Figure 1. Overview of the PneumaCult™ Culture System for Airway Research

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 1. Overview of the PneumaCult™ Culture System for Small Airway Research

Expansion of human small airway epithelial cells (HSAEC) in submerged culture is performed with PneumaCult™-Ex Plus Medium. During the early Expansion Phase of the air-liquid interface (ALI) culture procedure, PneumaCult™-Ex Plus 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 PneumaCult™-ALI-S is added to the basal chamber only. Differentiation into a mucociliary epithelium is obtained following 21+ days of incubation and can be maintained for more than one year.

Figure 1. Overview of the PneumaCult™ Culture System for Human Airway Organoid Generation

In the early two-dimensional expansion phase of the human airway organoid culture procedure, HBECs are expanded using PneumaCult™-Ex Plus Medium. The HBECs are then embedded into a Matrigel® dome and expanded for 4 - 7 days using PneumaCult™ Airway Organoid Seeding Medium. Following the expansion, the HBECs are differentiated using PneumaCult™ Airway Organoid Differentiation Medium for an additional 21+ days.

Figure 1. Generation of Lung Progenitor Cells from Human ES/iPS Cells with the STEMdiff™ Lung Progenitor Kit

Human PSC (hPSC) cultures progress through a simple three-stage process to generate lung progenitor cells. hPSC clumps are first seeded in mTeSR™1. On Day 1, differentiation is initiated with Medium DE-1 (STEMdiff™ Endoderm Basal Medium containing Supplement MR and Supplement CJ). Subsequently on day 2 and 3, the medium is changed to Medium DE-2 (STEMdiff™ Endoderm Basal Medium containing Supplement CJ) for definitive endoderm patterning. On Day 4, to initiate anterior foregut endoderm patterning, the endoderm monolayer is passaged in Medium LP-1 (STEMdiff™ Lung Basal Medium, Lung Supplement (10X), and Supplement 1) and Y-27832. Finally, at Day 7, the cells are differentiated into the lung progenitor stage with Medium LP-2 (STEMdiff™ Lung Basal Medium, Lung Supplement (10X), and Supplement 2).

Figure 1. Generation Of Branching Lung Organoids Using the STEMdiff™ Branching Lung Organoid Kit

(A) Human PSC cultures progress through a four-stage differentiation process to generate human branching lung organoids. By the end of stage 1 (day 3), cultures exhibit characteristics typical of definitive endoderm and anterior foregut differentiation is initiated. During stage 2 (days 3 - 6) anterior foregut endoderm buds are released from the monolayer, and are then suspended to form ventralized lung bud organoids in stage 3 (days 6 - 14). In stage 4, the lung bud organoids are embedded into Matrigel sandwich cultures to mature into branching lung organoids. (B) Morphological representation of the culture at different stages.

Figure 1. Generation of Apical-Out Airway Organoids Using the PneumaCult™ Apical-Out Airway Organoid Medium

In the two-dimensional expansion phase of the human apical-out airway organoid culture protocol, HBECs or HAECs are expanded using PneumaCult™-Ex Plus Medium. The HBECs or HAECs are then plated into an AggreWell™400 24-well plate and allowed to aggregate for 1 - 6 days using PneumaCult™ Apical-Out Airway Organoid Medium. Following the aggregation, the cells are dislodged from the microwells and the aggregate suspension is transfered to a 24-well flat-bottom plate and differentiated for 9 - 14 days using PneumaCult™ Apical-Out Airway Organoid Medium to generate apical-out airway organoids that display beating cilia.