Simplified Embryo and Oocyte Warming Protocols

Oocytes and Embryos

The following protocols and tips are for the vitrification of embryos from 2PN (two-pronuclear zygote) to blastocyst using our Vitrification Freeze Kit (Irvine Scientific, Catalog # 90133), Cryolock™(Catalog # CL-R-CT) or CryoTip® (Catalog # 40709) or HSV Security Vitrification Straws (Catalog # 25246-25251).

 

Protocols

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. 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.

Tips for Embryo and Oocyte Warming with CryoTip

  • Have all necessary material, tools and equipment ready and easily accessible before starting procedure.
  • Pre-equilibrate a dish of appropriate culture medium supplemented with SSS or DSS at 20% (v/v), or HSA at 12 mg/ml for final recovery of specimen.
  • CryoTips must remain submerged in LN2 until ready to warm. When transferring CryoTips from LN2 filled holding reservoir or between LN2 storage tanks, CryoTips should always be submerged in an LN2 filled goblet to prevent uncontrolled/premature warming in air.
  • Use sterile medical-grade sharp scissors.
  • Set up warming dish with drops of solutions (see step #3) prior to removing CryoTip from LN2.
  • Use a 37°C waterbath with a minimum volume of at least 500 mL.
  • Ensure that the plunger of the syringe has been withdrawn half way prior to attaching the CryoTip to the connector.
  • Rapid and controlled dispensing of contents from the CryoTip is essential and requires a secure seal between the CryoTip, Connector and syringe (or pipette).
  • Cut fine tip end over the dish in case of premature dispensing of contents. Carefully dispense contents as a drop (ideally hanging on edge of CryoTip before touching directly next to TS drop) to AVOID BUBBLES.
  • Limit exposure to light while moving the specimens through the solutions.
  • NOTE: Following complete recovery (2-4 hours post-warming), oocytes must be fertilized by ICSI due to zona hardening during vitrification.

 

Simplified Warming Protocol with HSV Device

STEPS 9-12 MUST BE PERFORMED AT ROOM TEMPERATURE (22-27°C).

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.

Tips for Embryo and Oocyte Warming with HSV Device

  • Have all necessary materials, tools and equipment ready and easily accessible before starting procedure.
  • Refer to Directions for Use or Product Insert that accompany the HSV Device for specific instructions.
  • Pre-equilibrate a dish of appropriate culture medium with 20% (v/v), or 12mg/mL protein for final recovery of specimen(s).
  • HSV Device must remain submerged in LN2 until ready to warm. When transferring HSV Device from LN2 filled holding reservoir, or between LN2 storage tanks, vitrification devices should always be submerged in an LN2 filled goblet to prevent uncontrolled/premature warming in air.
  • Set up warming dish with a minimum volume of 250 μL of TS for embryos, or a minimum volume of 1 mL of TS for oocytes, at least 30 minutes prior to beginning procedure.
  • Remove the TS dish from 37°C incubator or heating stage right before the warming procedure.
  • Immediately immerse the curved spatula (or gutter) into the 37°C TS after removed from LN2 (within 2 seconds).
  • Maintain proper function of your Knipex (or other wire cutter device) with regular monitoring and replacing when needed.
  • Gently swirl the gutter in TS to help detach the specimen(s) from the device.
  • Limit exposure to light while moving the specimens through the solutions.
  • If you choose to, you may overlay DS and WS drops with 8.0 mL to 8.5 mL of equilibrated mineral oil, at least 45 minutes prior to starting warming procedure.
  • NOTE: Following complete recovery (2-4 hours post-warming), oocytes must be fertilized by ICSI due to zona hardening during vitrification.

 

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