Table 1

Sequences of the primers for qPCR"

Table 2

Formulation conditions for collagenase Ⅱ and Ⅳ combination"

Table 3

Combined collagenase Ⅱ and Ⅳ digestion efficacy comparison"

Table 4

Treatment conditions for different groups"

Figure 1

Comparison of digestion time and cell yield (cell number/field) A, comparison of digestion time among multiple treatment groups (n=3); B, quantification of viable cells isolated using different methods following 72-hour culture (n=3). * P < 0.05, * * P < 0.01, * * * P < 0.001, optimized group vs. other groups; ### P < 0.001, #### P < 0.000 1, multiple comparisons."

Table 5

Concentration of digestive enzymes and treatment condition in each group"

Figure 2

Comparison of digestion time and cell yield (cell number/field) among the optimized group, 0.5× and 2× enzyme concentration group A, comparison of digestion time among the optimized group, 0.5× and 2× enzyme concentration group (n=3); B and C, after incubation for 72 h, the cells were photographed and analyzed by Operetta CLS High Content Imaging Analysis System (×10), and the number of cells in each field was counted (n=3). * P < 0.05, * * P < 0.01, optimized group vs. other groups; # P < 0.05, ## P < 0.01, multiple comparisons."

Figure 3

Comparison of digestion time and cell yield (cell number/field) between the optimized group and commercial kit group A, comparison of digestion time between the optimized group and the commercial kit group (n=3, * * * * P < 0.000 1); B, after 72 h of incubation, the cell yield of the optimized group and the commercial kit group was analyzed (n=3, * * P < 0.01); C, the obtained cells were photographed and analyzed by Operetta CLS High Content Imaging Analysis System (×10)."

Figure 4

Comparison of CFs purity between the optimized group and the commercial kit group A, immunofluorescence analysis of vimentin expression in CFs. Vimentin protein levels in CFs were assessed by immunofluorescence staining, with representative images captured using a high-content screening (HCS) system at 20× magnification. B, quantitative comparison of vimentin fluorescence intensity. Randomly selected microscopic fields (n=20 per group) with comparable cellular densities were subjected to HCS-based fluorescence quantification. No statistically significant intergroup differences were observed in vimentin intensity profiles (P>0.05). C, threshold-based vimentin-positive cell analysis. The percentage of DAPI (+) nuclei exhibiting vimentin fluorescence intensities exceeding 2 000 was calculated for each field. This threshold analysis revealed comparable proportions of vimentin (+) cells between experimental groups (P>0.05). CFs, cardiac fibroblasts; ns, no significance."

Figure 5

Evaluation of the trans-differentiation ability of CFs after TGF-β1 treatment A, after stimulation with TGF-β1, the viability of P1 generation CFs was evaluated through the CCK-8 assay (n=4); B and C, the protein expression levels of ColⅠ, α-SMA and FN were analyzed by Western blotting; D, the mRNA levels of ColⅠ, α-SMA and FN were detected by qPCR (n=3). * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.000 1. CFs, cardiac fibroblasts; TGF-β1, transforming growth factor β1; α-SMA, α-smooth muscle actin; ColⅠ, collagen typeⅠ; FN, fibronectin; qPCR, quantitative real-time PCR."