PneumaCult™ Alveolar Organoid Media

Cell culture media for expansion and differentiation of human alveolar organoids
Catalog #
100-0847_C
Cell culture media for expansion and differentiation of human alveolar organoids
From: 195 USD
Required Products

Overview

Grow, expand, and differentiate organoids from human alveolar epithelial cells. Unlike traditionally used animal models, alveolar organoids mimic the regional specificity of the in vivo alveoli, making them ideally suited for alveolar biology research, infectious disease studies, and drug screening. With the convenience of PneumaCult™ Alveolar Organoid Media, you can efficiently passage and expand human alveolar epithelial type II (ATII) cells long term as organoids, then further differentiate these ATII organoids to alveolar epithelial type I (ATI) cells for downstream applications.

Use PneumaCult™ Alveolar Organoid Expansion (AvOE) Medium to achieve a 10,000X cumulative-fold expansion of ATII cells as organoids within 10 passages; this high yield reduces the need to regularly source donor tissue. You can flexibly pause your cultures at any passage by cryopreserving organoids cultured in PneumaCult™ AvOE Medium as single cells. The medium’s three-component, serum-free format maximizes experimental reproducibility, and the alveolar organoids maintain properties indicative of the ATII cell phenotype, including the ability for self-renewal, expected marker expression (e.g. HT2-280 and SP-C), and lineage potential for differentiation to ATI cells.

Use PneumaCult™ Alveolar Organoid Differentiation (AvOD) Medium to differentiate ATII cells expanded as organoids in PneumaCult™ AvOE Medium. The two-component PneumaCult™ AvOD Medium enables in-dome differentiation of ATII organoid cultures to ATI cells in as few as 10 days, with over 85% purity. Differentiated cells show decreased ATII marker expression and strong up-regulation of ATI cell markers (RAGE/AGER, HT1-56, and GPRC5a).

PneumaCult™ AvOE and AvOD Media are compatible with primary isolated fresh or cryopreserved ATII cells, as well as high-quality commercially available alveolar epithelial cell sources.

Learn more about lung organoid cultures in our learning center.
Advantages
⦁ Model the human alveolar physiology in 3D with an in vitro culture system that recapitulates key features of ATII and ATI cells in vivo
⦁ Maximize yield from the initial sample with a medium that supports passaging and long-term expansion of ATII organoids
⦁ Cryopreserve and re-initiate organoid cultures with the biobanking capabilities of the alveolar media
⦁ Convenient format and easy-to-use protocol that generates mature ATII and fully differentiated ATI organoids
⦁ Standardized media with optimized formulation and rigorous quality control testing
Components
  • PneumaCult™ Alveolar Organoid Expansion Medium (Catalog #100-0847)
    • PneumaCult™ Alveolar Organoid Expansion Basal Medium, 450 mL
    • PneumaCult™ Alveolar Organoid Expansion 10X Supplement, 50 mL
    • PneumaCult™ Alveolar Organoid Expansion 100X Passage Supplement, 1.5 mL
  • PneumaCult™ Alveolar Organoid Differentiation Medium (Catalog #100-0861)
    • PneumaCult™ Alveolar Organoid Differentiation Basal Medium, 90 mL
    • PneumaCult™ Alveolar Organoid Differentiation 10X Supplement, 10 mL
Subtype
Specialized Media
Cell Type
Airway Cells, Epithelial Cells
Species
Human
Application
Cell Culture, Differentiation, Expansion, Maintenance, Organoid Culture
Brand
PneumaCult
Area of Interest
Disease Modeling, Drug Discovery and Toxicity Testing, Epithelial Cell Biology, Infectious Diseases, Organoids, Respiratory Research

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
PneumaCult™ Alveolar Organoid Expansion Medium
Catalog #
100-0847
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
PneumaCult™ Alveolar Organoid Differentiation Medium
Catalog #
100-0861
Lot #
All
Language
English
Document Type
Technical Manual
Product Name
PneumaCult™ Alveolar Organoid Media
Catalog #
100-0847
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
PneumaCult™ Alveolar Organoid Expansion Medium
Catalog #
100-0847
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
PneumaCult™ Alveolar Organoid Expansion Medium
Catalog #
100-0847
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
PneumaCult™ Alveolar Organoid Expansion Medium
Catalog #
100-0847
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
PneumaCult™ Alveolar Organoid Differentiation Medium
Catalog #
100-0861
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
PneumaCult™ Alveolar Organoid Differentiation Medium
Catalog #
100-0861
Lot #
All
Language
English

Educational Materials (7)

Brochure
Organoids
Protocol
Performing Immunocytochemical Staining of Epithelial Organoids
Wallchart
Cellular Organization and Biology of the Respiratory System
Wallchart
Dynamic Modeling in Organoids
Webinar
Organoid Expert Panel
52:35
Organoid Expert Panel
Webinar
HUB & STEMCELL Organoids as Models of Infectious Disease Mini-Symposium
1:10:09
HUB & STEMCELL Organoids as Models of Infectious Disease Mini-Symposium
Scientific Poster
Highly Expandable Human Alveolar Organoids to Study Respiratory Diseases

Product Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area
Workflow Stages

Data and Publications

Data

PneumaCult™ Alveolar Organoid Media workflow diagarm illustrating the expansion and differentiation stages of the protocol.

Figure 1. Generation of Alveolar Organoids Using PneumaCult™ Alveolar Organoid Media

The PneumaCult™ Alveolar Organoid Media workflow is a two-stage protocol. During the expansion stage, primary isolated or cryopreserved human ATII single cells are seeded in PneumaCult™ Alveolar Organoid Expansion (AvOE) Seeding Medium. On day 2 - 3, after a full-media change, cultures are expanded using PneumaCult™ AvOE Medium to obtain mature ATII organoids. In the differentiation stage, ATII organoids are cultured for 10 additional days using PneumaCult™ Alveolar Organoid Differentiation (AvOD) Medium to generate ATI organoids.

Brightfield image of alveolar organoids and a bar graph of organoid forming efficiencies of ATII organoid cultures.

Figure 2. PneumaCult™ Alveolar Organoid Expansion Medium Supports High Organoid Forming Efficiency

Organoid forming efficiency (OFE) serves as a functional measure of the health of an organoid culture and the performance of the media. ATII organoids were expanded in PneumaCult™ AvOE Medium (160 cells seeded per dome). (A) Brightfield image taken at day 9 using a 4X objective. The entire dome was captured in the image, each organoid formed was counted, and indicated (total 44; OFE=27.5%). (B) Bar graph showing OFE of ATII organoid cultures derived from three different donors (means ± SD). OFE = (Organoids Formed/Cells Seeded) x 100%.

Comparison of the cumulative doublings for alveolar organoids cultured in PneumaCult™ and published formulation.

Figure 3. PneumaCult™ AvOE Medium Supports Long-Term Expansion and Cryopreservation For Biobanking

(A) Box and whiskers plot of the cumulative doublings at each passage (n=4 independent donors) for organoids expanded in PneumaCult™ AvOE Medium (orange) and published formulation (gray). The means are connected by a line. Long-term passaging of alveolar organoids and over 10000x fold expansion was achieved by both PneumaCult™ AvOE Medium and published formulation. (B) Bar graph showing population doublings (mean ± SD) of organoids generated from cells previously cryopreserved at early and late passages, and expanded in PneumaCult™ AvOE Medium. The cultured organoids retained their expansion potential after thawing.

Plots showing ATII and ATI cell marker expression in alveolar organoids cultured in PneumaCult™ Media.

Figure 4. PneumaCult™ Alveolar Organoid Media Maintains High Levels of ATII and ATI Cell Marker Expression

(A) Flow cytometric gating of fluorescence minus one (FMO) controls for HT2-280 and HT1-56 markers (top), stained cells from organoids expanded in PneumaCult™ AvOE Medium (middle), and subsequently differentiated in PneumaCult™ AvOD Medium (bottom). PneumaCult™ AvOE Medium maintains both the ATII marker expression (HT2-280) and lineage potential to differentiate into ATI cells (HT1-56) in PneumaCult™ AvOD Medium. (B) Organoids derived from 4 independent donors, expressing high levels of HT2-280 were cultured in PneumaCult™ AvOE Medium or published formulation, and compared at each passage. Dashed line depicts 50% of starting HT2-280++%. (C) Scatter plot illustrating the percent change in HT2-280 expression between P0 and P5 from the same donors. Organoids cultured in PneumaCult™ AvOE Medium maintained higher levels of HT2-280 marker expression, compared to those cultured using published formulation. Mean is displayed as a solid line. * = p<0.05 in a one-tailed paired T-test (n=4).

Immunostained alveolar organoids showing expression of ATI and ATII markers.

Figure 5. Immunohistochemistry Confirms Expression of ATII and ATI Cell Markers in Alveolar Organoids

(A) Organoids from three donors expanded in PneumaCult™ AvOE Medium (top) express the ATII markers, HT2-280 (green) and pro-surfactant protein C (pro-SPC, yellow). When further differentiated in PneumaCult™ AvOD Medium (bottom), the levels of these ATII markers are down-regulated, while the ATI marker RAGE (red) is up-regulated. (B) Differentiated organoids also express high levels of ATI marker GPRC5a (yellow).

Immunostained alveolar organoids showing expression of SARS-CoV-2 entry markers.

Figure 6. Organoids Cultured in PneumaCult™ Alveolar Organoid Media Express TMPRSS2 and ACE2, Key Markers for SARS-CoV-2 Entry

Organoids from three donors expanded in PneumaCult™ AvOE Medium (top) and differentiated in PneumaCult™ AvOD Medium (bottom) express proteins associated with SARS-CoV-2 entry, TMPRSS2 and ACE2. While ACE2 was expressed in all donors and conditions, TMRPSS2 expression was donor-dependent.

Heatmap comparing gene expression in alveolar organoids cultured in PneumaCult™ and published formulation.

Figure 7. Gene Expression Analysis of Organoids Cultured in PneumaCult™ Alveolar Organoid Media Shows Presence of Alveolar Markers

Normalized gene expression heatmap of organoids expanded in PneumaCult™ AvOE Medium and differentiated in PneumaCult™ AvOD Medium compared to those grown using a published formulation. The presence of canonical ATII and ATI genes as well as SARS-CoV-2 key entry markers, ACE2 and TMPRSS2, are shown. Published RNA-Seq gene expression datasets of primary alveolar epithelial cells (including ATI and ATII cells, from Mikami et al. 2021, GSE186359) and primary ATII cells (Jacob and Kotton, 2017, GSE96642) were used as controls.

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