Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

Abstract

Rationale: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts.Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts.Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T31Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures.

Expansion of airway basal epithelial cells from primary human non‐small cell lung cancer tumors

“…We attempted cell culture from 12 patients (median age = 75 years, range = 58–90 years), whose surgically resected primary lung adenocarcinoma (LUAD) tumors (stage II [ n  = 9] or III [ n  = 3]) are being analyzed in the on‐going Tracking Cancer Evolution through Therapy (TRACERx) clinical study,14, 15 using a published protocol 17. We obtained primary epithelial cell cultures that could be passaged from 10 (83.3%) of these.…”

“…Protocol differences exist between the aforementioned studies: key studies have used inactivated human dermal fibroblasts7, 8 as feeder layers rather than the mouse embryonic fibroblasts often used in those that see normal cell expansion and it has also been possible to transition tumor cell cultures off feeder layers after 6 months of culture establishment 8. We have previously demonstrated that adult human lung fibroblasts do not support long‐term normal airway epithelial cell expansion in the same way as 3T3‐J2 cells17 so the use of adult and/or human feeder cells might favor tumor cell expansion. Relevant biological differences might also exist between mitotic inactivation using irradiation4, 9 rather than mitomycin C, as used here.…”

“…Tumors were digested to single cell suspensions in serum‐free RPMI medium (Gibco) containing 0.25 U/ml Liberase TM (Roche, UK) and 0.1 mg/ml DNAse I (Sigma) in a waterbath at 37°C for 2 hr with regular agitation. Tumor‐derived cells were plated on 3T3‐J2 feeder layers in epithelial cell culture medium containing EGF and Y‐27632 (3T3 + Y) as previously described 16, 17. Airway spheroids, or “tracheospheres,” were generated using a previously published protocol 16…”

Best of Milan 2017—ERS Lung Science Conference session “Lung tissue repair and remodeling in chronic lung diseases: mechanisms and therapeutic approaches”

“…However, for urgent re-cellularization of bioengineered scaffolds there is often insufficient time for isolation and expansion of autologous airway epithelial cells to obtain the required cell numbers by standard methods. Thus, Butler and colleagues have developed a novel and faster method to obtain larger numbers of functional airway basal cells (15). Based on existing work in the stem cell field (16), they compared a co-culture with mitotically inactivated 3T3-J2 fibroblast feeder cells in the presence of Rho-associated protein kinase (ROCK) inhibition (inhibitor Y27632) (3T3 + Y), to the classical expansion in bronchial epithelial growth medium (BEGM) (15).…”

“…Thus, Butler and colleagues have developed a novel and faster method to obtain larger numbers of functional airway basal cells (15). Based on existing work in the stem cell field (16), they compared a co-culture with mitotically inactivated 3T3-J2 fibroblast feeder cells in the presence of Rho-associated protein kinase (ROCK) inhibition (inhibitor Y27632) (3T3 + Y), to the classical expansion in bronchial epithelial growth medium (BEGM) (15). Basal cells cultured under 3T3 + Y conditions proliferated faster than those grown in BEGM, retained their normal karyotype after 6 weeks in culture, were able to differentiate into a pseudostratified epithelium at the air-liquid interface, as well as to form tracheospheres, also in late passages (15).…”