The enzymes DisoNNex and TrypLE are interchangeable as harvesting agents for adipose tissue-derived mesenchymal stromal cells.

Abstract

Cell therapies based on adipose tissue-derived mesenchymal stromal cells (ASCs) are promising candidates for the treatment of numerous diseases. Relevant to the potential of these therapies is the development of consistent and efficient ASC-manufacturing processes, with enzymatic cell dissociation being a critical step. DisoNNex, a novel cell dissociation enzyme based mainly on chymotrypsin activity, offers a Good Manufacturing Practice (GMP)-compliant alternative to existing dissociation methods; its impact on ASC characteristics remains yet to be explored. The performance of DisoNNex was compared with the current dissociation industry standard, TrypLE Select Enzyme 1x (TrypLE; Gibco/Thermo Fisher Scientific, Waltham, MA, USA). The DisoNNex concentration matching the dissociation kinetics of TrypLE was defined using time-lapse microscopy and image analysis. ASCs from five donors were harvested using either TrypLE or DisoNNex across two passages. Assays assessed immunophenotype, apoptosis, secretion of vascular endothelial factor, indoleamine 2,3-dioxygenase expression and suppression of lymphocyte proliferation. Multi-omics (bulk RNA sequencing, proteomics, metabolomics and lipidomics) were included in the study. DisoNNex at 15 µg/mL matched TrypLE in dissociation kinetics and maintained ASC viability. No significant differences were observed in functional assays. Multi-omics analysis revealed only four differentially expressed genes of 12509 protein-coding genes, and no significant differences in proteins, metabolites or lipids were found. The Multi-Omics Factor Analysis plot showed no clustering according to the dissociation enzymes used for harvest. DisoNNex is a relevant alternative to TrypLE for ASC harvest, resulting in equivalent yields and cell quality under the tested conditions. These findings support the potential of DisoNNex in GMP-compliant manufacturing of ASC-based cell therapies and provide starting conditions for its implementation in ASC processes. Nonetheless, full comparability studies within an established production process are pending.