Culture conditions
Below is an introduction provided by Dr. Lotte Stroebech of Stroebech Media, outlining the critical parameters and best practices for successful bovine in vitro embryo production. These guidelines emphasize the importance of maintaining precise temperatures, proper gas concentrations, and correct media handling procedures, ensuring optimal conditions for oocyte maturation, fertilization, and embryo culture. From selecting the right plastics to preventing contamination and toxicity, every detail is designed to support high-quality, repeatable results in the lab.
Culture conditions
Incubator temperature should be 38.8°C
All heated stages should be 35°C, not more!
Equilibrate all media in the incubator to 38.8°C Semen Wash Medium and OPU Medium should only be warmed
Gas concentrations
The media contain 25-27 mM bicarbonate (HCO3- ) corresponding to a pH 7.3-7.4 see appendix 2
Maturation (IVM) 6-6.5% CO2 humidified atmospheric air (21% O2)
Fertilization (IVF) 6-6.5% CO2 humidified atmospheric air (21% O2)
Culture (IVC) 6-6.5% CO2 and 6-9% O2 - Bovine embryos require low oxygen for IVC
If the lab is located above sea level, you will need to adjust the CO2/O2 concentrations accordingly - See appendix 3
Media dish preparations
Maturation: in 500 μl 4WP without oil overlay or in vials 800-1000 μl Note: Many vials are toxic
Fertilization: in drops (no less than 100 μl) with oil overlay or in 500 μl 4WP without oil overlay
Culture: in drops (no less than 100 μl) with oil overlay or in 500 μl 4WP with oil overlay and no change of media
Rinse dishes: always rinse oocytes/embryos once in the corresponding final medium in order not to dilute medium that is to be incubated
When oil overlay is used in 4WP, rotate the lid. Just one oil drop between the lid and the dish will seal them together and prevent CO2 equilibration of the medium resulting in embryo death. Remember: The quality of oil is key to success!
Do never aliquot any media into plastic vials for storage
Remove lid when warming and equilibrating media in glass bottles, except for Semen Wash Medium Media in 4WP and petri dishes should be prepared minimum 30 minutes prior to use for CO2/O2 equilibration
Micro drop (100 μl) preparation: first make a 10 μl drop medium, cover with plenty of oil overlay, then inside the drop under oil supplement the remaining medium amount. Drops will now be round and not flat Make sure there is a minimum of 3mm oil above the drop
Disposables
Use polystyrene plastics that are embryo-grade quality, cyto- and endotoxin, pyrogen- and RNase free. Note that cell culture grade is not good enough! Use 4-well plates (4WP) with a culture area per well of 1.9 cm2. Use only filtered pipette tips to avoid risk of contamination of pipettes, which may be a hidden source of continuous infection.
