OPU-IVF PROTOCOLS

Oocytes Aspiration Plus

SAR IVF

SAR IVC

 

IVF for Large Scale Embryo Production PROTOCOLS

SAR IVF

SAR IVC

 

IVF For Large Scale Embryo Production PROTOCOL

 

SAR IVC

 

A. Embryo in vitro culture (IVC) with SAR IVC medium

1. Aliquot 4 mL of SAR IVC medium into each 5 mL round-bottom tube (Cat# FALCON 352058, Fisher).

2. Loosen the cap, equilibrate the SAR IVC medium at 39C for 23 hours prior to culture.

3. Before culturing, prepare appropriate number of 35 mm petri dishes (Cat# FALCON 351008, Fisher). Add 7 droplets of SAR IVC to each petri dish. Slowly layer 33.5 mL of mineral oil (Cat# M8410, Sigma) over the droplets. Loosen the cap of the petri dish and set into the humidified, 5% CO2 incubator to pre-warm at 39C for 23 h prior to embryo culture.

4. At 18 h IVF, remove cumulus cells surrounding zona by 1.5 min of vortexing and then pipetting and washing zygotes in 10% fetal bovine serum (FBS) M199 medium (SAR cumulus removal medium) in a 35 mm petri dish (Cat# FALCON 351008, Fisher). After cumulus cell removal, wash zygotes in 10% FBS M199 medium at least times.

    5. Prepare an appropriate number of 60 mm petri dishes (Cat# FALCON 351007, Fisher) and add 3 drops (1 mL/3 drops) of SAR-IVC in each petri dish, using a 1 mL pipet. Wash 25 zygotes in each drop, a total 3 times, and then transfer the zygotes into the 50 L/droplet of SAR IVC to yield 2030 zygotes/droplet.

    6. Culture the embryo in the presence of a mixture of 5% CO2, 5% O2, and 90% N2, at 39C for 7 days.

    7. At 48 h culturing, check the cleavage rate; remove 1 cell and 2 cell embryos from the droplet. Remove 25 L of SAR IVC medium from the droplet, add 25 L of fresh pre-equilibrated SAR IVC medium.

    8. Change medium on Day 5 of embryo culture.

    9. Check blastocyst development on Day 7 of IVC.

     

    Note: For the ovum pick up (OPU)-IVF procedure, 520 embryos per 50 L droplet of SAR IVC medium are cultured at 39C in a humidified incubator at 5% CO2, 5% O2 and 90% N2 for 7 days. Half the volume (25 L) of fresh medium can be changed on Days 2 and 5 after culture by removing 25 L of old medium and replacing with 25 L of fresh medium. This culture is a serum free, cell co-culture free system.

     

If a large scale IVF/IVC is used, such as embryo production using slaughterhouse oocytes, about 400 L of IVC medium is added into each well of a NUNC 4-well culture dish, and covered with 200 L of mineral oil, and about 100 presumptive zygotes are transferred into each well. Embryos are cultured at 39C in a humidified incubator at with 5% CO2, 5% O2 and 90% N2 for 7 days. Half the volume (200 L) of fresh medium can be changed on Days 2 and 5 after culture by removing 200 L of old medium and replacing with 200 L of fresh medium. This culture is a serum free, cell co-culture free system. For a robotic embryo culture, it is also possible to culture embryos for the entire 7 days without medium change.

 

 

IVF For Large Scale Embryo Production Protocol (pdf format)