Simplified Embryo Vitrification Protocols

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

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

Note: Prior to vitrification of blastocysts, refer to Collapsing Protocol, Human Blastocysts.

Have all necessary materials, tools and equipment ready and easily accessible before starting procedure.

Simplified Embryo Vitrification with CryoTip

As an added precaution during the preparation procedure, carefully examine each CryoTip outside of the package. Prior to use, all CryoTips should be examined under a suitable magnification (40x power) for possible damage (such as tip breakages or cracks) that may have occurred during transport.

1. Aseptically dispense one (1) 50 μL drop of ES.

2. Transfer embryo(s) (2 maximum), to the ES drop and expose undisturbed for 6-10 minutes.

NOTE: The specimen(s) will shrink and then gradually return to original size, indicating that equilibration is complete.

3. During above equilibration in ES, aseptically dispense one (1) 50 μL drop of VS 2 minutes prior to complete equilibration.

4. Transfer embryo(s) with minimal volume of medium from ES to the VS drop for 30 seconds before loading.

5. Gently pipette embryo(s) once within VS drop to ensure complete rinse with VS.

NOTE: To minimize floating, after 10 seconds pipette the specimen(s) to the bottom center of the VS drop.

6. Load, seal and plunge CryoTip into LN2 within 80 seconds, not to exceed 110 seconds after initial exposure to VS.

7. Refer to CryoTip Loading Protocol diagram and product insert for detailed loading instructions.

 

Simplified Embryo Vitrification with HSV Device

1. Aseptically dispense one (1) 50 μL drop of ES.

2. Transfer embryo(s) (2 maximum), to theES drop and expose undisturbed for 6-10 minutes.

NOTE: The specimen(s) will shrink and then gradually return to original size, indicating that equilibration is complete.

3. During above equilibration in ES, aseptically dispense one (1) 50 μL drop of VS 2 minutes prior to complete equilibration.

4. Transfer embryo(s) with minimal volume of medium from ES drop to the VS drop for 30 seconds before loading.

5. Gently pipette embryo(s) at least once within VS drop to ensure complete rinse with VS.

NOTE: To minimize floating, after 10 seconds pipette the specimen(s) to the bottom center of the VS drop.

6. Load, seal and plunge HSV Device within 80 seconds, not to exceed 110 seconds after initial exposure to VS. 

NOTE: Load after specimen is completely dehydrated and stable at the bottom of the drop.

7. Refer to HSV Device Loading Protocol diagram and product insert for detailed loading instructions.

 

Tips for Embryo Vitrification

  • All procedures are to be done at ROOM TEMPERATURE (22-27°C). DO NOT USE HEATED STAGE.
  • Have all necessary material, tools and equipment ready and easily accessible before starting procedure.
  • CryoTips should be pre-labeled with patient information, and assembled with connector and syringe or pipette (for loading), prior to starting procedure. To protect the finely pulled tip from damage, keep it covered with metal cover sleeve until ready to load specimen(s).
  • HSV device should be pre-labeled with patient information, and the capillary tube should be connected with the longer end of the blue plastic insertion device, prior to starting procedure.
  • Where possible, select only the best quality embryos (2PN to Blastocyst) for vitrification.
  • The recommended CryoTip or HSV Device capacity is a MAXIMUM of 2 specimens.
  • Process only as many specimen(s) as will be loaded per CryoTip or HSV device at one time.
  • Minimize exposure of specimens to light during equilibration in ES and VS solutions.
  • Transfer specimens between drops using a minimal volume of medium.
  • The timing for exposure to VS is CRITICAL.
    • Maintain microscopic visualization of specimen(s) by adjusting focus as needed, during rapid exposure to VS (specimens will float in the drop).
    • Keep transfer pipette tip close to drop for quick manipulations.
    • Count the seconds mentally or with timer during 30 seconds exposure to VS.
    • Load, seal and plunge the CryoTip or HSV Device within 80 seconds, not to exceed 110 seconds after inital exposure to VS.

Related Protocol

Simplified Embryo and Oocyte Warming Protocols

Simplified MII Oocytes Vitrification Protocols