Rat albumin elisa quantification kit

1 mL of culture media were collected for urea and albumin synthesis measurements each day. Urea content in the culture media was measured using Urea Nitrogen Kit (Stanbio Laboratory, USA). Albumin concentration was measured using Rat Albumin ELISA Quantification Kit (Bethyl Laboratories Inc., USA). The absolute urea and albumin amounts were calculated based on media volume collected and was normalized against the cell number by the end of culture.

Culture medium was also collected at day 3, day 5 and day 7 for hepatocyte functional evaluation. Hepatocytes were also cultured in a sandwich configuration as a 3D control, the seeding density and media was the same as decellularized scaffolds culture. Briefly, ice-cold 0.25 mg/ml Matrigel^TM (BD Biosciences, Heidelberg, Germany) diluted in incubation media at 1.5 ml/well were added to the 6-well plates and then incubated at 37 °C for 2 h. Excess Matrigel^TM was removed by aspirating media and rinsing with PBS. Hepatocytes were then added onto the Matrigel^TM-coated bottom of the plates and allowed seeding for 24 h at 37 °C. Then the media was aspirated and replaced with another 1.5 ml/well Matrigel^TM solution, and the plates were incubated for 2 h before media change for the upper collagen layer to be fixed. The normal single layer culture was used as a 2D control. The media was replaced every day and the culture ended at day 7. Albumin was measured with a rat albumin ELISA quantification kit from Bethyl Laboratories (Montgomery, TX) according to the manufacturer’s instructions. Measurements of urea were performed using a QuantiChrom^TM DIUR-500 Urea Assay Kit (BioAssay System, Hayward, CA). All assays were performed in triplicate.

Functional capacity of cultured hepatocytes was evaluated by the amount of albumin secreted into the culture medium using Rat Albumin ELISA Quantification Kit (Bethyl Laboratories, Montgomery, TX).