2.4.4. Lactate Dehydrogenase (LDH) Cytotoxicity

LDH is an enzyme usually present in the cytoplasm of living cells and is released into the cell culture medium by the membrane permeabilization of severely affected or dead cells. The increased LDH activity in the culture supernatant is proportional to the number of lysed cells [14].

LDH levels were determined using the commercial kit MAK066 (Sigma-Aldrich, München, Germany). For the assay, the supernatant of the cell cultures was collected and centrifuged at 600 RCF for 10 min. Then, 10 µL of the supernatant was transferred to a 96-well plate with 40 µL/well of the LDH reaction solution. The mixture was incubated at 37 °C at 70 rpm for 30 min in the dark. The absorbance was measured in a spectrophotometer at a λ of 450 nm. The absorbance of the culture medium was used as a negative control and the medium of cells lysed with the kit’s lysis solution as a positive control.

LDH measurements were performed on healthy cells, cells under hypoxic conditions, and cells under hypoxic conditions intervened either with hydrogels, encapsulated cells, or under normoxic conditions. The cytotoxicity percentage was calculated using Equation (4):