The following protocols and tips are for the vitrification of embryos from 2PN (two-pronuclear zygote) to blastocyst using our Vitrification Freeze Kit, Cryolock or CryoTip or HSV Security Vitrification Straws.

• Simplified Warming for CryoTip (PDF / Video)
• Tips for Embryo and Oocyte Warming for CryoTip
• Simplified Warming for HSV Device (PDF / Video)  
• Tips for Embryo and Oocyte Warming for HSV Device
• Simplified Warming for Cryolock (PDF / Video)

Do not begin warming procedure until you have a pre-equilibrated dish of appropriate culture medium supplemented with SSS or DSS at 20% (v/v), or HSA at 12mg/ml for final recovery of specimen(s). Have all necessary material, tools and equipment ready and easily accessible before starting procedure.

Simplified Warming Protocol with CryoTip

ALL PROCEDURES MUST BE PERFORMED AT ROOM TEMPERATURE (22 - 27°C).

1. 1. Select CryoTip(s) to be warmed from LN₂ storage and quickly transfer to LN₂ filled holding reservoir in preparation for warming procedure.

   2. Place LN₂ filled holding reservoir close to 37° C waterbath (minimum volume 500mL) and microscope for subsequent rapid manipulation.

   3. To set up warming dish: aseptically dispense (as shown in diagram):

   • One (1) 50 μL drop of TS
   • One (1) 50 μL drop of DS

   NOTE: For oocytes, dispense a minimum of 100µL of TS

   4. Quickly remove CryoTip from LN₂ and within 1 second fully immerse the device in the 37° C waterbath (>500 mL) and gently swirl device for 3 seconds. Swirling the device is critical to ensure the most rapid warming rate (+24,000°C/min).

   5. Remove CryoTip from the waterbath and promptly remove metal cover sleeve from device by firmly grasping the lower end of the cover sleeve and pulling away from the CryoTip. Gently wipe away any water with a sterile dry tissue ensuring the tip of the device is dry.

   6. Using sterile medical grade sharp scissors make Cut #1 below seal at wide end of CryoTip.

   7. Withdraw the plunger of the syringe (with connector attached) to the half way position. Gently attach CryoTip to Connector and syringe (or pipette).

   8. Place fine tip end over the prepared warming dish and quickly make Cut #2 above the seal at the fine end.

9. While visualizing under the microscope, dispense contents of CryoTip as a small drop directly adjacent to TS drop. Once you visualize the specimen(s), touch the CryoTip contents drop to the TS drop with end of CryoTip to mix. Set timer for 1 minute and leave undisturbed.

10. Transfer specimen(s) to DS for 4 minutes. Gently pipette specimens once to ensure complete rinse in DS.

11. During the 4 minute exposure in DS aseptically dispense two (2) 50 μL drops of WS (WS1, WS2).

12. Transfer specimen(s) to WS1 then WS2 for 4 minutes each undisturbed.

13. Transfer warmed OOCYTE(S) to pre-equilibrated culture medium with 20% (v/v) protein supplement or 12 mg/mL for recovery (2-3 hours to allow time for spindle reformation) prior to subsequent manipulations.

14. There are two options for warmed EMBRYO(S):

a) For immediate transfer to patient: transfer EMBRYO(S) to pre-equilibrated ‘transfer’ medium containing 20% (v/v) protein supplement or 12 mg/mL.

b) For further culture: transfer EMBRYO(S) to pre-equilibrated culture medium containing 20% (v/v) protein supplement or 12 mg/mL for a 3 hour recovery period. After recovery period transfer EMBRYO(S) to culture medium with 10% (v/v) protein and incubate accordingly until desired developmental stage has been reached for transfer to patient.

1. Set up thawing dishes (as shown in diagram):

• At 37°C: Aseptically dispense a minimum volume of  250 μL of TS and warm to 37°C (incubator without CO2) or on a heating stage at least 30 minutes prior to starting warming procedure.

Note: For oocytes, dispense a minimum of 1 mL of TS

2. Identify HSV Straw(s) to be warmed from LN₂ storage and quickly transfer to an LN₂ filled holding reservoir in preparation for warming procedure.

3. Place LN₂ filled holding reservoir in close proximity to the working area and stage of the microscope in order to achieve subsequent rapid manipulation from reservoir to TS.

4. Remove TS dish from 37°C incubator or heating stage and place it under focus on top of the microscope stage.

5. Lift the straw with forceps enough to expose the colored handling rod. Make sure the end with the specimen(s) remains immersed in the LN₂.

6. Use a Knipex (or other wire cutter device) to cut the straw at the height of the colored handling rod. The red cut-length guide on the Knipex should be positioned in maximum length position or removed.

• Alternatively, use fingers and thumb to spin the straw while making cutting movements with scissors, 10 mm under the top of the colored handling rod.

7. With one swift but controlled motion, quickly grab the handling rod and extract it out of the straw.

8. Immediately plunge the curved spatula (or gutter) of the handling rod into the 37°C TS and gently swirl to detach specimens from device and leave the oocyte or embryo for a total of 1 minute in the TS. After 30 seconds following the initial plunge, gently pipette the specimen (if floating) and place at the bottom of the TS drop/well.

Steps 9-12 must be performed at room temperature (22-27°C).

• At room temperature: Aseptically dispense one (1) 50 μL drops of DS on a sterile Petri dish

9. Transfer specimen(s) to DS for 4 minutes. Gently pipette specimens once to ensure complete rinse in DS.

10. During the 4 minute exposure in DS, aseptically dispense two (2) 50 μL drops of WS (WS1, WS2) as shown in diagram.

11. Transfer specimen(s) to each WS1 then WS2 for 4 minutes each, undisturbed.

12. Transfer warmed OOCYTE(S) to pre-equilibrated culture medium with 20% (v/v) protein supplement or 12 mg/mL for recovery (2-3 hours to allow for spindle reformation) prior to subsequent manipulations.

13. There are two options for warmed EMBRYO(S):

a) For immediate transfer to patient: transfer EMBRYO(S) to pre-equilibrated ‘transfer’ medium containing 20% (v/v) protein supplement or 12 mg/mL.

b) For further culture: transfer EMBRYO(S) to pre-equilibrated culture medium containing 20% (v/v) protein supplement or 12 mg/mL for a 3 hour recovery period. After recovery period transfer EMBRYO(S) to culture medium with 10% (v/v) protein and incubate accordingly until desired developmental stage has been reached for transfer to patient.