Leveraging factors that control alveolar epithelial cell fate enables large-scale expansion for lung tissue engineering
Lauren K. Rochelle, Rachael S. Van, Richard J. Ottman, Daren F. Robinson, Ashley R. Dockham, Amy K. Smith, Daniel P. Keeley, Jia C. Wang, Darell W. McCoy, Tyler R. Zimmerman, Bryan A. Fioret, Ryan W. Bonvillain, Thomas H. Petersen, Sarah S. Hogan, Laila C. Roudsari
Lauren K. Rochelle, Rachael S. Van, Richard J. Ottman, Daren F. Robinson, Ashley R. Dockham, Amy K. Smith, Daniel P. Keeley, Jia C. Wang, Darell W. McCoy, Tyler R. Zimmerman, Bryan A. Fioret, Ryan W. Bonvillain, Thomas H. Petersen, Sarah S. Hogan, Laila C. Roudsari
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Research Article Cell biology Pulmonology

Leveraging factors that control alveolar epithelial cell fate enables large-scale expansion for lung tissue engineering

Abstract

Alveolar type 2 cells (AT2s) are critical to lung regeneration, and the absence of large-scale methods to expand AT2s has hindered regenerative medicine efforts. We report a microcarrier-based, large-scale expansion method that was used to generate hundreds of billions of human AT2s. Through our process, expanded AT2s largely retained their phenotype. Furthermore, we showed that culture medium, substrate composition, and stiffness are all critical to the maintenance of AT2s. Finally, we showed that expanded AT2s can differentiate into alveolar type 1–like cells, both in vitro and in a decellularized porcine lung, demonstrating the utility of these cells for lung tissue engineering.

Authors

Lauren K. Rochelle, Rachael S. Van, Richard J. Ottman, Daren F. Robinson, Ashley R. Dockham, Amy K. Smith, Daniel P. Keeley, Jia C. Wang, Darell W. McCoy, Tyler R. Zimmerman, Bryan A. Fioret, Ryan W. Bonvillain, Thomas H. Petersen, Sarah S. Hogan, Laila C. Roudsari

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Figure 1

Establishment of a scalable method to expand AT2s.

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Establishment of a scalable method to expand AT2s. (A) Schematic of AT2 ...
(A) Schematic of AT2 expansion. (B) Images of the culture systems used to expand AT2s: 250 mL spinner flask and 10 L bioreactor and controller. (C) Immunostaining for Ki-67 (green) alongside EdU (red), DAPI (blue), and phalloidin (magenta) on days 3 and 8 of P3 AT2s on microcarriers in T2-Max. Scale bar: 100 μm. (D) Immunostaining for AT2-specific proteins on AT2s on microcarriers on day 11 of P3: HT2-280 (green), pSP-C (white), DAPI (blue). Top left: maximum projection; top right: microcarrier cross-section, bright-field merge; lower left: microcarrier cross-section, without bright-field merge; lower right: microcarrier cross-section with pSP-C and DAPI only. Scale bars: 50 μm. (E) Transmission electron microscopy image of AT2s on a microcarrier (MC). Scale bar: 5 μm. (F) Growth curves from 3 successive cultures of AT2s isolated from donor 9VX and grown in 250 mL spinner flasks. (G) Growth curves from 3 successive cultures of AT2s isolated from donor 9VX and grown in 10 L bioreactors. (H) Fold change for P0 through P2 at 3 different scales (250 mL spinner, 3L bioreactor, and 10 L bioreactor), with each data point representing fold change for a different donor-derived cell bank. *P < 0.05, mixed effects analysis, Tukey’s multiple comparisons test. (I) Cell yields from 250 mL spinner flask, 3 L bioreactor, and 10 L bioreactor scales across 3 passages, with each data point representing yield from a different donor-derived cell bank. All bars represent mean ± SD. In H and I, 250 mL spinner: n = 12 donors, 3 L bioreactor: n = 9 donors, 10 L bioreactor: n = 10 donors.