Obstetrics, Gynecology & Reproductive Sciences

Human Ovarian Organ Culture

Reagents
	100% Ethanol 70% Ethanol Paraformaldehyde powder – kept at 4ƒ C NaOH pellets

Organ culture is a unique way to perform experiments on surgical tissues that can simulate the complete in vivo environment of the subject. In a cell line experiment, only one cell type is present unlike in the living subject where multiple cell types exist and interact with each other in a single tissue. In organ cultures, all the cells of the original environment are present and able to influence the others as occurs in vivo.

Buffers and Media
	Sterile 1X Phosphate Buffered Saline (PBS) DMEM F12 without phenol red, 10% charcoal serum, EGF (100ng/ml)
Paraformal dehyde Preparation
	1. Make fresh 4% paraformaldehyde and refrigerate. Label 100 ml glass bottle with “4% Paraformaldehyde” and with date. Do not use it if is older than one week 2. Fill bottle with 70 ml of ddH2O. Heat ddH2O to 60C in microwave. 3. Weigh 4 g of paraformaldehyde powder on scale. Place 1 pellet of NaOH into the heated ddH2O. Pour paraformaldehyde powder into bottle. 4. Close the bottle and shake until powder is dissolved. 5. Measure out 10 ml of 10X PBS (pH 7.4) and pour into bottle. Fill bottle to 100ml with ddH2O. 6. Fill glass vial with 4% paraformaldehyde and label as “Pre-culture control”. Store at 4(C for future use.
Tissue Preparation
	1. Obtain ovary tissue in media previously provided to surgical pathology in 50-ml Falcon tube. Begin tissue processing immediately. 2. Record surgical pathology number and age of patient in notebook. 3. Fill beaker 1/2 full of 100% Ethanol, place it under the hood with scissors and tweezers tips in the liquid. Flame instruments before use and periodically during use. Be sure to place beaker away from Bunsen burner to avoid any accidental fires. 4. Pour specimen and media into Petri dish. 5. After flaming instruments, begin to cut tissue into 1-2 mm3 (small enough to allow for adequate nutrition, yet large enough to use for immunohistochemistry). 6. After tissue is cut to appropriate size, remove media from Petri dish. Do so by tilting dish toward yourself and using a sterile pipette to scrape tissue pieces to bottom. Slowly tilt the dish away from yourself while removing the media away from the top of the dish being careful not to pipette any tissue pieces. Add 10 ml sterile PBS to dish of tissue.
Experimental Design and Hormonal Treatments
	1. Label 6 well plate with experimental design and record in notebook. It is best to label the bottom of the plate rather than the lid so there is no confusion when the lid is removed for media changes. 2. Add 5-ml media to each well. 3. Remove PBS from dish in the same fashion as the media removal. 4. Transfer tissue pieces into each well, making distribution as equal as possible. Make sure to reserve about 10-15 pieces of tissue for pre-culture control. 5. Hormone addition may be done directly to each well, or by creating a stock of media with the appropriate hormonal concentration. Make sure to use glass pipettes and to store stocks in sterile glass tubes when using steroid hormones.
Control Tissue
	1. Add ~ 10 pieces of tissue to the glass vial of paraformaldehyde and place on shaker in 4°C overnight. Make sure to flame tweezers after use. 2. Label cryotube as “Pre-culture Control” and add 3-4 small pieces of tissue. Place in liquid nitrogen tank for later RNA and protein isolation.
Storage and Maintenance
	1. Place on shaker in 37°C incubator with 5% CO2. The shaking motion allows for adequate nutrition, and it prevents tissue components such as fibroblasts from escaping. 2. Change media every 2 days. Do so by tilting plate towards yourself, using pipette to isolate tissue at bottom of well, tilting plate away, pipetting away old media from top of well.

Organ cultures should be kept no longer than 7 days. (See Note 1).