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 20 vials in liquid nitrogen. Next, we start the protocol to thaw the cells from the liquid nitrogen storage.
2. Place the cells from 1 vial into a 25 cm² Matrigel (1 mg/ml) coated tissue culture flask and add growth medium (GM) consisting of (335 ml DMEM advanced, 100 ml fetal bovine serum (FBS), 50 ml HS, 5 ml penicillin/streptomycin, 5 ml L-glutamin, 5 ml chicken embryo extract (CEE)).

Note: coat for 2 hr at Room Temperature and remove the Matrigel by aspiration.

3. Subculture the cells approximately 1:3 every 3 days.
   This means: on day 3: Passage cells from one 25 cm² to a 75 cm² flask, on day 6: passage cells from one 75 cm² flask to two 150 cm² flasks (with 30 min preplating in uncoated flasks). On day 9 transfer one 150 cm² flask to 1 triple 150 cm² flask, or if unavailable to three 150 cm² flasks (if needed preplate again depending on the phenotype of the cells). On day 12 the cells are ready for seeding into a muscle construct.

Notes: - Per triple 150 cm² the number of cells will be approximately 4.5 x 10⁶ cells.
- Use vials from different isolations to mix them to obtain a mixed population of cells at time of seeding.

Note: If you choose not to work with primary cells, C2C12 myoblasts are a good alternative.

2. Engineering Skeletal Muscle Tissues

1. Prepare silicon glue by mixing the"elastomer" with the "curing agent" (10:1). Cut +/- 5 mm square segments of Velcro with one triangular side (house-like shape; Figure 1). Glue the Velcro into the wells of a 6-well culture plate at a space of approximately 12 mm between the squares.

Notes: - Only use the soft side of the Velcro and face this side upwards.
- Make sure that the rooftops face each other.
- Only cover the Velcro with silicon glue, do not spread glue throughout the well.
- For later electrical stimulation it is relevant to align the constructs in vertical direction of the well plate (along the long axis).

2. Leave to dry overnight in vacuum oven, not heated, primarily to remove air bubbles. Sterilize by adding 70% EtOH to the wells and incubate for 15 min. Rinse 3x with PBS and put under UV for 15 min. Remove all PBS from the wells and the Velcro and place in incubator until use.
3. Thaw Matrigel solution in the fridge and prepare the collagen solution to the desired concentration shortly before making the 3D constructs. Dilute the stock collagen with sterile 0.02% acetic acid (final concentration 3.2 mg/ml).

Note: leave everything on ice.

4. Trypsinize the cells, resuspend in GM and count. Leave cells in centrifuge tube in the incubator.
5. Add the desired amount of Collagen stock solution for the numbers of constructs that you want to make to a tube (according to Table 1), add 0.5M NaOH to this collagen solution until a light pink color is indicating a pH of 7.5. Mix gently by pipetting up and down and avoid bubble formation. Then add the Matrigel and mix very gently but thoroughly. Finally, add the GM to the Collagen/Matrigel mixture (for the appropriate amounts see Table 1).

Notes: - Perform each step on ice! Matrigel and collagen will readily gel at increasing temperature.
- Mind that the color indeed is pink! An increase in pH will also induce rapid gelation.
- Final concentration of collagen: 1.6 mg/ml.

6. Centrifuge the desired amount of cells at 1,000 rpm for 5 min and remove the supernatant.

Note: - Depending on the activity of the cells the number of cells per construct needs to be adjusted. Typically, the number of cells lies between 750,000 and 1,250,000 cells per construct.

7. Use some of the gel mixture to resuspend the cell pellet and transfer the cells into the remaining mixture and mix thoroughly, but without introducing air bubbles.
8. Take the preheated well plates out of the incubator and pipette 0.35 -0.4 ml of the cell-gel mixture into the Velcro first. Then, begin to pipette the mixture from the center between the attachment sites and extend to the Velcro. Finally, use the remainder of gel to fill up the gap between the two Velcro anchors and pipette around the Velcro pieces.
9. Carefully check after 5-10 min if the gels are solid enough to be transferred, i.e. gently tap the dishes and inspect visually if the gel is rigid. If so, carefully place the dishes in an incubator. Usually, they can be picked up after ten minutes. Then, after 1-2 hr, gently overlay each gel with 4 ml of warm GM.

Note: -Do not make any vigorous movements when handling the plates.

10. Replace GM by differentiation medium (growth medium without chick embryo extract) after 24 hr, and replace with fresh medium every 2-3 days. On day 7 you should have obtained mature oriented muscle fibers, as can be evaluated with staining for cross striation development in the fused muscle fibers.

3. Electrical Stimulation

1. Sterilize the electrodes from the ionoptix plate (see table of reagents and equipment) before use with 70% ethanol and subsequently dry under UV in a safety cabinet. Place the plate with electrodes onto the culture plate with the constructs and cover with the lid from the culture plate and transfer to an incubator. Connect the Ionoptix C-pacer with the appropriate cables.

Note: Electrodes are placed parallel alongside the muscle construct (Figure 2).

2. Apply the stimulation protocol as anticipated.

Note: We generally use 4 V/CM, 6ms pulses at a frequency of 2Hz. Change culture medium every 24 hr during stimulation.

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