1. 피부 explants에서 섬유 아세포 문화의 확립 <ol><li> 100 단위 / ML 페니실린, 100 μg / ML 스트렙토 마이신, 및 0.25 μg / ML의 Fungizone을 보완 인간의 팔에서 얻은 4mm 펀치 biopsies는 가상에 배치됩니다. </li><li> 어떤 피하 지방을 트림과 가늘게 페트리 접시의 바닥에 대한 번호 24 메스 블레이드를 흔들하여 작은 조각으로 생검을 자르다. </li><li> 플라스크의 표면이 될 때까지 주요 섬유 아세포 성장 미디어 (가상 10 % FCS, 100 단위 / ML 페니실린, 100 μg / ML 스트렙토 마이신, 25 μg / ML Fungizone과 보충)와 25cm 두 조직 배양 플라스크에서 개최 조직 슬러리 추가 적용하지만 액체 깊이 조직이 떠 할 불충분합니다. </li><li> 5% CO 2 humidified 분위기 속에서 37 번 3 일 부화 ° C에 따라, 기본 섬유 아세포 성장 미디어의 3 ML를 추가합니다. </li><li> 신선한 주요 섬유 아세포 성장 미디어와 다른 3 일 배양 변경 미디어를 따라, 또는 cel 경우이게들이 새로운 미디어에 1시 4분을 분할 할 수 있습니다 거의 합류합니다. (Fungizone이 시점부터 생략 할 수 있습니다.) </li></ol> 2. I를 단계 - 쥐 꼬리에서 콜라겐 I의 작성을 참고 : 약 12​​-14 사춘기 (신선한 또는 냉동) 쥐 꼬리에 대한 프로토콜 <ol><li> 70 % 에탄올에 세척하여 쥐의 꼬리를 준비하고 다음과 같이 힘줄을 제거 : </li><li> 로 위에서 아래로 꼬리의 중간에 썰기하여 쥐의 꼬리에서 피부를 제거 메스와 꼬리의 길이를 따라 pealing. </li><li> 꼬리의 가까운 지역의 핵심에서 힘줄를 분리합니다. </li><li> 톱니 집게를 사용하여 피복을 피하기 꼬리 말초 지역으로 힘줄을 제거합니다. </li><li> 48 H에 대해 4 ° C에서 교반에 의해 힘줄의 1 G / 0.5 M 아세트산 250 ML 압축을 풉니 다. </li><li> 30 분의 추출물 (7,500 XG)를 원심 분리기와 펠렛을 폐기합니다. </li><li> 표면에 뜨는에 10 %의 동일한 볼륨 (w / V) NaCl을 추가하고 30-60 분 동안 저어. </li><l난 30 분에&gt; 원심 분리기 (10,000 XG), 표면에 뜨는을 버리고, 4 24 H에 감동하여 ~ 1시 1분 비율 0.25 M 아세트산에서 다시 분해 침전물 ° C. </li><li> 6L 6-8 변화에 대한 콜라겐 솔루션 ~ 17.5 MM 아세트산을 (냉수 리터 당 1 ML 빙하 아세트산, 변경이 X 매일) Dialyze. </li><li> 1.5 시간 30,000 XG에서 dialysed 콜라겐을 원심 분리기. </li><li> 멸균 플라스크의 표면에 뜨는 및 장소를 제거합니다. </li><li> ~ 2 MG / ML에 나는 집중 4에서 0.5 mm까지 아세트산을 사용하여 ° C. 콜라겐을 조정 </li></ol> 3. 단계 II - 내장 된 섬유 아세포 (8 일 계약을 체결 할 수 있도록)와 3D 매트릭스를 설정합니다. <ol><li> 미리 차가운 병에 4에서 추위 시약을 사용하여 ° C를 혼합 조립. 얼음에 콜라겐 I세요. </li><li> 합류 주요 섬유 아세포 중 하나 T75 플라스크를 들어 (~ 전화 번호 1 개 10 6) 사용 : <ul><li> 25 ML의 쥐 꼬리 콜라겐 (약 conc 2mg/ml) </li><li> 10X 3 ML가상 </li><li> 0.22 M NaOH : 첫 번째는, 2ml, 콜라겐이 주황색집니다 때까지 드롭 현명어진이 저어, 추가 </li><li> 하지만 핑크 (일반적으로 최대 3 ML). 대략 P H = 7.2. </li><li> 참고 : 조금이라도 알칼리성 조건에 노출하면 섬유 아세포가 젤 수축하지 않습니다 매체 / 젤은 중성 또는 약산성 상태를 유지하는 것이 중요합니다. </li></ul></li><li> , 섬유 아세포를 Trypsinise ​​5 분 400 XG에 돌리고 표면에 뜨는 제거합니다. </li><li> 5 분 동안 위에 스핀 : 12 X 35mm 플라스틱 요리를 준비 - 일반적으로 섬유 아세포 / 콜라겐 믹스 중 하나를 플라스크에서 12 요리를 얻을 수 있습니다. </li><li> 3 ML FCS에서 섬유 아세포를 다시 중단하고 즉시 콜라겐 믹스에 추가하고 저어. </li><li> 의 판금 약 2.5 ML 요리 당 콜라겐 / 섬유 아세포 가능한 한 빨리. 거품과 콜라겐 설정을 피하기 위해보십시오. </li><li> 콜라겐은 10 분에 공중에 5 % CO 2 humidified 분위기 속에서 37 ° C에서 설정할 수 있습니다. </li><li> 섬유 아세포의 1ml의 성장 추가일 미디어 (DMEM + 10% FCS). </li><li> 피펫을 사용하여 요리의 측면에서 콜라겐 / 섬유 아세포 매트릭스를 분리합니다. </li><li> 다음 날 : 섬유 아세포 성장 미디어 (DMEM + 10% FCS)의 1ml을 추가합니다. </li><li> 매일 미디어를 변경합니다. 그들은 24 잘 접시에 적합한까지 약 8 일간의 계약에 콜라겐 / 섬유 아세포 행렬을 허용 (직경 1.5 cm에 ~ 3.5 cm의 수축, 그림 1 참조). </li></ol> 참고 : 콜라겐 농도가 응용 프로그램에 맞게 조정해야합니다. 더 콜라겐 솔루션을 희석, 더 빨리는 섬유 아세포에 의해 계약을 체결합니다. 수축 속도에 약간의 차이가 같은 농도에서 콜라겐의 다른 일괄 적으로 경험 될 것입니다. 마찬가지로, 다른 섬유 아세포 문화가 서로 다른 가격으로 콜라겐 젤 수축되며, 더 많은 섬유 아세포가 제시 빠른 수축의 속도. 콜라겐 농도와 섬유 아세포 밀도 따라서에 의해 수축의 속도를 수정하는 조절 할 수 있습니다콜라겐 젤과 최종 밀도. 4. 단계 II - 모체의 상단에 관심 도금 세포 참고 : 사용하기 전에 에탄올 모든 포셉과 장비를 소독. <ol><li> 무딘 집게를 사용하여 부드럽게 24도 요리에 계약 매트릭스를 이동합니다. 이 배하지 않도록 확인하십시오. </li><li> 매트릭스의 꼭대기에서 약 4 × 10 4 / ML 및 플레이트 1ml (트립신 제거하는 trypsinising, 스핀 전지 후)에 관심 세포의 정지를 준비합니다. 필요한 셀의 실제 수는 사용하는 세포의 종류에 따라 달라집니다. 셀 매체의 관심 세포에 대한 일반적인 성장 매체해야합니다. </li><li> 세포가 모체의 상단 약 3-5일에 합류로 성장할 수 있습니다. </li></ol> 5. 단계 III - 침략을위한 그리드 (약 0~21일)에 매트릭스 전송 <ol><li> 삼각대를 생성하고 (그림 2 참조)을 사용하기 전에 압력솥에 스테인레스 스틸 격자를 잘라. </li><li> 에 멸균 그리드를 배치6cm 요리는 그리드 위의 수준 (약 10.5ml)에 성장 미디어를 추가 할 수 있습니다. 장소 그리드에 매트릭스와 매트릭스의 하단 미디어와 접촉하지만 침수되지 않도록 부드럽게 미디어를 대기음. 이것은 침략을 촉진 그라디언트를 만들어 공기 / 액체 인터페이스로 언급됩니다. 모든 매체 이틀 (그림 2 참조)로 대체합니다. </li><li> 셀을 라이브, 형광 세포를 사용하여 콘텐츠 영상이 단계 이전에 이미징 할 수 있습니다. 계약 또는 섬유 콜라겐은 I 제 2 고조파 세대 (SHG, 그림 3 참조)를 사용하여 이미지로 할 수 있습니다. 우리가 세포질 GFP를 표현 세포가 최소한 200 μm 깊이 이미징 할 수 있습니다 반면 SHG가 매트릭스에 최소한 100 μm를 이미징 할 수 찾을 폭 넓은 분야 감지와 결합 멀티 광자 여기를 사용합니다. </li></ol> 참고 : 침략의 정량화에 관하여, matricies이 격자에 배치되는 날은 날에게 공을 정의합니다. 격자에 게재은 침략의 정수를 촉진 세포 배양 매체의 그라디언트를 생성<html> 행렬을 O. 샘플은 다음 하나 이상의 이미징 할 수 있습니다 - 21 일 (또는 이상) 등 침입, 증식, 생존 또는 차별화 (참조 목록을 참조)과 같은 생물학적 과정을 평가합니다. 6. 단계 IV - 고정 <ol><li> 팔콘 튜브에 4% PFA의 5 ML을 추가합니다. </li><li> 평평한 표면에 매트릭스를 전송합니다. 새로운 깨끗한 메스 (당신은 단면 얼룩 질 줄 등) 반으로 행렬을 잘라, 밤 수정, 메스와 4% PFA로 송금 행렬을 올린다. </li><li> Organotypic 매트릭스 지금은 항체와 얼룩 / (그림 3 참조) 선택의 얼룩을 할 수 있습니다. </li></ol> 7. 대표 결과 : <img alt="그림 1" src="/files/ftp_upload/3089/3089fig1.jpg" /> 그림 섬유 아세포와 페트리 접시에서 분리하고 6~8일를 통해 계약을 체결 할 수 쥐 꼬리 콜라겐의 섬유 아세포 - 계약 콜라겐 I. 혼합물의 한 예. 그림 2 &quot;SRC =&quot;/ files/ftp_upload/3089/3089fig2.jpg &quot;/&gt; 이 Organotypic 분석의 진행을 그림. organotypic 함정과 진행 A, 설계도. B, 스테인레스 스틸, 공기 / 액체 인터페이스를 만들 수있는 살균 격자의 상단에 콜라겐 / 섬유 아세포 행렬의 예. <img alt="그림 3" src="/files/ftp_upload/3089/3089fig3.jpg" /> organotypic 검정 3 응용 프로그램을 그림. A, B 비 침습적 및 침입 췌장 ductal 암종 (PDAC) 전지는 organotypic 행렬과 시간에 폐를 끼치고. C는 섬유 아세포 - 계약 collagenous 피부 구성 요소에 층상 표피를 보여주는 피부에 상응하는 생활. D, C8161 흑색 증 세포는 침입 섬유 아세포에 collagenous 피부와 같습니다. E, organotypic 매트릭스 내에서 상호 작용과 섬유 아세포 (빨간색)와 침략 침략 PDAC 세포를 (녹색) 계약을 체결. F는 </strong&gt;, 세포 외 기질 (자주색)을 주변 저하와 상호 작용 침해 PDAC 세포 (녹색) multiphoton 기반의 초 고조파 세대를 사용하여 시각화.

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	<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li><li>Edward, M., Gillan, C., Micha, D., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+regulation+of+fibroblast+hyaluronan+expression:+a+mechanism+to+facilitate+tumour+growth+and+invasion.">Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion.</a> Carcinogenesis. 26, 1215-1223 (2005).</li><li>Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+breast+cancer+cells+in+vivo+requires+both+paracrine+and+autocrine+loops+involving+the+colony-stimulating+factor-1+receptor.">Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.</a> Cancer Res. 69, 9498-9506 (2009).</li><li>Serrels, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+study+of+E-cadherin+and+membrane+dynamics+in+living+animals:+implications+for+disease+modeling+and+drug+development.">Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.</a> Cancer Res. 69, 2714-2719 (2009).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li><li>Edward, M., Quinn, J. A., Pasonen-Seppanen, S. M., McCann, B. A., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Methylumbelliferone+inhibits+tumour+cell+growth+and+the+activation+of+stromal+hyaluronan+synthesis+by+melanoma+cell-derived+factors.">4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.</a> Br. J. Dermatol. 162, 1224-1232 (2010).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+characteristics+of+transformed+keratinocytes+from+mouse+and+human+skin+in+vitro+and+in+vivo.">Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.</a> J. Invest. Dermatol. 81, 168s-175s (1983).</li><li>Nystrom, M. L. <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+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J. Pathol. 205, 468-475 (2005).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, 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=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J. Cell. Biol. 185, 11-19 (2009).</li><li>Amjad, S. B., Carachi, R., Edward, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+regulation+of+TGF-beta+and+connective+tissue+growth+factor+expression:+a+role+in+suppression+of+scar+tissue+formation.">Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.</a> Wound Repair Regen. 15, 748-755 (2007).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nat. Cell. Biol. 9, 1392-1400 (2007).</li></ol>

우리는 공개 할 아무 것도 없습니다.

여기 침해 세포 이주 6-8 연구에 적합한 3 차원 매트릭스의 생산을위한 방법을 제시한다. 매트릭스는 기본 인간 표피 섬유 아세포로 몇 일 동안 계약 콜라겐 1 형 원 섬유로 구성되어 있습니다. 콜라겐은 폴리 펩타이드 끝에 반응성을 유지하면서 버퍼 조건 9 중화시 큰 집계에 콜라겐 원 섬유의 가교를 촉진 산성 추출,하지 효소 소화에 의해 준비가되어 있습니다. 더 자세히 생체에서 콜라겐의 기능을 모방하고 젤에 배양 세포의 침입 속성에 대한 중요한 결과를 가지고 다시 구성되는 젤에있는이 결과. 콜라겐 교차 연결은 어린 동물에 더 많은 불안정한 있기 때문에 신선한 또는 냉동 출발 물질을 사용 상관 있지만, 청소년 쥐 꼬리를 사용하지 않는 것이 중요합니다. 콜라겐 I 젊은 동물은 추출하는 것이 용이하며, w를 다시 구성i 번째 높은 충실도. 콜라겐 매트릭스는 고정과 스테인드 침해 종양 세포 또는 stromal 섬유 아세포 2,10 하나 향한 항체와와 침략 5,6을 억제 할 수 약물의 효능을 테스트하는 데 매우 유용합니다 수 있습니다. 이 프로토콜은 기본 인간의 피부 섬유 아세포의 사용을 제안하지만,이 조사중인 조직의 종류에 따라 다른 조직 유형에서 기본 섬유 아세포를 사용 가능합니다. 그것은 안정적으로 형광 단백질 conjugates와 transfected 된 세포를 포함 불후의 섬유 아세포를 사용 문화를 구축하는 것도 가능합니다. 이런 식으로 stromal와 종양 세포는 모두 침략 11시 세포 세포 상호 작용을 시각화하기 위해 표시 할 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 코멘트 (선택 사항) </th></tr><tr><td> 10X 가상 </td><td> Gibco </td><td> 21,430 </td><td> - </td></tr><tr><td> NaOH </td><td> 시그마 </td><td> 367176-500G </td><td> 물에 0.22 M 재고 준비 </td></tr><tr><td> FCS </td><td> PAA Laboratories의 </td><td> A15-101 </td><td> - </td></tr><tr><td> 35mm 요리 </td><td> 매 </td><td> 353001 </td><td> 단계 3.4에 대한 </td></tr><tr><td> 60mm 요리 </td><td> 매 </td><td> 353004 </td><td> 단계 5.2에 대한 </td></tr><tr><td> 봄 포셉 무딘 </td><td> Samco </td><td> E003/02 </td><td> 부드러운 아니라 톱니 </td></tr><tr><td> 16 % paraformaldehyde </td><td> 전자 현미경 서비스 </td><td> 15,710 </td><td> 사용 전에 PBS에 4 %로 희석 </td></tr><tr><td> CD-1, 크기 40 메쉬에 대한 화면 </td><td> 시그마 </td><td> S07707-5EA </td><td> 스테인리스 스틸 격자, 팩 당 5 </td></tr><tr><td> 투석 관 </td><td> Medicell 국제 </td><td> 7607 2295 </td><td> 12-14 KD </td></tr><tr><td> PBS </td><td> Oxoid </td><td> BR0014G </td><td> - </td></tr><tr><td> 아세트산 </td><td> 시그마 </td><td> 242853 </td><td> - </td></tr><tr><td> 24 잘 접시 </td><td> 매 </td><td> 353047 </td><td> 단계 4.1에 대한 </td></tr><tr><td> Fungizone </td><td> vitrogen에서 </td><td> 15290018 </td><td> - </td></tr></tbody></table>

organotypic collagen i assay: a malleable platform to assess cell behaviour in a 3-dimensional context

셀 마이그레이션 개발, 상처 치유, 면역 시스템의 세포 반응 및 종양 세포의 전이를 포함 생물학의 여러 측면들에게 필수적입니다. 마이그레이션은 밝은 현미경으로 조사 의무가 세포 역학을하기 위해 유리 coverslips에 연구되었습니다. 그러나, 세포 이주의 여러 측면이 충실 유리 및 플라스틱 1로 강성 2 차원 기판으로 표현되지는 탄성, 단백질 구성 및 기공 크기, 등의 지역 환경의 특징에 따라 분명이되었습니다. 또한, stromal 섬유 아세포 2, 면역 세포 3를 포함 해 다른 세포 유형, 상호 작용은 암 세포의 침략을 촉진에 중요한 역할을 보여왔다. 분자 수준에서 조사가 점점 더 비교했을 때 동일한 세포의 약물 치료에 반응을 포함한 분자 역학, 크게 다른 것으로 나타났습니다 <em&gt; 체외 및 생체 4인치 이상적으로,이 생활 생물에서의 자연적으로 발생하는 맥락에서 세포 이주를 공부하는 것이 바람직하다, 그러나이 항상 그렇지는 않습니다. 이러한 세포 유래 매트릭스, matrigel, organotypic 문화 (여기에 설명 된) 조직 explants, organoids 및 xenografts와 같은 중간 조직 배양 시스템은, 따라서 중요한 실험 중간체 있습니다. 이러한 시스템은 대략 특정 생체 환경에서의 측면 만은 안정적으로 transfected 세포 라인, 약물 치료 제도, 장기, 고해상도 영상의 사용으로 실험 조작에 더 의무가 있습니다. 이러한 중간 시스템은 프로브를 확인하고 이미지를 사전에 생체 5 이미지를 사업에 세포와 형광 기자의 동적 응답을에 필요한 매개 변수를 설립 근거를 증명 특히 유용합니다. 따라서, 그들은 리빙에서 실험에 대한 필요성을 감소에 중요한 역할을 제공 할 수g 동물.

Watch this Scientific Journal Video about Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context at JoVE.com

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

방법은 콜라겐 유형 I 및 기본 인간 섬유 아세포로 구성된 3 차원 매트릭스의 준비를 위해 설명되어 있습니다. 이 조직 기질의 기본 기능을 모방하고 현미경의 여러 형태의 의무이기 때문 organotypic 젤은 침입 세포 마이그레이션을 평가하는 유용한 기판 역할을합니다.

Organotypic 콜라겐 I 분석 : 3 차원 컨텍스트에서 셀 행동을 평가하기 위해 가단 플랫폼

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

organotypic-collagen-i-assay-malleable-platform-to-assess-cell

Research

JoVE Journal

Bioengineering

22.3K 조회수.  University of Glasgow. 방법은 콜라겐 유형 I 및 기본 인간 섬유 아세포로 구성된 3 차원 매트릭스의 준비를 위해 설명되어 있습니다. 이 조직 기질의 기본 기능을 모방하고 현미경의 여러 형태의 의무이기 때문 organotypic 젤은 침입 세포 마이그레이션을 평가하는 유용한 기판 역할을합니다.

비디오: Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Proteolytically 분해 3 차원 시스템에서 난소 대과의 캡슐화 및 문화에 대한 방법

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

연구

생체공학

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

단백질과 세포 교도소에 대한 2 차원 문양의 기판 만들기

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3 차원 자기 조립 펩티드 히드로겔에서 인간 신경 전구 세포의 배양

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3 차원 세포 배양을위한 자기보고 발판

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

피부에서 타입 I 콜라겐의 제조를위한 개선 된 방법

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

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 ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

평면 그라데이션 확산 시스템은 3D 콜라겐 매트릭스에 화성을 조사하기 위해

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

압축 된 콜라겐에 다진 조직 : 단일 무대 재건 복구를위한 세포 함유 Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

재조합 콜라겐 나 세포 확장과 생물에 셀 납품 시스템으로 그들의 잠재적인 사용에 대 한 펩 티 드 Microcarriers 모델

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.