Cryopreservation of Viable Human Lung Tissue for Versatile Post-thaw Analyses and Culture

Cryosubstitution to detect crystals and extracellular matrix distortion. Freshly-explanted pig lungs were expansion-perfused in standard (left) and pseudo-diaphragmatic expansion-cryoprotectant perfusion (PDX-CP) (right) cryoprotectant solution, frozen to −80°C at 1°C/min and subjected to substitution of aqueous with methanol, prior to processing for H&E staining. Much greater crystal formation and tissue distortion (contraction) were observed in standard cryoprotectant (panel A) than in PDX-CP (panel B). Green arrows show examples of crystal formation.

Lung tissue architecture maintained after thawing. Human lobectomy tissue cubes (IRB designation BAAT001 and SPYR008) were directly fixed in 4% paraformaldehyde at 4°C (A-C), fixed after expansion/perfusion with pseudo-diaphragmatic expansion-cryoprotectant perfusion (PDX-CP; 1× v/v, 4 cc/min; D-F) or fixed after cryopreservation, storage for a week at −80°C and thawing (G-I) and processed for hematoxylin and eosin staining of 6 μm sections. No loss of tissue/cellular architecture was observed in bronchoalveolar regions. Of note, some damage to ciliated epithelium in the upper airways of thawed tissues was observed (compare arrow regions in I to C, F). (Scale bars: black, 500 μm; green, 200 μm; red, 50 μm).

Protein integrity maintained in thawed lung tissue. ‘Fresh’ patient lobectomy tissue cubes, prior to preservation (“PreCryo”) and post-thaw (“Cryo1” and “Cryo2”) were homogenized at 4°C in isotonic phosphate buffered saline containing protease inhibitors, solubilized in 4× Laemli sodium docecyl sulfate-polyacrylamide gel electrophoresis buffer and 5 μg total protein per sample run on a 4-12% Bis-Tris gel at 200 V using 3-(N-morpholino)propanesulfonic acid MOPS buffer (bottom). Analysis of the protein profiles (middle, individually; top, overlay) showed only minor changes in protein profiles suggesting minimal degradation or protein modifications as a consequence of preservation and thawing.

Cell viability in fresh vs. thawed lung tissue. Human lung lobectomies (BAAT004, A and C; SPYR008, B and D) were enzymatically digested for cell isolation from fresh samples (A and B) or thawed after 14 days stored at −80°C samples (C and D). Cells from digested cubes were plated in suspension for 24 h prior to MitoTracker (Green) FM labeling of viable cells and nuclear counterstain (red). For BAAT004, we observed 81% viability and for SPYR008, 53% viability in thawed vs. fresh samples, with a greater fraction of dead cells in the latter (which may reflect more fibrotic tissue).

Viability of cryopreserved lung cells from a patient with idiopathic pulmonary fibrosis. Explanted lungs from a transplant patient with lung fibrosis were cryopreserved using pseudo-diaphragmatic expansion-cryoprotectant perfusion (PDX-CP). One cryovial containing three 125 mm3 cubes was thawed after 14 days in storage at −80°C and processed with 4% neutral buffered paraformaldehyde (PFA) fixation and H&E staining (A), or placed directly in a gelatin-coated dish for outgrowth culture (B), or enzymatically dissociated, plated in culture (C-E) and passaged (F, G) to expand multiple cell types. Panel H: Primary cells derived from PDX-CP cryopreserved tissue express cell-specific genes in culture. Reverse transcription polymerase chain reaction RT-PCR products separated by polyacrylamide gel electrophoresisPAGE. Samples were taken from a IPF patient explant during transplant surgery and cryopreserved; then later, thawed and enzymatically-dissociated before culturing. RNA was extracted from four samples for analyses: A: whole lung tissue (RNA extracted before cryopreservation); B, alveolar epithelial cell culture (SAEC medium, 1-week post-thawing/dissocation); C: alveolar cell culture (2-week post-thawing/dissociation); ADP-RT: housekeeping gene/normalization control (ARF1); SPB: surfactant protein B, type II pneumocyte marker (and possibly, bronchiolar Clara cells); SPC: surfactant protein C, type II pneumocyte marker; collagen: fibroblast marker (COL1A1); AQP5: aquaporin 5, type I pneumocyte marker. Last lane is a molecular weight marker. Panel I: Stem cells can be cultured from PDX-CP cryopreserved lung tissue. Enzymatically-dissociated cells (same IDF patient as Panel H) were cultured for seven days in mTeSR1 medium and RNA subsequently analyzed for expression of stem cell cell markers octamer-binding transcription factor 4 (OCT4) (2 separate primer sets used to amplify all transcript variants) or Nanog homeobox (NANOG)by RT-PCR. Prominent band in 25-bp marker (lane 1) is 125 bp.

Utility of differential gel electrophoresis (DIGE) to detect unique and minute differences in protein expression in lung cells as a function of specimen source or environment. A: Two-color image of 24 cm DIGE gel of porcine (Cy3-labeled, green) and pygmy sperm whale (Cy5, red) lung cells using pH 4-7 IPG strips. Expanded box indicates region of high protein profile similarity, and with a major significant difference in expression of one protein (red arrow). B and C: The expanded regions of porcine (below magenta bar) and pygmy sperm whale (below cyan bar) samples run in triplicate (3 DIGE gels) Protein spot represented in expanded box in A with significantly differing expression levels (D) between species is bordered with blue line.

N-Linked glycan expression in lung tissue as determined by matrix-assisted laser desorption mass spectrometric imaging. Lung tissue embedded in optimized cutting temperature compound was frozen-sectioned (10 μm) and mounted on ITO conductive slides, and processed with a series of ethanol washes. Processed slides were sprayed with protein N-glycanase F, incubated for 2 h at 37°C in a humidified chamber, followed by matrix application (2,5-dihydroxybenzoic acid (DHB) 30 mg/ml). Tissues were imaged using a Bruker Autoflex III mass spectrometer in reflection mode, and analyzed in FlexImaging (Bruker Daltonics). m/z Peak profiles for one tissue are shown (A: 1025.432 Da; B: 1046.394 Da; C: 1237.267 Da; D: 1449.516 Da).