In Vitro Assessment of Apoptosis and Necrosis Following Cold Storage in a Human Airway Cell Model

Abstract

As advances in medical technology improve the efficacy of cell and tissue transplantation, a void remains in our knowledge base as to the specific molecular responses of cells to low-temperature storage. While much focus has been given to solution formulation for tissue perfusion during storage, investigations into cold exposure-induced complex molecular changes remain limited. The intent of this study was to quantify the levels of cell death following hypothermic storage in a lung cell model, establishing a foundation for future in-depth molecular analysis. Normal human lung fibroblasts (IMR-90) were stored for 1 day or 2 days and small airway epithelial cells (SAEC) were stored for 5 days or 7 days at 4°C in complete media, ViaSpan, or ViaSpan + pan-caspase (VI) inhibitor. (Poststorage viability was assessed for 3 days using alamarBlue(™).) Sample analysis revealed that IMR-90 cells stored in ViaSpan remained 80% (±9) viable after 1 day of storage and 21% (±7) viable after 2 days of storage. SAEC cells stored in ViaSpan remained 81% (±5) viable after 5 days and 28% (±7) after 7 days. Microfluidic flow cytometry analysis of the apoptotic and necrotic populations in the ViaSpan-stored samples revealed that in the IMR-90 cells stored for 2 days, 7% of the population was apoptotic at 4-h poststorage, while ∼70% was identified as necrotic. Analysis of the SAEC cell system following 7 days of ViaSpan storage revealed an apoptotic peak of 19% at 4-h poststorage and a corresponding necrotic peak of 19%. Caspase inhibition during hypothermic storage increased viability 33% for IMR-90 and 25% for SAEC. Data revealed a similar pattern of cell death, through both apoptosis and necrosis, once the onset of cold storage failure began, implying a potential conserved mechanism of cold-induced cell death. These data highlight the critical need for a more in-depth understanding of the molecular changes that occur as a result of cold exposure in cells and tissues.

FIG. 1.

Viability following hypothermic storage. IMR-90 (1- or 2-day storage) and SAEC (5- or 7-day storage) cells were stored at 4°C in either ViaSpan or their respective complete media. Viability was measured using alamarBlue^™, following 24 h of recovery from cold storage and normalized to the prestorage control. Poor survival of IMR-90 cells stored in media after 1 day or 2 days of hypothermic storage. IMR-90 cells stored in Viaspan after 1 day and 2 days were 80.0% ± 9.0% and 20.9% ± 7.1 viable, respectively. Higher viabilities of SAEC in media were seen at 84.4% ± 7.2 (5 days) and 78.3% ± 5.7 (7 days). SAEC storage in ViaSpan had 81.1% ± 5.0 survival (5 days) that decreased to 27.6% ± 6.8 (7 days) (N = 3).

FIG. 2.

Fluorescent microscopy following hypothermic storage. IMR-90 (2 days) and SAEC cells (7 days) were stored at 4°C in either ViaSpan or their respective complete media. Cells were categorized as live (blue), apoptotic (green), and necrotic (red) using the Vybrant Apoptosis Assay. (A) IMR-90 cells stored in media for 2 days showed low cell yield with primarily necrotic labeled cells. ViaSpan-stored IMR-90 cells demonstrated a high level of necrosis for all time points assessed, while live cells decreased over the recovery interval. Apoptotic cells peaked at the 4- and 8-h intervals. (B) SAEC cells stored in media revealed a low level of both apoptosis and necrosis at all time intervals, with a high number of viable cells. ViaSpan-stored SAEC cells showed a large concentration of necrotic cells and low levels of viable cells throughout. A peak in apoptosis was observed at the 4- and 8-h intervals as well.

FIG. 3.

Quantification of cell death following hypothermic storage. Cold storage (4°C) of IMR-90 (2 days) and SAEC (7 days) in either ViaSpan or their respective complete media. Cells were probed for apoptosis (Yo-Pro-1) and necrosis (PI) and quantified via microfluidic flow cytometry. IMR-90 cells stored in media yielded an insufficient number of cells for meaningful analysis. IMR-90 cells in ViaSpan demonstrated a peak in apoptosis at the 4-h recovery interval (5.5% ± 0.6) that declined at 24 h (2.0% ± 0.6). Analysis of the necrotic population revealed an elevated level at 1 h of recovery that peaked at the 24-h interval (89.4% ± 1.7). SAEC cells stored in media demonstrated an apoptotic level of ∼5% across all time intervals. The necrotic population analysis revealed a similar pattern of ∼7.5% and a peak of 9.6% ± 2.5 (8 h). The ViaSpan-stored SAEC cells showed increased levels of apoptosis throughout recovery that peaked at 4 h (18.8% ± 2.3) and remained elevated at 24 h (12.8% ± 1.2). The initial level of necrosis at 1-h recovery (12.3% ± 1.5) rose to 18.9% ± 2.4 by the 24-h recovery interval (N = 4).

FIG. 4.

Addition of caspase inhibitor to ViaSpan during hypothermic storage. (A) IMR-90 (2 days) and SAEC cells (7 days) were stored at 4°C in either ViaSpan or ViaSpan + [10 μM] pan-caspase inhibitor and viability was measured using alamarBlue^™ following 24 h of recovery. Addition of caspase inhibitor showed minor improvements in the shorter time intervals for both cells types (3.7% for IMR-90 and 6.4% for SAEC). The longer storage intervals demonstrated a significant increase in viability with the inclusion of inhibitor (33.2% for IMR-90 cells and 25.0% for SAEC) (N = 3). (B) SAEC cells were stored at 4°C for 7 days in ViaSpan + [10 μM] pan-caspase inhibitor. SAEC cells were probed for apoptosis (Yo-Pro-1) and necrosis (PI) and quantified via microfluidic flow cytometry. Analysis revealed a high level of viable (unlabeled) cells, a significant level of apoptotic cells that peaked at 8 h of recovery (17.0% ± 1.9) and lower level of necrotic cells that showed a peak at 24 h of recovery (16.8% ± 2.3) (N = 4). (C) IMR-90 cells were stored at 4°C for 2 days in ViaSpan + [10 μM] pan-caspase inhibitor. IMR-90 cells were probed for apoptosis (Yo-Pro-1) and necrosis (PI) and quantified via microfluidic flow cytometry. Analysis revealed that a majority of the cell population remained viable (unstained) that reached its lowest level at 24 h of recovery (48.6% ± 2.6). The apoptotic population remained elevated above 7% for all observed time intervals with a peak at 4 h (9.6% ± 1.3), while the necrotic population had significantly higher numbers of labeled cells with a peak at 24 h (43.9% ± 3.6) (N = 4).

FIG. 4.

Addition of caspase inhibitor to ViaSpan during hypothermic storage. (A) IMR-90 (2 days) and SAEC cells (7 days) were stored at 4°C in either ViaSpan or ViaSpan + [10 μM] pan-caspase inhibitor and viability was measured using alamarBlue^™ following 24 h of recovery. Addition of caspase inhibitor showed minor improvements in the shorter time intervals for both cells types (3.7% for IMR-90 and 6.4% for SAEC). The longer storage intervals demonstrated a significant increase in viability with the inclusion of inhibitor (33.2% for IMR-90 cells and 25.0% for SAEC) (N = 3). (B) SAEC cells were stored at 4°C for 7 days in ViaSpan + [10 μM] pan-caspase inhibitor. SAEC cells were probed for apoptosis (Yo-Pro-1) and necrosis (PI) and quantified via microfluidic flow cytometry. Analysis revealed a high level of viable (unlabeled) cells, a significant level of apoptotic cells that peaked at 8 h of recovery (17.0% ± 1.9) and lower level of necrotic cells that showed a peak at 24 h of recovery (16.8% ± 2.3) (N = 4). (C) IMR-90 cells were stored at 4°C for 2 days in ViaSpan + [10 μM] pan-caspase inhibitor. IMR-90 cells were probed for apoptosis (Yo-Pro-1) and necrosis (PI) and quantified via microfluidic flow cytometry. Analysis revealed that a majority of the cell population remained viable (unstained) that reached its lowest level at 24 h of recovery (48.6% ± 2.6). The apoptotic population remained elevated above 7% for all observed time intervals with a peak at 4 h (9.6% ± 1.3), while the necrotic population had significantly higher numbers of labeled cells with a peak at 24 h (43.9% ± 3.6) (N = 4).