We would like to acknowledge the many patients at the Colorado Center for Reproductive Medicine (CCRM) that have graciously donated their embryos for research. We would also like to thank Karen Maruniak and the clinical laboratory at CCRM for their help in processing hCG samples, as well as Sue McCormick and her clinical IVF embryology team at CCRM for their help with embryo collection, storage, tracking, and donation. Funding was provided internally by CCRM.

All human embryos have been donated with consent for use in research. Protocols for extended human embryo culture have been approved by the Western Institutional Review Board (study no. 1179872) and follow international guidelines. Any use of human embryos must be reviewed by the appropriate ethics and governing bodies associated with the research institution using this protocol.
1. Preparation
<ol>
	<li>Prepare media and recovery plates one day prior to embryo warming in a sterile laminar f...

<ol>
	<li>Carter, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+placentation--a+review.">Animal models of human placentation--a review.</a> Placenta. , 41-47 (2007).</li><li>Carter, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+placentation+and+animal+models:+patterns+of+trophoblast+invasion+--+a+workshop+report.">Comparative placentation and animal models: patterns of trophoblast invasion -- a workshop report.</a> Placenta. 27, 30-33 (2006).</li><li>Pijnenborg, R., Robertson, W. B., Brosens, I., Dixon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+article:+trophoblast+invasion+and+the+establishment+of+haemochorial+placentation+in+man+and+laboratory+animals.">Review article: trophoblast invasion and the establishment of haemochorial placentation in man and laboratory animals.</a> Placenta. 2, 71-91 (1981).</li><li>Edwards, R. G., Bavister, B. D., Steptoe, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+of+fertilization+in+vitro+of+human+oocytes+matured+in+vitro.">Early stages of fertilization in vitro of human oocytes matured in vitro.</a> Nature. 221 (5181), 632-635 (1969).</li><li>Thomson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>O'Leary, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+progression+of+the+human+inner+cell+mass+during+embryonic+stem+cell+derivation.">Tracking the progression of the human inner cell mass during embryonic stem cell derivation.</a> Nature Biotechnology. 30 (3), 278-282 (2012).</li><li>O'Leary, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cells+using+a+post-inner+cell+mass+intermediate.">Derivation of human embryonic stem cells using a post-inner cell mass intermediate.</a> Nature Protocols. 8 (2), 254-264 (2013).</li><li>Shahbazi, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+the+human+embryo+in+the+absence+of+maternal+tissues.">Self-organization of the human embryo in the absence of maternal tissues.</a> Nature Cell Biology. 18 (6), 700-708 (2016).</li><li>Deglincerti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+the+in+vitro+attached+human+embryo.">Self-organization of the in vitro attached human embryo.</a> Nature. 533 (7602), 251-254 (2016).</li><li>Zhou, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+the+transcriptome+and+DNA+methylome+landscapes+of+human+implantation.">Reconstituting the transcriptome and DNA methylome landscapes of human implantation.</a> Nature. 572 (7771), 660-664 (2019).</li><li>Lv, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+RNA+sequencing+reveals+regulatory+mechanism+or+trophoblast+cell-fate+divergence+in+human+peri-implantation+conceptuses.">Single cell RNA sequencing reveals regulatory mechanism or trophoblast cell-fate divergence in human peri-implantation conceptuses.</a> PLoS Biology. 17 (10), 3000187 (2019).</li><li>West, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+trophoblast+differentiation+in+peri-implantation-stage+human+embryos.">Dynamics of trophoblast differentiation in peri-implantation-stage human embryos.</a> Proceedings of the Nationals Academy of Sciences U.S.A. 116 (45), 22635-22644 (2019).</li><li>Xiang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+landscape+of+3D-cultured+human+pre-gastrulation+embryos.">A developmental landscape of 3D-cultured human pre-gastrulation embryos.</a> Nature. 577 (7791), 537-542 (2020).</li><li>Mall, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+upon+the+collection+of+human+embryos+at+the+johns+hopkins+university.">Report upon the collection of human embryos at the johns hopkins university.</a> The Anatomical Record. 5 (7), 343-357 (1911).</li><li>Buettner, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Franklin+Paine+Mall+(1862-1917).">Franklin Paine Mall (1862-1917).</a> Embryo Project Encyclopedia. , (2007).</li><li>Carnegie Institution of Washington. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Contributions to embryology. , (1915).</li><li>Hertig, A. T., Rock, J., Adams, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+description+of+34+human+ova+within+the+first+17+days+of+development.">A description of 34 human ova within the first 17 days of development.</a> American Journal of Anatomy. 98 (3), 435-493 (1956).</li><li>Logsdon, D. M., Ermisch, A. F., Kile, R., Schoolcraft, W. B., Krisher, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg+cylinder+development+during+in+vitro+extended+embryo+culture+predicts+the+post+transfer+developmental+potential+of+mouse+blastocysts.">Egg cylinder development during in vitro extended embryo culture predicts the post transfer developmental potential of mouse blastocysts.</a> Journal of Assisted Reproduction and Genetics. 37 (4), 747-752 (2020).</li></ol>

The development of the protocol to culture human embryos through implantation has allowed scientists to explore a previously uncharted time in development8,9. Here, we use an extended culture system to culture human embryos and study early trophoblast differentiation before the formation of villous placenta. The methods described here allow us to collect different TB sublineages for use in downstream single-cell analysis. This work allows the scientific community...

Human implantation and the emergence of the early placenta are historically difficult to investigate and remain largely unknown because human tissues are inaccessible at this stage when pregnancy is clinically undetectable. Animal models are inadequate, as human placentation has its own unique features compared to other eutherian mammals. For example, human placenta invades deeply into the decidua with some trophoblast cells reaching at least the inner third of the uterine myometrium while other cells remodel the uterine spiral arteries. Even our closest evolutionary ancestors, the non-human primates, show differences in placental morphology and trophoblast interactions with the maternal decidual tissues1,2,3. Obtaining pre-implantation human embryos in vitro was not possible until in the 1980&#8217;s when clinical human in vitro fertilization (IVF) started as a routine practice for treating infertility4. Now, human blastocysts can be grown in vitro to allow for the selection of more viable embryos for transfer, as well as enable safe genetic testing. Improvement in embryo culture techniques, as well as the increasing use of IVF has yielded many surplus blastocysts which remains after patient&#8217;s treatment cycles have been completed. With patient consent, IRB approval, and with certain restrictions, these blastocysts may be utilized for research studies. They have become an invaluable resource that were used for the derivation of human embryonic stem cells5, understanding the transition of inner cell mass to embryonic stem cells6,7, and more recently, have been successfully cultured until day (D) 13 to remodel human implantation8,9. By utilizing recently developed single cell omics approaches, the access to these implantation stage human embryo tissues has offered unique opportunities to describe the molecular mechanisms that regulate this highly dynamic cell differentiation process, which were previously impossible to explore10,11,12,13.
Here, we describe the methods used in our recent publication characterizing the dynamics of trophoblast differentiation during human implantation12. This protocol includes the warming of vitrified blastocysts, extended embryo culture up to D12 post IVF, enzymatic digestion of the embryo into single cells, and cell collection for downstream assays (Figure 1). This extended culture system supports peri-implantation stage human embryo development without maternal input and recapitulates trophoblast differentiation that appears consistent with the observations made from histological specimens many years ago14,15,16,17. During implantation, the trophoblast population is comprised of at least two cell types: the mononucleated progenitor-like cytotrophoblast (CTB) and the terminally differentiated, multinucleated syncytiotrophoblast (STB). Upon trypsin digestion, the CTB are small, round cells that are morphologically indistinguishable from other cell lineages (Figure 2A, left panel). Separation of CTB from other cell lineages, such as epiblast and primitive endoderm, can be achieved by their distinct transcriptomic profiles revealed by single cell RNA sequencing. Syncytiotrophoblast cells can be easily identified as irregular shaped structures that are significantly larger than the other cell types and mainly located at the periphery of the embryo (Figure 2A, middle panel; Figure 2B, left panel). Migratory trophoblast cells (MTB) are another trophoblast sublineage found during embryo extended culture and can be recognized as seemingly moving away from the main body of the embryo (Figure 2A, right panel, Figure 2B, right panel). Migratory trophoblast, although expressing many of the same markers as extra villous trophoblast (EVT), should not be referred to as EVT, since the villous structures in the placenta have not emerged at this very early stage of development.
Experimentally, we were able to collect small CTB and large STB that are easily distinguishable after embryos are digested into single cells at D8, D10, and D12 (Figure 2A,B). Migratory trophoblasts arise at the later stages of extended embryo culture and can be collected at D12 before enzymatic digestion of the whole embryo (Figure 2A,B). By phenotypically separating these three trophoblast sublineages before single cell analysis, we can identify specific transcriptomic markers and define the biological role of each cell type. Cytotrophoblast are highly proliferative and act as progenitor cells in supplying the STB and MTB differentiated lineages10,11,12,13. Syncytiotrophoblast are involved in producing placental hormones to maintain pregnancy and may be also responsible for the embryos burrowing into the endometrium10,11,12,13. Migratory trophoblast has even stronger features of an invasive, migratory phenotype and are likely responsible for deeper and more extensive colonization of the uterine endometrium10,11,12,13. After defining the transcriptomic signature of each cell type, clustering analyses have also revealed two additional subsets of cells that were morphologically indistinguishable from CTB and had transcriptomes with features of STB and MTB, respectively12. These intermediate stage cells are likely in the process of differentiating from CTB to either the MTB or STB sublineages and would have been overlooked if embryos were blindly digested and cells were separated by transcriptome alone.
The protocol described here utilizes a two-dimensional (2D) culture system and may not be optimal for supporting three-dimensional (3D) structural development, as suggested by a recent publication describing a newly developed 3D culture system13. Nevertheless, in this 2D system the differentiation of early trophoblast seems to be consistent with observations made from in vivo specimens14,15,16,17. This protocol may also be easily adapted for the use in the recently described 3D culture system13 with minimal changes. All steps are carried out with a handheld micromanipulation pipette with commercially available disposable tips or a mouth pipette from a finely pulled glass pipette attached to rubber tubing, a filter, and a mouthpiece.

<table>
	<tbody>
		<tr>
			<td>3130 or 3110 Forma Series II water-jacketed CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-998-078</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Corning Primaria tissue culture dish</td>
			<td>VWR</td>
			<td>62406-038</td>
			<td>Case of 500</td>
		</tr>
		<tr>
			<td>5 mL snap cap tube</td>
			<td>VWR</td>
			<td>60819-295</td>
			<td>Pack of 25</td>
		</tr>
		<tr>
			<td>60 mm Corning Primaria tissue culture dish</td>
			<td>VWR</td>
			<td>25382-687</td>
			<td>Case of 200</td>
		</tr>
		<tr>
			<td>6-well dish</td>
			<td>Agtech Inc.</td>
			<td>D18</td>
			<td>Pack of 1, 10, or 50</td>
		</tr>
		<tr>
			<td>Acidic Tyrode&#39;s solution</td>
			<td>Millipore Sigma</td>
			<td>T1788</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Biotix 1250 &#181;L pipette tips</td>
			<td>VWR</td>
			<td>76322-156</td>
			<td>Pack of 960</td>
		</tr>
		<tr>
			<td>Blast, blastocyst culture media</td>
			<td>Origio</td>
			<td>83060010</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Dilution Solution</td>
			<td>Kitazato</td>
			<td>VT802</td>
			<td>1 x 4 mL</td>
		</tr>
		<tr>
			<td>Disposable Borosilicate Glass Pasteur Pipets</td>
			<td>Thermo Fisher Scientific</td>
			<td>1367820D</td>
			<td>5.75 in. (146mm); 720/Cs</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Millipore Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo culture paraffin oil OvOil</td>
			<td>Vitrolife</td>
			<td>10029</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Eppendorf PCR tubes 0.2 mL</td>
			<td>VWR</td>
			<td>47730-598</td>
			<td>Pack of 1,000</td>
		</tr>
		<tr>
			<td>Eppendorf PCR tubes 0.5 mL</td>
			<td>VWR</td>
			<td>89130-980</td>
			<td>Case of 500</td>
		</tr>
		<tr>
			<td>Fibronectin from human plasma. Liquid .1% solution</td>
			<td>Millipore Sigma</td>
			<td>F0895</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>Gilson 1 mL Pipetteman</td>
			<td>Thermo Fisher Scientific</td>
			<td>F123602G</td>
			<td>1 Pipetteman 200-1000 &#181;L</td>
		</tr>
		<tr>
			<td>Gilson 20 &#181;L Pipetteman</td>
			<td>Thermo Fisher Scientific</td>
			<td>F123602G</td>
			<td>1 Pipetteman 2-20 &#181;L</td>
		</tr>
		<tr>
			<td>Gilson 200 &#181;L Pipetteman</td>
			<td>Thermo Fisher Scientific</td>
			<td>F123602G</td>
			<td>1 Pipetteman 50-200 &#181;L</td>
		</tr>
		<tr>
			<td>G-MOPS handling media</td>
			<td>Vitrolife</td>
			<td>10129</td>
			<td>125 mL</td>
		</tr>
		<tr>
			<td>Handling media</td>
			<td>Origio</td>
			<td>83100060</td>
			<td>60 mL</td>
		</tr>
		<tr>
			<td>Ibidi 8 well chambered coverslip</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>15 slides per box</td>
		</tr>
		<tr>
			<td>IVC1/IVC2</td>
			<td>Cell Guidance Systems</td>
			<td>M11-25/ M12-25</td>
			<td>5-5mL aliquots</td>
		</tr>
		<tr>
			<td>K System T47 Warming Plate</td>
			<td>Cooper Surgical</td>
			<td>23054</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma Millex Sterile Syringe Filters with Durapore PVFD Membrane</td>
			<td>Fisher Scientific</td>
			<td>SLGVR33RS</td>
			<td>Pack of 50</td>
		</tr>
		<tr>
			<td>Mouth pieces</td>
			<td>IVF Store</td>
			<td>MP-001-Y</td>
			<td>100 pieces</td>
		</tr>
		<tr>
			<td>Oosafe center well dish</td>
			<td>Oosafe</td>
			<td>OOPW-CW05-1</td>
			<td>Case of 500</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage SPS</td>
			<td>Origio</td>
			<td>ART-3010</td>
			<td>12x 12 mL</td>
		</tr>
		<tr>
			<td>Rubber latex tubing for mouth pieces</td>
			<td>IVF Store</td>
			<td>IVFS-NRL-B-5</td>
			<td>5 ft.</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Stripper tips</td>
			<td>Cooper Surgical</td>
			<td>MXL3-275</td>
			<td>20/pk 275 &#181;m</td>
		</tr>
		<tr>
			<td>Thawing Solution</td>
			<td>Kitazato</td>
			<td>VT802</td>
			<td>2 x 4 mL</td>
		</tr>
		<tr>
			<td>The Stripper Micropipetter</td>
			<td>Cooper Surgical</td>
			<td>MXL3-STR</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604013</td>
			<td>1 x 100 mL</td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Millipore Sigma</td>
			<td>P1379-25ML</td>
			<td>25 mL bottle</td>
		</tr>
		<tr>
			<td>VWR 1-20 &#181;L pipette tips</td>
			<td>VWR</td>
			<td>76322-134</td>
			<td>Pack of 960</td>
		</tr>
		<tr>
			<td>VWR 1-200 &#181;L pipette tips</td>
			<td>VWR</td>
			<td>89174-526</td>
			<td>Pack of 960</td>
		</tr>
		<tr>
			<td>Washing Solution</td>
			<td>Kitazato</td>
			<td>VT802</td>
			<td>1 x 4 mL</td>
		</tr>
	</tbody>
</table>

single cell collection of trophoblast cells in peri-implantation stage human embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Healthy embryos exhibited continued proliferation over the course of extended culture (Figure 2B). Abnormal embryos began to retract from their outer edges and disintegrate (Figure 2C). From our experience, approximately 75% of embryos were attached to the bottom of the fibronectin coated dish at 48 h and the attachment increased to approximately 90% by 72 h in culture. The success of embryo attachment may be largely impacted by the initial quality of the blasto...

Watch this Scientific Journal Video about Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos at JoVE.com

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

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.

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

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.

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Research

JoVE Journal

Biology

6.1K visualizações.  Colorado Center for Reproductive Medicine. Muitas gestações falham no desenvolvimento de embriões humanos em torno dos sete ou oito pós-fertilização. Este protocolo oferece oportunidades aos investigadores para cultivar conceitos humanos in vitro durante esta misteriosa janela de implantação. Isso nos permite compreender os mecanismos que sustentam o sucesso da implantação humana e da formação placentária precoce.Usar um sistema de cultura estendido para cultivar embriões humanos em estágio de peri-implantação ...