Costar® 6.5 mm Transwell®, 0.4 µm Pore Polyester Membrane Inserts

Polystyrene plate with lid and polyester membrane inserts
Costar® 6.5 mm Transwell®, 0.4 µm Pore Polyester Membrane Inserts

Polystyrene plate with lid and polyester membrane inserts for cell culture that feed basolaterally

48 Inserts
Catalog # 38024
309 USD

Overview

Costar® 6.5 mm Transwell®, 0.4 µm Pore Polyester Membrane Inserts are recommended for culturing airway epithelial cells at the air-liquid interface in PneumaCult™-ALI Medium (Catalog #05001). These permeable inserts support the growth of both anchorage-dependent and anchorage-independent cells, including those that are fed at the basolateral surface or in co-culture systems.

48 Inserts; 4 x 24-well plates, each with 12 inserts
Species
Human, Mouse, Rat, Non-Human Primate, Other
Application
Cell Culture

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
Costar® 6.5 mm Transwell®, 0.4 µm Pore Polyester Membrane Inserts
Catalog #
38024
Lot #
All
Language
English

Educational Materials (3)

Brochure
Modeling Cystic Fibrosis Airway: PneumaCult™-Ex Plus and PneumaCult™-ALI
Brochure
PneumaCult™ Culture Media for Human Airway Epithelial Cells
Webinar
Studying Cystic Fibrosis Using Primary Human Nasal Epithelial Cells
51:30
Studying Cystic Fibrosis Using Primary Human Nasal Epithelial Cells

Data and Publications

Data

Figure 1. ALI Culture Morphology is Superior When Using TranswellⓇ Inserts Compared to Alternative Commercially Available Inserts

Human bronchial epithelial cells (HBECs), expanded in PneumaCult™ Ex-Plus Medium (Catalog #05040), were seeded onto inserts at an early (P3) or late passage (P6) and differentiated in PneumaCult™-ALI Medium (Catalog #05001) for 4 weeks. Greater differentiation (represented by brown areas of the culture), as well as mucus secretion and visibly beating cilia on the apical surface (not shown), were observed with cultures grown on TranswellⓇ Inserts when compared to other commercially available inserts of the same material and pore size. A lighter morphology, representative of a thinner epithelium and reduced mucus production, is expected and observed with increased number of passages. Scale bar = 500 µm.

Figure 2. ALI Cultures Form Thicker Pseudostratified Epithelium When Using TranswellⓇ Inserts Compared to Alternative Commercially Available Inserts

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 TranswellⓇ Inserts resulted in a pseudostratified columnar epithelium with visible cilia on the apical surface and this was of greater thickness when compared to alternative commercially available inserts of the same material and pore size. 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 of HBECs seeded at P3; (C) ALI culture thickness of HBECs seeded at P6 (n = 2 replicate wells from a single experiment). Scale bar = 20 µm.

Figure 3. ALI Cultures Generated Using TranswellⓇ 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 TranswellⓇ or Alternative Commercially Available Inserts Show Comparable Proportions of Ciliated Cells

ALI cultures differentiated from HBECs at an early passage (P3) were dissociated 4 weeks after air-lift. The total number of cells and the number of ciliated cells were counted using a hemocytometer. The percentage of ciliated cells within the ALI cultures were comparable when using TranswellⓇ or alternative commercially available inserts of the same material and pore size. Error bars represent standard deviation (n = 3 replicate wells from a single experiment).

Figure 5. ALI Cultures Generated Using TranswellⓇ or Alternative Commercially Available Inserts Show Comparable Expression of Large Airway Markers

ALI cultures, differentiated from HBECs at early passage (P3), were collected 4 weeks after air-lift. Expression of large airway markers for (A) ciliated (FOXJ1), (B) goblet (MUC5B), and (C) basal (P63) cells were assessed by qPCR and normalized to the housekeeping gene TBP. Expression is shown as a relative quantity to that of cultures grown on TranswellⓇ Inserts. Relative gene expression of all markers were comparable when using TranswellⓇ or alternative commercially available inserts of the same material and pore size. Error bars represent standard deviation (n = 2-3 replicate wells from a single experiment).

Figure 6. ALI Cultures Show Optimal and Comparable Barrier Function When Generated Using TranswellⓇ or Alternative Commercially Available Inserts

TEER measurements of ALI cultures differentiated from HBECs at an (A) early passage (P3) or (B) late passage (P6) were taken 4 weeks after air-lift. Values were corrected against blank wells. Average values of both donors were within the expected normal physiological levels (200 - 800 Ω x cm2) indicative of optimal culture differentiation in cultures generated using TranswellⓇ or alternative commercially available inserts of the same material and pore size. Error bars represent standard deviation (n ≥ 6 replicate wells from a single experiment).

Figure 7. ALI Cultures Generated Using TranswellⓇ 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 TranswellⓇ Inserts showed functional ion-transporters within the epithelial cell layer, as demonstrated by the addition of activating and inhibitory drugs. (A) Representative electrophysiological trace (n = 1 per donor); (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 8. Differentiated Intestinal Organoid-Derived Monolayers or Caco-2 Cells Reach Full Confluence When Using TranswellⓇ Inserts

Human intestinal organoids, previously maintained in IntestiCult™ Organoid Growth Medium (Human) (Catalog #06010), were dissociated, replated onto a TranswellⓇ 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 9. Barrier Function of Differentiated Intestinal Organoid-Derived Monolayers or Caco-2 Cells Using TranswellⓇ or Alternative Commercially Available Inserts

TEER measurements of human intestinal organoid monolayers or the epithelial cell line, Caco-2, were taken 7 days after plating. Intestinal organoid cultures demonstrate values within the expected range, indicative of optimal barrier function when using TranswellⓇ Inserts but not alternative inserts of the same material and pore size (n = 2 replicate wells from a single experiment).

Figure 10. FITC Dextran Permeability of Differentiated Intestinal Organoid-Derived Monolayers or Caco-2 Cells Using TranswellⓇ or Alternative Commercially Available Inserts

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. Intestinal organoid cultures demonstrate low permeability with the steady diffusion of FITC-dextran observed over time, indicative of a confluent monolayer when using TranswellⓇ Inserts but not alternative inserts of the same material and pore size (n = 2 replicate wells from a single experiment).

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