In vitro Immunization of Human Tonsilar Lymphocytes Using Slice Culture Sytem

It is difficult to immunize human lymphocytes in vitro by conventional cell culture methods. Activation of lymphocytes requires not only specific antigen stimulation but also delicate cell to cell interaction. If the cellular organization could be maintained in culture system, lymphocytes could be immunized in vitro with higher frequency. For the purpose of in vitro immunization of human lymphocytes, we used slice culture system which could maintain morphological and functional organization. Human tonsils resected from eleven-year old boy were evenly divided into two pieces, and one was cultivated in conventional cell culture and the other in slice culture system. In the former system, tonsilar mononuclear cells, separated by Ficoll-Hypaque density gradient centrifugation, were cultivated in RPMI 1640 supplemented with 10% human type AB serum in the cell density of 5x10⁶ /ml. In the latter, tonsillar tissues were sliced into small pieces of 8 mm³ , and were cultivated in Waymouth MB 752/1 medium supplemented with 10% Human type AB serum, gassed under 5% CO₂ and 95% O₂ at 37℃. After stabilized for one hour, each system wasw challenged with 50 µg/ml of KLH or 100 µg/ml of LPS. At 3, 6, 12 and 24 hours after antigen challenge, culture supernatants were assayed for the specific antibody by ELISA, and cells or tissues were analyzed for the expression of CD₂₃ by flow cytometry. The result showed that tonsilar B lymphocytes in slice culture system expressed CD₂₃ as early as 3 hours after antigen challenge, while those in cell culture system expressed CD₂₃ from 6 hours after challenge. Specific antibodies were detected only in supernatants of slice culture system from 6 hours after challenge. These results suggested thathuman lymphocytes could be immunized in vitro if the cellular organization was maintained.