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Summary

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.

Abstract

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.

Introduction

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 tissues^1,2,3. Obtaining pre-implantation human embryos in vitro was not possible until in the 1980’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’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 cells^6,7, and more recently, have been successfully cultured until day (D) 13 to remodel human implantation^8,9. 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 explore^10,11,12,13.

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 ago^14,15,16,17. 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 lineages^10,11,12,13. Syncytiotrophoblast are involved in producing placental hormones to maintain pregnancy and may be also responsible for the embryos burrowing into the endometrium^10,11,12,13. Migratory trophoblast has even stronger features of an invasive, migratory phenotype and are likely responsible for deeper and more extensive colonization of the uterine endometrium^10,11,12,13. 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 specimens^14,15,16,17. 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.

Protocol

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 Prepare media and recovery plates one day prior to embryo warming in a sterile laminar flow hood. Prepare 2 mL of blastocyst culture media (BM) with 10% v/v serum protein substitute (SPS). NOTE: Blastocyst culture media may or may not contain added protein. Here, we used a medium that must be supplemented with 10% of the indicated albumin protein. Check the manufacturer guidance for variation in this supplementation. All media should be filtered using a sterile 0.22 μM filter attached to a sterile syringe before equilibration. Fill two center-well organ culture wash dishes with 500 μL of BM with 10% v/v SPS covered with 500 μL of embryo culture grade oil. In a 60 mm tissue culture dish, layer 8 mL of embryo culture oil and then anchor 20 μL drops of BM with 10% v/v SPS to the bottom of the culture plate. Make 1 drop for each embryo warmed. NOTE: More media, drops, and wash dishes may be made as needed. Drops may also be added to the dish before the oil is layered. Anchoring drops under the oil will help reduce evaporation and any subsequent changes in osmolality. Equilibrate BM with 10% v/v SPS wash dishes and recovery plate in an incubator at 37 °C, 6% CO2, 5% O2 for at least 4 h. Thaw one vial of the first step of extended culture media (IVC1) in 4 °C or on the bench top. Prepare one wash dish of 500 μL of IVC1 with no oil overlay. Aliquot approximately 4 mL of IVC1 into a 5 mL snap cap tube. Equilibrate IVC1 wash dish and small snap cap tube of IVC1 in 37 °C, 6% CO2, and atmospheric O2 for at least 4 h. Prepare extended culture plates one day prior to embryo warming in a sterile laminar flow hood. Dilute fibronectin from human serum in phosphate buffered saline (PBS) to 30 μg/mL. 250 μL of 30 μg/mL fibronectin per embryo will be needed to coat the chambers. Open the 8 well chambered coverslip package under the hood while taking care not to touch the wells. Gently pipette 250 μL of 30 μg/mL fibronectin into each well. Return the lid to the chambered coverslip and place in 4 °C for 20-24 h. Prepare extended culture plate in the morning of embryo warming. Retrieve the chambered coverslip with fibronectin and place in laminar flow hood. Remove the fibronectin mixture with a 1 mL pipette and discard into the waste container. Retrieve warmed, equilibrated IVC1 media from small 5 mL snap cap tube. Pipette 300 μL of equilibrated IVC1 into each well. Be careful not to let any fluid touch the lid as this will increase the risk of contamination. Return the chambered coverslip with IVC1 to incubate in 37 °C, 6% CO2, and atmospheric O2 until removal of the zona pellucida in step 4. 2. Warming vitrified D5 human embryos NOTE: Other manufacturers may have slightly different protocols according to their own vitrification technology. See manufacturer’s instructions for use when applicable. Warm 3.0 mL of thawing solution (TS) in 35 mm dish to 37 °C. Bring 300 μL of dilution solution (DS) and two wells of 300 μL of washing solution (WS) in a 6 well plate to room temperature. Using forceps, carefully remove the cryo device sleeve while ensuring that the vitrified embryo always remains under liquid nitrogen. Quickly move the cryo device from liquid nitrogen and plunge the tip of the cryo device in TS at 37 °C. Set a timer for 1 min and keep the cryo device submerged until the embryo detaches into the TS. NOTE: Warm embryos one at a time to keep track of embryo identities as they may relate to patient demographic information in downstream analysis. During the 1 min incubation in TS, carefully remove the cryo device and gently pick up the embryo and move to the opposite side of the TS dish. NOTE: If culturing multiple embryos, be diligent to keep embryos separate to ensure proper identities are maintained. After 1 min, gently pick up the embryo with a small amount of TS, approximately 2 μL. Move the embryo to the bottom of the 300 μL DS well while covering the embryo in a thin layer of TS from the pipette. Set a timer for 3 min and place the 6-well plate on the bench top at room temperature. After 3 min, pick up the embryo with a small amount of DS, approximately 2 μL, and move the embryo to the bottom of the next well containing 300 μL of WS. Again, gently layer a small amount of DS from the pipette over the embryo. Set a timer for 5 min and return to the bench top. After 5 min, pick up the embryo with minimal volume of WS and move to the top of the final well of 300 μL WS. The embryo will slowly fall and wash through the WS to the bottom. Expel any retained WS from the pipette into an empty well. Pick up the embryo with minimal volume and return to the top of the same 300 μL well of WS. The embryo will once again fall to the bottom of the well. Set a timer for 1 min and return the 6-well plate to the bench top. 3. Recovery of warmed embryos One at a time, move warmed embryos into a center-well organ tissue dish containing 500 µL of equilibrated BM with 10% v/v SPS under 500 µL of equilibrated embryo culture grade oil. Wash embryos by picking up each embryo and moving them around to several areas in the dish while blowing out old media between moves. Pick up the embryo and move it to an individual 20 µL culture drop of equilibrated BM with 10% v/v SPS under oil. Let the warmed embryos recover for 2 h in an incubator at 37 °C, 6% CO2, 5% O2. 4. Zona removal After a 2 h recovery, assess embryos for re-expansion and take pictures of each embryo. Move embryos individually in 500 µL of a 3-(N-morpholino)-propanesulfonic acid (MOPS)-buffered handling medium with 5% (v/v) fetal calf serum (FCS) prior to the treatment with acidic Tyrode’s solution. Move one embryo quickly through 300 μL of warmed acidic Tyrode’s solution while actively watching through the microscope. The zona pellucida will visually start to dissolve. This will take approximately 5 s. Immediately move the embryo with dissolving zona into 300 μL of warmed MOPS buffered medium to quench the acid Tyrode’s solution. Move the embryo with minimal volume of MOPS buffered medium into a center-well organ tissue dish containing 500 µL of equilibrated BM with 10% v/v SPS under 500 µL of equilibrated embryo culture grade oil to wash. Return the embryo to the 20 μL recovery drop from step 3.2. NOTE: Upon visual examination following zona removal, any embryos with retained zona pellucidae may be further treated with acidic Tyrode’s solution if necessary, by repeating steps 4.2-4.6. Minimizing exposure to the Tyrode’s solution is desired. 5. Blastocyst extended culture Move the embryos individually to the wash dish with equilibrated IVC1 media from step 1.1.6. Carefully move one embryo to one well of the chambered coverslip with equilibrated IVC1 and maintain embryo identification. NOTE: This step needs to be finished as quickly as possible to minimize medium evaporation. Return chambered coverslip to an incubator set to 37 °C, 6% CO2, atmospheric O2 for 2 days. NOTE: Be sure to thaw and equilibrate media in 37 °C, 6% CO2, atmospheric O2 for the media exchange 4 h in advance. At outgrowth D2 (D7 post fertilization), carefully examine the attachment of embryos under the microscope and perform media exchange. Note which embryo is attached to the dish before exchanging media. Gently tap the plate and examine whether an embryo has securely attached to the plate under the microscope. Remove the lid and carefully remove 150 μL of IVC1 and discard while not disturbing the attached embryo. If an embryo has not yet attached to the plate, do not exchange the media, as the serum in IVC1 will aid in embryo attachment. Pipette 150 μL of equilibrated extended culture media, IVC2, slowly into each well and return the lid to the chambered coverslip. NOTE: Ensure that removing the lid from the chambered coverslip is minimized to avoid medium evaporation. Carefully return the chambered coverslip to the incubator without splashing any media on the lid. Repeat media exchange and attachment check every day of extended culture until embryos are ready for fixation or single cell digestion. 6. Optional collection of spent media Optionally, collect spent media during the exchange of culture media after D7 or any day thereafter. During step 5.3.2, rather than discarding the medium snap-freeze the 150 μL of removed IVC1 into a sterile, low-bind 0.5 mL tube for future analysis. 7. Optional fixation for immunofluorescence Use a 200 μL pipette to wash embryos with ½ media exchanges of PBS for 3x before fixation to remove any extracellular debris. Washing away excess debris and protein will help to optimize clarity and reduce background in images obtained with immunofluorescence. Remove all media and slowly add 200 μL of 4% paraformaldehyde (PFA) in PBS to the well. The embryo will want to stick to the surface of the fluid. Multiple 150 μL washes with 4% PFA before removing all fluid will help to minimize any damage to the embryo. Incubate the embryos in 4% PFA for 20 min for fixation. Wash embryos 3x for 10 min per wash with 200 μL of 0.1% v/v polysorbate 20 in fresh PBS. Store fixed embryos in 0.1% v/v polysorbate 20 in PBS at 4 °C before proceeding with the immunofluorescence protocol. 8. Single cell digestion with Trypsin NOTE: Fresh (not fixed) embryos are used for single cell digestion. Wash the embryo once with 200 μL of PBS and add 200 μL of trypsin solution to each well. Return the chambered coverslip to the incubator for 5 min. Remove the chambered coverslip from the incubator and examine the embryos under a stereoscope. Cells on the periphery of the embryo will start to retract and MTB should still be attached to the plate where they are remotely located from the embryo. Use a small pipette or finely pulled mouth pipette to pick up individual MTB before breaking apart the whole embryo. Skip ahead to step 9.1 to save MTB and return to step 8.4 after step 9.3. Use a handheld micromanipulation pipette or mouth pipette to gently dissociate the embryo by aspirating up and down. Cells will begin to dissociate from the whole embryo. Continue to aspirate the embryo gently and repeatedly using a smaller diameter pipette tip or mouth pipette until the embryo has been incubated for a total of 10 min in trypsin. 9. Single cell selection and sample collection With minimal trypsin, move the dissociated cells through three wash drops of 20 μL PBS + 0.1% polyvinylpyrrolidone (PVP) under embryo culture oil with 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 mL low-bind tube with minimal volume of PBS+0.1% PVP. Snap freeze single cells in liquid nitrogen (LN2), and store in -80 °C for future use.

Representative Results

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…

Discussion

The development of the protocol to culture human embryos through implantation has allowed scientists to explore a previously uncharted time in development^8,9. 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…

Materials

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