(630e) An Integrated Miniature Bioprocessing for Personalized Human Induced Pluripotent Stem Cell Expansion and Differentiation into Neural Stem Cells

Haishuang Lin, Qiang Li, Yuguo Lei

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

Introduction: Cell therapies offer hope to treat many human diseases and injuries that cannot be treated or their progression cannot be altered by current treatments. Autologous cells are ideal for cell therapies since they induce minimal or no immune rejection. However, it is very challenging to isolate sufficient numbers of autologous cells from the patient for an effective treatment. Furthermore, majority of human cell types exhibit limited growth and significant phenotype shifting when cultured, preventing generating the required numbers of autologous cells through in vitro expansion. In this study, we describe our effort to develop such a miniature bioprocessing for making NSCs from human iPSCs in the PNIPAAm-PEG hydrogel.

Materials and Methods: A biomaterial was used as the scaffold for the 3D culture system. With a syringe and needle, 4 °C PNIPAAm-PEG solution containing single iPSCs were injected into room temperature E8 medium in a 15 ml conical tube. Fibrous hydrogels were formed instantly. A long needle was inserted through the septum cap into the tube that its open reached the tube bottom. A Variable-Speed Peristaltic Tubing Pump (Control Company, USA) was used to continuously perfuse the culture medium into the tube through this long needle. With the assist of this hydrogel scaffold, integrated the bioprocessing including the iPSC expansion, iPSC differentiation into NSCs, the subsequent depletion of undifferentiated iPSCs from the product, and concentrating and transporting the produced cells to the surgery room into two closed, 15 ml conical tubes.

Results: With the assist of miniature cell culture system, single iPSCs in hydrogel fibers grew into iPSC spheroids on day 5, and then became NSC spheroids after additional 7 days differentiation. With initial seeding density at 1x10⁶ cells/ml, 25-fold expansion and 2.5x10⁷ cells/ml hydrogel were achieved on day 12. Total of 1.0x10⁸ cells were produced in 4 ml of hydrogel in a 15 ml conical tube. Cell viability was >95% on day 12. 2% of the day 12 cells were SSEA4+. Live/dead cell staining showed no or undetectable dead cells. After magnetic separation, the produced cells expressed PAX6 and NESTIN and OCT4+/NANOG+ cells were not detectable. Cells pull down by the magnetic beads expressed both OCT4 and NANOG. 7 days and 30 days after transplantation, large numbers of the human nuclear antigen positive (HuNu+) and TUJ-1+ cells were found in the rat brain.

Conclusions: In summary, we showed that, with the assist of appropriate hydrogel scaffolds, personalized iPSC expansion and differentiation, as well as the subsequent product purification could be integrated into miniature bioprocessing that can be automated. We believe this type of bioprocessing can help the advancement of iPSC-based personalized cell therapies. To our best knowledge, this is the first report on hydrogel-based miniature cell culture device / bioprocessing for personalized iPSC expansion and differentiation.