We have recurring failure growing frozen mouse embryos to the blastocyct stage. The mouse embryos are grown in human embryo media as a QA/QC procedure. The mouse embryos develop to the morula stage and then they blocked and degenerate.
Photos of the affected mouse embryos show a large void between the embryo and the zona. The cytoplasm is very fragmented and disorganized. They have confirmed that their protein passed their previous QC checks. Several different sources of protein were used. At fist it was though that globulins in the proteins were causing the problem. But recombinant albumin without globulins did not overcome the blockage. The lab has also reviewed other operational parameters including stage temperatures, incubator functions (carbon dioxide levels, pH, and temperature) and they are within nominal specifications. The blockage does not occur when the mouse embryos are grown in KSOM media.
Several experiments looking at plastic dishes were run. By changing their culture dishes from Nunc to GPS, they were able to overcome the problem of mouse embryo blockage. The surprise is that the lot of Nunc dishes was tested previously and passed. Now the same lot is failing. Coincidentally both dishes are made from the same material: polystyrene. |
Culture dishes
Patrick Quinn said on 17 October 2009
"Antonia, me again. What media were being tested and what stage were the mouse embryos at initiation of culture, 1- or 2-cell? Do fresh mouse embryos work? I believe they can be obtained from Embryotech if you don't have a mouse colony. The reason I ask about stage and media is that I have found, as have others, that 1-cell embryos do not grow well in complex media such as Blastocyst medium, or probably KSOM with all amino acids (Life Global medium?) because the essential amino acids have a negative effect if growing from the 1-cell stage but not the 2-cell stage. Let's Mike, you and I have a brainstorming session at ASRM on this topic offer some strong coffees,
Patrick"
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Culture dishes
Patrick Quinn said on 17 October 2009
"Antonia and Mike,
I am answering more on the section Mike wrote about using formalin in the AAB Proficiency Test of embryo medium. Yes, the adulterant is formalin and yes, without oil it can diffuse to other dishes; there was a paper on this a few years back now and I apologize for not remembering the authors name but it was in Fertil Steril and from the Cleveland Clinic showing the cross contaimination with formalin if oil not used.
To stop client complaints about not using a stong enough formalin concentration to get a well defined response of growth versus no growth of mouse embryos, human sperm motility and hamster sperm motility, the three major assays used in the AAB PT samples, we have to use a high concentration of formalin. Clinets and the AAB PT service miss the point entirely that what we are looking for in this PT is consistency between labs, not a growth/no growth situation. The AAB still fails to meausre things such as Z-scores that would illustrate variability between labs for each particular sample. Sorry Antonia, got off track and on to my soap box; I will rereard your original post and comment if I can.
Patrick"
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Mouse embyros failing in Nunc
Michael L. Reed said on 02 October 2009
"Hi Antonia. A good question - Dean Morbeck with Mayo has an abstract for the upcoming ASRM meeting about culturing mouse embryos either alone or in groups for QC purposes. These embryos were commercial in origin, one-cell I believe, and the sensitivity of the embryos grown individually was much greater, to toxins, than embryos grown in groups. Dean was using well dishes for his study.
How were the embryos grown in your scenario with the Nunc dishes?
Were they grown individually or in groups? I'm also assuming oil overlay.
The GPS dish allows for individual growth tracking and embryo cross-talking at the same time, so perhaps this is part of the solution to your question, if the embryos were grown individually. If not...
I did some titration work with the embryo toxicity testing medium from the AAB - and I was able to reduce the amount of toxin to levels that stopped the formation of the blastocyst - just as you described. A low level toxin leached perhaps from the dishes? The toxin in question, from the toxicity challenge, was, I believe, formaldehyde. Patrick Quinn could tell you for certain, and this was probably 6 or more years ago. I'll did around for the data.
I was also able to demonstrate movement of the toxin from wells without mineral oil, into the atmosphere, and into wells that did not have toxin, and that were also not covered by mineral oil. A four well dish with the cover in place. Very cool.
I'd love to hear more. Best wishes.
Mike"
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