Antibiotic antimycotic solution

The study was approved by the ethical committee of Motol University Hospital, Prague, Czech Republic. All surgical tissue samples were obtained from HNSCC patients after they signed the informed consent documents. Patients were completely clinically examined, and tumour samples were taken from verified HNSCC under general anaesthesia; the inclusion criteria were as follows: histologically confirmed squamous cell carcinoma and no previous oncologic treatment; each tissue specimen underwent pathology quality control. Hematoxylin and eosin-stained sections from each sample were subjected to independent pathology review to confirm squamous cell carcinoma histological consistence. Tumour samples with approximately >60% tumour nuclei and <20% necrosis were considered sufficient for the following analyses. The tissue material harvested during surgery was divided into two pieces, the first was stored in an RNAlater (Ambion, Austin, TX, USA) for later RNA analyses. The second portion of the sample was placed into the cultivation medium (MEM) with the addition of 1% trypsin and 1% antibiotic-antimycotic solution (Santa Cruz Biotechnology, Dallas, TX, USA). The material was maintained at cold temperatures for 16–24 h to enable trypsin diffusion. Primary cell lines were subsequently prepared.

B95-8 cells (EBV producer cell line) [2 (link)] were cultured in RPMI-1640 (GIBCO, USA), supplemented with 10% FBS (GIBCO, USA), 1% antibiotic antimycotic solution (Santa Cruz, UK), 50 μg/ml gentamycin (Hyclone, USA) and 1× glutamine (GIBCO, USA), as previously described [58 (link)]. Cells were cultured until they reached a density of approximately 5×10⁶ cells/ml. Culture supernatants were centrifuged to remove cells/cell debris and then filtered using a 0.45 μm filter. Fresh filtered virus preparations were used for inoculation of rabbits. EBV copy number in the inoculum was estimated using quantitative real-time PCR (qPCR) (see ahead). The infectivity of the virus was determined by in vitro immortalization of human PBMCs [40 ]. This was approved by the Al Ain Medical District Human Research Ethics Committee (Approval number AAMD HREC 14/13).

The tissue material was first rinsed with 50% ethanol (Merck, Darmstadt, Germany), followed by phosphate buffer (Invitrogen, Waltham, MA, USA). The necrotic and adipose tissues were then removed from the tissue using a sterile scalpel, and the tumour tissue was cut into 3 mm pieces. Next, samples in the trypsin media were kept at 37 °C to activate the trypsin. Subsequently, the tumour pieces were transferred to a sterile tube with phosphate buffer and centrifuged at 4 °C, 2700 rpm for 7 min. After removing the supernatant, the tumour pieces were placed into the culture medium (MEM) with added 10% FBS (Biochrom, Holliston, MA, USA), 1% antibiotic-antimycotic solution, 10 μg/mL gentamicin sulfate, and 10 μg/mL ciprofloxacin (Santa Cruz Biotechnology, Dallas, TX, USA) to prevent contamination. After 7 days the culture medium was replaced with MEM medium with 10% FBS, supplemented with antibiotics (penicillin 100 U/mL and streptomycin 0.1 mg/mL) (Biochrom, Holliston, MA, USA) and 0.4 μg/mL hydrocortisone (Merck, Darmstadt, Germany). Since we were primarily interested in the effect of TME-derived fibroblasts (CAFs), selection using population overgrowth was performed. The passages of CAFs ranged from 1 to 5.