Mechanical Dissociation or Enzymatic Digestion for the Isolation of Neural Stem Cells for Therapeutic Use

Abstract

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Introduction. Periodontitis affects >743 million people worldwide. Neural crest-derived stem cells (NC-SCs) hold the required multipotentiality to regenerate the damaged periodontium. These cells have been identified within adult tissue samples of adipose tissue, PDL and palatal tissue, reducing ethical problems associated with stem cell use. Typical enzyme isolation protocols are expensive, time consuming and may represent increased patient risk. Mechanical dissociation has been suggested as a method of generating cell suspensions required for stem cell isolation and proliferation. Material and methods. Samples of 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 shown to contain populations of NCSCs. We used the Rigenera-Human Brain Wave® prototype mincer to produce a healthy suspension of NC stem cells. We have compared successful cell cultures produced via mechanical dissociation and enzymatic dissociation, producing single cell suspensions suitable for Magnetic Active Cell Sorting (MACs) with human antigen marker CD271. Results. Despite Countess Automated Cytometry data identifying cell suspensions produced by mechanical dissociation (n=24) containing on average 26,8 times as many living cells as enzymatic cell suspensions (n=18), enzymatic suspensions produced more successful cell cultures. Spheroids and subsequent adherent cells formed from 4 enzymatic cell suspensions (44,4%) vs. 1 mechanical cell suspension (8.3%). Additionally, 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 NC-SCs from our previous studies. Conclusion. The results of this study have indicated the suitability of this prototype mincer for prospective experiments with palatal tissue. Although proliferative cultures of adherent cells were isolated using mechanical dissociation, this mechanical dissociation method does not represent a viable alternative to enzymatic digestion, suitable for use in the clinical setting.