[Establishment of an effective way for separating and culturing tumor infiltrating lymphocytes]

The key to preparing TILs for clinical application is to enhance its capacity of proliferation and cytotoxicity. For this purpose we have made a study of methodology and established an effective way consisting of 3 processes to separate and culture TILs. It included: (1) mechanical splitting; (2) digesting with 0.05% collagenase I and 0.003% DNAase I mixture in room temperature for 6 hrs and 18hrs in cool, and (3) centrifuging with 75% and 100% Ficoll non-continuous grade density. The separated TILs were suspended in conditional culture medium for proliferation. Most of them reached the amount of 10(9) at the end of culture, which was essential for clinical therapy. Their NK and LAK activities were 56.3 +/- 11.7% and 48.6% +/- 10.6% respectively, in which the CD6+ T cells played an important role. The results suggest this a perfect way for separating and culturing TILs.