In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44(+)/CD90(-), CD44(-)/CD90(-), CD44(+)/CD90(+), CD44(-)/CD90(-)). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90(+)) and epithelial (CD90(-)) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44(+) cells exhibited stronger migratory capacity than CD44(-) subpopulations. CD44(+) subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44(-) subpopulations. Furthermore, co-cultivation experiments revealed that CD44(-)/CD90(+) cells supported growth of epithelial tumor cells (CD44(+)/CD90(-)). On the contrary, factors released by CD44(+)/CD90(+) type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44(-)/CD90(+), but this subpopulation had a low migratory capacity.