FAQs on Human Airway Organoid Cultures

Find answers to frequently asked questions (FAQs) about culturing human airway organoids using PneumaCult™ Airway Organoid Kit.

General Information

Which PneumaCult™ media should I use for generating primary lung organoids?

The PneumaCult™ product line consists of robust cell culture media for culturing human airway and alveolar epithelial cells. For establishment and differentiation of airway organoids derived from human bronchial epithelial cells (HBECs), you can use PneumaCult™ Airway Organoid Kit. When fully differentiated, these organoids exhibit a centralized lumen surrounded by a polarized airway epithelial cell layer composed of differentiated cell types. To generate reproducible apical-out airway organoids from HBECs, use PneumaCult™ Apical-Out Airway Organoid Medium. With a Matrigel®-free protocol, this cell culture medium enables easy downstream processing.

If you are looking to model the human alveolar region, you can use PneumaCult™ Alveolar Organoid Media. With these expansion and differentiation media, you can efficiently passage and expand human alveolar epithelial type II (ATII) cells as organoids, and then further differentiate the ATII organoids to alveolar epithelial type I (ATI) organoids.

Prior to initiating airway organoid cultures using PneumaCult™ Airway Organoid Kit or PneumaCult™ Apical-Out Airway Organoid Medium, you can expand HBECs in 2D adherent cultures using the serum- and BPE-free PneumaCult™-Ex Plus Medium. To learn more about all the available PneumaCult™ cell culture media, visit our PneumaCult™ page or browse our brochure.

Still unsure which cell culture media to use? Try our product finder to find the right media for your pulmonary research >

What is the difference between PneumaCult™-ALI Medium and PneumaCult™ Airway Organoid Kit?

PneumaCult™-ALI Medium enables the culture of HBECs at the air-liquid interface (ALI) on Transwell® inserts, providing easy access to the apical and basolateral sides for downstream applications. In contrast, PneumaCult™ Airway Organoid Kit is designed for efficient establishment and differentiation of airway organoids derived from HBECs. In both culture systems, HBECs undergo extensive mucociliary differentiation to form a pseudostratified epithelium that exhibits morphological and functional characteristics similar to those of the human airway in vivo. When fully differentiated, the airway organoids exhibit a centralized lumen surrounded by a polarized airway epithelial cell layer composed of differentiated cell types, such as ciliated cells and goblet cells.

Can PneumaCult™ Airway Organoid Kit generate apical-out organoids?

PneumaCult™ Airway Organoid Kit was not formulated for generating apical-out organoids. A key factor driving apical-out or apical-in organoids is the presence of a matrix such as Matrigel®. To generate apical-out airway organoids from HBECs using a Matrigel®-free protocol, use PneumaCult™ Apical-Out Airway Organoid Medium.

Materials and Plate Preparation

Can airway organoids generated using PneumaCult™ Airway Organoid Kit be scaled up into 96-well plates for high-throughput applications?

PneumaCult™ Airway Organoid Kit has been developed using 24-well flat-bottom plates. A high-throughput workflow using 96-well plates is currently being developed by our Research and Development team.

Which cultureware can be used to initiate airway organoids using PneumaCult™ Airway Organoid Kit?

PneumaCult™ Airway Organoid Kit was optimized using tissue culture-treated 24-well flat-bottom plates which can be used to initiate airway organoids in the 3D expansion phase.

Which type of Matrigel® can be used to initiate airway organoids using PneumaCult™ Airway Organoid Kit?

PneumaCult™ Airway Organoid Kit has been optimized using Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-free, LDEV-free (Corning 356231).

Sample Preparation

Which primary airway epithelial cell vendors have been tested for establishing airway organoids using PneumaCult™ Airway Organoid Kit?

There are many commercially available sources for primary airway epithelial cells. We have tested human airway epithelial cells from Epithelix, which have resulted in the successful differentiation of organoids.

Does PneumaCult™ Airway Organoid Kit support the culture of rat or mouse airway epithelial cells?

Rat tracheal epithelial cells have been tested in-house and are shown to be compatible with PneumaCult™ Airway Organoid Kit. We have not tested the compatibility of mouse airway epithelial cells in-house.

Does PneumaCult™ Airway Organoid Kit support the culture and differentiation of distal alveolar cells (i.e. ATII cells)?

PneumaCult™ Airway Organoid Kit is not optimized for the differentiation of distal airway cells. We offer the PneumaCult™ Alveolar Organoid Media for passaging and long-term expansion of human alveolar epithelial type II (ATII) cells as organoids, and for further differentiation of these ATII cells to alveolar epithelial type I (ATI) organoids for downstream applications.

Does PneumaCult™ Airway Organoid Kit support the culture and differentiation of small airway cells?

We have not tested small airway epithelial cells with PneumaCult™ Airway Organoid Kit in-house; however, this can be trialed. Because PneumaCult™ Airway Organoid Kit is optimized for the differentiation of large airway cells, small airway organoids generated using PneumaCult™ Airway Organoid Kit might shift toward a large airway epithelial cell phenotype. For the culture of human small airway epithelial cells at the ALI, we offer the PneumaCult™-ALI-S Medium.

Does PneumaCult™ Airway Organoid Kit support the culture of HBECs derived from cystic fibrosis donors?

Commercially available HBECs from cystic fibrosis (CF) donors are compatible with PneumaCult™ Airway Organoid Kit. These organoids contain lumina with robust cilia beating and display similar morphology to normal donors in both the seeding and differentiation phases.

See Figure 3 and 4 in the PneumaCult™ Airway Organoid Kit product page to view data showing airway organoids derived from both healthy and CF donors.

Setup & Culture

When should I passage my 2D HBEC expansion cultures in PneumaCult™-Ex Plus Medium prior to initiating the airway organoid cultures using PneumaCult™ Airway Organoid Kit?

HBECs cultured in PneumaCult™-Ex Plus Medium should be passaged once they reach 50 to 60% confluence. For a visualization of the cellular morphology of HBEC cultures at this recommended confluence, see our protocol video on How to Model the Human Airway at the Air-Liquid Interface: Expansion of HBECs (from 02:45 min).

During the 2D expansion phase in PneumaCult™-Ex Plus Medium, at which passage should HBECs be used to initiate airway organoids using the PneumaCult™ Airway Organoid Kit?

Airway organoid cultures initiated using early-passage (2 or 3) HBECs expanded in PneumaCult™-Ex Plus Medium display the most optimal morphology. Although some donor variability is to be expected, using HBECs at a slightly later passage (4 or 5) can still generate differentiated airway organoids.

How do I initiate cultures using PneumaCult™ Airway Organoid Kit?

Here are the general steps for initiating organoid cultures using PneumaCult™ Airway Organoid Kit:

  • Passage HBECs cultured in PneumaCult™-Ex Plus Medium once they reach 50 to 60% confluence.
  • Using 24-well non-tissue culture-treated plates, seed 2 x 103 HBECs (based on passage 3 cultures) into 50 µL domes composed of 90% Matrigel® and 10% PneumaCult™ Airway Organoid Seeding Medium mix.
  • After the domes have solidified, add 500 to 750 µL of PneumaCult™ Airway Organoid Seeding Medium to each well containing a dome. Perform medium changes every 2 days for 4 to 7 days. The seeding phase may take longer for some donor cell populations.
  • For differentiation of the organoids generated during the seeding phase, add 750 to 1000 µL of PneumaCult™ Airway Organoid Differentiation Medium to each well. Perform media changes every 2 days for 21 to 28 days.

For the detailed protocol, please refer to the Product Information Sheet.

How can the HBEC seeding density be optimized in PneumaCult™ Airway Organoid Seeding Medium?

The HBEC seeding density in PneumaCult™ Airway Organoid Seeding Medium must be optimized based on the passage number for expansion in PneumaCult™ Ex Plus Medium. A higher HBEC seeding density with higher passage numbers often improves airway organoid differentiation. Optimize the seeding density by adding 2 x 103 HBECs per 50 µL Matrigel® dome based on expansion cultures at passage 3. We suggest adding 4000 cells per dome at passage 4 and 6000 cells per dome at passage 5. Under-seeding the cells may hinder organoid formation and over-seeding the cells may result in overly crowded domes.

How long can HBEC organoids remain in the seeding phase when cultured in PneumaCult™ Airway Organoid Seeding Medium?

HBECs are seeded into 50 µL domes and cultured for 4 to 7 days in PneumaCult™ Airway Organoid Seeding Medium, which allows for the initiation of 3D organoids. This time in culture can be optimized depending on the user requirements and cell source.

Are HBEC-derived organoids maintained in the PneumaCult™ Airway Organoid Seeding Medium differentiated?

HBEC-derived organoids maintained in the PneumaCult™ Airway Organoid Seeding Medium are composed of basal cells. They do not contain a lumen, nor appear to be polarized. The HBEC-derived organoids will differentiate only when cultured in the PneumaCult™ Airway Organoid Differentiation Medium. After 21 to 28 days, the differentiated organoids are circular and exhibit a centralized lumen surrounded by a polarized airway epithelial cell layer composed of differentiated cell types, including ciliated cells and goblet cells.

How long does it take for airway organoids generated using PneumaCult™ Airway Organoid Kit to become fully differentiated?

After HBEC organoids have been generated in PneumaCult™ Airway Organoid Seeding Medium for 4 to 7 days, the medium is switched to PneumaCult™ Airway Organoid Differentiation Medium for an additional 21 to 28 days. The total time in culture is donor- and passage- dependent and may span from 25 to 35 days.

Using PneumaCult™ Airway Organoid Kit, how many organoids can be generated, per 50 µL dome, after the seeding and the differentiation phases?

After the 3D HBEC organoid seeding phase in PneumaCult™ Airway Organoid Seeding Medium, one can expect between 400 - 600 organoids by days 4 to 7. When the 3D HBEC organoids are further differentiated using PneumaCult™ Airway Organoid Differentiation Medium, the expected number of fully differentiated organoids will decrease and vary between cultures depending on the donor and passage number.

When culturing organoids from the seeding phase in PneumaCult™ Airway Organoid Differentiation Medium, when do signs of differentiation first appear?

Signs of differentiation will become visible after approximately 14 days of culture in PneumaCult™ Airway Organoid Differentiation Medium, including the presence of a central lumen and differentiated cell types, notably ciliated cells and goblet cells. The PneumaCult™ airway organoids are fully differentiated after 21 - 28 days, at which point characterization assays may be performed.

See Figure 2 in the PneumaCult™ Airway Organoid Kit product page for bright-field and immunostained images of differentiated airway organoids.

What size are organoids when differentiated in PneumaCult™ Airway Organoid Differentiation Medium?

The size of the organoids differentiated in PneumaCult™ Airway Organoid Differentiation Medium are roughly 100 µm.

What is the distribution of airway epithelial cell types found in organoids differentiated using the PneumaCult™ Airway Organoid Kit?

Organoids generated using PneumaCult™ Airway Organoid Kit possess a central lumen and a polarized epithelium containing differentiated cell types, notably ciliated cells and goblet cells. Using immunocytochemistry, we have confirmed the presence of the goblet cell marker Muc5AC and the basal cell markers CD49f and CD271. The ciliated cell percentage was found to be approximately 40 - 50%.

Cryopreservation and Passaging

Can cryopreserved HBECs be washed and placed directly into Matrigel® domes in PneumaCult™ Airway Organoid Seeding Medium?

Cryopreserved HBECs should be passaged at least once in PneumaCult™ Ex Plus Medium prior to seeding into domes for the organoid seeding phase. HBECs used directly from cryopreservation may not perform as well.

Characterization

Are organoids generated using PneumaCult™ Airway Organoid Kit sensitive to the Forskolin Swelling Assay?

Airway organoids generated using PneumaCult™ can be treated with 20 μM amiloride, 10 μM forskolin, and 25 μM genistein for 6 hours to bring about forskolin-induced swelling. Forskolin-induced swelling is lost in PneumaCult™ airway organoids derived from CF donors but re-established in VX-809-treated airway organoids.

See Figure 3 in the PneumaCult™ Airway Organoid Kit product page to view the results from forskolin treatment of airway organoids derived from healthy and CF donors.

Can organoids generated using PneumaCult™ Airway Organoid Kit be dissociated into single cells for characterization and downstream applications?

PneumaCult™ airway organoids can be retrieved from the Matrigel® dome using Corning® Cell Recovery Solution and further dissociated into single cells using ACCUTASE™.

Can ciliated cells be quantified from organoids generated using PneumaCult™ Airway Organoid Kit?

To quantify the number of ciliated cells, PneumaCult™ airway organoids can be collected in cold Corning® Cell Recovery Solution followed by an enzymatic dissociation in ACCUTASE™ into a single-cell suspension. The total number of cells and the number of ciliated cells can be counted using a hemocytometer.

View Figure 4 in the PneumaCult™ Airway Organoid Kit product page to see the percentage of ciliated cells in airway organoids grown using PneumaCult™ Airway Organoid Kit.

Is there a suggested protocol for performing immunocytochemical staining on differentiated lung airway organoids?

Read our protocol on Performing Immunocytochemical Staining of Epithelial Organoids to learn about whole-mount fixation and immunostaining of epithelial organoids derived from different tissue types, including lungs. Suggested antibodies include ZO-1 (junction protein marker), MUC5AC (goblet cell marker), AC-Tubulin (ciliated cell marker), and DAPI (nuclear stain).

How can I characterize HBEC organoids generated after the seeding/expansion phase in PneumaCult™ Airway Organoid Seeding Medium?

For characterizing the HBEC organoids after the seeding phase, antibodies against basal cell markers such as p63, CD49f, and CD271 can be used.

How long can airway organoids be cultured in PneumaCult™ Airway Organoid Differentiation Medium following the 21 to 28 day differentiation protocol for downstream applications?

After day 28 of the differentiation protocol, the Matrigel® dome may start to lose its integrity, and the lumen may overfill with mucus. In-house, we have kept differentiated airway organoids in PneumaCult™ Airway Organoid Differentiation Medium for approximately 6 weeks.

Can't find the answer you are looking for? Reach out to us and one of our pulmonary specialists will get back to you.


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