Airway contractility in the precision-cut lung slice after cryopreservation

Abstract

An emerging tool in airway biology is the precision-cut lung slice (PCLS). Adoption of the PCLS as a model for assessing airway reactivity has been hampered by the limited time window within which tissues remain viable. Here we demonstrate that the PCLS can be frozen, stored long-term, and then thawed for later experimental use. Compared with the never-frozen murine PCLS, the frozen-thawed PCLS shows metabolic activity that is decreased to an extent comparable to that observed in other cryopreserved tissues but shows no differences in cell viability or in airway caliber responses to the contractile agonist methacholine or the relaxing agonist chloroquine. These results indicate that freezing and long-term storage is a feasible solution to the problem of limited viability of the PCLS in culture.

Figure 1.

Viability is preserved in frozen-thawed precision-cut lung slice (PCLS). (A) Lactate dehydrogenase assay shows no significant difference between frozen-thawed and never-frozen groups in the percentage of dead to live cells (n = 22 PCLSs in each group). (B) MTT assay showed a significant reduction in cell metabolic activity after freeze-thaw (*P < 0.05) but well within the acceptable range previously reported for other tissues. These data are reported as mean and standard deviation of formazan absorbance at 540 nm (n = 18 PCLSs in each group). (C) Images of distal airways from sections of never-frozen and frozen-thawed PCLSs immunostained for CC10 expression in the lung epithelium and α-smooth muscle actin (SMA) expression in the airway smooth muscle. Cell nuclei were counterstained by hemotoxylin. Scale bar, 50 μm.

Figure 2.

Cell death in PCLS is minimal and predominantly localized to airway epithelium. (A) Representative bright field and florescence images of live-dead stained never-frozen or frozen-thawed PCLSs (original magnification: ×10). Scale bar = 200 μm. (B) Representative bright field and fluorescence images of live-dead stained never-frozen or frozen-thawed PCLSs (original magnification: ×20). Scale bar, 200 μm.

Figure 3.

Airway contractility is preserved in frozen-thawed PCLSs. In the never-frozen group, the mean diameter was 151 μm (range, 88–389 μm); in the frozen-thawed group, the mean diameter was 175 μm (range, 77–415 μm). (A) Representative images of airway contraction to methacholine (MCh) (10⁻⁷ to 10⁻⁴ M) and airway relaxation to chloroquine (ChQ) (1 mM) in the never-frozen and frozen-thawed groups. Scale bar = 50 μm. (B) When pooled, airway contraction and relaxation in never-frozen (n = 57 airways) and frozen-thawed (n = 46 airways) PCLSs were not statistically different with MCh or ChQ (P = 0.2–0.4). Plotted are mean and SEM. (C, D) In both groups, individual airways contracted heterogeneously. To assess the role of initial airway size, we grouped airways into two bins: smaller than 200 μm (C) and larger than 200 μm (D). Within each airway size bin, never-frozen and frozen-thawed airways did not show significant differences in their responses to MCh and to ChQ (P = 0.1–0.5).