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 MII Oocyte Vitrification with CryoTip (PDF | Video}   
• Simplified Oocyte Vitrification with HSV Device (PDF / Video) 
• Tips for Oocyte Vitrification (PDF / Video)
• CryoTip Loading Protocol (PDF / VIdeo) 
• HSV Device Loading Protocol (PDF / VIdeo) 
• Cryolock Loading Protocol (USA) (PDF)
• Cryolock Loading Protocol (International) (PDF)

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

NOTE: As referenced in clinical literature, we recommend oocyte vitrification to be performed within 2 hours from time of oocyte retrieval¹.

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

Simplified MII Oocyte 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) 20 μL drop of H and
• three (3) 20 μL drops of ES (ES1, ES2 and ES3)

NOTE: ES1 and ES2 should be in close proximity to H (but not touching).

2. Place MII oocyte(s) (2 maximum), into H and expose undisturbed for 1 minute.

3. Merge ES1 with H. Allow spontaneous mixing for 2 minutes.

Use tip of transfer pipette to move ES1 towards H until drops merge.

4. Then merge ES2 with H+ES1. Allow spontaneous mixing for 2 minutes.

5. Transfer oocyte(s) from merged drop to ES3 and expose undisturbed for 6-10 minutes.

6. During the 6-10 minute exposure, aseptically dispense one (1) 50 μL drop of VS.

NOTE: Visually observe re-expansion (equilibration) of oocyte(s) to at least 80% of original volume.

7. Transfer oocyte(s) from ES3 to VS for 30 seconds before loading.

8. Gently pipette oocyte(s) once within VS drop to ensure complete rinse in VS.

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

9. Load, seal and plunge CryoTip® within 80 seconds, within 110 seconds after exposure to VS drop.

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

Simplified MII Oocyte Vitrification with HSV Device

1. Aseptically dispense:

• one (1) 20 μL drop of H and
• three (3) 20 μL drops of ES (ES1, ES2 and ES3)

NOTE: ES1 and ES2 should be in close proximity to H (but not touching).

2. Place MII oocyte(s) (2 maximum), into H and expose undisturbed for 1 minute.

3. Merge ES1 with H. Allow spontaneous mixing for 2 minutes. 

Use tip of transfer pipette to move ES1 towards H until drops merge.

4. Then merge ES2 with H+ES1. Allow spontaneous mixing for 2 minutes.

5. Transfer oocyte(s) from merged drop to ES3, and expose undisturbed for 6-10 minutes.

6. During above 6-10 minute exposure, aseptically dispense one (1) 50 μL drop of VS.

7. Transfer oocyte(s) from ES3 to VS for 30 seconds before loading.

8. Gently pipette oocyte(s) once within VS drop to ensure complete rinse in VS.

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

9. Load, seal and plunge HSV Device (per device instructions) within 80 seconds, not to exceed 110 seconds after exposure to VS.

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