1. 분석 설정 <ol><li> 4 ° C (약 2 시간)에 얼음 Matrigel의 작은 나누어지는 (&lt;500 μl)을 녹여. </li><li> 겔 제조법 (아래 표에서 예를 들어 볼륨을 참조) 준비 : 10X PBS (총 판매량 1X), 1N 수산화 나트륨을 (추가 콜라겐의 0.023 권에 상응하거나 콜라겐 제조 업체의 권장 사항마다 적절한 등), Matrigel과 콜라겐이에 나를 입력 1 MG / ML 및 1.3 밀리그램 / 각각 ML (다른 매트릭스 공법은 세포 종류와 실험에 따라 사용될 수있다)의 최종 농도. </li></ol> 예 겔 제조법 <table border="1"><tbody><tr><td> 구성 요소 </td><td> 주식 농도 </td><td> 최종 농도 </td><td> 볼륨을 추가 </td></tr><tr><td> 10X PBS </td><td> 10X </td><td> 1X </td><td> 0.090 ML </td></tr><tr><td> 멸균 물 </td><td></td><td></td><td> 0.346 ML </td></tr><tr><td> 1N NaOH </td><td></td><td></td><td> 0.008 ML </td></tr><tr><td> Matrigel </td><td> 9.90 밀리그램 / ML </td><td> 1 MG / ML </td><td> 0.101 ML </td></tr><tr><td> 쥐의 꼬리 타입 I의 콜라겐 </td><td> 3.66 밀리그램 / ML </td><td> 1.3 MG / ML </td><td> 0.355 ML </td></tr></tbody></table><ol start="3"><li> 4 ° C.에 1 시간을위한 위의하고 품어 최종 해결책 &#39;같은 순서대로 얼음 젤의 구성 요소를 섞어 우리의 경험에서 콜라겐의 더 많은 유니폼을 겔화의 세포 시딩 결과 이​​전 1 시간 보육. 항상 얼음 Matrigel과 콜라겐을 유지하고 겔화 방지를 위해 최대한 빨리 가능한 작동하도록해야합니다. </li><li> 소독 집게를 사용하여 12도 강판으로 장소 12mm 직경 8 μm의 기공 세포 배양 삽입합니다. </li><li> 5 X 10 6 세포 / ML (총 볼륨 겔 용액의 10 %이어야합니다)에 혈청이없는 미디어 셀 및 resuspend 세어보세요. </li><li> 세포 통화 100 μl 추가겔 용액 900 μl (최종 세포 농도가 5 × 10 5 세포 / ML)에 spension하며 위아래로 가볍게 pipetting에 의해 철저하게 섞는다. </li><li> 겔은 polymerizes 때까지 30 분 37 ° C에서 5 % CO 2 배양기에 각각 삽입 및 전송로 최종 혼합물의 150 μl를 추가합니다. </li><li> 혈청 프리 미디어를 사용하면, </li></ol><ul><li> 정적 상태에 삽입 이하 겔과 1,200 μl 위에 100 μl를 추가합니다. 우물에 삽입하고 외부 내부의 액체 레벨 겔없이 중간 흐름 전반에 걸쳐 최소한의 압력 차이가 발생, 약이어야합니다. </li><li> 유동 상태에 젤 위에 넣고 650 μl 아래 100 μl를 추가합니다. 그들이 이러한 위치에있는 멤브레인 통해 마이 그 레이션에서 세포를 차단하므로 삽입 아래에있는 공기 방울을 피하기 위해주의하십시오. 이러한 볼륨에 의해 생성되는 압력 차이는 약 1.3 cm H 2 O (또는 1mm HG)입니다. </li></ul><ol start="9"><li> 37의 장소 플레이트° C, 24 시간을위한 5% CO 2 배양기. </li></ol> 2. 세포 염색법과 집계 <ol><li> 500 1X PBS의 μl 당 자로 새로운 24 잘 플레이트를 추가, 이것은 삽입을 씻어 사용됩니다. </li><li> 흐름 transwells의 상단 부분에 남아있는 매체를 제거하고 볼륨을 결정합니다. 원래, 650 μl를 (이것은 아래 참조, 유량 계산에 사용됩니다) 추가된 총 볼륨에서 나머지 볼륨을 빼는 방법으로 총 eluted 볼륨을 계산합니다. 삽입에서 젤을 제거 및 비 침략 세포를 제거하는 멤브레인의 상단 표면을 닦아하기 위해 면봉을 사용합니다. 삽입을 씻어 15 s에 대한 1X PBS를 포함하는 24 잘 판에 삽입 놓으십시오. </li><li> PBS를 제거하고 transmigrated 세포를 해결하기 위해 상온에서 30 분간 각각 넣고 품어 밑에 4 % paraformaldehyde (PFA) 500 μl를 추가합니다. </li><li> PFA를 제거하고 잔류 정착액을 제거하기 위해 500 μl 1X PBS로 한번 헹굼. </li><li> 500 μl O를 추가하십시오세포를 permeabilize 위해 상온에서 10 분 삽입하고 품어 아래 F 0.5 % 트리톤 X-100 솔루션입니다. </li><li> 아래로 막 얼굴의 아래쪽에 배치하기 위해 신중하다고, 2 μg / 1X PBS 용액에 ML DAPI 100 μl로 면도날과 장소를 사용하여 삽입 세포막을 잘라 (transmigrated 세포 뒤집음). </li><li> 상온에서 30 분간 150 rpm으로 흔드는에 알루미늄 호일과 장소에서 랩 플레이트. </li><li> 흔드는 500 μl 1X PBS 무료 DAPI를 제거하는 (각 10 분 반복 3 회)에서 세포막을 씻으십시오. </li><li> 최대 직면한 transmigrated 전지와 유리 슬라이드에 플레이스 점막은 마운팅 솔루션과 커버 슬립을 추가합니다. </li></ol> 3. 데이터 분석 <ol><li> 각 멤브레인 5 임의로 선택된 위치 (거리 가장자리에서 머물면서 10X 또는 20X 객관 렌즈 사용)에 DAPI 묻은 핵을 세어보세요. </li><li> 평균 셀 카운트를 계산하고 다음 수식을 사용하여 %의 침공을 얻을 : %의 침공 = 100 % X (평균 세포 개수 X 막 면적) / (세포 씨앗의 현미경 이미지 X 번호의 표면적) %의 침공은 독립적인 실험 간의 비교를 가능하도록하기 위해서 몇 가지 제어 상태 (일반적으로 정적 상태)로 정규화 할 수 있습니다. </li><li> 배양 시간 (예, 24 시간)에 의해 유동 조건에서 총 eluted 볼륨을 분할 : 평균 유속을 계산합니다. </li><li> 평균 유속 계산 : 젤 / 멤브레인 (이 경우에는 60mm 2)의 단면적에 의한 평균 유속을 나눕니다. </li></ol> 4. 대표 결과 중간 흐름 미만의 종양 세포 침공을 측정하기 위해, 우리는 MDA-MB-435S 전이성의 흑색증 세포를 사용하여 우리의 3 차원 유동 침략 분석을 수행. 이러한 세포는 이전에 중간 유체 유동 13-14에 대한 응답으로 침략을 보여왔다. 전지는 1.3 밀리그램 / ML 쥐 타이 구성된 매트릭스에 박혀 있었다난 힘줄의 콜라겐 유형 I 및 5 × 10 5 세포 / ML의 최종 세포 농도 1 밀리그램 / ML Matrigel 지하 막 행렬. 두 가지 조건을 비교했다 : (1) 평균 중간 흐름 = 0.1 μm의들 -1 (2) 정적 상태 = 노 측정 가능한 유량 (그림 1). 24 시간 후, 멤브레인의 땀구멍으로 침입 세포는 세포 계수를 촉진하기 위해 DAPI 물들일되었다. 그림 2는 침략 세포의 대표적인 이미지를 보여줍니다. brightfield에서 막만을 모공도 볼 수 있습니다. 형광 사용 DAPI 묻은 핵은 세포 계수와 phalloidin 스테인드 F-actin 구조에 사용되었다는 세포 본문 (선택 사항) 시각화하는 데 사용되었습니다. 동등 변화하는 가정 학생의 T-테스트를 ​​사용하여, 우리는 그 중간 흐름이 크게 (그림 3) (P = 0.003) 정적 조건 이상의 2.3 배하여 MDA-MB-435S 세포 침공을 증가했다. 이 corrobora이 세포주 13-14를 사용하여 유사한 결과를 (하지만 다른 매트릭스 조건에 따라서 유량 판매율 포함) 오셨. <img alt="그림 1" src="/files/ftp_upload/4159/4159fig1.jpg" /> 그림 1. 3 차원 침입형 유체 유동 침략 분석의 도식. 먼저 적절한 농도와 볼륨을 사용하여 겔 용액을 준비합니다. 그런 다음 겔 용액에 세포를 추가하고 세포 배양 삽입에 전송할 수 있습니다. 마지막으로 각 조건에 미디어의 적절한 볼륨을 추가하고 알을 품다. 침입형 유체의 흐름은 유체 압력의 머리에 의해 주도된다. <img alt="그림 2" src="/files/ftp_upload/4159/4159fig2.jpg" /> 그림 2. 막에 MDA-MB-435S 세포를 Transmigrated. 명시야 이하 멤브레인의) 사진; 침략 세포 침입 세포 계수 촉진하기 위하여 DAPI와 알렉사 형석와의 중간 유체 흐름 침략 분석과 스테인드 488-복합 phalloidin 후 수정되었습니다 B) DAPI 역ined 핵 (파란색), C) 알렉사 형석 488-phalloidin의 스테인드 F-actin (녹색). 스케일 바는 50 μm의를 나타냅니다. <img alt="그림 3" src="/files/ftp_upload/4159/4159fig3.jpg" /> 그림 3. 흐름 이하 MDA-MB-435S 세포의 침공 증가했습니다. 24 시간 후 세포 침공은 크게 중간 유량 (P = 0.003)에 의해 향상. 결과는 평균 정적 상태로 정규화과 가치는 6 세포 배양 삽입의 평균 ± SEM을 대표하고 있습니다.

<ol>
	<li>Cichon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+influences+that+drive+progression+from+benign+breast+disease+to+invasive+breast+cancer.">Microenvironmental influences that drive progression from benign breast disease to invasive breast cancer.</a> J. Mammary Gland. Biol. Neoplasia. 15, 389-3897 (2010).</li><li>Proia, D. A., Kuperwasser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroma:+tumor+agonist+or+antagonist.">Stroma: tumor agonist or antagonist.</a> Cell Cycle. 4, 1022-1025 (2005).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment+and+progression.">Tumor microenvironment and progression.</a> J .Surg. Oncol. 103, 468-474 (2011).</li><li>Provenzano, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC Med. 6, 11 (2008).</li><li>Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Butler, T. P., Grantham, F. H., Gullino, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+transfer+of+fluid+in+the+interstitial+compartment+of+mammary+tumors.">Bulk transfer of fluid in the interstitial compartment of mammary tumors.</a> Cancer Res. 35, 3084-3088 (1975).</li><li>Dafni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+vascular+endothelial+growth+factor+165+drives+peritumor+interstitial+convection+and+induces+lymphatic+drain:+magnetic+resonance+imaging,+confocal+microscopy,+and+histological+tracking+of+triple-labeled+albumin.">Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin.</a> Cancer Res. 62, 6731-6739 (2002).</li><li>Chary, S. R., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+interstitial+convection+and+diffusion+of+albumin+in+normal+and+neoplastic+tissues+by+fluorescence+photobleaching.">Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 5385-5389 (1989).</li><li>Heldin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+interstitial+fluid+pressure+-+an+obstacle+in+cancer+therapy.">High interstitial fluid pressure - an obstacle in cancer therapy.</a> Nat. Rev. Cancer. 4, 806-813 (2004).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li><li>Shields, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chemotaxis+as+a+mechanism+of+tumor+cell+homing+to+lymphatics+via+interstitial+flow+and+autocrine+CCR7+signaling.">Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling.</a> Cancer Cell. 11, 526-538 (2007).</li><li>Shieh, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+invasion+is+promoted+by+interstitial+flow-induced+matrix+priming+by+stromal+fibroblasts.">Tumor cell invasion is promoted by interstitial flow-induced matrix priming by stromal fibroblasts.</a> Cancer Res. 71, 790-800 (2011).</li><li>Shi, Z. D., Wang, H., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycans+mediate+interstitial+flow+mechanotransduction+regulating+MMP-13+expression+and+cell+motility+via+FAK-ERK+in+3D+collagen.">Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.</a> PLoS One. 6, e15956 (2011).</li><li>Fleury, M. E., Boardman, K. C., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+morphogen+gradients+by+subtle+interstitial+flow+and+matrix+interactions.">Autologous morphogen gradients by subtle interstitial flow and matrix interactions.</a> Biophys J. 91, 113-121 (2006).</li><li>Miteva, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmural+flow+modulates+cell+and+fluid+transport+functions+of+lymphatic+endothelium.">Transmural flow modulates cell and fluid transport functions of lymphatic endothelium.</a> Circ. Res. 106, 920-931 (2010).</li><li>Ng, C. P., Hinz, B., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+fluid+flow+induces+myofibroblast+differentiation+and+collagen+alignment+in+vitro.">Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro.</a> J. Cell. Sci. 118, 4731-4739 (2005).</li><li>Kunder, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+particles+deliver+peripheral+signals+to+remote+lymph+nodes.">Mast cell-derived particles deliver peripheral signals to remote lymph nodes.</a> J. Exp. Med. 206, 2455-2467 (2009).</li><li>Helm, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+interstitial+flow+and+VEGF+directs+capillary+morphogenesis+in+vitro+through+a+gradient+amplification+mechanism.">Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 15779-15784 (2005).</li><li>McGuire, P. G., Seeds, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+plasminogen+activator+with+a+reconstituted+basement+membrane+matrix+and+extracellular+macromolecules+produced+by+cultured+epithelial+cells.">The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells.</a> J Cell Biochem. 40, 215-227 (1989).</li><li>Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+type+IV+procollagen,+laminin,+and+heparan+sulfate+proteoglycan+from+the+EHS+sarcoma.">Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.</a> Biochemistry. 21, 6188-6193 (1982).</li><li>Haessler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+dynamics+of+breast+cancer+cells+in+a+tunable+3D+interstitial+flow+chamber.">Migration dynamics of breast cancer cells in a tunable 3D interstitial flow chamber.</a> Integr. Biol. (Camb). , (2011).</li><li>Polacheck, W. J., Charest, J. L., Kamm, 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=Interstitial+flow+influences+direction+of+tumor+cell+migration+through+competing+mechanisms.">Interstitial flow influences direction of tumor cell migration through competing mechanisms.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 11115-11120 (2011).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

여기에서 우리는 세포 배양 삽입 내에 3 차원 매트릭스에 포함된 세포를 이용하여 종양 세포 침공에 침입형 흐름의 효과를 quantifying위한 방법론을 설명했습니다. 이것과 유사한 방식이 세포 유형에 13-15, 17의 다양한 침입형 흐름의 효과를 연구하는 데 사용되었습니다. 우리의 접근 방식은 일부 유형을 사용하여 종양의 매트릭스 microenvironment을 모방한 것이었 나 상피 조직과 주변 기질 ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 콜라겐 (쥐 꼬리) </td><td> BD </td><td> 354,236 </td><td> 무균 유지 </td></tr><tr><td> Millicell의 세포 배양 삽입 </td><td> Millipore </td><td> PI8P01250 </td><td> 8 μm의 기공 직경, 폴리 카보 네이트 멤브레인 </td></tr><tr><td> Matrigel </td><td> BD </td><td> 354,234 </td><td> 무균 유지 </td></tr><tr><td> PBS </td><td> 시그마 알드리치 </td><td> 100M-8202 </td><td> 단계를 빨 겔 솔루션 1X를 준비하기위한 10X </td></tr><tr><td> 수산화 나트륨, 1.0N 솔루션 </td><td> 시그마 알드리치 </td><td> S2770 </td><td> 무균 유지 </td></tr><tr><td> DMEM 1X </td><td> CellGro </td><td> 10-013 - 이력서 </td><td> 무균 유지 </td></tr><tr><td> 태아 소 혈청 </td><td> 애틀랜타 체액 </td><td> 511,150 </td><td> 무균 유지 </td></tr><tr><td> 페니실린의 스트렙토 마이신 </td><td> CellGro </td><td> 30002CI </td><td> 무균 유지 </td></tr><tr><td> 트리톤 X-100 </td><td> 시그마 알드리치 </td><td> X100-500 ML </td><td> PBS의 0.5 % </td></tr><tr><td> Paraformaldehyde </td><td> 피셔 과학 </td><td> 04042-500 </td><td> PBS의 4퍼센트 </td></tr><tr><td> 탈이온수 </td><td></td><td></td><td> 무균 유지 </td></tr><tr><td> 4 &#39;, 6 diaminido-2-phenylindole (DAPI) </td><td> MP의 Biomedicals </td><td> 0215757401 </td><td> 1 MG / ML 주식 솔루션 </td></tr><tr><td> 마운트 솔루션 </td><td> 써모 과학 </td><td> TA-030-FM </td><td></td></tr><tr><td> 트립신-EDTA (에틸렌 다이아 민 테트라 초산) </td><td> CellGro </td><td> 25-052-CI </td><td> 무균 유지 </td></tr></tbody> &lt;/ TABLE&gt;

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

대부분의 고체 종양의 성장과 진보는 암 세포와 기질 - 연관된 종양 microenvironment 1에서 신호에 대한 그들의 반응의 초기 변화에 따라 달라집니다. 이전에는 종양 microenvironment에 관한 연구는 주로 종양 - stromal 상호 작용 1-2에 주력하고있다. 그러나, 종양 microenvironment도 효과 저조한 이​​해 남아 biophysical 세력, 다양한 포함되어 있습니다. 이들 세력은 유전자 발현의 변화, 세포 분열, 분화 및 침공 3로 이어질 종양 성장의 biomechanical 결과입니다. 매트릭스 밀도 4, 강성 5-6, 6-7 및 구조, 중간 유체 압력 8, 침입형 유체의 흐름 8 모든 암 진행하는 동안 변경됩니다. 특히 침입형 유체의 흐름은 정상 조직의 8-10에 비해 종양으로 높다. 예상 침입형 유체 층오우 판매율은 측정 및 종양 크기와 차별화 9, 11에 따라 0.1-3 μm의들 -1의 범위에 있어야 발견되었다. 이것은 종양 유발 angiogenesis 및 증가된 혈관 투과성 12 시까지 인한 고가 중간 유체 압력에 의한 것입니다. 침입형 유체의 흐름은 암세포 13-14, 혈관 섬유아 세포 및 평활근 세포 15 침략을 향상을 보여줘왔다. 이 침공은 3-D 16 셀 주위 만들거나 모체 metalloproteinase (MMP) 표현 15, 케모카인 분비와 세포 유착 분자 발현 17 증가 autologous chemotactic의 그라디언트가 원인일 수 있습니다. 그러나 세포가 유체의 흐름을 감지하는 메커니즘이 잘 이해되지 않습니다. 종양 세포의 행동을 변화 이외에도, 중간 유체 흐름은 종양 microenvironment의 다른 세포의 활동을 modulates. 이것은 종양 - 홍보 myofibr으로 섬유아 세포의 (A) 운전 분화와 관련된oblasts 18 (b)는 항원과 림프절 19, 그리고 (c) modulating 림프 내피 세포 morphogenesis 20 ~ 다른 용해 요인에 호송. 여기에 제시된 기술은 체외에서 세포에 침입형 유체 흐름을 부과하고 침공 (그림 1)에 미치는 영향을 quantifies. 이 방법은 stromal 및 암 세포 침입 13-15, 17 일에 유체 흐름의 효과를 측정하기 위해 여러 연구에 게시되었습니다. 매트릭스 구성, 셀 유형, 및 세포 농도를 변경하면이 방법은 이러한 침략, 분화, 증식 및 유전자 발현 등의 세포 프로세스에 침입형 흐름의 효과를 연구하기 위해 다른 질환과 생리적 시스템에 적용할 수 있습니다.

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

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

침입형 유체의 흐름은 고체 종양에서 상승되며 종양 세포 침공을 조절하실 수 있습니다. 여기에서 우리는 매트릭스에 포함된 세포에 침입형 유체 흐름을 적용한 다음 세포 침입에 미치는 영향을 측정할 수있는 기법을 설명합니다. 이 기술은 쉽게 다른 시스템을 연구하는 데 적용할 수 있습니다.

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 ...

three-dimensional-cell-culture-model-for-measuring-effects

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JoVE Journal

Bioengineering

16.4K 조회수.  Drexel University. 침입형 유체의 흐름은 고체 종양에서 상승되며 종양 세포 침공을 조절하실 수 있습니다. 여기에서 우리는 매트릭스에 포함된 세포에 침입형 유체 흐름을 적용한 다음 세포 침입에 미치는 영향을 측정할 수있는 기법을 설명합니다. 이 기술은 쉽게 다른 시스템을 연구하는 데 적용할 수 있습니다.

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

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

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 ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

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 ...

연구

생체공학

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

모델 보컬 폴드 폴립에 의해 유도된 3차원 유동 분리 조사

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

암 세포의 침략에 응용 프로그램을 사용하여 종양의 기질 미시의 조직 공학

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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 ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

유 전적으로 변형 된 세포 이식을위한 미세 패턴 배양 플레이트에 회전 타원체 세포를 향해 유전자 형질

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

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 ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

환경 상처 3 차원 생체 외에서 내 셀 마이그레이션 특성화

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Keratinocyte 확산에 2-및 3 차원의 평가 유형 I 교원 질 기판

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

다층 하이드로겔 바이오잉크를 3차원 바이오프린팅에 사용하여 균일한 세포 분배

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

음향 분리기사용 CHO 수확세포 배양액의 1차 해명

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...