(127d) A Simple and Scalable Hydrogel-Based 3D System for Culturing Protein-Producing Cells

Qiang Li, Haishuang Lin, Ou Wang and Yuguo Lei

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

Introduction: Recombinant protein therapeutics have become important components of the modern medicine. Majority of them are produced with mammalian cells that are cultured either through adherent culture, in which cells are cultured on substrates, or suspension culture, in which cells are suspended and cultured in agitated cell culture medium in a culture vessel. The adherent cell culturing method is limited by its low yield. In suspension culturing, cells need extensive genetic manipulation to grow as single cells at high density, which is time- and labor-consuming. Here, we report a new method, which utilizes a thermoreversible hydrogel as the scaffold for culturing protein-expressing cells. The hydrogel scaffolds not only provide 3D spaces for the cells, but also act as physical barriers to prevent excessive cellular agglomeration and protect cells from the hydrodynamic stresses. As a result, cells can grow at high viability, high growth rate, and extremely high yield even without genetic manipulations. The method that can culture the Wnt3A-expressing cells at high density, while allow continuously collecting the conditioned medium in an easy manner is of great value for the cost-effective production of Wnt3A proteins.

Materials and Methods: We used the murine L cells (i.e. L-Wnt-3a-cells) expressing the human Wnt3A proteins as a model system to demonstrate this novel culture method. Single L-Wnt-3a-cells were mixed with 10% PNIPAAm-PEG solution at 4 ºC and casted on a 12-well plate, then incubated at room temperature or 37 ºC for 5 minutes to form a hydrogel before adding warm DMEM medium supplemented with 10% FBS. The conditioned medium was collected daily for evaluating the Wnt3A protein secreted. To passage cells, ice-cold PBS was added for 2 minutes to dissolve the hydrogel. Cell masses were collected by centrifuging at 100 g for 3 minutes and treated with 0.05% trypsin at 37 ºC for 5 minutes. Cells were dissociated into single cells with pipettes for the next passage. MDA-468 cells (ATCC® HTB-132â„¢) were stably transfected with a GFP reporter of the canonical Wnt signaling (Addgene, #24305) and used for qualitative detecting Wnt3A proteins. MDA-468 cells (ATCC® HTB-132â„¢) were stably transfected with a luciferase reporter of the canonical Wnt signaling (Addgene, #24308) and used for quantifying Wnt3A proteins.

Results and Discussion: The L-Wnt-3a-cells can be cultured in the thermoreversible PNIPAAm-PEG hydrogel at a high growth rate and volumetric yield. When seeded at 5x10⁵ cells/mL, cells expanded 6.9, 38.7, 97.1, and 132.5-fold to yield 3.4x10⁶, 1.9x10⁷, 4.9x10⁷, and 6.6x10⁷ cells/mL on day 3, 5, 7, and 9, respectively. The cell yield in the hydrogels is around 10 to 20 times of the suspension culturing. Next, we studied whether L-Wnt-3a-cells could be cultured in the hydrogel for long-term. During a 10-passage culture in the hydrogel, when seeded at 1x10⁶ cells/mL, L-Wnt-3a-cells consistently expanded ~37-fold per passage per 5 days with cell viability >98%. We found that the hydrogel supports long-term culturing of L-Wnt-3a-cells without significantly altering their phenotype. To analyze whether the cultured L-Wnt-3a-cells secreted biologically active Wnt3A proteins, we generated MDA-468 cells that stably expressed a GFP reporter and MDA-468 cells that were stably transfected with a luciferase reporter for the canonical Wnt signaling. We found that the protein productivity per cell per day in the hydrogel is much higher than the adherent culturing method.

Conclusion: In conclusion, a simple, efficient, and scalable 3D culture system based on thermoreversible hydrogel was developed for culturing protein-producing mammalian cells with high cell viability, growth rate, volumetric yield, and protein productivity. To the best of our knowledge, this is the first report using a 3D thermoreversible hydrogel for protein production. This simple method provides a valuable tool for research laboratories and pharmaceutical industry for producing therapeutic proteins.