Crl 4031

Cryopreserved immortalized RPTEC/TERT1 cells (ATCC® CRL-4031™) stocked at 10⁶ cell/vial with passage number less than 9 were thawed and subcultured in T25 cell culture flasks until they reach ∼90% confluency. During the subculture period that lasted 5 days, DMEM/F12 (Gibco 11320033) supplemented with hTERT immortalized RPTEC growth kit (ATCC® ACS4007™) was used as culture medium per manufacturer’s instructions. The cells were trypsinized and resuspended at 4 × 10⁶ cell/mL in REGM™ (Lonza CC-3190). The chip microchannels and reservoirs were sterilized with 70% ethanol, dried under clean bench, and exposed to UV light for 1 h. Prior to seeding, the membrane was coated with a cell adhesion agent, FNC Coating Mix® (Athena 0407), and incubated at 37 °C for 1 min to enhance the rate of cell attachment. After filling the bottom channel of each device with 200 μL of REGM, 30 μL of the cell suspension was gently injected into the top channel. The devices were incubated at 37 °C, in 5% CO₂ for 1 h until the RPTECs completely settle and attach to the membrane, after which 170 μL of REGM was added to the top channel. The devices were maintained in the incubator and monitored while the media in both channels were refreshed on a daily basis.

Changes in cytotoxicity caused by the adsorption of drugs to each material used in the MPS were measured using the lactate dehydrogenase (LDH) assay. To compare the adsorptivity and cytotoxicity of adhesive tapes, two different types of tapes (HJ-9150W, Nitto Denko, Tokyo, Japan, described as Tape A and NT-1001, As one, Japan, as Tape B) were tested. Each material was immersed in 500 μL of medium containing a specific concentration of each nephrotoxic drug, as described above, and incubated for four hours at room temperature with 1 rpm rotation. After incubation, each medium was collected, and applied to the RPTECs (CRL-4031, ATCC, Manassas, VA, USA) cultured in 96-well plates (167008, ThermoFisher, Waltham, MA, USA). After 96 h incubation without any replacement of medium, the cytotoxicity was determined using an LDH assay kit (CK12, Dojindo, Kumamoto, Japan). Moreover, to evaluate the cytotoxicity by the solvent in adhesive tapes, each tape was immersed into 500 μL of medium without any drug and incubated as described above. After incubation, each medium was collected and the cytotoxicity was measured. COP and PDMS slabs were not evaluated because they were not expected to have cytotoxic substances eluted to the medium.