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>

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

16.5K 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

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

인간의 골격근에서 기본 근육 조직 세포와 섬유 아 세포의 분리 및 정량 면역 세포 화학 특성

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

연구

발생학

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

인간의 Myoblast의 분리, Myogenic 분화의 평가 및 저장 운영 칼슘 항목 측정

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

신경 근육 장애에 대 한 치료를 조사 하기 위해 Myoblasts에 인간 섬유 아 세포의 직접 재프로그래밍

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

유전학

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

골격 근 생검 절차에서 인간 근육 선조 세포의 격리, 배양, 특성화 및 분화

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

마우스 골격 근 이식에서 기본 근구의 격리 및 분화

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

생물학

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

인간의 골격 근육에서 혈관 유래 다 능성 전구체의 분리

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

초기 산후 발달에 있는 에머리 드레이푸스 근 이영양증의 Murine 모형에서 단 근섬유 격리 및 문화

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

의학

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA 일렉트로, 근섬유의 분리 및 이미징

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...