Method to evaluate the proliferation of activated lymphocytes in a three-dimensional collagen matrix

It is well known that the enhancement of the cell-matrix interactions represents one of the early steps in the process of lymphocyte activation. However, the information regarding the role of these interactions in the late stages of lymphocyte activation (in particular, the proliferation) is still controversial. This is basically due to the absence of adequate experimental models. In the present work we carried out a step-by-step modification of a well-studied model of mitogen-stimulated lymphocyte activation, adjusting it to the conditions of a three-dimensional collagen matrix (3D-CM). All the changes added to the standard procedure in the process of this modification were rigorously controlled using various experimental models. The final version of the method includes the following steps: (i) 24-h lymphocyte (lymphocyte fraction from mouse spleen) preincubation with mitogens (Con A or LPS) with a subsequent cell wash (parameters being controlled: irreversible lymphocyte activation, independence of the proliferation from cell-cell interactions); (ii) transfer of the activated lymphocytes to (3)H-thymidine containing 3D-CM and incubation for 48 h (controlled parameters: distribution of the radioactive label within the 3D-CM and its biological accessibility to lymphocytes); (iii) degradation of the 3D-CM with bacterial collagenase and cell transfer onto glass fiber filters (controlled parameters: cell viability after cultivation in the 3D-CM and treatment with the collagenase). With this method we found that the proliferation of the Con A- and LPS-stimulated lymphocytes in 3D-CM was dramatically inhibited (by 66.5 +/- 14.9% and by 88.1 +/- 10.2%, respectively). The discovered inhibition of the lymphocyte proliferation was not a consequence of either the ineffectiveness of the mitogens, the disruption of the cell-cell interactions, an insufficient inclusion of the radioactive label into cells, or of a decreased cell viability.