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

Abstract

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.

Keywords: Vibrio alginolyticus; adipose tissue; collagenase; disaggregation; regenerative medicine.

Figure 7

(A) Cellular yield, (B) clonogenic potential, and (C) cellular growth of extracted cells after enzymatic digestion with the optimized V. alginolyticus collagenase compared with Collagenase Type I and the C. histolyticum blend. The results are shown as means ± standard errors, indicating the significant statistical differences (*: p-value < 0.05).

Figure 1

Experimental plan for the novel collagenase optimization method and comparison with commercial enzymes.

Figure 2

(A) Cellular yield, (B) clonogenic potential, and (C) cellular growth of extracted cells after enzymatic digestion with V. alginolyticus collagenase in varying concentrations and with different incubation times. The results are shown as means ± standard errors, indicating the significant statistical differences (*: p-value < 0.05, **: p ≤ 0.01, ***: p ≤ 0.001).

Figure 3

(A) Viability and (B) proliferation capacity of extracted cells after enzymatic digestion with V. alginolyticus collagenase in varying concentrations and with different incubation times. The results are shown as means ± standard errors, indicating the significant statistical differences (**: p ≤ 0.01, ****: p ≤ 0.0001).

Figure 4

Immunophenotyping of the extracted cells with the optimized protocol of V. alginolyticus collagenase after adipose tissue enzymatic digestion (P0).

Figure 5

Immunophenotyping of extracted cells with the optimized protocol after the culture procedure until the cells reached passage 4 from the adipose digestion.

Figure 6

(A) Optical microscopy images of extracted cells with the V. alginolyticus optimized protocol compared with extracted cells with Col. Type I after being induced with differentiation medium (adipocytes present lipid droplets identified with black arrows, chondrocytes in blue, and osteocytes in red) in comparison with uninduced cells (CTRL-). Statistical graphs of semi-quantitative analysis of (B) lipid droplet number for adipogenic differentiation, (C) the cartilage-like matrix area for chondrogenic differentiation, and (D) calcium deposit areas for osteogenic differentiation. The results are shown as means ± standard errors, indicating the significant statistical differences (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 8

(A) Viability and (B) proliferation capacity of extracted cells after enzymatic digestion with the optimized V. alginolyticus collagenase compared with Collagenase Type I and the C. histolyticum blend. There are no statistical differences among the data.

Figure 9

Viability percentage of expanded cells placed in contact with the optimized V. alginolyticus collagenase compared with Collagenase Type I and the C. histolyticum blend for 20 min evaluated with the (A) trypan blue exclusion test and (B) MTT test. The results are shown as means ± standard errors, indicating the significant statistical differences (***: p ≤ 0.001, ****: p ≤ 0.0001).

Figure 10

Representative images of TEM analysis of P4 cells treated with the optimized V. alginolyticus collagenase compared with Collagenase Type I and the C. histolyticum blend for 20 min. Cells placed in agitation for 20 min acted as the control group.