collagenase type ii solution  (Thermo Fisher)

Journal: Regenerative Therapy

Article Title: Generation of implant-type tissue-engineered cartilage from human embryonic stem cell-derived chondrocytes

doi: 10.1016/j.reth.2025.10.012

Figure Lengend Snippet: Isolation, expansion, and characterization of cells isolated from human embryonic stem cell-derived cartilage (hESC-Cart cells) and hACs. (a) Cartilage tissue derived from hESCs after induction. (b) Cartilage tissue after perichondrium removal, minced into small fragments. (c) Isolated single cells obtained by collagenase type II digestion. Scale bar: 100 μm. (d) Cumulative cell expansion curves of hACs (gray line) and hESC-Cart cells (black line) during serial passaging (n = 3 per group). hESC-Cart cells showed significantly greater proliferative capacity compared with hACs over 20 passages (p < 0.05 by unpaired t -test). Data are shown as mean ± standard deviation (SD). (e) Phase-contrast images of hACs (left) and hESC-Cart cells (right) at passages P3, P6, P12, and P20, showing morphological differences and gradual changes with increasing passage number. Images were captured using a 4 × objective. Scale bar: 100 μm. (f) Relative expression of representative cartilage markers (ACAN, COL2A1, SOX9, Vimentin) and pluripotency markers (OCT3/4, NANOG) for P3 hACs and P3 hESC-Cart cells. Relative expression levels were calculated using the ΔΔCt method, with all values normalized to hACs (set as 1.0). Data are presented as mean ± SD (n = 3). No statistically significant differences were observed (ACAN: p = 0.1263, COL2A1: p = 0.1457, SOX9: p = 0.2967, Vimentin: p = 0.9837, OCT3/4: p = 0.9775, NANOG: p = 0.9666; unpaired t -test).

Article Snippet: The cartilage fragments were digested in a 0.3 % type II collagenase solution (Worthington, Freehold, NJ) for 1 h. The released cells were washed with phosphate-buffered saline (PBS, FUJIFILM Wako Chemicals) and cultured in dishes.

Techniques: Isolation, Derivative Assay, Passaging, Standard Deviation, Expressing