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

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

[STEPS]
None. d.     Lower the mold slowly into liquid nitrogen until the whole block freezes, then store in -80C freezer
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.