Table 1.

Recent studies exploiting CRC HAE co-culture method with feeder cells

CRC HAE culture with feeder cells
Publication	Cell type, sample type, age group	Feeder cells? /Cell stasis factor	Expansion	Differentiation	Culture length:	Major findings:	Limitations
Suprynowicz [28]	HTE epithelium	Yes, IR	Submerged culture, KSFM or F-medium, 10uM Y27632; Vessels: no coating	ALI culture, Until confluence- CELLnTEC CnT57 medium + 10 μM Y-27632 Later: CELLnTEC CnT-02–3D differentiation medium, 14 days	Passage number N/A, forms cilia in ALI	1. Stable karyotype, non-tumorigenic 2. Rapid and reversible induction of cell divisions 3. Maintenance of lineage characteristics	Unknown maximal number of passages for HAE culture
Butler [66]	HBE epithelium (biopsies)	Yes, IR or mitomycin C	Submerged culture: DMEM- F12 (3:1 ratio), 5% FBS, 5 uM Y-27632, hydrocortisone (25 ng/ml), epidermal growth factor (0.125 ng/ml) insulin (5 mg/ml), 0.1 nM cholera toxin, amphotericin B (250 mg/ml) and gentamicin (10 mg/ml), pen-strep (1x) Vessels: no coating	Differentiation: suspension culture (tracheospheres) or ALI	Expansion for > 3 months (split ratio 1:5), cells expanded for > 6 weeks can differentiate (tracheospheres, ALI)	1. CRC cells are non-tumorigenic and react to contact inhibition 2. Cell yield sufficient for tissue engineering/ regeneration (> 1 × 107 cells obtained in < 4 weeks)	n/a
Reynolds [27]	HNE brushings of healthy adult donors	Yes, IR	Submerged culture: DMEM:F12 (3:1) + 7,3% FBS, hydrocortisone/EGF mix, Cholera toxin 8,6 ng/ul, Adenine 24 ng/ul, Insulin 10ug/ml Vessel coating: rat tail collagen type I (30 µg/ml) Conditions compared with regular BEGM culture	ALI culture, organotypic raft cultures or 3D tracheospheres in Matrigel	CRC expanded HNE cells could be differentiated at P4, in contrast to BEGM-expanded HNE cells.	1. Increased clone formation 2. Increased cell yield compared to BEGM (~ 400 fold) 3. Mechanism of Y-27632 action: (a) promotion of the basal cell phenotype; (b) stimulation of basal cell–specific cell–cell and cell–ECM interactions; and (c) suppression of differentiation to an airway luminal cell phenotype	1) For some donors—almost no MCC visible (MCC number: from < 10% to ~ 40%); 2) Limitated  proliferative lifespan of HNEs expanded in CRC or BEGM, visible already after 2 passages (less pronounced in CRCs)
Wolf [65]	HNE and HBE cells of healthy donors at different ages (from newborns to adults)	conditioned medium from IR fibroblasts	Submerged culture: BEGM medium conditioned by 72 h incubation with NIH-3T3 fibroblasts irradiated with 30 Gy and frozen in aliquots.  Conditioned BEGM mixed with fresh BEGM (3:1 v/v); supplemented with 10 µM Y-27632 Vessels coating: human placental collagen type IV	ALI culture; Conditioned BEGM medium + Y-27632 to the basolateral compartment only	~ 13–14 PDs within 30 days (split ratio 1:4)	1. Increased proliferation and extended proliferative lifespan 2. Maintenance of lineage characteristics after multiple passages, 3. Physiological airway responses to dsRNA (induction of antiviral genes, IFN λ1 production, inflammatory and remodeling responses)	1) Weak differentiation at ALI for some donors; 2) Not sure whether the anti-viral responses observed in CRCs are identical to the in vivo HAE responses (CRCs induction changes expression of some inflammatory genes)
Brewington [57]	HNE samples from healthy or CF patients (curettage, from newborns to adults)	YES, IR (commercially available, frozen)	Submerged culture: DMEM/F12 + 10% FBS, cholera toxin (0,01 mg), EGF (0,04ug), HC (0,4ug/500 ml), Adenine (24 mg), Y-27632 (3,2 mg) + antibiotics Vessel coating: VitroCOl	Suspension culture (spheroids): DMEM/F12 + Ultroser G, 20 ml Fetal Clone II, Bovine Brain Extract, Transferrin, epinephrine, ethanolamine, insulin, HC, RA, triiodothyronine, phosphorylethanolamine	spheroids differentiate within 10 days	1. Faster differentiation of spheroids than ALI cultures 2. Spheroid swelling is a quick and robust assay for CFTR activity	1) Short availability of spheroids for analysis 2) No information on the differentiation capacity beyond passage 2 3) Preclinical utility not fully clear (drug testing in CF patients?)
Peters- Hall [69]	bronchial biopsy	YES, IR	Submerged culture: P0-P3: BEGM, 0.5 ng/ml EGF, 2% O2 P3 -5: (CRC culture); BEGM, 5% FBS, 10uM Y-27632, no coating, 2% O2 Vessel coating: P0-P3: Porcine gelatin, P3-P5: no coating	ALI culture, P5 + (Transwells, expansion): BEGM + 10uM Y-27632, no coating, 21% O2 P5 + (ALI): BEGM, 0.5 ng/ml EGF, 0.11 mM CaCl2, 0.1 mg/ml BPE, 21% O2	up to 47 PDs (passage 15)	1. Increased ciliation of MOD CRCs at P5 and P10 (~ 33–38%) 2. CFTR activity probably retained at later passages 3. Increased HBECs lifespan allowed CRISPR-Cas9 gene edition 4. Mod CRC conditions reduce cellular stress in HBECs from non-CF donors and prevent premature cellular senescence	Requires various media, complicated procedure