(702e) A Cell-Friendly 3D Culture System for Scalable Culturing of Primary Human Glioblastoma Tumor-Initiating Cells

Qiang Li, Haishuang Lin, Ou Wang and Yuguo Lei

Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, Nebraska, USA

Introduction: Glioblastoma is the most aggressive and deadly brain cancer. Studies attribute the high drug resistance and recurrence of glioblastoma to the existence of a small population of cells termed the tumor-initiating cells (TICs) within the tumor mass. These TICs are very valuable for developing new drugs for treating glioblastoma. Massive numbers of TICs are needed to deliver on the biomedical promise of these cells. However, the cost-effective production of large numbers of high-quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. In vivo, glioblastoma TICs reside in 3D microenvironments that have plenty of cell-cell and cell-ECM (extracellular matrix) interactions, sufficient supply of nutrients, oxygen, and growth factors, and no or minimal hydrodynamic stresses. We designed the AlgTubes to mimic these in vivo microenvironments for culturing glioblastoma TICs. The hydrogel tubes created cell-friendly microspaces that allowed TICs to interact with each other and expand. Meanwhile, the tubes protected TICs from hydrodynamic stresses in the culture vessel and confined the cell mass less than 400 µm (in radial diameter) to ensure efficient mass transport during the entire culture. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery.

Materials and Methods: To establish these cell lines, the tumor samples were dissociated into single cells with 0.5% trypsin and then cultured in Neurocult^TM medium supplemented with bFGF, EGF, and heparin. A micro-extruder was fabricated to process AlgTubes. 2% Hyaluronic acid (HA) solution containing single glioblastoma TICs is pumped into the central channel and 1.5% alginate solution in 145 mM NaCl is pumped into the side channel of the micro-extruder. The corresponding coaxial core-shell flow is extruded into a 100 mM Ca²⁺ buffer, which instantly crosslinks the alginates solution to form a hydrogel shell to make AlgTubes. Subsequently, cells laden AlgTubes were transferred to 6-well plate for culturing with the medium. The medium was changed daily. To passage cells, the AlgTubes were dissolved with 0.5 mM EDTA for 5 minutes. The released cell mass was collected and treated with 0.05% trypsin at 37 ºC for 5 minutes and dissociated into single cells for the following culture.

Results and Discussion: Primary glioblastoma TICs (L0, L1, and L2) from different donors could be efficiently cultured in AlgTubes with high growth rate (~700-fold expansion/14 days) and high volumetric yield (~3.0x10⁸ cells/mL). With this yield, it will take about 30 milliliters of AlgTubes to generate ~1×10¹⁰ glioblastoma TICs. To assess whether the AlgTubes support long-term culturing of glioblastoma TICs, all the three lines (L0, L1, and L2) were continuously cultured for 10 passages (e.g. about two months). After the long-term culture in AlgTubes, these cells still expressed the TIC markers Nestin, Sox2, CD44, Olig2, CD133, and CD15. Our results showed TICs cultured for long-term in AlgTubes retained their capability to differentiate into neurons and glia cells or re-initiate tumors in vivo. Additionally, AlgTubes-based bioreactors could be readily built for the scalable production of TICs. Our comparative studies showed the AlgTubes offered significant advantages over the 2D, static 3D, and dynamic 3D suspension culturing in terms of cell growth rate and yield. To our best knowledge, this is the first that utilizes alginate hydrogel tubes for successful TICs expansion.

Conclusion: In conclusion, we developed a new method for culturing glioblastoma TICs with high efficiency. It is simple, scalable and cost-effective. We believe the technology is a valuable tool for developing therapies targeted to TICs. The AlgTubes will be of broad interest to individual laboratories, institutions, and biotechnology companies working on developing new cancer therapies.