Hi, does anybody know if it is important to use a gassed medium (such as M16 or T6) for embryo culture if I want to go only up to the 2-cell stage? Is it possible to use M2 medium instead? How important is the CO2 at that stage? (the problem being that I need to image and there is no CO2 at the microscope).
Thanks!
hello
if you want go of 2-cell stage , must be use M16
also , There are a lot of factors affecting the efficiency of in vitro development of mouse embryos during cultivation, influencing the embryo interaction with soluble factors.
in attachment very important reference , will be help you .
good luck
Hello,
The main function of CO2 is buffering.I regularly do prolonged embryo manipulation outside CO2incubator.In that case the practical idea is to use a HEPES buffered medium(its very effective) and do the manipulation under pre gassed mineral oil maintaining proper temperature .I think theses steps can help to keep the embryos viable for short term manipulation or imaging.
Cheers
I cannot imagine why anyone would use M2/M16 medium for either the culture or investigation of preimplantation mouse embryos. The field of culture media, particularly mouse culure media has made tremendous advances in the last 20 years. I suggest KSOMAA with or without Hepes depending on the time spend outside the incubator. If the embryos are cultured under oil you have more time to examine the embryos...
Hello, thanks for your answers. Actually most of the protocoles I have seen around use either M16 or M2, but it is possible that they are quite old. Doing it with M2 is just the simplest (and cheapest...) way since we actually already use this medium for oocyte culture in the lab. I will take a look at KSOMAA!
M2 is HEPES based medium. This does not require carbon dioxide incubator for short term manipulations, e.g., recovery of embryos etc. Since its pH will not change.
M16 is a sodium bicarbonate containing medium. It needs continuous carbon dioxide presence to keep the right pH.
If you are taking longer time for pictures, you may transfer in M2 medium momentarily.
If you are using under oil and for very short term, M16 should be OK
I work in human IVF field. To culture mouse embryos as our quality control is routine. M6 or M2 could encounter 2-cell block in some strain of mice. As Michael Summers described, it is better to use KSOMAA with Hepes or use modified Human Tubal Fluid (with Hepes). Both media are more advance and do not need CO2. Good luck.
If you overlay your culture with oil (specific for culture) you can do imaging. Ham's F-10 very affordable and can support mouse embryos.
Hi, I guess you already got all the important answers concerning embryo media. Just to add a little piece of information, I would say that if you have M2/M16 media available and you only need to culture embryos to the two-cell stage, they are fine.
To overcome the setting problem you can try to make your own microscope 5% CO2 flow apparatus by using a glass 9 cm Petri dish on the edge of which a nozzle is sealed, which in turn is connected by rubber tubing to a tank filled with 5% CO2 in air. You will use the modified Petri to cover your embryo-containing dish under the microscope (it's a little incubation chamber) and even perform continuous culture in these conditions.
It is relatively cheap and works well.
Good luck.
Hi, Like a few others mentioned, I also culture embryos using KSOMAA and they work perfectly. Also better than M2/M16 in my hands.
Good luck!
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