Name: engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation
Uploaded: 2013-03-19T12:00:00
Description: Watch this Scientific Journal Video about Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation at JoVE.com

The authors want to thank Yabin Wu for culturing the tissues presented in Figure 2, the picture was taken by Bart van Overbeeke. The work was financially supported by SenterNovem, grant ISO 42022.

1. Culture of Murine Myoblast Progenitor Cells or C2C12 Cells
<ol>
	<li>Isolate cells according to the protocol initially published by Shefer and colleagues 10 and later adapted by Collins et al. 11 and Boonen et al. 12 and store these in liquid nitrogen. This requires mice, e.g. C57Bl/6. Alternative methods are used in other labs, e.g. a method published in Journal of Visualized Experiments by Li Y et al. 13. For the reagents and ...

<ol>
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Part A. , (2011).</li><li>Darabi, R., Pan, W., Bosnakovski, D., 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=Functional+myogenic+engraftment+from+mouse+iPS+cells.">Functional myogenic engraftment from mouse iPS cells.</a> Stem Cell Rev. 7 (4), 948-957 (2011).</li><li>Langelaan, M. L. P., Boonen, K. J. M., Rosaria-Chak, K. Y., 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=Advanced+maturation+by+electrical+stimulation:+Differences+in+response+between+C2C12+and+primary+muscle+progenitor+cells.">Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells.</a> J. Tissue Eng. Regen. Med. 5 (7), 529-539 (2011).</li><li>van der Schaft, D., van Spreeuwel, A. C., van Assen, H. C., Baaijens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanoregulation+of+vascularization+in+aligned+tissue+engineered+muscle;+a+role+for+VEGF.">Mechanoregulation of vascularization in aligned tissue engineered muscle; a role for VEGF.</a> Tissue Eng. Part A. , (2011).</li><li>Shefer, G., Wleklinski-Lee, M., Yablonka-Reuveni, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+satellite+cells+can+spontaneously+enter+an+alternative+mesenchymal+pathway.">Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway.</a> J. Cell. Sci. 117 (Pt. 22), 5393-5404 (2004).</li><li>Collins, C. A., Olsen, I., Zammit, P. S., 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=Stem+cell+function,+self-renewal,+and+behavioral+heterogeneity+of+cells+from+the+adult+muscle+satellite+cell+niche.">Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche.</a> Cell. 122 (2), 289-301 (2005).</li><li>Boonen, K. J. M., Rosaria-Chak, K. Y., Baaijens, F. P. T., van der Schaft, D. W. J., Post, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+environmental+cues+from+the+satellite+cell+niche:+optimizing+proliferation+and+differentiation.">Essential environmental cues from the satellite cell niche: optimizing proliferation and differentiation.</a> Am. J. Physiol. Cell Physiol. 296 (6), C1338-C1345 (2009).</li><li>Li, Y., Pan, H., 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=Isolating+Stem+Cells+from+Soft+Musculoskeletal+Tissues.">Isolating Stem Cells from Soft Musculoskeletal Tissues.</a> J. Vis. Exp. (41), e2011 (2010).</li><li>Boonen, K. J. M., Langelaan, M. L. P., Polak, R. 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=Effects+of+a+combined+mechanical+stimulation+protocol:+Value+for+skeletal+muscle+tissue+engineering.">Effects of a combined mechanical stimulation protocol: Value for skeletal muscle tissue engineering.</a> J. Biomech. 43 (8), 1514-1521 (2010).</li><li>Gawlitta, D., Boonen, K. J. M., Oomens, C. W. J., Baaijens, F. P. T., Bouten, C. V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+serum-free+culture+conditions+on+skeletal+muscle+differentiation+in+a+tissue-engineered+model.">The influence of serum-free culture conditions on skeletal muscle differentiation in a tissue-engineered model.</a> Tissue Eng. Part A. 14 (1), 161-171 (2008).</li><li>Koning, M., van Luijn, M., van der Schaft, D. W. 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=Human+skeletal+muscle+formation+and+engraftment+In+vivo+is+independent+of+preconditioning+In+vitro+with+HUVEC.">Human skeletal muscle formation and engraftment In vivo is independent of preconditioning In vitro with HUVEC.</a> , (2013).</li><li>Levenberg, S., Rouwkema, J., Macdonald, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vascularized+skeletal+muscle+tissue.">Engineering vascularized skeletal muscle tissue.</a> Nat. Biotechnol. 23 (7), 879-884 (2005).</li><li>Koffler, J., Kaufman-Francis, K., Yulia, S., 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=Improved+vascular+organization+enhances+functional+integration+of+engineered+skeletal+muscle+grafts.">Improved vascular organization enhances functional integration of engineered skeletal muscle grafts.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (36), 14789-14794 (2011).</li></ol>

The engineering of muscle tissues has great potential for the use as a disease model, for drug screening, in regenerative medicine and for meat production. However, the requirements for these applications vary. We chose to work with a combination of collagen and matrigel, because collagen allows for cell alignment and because the myoblast progenitor cells require the presence of basement membrane derived proteins as determined in previous 2D studies 12. Moreover, fibrin gels have been tested in our labo...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Matrigel-growth factor reduced</td>
 <td>Beckton and Dickinson</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM (high glucose)*</td>
 <td>Gibco</td>
 <td>42430</td>
 <td></td>
 </tr>
 <tr>
 <td>Advanced DMEM</td>
 <td>Gibco</td>
 <td>12491</td>
 <td></td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>Gibco</td>
 <td>65050-122</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Greiner</td>
 <td>758075</td>
 <td></td>
 </tr>
 <tr>
 <td>0.45 and 0.22 &mu;m syringe filter*</td>
 <td>Whatmann (Schleicher and Scheull)</td>
 <td>10462100</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030024 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin</td>
 <td>Gibco</td>
 <td>10378016 </td>
 <td></td>
 </tr>
 <tr>
 <td>Amphotericin</td>
 <td>Gibco</td>
 <td>15290-018 </td>
 <td></td>
 </tr>
 <tr>
 <td>Culture plastic</td>
 <td>Greiner</td>
 <td></td>
 <td>Includes culture flasks and pipettes</td>
 </tr>
 <tr>
 <td>Chick embryo extract</td>
 <td>United States Biological</td>
 <td>C3999</td>
 <td></td>
 </tr>
 <tr>
 <td>Pasteur pipette*</td>
 <td>Hilgenberg</td>
 <td></td>
 <td>Pasteur pipettes, with constriction, with cotton, open tip L: 230 mm with tip diameter of 0,9 - 1,1 mm </td>
 </tr>
 <tr>
 <td>Pasteur pipette*</td>
 <td>Hilgenberg</td>
 <td></td>
 <td>Pasteur pipettes, with constriction, with cotton, open tip L: 230 mm with tip diameter of 1,4 - 1,6 mm </td>
 </tr>
 <tr>
 <td>Pasteur pipette</td>
 <td>VWR</td>
 <td>612-1702</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase type I*</td>
 <td>Sigma</td>
 <td>C0130-16</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;m cell strainer*</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td></td>
 </tr>
 <tr>
 <td>19G needle</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Elastomer</td>
 <td>Dow Corning corporation</td>
 <td>3097358-1004</td>
 <td>Silastic MDX 4-4210#</td>
 </tr>
 <tr>
 <td>Curing agent</td>
 <td>Dow Corning corporation</td>
 <td></td>
 <td>Silastic MDX 4-4210#</td>
 </tr>
 <tr>
 <td>Velcro</td>
 <td>Regular store</td>
 <td></td>
 <td>You can buy this at a regular store, only use the soft side</td>
 </tr>
 <tr>
 <td>Collagen type I, rat tail</td>
 <td>BD Biosciences</td>
 <td>3544236</td>
 <td></td>
 </tr>
 <tr>
 <td>C-Pace EP Culture Pacer </td>
 <td>Ionoptix</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>6-well culture dishes for electrical stimulation </td>
 <td>Beckton Dickinson-Falcon</td>
 <td>BD Falcon #353846 </td>
 <td></td>
 </tr>
 <tr>
 <td>C-Dish culture dish electrodes</td>
 <td>Ionoptix</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>* Needed for the isolation of cells (point 1.1) 
# Together in one kit</td>
 </tr>
 </tbody>
</table>

engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

The end product will be muscle constructs as indicated in Figure 3. The size of the tissue will be approximately 8 mm long, 2 mm wide and 0.5 mm thick. Electrical stimulation during differentiation will change the expression of myosin heavy chain isoforms, but does not greatly enhance the differentiation process as induced by the differentiation medium 8, but electrical stimulation can also be applied at the end of the process to check for functionality of the muscle, because a muscle with fully d...

Watch this Scientific Journal Video about Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation at JoVE.com

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

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