Improved Expansion and Maintenance of the Differentiation Potential of Primary Human Airway Epithelial Cells
Bibliographic record
Abstract
Abstract RATIONALE Primary airway basal cells and their differentiation into mature epithelia at the air-liquid interface (ALI) and as organoids are useful models for studying airway pathophysiology and drug testing. Culturing these cells in currently available media carries limitations for their growth and differentiation potential, especially when starting with low cell numbers or precious samples. Therefore, we developed PneumaCult™ Next Generation Expansion Medium (NGEx), an optimized medium for robust passaging and biobanking.METHODS Commercially available or freshly isolated healthy human bronchial (HBEC), small airway (SAEC), and nasal cells (HNEC) were expanded until 80% confluent in PneumaCult™-NGEx, PneumaCult™-Ex Plus, or other commercially available media, then serially passaged until senescence. Cells were assessed for basal cell identity and seeded into downstream assays to evaluate differentiation and function. Assays included ALI differentiation using PneumaCult™-ALI or -ALI-S Media or organoid differentiation using PneumaCult™ Airway Organoid Kit or PneumaCult™ Apical-Out Airway Organoid Medium. RESULTS Airway basal cells expanded in PneumaCult™-NGEx displayed the desired cobblestone morphology. HBECs expanded in PneumaCult™-NGEx for 20 additional population doublings (PDs) compared to PneumaCult™-Ex Plus. A high percentage (>90%) of PneumaCult™-NGEx-expanded cells expressed basal cell markers (CD49f, CD271, KRT5, or p63) throughout long-term passaging. HBECs efficiently differentiated at higher PDs (> 45 PDs) compared to other tested media (n ≥ 4 donors). After 30 PDs, PneumaCult™-Ex Plus and other media failed to differentiate in ALI cultures, whereas, HBECs expanded in PneumaCult™-NGEx successfully formed pseudostratified epithelia with beating cilia and secreted mucus, with an appropriately tight epithelial barrier (TEER ≥ 300 Ω x cm2), and responded to CFTR modulators as assessed by Ussing chambers. RNASeq analysis demonstrated that basal cells expanded in PneumaCult™-NGEx- or PneumaCult™-Ex Plus, and subsequently differentiated, had similar gene expression profiles. At later passages, PneumaCult™-NGEx-expanded cells differentiated into ciliated- and goblet cell-containing apical-in organoids with higher efficiencies and better preserved CFTR function, as assessed by the forskolin-induced swelling assay, compared to cells expanded in PneumaCult™-Ex Plus. PneumaCult™-NGEx-expanded cells successfully generated apical-out airway organoids at a higher PD than PneumaCult™-Ex Plus. SAECs and HNECs cultured in PneumaCult™-NGEx produced an additional 25 and 10 PDs, respectively, compared to cells cultured in PneumaCult™-Ex Plus. These cells generated mature ALI and organoid cultures at later passages. CONCLUSIONS PneumaCult™-NGEx outperformed all tested media in enhancing the expansion of primary airway basal cells while maintaining their differentiation potential at higher passages. Thus, it supports generating robust, functional, and physiologically relevant airway models suitable for studying airway physiology.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".