Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.
Many pregnancies fail early in human embryo development around the seven or eight post-fertilization. This protocol offers opportunities to investigators to grow human conceptors in vitro during this mysterious implantation window. This allows us to understand the mechanisms that underpin the success of human implantation and early placental formation.
Using an extended culture system to grow peri-implantation stage human embryos in vitro is probably the only way to obtain authentic materials to study implantation and early trophoblast differentiation. This straightforward technique is a powerful tool that allow us to answer many fundamental questions about human early development. Research conducted with the use of this protocol will largely contribute to the understanding of early pregnancy loss, recurrent implantation failure and the placenta pathologies.
Demonstrating the procedure will be Deirdre Logsdon, the PhD student from a laboratory. One day prior to embryo warming, prepare media and recovery plates in a sterile laminar flow hood. Fill two center well organ culture wash dishes with 500 microliters of BM with 10%SPS, then cover the BM with 500 microliters of embryo culture grade oil.
In a 16 millimeter tissue culture dish, layer eight milliliters of embryo culture oil and anchor 20 microliter drops of BM with 10%SPS to the bottom. Equilibrate the wash dishes and recovery plate in an incubator at 37 degrees Celsius, 6%carbon dioxide and 5%oxygen for at least four hours. Aliquot approximately four milliliters of IVC 1 into a five milliliter snap cap tube and prepare one wash dish with 500 microliters of IVC 1 with no oil overlay.
Equilibrate the dish and tube in the incubator for at least four hours. Dilute fibronectin from human serum in PBS to 30 micrograms per milliliter. Open the eight well chambered coverslip package taking care not to touch the wells, then gently pipette 250 microliters of the fibronectin into each well.
Replace the lid on the coverslip and incubate it at four degrees Celsius for 20 to 24 hours. Prepare the extended culture plate on the morning of embryo warming. Retrieve the chambered cover slip with fibronectin and place it in the laminar flow hood.
Then remove the fibronectin mixture with a one milliliter pipette and discard it. Pipette 300 microliters of the equilibrated IVC 1 into each well and place the coverslip into the incubator until removal of the zona pellucida. After warming and recovery of the D5 human embryos, assess them for re-expansion and take pictures of each embryo.
Move each embryo to 500 microliters of a MOPS buffered handling medium with 5%FCS. Then treat it with acidic Tyrode's solution as described in the text manuscript. Immediately move the embryo with the dissolving zona into 300 microliters of warmed MOPS buffered medium to quench the Tyrode's solution.
Then move the embryo into a central well organ tissue dish containing the equilibrated BM with 10%SPS under the embryo culture grade oil. Then return the embryo to the 20 microliter recovery drop. Individually move the embryos to the wash dish with the equilibrated IVC 1 media, then carefully move each embryo to a well of the chambered coverslip, making sure to keep track of the embryo identification.
Return the chambered coverslip to an incubator set to 37 degrees Celsius, 6%carbon dioxide and atmospheric oxygen for two days. At outgrowth day two, carefully examine the attachment of the embryos under the microscope and exchange the media. Know which embryo is attached to the dish by gently tapping the plate.
To change the media, remove the lid and carefully aspirate 150 microliters of IVC 1, taking care to not disturb the attached embryo. If an embryo has not yet attached to the plate, do not exchange the media because the serum in IVC 1 will aid in attachment. Slowly pipette 150 microliters of equilibrated extended culture media, IVC 2 into each well and Replace the lid on the coverslip.
Carefully returned the chambered coverslip to the incubator. Repeat media exchange and attachment check every day until embryos are ready for fixation or single celled digestion. If collection of spent media is necessary for further analysis, snap freeze the 150 microliters of removed IVC 1 into a sterile low bind 0.5 milliliter tube.
Wash each embryo once with 200 microliters of PBS, then add 200 microliters of trypsin solution to each well. Return the chambered coverslip to the incubator for five minutes. Use a small pipette or finely pulled mouth pipette to pick up an individual MTB then use a larger pipette to gently dissociate the embryo by aspirating up and down.
Continue aspirating the embryo gently and repeatedly using a smaller diameter pipette tip until the embryo has been incubated for a total of 10 minutes in trypsin. To perform single cell selection, move the dissociated cells through 320 microliter wash drops of PBS and 0.1%PVP under embryo culture oil, taking care not to lose any cells. After washing the cells use a finely pulled glass pipette to select one cell.
Carefully pipette the single cell into a sterile 0.2 milliliter low bind tube with a minimal volume of PBS and PVP. Snap free single cells in liquid nitrogen and store them at negative 80 degrees Celsius for further use. Healthy embryos exhibited continued proliferation over the course of extended culture, while abnormal embryos began to retract from their outer edges and disintegrate.
At day eight post fertilization, most cells in the embryos were cytotrophoblast or CTBs, that were positive for trophoblast marker GATA-3. On the periphery of the embryo, the CTBs were already differentiating into multi-nucleated syncytiotrophoblasts, which had a sheet like appearance and stained positive for humans CGB. At day 10, the formation of CGB positive migratory trophoblast cells was at a maximum, which was confirmed by the upsurge of HCG production at this time.
Migratory trophoblasts which stained positive for HLA-G, also began to emerge and migrate away from the embryo body. By day 12, STB differentiation was in decline and MTB production became more prominent, suggesting a shift of emphasis from hormone production on day 10 to cell migration on day 12. These changes can be observed in a time lapse video of the peri-implantation period.
The video demonstrates the collapse of the blastocele, the formation of the STB, and the eventual differentiation and migration of MTB. The most important thing to keep in mind when completing this procedure is that you are working with live embryos that are sensitive to changes in temperature, osmolality and pH. Minimizing the amount of time that the dishes are out of the incubator is critical to maintaining embryo health.
After completing this procedure, investigators can use isolated single cells for downstream single cell omics assays, such as single cell RNA sequencing and whole genome bisulfite sequencing to better understand epigenetic and transcriptomic changes during human implantation and early placenta formation.
