In Vitro Study of a Novel Vibrio alginolyticus-Based Collagenase for Future Medical Application

Mesenchymal stem cells extracted from adipose tissue are particularly promising given the ease of harvest by standard liposuction and reduced donor site morbidity. This study proposes a novel enzymatic method for isolating stem cells using Vibrio alginolyticus collagenase, obtaining a high-quality product in a reduced time. Initially, the enzyme concentration and incubation time were studied by comparing cellular yield, proliferation, and clonogenic capacities. The optimized protocol was phenotypically characterized, and its ability to differentiate in the mesodermal lineages was evaluated. Subsequently, that protocol was compared with two Clostridium histolyticum-based collagenases, and other tests for cellular integrity were performed to evaluate the enzyme's effect on expanded cells. The best results showed that using a concentration of 3.6 mg/mL Vibrio alginolyticus collagenase allows extracting stem cells from adipose tissue after 20 min of enzymatic reaction like those obtained with Clostridium histolyticum-based collagenases after 45 min. Moreover, the extracted cells with Vibrio alginolyticus collagenase presented the phenotypic characteristics of stem cells that remain after culture conditions. Finally, it was seen that Vibrio alginolyticus collagenase does not reduce the vitality of expanded cells as Clostridium histolyticum-based collagenase does. These findings suggest that Vibrio alginolyticus collagenase has great potential in regenerative medicine, given its degradation selectivity by protecting vital structures for tissue restructuration.

Automated summary

This study proposes a novel enzymatic method using Vibrio alginolyticus collagenase to isolate mesenchymal stem cells from adipose tissue, which can be harvested easily and with reduced donor site morbidity. The optimized protocol of using 3.6 mg/mL Vibrio alginolyticus collagenase for a 20-minute enzymatic reaction showed comparable results to using Clostridium histolyticum-based collagenases for a 45-minute reaction. The extracted cells using Vibrio alginolyticus collagenase maintained the phenotypic characteristics of stem cells after culture conditions, and the enzyme did not reduce the vitality of expanded cells.