从肌肉活检患者来源的细胞系疾病建模隔离和永生化

摘要

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.

摘要

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’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.

引言

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.

研究方案

注:协议收集人体组织必须审查和批准的机构IRB委员会。收集丢弃，去识别人体组织已经批准了波士顿儿童医院和布里格姆妇女医院IRB委员会。以下描述的方法已经应用于用于从去标识肌细胞的分离，丢弃的组织。所描述的方法适用于组织从患者同意收集材料。

1.细胞分离

1. 肌肉活检和肌原细胞的纯化的离解
   1. 预先称重10cm的组织培养板在组织培养生物安全罩，然后重新称重含有肌肉活检来计算组织的量的板被解离。
   2. 使用无菌手术刀，肉的肌肉组织和细加几滴无菌1X HBSS以防止组织干燥。
   3. 添加每个迪为3.5mlspase II和每克肌肉组织胶原酶ð被消化。通过无菌25毫升吸管几次吸取的组织糜和酶溶液。孵育板在组织培养培养箱中于37℃，5％CO ₂的15分钟和消化组织直至浆料容易传递虽然灭菌的5毫升吸管。
      注:组织离解，通常通过酶消化内45-90分钟达到。请参阅'代表结果"部分了解更多详细信息。
   4. 添加2倍体积的无菌生长培养基分离的组织，滤波器的通过100微米的细胞过滤过50ml锥形管中，沉淀的细胞10分钟，在329×g离心（〜1100转），室温。请参考材料表介质组合物。
   5. 重悬沉淀在1体积的无菌生长培养基并加入7倍体积的红血细胞溶解液。过滤通过40微米的细胞过滤的溶液中，然后沉淀吨他的细胞10分钟，在329×g离心，室温。
   6. 计数细胞在血细胞计数器和重悬细胞于1×HBSS中的0.5％BSA的1×10 ^6个细胞/ 100微升的浓度。预留〜250,000个细胞在单管，将用作为负（未染色）的控制。预留额外的单色染控制管碘化丙啶和CD56，这是必需的CD56阳性细胞的FACS分选合适的门。请参考细胞分选说明书或FACS分拣核心设施的专家，以确保适当的控制包括咨询。
   7. 染色的细胞进行排序与5微升/ 10 ⁶细胞抗CD56抗体。在冰上孵育所有样品（包括对照）处理30分钟。
   8. 在10ml 1×HBSS洗涤样品并沉淀细胞在329×g离心（〜1100转）在冷冻离心机，4℃下10分钟。
   9. 以1微克/ ml的终浓度添加碘化丙啶至样品为SOR泰德排除死细胞。净化从使用荧光激活细胞分选仪的非肌细胞肌CD56阳性细胞。
2. 皮肤活检的解离
   注:真皮成纤维细胞可以从皮肤拳打患者进行隔离时，肌肉活检不可用。真皮成纤维细胞可以被用作生物材料的许多研究，包括转导的MyoD，以产生肌原细胞。另外，皮肤成纤维细胞可用于产生iPS细胞，这可以分化成为进一步研究各种细胞类型。
   1. 运输皮肤活检在输送介质实验室。一旦接收样本，尽快进行原代培养。如果主培养不能在同一天被建立，存储所述样品在室温下过夜。
   2. 转移的皮肤活检在层流罩中的无菌35mm平皿。
   3. 冲洗皮肤活检在培养皿用无菌1X PBS以清除血液和碎片。用无菌解剖刀除去脂肪组织。
   4. 加入2 ml胶原酶溶液和剁碎用手术刀将组织，孵育在37℃下45分钟到1小时，这取决于组织的大小。
   5. 转移消化的组织到15ml锥形管中，冲洗培养皿以2 ml成纤维细胞培养基中两次，并收集在同一个管中的介质。
   6. 沉淀细胞，在200×g离心5分钟，在室温下进行。
   7. 弃去上清液，并用3 ml成纤维细胞培养基洗涤沉淀，再次除去胶原酶，然后沉淀细胞。请参考材料表介质组合物。
   8. 重复步骤1.2.7一次。
   9. 重新悬浮的沉淀在5ml成纤维细胞培养基和细胞板到一个T25的无菌组织培养烧瓶中。孵育烧瓶在37℃，5％的CO _2。
   10. 评价培养成纤维细胞附着和生长在未来1-3天。
       注:一些小的组织块也可以连接到板和成纤维细胞移植这些组织片段。
   11. 维持培养相同的条件下，直至成纤维细胞生长至约80％汇合。
   12. 通过胰酶消化收集成纤维细胞和转移到新鲜培养瓶中额外的扩展。通过洗涤的文化在1X PBS 3次游离Ca ⁺⁺和Mg ⁺⁺的执行胰酶消化。加入胰蛋白酶-EDTA（见材料表），以将细胞（2毫升/ T25培养瓶中）为2分钟，在37℃。
   13. 分裂细胞分离到额外的瓶中。如果一些组织片保持附着到原始T25烧瓶，加入5 ml新鲜培养基到该烧瓶中，并更成纤维细胞会迁移出不断。
       注:扩展成纤维细胞培养物可以被冷冻下来为P1，并存储在液氮中未来的实验。

2.永生肌细胞

板5000000菲尼克斯亲嗜性包装细胞（PE）的过夜在10cm的无菌组织培养板中的DMEM和培养基199中的4:1的比例，补充有10％小牛血清。
1. 料的细胞30分钟前，转染用5ml新鲜培养基补充了10mM咖啡因。
2. 从一个MIDI准备（CDK4或hTERT基因的质粒）和均匀的PolyJet的2微克质粒DNA的混合物中，并在室温下孵育15分钟，如制造商推荐的。
3. 质粒/混合的PolyJet添加到PE细胞过夜。
4. 喂细胞用新鲜的培养基（DMEM培养基和199中的4:1的比例，补充有10％小牛血清）。 12小时后，收集含病毒的上清液，并通过0.45μm孔径过滤器过滤。
5. 使用1毫升回收的上清的感染过夜嗜包装细胞系PA317 ⁵和选择后获得稳定的病毒产生细胞系与任一为0.5mg / ml的新霉素（G418），用于CDK4或0.5毫克/毫升潮霉素为端粒酶。
6. 准备通过发展稳定的包装细胞融合附近，然后收获上清液每天早晨，傍晚和清晨的三丰收工作病毒上清。
7. 过滤病毒上清，要么在-80直接使用或把它们分为1​​ml等份并储存℃下以备后用。需要注意的是病毒上清失去50％的感染效率与每个冻融。记得漂白洗丢弃一切已经触及病毒颗粒之前。
   注:稳定PA317病毒产生细胞系可以被冻结，并保持在-150℃下的永久存储（冷冻培养基在10％的DMSO; 90％血清）。
8. 板的FACS纯化的肌细胞以5×10 ⁴个细胞/孔在6孔板涂有0.1％明胶的密度（1.1-1.9中的步骤描述）。确保细胞被附着在板继续进行病毒感染之前。
9. 加入400微升过滤，女reshly制备病毒上清或冷冻等分试样，以每6孔板过夜（保持2孔作为对照）。
10. 通过用2.5ml /孔的新鲜肌肉媒体（4喂食细胞改变介质:; 0.02M的HEPES缓冲液; 1.4毫克/升维生素B12; 1 Dulbecco改进的Eagle培养基（DMEM）和培养基199补充有15％胎牛血清0.03毫克/ L的硫酸锌，0.055毫克/ L的地塞米松，2.5微克/升的肝细胞生长因子与10μg/ L的碱性成纤维细胞生长因子）。丢弃含有漂白剂容器中的病毒颗粒的媒体和吸管。离开细胞在相同培养基中3天，以从感染中恢复，然后用任一400微克/ ml的新霉素（CDK4感染）或300微克/毫升潮霉素（hTERT的感染）治疗供选择。
11. 保持在药物选择细胞直至细胞在对照培养皿模具（1-2周）。
12. 传代细胞，才成为汇合（60-80％汇合;采用0.05％胰蛋白酶的EDTA），即使是在selecti上期。 Replate细胞多10cm培养皿新鲜的成肌细胞的培养基（如2.11所述）补充与选择的药物。保持永生选择细胞作为异质群体或克隆，以获得完全均匀的遗传背景（转基因的每一个细胞都在相同的插入）。
13. 使用以下步骤进行克隆选择:
    1. 种子的细胞以低密度（ 例如 300到500个细胞于10cm菜），并保持它们为约两周，直到小菌落形成（10-20个细胞）。
    2. 除去大部分介质在这一点上，只留下一薄膜，防止细胞干燥。
    3. 将一个克隆环（有机硅真空硅脂一端蘸上）在每个需要的克隆，并添加几滴胰蛋白酶/ EDTA的。
    4. 收获细胞一旦成为用1毫升提示或巴斯德吸管吸仔细的细胞，并将它们转移到最小可用多孔解放军杀进德（96或48，24或12孔板）。
    5. 根据需要，以防止局部融合展开克隆。

结果

图1示出的一些参与了主组织离解的关键步骤:组织的确切量在无菌组织培养培养皿称重（ 图1A，B）。然后将组织细使用无菌手术刀切碎，直到组织浆料得到（ 图1C，D）。下列另外的消化酶，主要肌肉组织离解，通常通过酶消化内45-90分钟达到。组织消化的进展通常监视每15-20分钟，以防止过量酶切和细胞死亡。酶消化可以轻轻吸入混合几次每次15-20分钟。在?...

讨论

细胞作为一种有用的资源

建立疾病表型或疾病的体外模型时，肌细胞群的分离和培养是非常有用的。此处所描述的生肌细胞分离过程允许从骨骼肌标本，然后可以被传播，分化，或立即分析成肌细胞和成纤维细胞的分离。成肌细胞的结构和功能可以通过显微镜检查来评价，细胞存活，细胞融合的评价的评价，或者通过RNA或蛋白质表达的分子研究。此外，成肌细胞...

材料

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