우리는 우리의 진심으 감사 닥터 벤 파울러에 대 한 그의 기술 편 실험을 확장 하 고 싶습니다.

이 프로토콜은 오클라호마 보건 과학 센터의 대학/기관 검토 위원회 (IRB #4509)의 지침을 따릅니다. 프로토콜의 모든 부분 헬싱키의 선언의 신조를 만났다. 각 막 샘플 국가 개발 및 연구 연구소 (NDRI)와 오클라호마 라이온 스 안구 은행에서 얻은 했다.
1입니다. 1 차 셀의 격리
<ol><li>21.5 cm2 무 균 Dulbecco 인산 염 버퍼 식 염 수 (DPBS)의 2 개 mL를 포함 하는 배양 접시에 조직 인간 각 막 조직 샘플을 접수 한 후, 전송 (1 x).</li>
	<li>긁 고 막 돔 얼굴 위로 배치 하 여 각 막 상피를 제거 합니다. 완전 한 제거를 보장 하기 위해, 철저 하 게 45 ° 각도로 면도날을 사용 하 여 상피 층을 다쳤어요. 다음, 각 막 돔 얼굴 다운 플립 하 고 철저 하 게 완전 한 제거를 보장 하기 위해 45 ° 각도로 면도날으로 기질에서 endothelium을 다쳤어요. 워시 DPBS와 실질 조직.</li>
	<li>실질 조직 유지 하는 DPBS를 사용 하 여 모든 시간에 젖은 stromal 조각 10 외과 블레이드를 사용 하는 작은 조각으로 잘라. 미디어 (2 x 2 m m 조직 플라스 크 당 4 또는 5 조각) 없이 T25 문화 플라스 크에 소독 집게와 장소 stromal 조각을 선택 합니다.</li>
	<li>약 30-40 분 동안 37 ° C에서 플라스 크의 밑바닥에 explants 허용. 준수, 일단 천천히 추가 글의 최소 필수 매체 (EMEM) 10% 태아 둔감 한 혈 청 (FBS) 및 1% 포함 된 항생제/Antimycotic는 explants 방해 없이 플라스 크에.</li>
	<li>부드럽게 37 ° c, 5% CO2배양 기에 플라스 크를 배치 합니다. 세포 분리 시작 때까지 explants는 그대로 두고 플라스 크에서 마이그레이션. 약 2-4 주 후, 셀 100% 합류를 도달 한다. 100% 합류 시 조직 문화 미디어를 제거 하 여 세포를 통과 하 고 셀 DPBS 씻어.</li>
	<li>세포를 트립 신을 추가 하 고 37 ° c.에서 그들을 품 어 약 5 분 후 그들은 분리가 확인 가벼운 현미경에서 세포를 확인 하 고 신선한 미디어를 추가 하 여는 베이스를 중단.</li>
	<li>1000 x g.에서 원심 분리는 상쾌한은 펠 렛을 방해 하지 않고 신중 하 게 제거에 대 한 15 mL 원뿔 튜브에 세포 현 탁 액을 전송 하 고 중단 신선한 EMEM 셀 10% FBS 1% 항생제. 셀을 계산 하 고 씨앗 신선한 EMEM와 확장에 대 한 T75 문화 플라스 크에 약 1 x 106 셀 1% FBS 1% 항생제.</li>
</ol>2. 기본 HCFs 문화 및 3D 어셈블리 생성
<ol><li>3D 조립에서 구문을 explants 이전에 성장된 EMEM 10% FBS 1% 항생제/Antimycotic, 통로 및 수 HCFs.</li>
	<li>씨 약 1 x 106 셀/잘 신선한 EMEM의 1.5 mL 함께 3D 구조에서 0.4 µ m 모와 폴 리 카보 네이트 막 삽입에 기본 HCFs의 10% FBS 1% 항생제/Antimycotic 구성의 상단에. 각 구조물의 하단에 같은 미디어의 또 다른 1.5 mL를 추가 합니다.</li>
	<li>셀 시드의 24 시간 후 문화 EMEM 셀 10% FBS 1% 항생제/Antimycotic, 위에 추가 1.5 mL 및 각 구조물의 하단에 1.5 mL에 의해 0.5 m m 2-O-α-D-glucopyranosyl-L-ascorbic 산 (비타민 C) 자극.</li>
	<li>37 ° c, 5% CO2 3 주 동안 인큐베이터에 문화를 유지 하 고 전체 연구 기간 동안 매일 신선한 미디어를 공급. 아니 더 통로 필요 합니다.</li>
</ol>3. 세포 분화 및 공동 문화
<ol><li>3 주 후, 8 x 103 셀/잘 이전 조립된 3D 상단 SH-SY5Y 인간 신경 세포의 10%를 포함 하는 EMEM의 포함 1.5 mL를 구성 하는 것을 추가 FBS, 1% 항생제/Antimycotic, 그리고 0.5 m m 2-O-α-D-glucopyranosyl-L-ascorbic 산 (비타민 C)입니다.<ol>
<li>SH SY5Y 신경 세포 세포 분화를 시작 하기 전에 적어도 24 h에 대 한 각 막 실질 세포의 위에 성장 하는 것을 허용 한다.</li>
	</ol>
</li>
	<li>EMEM 1 %FBS 및 10 µ M의 1.5 mL로 세포를 자극 하 여 SH SY5Y 세포 분화를 시작 구성 위에 Retinoic 산. 구성의 하단에는 EMEM 10% FBS 1% 항생제/Antimycotic 0.5 m m 2-O-α-D-glucopyranosyl-L-ascorbic 산 (비타민 C)의 1.5 mL를 추가 합니다. 참고 Retinoic 산 단계 어둠 속에서 수행 되어야 합니다.<ol>
<li>5 일 동안이 차별화 미디어에 셀 문화를 계속 합니다.</li>
	</ol>
</li>
	<li>세포 분화 단계에 도달, 1.5 mL 혈 청 자유로운 매체 포함 된 2 셀 전환 두뇌 파생 된 neurotrophic 요인 (BDNF)와 1%의 nM 항생제/Antimycotic 구성의 상단에 추가 하 여 EMEM에 추가. 구성의 하단에는 EMEM 10% FBS 1% 항생제/Antimycotic 0.5 m m 2-O-α-D-glucopyranosyl-L-ascorbic 산 (비타민 C)의 1.5 mL를 추가 합니다. 참고 BDNF 단계 어둠 속에서 수행 되어야 합니다.</li>
	<li>표 1에 표시 된 대로 어떤 추가 분석에 대 한 처리 하기 전에 2 추가 일에 대 한 셀을 문화.</li>
</ol>

<ol>
	<li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. 96 (5), 614-618 (2012).</li><li>R&#246;nkk&#246;, S., Vellonen, K. -. S., J&#228;rvinen, K., Toropainen, E., Urtti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+cell+culture+models+for+drug+toxicity+studies.">Human corneal cell culture models for drug toxicity studies.</a> Drug Delivery and Translational Research. 6 (6), 660-675 (2016).</li><li>Ghezzi, C. E., Rnjak-Kovacina, J., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+tissue+engineering:+recent+advances+and+future+perspectives.">Corneal tissue engineering: recent advances and future perspectives.</a> Tissue Eng Part B Rev. 21 (3), 278-287 (2015).</li><li>Shafaie, S., Hutter, V., Cook, M. T., Brown, M. B., Chau, D. Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Cell+Models+for+Ophthalmic+Drug+Development+Applications.">In Vitro Cell Models for Ophthalmic Drug Development Applications.</a> BioResearch Open Access. 5 (1), 94-108 (2016).</li><li>Shaheen, B. S., Bakir, M., Jain, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+nerves+in+health+and+disease.">Corneal nerves in health and disease.</a> Surv Ophthalmol. 59 (3), 263-285 (2014).</li><li>Beuerman, R. W., Schimmelpfennig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+denervation+of+the+rabbit+cornea+affects+epithelial+properties.">Sensory denervation of the rabbit cornea affects epithelial properties.</a> Exp Neurol. 69 (1), 196-201 (1980).</li><li>Heigle, T. J., Pflugfelder, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+tear+production+in+patients+with+neurotrophic+keratitis.">Aqueous tear production in patients with neurotrophic keratitis.</a> Cornea. 15 (2), 135-138 (1996).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+3D+corneal+tissue+model+with+epithelium,+stroma,+and+innervation.">In vitro 3D corneal tissue model with epithelium, stroma, and innervation.</a> Biomaterials. 112, 1-9 (2017).</li><li>Ross, R. A., Spengler, B. A., Biedler, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+morphological+and+biochemical+interconversion+of+human+neuroblastoma+cells.">Coordinate morphological and biochemical interconversion of human neuroblastoma cells.</a> J Natl Cancer Inst. 71 (4), 741-747 (1983).</li><li>Griffith, L. G., Naughton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+-+Current+challenges+and+expanding+opportunities.">Tissue engineering - Current challenges and expanding opportunities.</a> Science. 295 (5557), (2002).</li><li>Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+characterization+of+organized+extracellular+matrix+deposition+by+ascorbic+acid-stimulated+human+corneal+fibroblasts.">Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts.</a> Invest Ophthalmol Vis Sci. 48 (9), 4050-4060 (2007).</li><li>Karamichos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+tissue+engineering:+current+and+future+directions.">Ocular tissue engineering: current and future directions.</a> J Funct Biomater. 6 (1), 77-80 (2015).</li><li>Karamichos, D., Brown, R. A., Mudera, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+stiffness+regulates+cellular+contraction+and+matrix+remodeling+gene+expression.">Collagen stiffness regulates cellular contraction and matrix remodeling gene expression.</a> J Biomed Mater Res A. 83 (3), 887-894 (2007).</li><li>Ruberti, J. W., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prelude+to+corneal+tissue+engineering+-+gaining+control+of+collagen+organization.">Prelude to corneal tissue engineering - gaining control of collagen organization.</a> Prog Retin Eye Res. 27 (5), 549-577 (2008).</li><li>Karamichos, D., Guo, X. Q., Hutcheon, A. E., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+fibrosis:+an+in+vitro+model.">Human corneal fibrosis: an in vitro model.</a> Invest Ophthalmol Vis Sci. 51 (3), 1382-1388 (2010).</li><li>Chen, F. M., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+biomaterials+of+human+origin+for+tissue+engineering.">Advancing biomaterials of human origin for tissue engineering.</a> Prog Polym Sci. 53, 86-168 (2016).</li><li>Priyadarsini, S., Sarker-Nag, A., Rowsey, T. G., Ma, J. X., Karamichos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+3D+In+Vitro+Model+to+Accelerate+the+Development+of+Human+Therapies+against+Corneal+Diabetes.">Establishment of a 3D In Vitro Model to Accelerate the Development of Human Therapies against Corneal Diabetes.</a> PLoS One. 11 (12), e0168845 (2016).</li><li>Karamichos, D., Hjortdal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratoconus:+tissue+engineering+and+biomaterials.">Keratoconus: tissue engineering and biomaterials.</a> J Funct Biomater. 5 (3), 111-134 (2014).</li><li>Wilson, S. L., Yang, Y., El Haj, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Stromal+Cell+Plasticity:+In+Vitro+Regulation+of+Cell+Phenotype+Through+Cell-Cell+Interactions+in+a+Three-Dimensional+Model.">Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model.</a> Tissue Engineering Part A. 20 (1-2), 225-238 (2014).</li><li>Proulx, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+a+human+cornea+by+the+self-assembly+approach+of+tissue+engineering+using+the+three+native+cell+types.">Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.</a> Molecular Vision. 16 (234-236), 2192-2201 (2010).</li><li>Gonzalez-Andrades, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+in+vitro+model+of+stratified+epithelial+wound+healing+with+barrier+function.">Establishment of a novel in vitro model of stratified epithelial wound healing with barrier function.</a> Sci Rep. 6, 19395 (2016).</li><li>Hopkins, A. M., DeSimone, E., Chwalek, K., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+in+vitro+modeling+of+the+central+nervous+system.">3D in vitro modeling of the central nervous system.</a> Prog Neurobiol. 125, 1-25 (2015).</li><li>Schulz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+Corneal+Cell-Based+Microenvironment+as+Prerequisite+for+Balanced+3D+Corneal+Epithelial+Morphogenesis:+A+Promising+Animal+Experiment-Abandoning+Tool+in+Ophthalmology.">Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology.</a> Tissue Engineering Part C-Methods. 20 (4), 297-307 (2014).</li><li>Gao, J., Wang, Y., Zhao, X., Chen, P., Xie, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204-5p-Mediated+Regulation+of+SIRT1+Contributes+to+the+Delay+of+Epithelial+Cell+Cycle+Traversal+in+Diabetic+Corneas.">MicroRNA-204-5p-Mediated Regulation of SIRT1 Contributes to the Delay of Epithelial Cell Cycle Traversal in Diabetic Corneas.</a> Invest Ophthalmol Vis Sci. 56 (3), 1493-1504 (2015).</li><li>Koulikovska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+regeneration+of+corneal+tissue+via+a+bioengineered+collagen+construct+implanted+by+a+nondisruptive+surgical+technique.">Enhanced regeneration of corneal tissue via a bioengineered collagen construct implanted by a nondisruptive surgical technique.</a> Tissue Eng Part A. 21 (5-6), 1116-1130 (2015).</li><li>Ljubimov, A. V., Saghizadeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+corneal+wound+healing.">Progress in corneal wound healing.</a> Progress in retinal and eye research. 49, 17-45 (2015).</li><li>Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+and+wound+healing.">Extracellular matrix and wound healing.</a> Curr Opin Ophthalmol. 12 (4), 237-241 (2001).</li><li>Karamichos, D., Hutcheon, A. E., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta3+regulates+assembly+of+a+non-fibrotic+matrix+in+a+3D+corneal+model.">Transforming growth factor-beta3 regulates assembly of a non-fibrotic matrix in a 3D corneal model.</a> J Tissue Eng Regen Med. 5 (8), e228-e238 (2011).</li><li>Karamichos, D., Lakshman, N., Petroll, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+for+assessing+fibroblast+migration+in+3-D+collagen+matrices.">An experimental model for assessing fibroblast migration in 3-D collagen matrices.</a> Cell Motil Cytoskeleton. 66 (1), 1-9 (2009).</li><li>Karamichos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+in+Vitro+Model+for+Keratoconus+Disease.">Novel in Vitro Model for Keratoconus Disease.</a> J Funct Biomater. 3 (4), 760-775 (2012).</li></ol>

저자 아무 경쟁 금융 관심사를 선언합니다.

여러 연구 결과 각 막 질환의 더 나은 이해를 개발 하는 데 도움이 뿐만 아니라 수 치료를 발견 하는 다양 한 동물 모델 개발에 집중 되었습니다. 그러나, 이러한 연구에서 인 간에 게 중요 한 가치는 확인 되지는 않았습니다. 날짜 하려면, 다양 한 생체 외에서 모델 개발 고 널리 그들의 놀라운 임상 중요성으로 조사 되었습니다. 우리의 이전 설립 3 차원 생체 외에서 모델은 새로운 시?...

인체 각 막에서는 가장 밀도가 innervated 조직입니다. 신경 터치, 고통, 온도, 다양 한 감각에 대 한 책임은 그리고 또한 상처 치유, 반사, 눈물 분 비5,,67및 생산에 필수적인 역할을 했습니다. 각 막, 실질 신경 줄기 limbal 총에서 발생 하 고 광선으로 주변 각 막 stroma를 입력 합니다. Stromal 신경 조직 콜라겐 lamellae 평행 이며, 그들은 더 작은 성숙기로 그들은 천박한 기질5,8쪽으로 진행으로 분기. 상피 층을 관통 하는 신경 섬유를 추가 하 고 따라서, 신경 분포 널리 퍼져 각 막 상피와 기질. 따라서, 신경 분포는 각 막의 건강 하 고 병에 걸린 상태에서 필수적인 역할을 하고있다. 이 프로토콜에서 우리는 vivo에서 stromal 신경 상호 작용을 모방 하는 종류의 첫 번째 모델 소설 3 차원 생체 외에서 의 발전을 공개. 그것은 가장 설립, 잘 특징이 라인 신경 성장을 공부 하는 데 사용 중 SH-SY5Y 셀 라인이이 연구를 위해 사용 했다. SH SY5Y 셀 라인 두 기판 부착 (S 형) 생산 설명 되었으며 neuroblastic (N 형) 세포 transdifferentiation9를 받을 수 있습니다. 그 결과,이 셀 라인은 N 형 셀의 트리플 연속 subclone 선택에서 파생 된, 비록 그것은 또한 포함 retinoic 산 및 두뇌 파생 된 사용을 통해 신경 세포로 분화를 겪고 수 S 형 세포의 작은 수 科 요소9. 이 당뇨병 성 망막 증 (DM) 및 다른 눈 질병와 관련 된 각 막 합병증의 더 나은 이해로 이어질 수 있는 도구를 제공 합니다. 하 고 눈 질병을 가진 환자에서 신경 세포 배양과 관련 된 어려움 때문에이 3D 생체 외에서 모델 신경 상호 작용을 공부 하 고 각 막 stroma로 신호에 상당한 영향을 제공 합니다.
질병 상태는 종종 매우 큰 규모, 손상 된 삶의 질으로 이어지는 신체의 다양 한 조직 영향을. 눈 dystrophies는 종종 조직의 질병 및 시력의 손실 또는 심지어 영구적인 시력 상실을 리드와 관련 된 일반적인 합병증. 포괄적인 연구는 종종 질병 상태 뿐만 아니라 기저 세포 수준에서 효과의 더 나은 이해를 위해 근본적 이다. 이러한 질병의 효과 연구, 다양 한 생체 외에서 그리고 vivo에서 모델 조직 엔지니어링 응용 프로그램의 도움으로 개발 되었습니다. 각 막 조직 공학 응용 과학10,11,12,,1314, 다양 한 분야에 걸쳐 큰 관심을 얻고 있다 하지만 여전히 주요 제한 중 실제 응용 프로그램을 거부, 감염과 흉터10,11,12,,1314이식 각 막 등. 여러 연구를 성공적으로 개발 하 고 다양 한 생체 외에서 모델3,15,,1617,18, 설립 있다 19,20,21,22,23,,2425,26. 3 차원 생체 외에서 모델은 가장 유망한와 큰 과학적 관심의. 3D 모델은 더 나은 vivo에서 세포 및 생리 적 이벤트 섬유 증 중 중요 하 고 상처 치유15,27,,2829거울 알려져 있습니다. 이러한 생체 외에서 모델 각 막 합병증 등 다른 질병 상태를 치료에 새로운 치료 접근을 찾는 중요 한 역할을. 각 막 기능에 신경 분포의 중요 한 역할에도 불구 하 고 약간의 노력이 했다 각 막 조직 설계 구조2,3내 주변 신경 확산을 촉진. 그러나, 제안 된 3D 생체 외에서 셀 구조는 원하는 조직 기능을 달성 하기 위하여 대상 조직 모방.
당뇨병 각 신경 결함 때문에 여기서 설명 하는 모델에 대 한 명백한 응용 프로그램은, 인간의 생체 외에서 모델 Keratoconus와 Fuchs dystrophies 등 누릴 수 있는 다른 여러 각 막 질환 있다. 우리의 3D 모델이이 전망에서 나온다 고 약 납품을 평가 하기 위해 각 막 직물의 생체 외에서 표현 및 새로운 눈 약의 안전의 개발을 제안.

<table>
	<tbody>
		<tr>
			<td>Healthy corneal tissue</td>
			<td>NDRI</td>
			<td></td>
			<td>Samples from donors with no ocular trauma or systemic disease</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Solution (1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td>Fischer Scientific</td>
			<td>13-812-42</td>
			<td>Fisherbrand Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr>
			<td>Single edge razor blades</td>
			<td>Personna</td>
			<td>270100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical scalpel blades No.10</td>
			<td>Feather Surgical Blade</td>
			<td>2976#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#8217;s Minimum Essential Medium</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td>10% FBS is required for media preparation</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco by Life Technologies</td>
			<td>15240-062</td>
			<td>1% Antibiotic-Antimycotic is required for media preparation</td>
		</tr>
		<tr>
			<td>0.05% Trypsin EDTA(1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane inserts with 0.4-&#956;m pores</td>
			<td>Corning Costar</td>
			<td>3412</td>
			<td></td>
		</tr>
		<tr>
			<td>2-O-&#945;-Dglucopyranosyl-L-ascorbic acid (Vitamin C)</td>
			<td>Sigma-Aldrich</td>
			<td>SMB00390-14</td>
			<td>A concentration of 0.5 mM should be used for the study</td>
		</tr>
		<tr>
			<td>Wax block</td>
			<td>VWR</td>
			<td>50-949-027</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-SY5Y Neuroblastoma cells</td>
			<td>ATCC</td>
			<td>SHSY5YATCC CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3014-10UG</td>
			<td>Final concentration of 10uM needs to be used</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-100MG</td>
			<td>Final concentration of 2nM needs to be used</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide(DMSO)</td>
			<td>VWR-Alfa Aesar</td>
			<td>67-68-5</td>
			<td>Ultra Pure Grade-Sterile DMSO to be used</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T25)</td>
			<td>Fisher Scientific</td>
			<td>12-565-351</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T75)</td>
			<td>Fisher Scientific</td>
			<td>12-565-349</td>
			<td></td>
		</tr>
	</tbody>
</table>

corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea

조직 공학 인간 각 막 교체 각 막 시력 손실1에서 고통을 전세계 약 10 백만 명에 대 한 높은 수요로 인해 상당한 인정을 받고 있다. 가능한 인간 각, 3 차원 (3D) 조직에 중요 한 진행에 대 한 수요를 해결 하기 위해 공학이 했다2,,34. 이러한 각 막 모델 에서부터 간단한 단층 시스템 3D 전체 두께 각 막 등가물2로 이어지는 다층된 모델.
그러나, 생체 외에서 질병 모델의 맥락에서 3 차원 조직 설계 각 막의 사용 날짜 부족 유사 다층된 3 차원 각 막 조직 구조, 기능, 및 서로 다른 세포 유형 (즉, 신경, 네트워킹을 공부 상피와 기질, 내 피)2,3. 또한, 생체 외에서 각 막 조직 모델에 대 한 수요는 제약 제품에 대 한 동물 실험을 줄이기 위해 시도에서 증가 했다. 따라서, 더 정교한 모델은 인간의 생리 적 요구 사항, 시스템 일치 하는 데 필요한 그리고 더 환자 인구에 관련 된 모델의 개발은 절대적으로 필요 하다. 각 막에 여러 세포 유형 영향을 받는 질환과 dystrophies Keratoconus, 당뇨병 각 Fuchs, 등을이 모델에 포함 된 기본 인간 각 막 섬유 아 세포 (HCFs) 건강 한 기증자와에서 뉴런에서의 3D 공동 문화 모델 SH SY5Y 셀 라인입니다. 인간의 각 막 조직 내에서 2 개의 세포 유형 사이 상호 작용을 조사 하기 위해 처음으로 수 있습니다. 우리는이 모델 잠재적으로 관련 된 신경 손상 하는 각 막 질환의 신경 기질 상호 작용 하는 근본적인 메커니즘을 해 부 수 믿습니다. 이 3D 모델 vivo에서 각 막 직물의 기본 해 부 및 생리 적 특성을 반영 하 고 수 도구로 사용 될 미래에는 각 막 결함 조사로 동물 테스트 하기 전에 다양 한 에이전트의 효능을 심사.

그림 1 은 작업 모델 3D 생체 외에서 단계별 대표 이미지입니다. 첫 번째 단계에서 세포 인간 각 막에서 격리 됩니다. 다음, 그들은 폴 리카 보 네이트 막에 성장 하 고 자기 secreted 3 차원 매트릭스를 비타민 C와 자극. 이 3D 구조 시스템 유도는 다층 세포 vivo에서의 합성-stromal 매트릭스 처럼. 그 후, 신경 세포는 신경 세포 분화를 시작 하 여...

Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

이 프로토콜에는 새로운 3 차원 생체 외에서 모델을, 어디 각 막 실질 세포와 분화 된 신경 세포 교양 함께 시험에는 두 종류의 세포 상호 작용의 이해를 돕기 위해 설명 합니다.

각 막 조직 공학:는 생체 외에서 의 모델 인간 각 막의 실질 신경 상호 작용

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

corneal-tissue-engineering-an-vitro-model-stromal-nerve-interactions

Research

JoVE Journal

Developmental Biology

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

8.7K 조회수.  University of Oklahoma Health Sciences Center. 이 프로토콜에는 새로운 3 차원 생체 외에서 모델을, 어디 각 막 실질 세포와 분화 된 신경 세포 교양 함께 시험에는 두 종류의 세포 상호 작용의 이해를 돕기 위해 설명 합니다.

비디오: Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

에반스 블루 염료와 Zebrafish의 애벌레 골격 근육 무결성 분석

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

연구

발생학

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

SH-SY5Y 인간 신경 모세포종 세포 라인의 차별화

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

멀티 컬러 형광 기자 마우스를 사용하여 각막 세포의 리니지 추적하는 방법

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

개발<em&gt; 체외</em&gt; 분석은 중간 엽 세포를 시행 한 상피 - 중간 엽 전환의 수축 기능을 평가하는

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

조직 공학 및 질병 조사에있는 사용을위한 소아 인간의 식도 상피 세포의 조건부 재 프로그래밍

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

연골 세포 분열 분열의 DNA 메틸화 수준을 정량화하는 5-mC Dot Blot Assay<em&gt; 체외에서</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

생체 외에서 마우스 시뮬레이션된 Microgravity에서 Preantral 모 낭의 성장

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Mouse Secondary Palate Fusion의 라이브 이미징

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

해 부 및 Immunofluorescent 얼룩의 버섯 몸과 성인 초파리 melanogaster 두뇌 신경 세포 포토 리셉터

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

트랜스와 초파리 노치의 바인딩을 평가를 집계 분석 셀-Ligands 및 Cis에 의해 그것의 억제-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...