A Novel Method for Thyroarytenoid Myofiber Culture

Objectives/Hypothesis Myofiber culture has been employed to investigate muscle physiology in vitro and is well‐established in the rodent hind limb. Thyroarytenoid (TA) myofiber culture has not been described, providing an opportunity to employ this method to investigate distinct TA myofiber functions. The purpose of this study was to assess the feasibility of a TA myofiber culture model. Study Design In vitro. Methods TA muscles from five Sprague Dawley rats were independently isolated and digested for 90 min. A smooth‐tip, wide‐bored pipette dissociated TA myofibers from cartilage, and the fibers were distributed on collagen‐coated dishes and incubated at 37°C, 5% CO2 for 2 h. Myofiber specificity was determined via immunolabeling for desmin and myosin heavy chain (MHC). Myofibers viability was assessed over 7 days via esterase assay. Additional myofibers were immunolabeled for satellite cell marker Pax‐7. Glucocorticoid (GC) receptor (GR) was immunolabeled following GC treatment. Results The harvest technique yielded ~120 myofibers per larynx. By day 7, ~60% of the fibers remained attached and were calcein AM‐positive/ethidium homodimer‐negative, indicating viability. Myofibers were positive for desmin and MHC, indicating muscle specificity. Cells surrounding myofibers were positive for Pax‐7, indicating the presence of myogenic satellite cells. Myofibers also responded to GC treatment as determined by GR nuclear translocation. Conclusion TA myofibers remained viable in culture for at least 7 days with a predictable response to exogenous stimuli. This technique provides novel investigative opportunities regarding TA structure and function. Level of Evidence N/A Laryngoscope, 133:3109–3115, 2023 Primary isolated myofiber culture is considered an optimal model to investigate pathological/treatment conditions in mature myofibers in vitro but has not been conducted in thyroarytenoid (TA) myofibers. This paper provides methodology for a feasible TA myofiber culture model.