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bioRxiv · 10.1101/2025.06.23.661031

Dendritic Polyglycerol Amine Substrate Extends the Viability of Mixed Glial Cultures for Repeated Isolation of Immature Oligodendrocyte Lineage Cells

Uccelli, N. A.·Chitsaz, D.·Gothie, J.-D.·Kakkar, D.·Mohammadifar, E.·Antel, J. P.·Haag, R.·Kennedy, T. E.
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Abstract

Primary mixed glial cultures are key tools to isolate and study astrocytes, microglia and oligodendrocytes. Cell-substrate adhesion is critical for neural cell survival and differentiation. Cationic polymers like poly-D-lysine (PDL) are widely used to promote cell adhesion to cell culture substrates, however, PDL is not stable long-term, with cultured cells often detaching (peeling) after 2-3 weeks. Dendritic polyglycerol amine (dPGA) is a synthetic polycationic non-protein polymer biomimetic of poly-lysine that is highly resistant to degradation by cellular proteases. Substrates coated with dPGA promote cell adhesion and improve survival in long-term neuronal cultures. Here we assessed dPGA as a substrate coating to provide long-term support for mixed glial cultures. Oligodendrocyte precursor cells (OPCs) were isolated weekly by differential adhesion from cultures grown in T75 flasks with PDL or dPGA-coated substrates. Following two "shake-off" isolations, the cell layer in most PDL-coated flasks fully detached, rendering these flasks unusable for further culture. In contrast, dPGA-coated flasks consistently yielded cells for six or more sequential isolations over seven weeks in culture. dPGA-coated flasks produced more cells, a greater percentage of O4+ cells, and maintained similar proportions of OPCs and MBP-positive cells as when isolated from a PDL-coated substrate. dPGA is cyto-compatible, functionally superior, easy to use, low cost and a stable alternative to conventional cell substrate coatings. The enhanced long-term stability of mixed glial cultures grown on a dPGA substrate has the capacity to increase cellular yield, reduce animal use, and facilitate studies of oligodendrocyte cell biology.

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Uccelli, N. A., Chitsaz, D., Gothie, J.-D., Kakkar, D., Mohammadifar, E., Antel, J. P., Haag, R., Kennedy, T. E.. 2025-06-23. Dendritic Polyglycerol Amine Substrate Extends the Viability of Mixed Glial Cultures for Repeated Isolation of Immature Oligodendrocyte Lineage Cells. https://doi.org/10.1101/2025.06.23.661031

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