Cold storage of human precision-cut lung slices in TiProtec preserves cellular composition and transcriptional responses and enables on-demand mechanistic studies

Abstract

Background: Human precision-cut lung slices (hPCLS) are a unique platform for functional, mechanistic, and drug discovery studies in the field of respiratory research. However, tissue availability, generation, and cultivation time represent important challenges for their usage. Therefore, the present study evaluated the efficacy of a specifically designed tissue preservation solution, TiProtec, complete or in absence (-) of iron chelators, for long-term cold storage of hPCLS.

Methods: hPCLS were generated from peritumor control tissues and stored in DMEM/F-12, TiProtec, or TiProtec (-) for up to 28 days. Viability, metabolic activity, and tissue structure were determined. Moreover, bulk-RNA sequencing was used to study transcriptional changes, regulated signaling pathways, and cellular composition after cold storage. Induction of cold storage-associated senescence was determined by transcriptomics and immunofluorescence (IF). Finally, cold-stored hPCLS were exposed to a fibrotic cocktail and early fibrotic changes were assessed by RT-qPCR and IF.

Results: Here, we found that TiProtec preserves the viability, metabolic activity, transcriptional profile, as well as cellular composition of hPCLS for up to 14 days. Cold storage did not significantly induce cellular senescence in hPCLS. Moreover, TiProtec downregulated pathways associated with cell death, inflammation, and hypoxia while activating pathways protective against oxidative stress. Cold-stored hPCLS remained responsive to fibrotic stimuli and upregulated extracellular matrix-related genes such as fibronectin and collagen 1 as well as alpha-smooth muscle actin, a marker for myofibroblasts.

Conclusions: Optimized long-term cold storage of hPCLS preserves their viability, metabolic activity, transcriptional profile, and cellular composition for up to 14 days, specifically in TiProtec. Finally, our study demonstrated that cold-stored hPCLS can be used for on-demand mechanistic studies relevant for respiratory research.

Keywords: 3R; Fibrosis; Human lung models; Human precision-cut lung slices (hPCLS); Long-term cold storage; TiProtec; Tissue preservation.

Fig. 1

Viability and metabolic activity after long-term cold storage. After preparation, hPCLS were cold stored in DMEM/F-12, TiProtec, or TiProtec (-), at 4 °C for up to 28 days. A) Viability changes were assessed by Calcein AM and EthD-1 staining. Viability is expressed as a percentage of the baseline viability of freshly sliced hPCLS at day 0 (100%). A positive control was also measured, after treating hPCLS with 1% Triton-X. Data set was analyzed using a RM two-way ANOVA followed by a Tukey´s multiple comparison test: * simple effects of the medium and # simple effects of storage time. Data are presented as means ± SEM (n = 4 patients, single dots represent average activity from 3 technical replicates per patient). Asterisks indicate significant differences (** p < 0.01, # p < 0.05, ## p < 0.01). B) Representative images of Live/Dead™ staining of hPCLS at baseline (T0) and after long-term cold storage. C) Quantification of Alamar Blue assay. The baseline metabolic activity was measured on day 0 and set as 100%. A negative control with only medium was also included. Metabolic activity changes are presented as a percentage of the baseline activity of freshly cut hPCLS (T0). Data set was analyzed using a Mixed-effects model followed by a Tukey´s multiple comparison test: * simple effects of the medium and # simple effects of storage time. Data are presented as means ± SEM (n = 4–6 patients, single dots represent averaged activity from 3 technical replicates per patient). Asterisks indicate significant differences (*p < 0.05, # p < 0.05, ## p < 0.01, ### p < 0.001)

Fig. 2

Transcriptional changes in hPCLS after cold storage. A) Principal component analysis for freshly cut hPCLS (T0) and after 7 days of cold storage in DMEM/F-12, TiProtec (-), and TiProtec. Shapes show three (DMEM/F-12) or four (T0, TiProtec (-), TiProtec)) different biological replicates and colors indicate the cold storage solution. B) Venn diagram of differentially expressed genes (DEG) after 7 days of cold storage when compared to T0 baseline control (LFC > 0). C) Cell type signature enrichment analysis of main cellular compartments in hPCLS based on DEG displayed in B. D) Heatmap of DEG after 7 days of cold storage in TiProtec (-) or TiProtec when compared to T0 baseline control (LFC > 1). E) Deregulated pathways in hPCLS after 7 days of cold storage in TiProtec or TiProtec (-) based on DEG from D. F) Deregulated pathways in hPCLS after 7 and 14 days of cold storage in TiProtec based on DEG in comparison to T0 control (LFC > 1).

Fig. 3

Transcriptional changes induced by TiProtec and TiProtec (-) in comparison to DMEM/F-12 after 7 days of cold storage. A) Heatmap of differentially expressed genes (DEG) after 7 days of cold storage when compared to DMEM/F-12 in hPLCS obtained from three (DMEM/F-12) or four (TiProtec (-), TiProtec) different donors. B) Gene set enrichment analysis of deregulated pathways in hPCLS after cold storage based on DEG from A. C) Normalized counts of up- and -downstream regulators of oxidative stress-associated pathways after 7 days of cold storage in DMEM/F-12, TiProtec (-), or TiProtec. Single points with different shapes represent three (DMEM/F-12) or four (TiProtec solutions) different biological replicates. D) Cell type signature enrichmentanalysis in hPCLS after cold storage with TiProtec or TiProtec (-) when compared to DMEM/F-12 for 7 days

Fig. 4

Senescence response in cold-stored hPCLS. A) Normalized counts for senescence-associated genes: CDKN1A/P21, GDF-15, and TP53. Single points represent independent donors (N = three (DMEM/F-12)) or four (T0, TiProtec (-), TiProtec)). B) Heatmap of enrichment scores after GSEA for senescence-related pathways of DEG from day 7 cold storage in TiProtec (-) or TiProtec in comparison to DMEM/F-12. C) Quantification of P21/CDKN1A + nuclei in hPCLS after standard cell culture for 7 days or cold storage in DMEM/F-12, TiProtec (-), or TiProtec for 7, 14, and 21 days. Single points represent independent donors (N = 3). D) Representative images of immunostaining for P21/CDKN1A and Podoplanin (PDPN, structural marker for alveolar region) in hPCLS after cold storage in DMEM/F-12, TiProtec (-), or TiProtec for 7, 14, and 21 days. Scale bar = 100 μm.

Fig. 5

Evaluation of early-fibrotic changes in cold-stored hPCLS after fibrotic stimulation. A) RT-qPCR of the extracellular matrix-related genes COL1A1 and FN1 and the myofibroblasts marker, ACTA2, in hPCLS treated with control (CC) or fibrotic cocktail (FC) at baseline or after 7 and 14 days of cold storage with TiProtec or TiProtec (-). Single points with different shapes represent independent biological replicates (N = 4). * p < 0.05, ** p < 0.01 after unpaired-t-test (baseline) or Kruskal-Wallis test followed by Dunn´s multiple comparison test (day 7, day 14). B) Representative images of immunostaining for fibrosis-related proteins (aSMA and FN1) in hPCLS treated with CC or FC at baseline or after 7 days of cold storage with TiProtec or TiProtec (-). Scale bar = 100 μm.