on transwell and insert this OECs culturing were co-cultured with BMSCs attached on 6 well bottom. Another experimental group cultured BMSCs in OEC-derived conditioned medium. Cultured cells were fixed at co-culture day 3, 7, 14 and 21. The expression of GFAP and P75 were characterized by immunocytochemistry. RT-PCR was used to evaluate the gene expression of P75, NSE and NF in BMSCs co-cultured with OECs. Our results showed that BMSCs able to differentiate into OEC-like cells using co-culture system in vitro. These findings could be helpful for the development of the cell-based therapeutic strategies for CNS repair.