Figure 2. Meniscal viability and metabolic activity assessed by fluorescence live/dead imaging and alamarBlue assays.

(A) Fresh, slow-frozen (SF), and vitrified meniscal specimens (1 mm and 3.5 mm thick). Each 3.5 mm specimen’s sagittal section is represented through three images: top, middle, and bottom. The groups include fresh, SF, vitrified after the 1.5-hour VS55 loading (1.5 Hr Vit), 3-hour VS55 loaded and unloaded without vitrification (3Hr CPA), and vitrified after the 3-hour VS55 loading (3 Hr Vit). Scale bars represent 200 μm. (B) Cell viability quantified in the middle images of 3.5 mm specimens from fresh (N=n =5) and preserved groups at day 0 post-warming including SF (N=n=4), 1.5 Hr Vit (N=n=6), 3 Hr CPA (N=n=3), and 3 Hr Vit (N=n=4). p-value was determined with a two-sided t-test. (C) Metabolic activity in 1 mm and 3.5 mm thick vitrified tissues compared to SF 3.5 mm thick tissue and the fresh 3.5 mm thick control group, over 0 to 4 days post-warming. Results were normalized to respective specimens (N=3–4; n=6–12) before CPA loading. Results from fresh control samples were normalized to their average values on day 0 (N=3; n=8). A black dashed line at 100% recovery indicates cell metabolic activity of fresh meniscal specimens. N = number of independent menisci; n = number of specimens. p-value determined via two-way ANOVA. Data are mean ± SD.