Hybrid Three-Dimensional Lung Tissue Cultures to Improve Ex Vivo Models of Chronic Obstructive Pulmonary Disease.

RATIONALE: Continued reliance on preclinical animal models and traditional human cell culture experiments conducted on supraphysiologically stiff surfaces in two dimensions often limits the translation of cellular and molecular mechanistic findings into effective therapies for patients suffering from chronic obstructive pulmonary disease (COPD). COPD ranks as the fourth highest cause of mortality in the United States, and thus it is imperative to improve ex vivo models of this devastating disease. One emerging novel model of study involves using precision-cut lung slices as three-dimensional lung tissue culture (3D-LTC) platforms. Although this model retains the complex pulmonary architecture required for more precise ex vivo modeling, it is limited by short-term viability (∼5-7 d).

OBJECTIVES: We hypothesized that this decrease in viability is related to slices losing the extracellular matrix adhesion foundation critical for normal cellular functioning and that encapsulation of slices in custom hydrogel microenvironments would improve maintenance of a stable phenotype for longer-term investigational studies.

METHODS: The 3D-LTC slices tested here were created according to standard protocols from healthy mouse lungs and from the lungs of a patient with COPD. We synthesized poly(ethylene glycol)-based polymers to create custom hydrogels for encapsulation of 3D-LTCs with mechanical properties that match each type of lung tissue. The Young's modulus (E) or stiffness of hydrogels was measured with a parallel-plate rheometer. Lung slices were analyzed for viability with a colorimetric assay that measures cellular metabolism (WST-1; Millipore Sigma) and by staining for cell membrane integrity (Invitrogen LIVE/DEAD viability kit; Thermo Fisher Scientific). All fluorescently labeled samples were imaged by spinning disk confocal microscopy, and cell viability at all time points was analyzed with ImageJ software.

RESULTS: The average percent viability of cells within hybrid 3D-LTCs was consistently higher than control 3D-LTCs on Day 1 (35% increase; P < 0.001) and Day 7 (55% increase; P < 0.001) according to LIVE/DEAD staining results. The WST-1 assay further showed no significant difference in viability between the two systems up to Day 18 in culture.

CONCLUSIONS: We have demonstrated that it is possible to create customized hydrogel platforms that replicate the stiffness of healthy mouse and human COPD lung tissues. Precision cut lung slices embedded in these materials maintain higher levels of viability compared with controls in culture. Further optimization of this technology platform is ongoing and will facilitate the development of more organotypic ex vivo models of chronic pulmonary disease.