रोग मॉडलिंग के लिए पेशी बायोप्सी से मरीज व्युत्पन्न सेल लाइनों के अलगाव और अमरता
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
एक उपयोगी संसाधन के रूप में कोशिकाओं
रोग के इन विट्रो मॉडल में या रोग फेनोटाइप की स्थापना जब myogenic सेल आबादी के अलगाव और संस्कृति अत्यंत उपयोगी है। यहाँ वर्णित myogenic सेल अलगाव की प्रक्रिय?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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 | |
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