Kaefer Samuel L, Zhang Lujuan, Brookes Sarah, Morrison Rachel A, Voytik-Harbin Sherry, Halum Stacey
Indiana University School of Medicine (IUSM) Indianapolis Indiana USA.
Department of Otolaryngology-Head and Neck Surgery IUSM Indianapolis Indiana USA.
Laryngoscope Investig Otolaryngol. 2024 Dec 8;9(6):e1259. doi: 10.1002/lio2.1259. eCollection 2024 Dec.
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.
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 × 10 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.
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.
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.
最近，我们实验室发现了一种自我神经支配的肌肉细胞群体，称为运动终板表达细胞（MEEs）。当用作注射治疗时，这些细胞会向微环境中天然释放多种神经营养因子，促进神经支配。与其他干细胞不同，MEEs的治疗潜力取决于细胞维持特定运动终板（MEPs）中表型细胞表面蛋白的能力。本研究的目的是确定能保持MEEs活力和自我神经支配功能的运输条件。
培养成年尤卡坦猪的肌肉祖细胞（MPCs）并诱导生成MEEs。研究了短期冷冻保存方法对MPC和MEE阶段的影响。研究了一种最低限度补充培养基用于悬浮介导运输的情况，并将MEEs以恒定浓度（1×10⁶细胞/mL）装入塑料注射器。样本分别置于不同温度（4、22和37°C）和持续时间（6、18、24和48小时）下，随后对活力进行统计分析。检查维持细胞治疗可接受活力的运输条件下的凋亡率以及所需的成肌、神经营养、神经肌肉接头和血管生成基因的表达。
冷冻保存对我们的细胞群体有害。然而，确定了一种理论上注射安全的最低限度补充培养基。运输温度和持续时间影响细胞活力，在研究结束前，温度越高导致死亡率越快。运输条件似乎不影响凋亡率。所需基因的任何表达变化都在可接受范围内。
在可注射肌肉细胞运输过程中，必须考虑运输状态、培养基、持续时间和温度，因为它们会影响细胞活力和完整基因的表达。这些所述因素在一般细胞运输规划中不可或缺，当用于喉部注射的细胞群体来自患者自身最初的肌肉活检时，可能同样重要。
