Optimization of Single Myofiber Isolation Protocol from the Flexor Digitorum Brevis Muscle to Study Satellite Cells

Ahmed, Ahmed; Hussein, Hoda H.; Mahmoud, Omayma M.; Abdel-Alrahman, Gamal; Kyba, Michael; Fareed, Shimaa A.

doi:10.21608/scumj.2023.311155

Optimization of Single Myofiber Isolation Protocol from the Flexor Digitorum Brevis Muscle to Study Satellite Cells

Document Type : Original Article

Authors

¹ Department of Anatomy, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

² \University of Minnesota Twin Cities, Minneapolis, Minnesota, USA

³ University of Minnesota Twin Cities, Minneapolis, Minnesota, USA

10.21608/scumj.2023.311155

Abstract

Background: Satellite cells, which are located between the basal lamina and sarcolemma of the myofiber, have a tremendous ability to repair damaged skeletal muscle. Although they remain quiescent in their niche, they activate rapidly following injury; understanding the mechanisms underlying this activation process has the potential to open new avenues for muscle regenerative therapies. The flexor digitorum brevis muscle “FDB” is one of the best candidates to study satellite cell physiology in the unperturbed myofiber, as it is composed of relatively short myofibers attached to the muscle tendon in an oblique manner, enabling a high rate of intact fiber isolation compared to muscles with longer fibers. Aim: To optimize the preparation process to avoid maldigestion that renders broken and contracted myofibers not suitable for studying quiescent satellite cells and the subsequent activation processes. Material and Methods: FDB muscles from both sides from C57BL/6 male mice were used. The muscles were digested with collagenase digestion solution then cultured in myofiber culture medium in low CO₂ incubators at 37⁰C. Results: This study revealed that a short incubation period in the digestion solution of two hours is optimum for harvesting the largest number of viable myofiber. Dishes coated with laminin were used to allow the attachment of the single myofibers and further facilitate the immunofluorescence staining process. Conclusion: The used method provides a better tool to isolate the single myofibers from the FDB muscle and hence better analysis of satellite cell activation.

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