TITLE: Human ovarian tissue explant cultures (static or fluidic conditions)
AUTHORS: hannah.anvari, pooja.devrukhkar, francesca.e.duncan francesca.duncan

[DESCRIPTION]
The Cellular Senescence Network (SenNet) was recently established to map senescent cells in the human body. As part of this initiative, our goal is to profile senescent cells in the human ovary in vivo as well as ex vivo using a microphysiological platform called LATTICE. Human ovarian tissue is collected through the Northwestern University Reproductive Tissue Library.  Tissue is collected from women undergoing bilateral salpingo-oophorectomies or hysterectomies with absence of ovarian neoplasia. With this model, we are generating a molecular signature of Doxorubicin-induced cellular senescence in static and fluidic culture which will then be mapped onto human ovaries across an aging series.  To do this, human ovarian explants (cortex and medulla) are cultured in Doxorubicin (0.1 μg/ml) for 24 hours followed by up to 11 days of culture.       

[GUIDELINES]
Researchers working with human specimens will adhere to all safety and training protocols required by the institution (Northwestern Medicine/Northwestern University) including but not limited to:    

Biosafety Certification
Bloodborne Pathogens Certification
Working with Formaldehyde Certification
Collaborative Institutional Training Initiative (CITI program) certification

[STEPS]
SECTION: Processing human ovarian tissue for ovarian explant culture
1.  
SECTION: Processing human ovarian tissue for ovarian explant culture
2. Ovarian tissue samples are collected from the Northwestern Pathology department and placed immediately on ice following collection. The research coordinator will bring research specimens to the lab on ice (2-4 °C). Samples should be transported in a blue biohazard labeled cooler bag (Fig. 1A, B). Tissue should be kept at 2-4°C throughout all processing steps and should be processed within 24 hours of procurement. Ovarian tissue is kept in ORIGIO Handling medium throughout procurement and processing up until the transfer to culture growth media (Fig. 1C).
SECTION: Processing human ovarian tissue for ovarian explant culture
3. On arrival, ovarian tissue should be measured for length, width, and thickness (Fig. 1D). Also note the approximate anatomy of the cortex and the medulla (Fig. 1E). The cortex should be on the outermost edge surrounding the entire tissue piece and will have a glossy, somewhat translucent appearance. The medulla should be the innermost portion if the ovarian tissue slice is lying flat and will likely contain fibrotic-looking, white tissue, especially in postmenopausal ovaries.
SECTION: Processing human ovarian tissue for ovarian explant culture
4. Cut the ovarian tissue along the cortex and medulla region border, leaving approximately 2-3 mm between the edge of the piece and the cut surface (Fig. 1F). This cut should result in a piece containing both the cortex (the outer translucent looking layer), and the medulla which will be softer and less rigid when held with forceps.
SECTION: Processing human ovarian tissue for ovarian explant culture
5. Place the cortex-containing portion flat on the center well of the Stadie-Riggs tissue slicer with the cortex side facing up (Fig. 1G).
SECTION: Processing human ovarian tissue for ovarian explant culture
6. Smoothly run the blade through the Stadie-Riggs tissue slicer (Fig. 1H). Avoid jagged movements as much as possible as it will result in uneven tissue slices. The first slice will yield one 500 µm slice of the ovarian cortex (Fig. 1I). The second slice can be discarded as this consists of both cortex and medulla tissue. The third slice is the ovarian medulla and can be treated like the first slice for culture preparations. Additional medulla slices and remaining tissue can also be fixed for histology.
SECTION: Processing human ovarian tissue for ovarian explant culture
7. Add 2-3 mL of ORIGO Handling Medium, to a petri dish containing a tissue-slicing plastic disc (Fisher Scientific, 1018001) (Fig. 1J).
SECTION: Processing human ovarian tissue for ovarian explant culture
8. At room temperature under a dissection scope, manually cut the ovarian tissue slice into 1mm x 1mm squares in the droplet of ORIGO Handling Medium (Fig. 1J)
SECTION: Processing human ovarian tissue for ovarian explant culture
9. Once all ovarian tissue pieces (1 mm x 1 mm x 0.5 mm) are cut, submerge them in sufficient ORIGIO Handling medium to ensure they do not dry out during culture plate preparation.
SECTION: Processing human ovarian tissue for ovarian explant culture
10. Cut ovarian pieces can be transferred to equilibrated α-MEM-based media with supplements and then should be loaded onto cell culture inserts (Millipore Sigma, PICM01250) in groups of three separately as cortex (Fig. 1K) and medulla (Fig. 1L). Follow detailed procedure outlined for ovarian explant cultures to load tissues and maintain culture.
SECTION: Human ovarian explant culture
39.   

SECTION: Human ovarian explant culture
0.39. Before loading tissues into a standard 24-well plate add 400 µL of growth media to each well the tissue will be cultured. Any remaining wells should be filled with equal amounts of 1 x PBS. This can be done either at the time of culture if media is equilibrated in 60 mm dishes or ahead of time when equilibrating the media.
SECTION: Human ovarian explant culture
0.39. Once 1 mm x 1 mm x 0.5 mm pieces of ovarian cortex and medulla are cut, transfer to a new 60 mm dish with ORIGIO handling media such that the tissue pieces is completely submerged/floating in media.
SECTION: Human ovarian explant culture
0.39. Cut the tip (approximately 1 cm) off of a 1000 µL pipette tip using a No. 10 scalpel.
SECTION: Human ovarian explant culture
0.39. Carefully load three pieces of tissue to each culture transwell (Millipore Sigma, PICM01250) as follows: sequentially take up some media (~100 µL) followed by a tissue piece with some media, and then take up an additional 100-200 µL of media into the pipette (Fig. 2A). Hover the pipette tip over the base of the transwell membrane and flick the edge so that the tissue pieces gather at the very bottom of the pipette tip in a bubble of media as seen in Fig. 2A. Gently touch the media/tissue bubble that bulges out of the tip (not the actual pipette tip as this could perforate the transwell and result in flooding of the tissues) to release a droplet with minimal media and tissue pieces in the center of the well. 
SECTION: Human ovarian explant culture
0.39. If necessary, remove any excess media using a 200 µL pipette making sure to never touch the transwell with the pipette tip as it may perforate the membrane. 
SECTION: Human ovarian explant culture
0.39. After placing the ovarian pieces into the transwell the tissue should be resting in the center of the wells with a small droplet of media covering the tissues in each well of a 24-well plate (Fig. 2B-C). The droplet should not exceed 50 µl and should not extend more than 1-2 mm beyond the tissue edges.
SECTION: Human ovarian explant culture
0.39. Culture the tissues at 37°C with 5% CO2 (atmospheric O2 about 21% )for anywhere from one to eleven days.
SECTION: Human ovarian explant culture
0.39. For induced senescence with doxorubicin, doxorubicin is added to the culture media at a concentration of  0.01 µg/ml (Fisher Scientific, 22-521-0) for the first 24 hours of culture, regardless of the culture endpoint.
SECTION: Human ovarian explant culture
0.39. After 24 hours, remove the plate from the incubator and wash twice with standard growth media. Washes can be performed by lifting the transwell with curved forceps, make sure not to puncture the membrane when lifting. Then remove the full volume of media, ~400 µL. Replace with 400 µL of equilibrated growth media without doxorubicin. Repeat twice and then return to the incubator for the required time points. Ensure at this point that the droplet is intact, if the well has flooded, remove excess media with a 200 µL pipette without touching the surface of the membrane. If the tissue is dry add a small droplet of media, no more than 50 µL in volume.
SECTION: Human ovarian explant culture
0.39. Half volume changes of media should be performed every other day (200 µL) in each well containing tissue. This can be done as detailed in step 10.
SECTION: Human ovarian explant culture
0.39. When the culture period ends, remove the tissues from the inserts by carefully peeling away the mesh bottom with sterile forceps. With curved forceps, hold the edge of a transwell and with straight forceps, pierce the edge of the membrane so it breaks away from the plastic edge. Peel the remaining membrane away from the edge.
SECTION: Human ovarian explant culture
0.39. Add the mesh membrane with the tissue pieces adhered to the center to a 1.5 mL microcentrifuge tube with 1 mL of Modified Davidson’s Fixative Solution (Cat. #64133-50; Electron Microscopy Sciences, Hatfield, PA). If freezing the tissues, place the mesh bottom into a dish of PBS and then carefully move the tissue pieces to a dry 1.5 mL microcentrifuge tube using a similar technique used to load the tissues onto the wells: take up some PBS and tissue (100-200 µL) into the cut tip of a 1000 µL pipette tip. Expel the contents into a dry 1.5 mL microcentrifuge tube and then remove excess PBS with a 200 µL pipette tip before placing them on dry ice. Store at -80°C.
SECTION: Ovarian explant culture - Media and plate preparation
11. Growth media should be equilibrated for 4-6 hours before the tissue is received (Media contains MEM Alpha (1X) + GlutaMAX (Thermo, 32561037), 20 mIU ml−1 recombinant Follicle-Stimulating Hormone, 1 mg/mL fetuin, 1 μg/mL Insulin-Transferrin-Sodium Selenite supplement, and 30 mg/10mL human serum albumin.
SECTION: Ovarian explant culture - Plate loading and culture
13. Once 1 mm x 1 mm x 0.5 mm pieces of ovarian cortex and medulla are cut, transfer to a new 60 mm dish with ORIGIO handling media such that the tissue pieces is completely submerged/floating in media.
SECTION: Ovarian explant culture - Plate loading and culture
16.   

SECTION: Ovarian explant culture - Media and plate preparation
12. Before loading tissues into a standard 24-well plate add 400 µL of growth media to each well the tissue will be cultured. Any remaining wells should be filled with equal amounts of 1 x PBS. This can be done either at the time of culture if media is equilibrated in 60 mm dishes or ahead of time when equilibrating the media.
SECTION: Ovarian explant culture - Plate loading and culture
14. Cut the tip (approximately 1 cm) off of a 1000 µL pipette tip using a No. 10 scalpel.
SECTION: Ovarian explant culture - Plate loading and culture
15. Carefully load three pieces of tissue to each culture transwell (Millipore Sigma, PICM01250) as follows: sequentially take up some media (~100 µL) followed by a tissue piece with some media, and then take up an additional 100-200 µL of media into the pipette (Fig. 2A). Hover the pipette tip over the base of the transwell membrane and flick the edge so that the tissue pieces gather at the very bottom of the pipette tip in a bubble of media as seen in Fig. 2A. Gently touch the media/tissue bubble that bulges out of the tip (not the actual pipette tip as this could perforate the transwell and result in flooding of the tissues) to release a droplet with minimal media and tissue pieces in the center of the well.

SECTION: Ovarian explant culture - Plate loading and culture
17. If necessary, remove any excess media using a 200 µL pipette making sure to never touch the transwell with the pipette tip as it may perforate the membrane. 
SECTION: Ovarian explant culture - Plate loading and culture
18. After placing the ovarian pieces into the transwell the tissue should be resting in the center of the wells with a small droplet of media covering the tissues in each well of a 24-well plate (Fig. 2B-C). The droplet should not exceed 50 µl and should not extend more than 1-2 mm beyond the tissue edges.
SECTION: Ovarian explant culture - Plate loading and culture
19. Culture the tissues at 37°C with 5% CO2 (atmospheric O2 about 21% )for anywhere from one to eleven days.

SECTION: Ovarian explant culture - Plate loading and culture
20. For induced senescence with doxorubicin, doxorubicin is added to the culture media at a concentration of  0.1 µg/ml (Fisher Scientific, 22-521-0) for the first 24 hours of culture, regardless of the culture endpoint.
SECTION: Ovarian explant culture - Plate loading and culture
21. After 24 hours, remove the plate from the incubator and wash twice with standard growth media. Washes can be performed by lifting the transwell with curved forceps, make sure not to puncture the membrane when lifting. Then remove the full volume of media, ~400 µL. Replace with 400 µL of equilibrated growth media without doxorubicin. Repeat twice and then return to the incubator for the required time points. Ensure at this point that the droplet is intact, if the well has flooded, remove excess media with a 200 µL pipette without touching the surface of the membrane. If the tissue is dry add a small droplet of media, no more than 50 µL in volume.

SECTION: Ovarian explant culture - Plate loading and culture
22. Half volume changes of media should be performed every other day (200 µL) in each well containing tissue. This can be done as detailed in Step 10.
SECTION: Ovarian explant culture - Plate loading and culture
23. When the culture period ends, remove the tissues from the inserts by carefully peeling away the mesh bottom with sterile forceps. With curved forceps, hold the edge of a transwell and with straight forceps, pierce the edge of the membrane so it breaks away from the plastic edge. Peel the remaining membrane away from the edge

SECTION: Ovarian explant culture - Plate loading and culture
24. Add the mesh membrane with the tissue pieces adhered to the center to a 1.5 mL microcentrifuge tube with 1 mL of Modified Davidson’s Fixative Solution (Cat. #64133-50; Electron Microscopy Sciences, Hatfield, PA). If freezing the tissues, place the mesh bottom into a dish of PBS and then carefully move the tissue pieces to a dry 1.5 mL microcentrifuge tube using a similar technique used to load the tissues onto the wells: take up some PBS and tissue (100-200 µL) into the cut tip of a 1000 µL pipette tip. Expel the contents into a dry 1.5 mL microcentrifuge tube and then remove excess PBS with a 200 µL pipette tip before placing them on dry ice. Store at -80°C.