Reagent b

Daily imaging was done by phase contrast microscopy. Cell counts and viability were performed using the Nucleocounter NC3000 (Chemometec) as per manufacturer's instructions for two separate samples. Counts were performed directly on the microcarriers using the reagent A100 and reagent B protocol. Briefly, the cell‐microcarrier suspension was diluted to a 1:1:1 ratio with reagent A100 and reagent B (Chemometec); reagent A100 lyses the cells from the microcarriers releasing the nuclei, while reagent B stabilizes the suspension. The resulting suspension was loaded onto a Nucleocassette Via‐1, preloaded with acridine orange and DAPI and the cassette then transferred Nucleocounter NC3000 machine for processing.
Spent medium samples were collected before and after medium exchanges in the bioreactor and were analyzed for glucose and lactate concentrations on an AccuTrend Plus meter (Roche). Fresh growth medium was used as baseline control.
Based on cell counts, the following parameters were calculated:

Specific growth rate
(1)μ=lnCxtCx0∆t,where μ is the specific growth rate (h⁻¹), Cx(t) and Cx(0) represent cell numbers at the end and start of the culture, t represents time in culture (h).

Doubling time
(2)td=ln2μ,where t_d is doubling time (h) and μ is the specific growth rate (h⁻¹).

Fold increase
(3)FI=CxtCx0,where Cx(t) represents the maximum cell number and Cx(0) is the initial cell number.

The effect that the compounds 1a–6a exert on the melanoma cell number (in cell culture) was tested on a MEW155 human melanoma tumor cell line obtained from the Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology in Warsaw. The cells were plated in 24-well plates (5000 cells per well) and incubated for 24 h in Eagle’s medium containing 10% fetal bovine serum and 1% solution of penicillin and streptomycin. After the cells had reached 80% confluence, the tested compounds (dissolved in 1% DMSO) were added at a concentration of 50 μM. Control cells were treated with 1% DMSO only. After 96 h of incubation, the cell suspension was first lysed in lysis buffer (ReagentA, ChemoMetec A/S) and then stabilized in stabilization buffer (ReagentB, ChemoMetec A/S). The number of dead cells was quantified in a propidium iodide cassette with the use of a NucleoCounter^® NC-100™ (ChemoMetec A/S) cell counter. The tests were performed in triplicate. The results were normalized so that the values obtained for the control were 100%.