우리는 깊이 열성 미세 패턴 배양 플레이트뿐만 아니라 과학적인 조언을 제공하기 위해 토요 고세, 도쿄, 일본에서 박사 타케시 Ikeya 및 기술 직원을 주셔서 감사합니다. 우리는 또한 동물 실험에 대한 기술 지원을 위해 양 사토미 오구라 씨 성폭력 스즈키 씨 아스카 미요시 씨와 Katsue Morii 감사합니다. 이 작품은 재정적으로 JSP를 KAKENHI ​​보조금 에이드에서 과학 연구에 의해 부분적으로 지원, 혁신 (COI) 프로그램의 중심과 일본 과학 기술 진흥기구 (JST)의 S-혁신 프로그램 및 JSP를 코어 - 에 코어 프로그램, A. 고급 연구 네트워크.

모든 동물 실험은 도쿄 대학의 동물 관리 및 사용위원회, 일본 도쿄의 승인을 실시 하였다. 1. 셀 준비 <ol><li> 차 간세포를 들어, 수정 두 단계 콜라겐 분해 공정 (13, 14)에 의해 쥐의 간세포 분리를위한 프로토콜을 따릅니다. <ol><li> 이소 플루 란과 흡입 마취 스프 라그 돌리 (SD) 쥐 (남, 5 주 이전)를 마취. 챔버에 대한 이소 플루 란을 제공하기 위해 마취 기계에 연결된 실에 쥐를 놓습니다. 잠들기 후 쥐를 꺼내, 그리고 마스크를 사용하여 환기가 운영 테이블에 설정합니다. 쥐의 상태를 확인하여 0.7 L / 분 - 이소 플루 란은 0.4에서 약 흐름을 제어 할 수 있습니다. 8g / L 염화나트륨 이루어지는 특수 용액 (염화나트륨), 400 ㎎ / ℓ 염화칼륨 (KCl), 78 mg의로 간문맥으로부터 스프 라그 도울 리 (Sprague Dawley, SD) 랫트 (수컷 5 주령)의 간을 관류 / L소듐 디 하이드 로젠 포스페이트 디 하이드레이트 (의 NaH 2 PO 4 · 2H 2 O), 151 밀리그램 / L 나트륨, 인산 일 수소 칼슘 수화물 (NA 2 HPO 4 · 12H 2 O), 2.38 g / L 2- [4- (2- 히드 록시 에틸) -1- 피 페라 지닐] 에탄 설 폰산 (HEPES), 190 ㎎ / ℓ, 에틸렌 글리콜 디아민 테트라 아세트산 (EGTA), 350 밀리그램 / L 탄산 수소 나트륨 (NaHCO3를) 및 900 ㎎ / ℓ 글루코스. </li><li> 간 콜라게나 통해 용액을 순환시켜. NOTE는 : 용액 500 ㎎ / ℓ의 콜라게나 제, 9.8 g / L 행크 완충 염으로 구성되어 2.38 g / ㎖ HEPES, 556 ㎎ / ㎖ 염화 칼슘 수화물 (염화칼슘 2 · H 2 O), 350 ㎎ / ℓ의 NaHCO3, 상기 pH가 50 ㎎ / ℓ 트립신 억제제, 7.2로 조정. </li><li> 신중 간을 제거하고, 메스 블레이드를 사용하여 접시에 부드럽게 말하다 추가, 둘 베코 변형 이글 배지에 10 % 소 태아 혈청 (FBS)으로 보충 (DMEM), 및 100 ㎛를 통해 세포 현탁액을 필터나일론 메쉬. 추가 파편을 제거하기 위해 1 분 동안 20 × g에서 세포 현탁액을 원심 분리기. 이 단계에서 간세포 상등액에있다. </li><li> 두 번 원심 분리 단계 (세 회 원심 총)을 반복합니다. 3 분 펠릿의 형태들을 복구하기 위해 마지막으로, 50 × g에서 원심 분리 간세포. </li><li> 10 % FBS가 보충 된 DMEM으로 이루어지는 특수 배지에서 4 × 105 세포 / ml의 농도, 1 % 펜 연쇄상-공급 과잉 (PSQ), 1 % 디메틸 설폭 사이드에 간세포를 다시 일시 정지 (DMSO), 0.1 μmol / L 덱사메타손, / ㎖ 인슐린 0.5 μg의 10 밀리몰 / L의 니코틴 아미드, 0.2 밀리몰 / L 아스코르브 인산화 (ASC-2P) 및 10 NG / ㎖ 인간 표피 성장 인자 (hEGF) 15. 이 특수 매체는 시험관 조건에서 간 기능을 유지하기위한 필수입니다. </li></ol></li><li> 쥐 중간 엽 줄기 세포를 얻기위한, isoflur의 과도한 관리에 의해 스프 라그 돌리 (SD) 쥐 (남, 5 주 이전)를 안락사메탄. 대퇴골과 경골을 절제하고, 10 ml의 DMEM + 10 % FBS가 보충 된 이물질을 세정하는 뼈의 축으로 22 G 바늘을 삽입하여 골 수집 된 marrows. 100 ㎛ 나일론 메쉬를 통한 여과에 의해 세포를 수집. </li><li> DMEM은 10 % FBS와 1 % 페니실린 / 스트렙토 마이신을 함유하여 10cm 배양 접시 상에 세포를 시드. 회전 타원체의 실험을 위해, 5 통로 내에서 중간 엽 줄기 세포를 사용합니다. </li></ol> 3D 셀 회전 타원체 2. 준비 <ol><li> 상업적으로 세포 접착 영역이 규칙적으로 PEG 매트릭스에 의해 피복 된 비 접착 구역에 의해 둘러싸인 2 차원 방식으로 100 μm의 직경에 배열 된 미세 패턴 배양 플레이트를 얻었다. 주 : 세포 이식, 감온 중합체 PIPAAm 함께 추가적인 코팅 플레이트를 냉각하여 세포 분리를 허용 할 필요가있다 (단계 5.1 참조). 온도 변동이 중합체의 화학적 변화를 유도 할 수있다8-10. 37 ° C에서 PIPAAm는 세포가 정상 조건에서 배양 할 수 있도록 약간 소수성이다. 세포의 자연 박리에 이르는 빠른 수화 중합체의 32 ° C의 결과를 이하의 온도에서 감소. </li><li> 또한, 5 % CO 2를 함유하는 가습 분위기하에 37 ℃에서 그들을 andincubate / 4 × 105 세포의 밀도로 12- 웰 플레이트에 미세 패턴 간세포 또는 중간 엽 줄기 세포를 시드. 세포는 미세 패턴 접착 분야에 축적 점차 2 일 내에 둥근 회전 타원체를 형성 할 것이다. 주 : 단층 배양에서 대조군 세포의 제조, 통상 12 웰 플레이트를 사용하여 상술 한 것과 유사한 절차에 따라, 동일한 밀도로 세포를 시드. </li></ol> 폴리 플렉스 Nanomicelles 3. 준비 <ol><li> (PEG Mw가 = 12 블록 공중 합체, PEG-PAsp (DET) (폴리 [(2- 아미노 에틸) -2- 아미노 에틸] aspartamide - - [N N &#39;])을 합성000, 중합도 PAsp (DET) 세그먼트 (DP) = 59), 및 이전에 저자 (11)에 의해 기술 된 절차에 따라, 단 양이온 성 세그먼트 [PAsp (DET) (DP = 55)로 구성되는 단일 중합체, 16, 17. </li><li> 33.3 μg의로 중합체 농도를 조절하여 10 mM의 HEPES 완충액 (pH 7.3)에서 잔류 아미노기 동등한 몰비 PEG-PAsp (DET) 블록 공중 합체의 혼합 용액과 PAsp (DET) 호모 폴리머를 준비 / ㎖ 및 각각 19.1 μg의는 / ㎖,. 주의 : 전술 한 중합체의 농도 변화 할 수있다 (10) N / P 비율의 pDNA (N / P 비율)에 인산기로 두 중합체의 총 아미노기의 잔류 몰비 nanomicelles 제조 대표 값은 (자세한 내용은 설명을 참조) 전지 종류 및 목적에 따라. 두 중합체의 병용은 엔도 좀을 향상시키기 위해 효과적인 차폐 및 PEG PAsp (DET)의 기능을 모두 달성 할 수있다탈출 (18) (자세한 내용은 토론 참조). </li><li> pCAG-GS 플라스미드에 각각의 유전자를 발현하는 세그먼트 (http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html을 복제하여의 pDNA 인코딩 Gaussia 루시 페라 제, GL4 루시 페라 제, 또는 에리트로 포이 에틴을 준비 ) 제조사의 프로토콜에 따라 상업적 키트를 사용 CAG 프로모터 / 인핸서 하에서 발현을 얻었다. 유능한 대장균 균주에서의 pDNA를 증폭하여 엔도톡신 무 플라스미드 DNA 정제 시스템을 사용하여 정제. 10 mM의 HEPES 완충액 (pH 7.3)에 150 μg의 / ml의 용액을 얻었다 260 nm의 흡광도에서의의 pDNA의 농도를 결정한다. </li><li> (부피 기준) 1 : 폴리 플렉스 nanomicelles을 준비하기 위해 철저의 pDNA 용액 (10 mM의 HEPES 완충액에 150 μg의 / ㎖)과 (2)의 비율로 두 중합체의 혼합 용액을 혼합한다. </li></ol> 회전 타원체로 4. 유전자 형질 <ol><li> (간세포를 세포를 품어성숙한 회전 타원체의 형성이 가능하도록 미세 패턴 판에 파종 후 72 시간 또는 중간 엽 줄기 세포). 유전자 형질 전환을 위해, 신선한 배지 1 ml의 배양 배지를 교체 한 후, 각 웰에 (의 pDNA의 10 μg의 함유) 폴리 플렉스의 nanomicelle 용액 100 μL를 추가한다. 24 시간 동안 nanomicelle 솔루션 배양을 계속합니다. </li><li> 지질 계 형질 감염 시약을 이용하여 제어를 들어, 지질 계 시약 nanomicelle 방법 모두에 대해 동일하게 pDNA를 최종 투여 량을 조절 (3)의 중량비 시약 / pDNA에에서 시약의 pDNA 용액을 혼합한다. </li></ol> 셀 회전 타원체 5. 복구 및 이식 <ol><li> 냉장 PBS 200 μL와 배지를 교체하고 얼음에 접시를 놓습니다. 주 : 일반적으로, 회전 타원체 약 15 분으로 분리하고 이식 현탁액의 형태로 회수 될 수있다. </li><li> 부드럽게 200 μL의 세포를 대기음생체 내 주사에 대한 23 G 또는 27 G 바늘과 주사기를 사용하여 정지. </li></ol> 형질 전환 유전자 발현 6. 평가 <ol><li> 정확히 24 시간 신선한 배지로 교체 한 후 배지 100 ㎕ - 배지에서 분비 Gaussia 루시페라아제의 발현의 시험 관내 평가를 위해, 50 모은다. 상업적 레 닐라 루시퍼 라제 분석 시스템 및 제조자의 프로토콜에 따라 루미를 사용 루시페라아제 발현을 추정한다. 주 : Gaussia 루시 페라 일주일 이상 배지에서 안정적으로 유지. 따라서, 유전자 발현을 실시간으로 효율을 평가 샘플 매체를 수집하기 전에 하나 신선한 배지를 교체한다. 매체 변화의 타이밍은 유연 할 수있다. </li><li> 세포 이식을 다음 숙주 동물에서 유전자 발현의 생체 내 평가를 들어, BALB / C 누드 마우스 (암컷 마취; 7주이소 플루 란과 흡입 마취 이전). <ol><li> 챔버에 대한 이소 플루 란을 제공하기 위해 마취 기계에 연결된 챔버에 마우스를 놓습니다. 잠들기 후 마우스를 꺼내, 그리고 마스크를 사용하여 환기가 운영 테이블에 설정합니다. 마우스의 상태를 확인하여 0.5 L / 분 - 이소 플루 란은 0.2에서 약 흐름을 제어 할 수 있습니다. </li><li> 복부의 피하 조직으로 (4.1에서 설명) GL4 루시페라아제 발현 형질 타원체의 pDNA를 포함하는 세포 현탁액 200 μl를 주입한다. </li><li> (150 ㎎ / ㎏, 정맥 내 경로) 즉시 D-루시페린을 주입 한 후, 제조자의 프로토콜에 따라 IVIS 이미징 시스템을 사용하여 루시 페라 제 발현을 측정한다. </li></ol></li><li> 세포 이식의 치료 효과를 평가하기위한, 에리트로 포이 에틴으로 코딩하는 pDNA를 형질 스페 로이드를 함유하는 세포 현탁액 200 μl를 주입복부의 피하 조직. <ol><li> 혈액 (19)의 약 200 μl를 얻기 위해 턱밑 출혈로 혈액 샘플을 수집합니다. 혈액 시료 분석기를 이용하여 헤모글로빈 및 헤마토크리트를 측정한다. 참고 : 이식 세포 수는 판에 시드 숫자에 의해 조절된다. 불행히도, 타원체 내부 개수를 측정 할 수 없기 때문에 정확한 세포 수를 확인하는 것은 곤란하다. </li></ol></li><li> 실험 후 마취에서 각성 할 때까지 온도 컨트롤러와 연결된 가열 패드에 마우스를 놓습니다. </li></ol>

<ol>
	<li>Landry, J., Bernier, D., Ouellet, C., Goyette, R., Marceau, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroidal+aggregate+culture+of+rat+liver+cells:+histotypic+reorganization,+biomatrix+deposition,+and+maintenance+of+functional+activities.">Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.</a> J Cell Biol. 101 (3), 914-923 (1985).</li><li>Yuasa, C., Tomita, Y., Shono, M., Ishimura, K., Ichihara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+cell+aggregation+for+expression+of+liver+functions+and+regeneration+demonstrated+with+primary+cultured+hepatocytes.">Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes.</a> J Cell Physiol. 156 (3), 522-530 (1993).</li><li>Otsuka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+multiarray+formation+of+hepatocyte+spheroids+on+a+microfabricated+PEG-brush+surface.">Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface.</a> Chembiochem. 5 (6), 850-855 (2004).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+spheroid+culture+system+on+micropatterned+substrates+for+improved+differentiation+efficiency+of+multipotent+mesenchymal+stem+cells.">3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells.</a> Biomaterials. 30 (14), 2705-2715 (2009).</li><li>Bartosh, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+of+human+mesenchymal+stromal+cells+(MSCs)+into+3D+spheroids+enhances+their+antiinflammatory+properties.">Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties.</a> Proc Natl Acad Sci U S A. 107 (31), 13724-13729 (2010).</li><li>Frith, J. E., Thomson, B., Genever, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+three-dimensional+culture+methods+enhance+mesenchymal+stem+cell+properties+and+increase+therapeutic+potential.">Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential.</a> Tissue Eng Part C Methods. 16 (4), 735-749 (2010).</li><li>Nakasone, Y., Yamamoto, M., Tateishi, T., Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+spheroids+underlayered+with+nonparenchymal+cells+for+biomedical+applications.">Hepatocyte spheroids underlayered with nonparenchymal cells for biomedical applications.</a> IEICE Transactions on Electronics. E94, 176-180 (2011).</li><li>Nishida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+reconstruction+with+tissue-engineered+cell+sheets+composed+of+autologous+oral+mucosal+epithelium.">Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium.</a> N Engl J Med. 351 (12), 1187-1196 (2004).</li><li>Ohashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+functional+two-+and+three-dimensional+liver+systems+in+vivo+using+hepatic+tissue+sheets.">Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets.</a> Nat Med. 13 (7), 880-885 (2007).</li><li>Sekine, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+cell+sheet+transplantation+improves+damaged+heart+function+via+superior+cell+survival+in+comparison+with+dissociated+cell+injection.">Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection.</a> Tissue Eng Part A. 17 (23-24), 2973-2980 (2011).</li><li>Itaka, K., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+prospects+of+polyplex+nanomicelles+for+plasmid+DNA+delivery.">Progress and prospects of polyplex nanomicelles for plasmid DNA delivery.</a> Curr Gene Ther. 11 (6), 457-465 (2011).</li><li>Endo, T., Itaka, K., Shioyama, M., Uchida, S., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfection+to+spheroid+culture+system+on+micropatterned+culture+plate+by+polyplex+nanomicelle:+a+novel+platform+of+genetically-modified+cell+transplantation.">Gene transfection to spheroid culture system on micropatterned culture plate by polyplex nanomicelle: a novel platform of genetically-modified cell transplantation.</a> Drug Deliv and Transl Res. 2 (5), 398-405 (2012).</li><li>Howard, R. B., Christensen, A. K., Gibbs, F. A., Pesch, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enzymatic+preparation+of+isolated+intact+parenchymal+cells+from+rat+liver.">The enzymatic preparation of isolated intact parenchymal cells from rat liver.</a> J Cell Biol. 35 (3), 675-684 (1967).</li><li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> J Cell Biol. 43 (3), 506-520 (1969).</li><li>Tateno, C., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cultivation+of+adult+rat+hepatocytes+that+undergo+multiple+cell+divisions+and+express+normal+parenchymal+phenotypes.">Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes.</a> Am J Pathol. 148 (2), 383-392 (1996).</li><li>Kanayama, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PEG-based+biocompatible+block+catiomer+with+high+buffering+capacity+for+the+construction+of+polyplex+micelles+showing+efficient+gene+transfer+toward+primary+cells.">A PEG-based biocompatible block catiomer with high buffering capacity for the construction of polyplex micelles showing efficient gene transfer toward primary cells.</a> ChemMedChem. 1 (4), 439-444 (2006).</li><li>Itaka, K., Ishii, T., Hasegawa, Y., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polyamino+acid-based+polycations+as+safe+and+effective+gene+carrier+minimizing+cumulative+toxicity.">Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity.</a> Biomaterials. 31 (13), 3707-3714 (2010).</li><li>Uchida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated+Polyplex+With+Optimized+PEG+Shielding+Enhances+Gene+Introduction+in+Lungs+by+Minimizing+Inflammatory+Responses.">PEGylated Polyplex With Optimized PEG Shielding Enhances Gene Introduction in Lungs by Minimizing Inflammatory Responses.</a> Mol Ther. 20 (6), 1196-1203 (2012).</li><li>Golde, W. T., Gollobin, P., Rodriguez, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+simple,+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet.">A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.</a> Lab Anim (NY). 34, 39-43 (2005).</li><li>Uchida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+injectable+spheroid+system+with+genetic+modification+for+cell+transplantation+therapy.">An injectable spheroid system with genetic modification for cell transplantation therapy.</a> Biomaterials. 35 (8), 2499-2506 (2014).</li></ol>

The authors declare that they have no competing financial interests.

이 프로토콜에서, 유전자 도입 및 회전 타원체 회복 단계 동안 회전 타원체의 3 차원 구조를 유지하는 것이 중요하다. 세포는 세포 사멸 또는 세포 활성의 손실을 방지하는 것이 유리한 미세 환경을 유지하는 것이 필수적이다. 기존의 단일 층 문화의 간세포가 급속하게 (12) 파종 후 몇 일 자신의 분비 능력을 잃게 예를 들어, 알부민 분비, 간세포의 대표 타고난 기능은 물론, 간세포 구 상체...

세포 이식 치료는 다양한 난치성 질환을 치료하기 위해 널리 주목 받고있다. 이식 된 세포에 의해 분비 활성 및 생체 활성 인자의 반감기는 세포 이식 시스템의 개선 된 치료 효과에 필수적이다. 이식 이전에 세포의 유전 적 변형을 조절하고 생체 활성 인자의 분비를 포함한 세포 기능을 조작하는 유리한 기술이다. 또한 세포 사멸 또는 세포 활성의 손실을 방지하기위한 셀 유리한 미세 환경을 유지하는 것도 중요하다. 세포 간 상호 작용이 잘 보존되어있는 3 차원 (3D) 회전 타원체 세포 배양은, (일차 간세포의 알부민의 분비를 개선하고, 중간 엽 줄기 세포에서 다중 계통 분화 촉진을 위해, 예를 들어,이 목적을 위해 중간 엽 줄기 세포를 유망한 ) 1-7. spheroi이 연구에서는, 신규 한 콤비네이션 시스템D 문화와 유전자 형질이 유전자 변형 세포 이식을위한 플랫폼 역할을하는 데 사용됩니다. 회전 타원체 세포를 만들기위한, 미세 패턴 배양 플레이트에 회전 타원체 배양 시스템이 사용된다. 이 판에, 직경 100 ㎛의 세포 접착 영역은 규칙적으로 이차원으로 배열되고, PEG 행렬 (3)에 의해 피복 된 비 접착 영역에 의해 둘러싸여있다. 세포의 적절한 수를 파종하여, 직경 100 ㎛의 3 차원 회전 타원체의 배열은 미세 패턴 문화 침대에 대응하여 형성된다. 회전 타원체는 온도 감응성 고분자, 폴리 (이소 프로필 아크릴)로 코팅 된 열성 세포 배양 플레이트를 사용하여 자신의 3D 구조 (PIPAAm) 8-10을 방해하지 않고 복구됩니다. 미세 패턴 구조는 열성 판에 구성된다 (주문 제작). 단순히 플레이트의 온도를 낮춤으로써, 타원체는 배양 베드로부터 분리되어 분산되어인산 D는 식염수 (PBS)를 버퍼. 따라서, 100 ㎛의 균일 한 크기와 타원체의 다수 주사 현탁액의 형태로 얻을 수있다. <img alt="그림 1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> 도 1 미세 패턴 플레이트에 회전 타원체 배양 시스템의 도식 표현. 유전 적 변형이 원래 비 바이러스 유전자 전달체, nanomicelle 폴리 플렉스를 사용하여 유전자 형질 전환에 의해 달성된다. 이는 플라스미드 DNA (pDNA에) 및 폴리에틸렌 글리콜 (PEG) -polycation 블록 공중 합체 (11)로 구성된다. 이러한 특성 코어 - 쉘 (core-shell) 구조, PEG 쉘과 농축의 pDNA의 내부 코어로 구성된 치료 목적 (11)에 대한 세포로 안전하고 효과적인 유전자 도입을 허용있다. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">생의 더 큰 버전을 보려면 여기를 클릭하십시오를</a>의 그림입니다. <img alt="그림 2" src="/files/ftp_upload/52384/52384fig2.jpg" /> 도 핵산 및 PEG-블록 폴리 양이온 블록 공중 합체의 복합체에 의해 형성된 폴리 플렉스 nanomicelle 2. 구조.이 연구에서,이 기술의 주요 장점은 회전 타원체 구조 nanomicelles 의한 유전자 형질 전환시에 파괴되지 않는다는 것이다. 일차 래트 간세포 구 상체의 nanomicelle - 매개 형질 감염 후 장기간 유전자 발현은 형질 감염되지 않은 구 상체 (12)의 것과 유사한 수준으로 간세포에서 연속 알부민 분비 개월 이상이 얻어진다. 회전 타원체에서 유전자 발현과 알부민 분비 또한 열성 판에서 회복 후 유지됩니다. 그것은 nanomicelles 안전하게 형 간염의 타고난 기능을 손상시키지 않고 유전자 도입을 용이하게 할 수 있음을 알 수있다atocytes. 따라서, 유전자 도입 nanomicelles를 사용하여 유전자 변형 세포 이식을위한 유망한 플랫폼과 열성 미세 패턴 접시에 배양 회전 타원체 세포의 결합. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

세포 이식의 치료 효과를 향상시키기 위해, 유 전적으로 변형 된, 주사 타원체의 이식 시스템이 개발되었다. 셀 타원체가 열성 중합체로 코팅 미세 패턴 플레이트에 배양 시스템에서 제조된다. 구 상체의 수는 폴리에틸렌 글리콜 (PEG) 매트릭스에 의해 피복 비 접착 영역에 의해 둘러싸여 정기적 이차원으로 배열되어 100 ㎛ 직경의 세포 접착 부분에 대응하는, 플레이트 상에 형성된다. 스페 로이드 쉽게 플레이트의 온도를 낮춤으로써 액체 현탁액으로서 회수 할 수 있고, 그 구조는 잘 (27 G 이상)를 충분히 큰 구경으로 주사 바늘을 통해 그들을 통과시킴으로써 유지된다. 유전 적 변형은 구형 구조를 방해하지 않으면 서 세포에 유전자를 도입 할 수있는 원래의 비 바이러스 성 유전자 전달체, nanomicelle 폴리 플렉스를 사용하여 유전자 형질 전환에 의해 달성된다. 꼼꼼한 들어알부민 식으로 나타낸 바와 같이 루시페라아제 발현 유전자로 형질 진 간세포 타원체는 루시퍼 라제는 지속 보존 간세포 기능과 함께 이식 된 동물에서 얻어진다. 이 시스템은 중간 엽 줄기 세포를 포함한 다양한 종류의 세포에 적용 할 수있다.

Gaussia 루시페라아제 발현의 pDNA 유전자 형질 전환은 폴리 플렉스 nanomicelles 또는 제어 지질 계 형질 감염 시약 (12)을 이용하여 간세포 또는 MSC들에 의해 형성된 회전 타원체 수행 하였다. 제어 시약 크게 형질 (도 3) 한 후 하루 구조를 파괴하는 반면 nanomicelles은, 미세 패턴 플레이트에 비 형질 구 상체에 비해 회전 타원체 구조에서 거의 변화가 유발하지 않는다. nanomicelles, 일관?...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

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

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. ...

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K 조회수.  The University of Tokyo. 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.

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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned 표면은 암세포와 Hyaluronic 산성 상호 작용을 연구하기 위해

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

연구

생체공학

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Micropatterned 세포를 사용하여 약물의 영향 향상된 시각화 및 정량 분석

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

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

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

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

동적 마스크 프로젝션 석판술를 사용하여 신경 문화 시스템의 Micropatterned Hydrogels의 제작

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

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

인간 세포 배양을위한 회전 세포 배양 시스템 : 모델로 인간 Trophoblast 셀

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

대량 세포 계측법 : CyTOF 질량 Cytometer에 매일 조정하고 실행 세포 샘플에 대한 프로토콜

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

PDMS 막에 마이크로 패턴 세포를 스트레칭

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

미세 패턴 혈관 근육 박막의 확장 문화 미세 유체 Genipin 증착 기술

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

메카노생물학적 연구를 위한 궤도 셰이커에 6웰 플레이트에서 내피 세포의 분할 성장

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

정지 인간 조혈 줄기 세포를 유지하는 배양 방법

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...