TITLE: OSU TriState SenNet Processing and Storing of Explanted IPF Lungs
AUTHORS: Sean D. Stacey, Brenda F. Reader, Lorena Rosas, Victor Peters, Ana L Mora, Mauricio Rojas

[DESCRIPTION]
This protocol describes the processing and storing of explanted idiopathic pulmonary fibrosis (IPF) lungs by the Comprehensive Transplant Center (CTC) Human Tissue Biorepository before arrival at the TriState SenNet Biospecimen Core of The Ohio State University that functions as a part of the Cellular Senescence Network Program (SenNet). This protocol is based on the CTC Human Tissue Biorepository's standard operating procedure established for tissue collection for explant and donor samples.  We follow routine laboratory specimen processing guidelines to ensure the highest biospecimen quality.

[STEPS]
SECTION: Objective
1. To preserve lung tissue for further downstream cellular, protein, RNA, or DNA analyses.
SECTION: Preparation
2. Use appropriate PPE that includes nitrile gloves and disposable gown or washable lab coat.
SECTION: Preparation
3. Place three underpads and all needed equipment, including biohazard, receptacles, surgical kit, and specimen holders in the biosafety cabinet (BSC) and turn on. 
SECTION: Preparation
4. Lungs come from the OR separated left from right lungs in sterile plastic containers. Place container inside the BSC, remove lungs from containers, place on the underpad, and photograph lungs prior to sampling. The photograph should show both lungs with their respective lobes, front and back. 
SECTION: Preparation
5. Samples are collected from the area most representative to the disease. IPF lung tissue is fibrotic and this is characterized by the thickening and scarring of the tissue that feels hard and stiff to the touch. Identify the most representative areas in the upper and lower lobes of a lung. 
SECTION: Preparation
6. Resect a 10 cm x 10 cm x 2 cm (L x W x H) piece of parenchymal tissue from the representative sections of the upper and lower lobes and set aside.
SECTION: Preparation
7. Collect four pulmonary lymph nodes around the primary bronchus in 5mL cryovials.
SECTION: Freezing and storing
8. The following procedure is performed for the upper lobe and the lower lobe.
SECTION: Freezing and storing
9. Cut six small pieces of tissue (4mm3) from each piece of parenchyma and place onto the internal sides of a labeled cryovial using forceps. Collect ten cryovials of parenchyma from the upper and lower lobes.
SECTION: Freezing and storing
10. Lower the cryovials into liquid nitrogen and leave for 5 minutes. 
SECTION: Freezing and storing
11. Remove the cryovials from liquid nitrogen and store in the -80°C freezer.
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
12. The following procedure is performed for the upper lobe and the lower lobe.
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
13. For FFPE preserved samples: 
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
13.1. Add 3mL 10% neutral buffered formalin (NBF) to the 5mL cryovial containing lymph nodes
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
13.2. Cut four 1.25 cm3 pieces of tissue from each piece of parenchyma and place into a labeled 20 mL glass scintillation vial filled halfway with NBF. Top off NBF if needed and protect from light. Samples must be submerged for at least 24 hours and no longer than 72 hours to ensure complete saturation and to prevent overfixation.
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
13.3. Remove tissue from NBF and use a scalpel to cut the tissue into <2mm thickness pieces that are small enough to fit into the tissue cassette.
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
13.4. Enclose tissue into the cassettes and then submerge in 70% ethanol. Histology is performed using standard methods.
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
14. For OCT preserved samples: 
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
14.1. Cut tissue into 1.25 cm3 pieces and transfer tissue into 4% paraformaldehyde for 24 hours. 
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
14.2. Transfer to 30% of sucrose for 24 hours and then embed in a mold with OCT and dry ice.
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
14.3. Store in -80°C freezer.



SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
15. For Lipidomics and Proteomics analyses:
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
15.1. Cut the tissue into 1 x 1 cm pieces for freezer preservation in 75% of Hydroxypropyl
methylcellulose (HPMC) and 2.5% of polyvinylpyrrolidone (PVP) at -80°C. 
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
15.2. Embed in a mold with HPMC-PVP.
SECTION: Processing formalin fixed paraffin embedded (FFPE) and OCT samples
15.3. Store in a -80°C freezer.