This publication is funded through Cure CMD, an Association Contre Les Myopathies (AFM) grant (project 16297), and by the National Institutes of Health (grant numbers K08 AR059750, L40 AR057721 and 2R01NS047727).

참고 : 인체 조직의 수집을위한 프로토콜을 검토하고 기관 IRB위원회의 승인을 받아야한다. 폐기, 드 확인 된 인체 조직의 컬렉션은 보스턴 아동 병원과 브리검 여성 병원 IRB위원회에 의해 승인되었습니다. 후술하는 방법은 디 식별에서, 근원 세포의 분리를 위해 적용되어, 조직을 폐기. 설명 방법은 동의 환자 물질에​​서 수집 한 조직에 적용 할 수있다. 1. 세포 분리 <ol><li> 근육 조직 검사 및 근육 조직 세포의 정화의 해리 <ol><li> 조직의 양을 계산하는 근육 생검을 함유하는 접시를 다시 무게 - 후 해리를 위해 조직 배양 생물 안전성 후드에서 10cm 조직 배양 플레이트를 사전 달아. </li><li> 미세하게 멸균 메스, 말하다 근육 조직을 사용하고 마르지 조직을 방지하기 위해 멸균 1X HBSS 몇 방울을 추가합니다. </li><li> 3.5 ml의 디 각각 추가spase II 및 근육 조직의 그램 당 D 콜라게나 제 소화한다. 멸균 25 ML의 피펫을 통해 몇 번을 다진 조직과 효소 솔루션을 피펫. 15 분 동안 5 % CO 2, 37 ℃에서 조직 배양 인큐베이터에서 인큐베이션 플레이트를 슬러리 쉽게 5 ㎖ 멸균 피펫 비록 통과 할 때까지 조직을 소화. 주 : 조직 해리 보통 45-90 분 이내에 소화 효소에 의해 달성된다. 자세한 내용은 &#39;대표 결과&#39;섹션을 참조하십시오. </li><li> 329 XG (~ 1,100 RPM)을 첨가하고 실온에서 10 분 동안 50 ㎖ 원뿔형 튜브 및 세포 펠렛 위에 100 ㎛ 세포 스트레이너를 통해 해리 조직, 필터 멸균 성장 배지 2 볼륨을 추가. 미디어 컴포지션 재료 표를 참조하십시오. </li><li> 멸균 된 성장 배지의 1 부피에 펠렛을 재현 탁하고 7 볼륨을 적혈구 세포 용해 액을 합한다. 40 μm의 셀 스트레이너를 통해 솔루션을 필터링 한 후 t 펠렛329 XG에 10 분, 실내 온도 그는 세포. </li><li> 혈구 세포의 개수를 1 × 106 세포 / 100 ㎕의 농도로 HBSS 1X 0.5 % BSA에서 세포를 재현 탁. 따로 설정 ~ 음 (흠) 제어에 사용되는 단일 관에서 25 만 셀. FACS 정렬하여 CD56 양성 세포의 적절한 게이팅에 필요한 요오드화 프로피 듐 및 CD56 따로 추가 단일 색 스테인드 제어 튜브를 설정합니다. 정렬 매뉴얼 셀 또는 적절한 컨트롤을 보장하는 핵심 설비 전문가를 정렬 FACS가 포함되어 있습니다에 문의하기를 참조하십시오. </li><li> 얼룩 세포는 항 CD56 항체의 5μL / 10 6 세포를 정렬 할 수 있습니다. 30 분 동안 얼음에 (통제를​​ 포함한) 모든 샘플을 품어. </li><li> 10 ㎖의 1X HBSS에 워시 샘플 및 펠릿 세포 냉장 원심 분리기 329 XG (~ 1,100 RPM), 4 ° C의 온도에서 10 분 동안. </li><li> SOR되도록 시료 1μg / ml의 최종 농도로 요오드화 프로피 듐 추가죽은 세포의 배제 테드. 형광 활성화 된 세포 선별기를 이용하여 비 - 근육 조직 세포로부터의 근원 성 CD56 양성 세포를 정제 하였다. </li></ol></li><li> 피부 생검의 해리 참고 : 근육 조직 검사를 사용할 수없는 경우 피부 섬유 아 세포는 환자의 피부 펀치에서 고립 될 수있다. 피부 섬유 아세포는 근육 조직 세포를 생성하는 형질 MyoD 함께 포함하여 많은 연구를위한 생체 물질로서 사용될 수있다. 또한, 피부 섬유 아세포는 더 깊은 연구 다양한 세포 유형으로 분화 될 수 IPS 세포를 생성하는데 사용될 수있다. <ol><li> 교통 매체의 실험실로 피부 생검을 전송. 샘플이 수신되면, 가능한 빨리 초대 배양을 수행한다. 일차 배양 당일에 확립 될 수없는 경우, 실온에서 밤새 샘플을 저장한다. </li><li> 층류 후드 멸균 35mm 페트리 접시에 피부 생검을 전송합니다. </li><li> 멸균 1 페트리 접시에 피부 조직 검사를 씻어X PBS는 혈액과 이물질을 제거합니다. 멸균 메스 지방 조직을 제거합니다. </li><li> 2 ㎖의 콜라게나 제 용액을 첨가하고 메스 말하다 조직, 조직의 크기에 따라 1 시간 45 분 동안 37 ° C에서 배양한다. </li><li> , 15ml의 원뿔형 튜브에 소화 조직 전송 배 섬유 아세포 배양 배지 2 ㎖로 배양 접시 린스하고 동일한 튜브에서 매체를 수집한다. </li><li> 실온에서 5 분 동안 200 XG에서 세포 펠렛. </li><li> 상층 액을 제거하고 다시 다음, 펠렛 세포를 콜라겐을 제거하는 섬유 아세포 매체의 3 ml의 펠렛을 씻는다. 미디어 컴포지션 재료 표를 참조하십시오. </li><li> 단계 1.2.7 한 번 더 반복합니다. </li><li> T25 무균 조직 배양 플라스크 위에 5 ml의 섬유 아세포 매체와 플레이트 세포 펠렛을 다시 일시 중지합니다. 5 % CO 2와 37 ° C에서 플라스크를 품어. </li><li> 다음 1~3일을 통해 섬유 아세포의 부착과 성장을위한 문화를 평가합니다. &lt;BR /&gt; 참고 : 일부 작은 조직 조각은 또한 판에 부착 할 수 있고 섬유 아세포이 조직의 조각을 이동한다. </li><li> 섬유 아세포는 약 80 %의 합류로 성장 될 때까지 동일한 조건에서 문화를 유지한다. </li><li> 트립신에 의해 섬유 아세포를 수집하고 추가 확장을위한 신선한 배양 플라스크에 전송합니다. 칼슘 ++ 및 마그네슘 ++의 무료 문화를 1X PBS에서 3 회 세척하여 트립신을 수행합니다. 37 ° C에서 2 분 동안 세포 (2 ㎖ / T25 플라스크)에 트립신 - EDTA (참조 자료 표)를 추가합니다. </li><li> 추가 플라스크에 분리 된 세포를 분할합니다. 일부 조직 조각이 원래 T25 플라스크에 연결된 상태를 유지하는 경우,이 플라스크에 5 ml의 신선한 문화 매체를 추가하고 더 많은 섬유 아세포는 지속적으로 밖으로 이동합니다. 참고 : 확장 된 섬유 아세포 문화 P1로 아래로 냉동 및 미래의 실험을 위해 액체 질소에 저장할 수 있습니다. </li></ol></li></ol> 근육 조직 세포의 2 멸화 <ol><lI&gt; 4 박의 비율 DMEM 중소기업 199에서 10cm 무균 조직 배양 플레이트에 플레이트 5 백만 피닉스 Ecotropic 포장 세포 (PE) : 10 % 송아지 혈청 1. </li><li> 10 mM의 카페인 보충 신선한 매체의 5 mL를 형질에 30 분 전에 피드 세포. </li><li> MIDI 준비 (CDK4 또는 플라스미드는 hTERT) 및 polyjet으로부터 플라스미드 DNA의 2 μg의 혼합물을 균질화하고 제조업체에서 권장, 15 분 동안 실온에서 배양한다. </li><li> 밤새 PE 세포에 플라스미드 / polyjet 혼합물을 추가합니다. </li><li> 신선한 배지로 피드 세포 (DMEM 및 (4)의 중간 (199) 비율 : 10 % 송아지 혈청 1). 12 시간 후, 바이러스 - 함유 상등액을 모으고 0.45 ㎛의 공극 크기의 필터를 통해 필터링. </li><li> (G41 밤새 amphotropic 패키징 세포주 PA317 5를 감염 수집 상청액 1 ㎖를 사용하거나, 0.5 ㎎ / ㎖로 네오 마이신 선택 후 안정한 바이러스 - 생산 세포주를 얻기8) CDK4 또는 0.5 mg을 위해 /의 hTERT에 대한 하이 그로 마이신 ㎖로. </li><li> 다음 세 가지 수확의 상층 액을 매일 아침, 저녁과 아침을 수확, 근처 포화 상태에 안정적으로 포장 세포를 성장시켜 바이러스 상층 액을 작업 준비합니다. </li><li> 바이러스 상층 액을 여과하고, 직접 나중에 사용하기 위해 -80 ° C에서 1 ml의 분취 량 및 저장에 사용하거나 나눕니다. 바이러스 상등액을 각각 동결 - 해동로 50 %의 감염 효율이 없어집니다. 표백 세척을하기 위해 바이러스 입자를 만진 모든 것을 폐기하기 전에 기억하십시오. 주 : 안정 PA317 바이러스 생산 세포주 냉동 영구 저장 -150 ° C로 유지 될 수있다 (중간 동결하는 10 % DMSO이다 혈청 90 %). </li><li> 플레이트 FACS 정제 근원 성 세포 / 웰을 0.1 % 젤라틴으로 코팅 된 6- 웰 플레이트에 5 × 104 세포의 밀도 (1.1-1.9 단계에서 설명). 세포가 바이러스 감염으로 진행하기 전에 판에 제대로 연결되어 있는지 확인하십시오. </li><li> F, 필터링 400 μl를 추가reshly 하룻밤 (컨트롤로이 우물을 유지) 각 여섯 웰 플레이트에 바이러스 상층 액 또는 냉동 분량을 생산했다. </li><li> 2.5 ㎖ / 웰의 신선 근육 매체 (4 세포를 공급함으로써 변화 매체 : 0.02 M HEPES 완충액; 1.4 ㎎ / L 비타민 B12, 15 % 소 태아 혈청으로 보충 한 둘 베코 변형 이글 배지 (DMEM) 및 매체 (199) 0.03 mg의 / L의 ZnSO4, 0.055 ㎎ / L 덱사메타손, 2.5 ㎍ / L 간세포 성장 인자 및 10 ㎍ / L 염기성 섬유 아세포 성장 인자). 표백제 용기에 바이러스 입자를 함유하는 매체 및 피펫 버린다. 삼일 후, 감염에서 회복 400 ㎍ / ㎖의 네오 마이신 (CDK4 감염) 또는 300 ㎍ / ml의 하이 그로 마이신 (의 hTERT 감염)을 사용하여 선택을 치료하는 데에 동일한 배지에서 세포를 남겨주세요. </li><li> 제어 요리 다이의 셀 (1-2 주)까지 약물 선택에 따라 세포를 유지한다. </li><li> 심지어 selecti 동안 (EDTA 0.05 % 트립신을 사용하여 컨 플루를 60 내지 80 %의 밀집 상태) 통로 세포들은 합류되기 전에기간에. 선택 약물로 보충 신선한 근원 세포 매체와 여러 10cm 요리를 Replate 세포 (2.11에서 설명). 이종 집단으로 불멸화 선택된 셀을 유지하거나 완전히 균질 유전 적 배경 (모든 셀에서 동일한 트랜스 진의 삽입)을 얻었다 복제. </li><li> 다음 단계를 사용하여 클론 선택을 수행합니다 : <ol><li> 작은 식민지가 (10 ~ 20 세포)이 형성 될 때까지, 저밀도 (예 : 10cm 요리를 300-500 세포)에서 세포를 씨와 약 2 주 동안을 유지한다. </li><li> 마르지 않도록 세포 박막만을 남겨이 시점에서 매체의 대부분을 제거한다. </li><li> (일단 실리콘 진공 그리스에 담근에) 원하는 각 복제를 통해 복제 링을 놓고 트립신 / EDTA 몇 방울을 추가합니다. </li><li> 수확 세포들은 조심스럽게 1 ml의 끝이나 파스퇴르 피펫을 사용하여 세포를 흡입하고 사용할 수있는 가장 작은 멀티 웰 괞 찮아로 전송 반올림이 되시면테 (96 또는 48, 24 또는 12 웰 플레이트). </li><li> 지역의 포화 상태를 방지하기 위해 필요에 따라 복제를 확장합니다. </li></ol></li></ol>

<ol>
	<li>Flanigan, 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=The+muscular+dystrophies.">The muscular dystrophies.</a> Semin Neurol. 32, 255-263 (2012).</li><li>Mercuri, E., Muntoni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophies.">Muscular dystrophies.</a> Lancet. 381, 845-860 (2013).</li><li>Sharples, A. P., Stewart, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+models+of+skeletal+muscle+hypertrophy+and+atrophy.">Myoblast models of skeletal muscle hypertrophy and atrophy.</a> Curr Opin Clin Nutr Metab Care. 14, 230-236 (2011).</li><li>Tran, T., Andersen, R., Sherman, S. P., Pyle, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+skeletal+muscle+development+and+applications+in+regenerative+medicine.">Insights into skeletal muscle development and applications in regenerative medicine.</a> Int Rev Cell Mol Biol. 300, 51-83 (2013).</li><li>Miller, A. D., Buttimore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redesign+of+retrovirus+packaging+cell+lines+to+avoid+recombination+leading+to+helper+virus+production.">Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.</a> Mol Cell Biol. 6, 2895-2902 (1986).</li><li>Schubert, W., Zimmermann, K., Cramer, M., Starzinski-Powitz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+antigen+Leu-19+as+a+molecular+marker+of+regeneration+in+human+skeletal+muscle.">Lymphocyte antigen Leu-19 as a molecular marker of regeneration in human skeletal muscle.</a> Proc Natl Acad Sci U S A. 86, 307-311 (1989).</li><li>Mechtersheimer, G., Staudter, M., Moller, P. <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+natural+killer+(NK)+cell-associated+antigen+CD56(Leu-19),+which+is+identical+to+the+140-kDa+isoform+of+N-CAM,+in+neural+and+skeletal+muscle+cells+and+tumors+derived+therefrom.">Expression of the natural killer (NK) cell-associated antigen CD56(Leu-19), which is identical to the 140-kDa isoform of N-CAM, in neural and skeletal muscle cells and tumors derived therefrom.</a> Ann N Y Acad Sci. 650, 311-316 (1992).</li><li>Pavlath, G. K., Gussoni, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+myoblasts+and+muscle-derived+SP+cells.">Human myoblasts and muscle-derived SP cells.</a> Methods Mol Med. 107, 97-110 (2005).</li><li>Boldrin, L., Muntoni, F., Morgan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+human+and+mouse+satellite+cells+really+the+same.">Are human and mouse satellite cells really the same.</a> J Histochem Cytochem. 58, 941-955 (2010).</li><li>Belles-Isles, M., 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=Rapid+selection+of+donor+myoblast+clones+for+muscular+dystrophy+therapy+using+cell+surface+expression+of+NCAM.">Rapid selection of donor myoblast clones for muscular dystrophy therapy using cell surface expression of NCAM.</a> Eur J Histochem. 37, 375-380 (1993).</li><li>Meng, J., Adkin, C. F., Xu, S. W., Muntoni, F., Morgan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+human+muscle-derived+cells+to+skeletal+muscle+regeneration+in+dystrophic+host+mice.">Contribution of human muscle-derived cells to skeletal muscle regeneration in dystrophic host mice.</a> PLoS One. 6, e17454 (2011).</li><li>Zhu, C. 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=Cellular+senescence+in+human+myoblasts+is+overcome+by+human+telomerase+reverse+transcriptase+and+cyclin-dependent+kinase+4:+consequences+in+aging+muscle+and+therapeutic+strategies+for+muscular+dystrophies.">Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies.</a> Aging Cell. 6, 515-523 (2007).</li><li>Mamchaoui, 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=Immortalized+pathological+human+myoblasts:+towards+a+universal+tool+for+the+study+of+neuromuscular+disorders.">Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.</a> Skelet Muscle. 1, 34 (2011).</li><li>Sigmund, C. D., Stec, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetic Manipulation of the Renin-Angiotensin System Using Cre-loxP-Recombinase. Methods Mol Med. 51, 53-65 (2001).</li><li>Ludlow, A. T., 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=Quantitative+telomerase+enzyme+activity+determination+using+droplet+digital+PCR+with+single+cell+resolution.">Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.</a> Nucleic Acids Res. 42, e104 (2014).</li><li>Jankowski, R. J., Haluszczak, C., Trucco, M., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+myogenic+cell+populations+obtained+via+the+preplate+technique:+potential+for+rapid+isolation+of+muscle-derived+stem+cells.">Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells.</a> Hum Gene Ther. 12, 619-628 (2001).</li><li>Gharaibeh, B., 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=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu, Z., 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=Development+of+approaches+to+improve+cell+survival+in+myoblast+transfer+therapy.">Development of approaches to improve cell survival in myoblast transfer therapy.</a> J Cell Biol. 142, 1257-1267 (1998).</li><li>Choi, 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=MyoD+converts+primary+dermal+fibroblasts,+chondroblasts,+smooth+muscle,+and+retinal+pigmented+epithelial+cells+into+striated+mononucleated+myoblasts+and+multinucleated+myotubes.">MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes.</a> Proc Natl Acad Sci U S A. 87, 7988-7992 (1990).</li><li>Huard, C., 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=Transplantation+of+dermal+fibroblasts+expressing+MyoD1+in+mouse+muscles.">Transplantation of dermal fibroblasts expressing MyoD1 in mouse muscles.</a> Biochem Biophys Res Commun. 248, 648-654 (1998).</li><li>Lattanzi, L., 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=High+efficiency+myogenic+conversion+of+human+fibroblasts+by+adenoviral+vector-mediated+MyoD+gene+transfer.+An+alternative+strategy+for+ex+vivo+gene+therapy+of+primary+myopathies.">High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.</a> J Clin Invest. 101, 2119-2128 (1998).</li><li>Park, J. I., 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=Telomerase+modulates+Wnt+signalling+by+association+with+target+gene+chromatin.">Telomerase modulates Wnt signalling by association with target gene chromatin.</a> Nature. 460, 66-72 (2009).</li></ol>

The authors have nothing to disclose.

유용한 자원으로 세포 질병의 체외 모델에서 질병의 표현형을 설정할 때, 근원 세포 집단의 분리 및 배양은 매우 유용합니다. 여기에 설명 근원 성 세포 분리 과정은, 전파 분화, 또는 즉시 분석 할 수 골격근 시험편 근모 섬유 아세포의 분리를 허용한다. 근원 세포의 구조와 기능은 세포 생존, 세포 융합의 평가의 평가 또는 RNA 또는 단백질 분자의 발현 연구를 통해, 현...

A correction was made to the authors section in: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
One of the authors names was corrected from:
Stacy C. Cossette
to:
Stacy A. Cossette

A correction was made to the authors section in: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Erratum: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Molecular diagnostics has dramatically evolved in the past 20 years. Genomic DNA is now routinely isolated from sputum or cheek swab, while in the past it required a blood draw. With the current fast turnaround time and ease of gene sequencing, many disease mutations are routinely identified with no need of additional testing. In the case of muscle disease diagnostics, identification of dozens of new genes in the past decade responsible for either muscular dystrophy or myopathy have dramatically changed the ways these diseases are diagnosed 1,2. Currently, there are dozens of genes that have been identified as causes of muscular dystrophy and congenital myopathy, although the mechanisms by which many of these genes produce disease remain unclear. In particular, rare diseases constitute a challenge due to the small size of the patient populations. For these cases, as well as for more common diseases, the generation of stable tools that facilitate studies on the mechanism of pathogenesis and screening of therapeutic drugs is highly desirable. 
Despite dramatic progress in DNA diagnostics, muscle biopsies are still performed to establish the primary diagnosis in many patients in whom primary metabolic or muscle disease is suspected. When a muscle biopsy is necessary, it offers the opportunity for additional diagnostic and research tissue collection with minimal additional morbidity risk for the patient. As there are a number of uses for each tissue specimen, it is highly desirable to establish techniques for primary cell culture using surgical tissue that are straightforward, efficient, and require minimal amounts of tissue. Proper triage of muscle or skin biopsies is required to maximize isolation of primary cells from tissue and long-term storage of live material. Additionally, stem cell research and drug screening holds great promise for developing therapies for many diseases using cell-based assays 3,4.
We herein describe methods for primary cell isolation from human muscle or skin biopsies. Additionally, we include a protocol for immortalization of myogenic cells, which is useful for generating large numbers of cells from an individual. These cells can be used for downstream applications, such as custom drug-screenings, which are otherwise unachievable with the overall low number of cells obtained from primary tissue.

<table border="0" cellpadding="0" cellspacing="0" width="1197">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"><a name="RANGE!A1:D52">Name of Material/ Equipment</a></td>
			<td style="width:187px;">Company</td>
			<td style="width:207px;">Catalog Number</td>
			<td style="width:484px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Equipment</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Tissue&#160;culture biosafety hood</td>
			<td style="width:187px;">Baker Company, Inc.</td>
			<td style="width:207px;">Model SterilGuard Hood</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:320px;">Benchtop centrifuge, such as Beckman Model&#160;</td>
			<td style="width:187px;">Beckman Coulter</td>
			<td style="width:207px;">Model Allegra 6R</td>
			<td>If cell sorting is performed, a centrifuge with refrigeration is preferred</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Microscope</td>
			<td style="width:187px;">Nikon</td>
			<td style="width:207px;">Model Eclipse TS100</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:320px;">Tissue culture incubator, connected to a CO2 source</td>
			<td style="width:187px;">Forma Scientific&#160;</td>
			<td style="width:207px;">Series II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Fluorescence-activated cell sorter (FACS)</td>
			<td style="width:187px;">Becton Dickinson</td>
			<td style="width:207px;">Model Aria</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:320px;">Reagents for isolation of primary myoblasts from tissue</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispase II</td>
			<td style="width:187px;">Roche Applied Science</td>
			<td style="width:207px;">#04942078001</td>
			<td>Prepare a sock solution of 2.4U/ml in DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase D&#160;</td>
			<td style="width:187px;">Roche Applied Science</td>
			<td style="width:207px;">#088882001</td>
			<td>Prepare a stock solution of 10 mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1x Sterile Hank&#8217;s Balanced Saline Solution (HBSS), calcium and magnesium free</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">#14185-052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin, fraction V</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">#05470</td>
			<td>Prepare a sterile&#160; solution of 1X HBSS 0.5% BSA for FACS sorting</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Sterile growth medium for myoblasts: Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) with high glucose (4.5g) supplemented with 30% fetal bovine serum</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">11965-092</td>
			<td>Contains L- glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RBC lysis solution</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:207px;">158904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Propidium Iodide stock 10mg/mL</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">P4170</td>
			<td>Prepare the stock solution by diluting the powder in sterile distilled water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Anti-human CD56 antibody for flow cytometry</td>
			<td style="width:187px;">Biolegend</td>
			<td>318310</td>
			<td>APC-conjugated antibody, other labels are avaialable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Reagents for immortalization of primary myoblasts</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ecotropic packaging cell line PE</td>
			<td style="width:187px;">Cell Biolabs</td>
			<td style="width:207px;">RV-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Amphotropic packaging cell line PA3174</td>
			<td style="width:187px;">Cell Biolabs</td>
			<td style="width:207px;">RV-102</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Pig skin gelatin</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">G1890-500g</td>
			<td style="width:484px;">Prepare a stock solution of 0.1% gelatin in water. Coat the dish with the solution at 37&#176;C for one hour. Remove the solution and add medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">PolyJet&#160;</td>
			<td style="width:187px;">Signagen</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">G418</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:207px;">345812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hygromycin</td>
			<td style="width:187px;">EMD Biosciences</td>
			<td style="width:207px;">400051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">pBabe plasmids containing mCDK4 and hTERT</td>
			<td style="width:187px;">not commercially available</td>
			<td style="width:207px;"></td>
			<td>Stadler et al, 2013</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Qiagen plasmid midiprep kit</td>
			<td style="width:187px;">Qiagen</td>
			<td>12143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Medium 199</td>
			<td style="width:187px;">Life Technologies&#160;</td>
			<td>Medium 199 (31150022)</td>
			<td style="width:484px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:187px;">Life Technologies&#160;</td>
			<td>DMEM (11965-092)</td>
			<td style="width:484px;">Mix 4:1 DMEM:199</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:320px;">Myoblast growth medium: 4:1 Dulbecco&#8217;s modified Eagle medium (DMEM) and Medium 199 supplemented with 15% fetal bovine serum; 0.02 M HEPES buffer; 1.4 mg/l vitamin B12; 0.03 mg/l ZnSO4, 0.055 mg/l dexamethasone, 2.5 &#956;g/l hepatocyte growth factor and 10 &#956;g/l beta fibroblast growth factor.</td>
			<td style="width:187px;">Life technologie (DMEM, F199); Atlanta Biological (FBS); Invitrogen (Hepes); Fisher (ZincSulfate); Sigma (Vit.B12, Dexamethasone); Chemicon international (HGF); Biopioneer (betaFGF)&#160;</td>
			<td style="width:207px;">#15630-080 (Hepes); #Z68-500 (ZnSO); #V2876.#D4902 (Vit.B12, Dex); GF116 (HGF); HRP-0011 (bFGF).&#160;</td>
			<td style="width:484px;">Prepare media and stock solution Vit. B12 (20mg/ml); ZincSulfate (60&#181;g/ml); Dex (55&#181;g/&#181;l) separately for easier use. HGF stock solution (5&#181;g/ml) and FGF (20&#181;g/ml) should be added freshly every week at the final working concentration.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">TrypLE express</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">12605-010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Myosin heavy chain antibody for immunostaining</td>
			<td style="width:187px;">Developmental Hybridoma Bank</td>
			<td style="width:207px;">MF20</td>
			<td>Clone MF20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Desmin Antibody for immunostaining</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>MS-376-S0</td>
			<td>Clone D33</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Horse serum</td>
			<td style="width:187px;">Invitrogen</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Reagents for primary skin fibroblast isolation</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Transport medium: RPMI 1640 supplemented with 10% fetal bovine serum and 0.2% penicillin/streptomycin</td>
			<td style="width:187px;">RPMI medium 1640: GIBCO Life Technologies</td>
			<td style="width:207px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Human primary fibroblast culture medium: RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin</td>
			<td style="width:187px;">Fetal bovine serum: Thermo Scientific</td>
			<td style="width:207px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Collagenase solution: Collagenase type 2 (100mg) resuspended in 12.5 ml of fibroblast culture medium and filter-sterilized</td>
			<td style="width:187px;">Worthington</td>
			<td style="width:207px;">4176</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Sterile 1X Phosphate Buffer Saline (PBS) calcium and magnesium free</td>
			<td style="width:187px;">Lonza</td>
			<td style="width:207px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for isolation of primary myoblasts</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">50ml and 15ml sterile conical tubes</td>
			<td style="width:187px;">GeneMate/Bioexpress</td>
			<td style="width:207px;">C-3394-4 (50ml) ; C3394-1 (15ml))</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile scalpels</td>
			<td style="width:187px;">Aspen Surgical</td>
			<td style="width:207px;">372610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:187px;">Hausser Scientific</td>
			<td style="width:207px;">1492</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 5, 10 and 25 ml pipettes</td>
			<td>Bellco glass</td>
			<td>1226-05010 (5ml); 1200-10010 (10ml) ;1228-25050 (25ml)</td>
			<td>Reusable pipettes are washed, cotton plugged and autoclaved before use</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile tissue culture-treated plastic dishes (10cm)</td>
			<td style="width:187px;">BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile nylon cell strainers (100&#181;m and 40&#181;m size)</td>
			<td style="width:187px;">BD Falcon</td>
			<td colspan="2">352340 (40&#181;m); 352360 (100&#181;m)</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">&#160;Materials for myoblast immortalization</td>
			<td style="width:187px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 0.45&#181;m filters</td>
			<td>Millipore</td>
			<td>SLHV013SL</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Cloning rings</td>
			<td style="width:187px;">Corning</td>
			<td>#3166-8</td>
			<td>To be cleaned and autoclaved before and after use</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Materials for fibroblast cell lines&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 35mm tissue culture-treated plastic dishes</td>
			<td>Greiner bio-one</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile scalpels</td>
			<td>DeRoyal</td>
			<td>D4510A</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile T25 tissue culture flasks</td>
			<td>Techno Plastic Product, TPP</td>
			<td>90026</td>
			<td>sold in the USA by MIDSCI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">TrypLE express</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">12605-010</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and immortalization of patient-derived cell lines from muscle biopsy for disease modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

도 1은 주 조직 분해에 관여하는 주요 단계들을 도시한다 : 조직의 정확한 양은 (도 1A, B) 멸균 조직 배양 용 배양 접시에 칭량된다. 티슈 슬러리 (도 1C, D)를 구할 때까지 조직이어서 미세 멸균 다진 메스를 사용한다. 분해 효소를 첨가 한 다음에, 주 근조직 해리는 대개 45-90 분 이내에 소화 효소에 의해 달성된다. 소화 조직의 진행은 통상적 overdigestion 및 세포 ?...

Watch this Scientific Journal Video about Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling at JoVE.com

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

질병 모델링을위한 근육 조직 검사에서 환자 유래 세포주의 분리 및 멸화

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

isolation-immortalization-patient-derived-cell-lines-from-muscle

Research

JoVE Journal

Medicine

16.4K 조회수.  UT Southwestern Medical Center. This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

비디오: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

모델링 및 3 차원 이미징 단체 셀 침략

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

연구

의학

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Proteomics에서 골격근 성별 동종 이형

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

사후 조직에서 Cardiomyocyte 핵의 분리

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

수정 된 버그 스트롬 기술을 이용하여 인간의 골격 근육 생검 절차

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

인간 세포주에서 Chemoresistant 난소 암 줄기 세포에 대한 심화 학습

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

경피적 바늘 생검에 의해 획득 골격근 조직에서 고립 된 미토콘드리아의 준비 및 호흡계 평가

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

골격근 질환의 정량적 자기 공명 영상

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

전체 조직과 조직 검사에서 인간 상피 Enteroids과 Colonoids 설립

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

인간 유방암 조직과 세포주에서 Mammosphere 형성 분석

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Methods Article

질병 모델링을위한 근육 조직 검사에서 환자 유래 세포주의 분리 및 멸화