Abstract 199: A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments

Abstract Mantle cell lymphomas (MCL) are rare B-cell neoplasms belonging to the non-Hodgkin’s lymphomas and often show an aggressive clinical course. MCL respond relatively briefly to conventional chemotherapy and are characterized by frequent relapses (Dreyling et al., Ann Oncol, 2017). Therapy for MCL remains a challenge also due to limited cultivation time of primary cells. In vitro suspension culture of primary human MCL cells is not easily feasible (Bryant et al., Lab Invest, 2000) and cells undergo spontaneous apoptosis ex vivo (Zhang et al., Blood, 2012; Medina et al., Heamatologica, 2012). Useful in vitro MCL models that allow long-term culture of primary MCL cells could be helpful in the biological and therapeutic study of MCL. For this purpose, we established a 3D hydrogel culture model for cultivation of primary MCL cells that were first tested with cell lines. After 3 days of culture, the cells cultured in the hydrogel can successfully be analysed for cell viability (live-dead confocal laser scanning microscope-based assay, flow cytometry), metabolism (pH, glucose, lactate), and biomarker expression by immunohistochemistry. The 3D hydrogel cultivation model allows a variety of analytical methods, also because it is reversible and the cells can easily be separated as single cell suspension from hydrogel for further analytical purposes, such as flow cytometric analyses. Next, we cultured primary MCL cells in the alginate hydrogel 3D culture system. MCL cells were isolated from blood of different patients and separated by Ficoll centrifugation. The isolated cell population was identified as MCL cells by double-staining of anti-CD20 and anti-CD5 followed by flow cytometric analysis. Cells were further cultured in medium as conventional suspension culture or in alginate hydrogel as 3D culture. Cell death was assessed with AnnexinV by staining phospholipids of apoptotic cells, allowing flow cytometric quantification. All samples had less than 6% AnnexinV positive (+) cells at day 0 before the experiment started. After 2 days cultivation, primary MCL cells showed a strong induction of cell death in suspension culture with 65.5% (±4.2%) AnnexinV+ cells versus 21.9% (±0.6%) AnnexinV+ cells in 3D hydrogel culture. Subsequently after 3 days cultivation, 74.2% (±12.2%) AnnexinV+ cells in suspension versus 33.5% (±1.3%) AnnexinV+ cells in the hydrogel 3D culture were determined. Therefore, we conclude that our novel 3D culture system might be a more suitable cultivation method for primary MCL cells than conventional suspension culture in cell culture medium. We have developed a 3D cultivation system with alginate hydrogel that allows nearly tripling the in vitro cultivation time of primary MCL. This could pave the way for new long-term treatments in research in the future and serve a better understanding of the cells. Citation Format: Kathrin Böpple, Annette M. Staiger, Heike Horn, German Ott, Walter E. Aulitzky, Meng Dong. A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 199.