Isolation of Skeletal Muscle-derived Cells Modeling Neural Crest-Derived Stem Cells for Therapeutic Use in Regenerative Periodontology
2020
Abstract Periodontitis is microbial infection affecting periodontium, the tooth supporting structure and affects >743 million people
worldwide. Neural crest-derived stem cells (NCSCs) hold the promise to regenerate the damaged periodontium. These cells
have been identified within adult adipose tissue, periodontal ligament, and palatal tissue. Typical enzymatic isolation protocols
are expensive, time consuming and often not clinically compliant. Enzyme-free, mechanical dissociation has been suggested
as an alternative method of generating cell suspensions required for cell separation and subsequent expansion ex vivo. In our
study, samples of rat skeletal muscle tissue were used to appraise the suitability of a novel mincing method of mechanical
dissociation against enzymatic digestion with collagenase and dispase. Skeletal muscle is readily available and has been shown
to contain NCSC populations. We used a Rigenera-Human Brain Wave® prototype mincer to produce a suspension of skeletal
muscle-derived cells modeling NCSCs. We have compared the resulting cell cultures produced via mechanical dissociation
and enzymatic dissociation, producing single cell suspensions suitable for Magnetic Cell Sorting (MACs) and Fluorescence-activated
cell sorting (FACS). Despite the Countess Automated Cytometry data demonstrating that cell suspensions produced
by mechanical dissociation (n=24) contain on average 26.8 times as many viable cells as enzymatic cell suspensions (n=18),
enzymatic suspensions produced more successful cell cultures. Spheroids and subsequently adherent cells formed from 4
enzymatic cell suspensions (44.4%) vs. 1 mechanical cell suspension (8.3%). Enzymatic digestion protocols formed spheroids
faster and more plentifully than mechanical cell suspensions. Adherent cells and spheroids isolated via both methods appear
morphologically similarly to NCSCs from our previous studies. Keywords Isolation; skeletal muscle-derived cells; Neural crest-derived stem cells; Mechanical dissociation; Regenerative
periodontology; Enzymatic digestion
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