Optimizing transport methods to preserve function of self-innervating muscle cells for laryngeal injection

Abstract

Objectives

Recently, our laboratory has discovered a self-innervating population of muscle cells, called motor endplate-expressing cells (MEEs). The cells innately release a wide variety of neurotrophic factors into the microenvironment promoting innervation when used as an injectable treatment. Unlike other stem cells, the therapeutic potential of MEEs is dependent on the cells' ability to maintain phenotypical cell surface proteins in particular motor endplates (MEPs). The goal of this study is to identify transport conditions that preserve MEE viability and self-innervating function.

Methods

Muscle progenitor cells (MPCs) of adult Yucatan pigs were cultured and induced to generate MEEs. Effects of short-term cryopreservation methods were studied on MPC and MEE stages. A minimally supplemented medium was investigated for suspension-mediated transport, and MEEs were loaded at a constant concentration (1 × 10⁷ cells/mL) into plastic syringes. Samples were subjected to varying temperatures (4, 22, and 37°C) and durations (6, 18, 24, and 48 h), which was followed by statistical analysis of viability. Transport conditions maintaining viability acceptable for cellular therapy were examined for apoptosis rates and expression of desired myogenic, neurotrophic, neuromuscular junction, and angiogenic genes.

Results

Cryopreservation proved detrimental to our cell population. However, a minimally supplemented medium, theoretically safe for injection, was identified. Transport temperature and duration impacted cell viability, with warmer temperatures leading to faster death rates prior to the end of the study. Transport conditions did not appear to affect apoptotic rate. Any expression change of desirable genes fell within the acceptable range.

Conclusions

Transport state, medium, duration, and temperature must be considered during the transport of injectable muscle cells as they can affect cell viability and expression of integral genes. These described factors are integral in the planning of general cell transport and may prove equally important when the cell population utilized for laryngeal injection is derived from a patient's own initial muscle biopsy.