이 작품은 난소암 연구 기금에 의해 지원되었다 [부여 LT/UIC/01.2011] 임상 및 Translational 과학, UIC 암 센터, 보건 부여 C06RR15482 국립 연구소에 대한 UIC 센터. 동물 실험은 UIC의 동물 케어 및 사용위원회의 승인을 동물 관리 프로토콜에 따라 AAALAC에 의해 명시된 지침과 규정에 따라 수행되었다. 이 프로젝트에 대해 동물 시카고 일리노이 대학에서 생물 자원 연구소 (BRL)에 장벽 장소에 있습니다. BRL 적어도 두 번 매일 동물을 모니터링하고 동물 보호에 대한 조언을 제공하는 완전히 수의과 직원이 있습니다.

1. 난소 해부와 조직 분리 <ol><li> 나트륨 알지네이트요의 0.5 % (W / V) 솔루션을 준비, 프로토콜의 수정된 버전에서 준비 살균 PBS 37에 열을 ° C.를 사용하여 이전 5 설명 역전이나 락을로 믹스지만, 와동하지 않습니다. 25 G 바늘로 1 ML의 주사기에 알지네이트요를 그려 37에서 유지 ° C. </li><li> crosslinking 조직 캡슐에 알지네이트요 37에 따뜻한 ° C.에 대한 10mM CaCl 2 무균 용액 준비 </li><li> 문화 매체 (αMEM + 5 단위의 페니실린 및 5 μg / ML 스트렙토 마이신) 당 잘 각 실험 24 잘 접시의 1mL를 준비합니다. 문화 매체의 다양한 약물, 펩티드, 또는 바이러스를 포함합니다. 이전 기관의 캡슐에 transfection을 위해, 37 ° C.에서 4 시간 동안 Lipofectamine 2000 및 플라스미드 DNA와 조직을 품어 </li><li> 동물의 장기를 절개하다, 어떤 지방이나 잔여 조직을 제거 부드럽게 난소를 둘러싼 부르사 막 해내, 그리고 오르간을 bisecting × 4 조각으로 난소 또는 수란관 잘라. 동물은 자궁 경부 전위 다음 CO 2 질식에 의해 euthanized되었다. 수란관 들어, 부드럽게 연결 세포막을 분리 당기 및 절단하기 전에 집게를 사용하여 반대 방향으로 튜브의 끝부분을 확장하여 조직을 풀다. </li><li> 멸균 메쉬 섬유에 하나 알지네이트요의 비말을 (~ 1-3 μL) 장소. </li><li> 무균 집게를 사용하여 알지네이트요 비말 및 센터 포셉 또는 멸균 바늘을 사용하여 작은 물방울을 장기로의 조각을 이동합니다. </li><li> 무균 포셉와 메쉬 섬유 쥐고과 중력이 매달려 드롭에 알지네이트요을 강제 수 있도록 거꾸로 그것을 전환하여 장기 조각 (organoid)이있는 비말을 반전. </li><li> 드롭 솔루션으로 떨어지는 있도록 10mM CaCl 2 용액이 들어있는 10cm 페트리 접시의 가장자리에있는 집게를 누릅니다. </li><li> 2 분 동안 품어, 문화 매체에 추가 10mM CaCl 2 및 전송을 공개하기 위해 모공을 포함하는 멸균 스푼으로 제거합니다. 최대 네 개까지 알지네이트요의 젤로가 문화 매체를 포함하는 24 잘 접시의 단일 잘으로 전송할 수 있습니다. 정의된 기간 동안 문화. </li></ol> 2. 세포의 알긴산과 수집 저하 <ol><li> 알지네이트요 젤의 solubilization 들어, ° C 30 분 또는 때까지 젤이 완전히 해소됩니다 37 Leibovitz의 L - 15 미디어에 녹아있는 알지네이트요의 lyase (3.4 MG / ML)의 문화를 품다. </li><li> 알지네이트요의 lyase에서 조직을 제거합니다. </li><li> 오세 수집 1 시간 8 Leibovitz의 L - 15 미디어에서 collagenase 500 단위로 organoid을 품어. 1 분 일초 펄스 (1 초 와동과 일초 나머지) 다음 1 분 중간 전원 이초 펄스 (이초 와동과 이초 휴식)에 소용돌이. 장기 조각이 신선한 불임 microcentrifuge 관에 microcentrifuge 관 및 피펫 뜨는의 하단에 가을 보자. 펠렛의 난소 표면 상피에 3,000 rpm으로 3 분 원심 분리기. </li><li> 투발 상피를 수집, 5 단위 페니실린 및 5 μg / ML의 스트렙토 마이신과 4 % (V / V) FBS과 DMEM의 조직 문화 플라스틱에 길이 튜브를 잘라 놓으십시오. 3~5일 동안 세포는 같은 민족인 기질에서 &quot;크롤링&quot;것이며,​​ 조직 문화 plastic9에서 성장. </li></ol> 3. 파라핀 섹션에 대한 알지네이트요 캡슐 장기 문화의 고정 <ol><li> 선택한 시점에서 배지에서 비드 캡슐 알지네이트요를 수집합니다. </li><li> 2 분 동안 멸균 10mM CaCl 2 용액으로 문화를 놓아 젤 등귀하다하는 알지네이트요을 Recrosslink. </li><li> 젤 고체 유지하고 완벽하게 고정하는 동안 조직을 캡슐 수 있도록 50 MM의 자당, 10 MM CaCl 2 50 MM 나트륨 cacodylate를 포함하는 2 % paraformaldehyde에 알지네이트요 캡슐 오르간을 수정. </li><li> 조직 처리에 따라, 파라핀, 5 μm의 섹션을 생성 마이크로톰과 함께 부분에 전체 알지네이트요 - 캡슐 장기 문화 부분을 포함. </li><li> 알지네이트요는 해산을 기반으로 항원 검색을 열로 인해 immunohistochemistry를 수행하면 슬라이드에서 제거됩니다. 알지네이트요는 남아 hematoxylin 및 eosin의 얼룩 서 아래 eosin에 대한 얼룩 것입니다. </li></ol> 4. 대표 결과 : 난소와 수란관의 3 차원 장기 문화의 예제는 그림 1에 표시됩니다. 난소는 다른 크기의 조각으로 잘라, 그리고 오세은 세포 분열 따위에 의해 번식하고 메스로 절개의 상처를 입었다고 표면을 복구하기 위해 마이 그 레이션 할 수 있습니다. 이 과정에서, 기본 기질에 상대적으로 오세의 올바른 방향은 난소를 캡슐 오세와 함께 유지됩니다. 그림 2에 표시된 구역 또는 eighths을 침범한 난소에 비해, 반으로 난소의 오세 더 완전히 난소를 캡슐. 난소의 상처 수리표면은 전체 난소의 맥락에서 연구 수 있으며, 배란과 표면 수리가 난소 표면 변형을 유도하는 방법에 대해 설명해 주실 수 있습니다. 실험의 종점은 필요한 정보에 따라 달라집니다. 조직 구조, 세포 형태학 및 특정 세포 성장 속도를 쉽게 파라핀 섹션을 사용하여 모니터링할 수 있습니다. 7 일 동안 5 μg / ML 인슐린 이외의 난소 표면 상피와 여러 계층의 형성의 hyperproliferation을 유도하면서 예를 들어, 난소 표면 상피의 단일 계층은 7 일 (그림 3A)에 대한 혈청 무료 αMEM에 culturing 후 관찰 세포 (그림 3B). 이전 고정 10 μm의 24 시간의 최종 농도에서 문화 bromodeoxyuridine를 추가하여, 세포의 증식이 (삽입된 페이지 3A 및 3B) 측정할 수 있습니다. granulosa 세포의 큰 비율 분아 따위에 의해 번식하는 동안 혈청 무료 αMEM 유지 문화, 오세 약 3~5%은 24 시간 라벨 기간 (삽입된 페이지 3A) 동안 증식을 받고있다. 문화 미디어 인슐린 또한 총 오세의 약 30 % (삽입된 페이지 3B)으로 오세를 proliferating의 비율을 증가시킵니다. 녹색 형광 단백질 (GFP)의 Immunofluorescence은 cDNA transfection (그림 3C) 다음과 같은 표준 현미경으로 알지네이트요 젤을 통해 시각 수 있습니다. 알지네이트요 오르간 문화가 난소 표면 상피 및 기본 머리카락 성장과 분석을 위해 제공하고 있지만, 그것은 성숙, 보조 모공이 문화 시스템 (그림 3D)에 생존하지 않을 것을인지해야합니다. 알지네이트요의 히드로겔 매트릭스 내에서 성숙 개별 모공의 문화가 다른 열 설명되었습니다. 단백질이나 RNA의 변경은 전체 기관 또는 효소 분해 (그림 4)를 사용하여 특정 세포 유형에서 분석할 수 있습니다. 일부 stromal 세포도 (그림 4D) 절연 있지만 collagenase와 교양 난소 organoids의 치료, 펠렛은 다음과 같은 치료를 얻은 세포의 오세의 농축을 허용하는 동시에 (그림 4C) 그대로 기본 기질을 떠납니다. <img alt="그림 1" src="/files/ftp_upload/2804/2804fig1.jpg" /> 입힐 수있는 총알 후 오세을 전파하기 위해 사용 그림 1. 마우스 난소 입체 문화 시스템. A) 현미경, 부르사 및 주변 지방 패드가 삭제되었습니다. B) 손상 oviductal 튜브와 난소 (O) (T) 및 지방 패드 (F). C) 부르사 지방 패드와 난소가 제거 (왼쪽)와 uncoiled 수란관 (오른쪽). D) 난소 또는 oviductal organoids는 알지네이트요 비말에 배치했다. E) 알긴산 쌌다 organoids은 CaCl 2 용액 crosslinking 젤 형태 알지네이트요로 감소했다. F) 알긴산은 난소 organoid를 캡슐. G) 알긴산은 oviductal의 organoid를 캡슐. H) 알긴산 캡슐 organoids 문화 매체에 incubated. <img alt="그림 2" src="/files/ftp_upload/2804/2804fig2.jpg" /> 그림 2. 표면적 cytokeratin 8 시까지 측정 난소 표면에 의해 생성. eighths, 분기, 그리고 반쪽, BSA 매체에서 교양 및 cytokeratin 8 일 (CK8) 항체를 사용 immunohistochemical 분석하여 측정을 침범한 후에 오세에 의해 난소 organoids의 캡슐. <img alt="그림 3" src="/files/ftp_upload/2804/2804fig3.jpg" /> 그림 3. 알긴산 문화 시스템은 성장 요인의 통과와 오세로 cDNA의 transfection을 허용하지만, 모공의 급속한 성장과 성숙을 지원하지 않습니다. A) 난소 organoid은 오세을 표시 CK8 표현에 대한 분석 기초 매체 7 일 교양. 삽입된 페이지, 시리얼 섹션 BrdU의 설립을 위해 얼룩. B) 문화 미디어에 인슐린의 추가는 오세의 증식을 유도. 삽입된 페이지, 시리얼 섹션 BrdU의 설립을 위해 얼룩. C) 난소 organoid는 GFP를 표현하기 위해 cDNA와 transfected. D) 알긴산 문화 시스템은 원시 모공 (빨간색 화살촉)의 성장을 지원하지만 성숙하지 보조 머리카락 (검은색 화살표). <img alt="그림 4" src="/files/ftp_upload/2804/2804fig4.jpg" /> 그림 4. 오세은 분석을 다음과 문화 격리 수 있습니다. A) Organoid가 이주하는 매체 교양. B) 캡슐의 organoid는 organoid 그대로두고 알지네이트요의 lyase와 함께 처리됩니다. 난소는 마르크 오세에 CK8 물들일 것입니다. C) Organoid는 근본적인 기질과는 별도로 오세하는 collagenase로 처리됩니다. 남은 조직의 일부가 난소 표면의 제거를 강조 CK8 물들일했다. CK8 (적색)에 대한 스테인드로 D) 치료를 다음과 수집된 세포 오세에 대한 풍부합니다. 세포는 핵을 표시 DAPI로 counterstained 있습니다.

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C., Wake, M. C., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Hydrogels+for+cell+immobilization.">Review: Hydrogels for cell immobilization.</a> Biotechnol Bioeng. 50, 357-364 (1996).</li><li>Iwamoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+bifunctional+alginate+lyase+from+Alteromonas+sp.+strain+no.+272+and+its+action+on+saturated+oligomeric+substrates.">Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates.</a> Biosci Biotechnol Biochem. 65, 133-142 (2001).</li><li>Auersperg, N., Maines-Bandiera, S. L., Dyck, H. G., Kruk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cultured+human+ovarian+surface+epithelial+cells:+phenotypic+plasticity+and+premalignant+changes.">Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes.</a> Lab Invest. 71, 510-518 (1994).</li><li>Auersperg, N., Ota, T., Mitchell, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+events+in+ovarian+epithelial+carcinogenesis:+progress+and+problems+in+experimental+approaches.">Early events in ovarian epithelial carcinogenesis: progress and problems in experimental approaches.</a> Int J Gynecol Cancer. 12, 691-703 (2002).</li><li>Kruk, P. A., Uitto, V. J., Firth, J. D., Dedhar, S., Auersperg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+interactions+between+human+ovarian+surface+epithelial+cells+and+adjacent+extracellular+matrix.">Reciprocal interactions between human ovarian surface epithelial cells and adjacent extracellular matrix.</a> Exp Cell Res. 215, 97-108 (1994).</li><li>West, E. R., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+alginate+hydrogels+and+their+effects+on+in+vitro+follicle+development.">Physical properties of alginate hydrogels and their effects on in vitro follicle development.</a> Biomaterials. 28, 4439-4448 (2007).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocr Rev. 22, 255-288 (2001).</li><li>Choi, J. H., Wong, A. S., Huang, H. F., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotropins+and+ovarian+cancer.">Gonadotropins and ovarian cancer.</a> Endocr Rev. 28, 440-461 (2007).</li><li>Fathalla, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incessant+ovulation--a+factor+in+ovarian+neoplasia.">Incessant ovulation--a factor in ovarian neoplasia.</a> Lancet. 2, 163-163 (1971).</li><li>Espey, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+the+hypothesis+that+mammalian+ovulation+is+comparable+to+an+inflammatory+reaction.">Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction.</a> Biol Reprod. 50, 233-238 (1994).</li></ol>

공개 아무것도 없습니다.

알지네이트요의 hydrogels를 이용한 3 차원 오르간 문화 시스템의 개발은 생물의 다양한에서 여러 가지 조직 유형을 분석하는 다양한 방법을 나타냅니다. 그것이 세포가 11 성장할 수있는 발판을 제공과 같은 3D 문화의 사용은 조직 공학 및 재생 의학 분야로 확장될 수 있습니다. 현재, 저희 연구실은 인간의 난소와 나팔관 조직을 사용하여 초기 연구를 겪고 있지만, 인간이 아닌 인간 영장류 ...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> Leibovitz L - 15 배지 </td><td> Gibco </td><td> 11,415 </td><td></td></tr><tr><td> αMEM 매체 </td><td> Gibco </td><td> 12000-022 </td><td></td></tr><tr><td> 나트륨 알지네이트요 </td><td> Novamatrix </td><td></td><td></td></tr><tr><td> 나트륨 cacodylate </td><td> 시그마 알드리치 </td><td> C0250 </td><td></td></tr><tr><td> 폴리 프로필렌 메쉬 섬유 </td><td> 맥매스터 카 </td><td></td><td> 45 &quot;와이드 롤, 0.0041&quot;오프닝 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11,668 </td><td></td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> Gibco </td><td> 15070-063 </td><td></td></tr><tr><td> 알긴산의 lyase </td><td> 시그마 알드리치 </td><td> A1603 </td><td></td></tr><tr><td> A1603 </td><td> 시그마 알드리치 </td><td> C9697 </td><td> 클로 스트 리듐 histolyticum에서 </td></tr><tr><td> 소 혈청 알부민 </td><td> MP의 Biomedicals </td><td> 103700 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> Gibco </td><td> Gibco </td><td></td></tr><tr><td> ITS 솔루션 </td><td> 로체 </td><td> 11074547001 </td><td> 인슐린 / 트랜스페린 / 나트륨 selenite </td></tr><tr><td> Cytokeratin 8 항체 </td><td> 발달 연구 브리 도마 뱅크 </td><td> TROMA - 1 </td><td> 1:200에​​서 사용 </td></tr><tr><td> DAPI </td><td> 벡터 실험실 </td><td> H1200 </td><td></td></tr><tr><td> Bromodeoxyuridine </td><td> 시그마 </td><td> B50002 - 1G </td><td> 최종 농도 10 μm의 </td></tr></tbody></table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

난소암은 여성 암 사망의 다섯 번째 주요 원인이며, 미국 1 63% 사망률이 있습니다. 난소 암에 대한 원산지 세포 유형은 문제가 여전히하거나, 난소 표면 상피 (오세) 또는 나팔관의 fimbriae 2,3의 말초 상피 수 있습니다. 체외의 기본 문화로 정상 세포 Culturing 과학자함으로써 definitively 원산지의 세포 유형을 결정, 뚜렷한 상피에서 난소암으로 이어질 수도 있습니다 특정 변경 내용을 모델로 사용됩니다. 이것은이 질병을 대상으로보다 정확한 생체, 조직 특정 유전자의 변화와 동물 모델, 그리고 더 나은 예방 전략의 개발을하실 수 있습니다. 알지네이트요의 hydrogels에서 정상 세포를 유지하는 것은 자신의 입체적인 맥락에서 세포의 체외 배양에 단기을 촉진하고 플라스미드 DNA, siRNA, 작은 분자의 도입을 수 있습니다. 하나의 동물에서 실험의 수를 증가 하나 장기 생성하실 수 있습니다 메스, 여러 문화를 사용하는 전략 상처에서 파생됩니다 조각 culturing 기관에 의해. 이것은 난소암의 형성과 관련된 오세의 확산에 선도적인 배란의 모델 측면을 인하. 이러한 플라스틱 표면에 잘 성장하지 않는 오세으로 셀 형식은이 방법을 사용하여 교양과 정상 세포의 프로세스 또는 암 형성 4에서 가장 오래된 행사로 조사를 용이하게하실 수 있습니다. 알긴산의 hydrogels는 조직 5 다양한 종류의 성장을 지원하기 위해 사용할 수 있습니다. 알긴산은 손상 조직 6,7하지 않는 부드러운 겔화 작업의 결과, β - D - mannuronic의 산성과 칼슘 이온과 가교 수있는 α - L - guluronic 수 소산의 단위를 반복 구성된 선형 다당류이다. 같은 Matrigel 같은 다른 세 차원 세포 배양 매트릭스와 마찬가지로 알지네이트요 티슈에 대한 기계적 지원을 제공하지만, 단백질은 알지네이트요 매트릭스와 반응하지 않습니다, 따라서 세포가 5 신호 시작하지 않는 합성 세포외 기질로 알지네이트요 기능. 알지네이트요의 히드로겔 표준 세포 배양 매체 수레와 체외에서 조직 성장의 아키텍처를 지원합니다. 방법을 준비, 분리, 최대 2 주가에 대한 문화의 유지 수 알지네이트요의 hydrogels로 난소 및 oviductal 오르간 작품의 포함을 위해 제공됩니다. 장기 문화 단백질과 RNA 샘플의 분석을 위해 세포의 효소 출시도 설명합니다. 마지막으로, 기본 세포 유형의 성장은 생쥐에서 유전자 불멸화없이 가능하며 한방과 유전자 변형 마우스를 사용하는 수사관을 수 있습니다.

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

그들의 입체적인 맥락에서 정상 세포의 문화 세포 변형 및 tumorigenesis에 필요한 초기 이벤트를 연구하는 다른 방법을 나타냅니다. 이 방법은 난소암 형성 초기에 이벤트를 연구 정상 난소와 oviductal 세포를 성장하는 데 사용됩니다.

난소와 Oviducts의 3 차원 조직 문화 알긴산의 Hydrogels

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K 조회수.  University of Illinois at Chicago. 그들의 입체적인 맥락에서 정상 세포의 문화 세포 변형 및 tumorigenesis에 필요한 초기 이벤트를 연구하는 다른 방법을 나타냅니다. 이 방법은 난소암 형성 초기에 이벤트를 연구 정상 난소와 oviductal 세포를 성장하는 데 사용됩니다.

비디오: Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

전도성 잉크의 평면 및 3 차원 인쇄

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

연구

생체공학

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

조직 문화에 대한 손상 소의 꼬리 척추 디스크의 준비

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

종양 세포 침략에 중간 유체 흐름의 효과를 측정하는 세 가지 차원 세포 배양 모델

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

세 가지 차원 콜라겐 매트릭스 내에서 세포의 이동 분석

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

세 가지 차원 생체 모방 기술 ​​: 소설 Biorubber는 콜라겐 하이드로 겔에 마이크로 및 매크로 스케일 아키텍처를 정의 작성합니다

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

반전 콜로이드 결정체 폴리 (에틸렌 글리콜) 비계의 제작 : 간 조직 공학을위한 삼차원 세포 배양 플랫폼

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

계층화 된 알긴산를 구축 : 이기종 세포 인구의 공동 문화를위한 플랫폼

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

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 ...