우리는 그대로 돼지의 폐 조직 기증에 대한 스미스 필드 팜을 감사드립니다. 우리는 또한 우리 자신의 장비를 사용할 수 있도록 박사 후 양 박사 크리스티나 당나라와 VCU 성형 외과 부서에 감사의 말씀을 전합니다. 히드로 겔 및 조직 샘플은 자금 조달 형태 NIH-NCI 암 센터 지원 그랜트에 의해 부분적으로 NIH - NINDS 센터 코어 그랜트 (5) P30 NS047463에서 자금에 의해 부분적으로 지원되는 해부학 및 신경 생물학 현미경 시설의 VCU 부서에서 SEM을 준비했다 P30의 CA016059. VCU 나노 기술 핵심 특성화 시설 (NCC)에서 샘플의 SEM 영상. 이 작품은 국립 과학 재단 (National Science Foundation), CMMI 1351162에 의해 투자되었다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 해결책 </td><td> 살균 필터 </td><td> 지도 </td></tr><tr><td> DiH 2 O </td><td> 예 </td><td> DiH 2 O; 무균 여과 </td></tr><tr><td> 0.1 % 트리톤 X-100 솔루션 </td><td> 예 </td><td> 흄 후드에서 DiH 2 O 100 ㎖에 100 μL 트리톤-X 100 솔루션을 추가하고 용해 될 때까지 교반; 살균 필터. </td></tr><tr><td> 2 % 데 옥시 콜레이트 솔루션 </td><td> 예 </td><td> 흄 후드에서 2g 나트륨 데 옥시 콜레이트 솔루션을 추가 100 ㎖ DiH 2 O 용해 될 때까지 교반 당; 살균 필터 </td></tr><tr><td> 1 M의 NaCl </td><td> 예 </td><td> DiH 2 O 1 L에 58.44 g의 NaCl을 추가; 용해 될 때까지 교반; 살균 필터 </td></tr><tr><td> DNase의 솔루션 </td><td> 예 </td><td> 12,000 추가단위 DNase의 1 L DiH 2 O에; 0.156 g 황산 추가 (무수물), 및 0.222 g의 CaCl2를; 용해 될 때까지 교반; 살균 필터 </td></tr><tr><td> PBS </td><td> 예 </td><td> , 27.2 g 나 2 HPO 4 · 7H 2 O (이염 수화물)를 결합 80g의 NaCl, 10 L DiH 2 O와 2g의 KCl; 용해 될 때까지 교반; 7.4으로 산도를 조정; 살균 필터 </td></tr></tbody></table> 표 1 : 조직 탈세 포화에 필요한 솔루션. 탈세 포화 프로세스에 대한 위의 솔루션을합니다. 4 ℃에서 보관하십시오. 약 2.5 L은 한 돼지의 폐를 위해 필요합니다. 1. 하이드로 겔 형성 <ol><li> (참조 9,10에서 적응) 돼지 폐 탈세 포화 <ol><li> 마음과 vasc으로, 도축장에서, 블록 엔 정상 돼지의 폐를 구합니다ulature 그대로. </li><li> 마음에 최대한 가까운 혈관을 절단, 마음을 멀리 해부 조직 가위 또는 메스를 사용, 나머지 혈관이 관류에 대한 이후 단계에서 사용됩니다. </li><li> 조심스럽게 메스 나 가위를 사용하여 기관, 기관지 및 혈관 주위의 결합 조직을 멀리 해부하다. 참고 : 탈세 포화 과정의 효과를 방해 할 수 큰 상처 또는 천공 늑막 통해 무기폐 또는 혈관 폐색 많은 양의없이 폐를 선택하여 절차의 나머지에 사용할 최적의 폐를 결정합니다. </li><li> (처분 할 수있다 심장, 결합 조직 및 제거 된 폐의 로브) 가능한 한 기관에 부착만큼 기관지를 떠나 최적 폐를 버려야. 하나의 폐는 기관지와 혈관에 부착 된 남아가 있어야합니다. 참고 : 원하는 경우 조직학에 대한 섹션을 유지. 9 </li><li> 페이지에 클램프 또는 봉합와 연결이 끊어진 기관지를 닫습니다하지 못하 초과 역류. </li></ol><ol start="6"><li> (표 1) 필요할 때까지 4 ° C에서 탈세 포화 솔루션과 냉장을 준비합니다. </li><li> 폐동맥 맞게 폐동맥 및 기관 모두를 통해 폐 조직을 탈 이온수로 3 회 관류하는 유관 핸드 펌프를 사용. 제마다 혈관을 관류. 혈관에 약 1 L 각 관류에 대한 기관에 1.5 L로 시작하지만, 세포 파편 제거로 더 많은 액체가 시스템을 통해 관류 할 수 있습니다. </li><li> 트리톤 X-100 용액을 모두 혈관과 기관을 관류. </li><li> 4 ℃에서 24 시간 동안 트리톤 X-100 용액에 폐 조직을 잠수함. </li><li> DI 워터와 관류의 혈관과 기관 3 회 씻어합니다. </li><li> 옥시 콜레이트 솔루션을 모두 혈관과 기관을 관류. </li><li> 4 ℃에서 24 시간 동안 옥시 콜레이트의 조직 섹션 잠수함. </li><li> DI 워터와 관류의 혈관과 기관 3 회 씻어합니다. </li><li>NaCl 용액으로 모두 혈관과 기관을 관류. </li><li> 4 ℃에서 1 시간 동안 여과 NaCl 용액에 조직을 잠수함. </li><li> DI 워터와 관류의 혈관과 기관 3 회 씻어합니다. </li><li> DNase의 솔루션을 모두 혈관과 기관을 관류. </li><li> 4 ℃에서 1 시간 동안 여과 DNase의 솔루션에서 조직을 잠수함. </li><li> 혈관 및 기관 PBS로 5 회를 모두 관류. </li><li> 눈에 띄는 연골 조직, 기관 만 호흡 영역 (주로 주변 지역)을두고기도를 수행에서 (주로 폐문과 폐의 중간 부분 주위에 있음) 직경의 모든 세관 2mm 이상을 멀리 해부하다. </li><li> 1 인치 절 이하에 조직을 해부하다. 조직의 방향은이 단계에서 문제가되지 않습니다. </li><li> 50 ML 원뿔 튜브를 초과하는 액체와 장소 조직을 따르십시오. -80 ° C에서 조직을 동결. 참고 : 조직학 세포 및 세포 debri의 제거를 보장하는 섹션을 유지S 원한다면. 우리는 H &amp; E를 사용, α - 갈 락토시다 아제, picogreen, 하이드 록시 프롤린, 닌히 드린, 알 시안 블루, SDS-PAGE 및 질량 분석의 특징입니다. 9 </li></ol></li><li> 폐 처리 <ol><li> decellularized 폐 조직을 포함하는 냉동 튜브의 뚜껑을 제거합니다. </li><li> 튜브 입구에 필터 종이를 놓고 고무 밴드로 고정합니다. 참고 : 튜브 및 내용은 여전히 ​​고정 될 때까지 그렇지 않으면 -80 ° C에 배치 동결해야합니다. </li><li> 모든 초과 액체 제조 업체의 지시에 따라 동결 건조기를 사용하여, 사라 될 때까지 조직을 동결 건조. 밀 준비가 될 때까지 -80 ° C에서 보관하십시오. </li><li> 근로 조건에 절연 및 내부 부품의 온도를 냉장고 밀에 액체 질소를 추가 시작하기 전에. </li><li> 밀 튜브에서 자기 밀 줄을 제거하고 바닥을 충당하기 위해 조직을 추가합니다. </li><li> 밀 줄을 교체하고 느슨하게 포장 조직을 추가합니다. 밀 바는 여전히 자유롭게 이동해야합니다. </li><li> 밀 튜브와 괞 찮아을 닫습니다냉동 공장에서 가전. </li><li> 최대 적재 선에 액체 질소를 입력합니다. </li><li> 냉동기 밀 미분말에 모든 조직 (약 5 분 ~ 600 분의 충돌 속도 -1)와, 스테인레스 스틸 충격기 폴리 카보네이트 실린더뿐만 아니라 제조자 지시에 따라 냉동기 밀을 사용하여 스테인리스 단부 플러그이다. 사용 전까지 -80 ° C에서 보관하십시오. </li></ol></li><li> 마이크로 다공성 겔 형성 (8 ㎎ / ㎖) (참조 각색 9,11) <ol><li> 1 % 추가 (W / V)를 decellularized 분말 및 0.1 % (w / v)의 0.01 M 염산을 펩신, 일정한 교반하에 들면, 실온에서, (액체의 최상위에 흐름을 볼 수 있어야한다) 48 시간. 주 : 분말 정적 분말이 튜브 벽면에 과잉 세척 할 기회를 제공 한 후이므로 염산을 추가 충전된다. </li><li> 48 시간 동안 소화 한 후, 5 분 동안 얼음에 솔루션 및 시약을 배치합니다. </li><li> 냉장 10 % 사용 (v / v)의 0.1 M NaOH를,D 11.11 % (v / v)의 10 배 PBS는 7.4의 생리적 산도에 소화 단백질 용액을 가지고 (농도를 1 배 전체 솔루션을 가지고). 참고 :이 솔루션은 이제 일주까지 4 ° C에 저장할 수 있습니다. 원하는 경우 유변학 (9)를 사용하여 겔화 반응 속도를 수행합니다. </li></ol></li></ol> 2. 기계적 강도를 개선하기 위해 하이드로 겔 가교 <ol><li> 1 %, 0.1 % 및 V genipin 가교 용액 / w 0.01 % 용액을 10 % DMSO에 genipin 분말의 필요량을 용해시켜 제조 하였다. </li><li> 1 시간 동안이 용액을 15 분마다 와동. </li><li> 이전 부분 1.3.4에서 조립 된 ECM 하이드로 겔 (96 웰 플레이트의 각 웰에 100 μL)를 커버하는 genipin 솔루션을 추가합니다. </li><li> 37 ° C에서 24 시간 동안 가교 각 ECM 하이드로 겔을 둡니다. </li><li> 세정액이 더 이상 청색까지 ECM 하이드로 겔을 PBS로 3 회 씻어 없다. 가교 하이드로 겔은 더 특성화 및 CE에 대한 준비가 될 것입니다LL 문화 연구. </li></ol> 미세 다공성 젤 3. 세포 배양 <ol><li> 이차원 코팅 <ol><li> 1.3.3에서 용액을 사용하여 96 웰 비 - 처리 된 조직 배양 플레이트의 각 웰에 프리 겔 용액 20 μL를 추가한다. </li><li> 4 ° C에서 냉장 하룻밤 단백질 흡착 플레이트 할 수 있습니다. </li><li> 기음 프레 겔 용액 PBS로 씻어. </li><li> 통로 세포 (9). </li><li> 우물에 10,000 세포 / cm 2를 추가합니다. </li><li> 물론 당 100 μL 미디어를 높이고 37 ° C에서 품어. </li></ol></li><li> 삼차원 세포 배양 9 <ol><li> 1.3.3에서 용액을 사용하여, 프리 겔 용액을 100 세포 / ml의 농도로 세포 펠렛을 재현 탁. </li><li> 신속 3D 세포 배양 용 ~ 500 μm의 두께 하이드로 겔을 형성하기 위해, 96 웰 플레이트의 각 웰에 프레 겔 세포 현탁액 16 μL 분주. </li><li> 30, 37 ° C에서 품어젤 분 형성한다. </li><li> 형성된 하이드로 겔의 상단에 미디어 100 μl를 추가하고 37 ° C에서 품어. </li></ol></li><li> 가변 강성 젤에 세 차원 세포 배양 <ol><li> 96 웰 플레이트의 각 웰에 피펫 1.3.3에서 프리 겔 용액을 100 μL 용액을 사용. </li><li> 겔을 형성하도록 30 분 동안 37 ℃에서 인큐베이션. 참고 : 여기에 사용할 수있는 2 부에서 단계를 상호 결합. </li><li> 통로 세포 (9). </li><li> 우물에 10,000 세포 / cm 2를 추가합니다. </li><li> 물론 당 100 μL 미디어를 높이고 37 ° C에서 품어. </li></ol></li></ol>

<ol>
	<li>Lee, J., Cuddihy, M., Kotov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Three-Dimensional Cell Culture Matrices: State of the Art. 14, (2008).</li><li>Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 3D Cell Culture. , (2011).</li><li>Walker, J. M., Ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Epithelial cell culture protocols. , (2012).</li><li>Badylak, S. F., Freytes, D. O., Gilbert, T. W. <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+as+a+biological+scaffold+material:+Structure+and+function.">Extracellular matrix as a biological scaffold material: Structure and function.</a> Acta Biomater. 5 (1), 1-13 (2009).</li><li>Koo, H. -. J., Lim, K. -. H., Jung, H. -. J., Park, E. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+evaluation+of+gardenia+extract,+geniposide+and+genipin.">Anti-inflammatory evaluation of gardenia extract, geniposide and genipin.</a> J. Ethnopharmacol. 103 (3), 496-500 (2006).</li><li>Jeffords, M. E., Wu, J., Shah, M., Hong, Y., Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+Material+Properties+of+Cardiac+Matrix+Hydrogels+to+Induce+Endothelial+Differentiation+of+Human+Mesenchymal+Stem+Cells.">Tailoring Material Properties of Cardiac Matrix Hydrogels to Induce Endothelial Differentiation of Human Mesenchymal Stem Cells.</a> ACS Appl. Mater. Interfaces. 7 (20), 11053-11061 (2015).</li><li>Suckow, M. A., Voytik-Harbin, S. L., Terril, L. A., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+bone+regeneration+using+porcine+small+intestinal+submucosa.">Enhanced bone regeneration using porcine small intestinal submucosa.</a> J. Invest. Surg. 12 (5), 277-287 (1998).</li><li>Brown, B. N., Badylak, S. F. <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+as+an+inductive+scaffold+for+functional+tissue+reconstruction.">Extracellular matrix as an inductive scaffold for functional tissue reconstruction.</a> Transl. Res. J. Lab. Clin. Med. 163 (4), 268-285 (2014).</li><li>Pouliot, R. A., 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=Development+and+characterization+of+a+naturally+derived+lung+extracellular+matrix+hydrogel.">Development and characterization of a naturally derived lung extracellular matrix hydrogel.</a> J.Biomed.Mater.Res.A. , (2016).</li><li>Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Part A. 16 (8), 2581-2591 (2010).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29 (11), 1630-1637 (2008).</li><li>Tilghman, R. W., 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=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PloS One. 5 (9), e12905 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials:+where+we+have+been+and+where+we+are+going.">Biomaterials: where we have been and where we are going.</a> Annu. Rev. Biomed. Eng. 6, 41-75 (2004).</li><li>Fridman, R., Benton, G., Aranoutova, I., Kleinman, H. K., Bonfil, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+initiation+and+growth+of+tumor+cell+lines,+cancer+stem+cells+and+biopsy+material+in+mice+using+basement+membrane+matrix+protein+(Cultrex+or+Matrigel)+co-injection.">Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection.</a> Nat Protoc. 7 (6), 1138-1144 (2012).</li><li>Zhang, W. -. J., 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=The+reconstruction+of+lung+alveolus-like+structure+in+collagen-matrigel/microcapsules+scaffolds+in+vitro.">The reconstruction of lung alveolus-like structure in collagen-matrigel/microcapsules scaffolds in vitro.</a> J. Cell. Mol. Med. 15 (9), 1878-1886 (2011).</li><li>Booth, A. J., 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=Acellular+Normal+and+Fibrotic+Human+Lung+Matrices+as+a+Culture+System+for+In+Vitro+Investigation.">Acellular Normal and Fibrotic Human Lung Matrices as a Culture System for In Vitro Investigation.</a> Am. J. Respir. Crit. Care Med. 186 (9), 866-876 (2016).</li><li>Matsuda, A., 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=Evidence+for+human+lung+stem+cells.">Evidence for human lung stem cells.</a> N. Engl. J. Med. 364 (19), 1795-1806 (2011).</li><li>Zuo, W., 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=p63+Krt5++distal+airway+stem+cells+are+essential+for+lung+regeneration.">p63+Krt5+ distal airway stem cells are essential for lung regeneration.</a> Nature. 517 (7536), 616-620 (2015).</li><li>Keane, T. J., Londono, R., Turner, N. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ineffective+decellularization+of+biologic+scaffolds+on+the+host+response.">Consequences of ineffective decellularization of biologic scaffolds on the host response.</a> Biomaterials. 33 (6), 1771-1781 (2012).</li><li>Neill, J. 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=Decellularization+of+human+and+porcine+lung+tissues+for+pulmonary+tissue+engineering.">Decellularization of human and porcine lung tissues for pulmonary tissue engineering.</a> Ann Thorac Surg. 96 (3), 1046-1055 (2013).</li></ol>

저자는 공개 아무것도 없어.

생물학 적분 측면 중 하나는 특정한 작업을 수행하는 계층 적 구조로 분자의 자기 조직이다. 연구실에서 (13)는, 자기 조립은 염 농도, 산도, 소화 기간 등 다양한 요인에 따라 달라집니다. 도시 된 바와 같이, 자기 조직화 하이드로 폼 가용화 된 단백질은 생리 학적 온도로 돌아 오면. 형성된 하이드로 겔은 생체 외에서 세포의 부착과 증식을 촉진 할 수있다. ECM?...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td style="width:371px;">Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Deoxycholate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6750-100g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>D5025-150KU</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepsin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Genipin</td>
			<td style="width:135px;">Wako Chemicals</td>
			<td style="width:119px;">078-03021</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10x</td>
			<td style="width:135px;">Quality Biological</td>
			<td>119-069-151</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:135px;">VWR</td>
			<td>45000-448</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Paper</td>
			<td>Whatman</td>
			<td align="right">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hand pump</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>10-239-1</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graduate Beaker</td>
			<td style="width:135px;">VitLab</td>
			<td align="right" style="width:119px;">445941</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomill</td>
			<td>SPEX</td>
			<td>6700</td>
			<td>Use cryogloves and eye protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>FTS FlexiDry</td>
			<td></td>
			<td>Use gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheometer</td>
			<td>Discovery</td>
			<td>HR-2</td>
			<td>Use gloves and eye protection.</td>
		</tr>
	</tbody>
</table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

여기에서는 시험 관내 폐 세포 배양 여러 컴포넌트 세포 배양 하이드로 겔을 형성하기위한 방법을 제시한다. 돼지, 쥐, 또는 마우스에서 블록의 폐 조직 엔 건강을 시작으로, 조직은 관류 및 세포 파편을 제거하기 위해 후속 화학 세제에 잠겨있다. 처리 전후의 조직 학적 비교는 이중 가닥 DNA 및 알파 갈 락토시다 제 염색을 95 % 이상 제거하는 것을 확인 세포 파편의 대부분이 제거되어 나왔다. 탈세 포화 후, 조직을 동결 건조 후 분말로 cryomilled이다. 매트릭스 분말 산성 펩신 소화 용액에 48 시간 동안 절단 한 후, 프리 겔 용액을 형성하기 위해 중화된다. 프레 겔 용액의 겔화 37 ° C에서 배양하여 유도 할 수 있고, 곧바로 다음 중화 사용 또는 최대 2 주 동안 4 ℃에서 저장 될 수있다. 코팅은 C에 대한 비 처리 판의 프리 겔 용액을 사용하여 형성 될 수있다엘 첨부. 세포는 세포 골격을 통해 이전 또는 코팅에 도금 할 수있는 형성된 겔의 표면 상에 도금 된 3 차원 배양을 달성하기 위해 자기 조립 전에 프레 겔에 현탁 될 수있다. 전략에 변화가 겔화 온도, 강도, 또는 단백질 단편의 크기에 영향을 미칠 수 있습니다 발표했다. 하이드로 겔 형성 외에도 하이드로 겔 강성 genipin을 사용하여 증가 될 수있다.

이 방법을 사용하여, 우리는 정상 돼지, 래트, 마우스 폐 (도 1)로부터의 하이드로 겔을 제조 하였다. 폐 처리는 각각 5 ㎎, 40 ㎎, 및 ECM 분말 10g을 추정 제공한다. 공정의 개요는도 2에 도시되어있다. 과정에서 주요 시각화은 다음과 같습니다 : 데 옥시 콜레이트 세척 후 폐의 흰색 외관; 프레 겔을 형성 한 후에, 용액을 은폐되어야하고 4 ℃에서 저장...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

이 폐 세포 외 기질에서 3 차원 세포 배양 지지체를 제작하는 방법이다. 그대로 폐 세 차원 세포의 성장을 지원할 수있는 하이드로 겔로 처리된다.

3 차원 세포 배양을위한 폐 세포 외 매트릭스에서 조정 히드로 겔

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Research

JoVE Journal

Bioengineering

11.5K 조회수.  Virginia Commonwealth University.  이 폐 세포 외 기질에서 3 차원 세포 배양 지지체를 제작하는 방법이다. 그대로 폐 세 차원 세포의 성장을 지원할 수있는 하이드로 겔로 처리된다. 

비디오: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

세포 배양 응용 프로그램에 대한 히드로 겔의 빛 매개 형성 및 패터닝

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

연구

생체공학

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

조직 특이 적 세포 배양 및 전달 플랫폼과 같은 세포 외 기질 유래 거품과 마이크로 담체의 제조

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

듀얼에 대 한 생체 모방 지하실 멤브레인으로 ultrathin Porated 탄성 Hydrogels 세포 배양

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

날조는 신장 피 질 세포 외 매트릭스 파생 하이드로 겔

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

희생 빈 섬유 막 세포 배양을 통해 기질 섬유의 생산

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

글로벌 세포 매트릭스 Metalloproteinase 및 3D Hydrogels 신진 대사 활동 측정

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

인쇄 3D 세포 -라덴 췌장 조직 구성을 인쇄를위한 췌장 조직 유래 세포 외 매트릭스 바이오 잉크

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

매트릭스 세포 대사 크로스토크 연구를 위한 인간 3D 세포외 세포외 매트릭스-세포세포 배양 모델

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

슈완 세포 표현형 사양을 평가하기 위한 튜블드 세포 외 세포 매트릭스 미세 환경의 준비

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

라이브 현미경 이미징 에서 3D 하이드로겔의 제어 변형

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...