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).

NOTE: Protocols for collection of human tissue must be reviewed and approved by the Institutional IRB committee. Collection of discarded, de-identified human tissue has been approved by the Boston Children&#8217;s Hospital and Brigham and Women&#8217;s Hospital IRB Committees. The methods described below have been applied for the isolation of myogenic cells from de-identified, discarded tissue. The described methods are applicable to tissue collected from consented patient material.
1. Cell Isolation
<ol>
	<li>Dissociation of muscle biopsy and purification of myogenic cells
	<ol>
		<li>Pre-weigh a 10 cm tissue culture plate in a tissue culture biosafety hood, and then re-weigh the plate containing the muscle biopsy to calculate the amount of tissue to be dissociated.</li>
		<li>Using sterile scalpels, mince muscle tissue finely and add a few drops of sterile 1x HBSS to prevent tissue from drying out.</li>
		<li>Add 3.5 ml each of dispase II and collagenase D per gram of muscle tissue to be digested. Pipette the minced tissue and enzyme solution through a sterile 25 ml&#160;pipette a few times. Incubate the plate in a tissue culture incubator at 37 &#176;C with 5% CO2 for 15 min&#160;and digest tissue until the slurry easily passes though a sterile 5 ml pipette. 
		NOTE: Tissue dissociation is usually achieved by enzymatic digestion within 45-90 min. Please refer to the &#8216;Representative Results&#8217; section for additional details.</li>
		<li>Add 2 volumes of sterile growth medium to dissociated tissue, filter through a 100 &#181;m cell strainer over a 50 ml&#160;conical tube and pellet cells for 10 min&#160;at 329 x g (~1,100 rpm), room temperature. Please refer to the Materials Table for media composition.</li>
		<li>Resuspend the pellet in 1 volume of sterile growth medium and add 7 volumes red blood cell lysis solution. Filter the solution through a 40 &#181;m cell strainer, then pellet the cells for 10 min at 329 x g, room temperature.</li>
		<li>Count the cells in a hemocytometer and resuspend the cells in 1x HBSS 0.5% BSA at a concentration of 1 x 106 cells/100 &#181;l. Set aside ~250,000 cells in a single tube that will be used as a negative (unstained) control. Set aside additional single-color stained control tubes for propidium iodide and for CD56, which are required for proper gating of CD56 positive cells by FACS sorting. Please refer to cell sorting manuals or consult with FACS sorting core facility experts to ensure appropriate controls are included.</li>
		<li>Stain the cells to be sorted with 5&#181;l/106 cells of anti CD56 antibody. Incubate all samples (including controls) on ice for 30 min.</li>
		<li>Wash samples in 10 ml 1x HBSS and pellet cells for 10 min&#160;at 329 x g (~1,100 rpm) in a refrigerated centrifuge, 4&#176;C temperature.</li>
		<li>Add propidium iodide at a final concentration of 1&#181;g/ml to the sample to be sorted for exclusion of dead cells. Purify myogenic CD56 positive cells from non-myogenic cells using the fluorescence activated cell sorter.</li>
	</ol>
	</li>
	<li>Dissociation of skin biopsy 
	NOTE: Dermal fibroblasts can be isolated from a skin punch from patients when muscle biopsies are not available. Dermal fibroblasts can be used as biomaterial for many studies, including transduction with MyoD to generate myogenic cells. Additionally, dermal fibroblasts can be used to generate iPS cells, which can be differentiated into various cell types for further study.
	<ol>
		<li>Transport the skin biopsy to the laboratory in transportation medium. Once the sample is received, perform the primary culture as soon as possible. If the primary culture cannot be established on the same day, store the sample at room temperature overnight.</li>
		<li>Transfer the skin biopsy to a sterile 35 mm petri dish in the laminar flow hood.</li>
		<li>Rinse the skin biopsy in a petri dish with sterile 1x PBS to remove blood and debris. Remove the adipose tissue with a sterile scalpel.</li>
		<li>Add 2 ml collagenase solution and mince the tissue with scalpel, incubate at 37 &#176;C for 45 min to 1 hr, depending on the size of the tissue.</li>
		<li>Transfer the digested tissue to a 15 ml conical tube, rinse the petri dish with 2 ml of fibroblast culture medium twice, and collect the medium in the same tube.</li>
		<li>Pellet cells at 200 x&#160;g for 5 min&#160;at room temperature.</li>
		<li>Discard the supernatant and wash the pellet with 3 ml of fibroblast medium to remove the collagenase, then pellet cells again. Please refer to the Materials Table for media composition.</li>
		<li>Repeat the step 1.2.7 one more time.</li>
		<li>Re-suspend the pellet in 5 ml fibroblast medium and plate cells onto a T25 sterile tissue culture flask. Incubate the flask at 37 &#176;C with 5% CO2.</li>
		<li>Evaluate the culture for fibroblast attachment and growth over the next 1-3 days. 
		NOTE: Some small tissue pieces may also attach to the plate and fibroblasts migrate out of these tissue pieces.</li>
		<li>Maintain the culture under the same conditions until fibroblasts are grown to approximately 80% confluence.</li>
		<li>Collect fibroblasts by trypsinization and transfer onto fresh culture flasks for additional expansion. Perform trypsinization by washing the cultures 3 times in 1x PBS free of Ca++ and Mg++. Add Trypsin-EDTA (see Materials Table) to the cells (2ml/ T25 flask) for 2 min&#160;at 37 &#176;C.</li>
		<li>Split detached cells into additional flasks. If some tissue pieces remain attached to the original T25 flasks, add 5 ml fresh culture medium to this flask and more fibroblasts will migrate out continually. 
		NOTE: The expanded fibroblast culture can be frozen down as P1 and stored in liquid nitrogen for future experiments.</li>
	</ol>
	</li>
</ol>
2. Immortalization of Myogenic Cells
<ol>
	<li>Plate 5 million Phoenix Ecotropic packaging cells (PE) overnight in a 10 cm sterile tissue culture plate in DMEM and Medium 199 in a ratio of 4:1, supplemented with 10% calf serum.</li>
	<li>Feed cells 30 min prior to transfection with 5 ml of fresh media supplemented with 10 mM caffeine.</li>
	<li>Homogenize a mixture of 2 &#956;g of plasmid DNA from a midi prep (CDK4 or hTERT plasmid) and polyjet and incubate at room temperature for 15 min, as recommended by the manufacturer.</li>
	<li>Add the plasmid/polyjet mixture to the PE cells overnight.</li>
	<li>Feed cells with fresh medium (DMEM and Medium 199 in a ratio of 4:1, supplemented with 10% calf serum). Twelve hours later, collect the virus-containing supernatant and filter it through a 0.45 &#956;m pore size filter.</li>
	<li>Use 1 ml of collected supernatant to infect overnight the amphotropic packaging cell line PA317 5 and obtain a stable virus-producing cell line after selection with either 0.5 mg/ml neomycin (G418) for CDK4 or 0.5 mg/ml hygromycin for hTERT.</li>
	<li>Prepare working viral supernatants by growing the stable packaging cells to near confluency, then harvesting the supernatant each morning, evening and morning for three harvests.</li>
	<li>Filter the viral supernatants and either directly use or divide them into 1 ml aliquots and store at -80 &#176;C for later use. Note that viral supernatant loses 50% infection efficiency with each freeze-thaw. Remember to bleach-wash before discarding everything that has touched the viral particles. 
	NOTE: The stable PA317 virus-producing cell line can be frozen and maintained at -150 &#176;C for permanent storage (freezing medium is 10% DMSO; 90% Serum).</li>
	<li>Plate FACS-purified myogenic cells (described in steps 1.1-1.9) at a density of 5 x 104 cells/well in 6-well plates coated with 0.1% gelatin. Ensure that cells are attached to the plate before proceeding with the viral infection.</li>
	<li>Add 400 &#956;l filtered, freshly produced viral supernatant or frozen aliquots to each six-well plate overnight (keep 2 wells as controls).</li>
	<li>Change medium by feeding cells with 2.5 ml/well of fresh muscle media (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&#160;vitamin B12; 0.03 mg/L&#160;ZnSO4, 0.055 mg/L&#160;dexamethasone, 2.5 &#956;g/l hepatocyte growth factor and 10 &#956;g/L&#160;basic fibroblast growth factor). Discard the media and pipettes containing the viral particles in a bleach container. Leave cells in the same medium for 3 days to recover from infection, then treat for selection using either 400 &#956;g/ml neomycin (CDK4 infection) or 300 &#956;g/ml hygromycin (hTERT infection).</li>
	<li>Maintain cells under drug selection until the cells in the control dish die (1-2 weeks).</li>
	<li>Passage cells before they become confluent (60-80% confluency; using 0.05% trypsin EDTA), even during the selection period. Replate cells in multiple 10cm dishes with fresh myoblast medium (as described in 2.11) supplemented with the selection drug. Maintain immortalized selected cells as a heterogeneous population or clone to obtain a completely homogeneous genetic background (same insertion of the transgene in every cell).</li>
	<li>Perform clonal selection using the following steps:
	<ol>
		<li>Seed the cells at low density (e.g. 300 to 500 cells in 10 cm dishes) and maintain them for approximately two weeks, until small colonies are formed (10-20 cells).</li>
		<li>Remove most of the medium at this point, leaving only a thin film to prevent the cells from drying out.</li>
		<li>Place a cloning ring (with one end dipped in silicone vacuum grease) over each desired clone and add a few drops of trypsin/EDTA.</li>
		<li>Harvest cells once they become rounded by carefully aspirating the cells using a 1 ml tip or a Pasteur pipet and transferring them to the smallest available multiwell plate (96 or 48, 24 or 12 well plates).</li>
		<li>Expand clones as needed to prevent local confluency.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Cells as a Useful Resource
The isolation and culture of myogenic cell populations is extremely useful when establishing disease phenotypes or in vitro models of disease. The myogenic cell isolation procedure described here allows the isolation of myoblasts and fibroblasts from skeletal muscle specimens, which can then be propagated, differentiated, or immediately analyzed. Myoblast structure and function can be assessed through microscopic examination, evaluation of cell survival, eva...

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.

Figure 1 illustrates some of the key steps involved in the primary tissue dissociation: the exact amount of tissue is weighed in a sterile tissue culture petri dish (Figure 1A, B). The tissue is then finely minced using sterile scalpels, until a tissue slurry is obtained (Figure 1C, D). Following addition of the digestion enzymes, primary muscle tissue dissociation is usually achieved by enzymatic digestion within 45-90 min. The progression of tissue digestion is typical...

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.

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 Views.  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.