Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies

Abstract

Introduction: Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study skeletal muscle energy metabolism. However, the transferability of the results to in vivo might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism. Methods and results: We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10 days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid. Discussion and conclusion: These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.

Keywords: 3D cell model; energy metabolism; metabolic disorders; muscle spheroid; myosphere; skeletal muscle.

FIGURE 1

Evaluation of 3D morphological parameters and comparison of muscle cell differentiation markers in 2D and 3D models. Myospheres were formed in the ultra-low attachment treatment (ULA) 96-well plate system and differentiation was carried out between 0 and 21 days. After 10 days of differentiation, mRNA was isolated, and gene expression was analyzed by qPCR. (A) Phase-contrast photos of myospheres during 3, 5, 10 and 12 of differentiation. Diameter (B) was analyzed by AnaSP for up to 21 days of differentiation. (C) mRNA expression of the muscle differentiation markers MYOD, MYOG, MYH2, MYH7, and SLC2A4 before (day 0) and after differentiation (day 10), normalized to housekeeping gene (RPLP0). Scale bar = 200 μm. Results are presented as mean ± SEM. *p < 0.05 **p < 0.01 ****p < 0.0001 by ordinary one-way ANOVA test.

FIGURE 2

Evaluation of cell viability and hypoxia core in 3D muscle spheroids. Myospheres were formed in the ultra-low attachment treatment (ULA) 96-well plate system and differentiation was carried out between 0 and 10 days. (A) Myospheres viability was monitored as relative ATP content (%) (normalized to day 0) up to 10 days of differentiation by luminescence assay CellTiter-Glo 3D^® cell viability. (B) Area percentage (%) of dead (ethidium red) cells normalized to the area of live cells (calcein AM green) (ethidium area*100/calcein AM area) analyzed at days 0 and 10 of differentiation by the LIVE/DEAD^® Viability/Cytotoxicity Assay and representative images (scale bar = 200 µm). (C) % of hypoxia levels (Image-IT™ (magenta)) per spheroid’s area and representative images of the Image-IT™ dye staining (scale bar = 100 µm). Results are presented as mean ± SEM. #p < 0.05 by unpaired t-test.

FIGURE 3

Characterization of the internal muscle spheroid structure. Myospheres were formed, differentiated up to 10 days, and fixed in 4% of paraformaldehyde (PFA) previous staining. (A) Analysis of structural markers represented the percentage (%) of dye in the total spheroid area over culture time. (B) Representative maximum intensity projection of the structural markers: F-actin (green; actin filaments), collagen (magenta; matrix production), and vimentin (yellow; vimentin filaments). (C) Representative image of F-actin before and after differentiation (days 0 and 10) (merge between nuclei (blue) and F-actin (green)). (D) Analysis of nuclei circularity (Hoechst) at 0 and 10 days of differentiation. (E) Representative fluorescence image sections from two different donors of nuclei staining at 0 (single rounded nuclei) and 10 (fused/elongated nuclei) days of differentiation. Scale bar = 100 µm. Results are presented as mean ± SEM. *p < 0.05 by ordinary one-way ANOVA test. #p < 0.05 by unpaired t-test.

FIGURE 4

Comparison of glucose and oleic acid metabolism between 2D myotube cultures and 3D muscle spheroids. Myospheres were differentiated for 10 days and D-[¹⁴C (U]glucose (0.5 μCi/mL, 200 µM) or [1-¹⁴C]oleic acid (0.5 μCi/mL, 100 µM), respectively, were used in 4 h substrate oxidation assay to assess the metabolic profile. (A) Complete oxidation and total D-[¹⁴C (U]glucose uptake in 2D and 3D cell models. (B) mRNA expression of solute carrier family 2 member 1 (SLC2A1) (glucose transporter) at days 0 and 10 of differentiation, normalized to housekeeping gene (RPLP0). (C) [1-¹⁴C]oleic acid complete oxidation and uptake in 2D and 3D cell model’s metabolism. (D) mRNA expression of the fatty acid transporter CD36 at days 0 and 10 of differentiation, normalized to housekeeping gene (RPLP0). (E) Fractional oxidation (ratio of substrate oxidation/uptake) of glucose and oleic acid in 2D and 3D cell models. Results are represented as mean ± SEM. #p < 0.05 by unpaired t-test; *p < 0.05 by ordinary one-way ANOVA test.

FIGURE 5

Pathway representation of glucose and oleic acid metabolism between 2D myotube and 3D myospheres culture. The 2D myotubes model had a preference to oxidize glucose while the 3D myospheres showed a preference towards lipid oxidative metabolism over glucose Made with Biorender.com.