Figure 1. ALI Cultures Generated Using CELLTREATⓇ Inserts Are Highly Differentiated

Human bronchial epithelial cells (HBECs), expanded in PneumaCult™ Ex-Plus Medium (Catalog #05040), were seeded onto CELLTREATⓇ Inserts at an early (P3) or late passage (P6) and differentiated in PneumaCult™-ALI Medium (Catalog #05001) for 4 weeks. A high level of differentiation (represented by brown areas of the culture), as well as mucus secretion and visible cilia beating on the apical surface (not shown), were observed with cultures grown on CELLTREATⓇ Inserts. Scale bar = 500 µm.

Figure 2. ALI Cultures Generated Using CELLTREATⓇ Inserts Form Pseudostratified Columnar Epithelium

ALI cultures, differentiated from HBECs at an early (P3) or late passage (P6), were fixed 4 weeks after air-lift. They were then paraffin embedded, sectioned, and stained with H&E. Cultures generated using CELLTREATⓇ Inserts resulted in a pseudostratified columnar epithelium with visible cilia at the apical surface. An expected reduction in ALI culture thickness is observed with increasing HBEC passage number. (A) Representative H&E stained cross-sections; (B) ALI culture thickness (n = 2 replicate wells from a single experiment). Scale bar = 20 µm.

Figure 3. ALI Cultures Generated Using CELLTREATⓇ Inserts Enable Differentiation of HBECs to Cell Types Present in the Large Airway Epithelium

ALI cultures differentiated from HBECs at an early passage (P3) were fixed and stained with antibodies for ciliated cells (AC-tubulin; green), cell junctions (ZO-1; red), and goblet cells (Muc5AC; white). The nuclei were counterstained with DAPI (blue). Pseudostratified mucociliary differentiation is indicated by high expression of markers of the large airway. Scale bar = 50 µm.

Figure 4. ALI Cultures Generated Using CELLTREATⓇ Inserts Show High Expression of Large Airway Markers

ALI cultures, differentiated from HBECs at an early passage (P3), were collected 4 weeks after air-lift. Expression of large airway markers for ciliated (FOXJ1), goblet (MUC5B), and basal (P63) cells were assessed by qPCR and normalized to the housekeeping gene TBP. Error bars represent standard deviation (n = 3 replicate wells from a single experiment).

Figure 5. ALI Cultures Generated Using CELLTREATⓇ Inserts Show Optimal Barrier Function

TEER measurements of ALI cultures differentiated from HBECs at an early (P3) or late passage (P6) were taken 4 weeks after air-lift. Values were corrected against blank wells. Average values of donors were within the expected normal physiological levels (200 - 800 Ω x cm²), indicative of optimal culture differentiation. Error bars represent standard deviation (n ≥ 9 replicate wells from a single experiment).

Figure 6. ALI Cultures Generated Using CELLTREATⓇ Inserts Show Normal Electrophysiological Activity

ALI cultures differentiated from HBECs at an early passage (P3) were assessed using Ussing Chamber analysis 6 months after air-lift. Cultures generated using CELLTREATⓇ Inserts showed functional ion-transporters within the epithelial cell layer, as demonstrated by the addition of activating and inhibitory drugs. (A) Representative electrophysiological trace of two independent donors; (B) CFTR-inducible and inhibitable short-circuit current (n = 2 donors). A = Amiloride (ENaC inhibitor); I+F = IBMX and Forskolin (CFTR activators); G = Genistein (CFTR potentiator); C = CFTRinh-172 (CFTR inhibitor); U = UTP (calcium-activated chloride channels (CaCCs) activator).

Figure 7. Differentiated Intestinal Organoid-Derived Monolayers or Caco-2 Cells Reach Full Confluence When Using CELLTREATⓇ Inserts

Human intestinal organoids, previously maintained in IntestiCult™ Organoid Growth Medium (Human) (Catalog #06010), were dissociated, replated onto CELLTREATⓇ Inserts, and differentiated using IntestiCult™ Organoid Differentiation Medium (Human) (Catalog #100-0214). The Caco-2 epithelial cell line from a colorectal adenocarcinoma was used as a control. Between 3 to 7 days of culture, monolayers from two different ileal donors and Caco-2 cells reached full confluence. Scale bar = 250 µm.

Figure 8. Differentiated Intestinal Organoid Derived Monolayers or Caco-2 Cells Generate Using CELLTREATⓇ Inserts Demonstrate Optimal Barrier Function

TEER measurements of human intestinal organoid monolayers or the epithelial cell line, Caco-2, were taken 7 days after plating. Cultures generated using CELLTREATⓇ Inserts demonstrate values within the expected range of over 200 Ω x cm², indicative of optimal barrier function (n = 2 replicate wells from a single experiment).

Figure 9. Differentiated Intestinal Organoid Derived Monolayers or Caco-2 Cells Generated Using CELLTREATⓇ Inserts Show Low Permeability to FITC-Dextran

The permeability of human intestinal organoid monolayers or the epithelial cell line, Caco-2, were measured 7 days after plating. FITC-dextran was added apically and sampled from the basolateral chamber after 1, 3, and 24 h. All cultures generated using CELLTREATⓇ Inserts demonstrate low permeability with the steady diffusion of FITC-dextran observed over time, indicative of a confluent monolayer (n = 2 replicate wells from a single experiment).