저자는 기술 지원을 위해 박사 Dongxuan 지아 감사하고 싶습니다. 이 문서에 나타나는 이미지 중 일부는 박사 말콤 S. 스타 인 버그, 분자 생물학의학과, 프린스턴 대학교와 공동으로했다. 저자는 또한 국방 전립선암 연구 프로그램 (보조금 PC - 030482와 PC - 991552)의 계열 NCI / NIH (부여 R01CA118755)은 관대한 지원을 감사드립니다.

1. 단일 세포 현탁액의 준비 <ol><li> 자기편 세포 배양은 90 % 합류로 성장해야 뭘로 monolayers는 PBS로 두 번 씻어서해야합니다. 잘 배수 후, 0.05 % 트립신 - 1 MM EDTA (에틸렌 다이아 민 테트라 초산) 2 MLS를 (100mm 접시)을 추가하고, 37 품어 ° C를 세포가 분리까지. 전체 중간 2 MLS를 추가하고 부드럽게 세포가 정지 때까지 혼합물을 씹다에 5 ML의 피펫을 사용하여 trypsinization를 중지합니다. 15 ML 원뿔 튜브에 세포를 전송합니다. </li><li> RT에서 5 분 10 MG / ML DNAse 주식 품어 40 μl를 추가합니다. 5 분 200 XG에 소용돌이 짧게하고 원심 분리기. </li><li> 뜨는 취소 1 ML 전체 조직 문화 매체와 펠렛을 씻으십시오. 반복 후, 전체 조직 문화 매체 2 MLS에 resuspend 세포. </li><li> hemacytometer, 또는 자동 셀 카운터를 사용하여 셀을 카운트 및 2.5 X 10 6 세포 / ML로 농도를 조정합니다. 이 예제에 대한 BioRad TC10 자동 세포 계수기가 사용되었습니다. </li></ol> 2. 공중 드랍스의 형성. <ol><li> 60mm 조직 문화 요리와 접시의 바닥에서 개최 PBS 5 MLS을에서 뚜껑을 제거합니다. 이것은 수화 챔버 역할을합니다. </li><li> 뚜껑을 반전하고 뚜껑의 하단에 10 μl 방울을 입금 20 μl pipettor을 사용합니다. 만지지만큼 방울은 너무 충분히 떨어져 배치되었는지 확인합니다. 접시 당 최소한 20 방울을 배치하는 것이 가능합니다. </li><li> 37 PBS - 채워진 바닥 챔버와 부화 ° C / 5 % CO 95분의 2 %의 습도에 뚜껑을 반전 매일 방울 모니터와 세포 시트 또는 집계 중 하나가 형성 때까지 알을 품다. 스테레오 현미경은 집계 형성을 평가하는 데 사용할 수 있습니다. </li><li> 일단 시트 양식, 그들이 둥근 바닥 유리 쉐이크로 전송할 수 있습니다 전체 미디어 중 3 MLS가 포함된 37 흔들어 물을 욕조에 incubated flasks ° C와 spheroids 양식까지 5% CO 2. </li></ol> 3. 노트 <ol><li> 셀 크기에 따라 농도는 위 또는 아래로 조정해야 할 수 있습니다. </li><li> 세포 멤브레인가 걸려 드롭 형성 이전에 형광 염료를 intercalating 물들일 수 있습니다. </li><li> 서로 다른 두 가지 세포 유형이 differentially 매달려 방울의 형성 이전에 1:1 (또는 다른) 비율 스테인드 및 혼합 수 있습니다. 이들은 암, stromal 세포, 배아 조직, 또는 유전적으로 특정 접착제 서명을 가지고 설계 세포 수 있습니다. </li><li> 세포 cadherin 기능을 보존하기 위해 0.05 %의 트립신 / 2 MM의 칼슘을 사용하여 조직 문화 요리에서 분리 수 있습니다. </li><li> 실험에 따라 시트 크기는 어느 측정할 수있다, 또는 집계는 기계적 시험 또는 생화 학적 또는 분자 분석을 위해 사용될 수 있습니다. </li></ol> 4. 대표 결과 : 시트 또는 회전 타원체 형성은 일반적으로 24 시간 이내에 발생하지만 세포 유형에 따라 더 오래 걸릴 수도 있습니다. 그림 1은 치료 전립선암 MAT - LyLu (MLL) 드롭 문화를 걸려의 세포 (그림 1A) 또는 25 μm의 MEK 억제제 PD98059로 치료 세포의 배양 18 시간 후 촬영한 이미지를 나타냅니다. 세포 시트의 표시 압축에 MEKi 처리 결과를 참고. 압축은 10-20의 크기 (또는 이상) 방울에 걸려 치료 그룹 사이의 평균 크기를 비교 측정하여 계량하실 수 있습니다. 우리는 픽셀의 전체 이미지 영역을 나타내기 위해 입자 분석을 적용 뭘로 우리는 일반적으로 이진 모드로 이미지를 다음 각 이미지를 thresholding 변환하여 ImageJ 소프트웨어를 사용하여 이미지를 분석합니다. 이것은 그때 쉽게 평방 미크론으로 변환할 수 있습니다. 그림 2는 치료와 MEKi - 대우 MLL 세포의 크기 비교를 나타냅니다. 학생의 T - 테스트 (P는 &lt;0.0001)에 의해 비교처럼 언급, MEKi 치료는 크게 시트 크기를 줄일 수 있습니다. 이 비교를 위해, 치료 및 치료 세포의 각 10 집계가 측정되었다. 그림 3은 드롭 문화를 걸려 24 시간 쉐이크 목욕 2 일 후에 형성된 여자 배아 간 회전 타원체를 나타냅니다. 매달려 드롭 분석도 공간적 위치가 채택 나타내기 위해 하나 이상의 셀 유형을 포함하도록 수정할 수 있습니다. 예를 들어, 병아리 배아 간세포는 형광 막 - intercalating 염색 물들일하고 다른 형광 마커 물들일되었습니다 여자는 배아 심장 세포와 혼합, 격리 수 있습니다. 대신 나머지보다 intermixed되는 그림 4A 쇼, 간, 심장 세포가 한 다른에서 &quot;정렬 아웃&quot;하는 경향과 채택 영역 - 내 - 어 - 구형의 심장 세포가 간 세포에 의해 싸여있는 구성. 이 구성은 간장과 심장 세포 사이의 세포 접착의 차이에 의해 설명하실 수 있습니다. 이 개념은 췌장 아일릿 세포 조직 5와 세포가 동일한 I이 세포 집단 사이의 정렬, 수륙 양용 3 teleost 4 배아의 배아 배아 계층 조직 사이의 동작을 정렬 세포를 설명하는 데 사용되었습니다 차동 접착 가설에 codified입니다N cadherin (6 그림 4B)의 같은 종류의 표현 수준 이외의 모든 존중합니다. 이 결과는 세포 집단 사이의 접착에 간단한 변화를 기술하는 방법 보여주는 경우에 특히 중요한 것은 예를 들어, 장기 구조의 생성을 초래할 수, 췌장 아일릿 5. <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="그림 1" /> 그림 1.하거나 부재 (A) 또는 앞에서 18 시간 동안 드롭 문화를 걸려에서 incubated 세포의 이미지 (B) MEK 억제제 PD98059 25 μm의 중. 이미지는 니콘 이클립스 TE 300 epifluorescence 현미경 및 Photometrics CoolSnap ES 디지털 카메라를 사용하여 캡처한되었습니다. <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="그림 2" /> 그림 2. 쥐의 전립선 암 세포의 MLL MEKi 유발 압축의 양을 정함. 위에서 설명한대로 집계 크기가 결정되었습니다 뭘로 치료 및 MEKi - 대우 MLL 세포 매달려 놓기 문화가 18 시간 동안 incubated되었습니다. 골재 크기의 통계적 분석은 학생의 T - 테스트로했다. 별표 상당히 더 작고 컴팩트 집계에 치료 및 MEKi - 치료 세포 및 제안 MEKi - 치료 결과 사이의 집합 크기에 통계적으로 의미 차이 (P는 &lt;0.0001)을 나타냅니다. <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="그림 3" /> 그림 3. 인큐베이터에서 다음 18 시간 동안 드롭 문화를 걸려 / 48 시간 동안 목욕을 흔들어에 여자는 배아 간세포 잠복기에 의해 형성 회전 타원체. 이 이미지는 니콘 SMZ800 스테레오 현미경과 소니 블랙 CCD 카메라를 사용하여 캡처한되었습니다. <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="그림 4" /> 그림 4. 드롭 문화 식이란 다른 세포 유형 간의 행동 아웃 정렬 보여줍니다. 패널에서이 병아리 배아 간세포는 PKH - 2 막 intercalating 염색 물들일되었으며 PKH - 26 묻은 여자는 배아 심장 세포와 같은 비율로 혼합. 패널 B에서는 2.4:1의 비율에서 표면에 N - CAD 표현이 N - cadherin - transfected L 세포 클론은 (LN4 및 LN2a), 또한 형광 막 intercalating 염료 PKH - 2 및 PKH - 26 물들일되었습니다 (시그마 - 알드리치), 동등한 비율로 혼합하여 방울 7 걸려으로 교양. 문화 18 시간 후, 세포 시트 떨고 flasks로 전송된 다른 48 시간 동안 incubated. 집계가의 2 % paraformaldehyde에서 수정되었습니다 생리 인산염 버퍼와 니콘 Optiphot - 2 현미경에 부착된 BioRad MRC600 스캐닝 레이저 공촛점 현미경 시스템을하셨습니다. 녹색과 적색 두 채널의 광학 부분이 수집되었으며 바이탈 이미지를 갖춘 실리콘 Graphix 크림슨 VGX 워크 스테이션은 Voxel보기 울트라 이미징 소프트웨어는 생성된 해부 구성을 공개, 두 채널에 잘못된 색상을 지정하고 이미지를 병합하는 데 사용되었습니다. (A) pseudocolor 파란색으로 간세포 (8)에 의해 심장 세포의 집합 보여주는 봉함을 통해 공촛점 광학과 (여기 의사 - 색상 노란색). N - cadherin (적색) (6)의 낮은 수준을 표현하는 사람들에 의해 높은 N - cadherin 표현 전지 (녹색)의 집계 보여주는 봉함을 통해 (B) 공촛점 광학 섹션을 참조하십시오.

<ol>
	<li>Cukierman, E., Pankov, R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+interactions+with+three-dimensional+matrices.">Cell interactions with three-dimensional matrices.</a> Curr Opin Cell Biol. 14, 633-639 (2002).</li><li>Nelson, C. M., Bissell, 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=Of+extracellular+matrix,+scaffolds,+and+signaling:+tissue+architecture+regulates+development,+homeostasis,+and+cancer.">Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer.</a> Annu Rev Cell Dev Biol. 22, 287-309 (2006).</li><li>Davis, G. S., Phillips, H. M., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-layer+surface+tensions+and+&amp;quot;tissue+affinities&amp;quot;+in+Rana+pipiens+gastrulae:+quantitative+measurements.">Germ-layer surface tensions and &amp;quot;tissue affinities&amp;quot; in Rana pipiens gastrulae: quantitative measurements.</a> Dev Biol. 192, 630-644 (1997).</li><li>Schotz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+differences+in+tissue+surface+tension+influence+zebrafish+germ+layer+positioning.">Quantitative differences in tissue surface tension influence zebrafish germ layer positioning.</a> HFSP J. 2, 42-56 (2008).</li><li>Jia, D., Dajusta, D., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+surface+tensions+guide+in+vitro+self-assembly+of+rodent+pancreatic+islet+cells.">Tissue surface tensions guide in vitro self-assembly of rodent pancreatic islet cells.</a> Dev Dyn. 236, 2039-2049 (2007).</li><li>Foty, R. A., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differential+adhesion+hypothesis:+a+direct+evaluation.">The differential adhesion hypothesis: a direct evaluation.</a> Dev Biol. 278, 255-263 (2005).</li><li>Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+casein+kinase+2+subunits+in+Chinese+hamster+ovary+and+3T3+L1+cells+provides+information+on+the+role+of+the+enzyme+in+cell+proliferation+and+the+cell+cycle.">Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.</a> J Biol Chem. 274, 32988-32996 (1999).</li><li>Foty, R. A., Pfleger, C. M., Forgacs, G., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+tensions+of+embryonic+tissues+predict+their+mutual+envelopment+behavior.">Surface tensions of embryonic tissues predict their mutual envelopment behavior.</a> Development. 122, 1611-1620 (1996).</li><li>Wendt, D., Riboldi, S. A., Cioffi, M., Martin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+and+bottlenecks+of+bioreactors+in+3D+cell+culture+and+tissue+manufacturing.">Potential and bottlenecks of bioreactors in 3D cell culture and tissue manufacturing.</a> Adv Mater. 21, 3352-3367 (2009).</li><li>Berry, D. P., Harding, K. G., Stanton, M. R., Jasani, B., Ehrlich, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+wound+contraction:+collagen+organization,+fibroblasts,+and+myofibroblasts.">Human wound contraction: collagen organization, fibroblasts, and myofibroblasts.</a> Plast Reconstr Surg. 102, 124-131 (1998).</li><li>Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+biology+in+three-dimensional+collagen+matrices.">Fibroblast biology in three-dimensional collagen matrices.</a> Trends Cell Biol. 13, 264-269 (2003).</li><li>Wang, F. <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+beta1-integrin+and+epidermal+growth+factor+receptor+in+three-dimensional+basement+membrane+breast+cultures:+a+different+perspective+in+epithelial+biology.">Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology.</a> Proc Natl Acad Sci U S A. 95, 14821-14826 (1998).</li><li>Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> J Cell Biol. 137, 231-245 (1997).</li><li>Boudreau, N., Werb, Z., Bissell, 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=Suppression+of+apoptosis+by+basement+membrane+requires+three-+dimensional+tissue+organization+and+withdrawal+from+the+cell+cycle.">Suppression of apoptosis by basement membrane requires three- dimensional tissue organization and withdrawal from the cell cycle.</a> Proc Natl Acad Sci U S A. 93, 3509-3513 (1996).</li><li>Croix, B., Rak, J. W., Kapitain, S., Sheehan, C., Graham, C. H., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+by+hyaluronidase+of+adhesion-dependent+multicellular+drug+resistance+in+mammary+carcinoma+cells.">Reversal by hyaluronidase of adhesion-dependent multicellular drug resistance in mammary carcinoma cells.</a> J. Nat. Cancer Inst. 88, 1285-1296 (1996).</li><li>Bissell, 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=Tissue+structure,+nuclear+organization,+and+gene+expression+in+normal+and+malignant+breast.">Tissue structure, nuclear organization, and gene expression in normal and malignant breast.</a> Cancer Res. 59, 1757-1763 (1999).</li><li>Marrero, B., Messina, J. L., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+tumor+spheroid+in+a+microgravity+environment+as+a+3D+model+of+melanoma.">Generation of a tumor spheroid in a microgravity environment as a 3D model of melanoma.</a> In Vitro Cell Dev Biol Anim. 45, 523-534 (2009).</li><li>Napolitano, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-free+three-dimensional+cell+culture+utilizing+micromolded+nonadhesive+hydrogels.">Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels.</a> Biotechniques. 43, 494-496 (2007).</li><li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture.">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnol Bioeng. 103, 655-663 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

연구는 3 차원 컨텍스트 (3D)에 culturing 세포는 독특한 세포 형태학 및 엄격한 2 차원 (2D) 문화 시스템 9 비교할 때 신호를 생성하는 것으로 나타났습니다. 예를 들어, fibroblast - 인구 콜라겐 젤는 3D에서 fibroblast의 형태는 2D 10,11에서 관찰와는 상당히 별개의 것을 보여줍니다. 마찬가지로, 3D 문화는 내유 상피 세포의 조직과 관련된 차별을 일으킬 수. 3D 문화 시스템은 정상 및 악성 세?...

<ul><li> 자동 세포 계수기 (BioRad TC10) </li><li> CO 2 게이블 표지 모델 3540 (랩 라인)과 물 목욕을 흔들어 </li></ul>

a simple hanging drop cell culture protocol for generation of 3d spheroids

세포 세포 결합 및 세포 - 하층 부착의 연구는 역사적으로 딱딱한 기판에 점착 성의 monolayer 문화에서 수행되었습니다. 조직 내에서 세포 그러나, 세포는 많은 가까운 이웃과 세포외 기질 구성 요소와 친밀한 연결을 설정하는 밀접하게 포장 조직 내에 대량 일반적으로 쌌다 있습니다. 따라서, 화학 환경 및 3D 조직 내에서 세포에 의해 경험 물리적 강제는 monolayer 문화에서 자란 세포에 의해 경험보다 근본적으로 다릅니다. 이것은 현저하게 세포 형태학 및 신호에 영향을 보여줘왔다. 여러 가지 방법이 콜라겐 젤 1 또는 biomaterial 공사장 공중 발판 2 세포의 캡슐을 포함하여 3 차원 세포 배양을 생성 고안되었습니다. 이러한 방법은 유용하면서 정상 조직에있는 친밀한 직접 세포 세포 접착 아키텍처를 요점을 되풀이하지 않습니다. 오히려, 그들은 더 밀접하게 대략적인 문화 시스템은있는 단일 세포는 느슨하게 ECM 제품의 3D meshwork 이내에 분산됩니다. 여기, 우리는 세포가 드롭 문화 매달려에 배치하고 그들이 세포가 서로와 세포외 기질 구성 요소와 직접 접촉하는 진정한 3D spheroids를 형성 때까지 생리 조건 하에서 incubated하는 간단한 방법을 설명합니다. 방법은 전문적인 장비를 필요로하지 않고 셀 - 셀 또는 셀 ECM 상호 작용에 영향을 elucidating에 관심이있을 것으로 매우 소량의 모든 생물 학적 요원의 추가 포함 적용할 수 있습니다. 방법은 또한 서로 다른 세포 인구가있는만큼 셀 사이의 공간 관계를 지정에서 셀 - 셀 또는 셀 - ECM 상호 작용의 역할을 명료하게하다하는 공동의 문화 두 (또는 그 이상)에 사용할 수 있습니다. 세포 세포 결합 및 세포 - ECM 부착은 악성 침입, 상처 치유, 및 조직 공학에 응용 프로그램을 위해 배아 개발, 종양 - stromal 세포 상호 작용 연구의 기초입니다. 이 간단한 방법은 biomechanical 속성 측정이나 생리학 관련 모델에서 분자와 생화 학적 분석을 위해 세포 집계와 같은 조직을 생성하는 수단을 제공합니다.

Watch this Scientific Journal Video about A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids at JoVE.com

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

우리는 세포 - 세포 상호 작용의 차이를 수치 3D 조직 같은 spheroids 및 잠재적인 응용 프로그램을 생성하는 간단하고 빠른 방법을 설명합니다.

3D Spheroids의 생성을위한 간단한 한깅 드롭 세포 배양 프로토콜

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

a-simple-hanging-drop-cell-culture-protocol-for-generation-3d

Research

JoVE Journal

Bioengineering

56.6K 조회수.  UMDNJ-Robert Wood Johnson Medical School. 우리는 세포 - 세포 상호 작용의 차이를 수치 3D 조직 같은 spheroids 및 잠재적인 응용 프로그램을 생성하는 간단하고 빠른 방법을 설명합니다.

비디오: A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

3D 섬유소의 준비는 줄기 세포 배양 용도 공사장 공중 발판

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

연구

생체공학

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

단계 성장 PEG-펩타이드 Hydrogels에서 β-세포 Spheroids의 생성 및 복구

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

관절 연골 세포 문화와 연골 생성을위한 3D 하이드로 겔 공사장 공중 발판

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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 ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

실시간 3D 단일 입자 추적에 대 한 프로토콜

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3 차원 동적 문화 시스템과 다층된 중간 엽 줄기 세포 시트의 건설

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

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

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

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

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

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

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

3D 바이오프린팅을 위한 지방유래 줄기세포 구상체의 대규모, 자동화 생산

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

삼차원 세포 배양 기질의 UV-광패터닝에 의한 다큐 세포 미세환경 생성

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

인간 유도 만능 줄기 세포 유래 심장 스페로이드의 생성, 고처리량 스크리닝 및 바이오뱅킹

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...