TITLE: Human Placenta Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC
AUTHORS: Valentina Stanley, Scott Lindsay-Hewett, Donald P. Pizzo, Louise Laurent, Mana Parast

[DESCRIPTION]
Human Placenta Tissue collection and storage protocol for HuBMAP's UCSD Female Reproductive TMC.

[STEPS]
SECTION: Preparation
1. Collect placenta from delivery room within 1 hour of delivery.
SECTION: Preparation
2. Place placenta into bucket and photograph prior to sampling.
SECTION: Preparation
3. Select 2 sites (A and B) from which samples will be collected. Aim for sites to be equidistant from the cord, avoid the edges, avoid areas with fibrin deposits/infarcts/necrosis and/or large blood vessels.
SECTION: Preparation
4. From site A, cut a circle all the way down from the fetal to the maternal surface, about one inch in diameter.
  

SECTION: Preparation
5. Wash the full-thickness core in cold PBS in a petri dish. Keep the fetal surface pointing up.
      

SECTION: Preparation
6. Divide the full core in half vertically, from fetal to maternal surface.
  

SECTION: Flash freezing and storing in RNAlater
7. With the left half of site A, divide the core horizontally into fetal (F), central (C) and maternal (M) regions.
   

SECTION: Flash freezing and storing in RNAlater
7.1. Cut each of the three regions from the previous step into 6 small chunks for a total of 18 chunks. Chunks should be small enough to just cover the bottom of a microfuge tube (~5mm x 5mm x 5mm). Collect tissue directly adjacent to the fetal and maternal membranes but do not cut from the membranes themselves.
  

SECTION: Storing in formalin and MACS
8. With the right half of site A, divide the tissue into 4 quadrants so that you have 2 fetal chunks and 2 maternal chunks, keeping all the membranes intact. Some of the central region tissue may need to be trimmed off if the core is very thick.
  

SECTION: Storing in formalin and MACS
9. Place one fetal piece and one maternal piece into the 10% formalin tubes (to be processed into FFPE blocks). Place the other fetal and maternal piece into cold MACS tissue buffer. Store in 4C fridge.
  

SECTION: Embedding in OCT
10. Alternatively, if not collecting into MACS tissue buffer, freeze the other fetal and maternal piece in OCT.
SECTION: Embedding in OCT
10.1. Fill plastic cryomold halfway with OCT compound being careful not to create bubbles.
SECTION: Embedding in OCT
10.2. Place fetal tissue (membrane side pointing up) in one mold and maternal tissue (membrane side pointing down) in another mold.
SECTION: Embedding in OCT
10.3. Cover tissue with a thin layer of OCT compound being careful not to create bubbles. 
11. Repeat Steps 4-10 with placenta Site B.
SECTION: Embedding in OCT
10.4. Slowly lower the mold into liquid nitrogen until the whole block freezes, then store in -80C freezer.
12. Take photos of the placenta after both sites (A and B) are removed. Measure the distance from the cord insertion to each sampling site.
SECTION: Flash freezing and storing in RNAlater
7.2. Dab the small chunks on a sterile gauze pad and place each one into a labeled microfuge tube.
SECTION: Flash freezing and storing in RNAlater
7.3. Place 9 small chunks (3 fetal, 3 central, 3 maternal) into the empty tubes to be snap frozen. Drop tubes into liquid Nitrogen. Leave for ~2-10 minutes. Remove and store in -80C freezer.
SECTION: Flash freezing and storing in RNAlater
7.4. Place the other 9 chunks (3 fetal, 3 central, 3 maternal) into the RNAlater filled tubes. Place RNAlater tubes into a 4C fridge. Allow to sit for 24-48 hours, then remove the RNAlater with a sterile transfer pipette and store in -80C freezer.
SECTION: FFPE processing
13. Tissue samples are removed from the formalin and rinsed in DI water. Water is changed multiple times until it is relatively clear and free of extraneous blood.
SECTION: FFPE processing
14. Place basket in Station 2 of the Leica Processor
SECTION: FFPE processing
15. Set Processor to program #1 (stations and timing listed below) and start time.
The following steps for dehydration and clearing are used:
NOTE: All steps are under vacuum

STATION           TIME    TEMPERATURE             SOLUTION
2                      1hr       Room temperature        PRO-SOFT DEHYDRANT
3                      1hr       Room temperature        PRO-SOFT DEHYDRANT
4                      1hr       Room temperature        PRO-SOFT DEHYDRANT
5                      1hr       Room temperature        PRO-SOFT DEHYDRANT
6                      1hr       Room temperature        PRO-SOFT DEHYDRANT
7                      1hr       Room temperature        PRO-SOFT DEHYDRANT
8                      1hr       Room temperature        PRO-PAR CLEARANT
9                      1hr       Room temperature        PRO-PAR CLEARANT
10                    1hr       Room temperature        PRO-PAR CLEARANT
11                     1hr       57C                                TYPE 3 WAX
12                     1hr       57C                                TYPE 3 WAX
SECTION: FFPE processing
16. Transfer the cassettes from the basket to the Leica embedding station
SECTION: FFPE processing
17. Choose an appropriately sized embedding mold from the embedding mold tray
SECTION: FFPE processing
18. Using forceps, place the sample with the face of interest at the bottom of the mold
SECTION: FFPE processing
19. Fill the mold with wax from the dispenser
SECTION: FFPE processing
20. Place the cassette on top of the embedding mold and fill the cassette with wax (Type 9)
SECTION: FFPE processing
21. Place the embedding mold containing the tissue and cassette onto the cold plate to solidify
SECTION: FFPE processing
22. Once the wax has solidified (~ 20 minutes), use a utility knife to remove the cassette from the mold
SECTION: FFPE processing
23. Scrape off excess wax on the side of the blocks; Blocks are now ready to be sectioned or stored