Xinxin DU1, Kristina Klaschik2,3, Peter Mallmann1, Evgenia Isachenko1, Gohar Rahimi1, Yue Zhao4, Christiane Bruns4, Vladimir Isachenko5. 1. Research Group for Reproductive Medicine, IVF-Laboratory and Department of Gynecology, University of Cologne, Cologne, Germany. 2. Center for Familial Breast and Ovarian Cancer, University Hospital Cologne, Cologne, Germany. 3. Center for Integrated Oncology, University Hospital Cologne, Medical Faculty, Cologne, Germany. 4. Department of General, Visceral and Cancer Surgery, University of Cologne, Cologne, Germany. 5. Research Group for Reproductive Medicine, IVF-Laboratory and Department of Gynecology, University of Cologne, Cologne, Germany v.isachenko@yahoo.com.
Abstract
BACKGROUND: Cryopreservation of ovarian tissue for patients with cancer comes with the concern for the existence of cancer cells in the grafts. Data about cancer cell viability after cryopreservation are currently lacking. For the experimental study of cryo-stability of cancer cells, different protocols of in vitro culture of various cell lines are used. The existing protocols are not able to mimic the effectiveness of in vivo/in situ development of cancer cells. This study aimed to test the new protocol for in vitro culture of breast cancer cells. MATERIALS AND METHODS: ZR-75-1 and MDA-MB-231 cells were in vitro-cultured in standard [RPMI 1640 and Dulbecco's modified Eagle's medium (DMEM), respectively] and experimental (AIM V) medium. Cell phenotypes were tested by CCK-8 cell proliferation measurement, wound-healing assay, transmembrane cell migration and invasion assay and immunofluorescent staining. A 10-day in vitro culture without cell passaging was conducted on both cell lines in culture medium to observe the generation of a cell layer as a model of solid tumor tissue. The density of the cell layers was revealed by immunofluorescent staining. RESULTS: AIM V significantly promoted continued proliferation of both cell lines. Cell motility of ZR-75-1 cells was increased considerably more in AIM V than in RPMI-1640. For MDA-MB-231 cells cultured in AIM V and DMEM, no significant differences in mobility and invasion were observed. Both cell lines maintained in AIM V medium generated a cell layer on day 7 and formed a compact structure on day 10 of in vitro culture. CONCLUSION: In vitro culture of ZR-75-1 and MDA-MB-231 cells in AIM V medium is more informative than culture in standard medium. The described protocol provides a means for the formation of compact structures from in vitro-cultured cancer cells as a model of solid tumor tissue. Copyright
BACKGROUND: Cryopreservation of ovarian tissue for patients with cancer comes with the concern for the existence of cancer cells in the grafts. Data about cancer cell viability after cryopreservation are currently lacking. For the experimental study of cryo-stability of cancer cells, different protocols of in vitro culture of various cell lines are used. The existing protocols are not able to mimic the effectiveness of in vivo/in situ development of cancer cells. This study aimed to test the new protocol for in vitro culture of breast cancer cells. MATERIALS AND METHODS: ZR-75-1 and MDA-MB-231 cells were in vitro-cultured in standard [RPMI 1640 and Dulbecco's modified Eagle's medium (DMEM), respectively] and experimental (AIM V) medium. Cell phenotypes were tested by CCK-8 cell proliferation measurement, wound-healing assay, transmembrane cell migration and invasion assay and immunofluorescent staining. A 10-day in vitro culture without cell passaging was conducted on both cell lines in culture medium to observe the generation of a cell layer as a model of solid tumor tissue. The density of the cell layers was revealed by immunofluorescent staining. RESULTS: AIM V significantly promoted continued proliferation of both cell lines. Cell motility of ZR-75-1 cells was increased considerably more in AIM V than in RPMI-1640. For MDA-MB-231 cells cultured in AIM V and DMEM, no significant differences in mobility and invasion were observed. Both cell lines maintained in AIM V medium generated a cell layer on day 7 and formed a compact structure on day 10 of in vitro culture. CONCLUSION: In vitro culture of ZR-75-1 and MDA-MB-231 cells in AIM V medium is more informative than culture in standard medium. The described protocol provides a means for the formation of compact structures from in vitro-cultured cancer cells as a model of solid tumor tissue. Copyright