title: Cryopreservation of viable human lung tissue for versatile post-thaw analyses and culture.
abstract: Clinical trials are currently used to test therapeutic efficacies for lung cancer, infections and diseases. Animal models are also used as surrogates for human disease. Both approaches are expensive and time-consuming. The utility of human biospecimens as models is limited by specialized tissue processing methods that preserve subclasses of analytes (e.g. RNA, protein, morphology) at the expense of others. We present a rapid and reproducible method for the cryopreservation of viable lung tissue from patients undergoing lobectomy or transplant. This method involves the pseudo-diaphragmatic expansion of pieces of fresh lung tissue with cryoprotectant formulation (pseudo-diaphragmatic expansion-cryoprotectant perfusion or PDX-CP) followed by controlled-rate freezing in cryovials. Expansion-perfusion rates, volumes and cryoprotectant formulation were optimized to maintain tissue architecture, decrease crystal formation and increase long-term cell viability. Rates of expansion of 4 cc/min or less and volumes ranging from 0.8-1.2 × tissue volume were well-tolerated by lung tissue obtained from patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis, showing minimal differences compared to standard histopathology. Morphology was greatly improved by the PDX-CP procedure compared to simple fixation. Fresh versus post-thawed lung tissue showed minimal differences in histology, RNA integrity numbers and post-translational modified protein integrity (2-dimensional differential gel electrophoresis). It was possible to derive numerous cell types, including alveolar epithelial cells, fibroblasts and stem cells, from the tissue for at least three months after cryopreservation. This new method should provide a uniform, cost-effective approach to the banking of biospecimens, with versatility to be amenable to any post-acquisition process applicable to fresh tissue samples.
authors: Department of Pediatrics, Medical University of South Carolina, Charleston, SC, U.S.A. baatzje@musc.edu.
authors: John E
authors: JE
authors: Baatz
authors: Department of Pediatrics, Medical University of South Carolina, Charleston, SC, U.S.A.
authors: Danforth A
authors: DA
authors: Newton
authors: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, U.S.A.
authors: Ellen C
authors: EC
authors: Riemer
authors: Department of Surgery, Medical University of South Carolina, Charleston, SC, U.S.A.
authors: Chadrick E
authors: CE
authors: Denlinger
authors: Department of Pharmacology, Medical University of South Carolina, Charleston, SC, U.S.A.
authors: E Ellen
authors: EE
authors: Jones
authors: Department of Pharmacology, Medical University of South Carolina, Charleston, SC, U.S.A.
authors: Richard R
authors: RR
authors: Drake
authors: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, U.S.A.
authors: Demetri D
authors: DD
authors: Spyropoulos
mesh_terms: D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods* / Q000592:standards; D003451:Cryoprotective Agents; D020869:Gene Expression Profiling; D006801:Humans; D008168:Lung; D055550:Protein Stability; D040901:Proteomics; D020871:RNA Stability; D014015:Tissue Banks; D046509:Tissue Culture Techniques
chemical_list: D003451:Cryoprotective Agents
keywords: Cryopreservation; biobank; biorepository; lung