Fig. 2

Functional assessment of cultured primary human hepatocytes (PHHs). a Photometric measurements of live cellular functions (transport, respiration, and viability) were imaged on days 2, 4, 8, 14, 21, 30 post seed revealing PHHs cultured in a 384-well plate remain stable for 30 days. b Condition of hepatocyte monolayer was determined by nuclei morphology, size, and fluorescence intensity, then classified as “healthy” and “unhealthy”. A steady adhered monolayer of ~11,000 PHHs and high albumin expression (ng ml⁻¹) persisted for the duration of 30 days. c, d Image analysis combining mean intensity of CellTracker^TM Green CMFDA with differential imaging filters allowed for bile canaliculi quantification, assignment of active vs. inactive transport, and identification of bile duct length (µm) where peak measurements were revealed on day 21 post seed. e The mean intensity of the fluorescent dye TMRM was used to measure mitochondrial activity with day 21 having the highest active mitochondrial sequestration. Graph bars represent means with s.d. for biological replicates (n = 3) and experimental replicates (n = 6, b, n = 3, c, e) or individual values were plotted (d). Statistical significance was calculated using an one-way ANOVA with Dunnett’s multiple comparisons test to day 2 where statistical significance values are represented as P < 0.0001 (***) and P < 0.0001 (****). White scale bars represent 500 µm and red represents 50 µm