Hacat immortalized human keratinocytes

The cell lines selected for this study, HaCaT—immortalized human keratinocytes, were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany), and RPMI-7951—human malignant melanoma cells (ATCC^® HTB-66™) were procured from American Type Culture Collection (ATCC, Lomianki, Poland). DMEM, containing 10% FBS and 1% Pen/Strep mixture (10,000 IU/mL), was used to culture healthy skin cells. RPMI-7951 cells were grown in EMEM supplemented with 10% fetal bovine serum and 1% antibiotic mixture. The experiments were performed under standard conditions: incubation at 37 °C in 5% CO₂ atmosphere.

The two chocolate products (VHC1 and VHC2) were subjected to in vitro toxicological tests, using healthy human cell lines (primary gingival keratinocytes, human skin keratinocytes—HaCat and human skin fibroblasts—1BR3). Human primary gingival keratinocytes cell line (ATCC^® PCS-200-014 ™) was purchased from ATCC (American Type Cell Collection), HaCat—immortalized human keratinocytes was purchased from CLS Cell Lines Service GmbH (catalog no.: 300493), while the human fibroblast cell line 1BR3 was acquired from ECACC (European Collection of Authenticated Cell Cultures, catalog no.: 90011801), in the form of frozen vials. The culture media and growth kits were purchased as follows: Dermal Cell Basal Medium (ATCC^® PCS-200-030 ™) and Keratinocyte Growth Kit (ATCC^® PCS-200-040 ™) from ATCC, Dulbecco’s modified Eagle medium (DMEM) with a high glucose content of 4.5 g/L, EMEM (Eagle’s minimum essential medium) as well as other necessary reagents for cell culture such as phosphate saline buffer (PBS), trypsin/EDTA solution, FBS (fetal bovine serum), penicillin/streptomycin solution and Trypan blue from Sigma Aldrich (St. Louis, MO, USA).