Figure 1. Generation of Alveolar Organoids Using PneumaCult™ Alveolar Organoid Media

The PneumaCult™ Alveolar Organoid Media workflow is a two-stage protocol. During the expansion stage, primary isolated or cryopreserved human ATII single cells are seeded in PneumaCult™ Alveolar Organoid Expansion (AvOE) Seeding Medium. On day 2 - 3, after a full-media change, cultures are expanded using PneumaCult™ AvOE Medium to obtain mature ATII organoids. In the differentiation stage, ATII organoids are cultured for 10 additional days using PneumaCult™ Alveolar Organoid Differentiation (AvOD) Medium to generate ATI organoids.

Figure 2. PneumaCult™ Alveolar Organoid Expansion Medium Supports High Organoid Forming Efficiency

Organoid forming efficiency (OFE) serves as a functional measure of the health of an organoid culture and the performance of the media. ATII organoids were expanded in PneumaCult™ AvOE Medium (160 cells seeded per dome). (A) Brightfield image taken at day 9 using a 4X objective. The entire dome was captured in the image, each organoid formed was counted, and indicated (total 44; OFE=27.5%). (B) Bar graph showing OFE of ATII organoid cultures derived from three different donors (means ± SD). OFE = (Organoids Formed/Cells Seeded) x 100%.

Figure 3. PneumaCult™ AvOE Medium Supports Long-Term Expansion and Cryopreservation For Biobanking

(A) Box and whiskers plot of the cumulative doublings at each passage (n=4 independent donors) for organoids expanded in PneumaCult™ AvOE Medium (orange) and published formulation (gray). The means are connected by a line. Long-term passaging of alveolar organoids and over 10000x fold expansion was achieved by both PneumaCult™ AvOE Medium and published formulation. (B) Bar graph showing population doublings (mean ± SD) of organoids generated from cells previously cryopreserved at early and late passages, and expanded in PneumaCult™ AvOE Medium. The cultured organoids retained their expansion potential after thawing.

Figure 4. PneumaCult™ Alveolar Organoid Media Maintains High Levels of ATII and ATI Cell Marker Expression

(A) Flow cytometric gating of fluorescence minus one (FMO) controls for HT2-280 and HT1-56 markers (top), stained cells from organoids expanded in PneumaCult™ AvOE Medium (middle), and subsequently differentiated in PneumaCult™ AvOD Medium (bottom). PneumaCult™ AvOE Medium maintains both the ATII marker expression (HT2-280) and lineage potential to differentiate into ATI cells (HT1-56) in PneumaCult™ AvOD Medium. (B) Organoids derived from 4 independent donors, expressing high levels of HT2-280 were cultured in PneumaCult™ AvOE Medium or published formulation, and compared at each passage. Dashed line depicts 50% of starting HT2-280++%. (C) Scatter plot illustrating the percent change in HT2-280 expression between P0 and P5 from the same donors. Organoids cultured in PneumaCult™ AvOE Medium maintained higher levels of HT2-280 marker expression, compared to those cultured using published formulation. Mean is displayed as a solid line. * = p<0.05 in a one-tailed paired T-test (n=4).

Figure 5. Immunohistochemistry Confirms Expression of ATII and ATI Cell Markers in Alveolar Organoids

(A) Organoids from three donors expanded in PneumaCult™ AvOE Medium (top) express the ATII markers, HT2-280 (green) and pro-surfactant protein C (pro-SPC, yellow). When further differentiated in PneumaCult™ AvOD Medium (bottom), the levels of these ATII markers are down-regulated, while the ATI marker RAGE (red) is up-regulated. (B) Differentiated organoids also express high levels of ATI marker GPRC5a (yellow).

Figure 6. Organoids Cultured in PneumaCult™ Alveolar Organoid Media Express TMPRSS2 and ACE2, Key Markers for SARS-CoV-2 Entry

Organoids from three donors expanded in PneumaCult™ AvOE Medium (top) and differentiated in PneumaCult™ AvOD Medium (bottom) express proteins associated with SARS-CoV-2 entry, TMPRSS2 and ACE2. While ACE2 was expressed in all donors and conditions, TMRPSS2 expression was donor-dependent.

Figure 7. Gene Expression Analysis of Organoids Cultured in PneumaCult™ Alveolar Organoid Media Shows Presence of Alveolar Markers

Normalized gene expression heatmap of organoids expanded in PneumaCult™ AvOE Medium and differentiated in PneumaCult™ AvOD Medium compared to those grown using a published formulation. The presence of canonical ATII and ATI genes as well as SARS-CoV-2 key entry markers, ACE2 and TMPRSS2, are shown. Published RNA-Seq gene expression datasets of primary alveolar epithelial cells (including ATI and ATII cells, from Mikami et al. 2021, GSE186359) and primary ATII cells (Jacob and Kotton, 2017, GSE96642) were used as controls.