TITLE: OSU TriState SenNet Processing and Storing of a Normal Donor Heart
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 normal donor heart by the Comprehensive Transplant Center (CTC) Human Tissue Biorepository and 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 heart tissue for further downstream cellular, protein, RNA, or DNA analyses.
SECTION: Preparation
2. In the biosafety cabinet (BSC), place three underpads and all needed equipment, including biohazard receptacles, surgical kits, and specimen holders. Before beginning processing samples, check that all appropriate personal protective equipment is donned and that BSC airflow is on.
SECTION: Preparation
3. A donor heart comes triple-bagged in HTK solution or saline from our local organ procurement organization (Lifeline of Ohio). Place the container holding the specimen inside the BSC, remove the heart from containers, and place the heart on the underpad.
SECTION: Preparation
4. For normal donor samples, the heart tissue is reddish-purple in color with white chordae located within. Healthy tissue feels firm and robust. When processing normal donor tissue, look for the healthiest region to sample. The donor’s CT or chest X-ray can provide information on selecting the healthiest regions. Select the sites from which samples will be collected and annotate them in the notebook.
SECTION: Preparation
5. From the heart, remove a 2 cm3 piece of tissue from the septum, left and right ventricles, and the left and right atrium. 
SECTION: Freezing and storing
6. Cut 7 small pieces of tissue (4mm3) from each region (septum, right and left ventricles, right and left atrium), and place them onto the internal sides of a labeled cryovial using forceps. Collect cryovials of heart tissue from each region.
SECTION: Freezing and storing
7. Lower the cryovials into liquid nitrogen and leave for 5 minutes. 
SECTION: Freezing and storing
8. After freezing, remove the cryovials from liquid nitrogen and promptly store them in a -80°C freezer.
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
9.  For FFPE sample storage: 
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
9.1. Add mL 4% paraformaldehyde (PFA) into a labeled 50ml conical tube, and place two 25 cm3 pieces of tissue from each region of the heart into separate tubes.
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
9.2. Keep samples away from light at 4°C for 24 hours. After 24 hours, remove the PFA and replace with 70% ethanol for another 24 hours. 
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
9.3. Remove the tissue from PFA and cut the tissue into a < 2mm thick piece that is small enough to fit into the tissue cassette. Tissue should be 1.5 cm x 1.25 cm.
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
9.4. Enclose the tissue into the cassette, and then fully submerge the cassette in a 50 mL conical with 70% ethanol.
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
9.5. Embedding and histology are performed using standard methods.
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
10. For OCT embedded samples:
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
10.1. Cut the tissue into 1 cm x 1 cm pieces. 
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
10.2. Transfer the piece of tissue into 4% PFA. Keep samples away from light at 4°C for 24 hours. 
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
10.3. After hours, transfer to a 30% sucrose and PBS solution at 4°C for 24 hours, and then embed in a mold with OCT.
SECTION: Storing for Formalin-fixed Paraffin-embedded (FFPE) and OCT sample processing
10.4. Store samples in a -80°C freezer.