TITLE: Slow freeze (cryopreservation) protocol for human ovarian tissue
AUTHORS: Elizabeth L Tsui, Monica M Laronda, Hannah McDowell

[DESCRIPTION]
Purpose: This protocol describes the procedure for cryopreservation of organ donor derived human ovarian tissue via slow freeze with a semi-automated controlled rate freezer in a research setting. 



[GUIDELINES]
**CRITICAL**Personnel who process and handle human specimens must adhere to Office of Research Safety guidelines at their institution. They must be up to date on their original and refresher courses for the following training (or equivalent):
1. Biosafety Certification
2. Bloodborne Pathogens Certification
3. Training in Filling and Maintaining Liquid Nitrogen Tanks (as appropriate for LN2 use)
4. Working with Formaldehyde Certification (as appropriate for fixation)
5. CITI training (highly encouraged)

[STEPS]
SECTION: Equipment set up
1. The controlled rate freezer has three components: computer, temperature controller - measures temperature of the tube rack, and cryobath (with internal tube rack suitable for cryovials)

Set up programmable freezer by opening the relevant freezer software and programming the controlled rate freezer as follows: 

1. Hold at 4 °C  for 5 min
2. Cool at 2 °C from +4 °C to -7 °C 
3. ALARM  "Seed Now." Press "pause" and allow for manual seeding (described below) before continuing with the protocol (usually indicated by pressing "run"). 
4. Hold for 10 min at -7 °C (Allowing for ice crystal formation)
5. Cool at -0.3 °C from -7 °C to -35 °C 
6. Alarm  "Plunge after -40 °C 


 


SECTION: Preparing the tissue for slow freeze
4. For adequate penetration of cryoprotectant, tissue pieces used for slow freeze should be no larger than   in any dimension. Prior to transfer to cryovials, tissue must be kept in appropriate holding media buffered for air, such as OFC Holding Media or Leibovitz-15, and at 2-4 °C to prevent the tissue from drying out. 

All following steps for this section should be performed at 2 °C to 4 °C . Maintain all steps at this temperature and reduce warming of tissues by handling cryovials with long forceps. 

SECTION: Equipment set up
3. Fill the cryobath with liquid nitrogen and gently lower in the internal tube rack to allow the internal tube rack to get to the protocol's starting temperature (4 °C ). 

Ensure that the cryobath has a minimum of 2 inches of liquid nitrogen at all times throughout the protocol to allow for appropriate cooling.  Keep a back up dewar filled with liquid nitrogen to aid with filling. 


 

SECTION: Equipment set up
2. Start the protocol using the controlled rate freezer software.
SECTION: Preparing the tissue for slow freeze
5. Prepare and label 2.0 mL cryovials with 1 mL 4 °C of cooled Freezing Media. Ensure that labels do not obscure the meniscus. 

SECTION: Preparing the tissue for slow freeze
6. Place the tissue pieces processed for slow freeze in the appropriate cryovials and verify that tissue sinks into the Freezing Media. 

A maximum of 4 tissue pieces processed for slow freeze can be stored in each vial, but ensure that anatomic position is accurately noted, and that there is at least 2x volume of Freezing Media to tissue volume. 
SECTION: Preparing the tissue for slow freeze
7. Allow tissue pieces to equilibrate in cryovial with 4 °C cold Freezing Media for 30 min. 


 


SECTION: Freeze program
8. When the internal tube rack has reached 4 °C  and the tissue in labeled cryovials have equilibrated for 30 min at 4 °C , add cryovials to internal tube rack, and begin the freeze protocol. 


 


SECTION: Freeze program
9. Allow freeze program to proceed as described above. 
SECTION: Freeze program
10. Seeding cryovials. 

At -7 °C , the freezer will alarm to alert the user to "SEED NOW." 

To "seed" cryovials means to generate a small piece of ice within the tube in order to allow for full ice crystal formation throughout the frozen vial.  
SECTION: Freeze program
10.1. Press "Pause" on the program to allow for time to seed. This should occur within 5 min

SECTION: Freeze program
10.2. Dip a long cotton swab into the liquid nitrogen, hold for a few seconds, then remove and press the cold swab to the side of cryovial at the meniscus. 

If the seed is successful, you will see a small ice crystal bloom at the meniscus. 

 


 

  

SECTION: Freeze program
11. Allow for the remainder of the freeze program to proceed. 


 

SECTION: Freeze program
10.3. Repeat for all remaining cryovials. 
SECTION: Freeze program
10.4. When finished, press "Run" to resume freeze program. 
SECTION: Freeze program
12. When the protocol is complete, the freezer will alarm, and the internal tube rack's temperature will freefall from -35 °C . Once the tube rack has reached -40 °C or colder, use long forceps to remove each cryovial and submerge each cryovial in liquid nitrogen by plunging liquid nitrogen. 

Repeat for all remaining cryovials. From this point on, the cryovials should be kept in liquid nitrogen storage.  

SECTION: Long-term storage
13. After all cryovials are plunged into liquid nitrogen, transfer to liquid nitrogen storage in a pre-cooled storage container. Cryovials should be stored in the liquid phase of the liquid nitrogen storage tank (or colder than -150 °C) for long-term storage. 

SECTION: Long-term storage
14. Allow controlled rate freezer to come to room temperature and dry prior to storage or next use.