Isolation and monolayer culture of human lymphocyte subpopulations

Objective: to create and evaluate the potential of an in vitro bioengineered model of the bone marrow endothelial niche for the support and activation of human hematopoietic stem cells. Material and methods. Human lymphocytes isolated from peripheral blood mononuclear cells were used. Experimental batch of platforms with various activated surfaces on the wells of 24-well plates were fabricated, namely: fibronectin only, poly-D-lysine only, fibronectin and poly-D-lysine, poly-D-lysine and anti-CD3 antibodies. During the 7-days cultivation, the cell populations state was inspected by direct microscopy, the viability was assessed via trypan blue staining, the adhesive activity assay was performed. Results. Under experimental conditions, lymphocyte viability was recorded at a high level of >80% for all test platforms and in control, respectively, with maintained typical for lymphocytes morphology. The use of the 24T_FN platform with the fibronectin activator showed non-uniform distributed cell islands and the formation of large conglomerates. The 24T_pDl platform with poly-D-lysine coating demonstrated a high degree of 2D uniformity with the formation of separate clusters within 50–100 cells. The adhesive activity on day 7 of the experiment for the platform 24T_FN and platform 24T_pDl was in the range of 60–70%, three times higher than the analogous data for control samples. The combined 24T_pDl@AbCD3 platform coated with poly-D-lysine in combination with anti-CD3 antibodies demonstrated the highest rates of cell survival, adhesion activity, and the 2-D uniformity. Conclusion. Herein, we demonstrated the capability of long-term monolayer subcultivation of human lymphocytes with high adhesive activity on the in vitro model synthetic platform based on the poly-D-lysine surface activator in combination with antibodies with affinity to CD3+ T cells.