hsaec medium  (PromoCell)

Journal: bioRxiv

Article Title: Dynamic Culture Improves the Predictive Power of Bronchial and Alveolar Airway Models of SARS-CoV-2 Infection

doi: 10.1101/2025.07.21.665885

Figure Lengend Snippet: (A) Schematic of the PhysioMimix® bronchial MPS, showing human primary bronchial epithelial cells cultured at air–liquid interface (ALI) under static and dynamic flow conditions. (B) Representative H&E-stained histological sections of bronchial tissues after 14 days of differentiation under static or MPS conditions. Scale bar, 50 µm. (C) Trans-epithelial electrical resistance (TEER) measurements over the 14-day ALI differentiation period. (D) Gene expression of Club cell (SCGB1A1) and Goblet cell (MUC5AC) markers in static versus MPS cultures after 14 days. GP = 0.0332 (*), 0.0021 (**). (E) Immunofluorescence staining of MPS bronchial tissue for acetylated α- tubulin (yellow), mucus (MUC5AC, green), actin (phalloidin, magenta), and nuclei (Hoechst 33342, blue). Scale bar, 100 µm.

Article Snippet: HPMECs, SAECs and NHBEs were maintained at 37°C, 5% CO 2 and 95% humidity in cell-type specific media (EGM-2 basal medium bullet kit [Lonza, CC-3162]; small airway epithelial cell media [PromoCell, C-211170]; PneumaCult-Ex Plus medium [STEMCELL Technologies, 05040], respectively) in T75 flasks until they reached 70% confluency.

Techniques: Cell Culture, Staining, Gene Expression, Immunofluorescence

Journal: bioRxiv

Article Title: Dynamic Culture Improves the Predictive Power of Bronchial and Alveolar Airway Models of SARS-CoV-2 Infection

doi: 10.1101/2025.07.21.665885

Figure Lengend Snippet: (A) Schematic of the PhysioMimix® alveolar MPS, showing human primary small airway epithelial cells cultured at air– liquid interface (ALI) under static and dynamic flow conditions. (B) Representative H&E-stained histological sections of alveolar tissues after 14 days of differentiation. Scale bar, 50 µm. (C) Trans-epithelial electrical resistance (TEER) measurements over the 14-day ALI differentiation period. (D) Gene expression of alveolar type I (AT1; AQP5) and alveolar type II (AT2; SFTPB) cell markers in static versus MPS cultures. GP = 0.0021 (**). (E) Immunofluorescence staining of alveolar MPS tissue for AT1 cells (RAGE, green), AT2 cells (SFTPB, yellow), actin (phalloidin, magenta), and nuclei (Hoechst 33342, blue). Scale bar, 100 µm.

Article Snippet: HPMECs, SAECs and NHBEs were maintained at 37°C, 5% CO 2 and 95% humidity in cell-type specific media (EGM-2 basal medium bullet kit [Lonza, CC-3162]; small airway epithelial cell media [PromoCell, C-211170]; PneumaCult-Ex Plus medium [STEMCELL Technologies, 05040], respectively) in T75 flasks until they reached 70% confluency.

Techniques: Cell Culture, Staining, Gene Expression, Immunofluorescence

Journal: bioRxiv

Article Title: Dynamic Culture Improves the Predictive Power of Bronchial and Alveolar Airway Models of SARS-CoV-2 Infection

doi: 10.1101/2025.07.21.665885

Figure Lengend Snippet: (A) Schematic of the PhysioMimix® lung MPS co-culture system. Epithelial cells were seeded on the apical side of the Transwell® insert and human pulmonary microvascular endothelial cells (HPMVECs) on the basolateral side, under air–liquid interface (ALI) and dynamic flow conditions. (B) Representative H&E and Alcian blue-stained histological section of bronchial MPS co-culture after 14 days. Mucus is stained blue; endothelial cells are indicated by white arrows. Scale bar, 50 µm. (C) TEER measurements comparing epithelial monoculture, endothelial monoculture, and epithelial–endothelial co-culture over 14 days under ALI conditions. (D) TEER comparison of bronchial and alveolar co-cultures grown under static or dynamic flow MPS conditions over the 14-day ALI differentiation period. (E) Gene expression of Club cell (SCGB1A1) and Goblet cell (MUC5AC) markers in NHBE cells before culture (NHBE pellet) and after 14 days of differentiation in MPS co- culture. (F) Gene expression of alveolar markers - AT1 (AQP5) and AT2 (SFTPB) - in SAEC cells before culture (SAEC pellet) and after 14 days of MPS co-culture. (G) Immunofluorescence staining of bronchial MPS co-culture tissue for acetylated α-tubulin (yellow), mucus (MUC5AC, green), actin (phalloidin, red), and nuclei (Hoechst 33342, blue). Top row shows epithelial layer; bottom row shows endothelial layer. Scale bar, 100 µm. (H) Immunofluorescence staining of alveolar MPS co-culture tissue after 14 days of differentiation, showing surfactant (SFTPB, green), actin (phalloidin, magenta), and nuclei (Hoechst 33342, blue). Endothelial cells are marked with white arrows. Scale bar, 20 µm.

Article Snippet: HPMECs, SAECs and NHBEs were maintained at 37°C, 5% CO 2 and 95% humidity in cell-type specific media (EGM-2 basal medium bullet kit [Lonza, CC-3162]; small airway epithelial cell media [PromoCell, C-211170]; PneumaCult-Ex Plus medium [STEMCELL Technologies, 05040], respectively) in T75 flasks until they reached 70% confluency.

Techniques: Co-Culture Assay, Staining, Comparison, Gene Expression, Immunofluorescence

Journal: Nature Communications

Article Title: Alveolar epithelial progenitor cells require Nkx2-1 to maintain progenitor-specific epigenomic state during lung homeostasis and regeneration

doi: 10.1038/s41467-023-44184-0

Figure Lengend Snippet: A Schematic of experimental design and overview. Live/CD31 − /CD45 − /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) were mixed with mouse lung fibroblasts from P28 mice and cultured for up to 35 days, followed by analysis via high content imaging. B H&E of 5 µm sections of FFPE day 35 Axin2 + organoids, showing cellular morphologies typical of both AT1 and AT2 cells. C – G Whole-mount immunofluorescence time course of Axin2 + organoids showing expansion of SFTPC + AT2 cells (red), increased differentiation into RAGE + AT1 cells (green) and increased structural complexity. H Imaris 3D reconstruction of day 35 Axin2 + organoid (z-depth = 174.13 µm) showing cellular arrangement/organization within mature organoids. I Click-iT EdU (green) whole-mount day 25 Axin2 + organoids, with proliferating cells primarily on outer edges or ‘buds’ growing outward from the organoid. J , K Electron microscopy of day 28 organoids. J Image of properly polarized AT2 cell with apical microvilli (black arrowhead) secreting surfactant (blue arrowhead) into a lumen. K Image of AT2 cell with lamellar bodies (black arrowhead) adjacent to an AT1 cell (green arrowhead, right). L , M Comparison of in vivo mouse lung (9-month C57BL/6J mouse) and in vitro day 25 Axin2 + organoids. Data throughout the figure represents at least 5 biological replicates with 3 technical replicates per experiment. [Scale bars = 50 µm, except for electron microscopy ( J , K ) scale bars = 2.5 µm]. (RAGE Receptor for Advanced Glycation End-products [AT1 cell marker], SFTPC Surfactant Protein C [AT2 cell marker], EdU 5-ethynyl-2’-deoxyuridine, FFPE formalin-fixed, paraffin-embedded). Schematics created with Biorender.com.

Article Snippet: To generate ‘spiked’ SAGM medium for mouse lung alveolar organoids, SABM Small Airway Epithelial Cell Growth Basal Medium (Lonza, CC-3119) was combined with the following additives: SAGM Small Airway Epithelial Cell Growth Medium SingleQuots Supplements and Growth Factors (using only the BPE [2 mL], Insulin [0.5 mL], Retinoic Acid [0.5 mL], Transferrin [0.5 mL], and hEGF [0.5 mL] aliquots) (Lonza, CC-4124), Heat Inactivated Fetal Bovine Serum (Corning, 35-011-CV, final concentration 5%), Antibiotic-Antimycotic (Gibco, 15240-062, final concentration 1x), Cholera Toxin from Vibrio cholerae (Sigma, C8052, final concentration 25 ng/mL).

Techniques: Cell Culture, Imaging, Immunofluorescence, Electron Microscopy, Comparison, In Vivo, In Vitro, Marker, Formalin-fixed Paraffin-Embedded

Journal: Nature Communications

Article Title: Alveolar epithelial progenitor cells require Nkx2-1 to maintain progenitor-specific epigenomic state during lung homeostasis and regeneration

doi: 10.1038/s41467-023-44184-0

Figure Lengend Snippet: A AAV6.2FF-Cre experimental set-up. Live/CD31 − /CD45 − /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) sorted from Axin2 creERT2-tdT ; R26R EYFP mice and Axin2 creERT2-tdT ; R26R EYFP ; Nkx2-1 fl/fl mice were treated with AAV6.2FF-Cre and plated with wild-type fibroblasts. B Comparison of brightfield and GFP whole-well images of organoids grown from control (AAV6.2FF-Cre-treated sorted R26R EYFP AEPs) and Nkx2-1 KO AEPs (AAV6.2FF-Cre-treated sorted R26R EYFP ; Nkx2-1 fl/fl AEPs) at day 28 of culture. Control (non-GFP) organoids with normal morphology are marked with a white asterisk. C – J H&E and immunofluorescence images of R26R EYFP ; Nkx2-1 fl/fl AEP-derived organoids that did ( F – J ) or did not ( C – E ) undergo recombination via AAV6.2FF-Cre. C – E Non-recombined organoids ( D ) express SPC (red) and Nkx2-1 (white) but do not express the YFP lineage label (green), whereas G recombined organoids do not express SPC or Nkx2-1 but do express the YFP lineage label. Non-recombined ( E ) and recombined ( H ) organoids maintain epithelial identity expressing CDH1. Nkx2-1 KO organoids express KRT8 and many proliferate and express Ki67 as late as day 40 of culture ( J - J ”). Data ( C – J ) represents 4 biological replicates with 3 technical replicates per experiment. K – R Integrated scRNAseq comparing epithelial cells from day 28 control organoids (Uninfected), AAV6.2FF-Cre-treated control organoids (AAV control), and AAV6.2FF-Cre-treated Nkx2-1 KO organoids (Nkx KO ). Nkx KO cells cluster separately from Uninfected and AAV control cells near Krt8 + cells ( K , L ), which make up a majority of cells in the Nkx KO condition ( M ). Marker genes for normal alveolar epithelium are lost and different markers gained ( N ) in Nkx KO . O – R Module scoring using published gene sets for AEPs ( O ), Krt8/PATS/DATP/ADI cells ( P ), lung cancer cells ( Q ), and foregut endoderm ( R ). Compare to Supplementary Fig. for marker gene analysis. [Scale bars = 50 µm]. Schematics created with Biorender.com.

Article Snippet: To generate ‘spiked’ SAGM medium for mouse lung alveolar organoids, SABM Small Airway Epithelial Cell Growth Basal Medium (Lonza, CC-3119) was combined with the following additives: SAGM Small Airway Epithelial Cell Growth Medium SingleQuots Supplements and Growth Factors (using only the BPE [2 mL], Insulin [0.5 mL], Retinoic Acid [0.5 mL], Transferrin [0.5 mL], and hEGF [0.5 mL] aliquots) (Lonza, CC-4124), Heat Inactivated Fetal Bovine Serum (Corning, 35-011-CV, final concentration 5%), Antibiotic-Antimycotic (Gibco, 15240-062, final concentration 1x), Cholera Toxin from Vibrio cholerae (Sigma, C8052, final concentration 25 ng/mL).

Techniques: Comparison, Control, Immunofluorescence, Derivative Assay, Expressing, Marker