Y32 - Optimizing Culture Media Conditions for Enhanced Myofiber Glucose Metabolism in 2D and 3D Systems

Introduction: : Skeletal muscle is a major site for systemic glucose metabolism, which is responsible for approximately 80% of postprandial, insulin-mediated glucose uptake. Impaired muscle glucose metabolism and insulin resistance are hallmark features of metabolic diseases such as Type 2 diabetes (T2D), making muscle an important target for disease modeling and therapeutic intervention. Current in vitro models often rely on 2D cultures supplemented with supraphysiological insulin concentrations. Many of these models primarily support GLUT1-mediated basal glucose uptake rather than insulin-stimulated GLUT4 responsive uptake. Given that GLUT4 translocation is critical for physiological glucose metabolism, insufficient GLUT4 expression in these models could lead to complication between intrinsic insulin resistance and experimentally induced metabolic defects. If muscle models are already insulin-resistant at baseline, it becomes challenging to accurately study diabetic-like phenotypes or evaluate therapeutic interventions.
Recent studies have highlighted the benefits of Insulin-like Growth Factor 1 (IGF-1) in enhancing myogenic differentiation, GLUT4 expression, and mitochondrial biogenesis.IGF-1 contributes to improved metabolic function. Furthermore, previous studies found that using low amino acid media, supplemented with L-carnitine and fatty acids (oleate, palmitate), promotes glucose metabolism and insulin sensitivity in skeletal muscle cultures. Thus, this study aims to optimize culture conditions by systematically comparing media supplemented with IGF-1, along with L-carnitine and fatty acids, in both 2D human skeletal muscle myotubes and 3D myobundles. We hypothesize that these strategies will improve glucose metabolism, promote insulin responsiveness, enhance GLUT4 expression, and eventually advance the physiological relevance of in vitro skeletal muscle models.

Materials and

Methods: : Primary human skeletal muscle cells (hSkM) were expanded to confluency under standard conditions. For 2D cultures (myotubes), cells were passaged onto Matrigel-coated well plates, while 3D myobundles were fabricated by encapsulating cells within a hydrogel composed of fibrinogen, matrigel, 3D human growth media (hGM), and thrombin, cast into custom molds. Both 2D and 3D cultures were grown for three days before undergoing differentiation. Cultures will then differentiate for 7 days using our lab’s standard differentiation media, DMEM supplemented with N-2, containing supraphysiological insulin (2,500 µg/mL). Differentiation was followed by a 7-day treatment period using four experimental media formulations: (1) standard DMEM + N2; (2) DMEM + IGF-1 with lipid supplementation; (3) MCDB105 (low amino acid medium) + N2 with lipid supplementation; and (4) MCDB105 + IGF-1 with lipid supplementation. The goal was to improve GLUT4-mediated glucose uptake and metabolic flexibility through media and supplement optimization.
At Day 17, cultures were subjected to glucose uptake assays, force testing, and RNA extraction for RT-PCR analysis. Glucose uptake was accessed by first starving cultures in glucose-free buffer for 3 hours, then sensitizing with insulin (10/100 nM). Quantification was done by using Promega Glucose Uptake-Glo™ Assay (J1341), based on calibration curves generated in each assay. Force testing was performed on myobundles by stimulating bundles once at 1 Hz for twitch force, 20 stimulations at 20 Hz for tetanus force, and 600 stimulations at 20 Hz for percentage fatigue. Total RNA was extracted post-treatment, followed by RT-PCR to assess GLUT1 and GLUT4 mRNA expression levels.

Results, Conclusions, and Discussions:: To optimize culture conditions for skeletal muscle models, we initially tested different combinations of base media and supplementations. Our initial goal was to confirm these media conditions with promising metabolic findings does not compromise myobundle functionality. We assessed myobundle development by measuring contractile properties through force tests, including twitch, tetanus, and fatigue assays (Figure 1). MCDB105-based formulations significantly impaired myobundle function likely due to inadequate nutrient content. Thereby, MCDB105 conditions were excluded from further studies.
Notably, IGF-1 supplementation supported contractile force development comparable to N-2 in DMEM, We then proceeded with DMEM supplemented with IGF-1 plus lipid supplementation to test their efficacy in supporting 2D myotube glucose metabolism (Figure 2). Using our standard differentiation media as a control, we observed significantly enhanced glucose uptake in the IGF-1-supplemented groups. This enhancement was most prominent under insulin sensitization at 100 nM, confirming IGF-1’s effectiveness in improving insulin responsiveness. Gene expression analyses revealed no statistically significant changes in GLUT1 levels across conditions, consistent with GLUT1’s role in basal glucose uptake. However, GLUT4 expression was unexpectedly higher in N2 groups, contrary to the glucose uptake results. This mismatch between GLUT4 mRNA and functional glucose uptake suggests that transcript levels may not directly correlate with protein abundance. Building on these positive results in 2D, we transitioned to 3D myobundle culture, which further improved glucose uptake responses under IGF-1 supplementation(Figure 2). A higher absolute glucose uptake values compared to 2D cultures and clearer insulin concentration-dependent trends were shown. Similar to the results from myotubes, GLUT1 showed no significant differences, while GLUT4 expression again did not correlate directly with glucose uptake. Thus, future western blot analyses will clarify GLUT4 protein localization and abundance.
Having established IGF-1’s beneficial effects in lipid-supplemented cultures, including enhanced myobundles’ contractile force and improved glucose uptake in all cultures, we investigated its potential to replace standard N2 supplementation during differentiation. Preliminary results indicated enhanced glucose uptake and improved insulin sensitivity with IGF-1, despite decreased GLUT1 and unchanged GLUT4 expression(Figure 3).. In the next step, we plan to evaluate novel human plasma-like media (HPLM) combined with IGF-1 plus lipid supplementation.

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