P140 Expanded primary cells for cell-based screenings

Astrid Noerenberg , upcyte technologies GmbH, Hamburg, Germany
Nils Runge , upcyte technologies GmbH, Hamburg, Germany
Torge Evenburg , upcyte technologies GmbH, Hamburg, Germany
Timo Johannssen , upcyte technologies GmbH, Hamburg, Germany
Primary cells are a valuable tool for R&D since they are more representative of the in vivo state when compared to cell lines. Their use in vitro is hampered by limited tissue availability, scarce cell yields and a restriction or even lack of proliferation. Taken together, these factors do significantly compromise the scope, length and reproducibility of experiments and often circumvent their use for extended cell-based screenings.

Here, we describe the controlled expansion of human primary cells, enabling production volumes of up to 2500 vials containing 5∙106 cells each. We first generated a library of lentiviral vectors carrying proliferation-inducing genes, allowing primary cells to bypass senescence. As a proof of principle, primary cells from several relevant target tissues were transduced, including liver sinusoidal endothelial cells (LSECs) & hepatocytes from various donors, keratinocytes, proximal tubular epithelial cells (PTCs) and bronchial epithelial cells (BEPCs).

As a result, we observed a marked increase in proliferation for all indicated cell types, including hepatocytes and LSECs which lack proliferation capacity in vitro. While we were able to expand the cells for up to 40 additional population doublings, upcyte® cells maintained several functional and phenotypic characteristics of mature cells. All cells exhibited expected morphology patterns and were restricted by the presence of specific growth factors, contact inhibition and anchorage dependence. Importantly, upcyte® cells maintained expression of respective marker proteins throughout the study. For example, upcyte® hepatocytes expressed cytokeratin 8/18, human serum albumin and alpha-1 antitrypsin. Accordingly, upcyte® LSECs were characterized by expression of CD31, mannose receptor and von-Willebrand-factor. Functional assays revealed primary-like activity of upcyte® cells, including phase I and II activities (hepatocytes), receptor-mediated endocytosis and tube formation (LSECs), wound healing (keratinocytes) and MMP secretion in response to the pollutant 2,3,7,8‑tetrachlorodibenzo-p-dioxin (BEPCs).

In conclusion, we developed a comprehensive platform enabling the controlled expansion of primary cells derived from various tissues. Obtained upcyte® cells exhibited a markedly extended lifespan while maintaining a primary-like phenotype, potentially facilitating throughput and reproducibility during biomedical research and drug discovery.