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>

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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><tbody><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher Scientific</td><td>BP151-100</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D6750-100g</td><td>Use in hood with eye protection and gloves.</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506-500g</td><td>None</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-500g</td><td>None</td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td>None</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>258148-500ML</td><td>Use with eye protection and gloves.</td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>BP359-500</td><td>Use with eye protection and gloves.</td></tr><tr><td>Genipin</td><td>Wako Chemicals</td><td>078-03021</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>PBS 10x</td><td>Quality Biological</td><td>119-069-151</td><td>None</td></tr><tr><td>PBS</td><td>VWR</td><td>45000-448</td><td>None</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Hand pump</td><td>Fisher Scientific</td><td>10-239-1</td><td>N/A</td></tr><tr><td>Graduate Beaker</td><td>VitLab</td><td>445941</td><td>N/A</td></tr><tr><td>Cryomill</td><td>SPEX</td><td>6700</td><td>Use cryogloves and eye protection.</td></tr><tr><td>Lyophilizer</td><td>FTS FlexiDry</td><td></td><td>Use gloves.</td></tr><tr><td>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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.6K 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

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

동적 마스크 프로젝션 석판술를 사용하여 신경 문화 시스템의 Micropatterned Hydrogels의 제작

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

연구

생체공학

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

셀 문화 응용 프로그램에 대 한 단백질 기반 Hydrogels 아키텍처의 쉬운 조작

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

생화학

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

미세 패턴 매크로 스케일 3D 셀룰러 아키텍처 모듈 형 하이드로 겔 시트의 건설

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting Cellularized는 조직 고유의 하이드로 겔 Bioink를 사용하여 구축

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

주 사용 재생 치료와 높은 처리량 마약 검사에 대 한 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

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 ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

캡슐화 및 3d에서 셀의 Immunostaining에 대 한 엘라 스 틴 같은 단백질 Hydrogels의 생산

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

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 ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

하이드로겔 어레이는 3D 종양 모델에서 매트릭스 성분 및 치료제의 스크리닝 효과를 위한 처리량 증가를 가능하게 합니다.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

ECM 경직에 대한 세포 반응을 프로브하기 위한 광조정 가능한 바이오프린팅 하이드로겔

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

저자 스포트라이트: 3D 프린팅 광조정 가능 하이드로겔을 사용한 만성 폐 질환 이해 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

섬유아세포 활성화를 연구하기 위한 3D 바이오프린팅 광조정 가능 하이드로겔

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...