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How to Generate Apical-Out Nasal Organoids

4 Parts 11 Materials Print

Apical-out nasal organoids provide a physiologically relevant, accessible model for studying the human nasal epithelium, particularly for applications such as drug screening, infectious disease modeling, and mucociliary function assays. Unlike traditional apical-in organoids, where the apical surface faces inward and is less accessible for experimental manipulation, apical-out organoids expose the apical membrane externally, enabling direct interaction with environmental stimuli, pathogens, or therapeutic compounds.

This model can be further optimized by inducing the differentiation of goblet cells through defined supplementation, allowing researchers to better replicate the cellular diversity of the native nasal epithelium. This protocol is based on the manuscript by , outlining a streamlined method (Figure 1) for generating assay-ready apical-out nasal organoids from primary human nasal epithelial cells (HNECs), and is designed to support consistent differentiation and robust epithelial polarization.

If you need support with this protocol, connect with our pulmonary specialists for additional guidance or troubleshooting.

Overview of the PneumaCult鈩 Apical-Out Nasal Organoid Workflow

Figure 1. Overview of the PneumaCult鈩 Apical-Out Nasal Organoid Workflow

Prior to generating apical-out nasal organoids, human nasal epithelial cells (HNECs) are expanded using PneumaCult鈩-NGEx (Catalog #100-1505) or PneumaCult鈩-Ex Plus (Catalog #05040) Medium in a two-dimensional expansion phase at 37掳C. The cells are then plated into an AggreWell鈩400 24-well plate (Catalog #34450) and allowed to aggregate and differentiate using PneumaCult鈩 Apical-Out Airway Organoid Medium (Catalog #100-0620) or PneumaCult鈩 Apical-Out Airway Organoid Secretory Medium (Catalog #100-2078) at 32.5掳C.

Materials

Protocol

Preparation of Reagents and Materials

Use sterile technique when preparing the following materials and media. If preparing volumes other than the indicated examples, adjust accordingly.

Complete PneumaCult鈩 Apical-Out Airway Organoid Medium (AOAOM)

The following example is for preparing 10 mL of complete PneumaCult鈩 AOAOM (PneumaCult鈩 Apical-Out Airway Organoid Basal Medium + PneumaCult鈩 Apical-Out Airway Organoid 10X Supplement + PneumaCult鈩 Apical-Out Airway Organoid 1000X Supplement + Heparin Solution + Hydrocortisone Stock Solution). If preparing other volumes, adjust accordingly.

  1. Thaw PneumaCult鈩 Apical-Out Airway Organoid 10X Supplement at 2 - 8掳C overnight. Mix the 10X Supplement gently by inverting the vial; do not vortex. Thaw PneumaCult鈩 Airway Organoid 1000X Supplement at room temperature (15 - 25掳C). Mix the 1000X Supplement thoroughly; do not vortex.
    NOTE: Once thawed, use immediately or aliquot and store supplements at 20掳C. Do not exceed the shelf life of the supplements. After thawing the aliquoted supplements, use immediately. Do not re-freeze.
  2. Combine the following:
    • 8.92 mL PneumaCult鈩 Apical-Out Airway Organoid Basal Medium
    • 1 mL PneumaCult鈩 Apical-Out Airway Organoid 10X Supplement
    • 10 渭L PneumaCult鈩 Apical-Out Airway Organoid 1000X Supplement
    • 20 渭L Heparin Solution
    • 50 渭L Hydrocortisone Stock Solution
    Mix thoroughly; do not vortex.
    NOTE: If not used immediately, store complete PneumaCult鈩 AOAOM at 2 - 8掳C for up to 2 weeks. Do not freeze.

Complete PneumaCult鈩 Apical-Out Airway Organoid Secretory Medium (AOAOSM)

The following example is for preparing 10 mL of complete PneumaCult鈩 AOAOSM (PneumaCult鈩 Apical-Out Airway Organoid Basal Medium + PneumaCult鈩 Apical-Out Airway Organoid 10X Supplement + PneumaCult鈩 Apical-Out Airway Organoid Secretory Supplement + Heparin Solution + Hydrocortisone Stock Solution). If preparing other volumes, adjust accordingly.

  1. Thaw PneumaCult鈩 Apical-Out Airway Organoid 10X Supplement at 2 - 8掳C overnight. Mix the 10X Supplement gently by inverting the vial; do not vortex. Thaw PneumaCult鈩 Apical-Out Airway Organoid Secretory Supplement at room temperature (15 - 25掳C). Mix the Secretory Supplement thoroughly; do not vortex.
    NOTE: Once thawed, use immediately or aliquot and store supplements at 20掳C. Do not exceed the shelf life of the supplements. After thawing the aliquoted supplements, use immediately. Do not re-freeze.
  2. Combine the following:
    • 8.83 mL PneumaCult鈩 Apical-Out Airway Organoid Basal Medium
    • 1 mL PneumaCult鈩 Apical-Out Airway Organoid 10X Supplement
    • 100 渭L PneumaCult鈩 Apical-Out Airway Organoid Secretory Supplement
    • 20 渭L Heparin Solution
    • 50 渭L Hydrocortisone Stock Solution
    Mix thoroughly; do not vortex.
    NOTE: If not used immediately, store complete PneumaCult鈩 AOAOSM at 2 - 8掳C for up to 2 weeks. Do not freeze.

Pre-Treating Plates

Using sterile technique, pre-treat the AggreWell鈩 24-well plate (for use in the Aggregation of Human Primary Nasal Epithelial Cells section of this protocol) and the tissue culture-treated 24-well flat-bottom plate (for use in the Differentiation to Apical-Out Nasal Organoids section of this protocol) as follows:

NOTE: Pre-treating plates with Anti-Adherence Rinsing Solution prevents cell adhesion and promotes efficient aggregate formation.
  1. Open the AggreWell鈩400 24-well plate and the 24-well tissue culture-treated flat-bottom plate in a biosafety cabinet.
    NOTE: Do not expose the plates to organic solvents, including ethanol and isopropanol.
  2. Pre-treat wells with Anti-Adherence Rinsing Solution as described below:
    1. Add 500 渭L Anti-Adherence Rinsing Solution to each well to be used.
    2. Swirl the cultureware to spread the solution evenly across the surface and the walls of the wells.
    3. Centrifuge the plate at 1300 x g for 10 minutes.
      NOTE: Plates must be well-balanced. Prepare balance plates using standard plates filled with water to match the weight and position of the 24-well plates.
    4. Remove Anti-Adherence Rinsing Solution from each well using an aspirator or a 1 mL pipettor.
  3. Rinse each well with 1 mL room temperature (15 - 25掳C) DMEM/F-12 with 15 mM HEPES.
  4. Fill each washed well with 0.5 mL of DMEM/F-12 with 15 mM HEPES and store the plate at 37掳C and 5% CO2. Treated plates can be stored for at least 5 days, but ideally should be used on the same day. Remove DMEM/F-12 immediately before use. Do not let the plate dry before use.

Aggregation of Human Primary Nasal Epithelial Cells

The following example is for passaging HNECs from a T-25 cm2 flask and plating them into a single well of an AggreWell鈩400 24-well plate for aggregation. If using more wells, adjust volumes accordingly.

  1. Warm sufficient volumes of D-PBS (Without Ca++ and Mg++), complete PneumaCult鈩 AOAOM (see Preparation of Reagents and Materials section), and the Animal Component-Free (ACF) Enzymatic Dissociation Solution and ACF Enzyme Inhibition Solution from the ACF Cell Dissociation Kit to room temperature (15 - 25掳C).
  2. Pre-treat an AggreWell鈩400 24-well plate with Anti-Adherence Rinsing Solution (see Pre-Treating Plates section).
  3. Wash human primary HNECs in a T-25 cm2 flask with 5 mL D-PBS (Without Ca++ and Mg++). Remove D-PBS.
  4. Add 2.5 mL ACF Enzymatic Dissociation Solution and incubate at 37掳C for 6 - 8 minutes, until cells can be dislodged with gentle tapping of the flask.
  5. Add 2.5 mL ACF Enzyme Inhibition Solution and transfer cells to a 15 mL conical tube.
  6. Centrifuge cell suspension at 350 x g for 5 minutes.
  7. Discard the supernatant and resuspend the cell pellet in 1 - 2 mL complete PneumaCult鈩 AOAOM.
    NOTE: HNECs suspended in complete PneumaCult鈩 AOAOM can be used for expansion in tissue culture flasks with PneumaCult鈩-Ex Plus Medium (Catalog #05040) or PneumaCult鈩-NGEx Medium (Catalog #100-1505) by centrifuging to remove PneumaCult鈩 AOAOM and resuspending the cells in the appropriate expansion medium.
  8. Perform a viable cell count using Trypan Blue and a hemocytometer.
  9. Add complete PneumaCult鈩 AOAOM to the AggreWell鈩400 well (prepared in step 3) to reach a total volume of 1 mL, and then transfer 360,000 cells to the well. Mix the suspension thoroughly.
  10. Centrifuge the AggreWell鈩400 plate at 100 x g for 3 minutes.
  11. Incubate at 32.5掳C and 5% CO2 to aggregate cells. If using cells expanded in PneumaCult鈩-NGEx Medium, proceed to Using HNECs Expanded in PneumaCult鈩-NGEx Medium section. If using cells expanded in PneumaCult鈩-Ex Plus Medium, proceed to Using HNECs Expanded in PneumaCult鈩-Ex Plus Medium section.
    NOTE: When aggregating and differentiating HNECs at 37掳C, extensive cell shedding is observed, which impacts the organoid generation efficiency.
Morphology of Apical-Out Nasal Organoids

Figure 2. Morphology of Apical-Out Nasal Organoids

Human nasal epithelial cells (HNECs) were expanded in PneumaCultTM-NGEx (Catalog #100-1505) or PneumaCultTM-Ex Plus (Catalog #05040) and aggregated in AggreWellTM 400 plates (Catalog #34450) using PneumaCult鈩 Apical-Out Airway Organoid Medium (AOAOM; Catalog #100-0620) or PneumaCult鈩 Apical-Out Airway Organoid Secretory Medium (AOAOSM; Catalog #100-2078). (A, B) Representative images of nasal epithelial cell aggregates at different magnifications following suspension from AggreWellTM after 4 days of incubation. Aggregates are defined by a cell-dense core that occasionally protrudes from the outer apical surface. (C, D) At the end of differentiation on Day 28 using PneumaCult鈩 AOAOM, apical-out nasal organoids have acquired the desired morphology, characterised by dense structures with clearly defined outer surface and minimal protrusions. (D) Beating cilia can be observed in the outer apical surface.


Differentiation to Apical-Out Nasal Organoids

Using HNECs Expanded in PneumaCult鈩-NGEx Medium

  1. Perform a partial medium change every 2 days at 32.5掳C using PneumaCult鈩 AOAOM by removing 0.5 mL of medium along the wall of the well.
  2. Slowly add 0.5 mL complete PneumaCult鈩 AOAOM, taking care not to suspend the aggregates. By Day 28, apical-out nasal organoids should display beating cilia, at which point, characterization assays can be performed.

Inducing Differentiation of Goblet Cells

PneumaCult鈩 Apical-Out Airway Organoid Secretory Medium (AOAOSM; Catalog #100-2078) and PneumaCult鈩 Apical-Out Airway Organoid Secretory Supplement (Catalog #100-1780) can be used with cells to further drive differentiation of goblet cells. Use of cells from early passages (up to passage 4) is recommended.

  1. On Day 6, remove 0.8 mL of medium along the wall of the well.
  2. Slowly add 1 mL of complete PneumaCult鈩 AOAOS Medium 2, taking care not to suspend the aggregates.
  3. Perform a partial medium change every 2 days using complete PneumaCult鈩 AOAOS Medium 2 for the remainder of the workflow at 32.5掳C.
    NOTE: If the assay includes washing the organoids, perform all washing steps in DMEM/F12 warmed to room temperature. Washing Apical-Out Nasal Organoids with D-PBS might have a detrimental effect on the structure of the organoids.

Inducing Differentiation of Goblet Cells

PneumaCult鈩 Apical-Out Airway Organoid Secretory Medium (AOAOSM; Catalog #100-2078) and PneumaCult鈩 Apical-Out Airway Organoid Secretory Supplement (Catalog #100-1780) can be used with cells to further drive differentiation of goblet cells. Use of cells from early passages (up to passage 4) is recommended.

  1. On Day 6, remove 0.8 mL of medium along the wall of the well.
  2. Slowly add 1 mL of complete PneumaCult鈩 AOAOS Medium 2, taking care not to suspend the aggregates.
  3. Perform a partial medium change every 2 days using complete PneumaCult鈩 AOAOS Medium 2 for the remainder of the workflow at 32.5掳C.
    NOTE: If the assay includes washing the organoids, perform all washing steps in DMEM/F12 warmed to room temperature. Washing Apical-Out Nasal Organoids with D-PBS might have a detrimental effect on the structure of the organoids.

Using HNECs Expanded in PneumaCult鈩-Ex Plus Medium

  1. Incubate the plate for 24 hours - 6 days.
    NOTE: Cells from most donors require 24 hours of aggregation. For adaptation of this step to each donor, refer to the Donor Optimization section.
    NOTE: If aggregation for more than 2 days is required, perform a partial medium change every 2 days using PneumaCult鈩 AOAOM by removing 0.5 mL of medium along the wall of the well. Slowly add 0.5 mL complete PneumaCult鈩 AOAOM, taking care not to suspend the aggregates. Continue feeds every other day.
  2. Pre-treat a tissue culture-treated 24-well flat-bottom plate with Anti-Adherence Rinsing Solution (see Pre-Treating Plates section).
  3. Add 1 mL complete PneumaCult鈩 AOAOM to each well of the AggreWell鈩 plate containing aggregates. Using a 1 mL pipettor, mix the medium to dislodge the aggregates from the microwell.
  4. Transfer 1mL of the aggregate suspension to the pre-treated 24-well flat-bottom plate (prepared in step 2). Two wells should be generated from each well of the AggreWell鈩 plate containing aggregates. Incubate at 32.5掳C and 5% CO2.
  5. Perform a partial medium change every 2 days as follows:
    1. Tilt the plate at a 25- to 30-degree angle and remove 0.5 mL of medium along the wall of the well, taking care not to remove suspended organoids.
    2. Add 0.5 mL complete PneumaCult鈩 AOAOM.
    3. Incubate at 32.5掳C and 5% CO2.
  6. By Day 28, apical-out nasal organoids should display beating cilia, at which point, downstream assays can be performed. Terminally differentiated apical-out nasal organoids are stable for at least two weeks.

Inducing Differentiation of Goblet Cells

PneumaCult鈩 Apical-Out Airway Organoid Secretory (AOAOSM; Catalog #100-2078) and PneumaCult鈩 Apical-Out Airway Organoid Secretory Supplement (Catalog #100-1780) can be used to further drive differentiation of goblet cells. Use of cells from early passages (up to passage 4) is recommended.

  1. On Day 6, remove 0.8 mL of medium along the wall of the well.
  2. Slowly add 1 mL of complete PneumaCult鈩 AOAOS Medium 2, taking care not to suspend the aggregates.
  3. Perform a partial medium change every 2 days using complete PneumaCult鈩 AOAOS Medium 2 for the remainder of the workflow.
NOTE: If the assay includes washing the organoids, perform all washing steps in DMEM/F12 warmed to room temperature. Washing apical-out nasal organoids with D-PBS might have a detrimental effect on the structure of the organoids.
Terminally Differentiated Organoids Exhibit Mature Polarized Nasal Epithelium

Figure 3. Terminally Differentiated Organoids Exhibit Mature Polarized Nasal Epithelium

(A) Day 28 apical-out nasal organoids cultured at 32.5oC in PneumaCult鈩 Apical-Out Airway Organoid Secretory Medium (AOAOM; Catalog #100-0620) contain ciliated cells, confirmed by the presence of acetylated tubulin (magenta) on the outward-facing apical cell surface, while keratin 5 (KRT5; green)-expressing basal cells were present alongside them. (B) Positive staining for MUC5AC (magenta) confirms the presence of multiple goblet cells in apical-out nasal organoids. (C) Cell-cell tight junction protein ZO-1 (red) can be readily visualized on the apical side of the organoid. Nuclei were visualized using DAPI (blue).

Gene Expression Analysis of Apical-Out Nasal Organoids

Figure 4. Gene Expression Analysis of Apical-Out Nasal Organoids

Heatmap showing fold changes in the RNA levels of common differentiation markers in apical-out nasal organoids cultured in PneumaCult鈩 Apical-Out Airway Organoid Medium (AOAOM ;Catalog #100-0620). Results were normalized against human nasal epithelial cells (HNECs) cultured in PneumaCult鈩-Ex Plus Medium and analyzed at passage 3. Ciliated cell markers, FOXJ1 and TUBB4B, are upregulated in terminally differentiated apical-out nasal organoids, and detection of MUC5AC indicates the presence of goblet cells. Data points represent the mean taken from 3 independent wells of a 24-well plate (n=3 donors).


Donor Optimization

Due to differences in aggregation time required between donors, it is recommended to first optimize the assay for each donor in order to maximize the efficiency. The following procedure is an example for optimizing the generation of apical-out nasal organoids using PneumaCult鈩 AOAOM for one new donor.

  1. Perform steps 1 - 11 in the Aggregation of Human Primary Nasal Epithelial Cells section, adding cells to at least 6 wells of an AggreWell鈩400 24-well plate in step 9.
  2. Centrifuge the AggreWell鈩400 plate at 100 x g for 3 minutes.
  3. Incubate the plate at 32.5掳C and 5% CO2 for 24 hours.
  4. Pre-treat a tissue culture-treated 24-well flat-bottom plate with Anti-Adherence Rinsing Solution (see Pre-Treating Plates section).
  5. Select one of the wells of the AggreWell鈩 plate containing cells and add 1 mL of complete PneumaCult鈩 AOAOM. Using a 1 mL pipettor, mix the medium to dislodge the aggregates from the microwell.
  6. Transfer 1 mL of the aggregate suspension to the 24-well flat-bottom plate (prepared in step 4). Two wells should be generated from each AggreWell鈩400 well.
  7. Incubate the 24-well flat-bottom plate and the AggreWell鈩400 plate at 32.5掳C and 5% CO2 for 24 hours.
  8. Observe the 24-well flat-bottom plate under the microscope for the presence of large or fused aggregates.
  9. If large aggregates have formed or fused, follow steps 5 - 8 again using another well from the AggreWell鈩400 plate.
  10. Repeat this daily transfer procedure of aggregates from AggreWell鈩400 wells to the 24-well flat-bottom plate until no fusion between aggregates is observed. The aggregation time in AggreWell鈩, where no aggregate fusion was observed in the 24 hours after suspension, should be used in future experiments using the donor tested.
    NOTE: Partial media changes should be performed every 2 days as outlined in the Differentiation to Apical-Out Nasal Organoids section, using HNECs expanded in PneumaCult鈩-Ex Plus Medium, Step 1.
Apical-Out Nasal Organoid Workflow Supports Efficient Generation and Differentiation of Organoids at Physiological Temperature

Figure 5. Apical-Out Nasal Organoid Workflow Supports Efficient Generation and Differentiation of Organoids at Physiological Temperature

Apical-out nasal organoids were generated by seeding 300 expanded passage 3 human nasal epithelial cells (HNECs) derived from 3 donors in PneumaCult鈩 Apical-Out Airway Organoid Medium (AOAOM;Catalog #100-0620). (A) Number of organoids per well of a 24-well plate at Day 28 shows improved organoid generation efficiency at 32.5oC compared to 37oC. (B) High numbers of apical-out nasal organoids can be detected at 32.5oC in successive passages at Day 28. Efficiency is expressed as the percentage of apical-out nasal organoids at the end of culture relative to the total number of aggregates used to seed the cultures. (C) Ciliated cell percentage of generated organoids at Day 28 indicates improved ciliogenesis at 32.5 oC. (D) This capacity is maintained at higher passage numbers. Data points represent independent wells of a 24-well plate.

Apical-Out Nasal Organoid Workflow Supports Standardized Generation and Differentiation of Organoids

Figure 6. Apical-Out Nasal Organoid Workflow Supports Standardized Generation and Differentiation of Organoids

(A) Aggregation using AggreWell鈩400 24-well plate and PneumaCultTM Apical-Out Airway Organoid Medium (AOAOM; Catalog #100-0620) allows for homogeneity of the culture and the standardized generation of apical-out nasal organoids that are highly similar in size and cellular composition. For each donor, at least 200 organoids generated at 32.5oC were counted from 3 separate wells at passage 3 to generate a histogram depicting the frequency of diameters in apical-out nasal organoids. The consistency of organoid diameter between donors indicates the homogeneity of the culture. (B) This homogeneity is also present in ciliated cell differentiation, with almost all (100% for Donors 1 and 2, and 97% for Donor 3) organoids displaying functional beating cilia on the outer surface, as well as goblet cell differentiation. (C, D) To measure the distribution of goblet cells, passage 3 apical-out nasal organoids were generated using PneumaCult鈩 AOAOM or Apical-Out Airway Organoid Secretory Medium (AOAOSM; Catalog #100-2078) at 32.5 oC, and following fixation, stained for MUC5AC on Day 28. The majority of organoids generated using PneumaCult鈩 AOAOM contain only 2 - 4 goblet cells, with 95.2% of the organoids having at least one goblet cell. In contrast, 100% of the organoids cultured using AOAOSM contained goblet cells, with the majority having between 5 and 8. These results demonstrate the uniformity of the resulting apical-out nasal organoids with consistent numbers of ciliated and goblet cells across various donors, and successful induction of goblet cell differentiation with PneumaCult鈩 AOAOSM. At least 170 organoids were imaged per donor (n = 3 donors) and the number of goblet cells per organoid was manually assessed.



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