Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Summary

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Abstract

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as 'MyoTACTIC', and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

Introduction

Skeletal muscle is one of the most abundant tissues in the human body and supports key body functions such as locomotion, heat homeostasis and metabolism¹. Historically, animal models and two-dimensional (2D) cell culture systems have been used to study biological processes and disease pathogenesis, as well as for testing pharmacological compounds in the treatment of skeletal muscle diseases^2,3. While animal models have greatly improved our knowledge of skeletal muscle in health and in disease, their translational impact has been hampered by high costs, ethical considerations and inters....

Protocol

1. PDMS MyoTACTIC plate fabrication

NOTE: PDMS MyoTACTIC plate fabrication requires a PU negative mold, which can be manufactured as previously described³⁰. The computer-aided design (CAD) SolidWorks file for the MyoTACTIC plate design has been made available on GitHub (https://github.com/gilbertlabcode/MyoTACTIC-SolidWork-CAD-file).

1. Prepare ~ 110 g of PDMS polymer solution in a disposable plastic cup at a 1:15 ratio of monomer to curing agent using the components in the silicone elastomer kit. Stir the polymer solution for 2-3 min using a 5 mL disposable serological pipette until completely mixed an....

Results

Described herein are methods to cast a 96-well PDMS-based MyoTACTIC culture platform from a PU mold, to fabricate arrays of hMMT replica tissues, and to analyze two aspects of hMMT function within the culture device-force generation and calcium handling. Figure 1 offers a schematic overview of the preparation of MyoTACTIC culture wells before hMMT seeding. PDMS is a widely used silicone-based polymer, that can be easily molded to create complex devices³². A PDMS-based.......

Discussion

This manuscript describes methods to fabricate and analyze a 3D hMMT culture model that can be applied to studies of basic muscle biology, disease modeling, or for candidate molecule testing. The MyoTACTIC platform is cost-friendly, easy to manufacture, and requires a relatively small number of cells to produce skeletal muscle microtissues. hMMTs formed within the MyoTACTIC culture platform are comprised of aligned, multinucleated, and striated myotubes, and respond to electrical stimuli by initiating calcium transients .......

Materials

Name	Company	Catalog Number	Comments
0.9% Saline Solution, Sterile	House Brand	1010	10 mL aliquots of the solution are made and stored at 4°C
25G Needle	BD, Medstore, University of Toronto	2548-CABD305127
6-Aminocaproic Acid, ≥99% (titration), Powder	Sigma - Aldrich	A2504-100G	A 50 mg / mL stock solution is generated by dissolving 5 mg of 6-aminocaproic acid powder in 100 mL of autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C
6.35 mm ID Tubing	VWR	60985-528
AB1167 Myoblast Cell Line	Institut de Myologie (Paris, France)
Arbitrary Waveform Generator	Rigol	DG1022Z
Basement Membrane Extract (Geltrex)	Thermo Fisher Scientific	A14132-02	Stored as aliquots of 50 µL or 100 µL at -80°C
Benchtop Vacuum Chamber	Sigma - Aldrich	D2672
BNC to Aligator Clip Cable			Ordered from Amazon
Culture Plastics	Sarstedt		Includes culture plates, serological pipettes, etc
Dimethyl Sulfoxide	Sigma - Aldrich	D8418-250ML
DPBS, Powder, No Calcium, No Magnesium	Thermo Fisher Scientific	21600069
Dulbecco's Modified Eagle Medium (DMEM) (1X)	Gibco	11995-065	This is a high glucose DMEM with L-glutamine and sodium pyruvate
Fetal Bovine Serum	Fisher Scientific	10437028
Fibrinogen from Bovine Plasma	Sigma - Aldrich	F8630-5G	Aliquots ranging from 7 - 10 mg of fibrinogen powder are made and stored at -20°C
Filtropur Syringe Filter, 0.22um Pore Size	Sarstedt	83.1826.001
Horse Serum	Gibco	16050-122
Human Recombinant Insulin	Sigma - Aldrich	91077C	Stock solution is 100X and made by dissolving 1 mg of human recombinant insulin in 1 mL of DMEM and 1 µL of NaOH 10N. Solution is filtered and stored as 1 mL aliquots at 4°C
Image Acquisition Software	Olympus	cellSens Dimension
Image Processing Software	National Institutes of Health	ImageJ
Isotemp Oven	Thermo Fisher Scientific	201
Microscope	Olympus	IX83
Microscope - Camera Mount	Labcam	Labcam for iPhone	Ordered from Amazon
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140-122
Plastic Disposable Syringes, 1cc	BD	2606-309659
Plastic Disposable Syringes, 50cc	BD	2612-309653
Pluronic F-127, Powder, BioReagent	Sigma - Aldrich	P2443-250G	A 5% stock solution of pluronic acid is made by dissolving 5 g of pluronic acid powder in 100 mL of chilled, autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C
Polydimethylsiloxane (Sylgard 184 Silicone Elastomer Kit)	Dow	4019862	Kits are also available at Thermo Fisher Scientific, Sigma - Aldrich, etc.
Polyurethane Negative Mold	In House
Release Agent	Mann Release Technologies	200
Rotary Vane Vacuum Pump	Edwards	A65401906
Scalpel	Almedic, Medstore, University of Toronto	2586-M36-0100
Single Edge Razor Blade	VWR	55411-050
Skeletal Muscle Cell Basal Medium	Promocell	C-23260	30 mL aliquotes are generated and at stored at 4°C.
Skeletal Muscle Cell Growth Medium (Ready-to-use)	Promocell	C-23060	42 mL aliquots are generated and stored at 4°C.
Smartphone (iPhone)	Apple	SE
Standard Duty Dry Vacuum Pump	Welch	2546B-01
Sterilization Bag	Alliance	211-SCM2
Thimble	Igege		Ordered from Amazon
Thrombin from human plasma	Sigma - Aldrich	T6884-250UN	100 units of thrombin is dissolved in 1 mL of a 0.1% BSA solution. 10 µL aliquots are prepared and stored at - 20°C.
Tin coated copper wire	Arco	B8871K48	Ordered from Amazon
Trypan Blue Solution, 0.4%	Thermo Scientific	15250061
Trypsin-EDTA, 0.25%	Thermo FIsher Scientific	25200072
Vacuum Chamber 2	SP Bel-Art	F42027-0000