PneumaCult™-ALI Medium

Serum- and BPE-free medium for human airway epithelial cells cultured at the air-liquid interface or as airway organoids

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PneumaCult™-ALI Medium with 12 mm Transwell® Inserts

Medium and Transwell® inserts for human airway epithelial cells cultured at the air-liquid interface

1 Kit
Catalog #05021
480 USD

PneumaCult™-ALI Medium with 6.5 mm Transwell® Inserts

Medium and Transwell® inserts for human airway epithelial cells cultured at the air-liquid interface

1 Kit
Catalog #05022
480 USD

PneumaCult™-ALI Medium

Serum- and BPE-free medium for human airway epithelial cells cultured at the air-liquid interface or as airway organoids

1 Kit
Catalog #05001
203 USD

Required Products


PneumaCult™-ALI Medium (Catalog #05001) is a serum- and BPE-free medium for the culture of human airway epithelial cells at the air-liquid interface (ALI). Airway epithelial cells cultured in PneumaCult™-ALI Medium undergo extensive mucociliary differentiation to form a pseudostratified epithelium that exhibits morphological and functional characteristics similar to those of the human airway in vivo. PneumaCult™-ALI Medium is also available in a kit that includes 12 mm Transwell® inserts (Catalog #05021) or 6.5 mm Transwell® inserts (Catalog #05022).

Additionally, PneumaCult™-ALI Medium supports the generation of differentiated airway organoids in a 3D culture system. For a detailed protocol, refer to the Technical Bulletin: A Sphere Culture Method for Mucociliary Differentiation of Primary Human Bronchial Epithelial Cells (Document #28216), available at or contact us to request a copy.

Together, PneumaCult™-ALI Medium and PneumaCult™-Ex Plus Medium (Catalog #05040) constitute a fully integrated BPE-free culture system for in vitro human airway modeling. This robust and defined system is a valuable tool for basic respiratory research, toxicity studies, and drug development.
• HBECs cultured with PneumaCult™-ALI undergo extensive mucociliary differentiation to form a pseudostratified epithelium that closely resembles the human airway
• PneumaCult™-ALI is serum-free and BPE-free to minimize variability
  • PneumaCult™-ALI Basal Medium, 450 mL
  • PneumaCult™-ALI 10X Supplement, 50 mL
  • PneumaCult™-ALI Maintenance Supplement (100X), 5 x 1 mL
Specialized Media
Cell Type:
Airway Cells
Cell Culture; Differentiation; Maintenance; Organoid Culture; Spheroid Culture
Area of Interest:
Disease Modeling; Drug Discovery and Toxicity Testing; Epithelial Cell Biology

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Figure 1. Overview of the PneumaCult™ Culture System

Expansion of human bronchial epithelial cells (HBECs) in submerged culture is performed with PneumaCult™-Ex Plus or PneumaCult™-Ex. During the early Expansion Phase of the ALI culture procedure, PneumaCult™-Ex Plus or PneumaCult™-Ex is applied to the apical and basal chambers. Upon reaching confluence, the culture is air-lifted by removing the culture medium from both chambers, and adding PneumaCult™-ALI to the basal chamber only. Differentiation into a pseudostratified mucociliary epithelium is obtained following 21-28 days of incubation and can be maintained for more than one year.

Figure 2. HBECs Cultured in PneumaCult™-Ex Successfully Differentiate into a Pseudostratified Mucociliary Epithelium with PneumaCult™-ALI

Early-passage (P1-3) HBECs cultured in PneumaCult™-Ex successfully differentiate when cultured at air-liquid interface with PneumaCult™-ALI for 28 days. H&E staining revealed the pseudostratifi ed structure of the epithelium with cilia present at the apical surface (A). Periodic acid-Schiff staining demonstrated the presence of goblet cells (B). The presence of ciliated and goblet cells was also demonstrated by immunofl uorescence staining of cilia marker acetylated (AC)-Tubulin (green; C) and the goblet cell marker Mucin5AC (green; D). Appropriate positioning of basal cells along the transwell insert was visualized by immunofl uorescence staining using the basal cell markers p75NTR (green) and p63 (red; E,F). A representative merged image indicates the apical cells, detected by DAPI alone, positioned along the epithelium and in close contact with the basal cells (detected by DAPI, p63 and p75NTR co-labeling) located along the insert (G).

Figure 3. Electrophysiological characterization of differentiated HBECs (P4) that were expanded in PneumaCult™-Ex Plus, PneumaCult™-Ex, and Bronchial Epithelial Growth Media

TEER (A) and representative characterization of the ion channel activities (B) for ALI cultures at 28 days post air-lift using HBECs expanded in PneumaCult™-Ex Plus, PneumaCult™-Ex, or Bronchial Epithelial Growth Media. Amiloride: ENaC inhibitor. IBMX and Forskolin: CFTR activators. Genistein: CFTR potentiator. CFTRinh-172: CFTR inhibitor. UTP: Calciumactivated Chloride channels (CaCCs) activator. All ALI differentiation cultures were performed using PneumaCult™-ALI.

Figure 4. Schematic of Sphere Culture Method Optimized for PneumaCult™-ALI Medium

Figure 5. Morphology of Bronchospheres Generated in PneumaCult™-ALI Medium

At Day 2, bronchospheres were small in size. By Day 17 the bronchospheres were larger in size with approximately 70% containing a visible lumen. By Day 28 almost all bronchospheres contained a lumen.


Cell stem cell 2018 MAY

Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium.

T. J. Lynch et al.


The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.
Cell stem cell 2018 MAY

Myoepithelial Cells of Submucosal Glands Can Function as Reserve Stem Cells to Regenerate Airways after Injury.

A. Tata et al.


Cells demonstrate plasticity following injury, but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing, we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically, SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly, we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways, they may serve as a reserve cell source for enhancing human airway regeneration.
Cellular microbiology 2018 MAR

Modelling persistent Mycoplasma pneumoniae infection of human airway epithelium.

Prince OA et al.


Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long-term carriage and extrapulmonary spread. The mechanisms of persistence and migration beyond the conducting airways, however, remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air-liquid interface culture, showing that M. pneumoniae gliding motility is essential for initial colonisation and subsequent spread, including localisation to epithelial cell junctions. We extended those observations here, characterizing M. pneumoniae infection of NHBE for up to 4 weeks. Colonisation of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation, barrier function remained largely intact. Desquamation was accompanied by epithelial remodelling that included cytoskeletal reorganisation and development of deep furrows in the epithelium. Finally, M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology, consistent with contrasting virulence in experimentally infected mice. In summary, this study reports pericellular invasion, NHBE cytoskeletal reorganisation, and tissue remodelling with persistent infection in a human airway epithelium model, providing clear insight into the likely route for extrapulmonary spread.
Human gene therapy 2018 JUN

Transducing Airway Basal Cells with a Helper-Dependent Adenoviral Vector for Lung Gene Therapy.

H. Cao et al.


A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases.
International journal of cancer 2018 JUL

Conditionally reprogrammed cells (CRC) methodology does not allow the in vitro expansion of patient-derived primary and metastatic lung cancer cells.

G. Sette et al.


Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently, conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However, the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here, we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay, monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly, pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast, brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion, patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions, limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus, CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells, as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells.