Efficient separation of human T lymphocytes from venous blood using PVP-coated colloidal silica particles (percoll)

In a comparative study, human peripheral T lymphocytes were separated as E rosettes by density centrifugation through various gradient media. Sheep red blood cells (SRBC) were removed by dissociation of the E rosettes at 37 degrees C with subsequent centrifugation on a similar density gradient prewarmed to 37 degrees C. In particular, gradients made of Ficoll Urovison were compared with Percoll gradients with regard to both separation steps. Using Percoll gradients, a maximal T cell recovery of 75% was obtained, whereas Ficoll separation yielded only 46%. T lymphocytes separated with Percoll exhibited equal viability compared to Ficoll isolated cells and consisted of 98% EAET-RFC. No inhibition of cellular function by Percoll treatment was detected, whereas Ficoll treatment led to an impaired mitogenic response. An inherent mitogenicity of Percoll was not observed. The method described results in considerably shortened centrifugation times due to the low viscosity of the Percoll medium and simultaneously seems to be less harmful to the rather fragile rosettes. Reproducibility was found to depend on careful control of density and osmolarity of the Percoll medium.