Journal of Neuro-Oncology 43: 1–10, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. Laboratory Investigation Improved technique for establishing short term human brain tumor cultures ∗ Maxine A. Farr-Jones 1 , Ian F. Parney 1,2 and Kenneth C. Petruk 1 1 Divisions of Neurosurgery and 2 Experimental Surgery, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada Key words: glioma, brain tumor, tissue culture, GFAP, vimentin, karyotype Summary Culturing human central nervous system tumors has been difficult compared to other neoplasms. We report improved success rates for establishing short term human brain tumor cultures using a modified tissue processing technique. Eighty-seven brain tumor specimens (56 glioblastomas, 8 mid grade astrocytomas, 8 oligodendrogliomas, 15 other) were obtained from June 1988 to March 1997. The first twenty-three samples were processed by dissection, partial enzyme dissociation, and filtration through a tissue culture sieve. Subsequent samples were processed identically except tumor cells were centrifuged on a density gradient prior to plating. Successful cultures were defined as those surviving greater than three passages in tissue culture and growing to sufficient numbers (>10 6 cells) to allow freezing. Success rate was 42% (10/23) using standard processing methods and 86% (55/64) with the addition of density gradient centrifugation. Glial fibrillary acidic protein (GFAP) and vimentin staining, karyotypes, and growth curves were obtained for representative glioma cultures. All cultures tested were positive for vimentin (29/29) while 62% (18/29) were positive for GFAP. Of four cultures karyotyped (two glioblastomas, two oligodendrogliomas), all but one oligodendroglioma culture exhibited clonal cytogenetic abnormalities. These immunohistochemical and karyotypic results are consistent with the malignant glial origin of these cells. Of note, low passage human glioma cultures grew slower and exhibited more contact inhibition than immortalized human glioblastoma cell lines. Nevertheless, this simple method for establishing short term human brain tumor cultures should aid in further developing human brain tumor pre-clinical models as well as enhancing clinical applications dependent on in vitro human brain tumor cell growth adjust. Introduction Many preclinical studies of primary central nervous system (CNS) neoplasms have relied on rodent tumor models such as the rat 9L gliosarcoma and the murine C6 glioma [1–5]. While these models have been useful, questions remain as to their validity when compared to human brain tumors such as malignant gliomas. Human tumor models (both in vitro and in vivo) would be more ideal. However, development of animal human tumor models is dependent on efficient human CNS tumor cell growth in vitro. ∗ M.A.F.J and I.F.P. contributed equally to the preparation of this manuscript. Recent interest in glioma immunogene therapy [5–7] has underscored the need to efficiently establish short term human glioma cell cultures. In this novel form of ex vivo gene therapy, anti-tumor vaccines are cre- ated using cultured autologous human glioma cells that have been genetically modified in vitro to increase their immunogenicity. Clearly, successful establish- ment of human glioma cultures is key to this treatment strategy. In this study, we report improved success in estab- lishing short term human brain tumor cultures using a modified tissue processing technique. In addition, the characteristics of these cells are described in terms of morphology, immunohistochemistry, cytogenetics, and growth kinetics.