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

Summary

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.

Abstract

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.

Protocol

1. Culture of Murine Myoblast Progenitor Cells or C2C12 Cells

1. 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 equipment you are referred to the table on page 7 and 8. From the muscle from one mouse you will generally obtain enough cells to cryopreserve about 2....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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