# Developmental-stage specific single-cell human embryo dissociation

Jenna Lammers, Sophie Loubersac, Laurent David, Thomas Freour, Arnaud Reignier

► To cite this version:

Jenna Lammers, Sophie Loubersac, Laurent David, Thomas Freour, Arnaud Reignier. Developmental-stage specific single-cell human embryo dissociation. STAR Protocols, 2023, 4 (3), pp.102363. 10.1016/j.xpro.2023.102363. inserm-04208736

HAL Id: inserm-04208736

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Submitted on 15 Sep 2023

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Protocol

Developmental-stage specific single-cell
human embryo dissociation

<figure><img src="image_2.png" /></figure>

Jenna Lammers,
Sophie Loubersac,
Laurent David,
Thomas Freour,
Arnaud Reignier

arnaud.reignier@
chu-nantes.fr

Highlights

Technique to isolate
embryo cells by
mechanical
dissociation

Technique suitable
from cell stage to
blastocyst

Isolation of individual cells ensures detailed analysis of human embryos and promotes our understanding of molecular mechanisms driving embryo development and cell specification. Here, we present a protocol for the processing of human embryos for single-cell analysis. We describe steps for growing embryos and individualizing cells from the polar and the mural parts of trophectoderm at the blastocyst stage using laser dissection. We then detail embryo dissociation followed by steps to pick, wash, and dispense cells in plates.

Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Protocol

# Developmental-stage specific single-cell human embryo dissociation

Jenna Lammers,<sup>1,2,3,4</sup> Sophie Loubersac,<sup>1,2,3</sup> Laurent David,<sup>1,2</sup> Thomas Freour,<sup>1,2</sup> and Arnaud Reignier<sup>1,2,5,*</sup>

## SUMMARY

Isolation of individual cells ensures detailed analysis of human embryos and promotes our understanding of molecular mechanisms driving embryo development and cell specification. Here, we present a protocol for the processing of human embryos for single-cell analysis. We describe steps for growing embryos and individualizing cells from the polar and the mural parts of trophectoderm at the blastocyst stage using laser dissection. We then detail embryo dissociation followed by steps to pick, wash, and dispense cells in plates.

## BEFORE YOU BEGIN

This protocol requires having access to a fully equipped *in vitro* fertilization (IVF) lab, in particular the micromanipulation workstation equipped with a laser. Operators must be specifically and properly trained for micromanipulation on those equipment.<fnref n="1" /> Of note, local regulations concerning research on human embryos can vary and should always be followed.

In order to have precise staging of the embryos, culture in a time-lapse incubator is definitely a plus, as it provides dynamic and continuous monitoring of embryo development in an undisturbed environment. Either fresh or frozen-thawed embryos can be used. Freezing/thawing procedures are not presented here but must be performed according to manufacturer's instructions. Embryo culture should be performed according to the European Society of Human Reproduction (ESHRE) Guidelines (1). As our procedures for single-cell isolation is not affected by the protocol and medium used for embryo culture, we will not detail these aspects. Specific detailed procedure for *in vitro* embryo culture is available from the manufacturers. For example, we use Vitrolife® G-TL® medium in our setting (https://www.vitrolife.com/products/ivf-media-oil/g-tl/). Embryo morphology assessment should be performed according the Istanbul Alpha - ESHRE consensus workshop on embryo assessment.<fnref n="2" />

Dissociation of human embryos into single cells depends of their developmental stage (Figure 1A). At 8-cell stage (Figure 1B) and morula stage (Figure 1C), the zona pellucida is laser-opened (step-by-step method details 1), and cells simply dissociated by gently pipetting the morula into Mg-Ca free decompaction medium (G-PGD®) buffer (step-by-step method details 4). At the early blastocyst stage (Figure 1D), the zona pellucida is laser-opened (step-by-step method details 1), and embryo dissociated with the use of Accutase®, a cell detachment solution of proteolytic and collagenolytic

<figure><img src="image_3.png" /></figure>

<footnote n="1"><sup>1</sup>Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, 44000 Nantes, France</footnote>

<footnote n="2"><sup>2</sup>Service de Médecine et Biologie de la Reproduction, CHU de Nantes, Nantes, France</footnote>

<footnote n="3"><sup>3</sup>These authors contributed equally</footnote>

<footnote n="4"><sup>4</sup>Technical contact: jenna.lammers@chu-nantes.fr</footnote>

<footnote n="5"><sup>5</sup>Lead contact</footnote>

<footnote>*Correspondence: arnaud.reignier@chu-nantes.fr
https://doi.org/10.1016/j.xpro.2023.102363</footnote>
A

<table><thead><tr><th>Developmental stage</th><th>Zona pellucida processing</th><th>Cell dissociation</th></tr></thead><tbody><tr><td>2 to 9+ cell stages</td><td>Laser opening</td><td>Ca-Mg free medium</td></tr><tr><td>Morula</td><td>Laser opening</td><td>Ca-Mg free medium</td></tr><tr><td>B1 & B2 blastocyst stages</td><td>Laser opening</td><td>Accutase + Ca-Mg free medium</td></tr><tr><td>B3 & B4 blastocyst stages</td><td>Laser dissection</td><td>Accutase + Ca-Mg free medium</td></tr><tr><td>Hatched blastocysts</td><td>-</td><td>Accutase + Ca-Mg free medium</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Figure 1. Typical embryonic morphologies (scale bar 30 µm)**</figcaption></figure>

(A) Detail of steps necessary for each developmental stage.

(B-E) Typical embryos morphology are shown for a 8-cell stage embryo B (cultured at 68h), a morula C ("MC" cultured 92h), a blastocyst D ("B2" cultured 108h), and an expanded blastocyst E ("B4" cultured 116 h).

enzymes (step-by-step method details 5 to 7). At the expanded blastocyst stage (Figure 1E), the embryos are laser-dissected to separate the mural from the polar side (step-by-step method details 2, 3). Cells are then dissociated with Accutase® (step-by-step method details 5-7). At the hatched blastocyst stage, the cells are dissociated with Accutase® (step-by-step method details 5-7).

## Mg-Ca free decompaction medium supplementation with HSA

Timing: 5 min

1. Add 10 µL of HSA in 190 µL of G-PGD® (Mg-Ca free decompaction medium following manufacturer's instructions).

## Biopsy medium warming

Timing: 1 h

2. Place bottle of mineral oil, G-PGD® and Accutase® at 37°C for 1 h.

## Micromanipulation workstation set up

Timing: 5 min

3. Install properly holding and biopsy pipettes on micromanipulation workstation.

Δ CRITICAL: Make sure the heating stage has reached 37°C.

## Micromanipulation dish preparation

Timing: 5 min on heating stage at 37°C

4. For 1 blastocyst, deposit 2 × 10 µL droplets of the mix (G-PGD + HSA) in the center of the dish, covered with warmed mineral oil. Add an additional droplet per additional blastocyst.

5. Put 1 blastocyst in 1 droplet with a 290 µm tips.
## Single cell isolation dish preparation

◎ Timing: 5 min at room temperature, when blastocyst dissection is done

6. 1 × 30 µL G-PGD® + HSA droplet.

7. 3 × 30 µL Accutase® droplets.

8. 15 × 10 µL GTL® droplets.

9. All droplets covered with mineral oil.

△ CRITICAL: Prepare the dish, just before cell isolation, when blastocyst dissection is done; make sure the heating stage is off to work at room temperature.

## KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Biological samples</td></tr><tr><td>Human embryos</td><td>N/A</td><td>N/A</td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>Human serum albumin solution (100 mg/mL)</td><td>Vitrolife</td><td>10064</td></tr><tr><td>G-PGD®</td><td>Vitrolife</td><td>10074</td></tr><tr><td>Accutase®</td><td>STEMCELL Technologies</td><td>07920</td></tr><tr><td>Ovoil®</td><td>Vitrolife</td><td>10029</td></tr><tr><td>G-TL®</td><td>Vitrolife</td><td>10145</td></tr><tr><td colspan="3">Other</td></tr><tr><td>Inverted microscope</td><td>Leica</td><td>AM6000</td></tr><tr><td>Micromanipulation station</td><td>Eppendorf</td><td>Cell Tram</td></tr><tr><td>Incubator</td><td>Panasonic</td><td>MCO-5M</td></tr><tr><td>Objective 40x with laser</td><td>Hamilton Thorne</td><td>Zilos-TK laser desktop</td></tr><tr><td>Heating stage</td><td>Leica</td><td>MATS Type I2</td></tr><tr><td>Stereo microscope</td><td>Leica</td><td>M125</td></tr><tr><td>ICSI dish</td><td>Vitrolife</td><td>16006</td></tr><tr><td>290 µm tips</td><td>Origio - Cooper Surgical</td><td>7-72-2290/5</td></tr><tr><td>170 µm tips</td><td>Origio - Cooper Surgical</td><td>7-72-2170/5</td></tr><tr><td>135 µm tips</td><td>Origio - Cooper Surgical</td><td>7-72-2135/5</td></tr><tr><td>75 µm tips</td><td>Origio - Cooper Surgical</td><td>7-72-2075/5</td></tr><tr><td>Stripper</td><td>Origio - Cooper Surgical</td><td>MXL3-STR</td></tr><tr><td>100 µL tips</td><td>Thermo Scientific</td><td>10287524</td></tr><tr><td>P100 pipet</td><td>BIOHIT Proline</td><td>NA</td></tr><tr><td>Pasteur pipet</td><td>Dominique Dutscher</td><td>043262S</td></tr><tr><td>Holding micropipet</td><td>Origio - Cooper Surgical</td><td>MPH-MED-30</td></tr><tr><td>Biopsy micropipet</td><td>Origio - Cooper Surgical</td><td>MBB-FP-SM-30</td></tr><tr><td>60 × 15 mm Dish</td><td>Starlab</td><td>CC7672-3359</td></tr></tbody></table>

## STEP-BY-STEP METHOD DETAILS

### Zona pellucida removal (for cleaved embryos, morula, early blastocyst stages (B1/B2 according to Gardner's classification)

◎ Timing: 5-15 min

The goal is to retrieve the embryo from the zona pellucida.
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Single-cell picking at each developmental stage (scale bar 30 µm)</figcaption></figure>

(A) Sample images of laser opening of zona pellucida at indicated developmental stages.

(B) Snapshots showing an expanded blastocyst before, during and after laser dissection.

(C) Snapshots showing a 10-cell embryo before, during and after biopsy.

(D) Snapshots showing a morula before and after biopsy.

This step is only performed if processing cleavage stage, morula stage and early blastocyst stage embryos (B1/B2). The operator performs brief laser impulses (around 9) to open the zona pellucida.

1. In the GPGD/HSA droplet hold and immobilize the embryo with the holding micropipet.

2. Use the laser to perform the hole, shoot the zona pellucida as far as possible of cells to open a hole large as 1.5x the biopsy micropipet (with a ZILOS-tk Hamilton-Thorne we use a laser power at 100% for 300 µs). A hole is visible in the zona pellucida (Figure 2A and Methods video S1: Zona pellucida removal for cleaved embryos).

## Mural / polar laser dissection

Timing: 5-15 min

This step is specifically for expanded blastocysts (B3/B4 according to Gardner's classification³). At this stage, dissociating polar vs. mural is critical, either to focus the analysis on the polar side, that contains the 3 main cell types of interest: epiblast (EPI), primitive endoderm (PrE) and trophectoderm (TE), or to capture differential-maturation events between mural and polar TE.

The operator starts by performing brief laser impulses to separate the polar TE and Inner cell mass (ICM) from the mural TE of the blastocyst (Figure 2B and Methods video S2: Mural / polar laser dissection).
A

<figure><img src="image_6.png" /></figure>

Good quality, embryo
amenable to polar/mural
dissection and single cell
isolation

<figure><img src="image_7.png" /></figure>

Medium quality, embryo
amenable to polar/mural
dissection

<figure><img src="image_8.png" /></figure>

Poor quality, use embryo
for bulk analysis of fixation

B

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 3. Trouble shooting examples (scale bar 30 µm)**</figcaption></figure>

(A) Embryo morphology associated with recommended processing.

(B) Examples of retracted, hatching and hatched blastocysts.

3. In the droplet, with the help of micropipettes, rotate the embryo in order to have the polar side close to the holding micropipet.

a. Hold and immobilize the blastocyst to strongly maintain the polar side in front of the holding micropipet.

b. Place the biopsy micropipet as close as possible to the mural trophectoderm, at the complete opposite side and apply a gentle suction pressure.

4. Use the laser to perform the dissection.

a. Shoot the zona pellucida and the trophectoderm as close as possible to the ICM, in each visible cell junction from one side to the other (up and down on the screen, 3 times, with a ZILOS-tk Hamilton-Thorne we use a laser power at 100% for 700 µs).

b. Simultaneously separate the 2 parts progressively by pulling each micropipet apart.

c. Use the laser until the 2 parts (mural and polar) are fully separated.

△ CRITICAL: Aspiration / holding setup should be equilibrated. Embryo should be placed at the bottom of the dish to ensure stability and homogeneous laser power.

**Single cell dissociation at cleavage stage and morula stage**

◎ Timing: 5-20 min

Once extruded or aspirated through the zona pellucida hole, cells should be gently pipetted in and out with 135 µm tips in G-PGD/HAS solution at 37°C to be individualized (Figure 2C and Methods video S3 Single cell dissociation at morula stage).

5. Perform the cell dissociation:

a. Triturate with gentle pipette flush under stereo microscope. G-PGD-HAS solution allows very easy dissociation of the cells.

b. Single cell can be isolated by aspiration and flushing.
**Note:** It is important to respect tips size to avoid breaking cell membrane.

c. Washing may be necessary depend on the analysis performed. Three washing steps in individual droplets of appropriate medium (often PBS) is recommended.

d. Replace cell isolated in the adapted container for your analysis.

## Single cell dissociation at B1/B2 stage, B3/B4 or B5/B6 hatched blastocysts

### Timing: 15-60 min

Embryos or embryo sections were incubated in G-PGD/HSA and transferred in Accutase. Embryos were gently pipetted in and out under stereo microscope. Single-cells/clumps were washed in GTL and gently pipetted in and out again if necessary. Single-cells were picked one by one upon dissociation (Figure 2D).

6. The mural or polar section, or the entire blastocyst, is incubated in the GPGD droplet for 10 min at 37°C with a 170 µm tips.

**Note:** Repeated aspirations and flush helps homogeneous diffusion of the media.

7. Place the section or the entire blastocyst in the accutase droplet for 3-4 min at room temperature with a 135 µm tips.

a. Pipet in and out around thirty to fifty times the cell cluster in the droplet with gentle pipette flush under stereo microscope.

b. During pipetting, to avoid, bubbles formation or cell lost, use the three droplets.

c. Single cell can be picked as soon as they individualize (Methods video S4 Isolation of single cells after accutase treatment of blastocysts).

8. Place the single cell or the clumps in the first GTL droplet at room temperature with a 75 µm tips.

a. Triturate with gentle pipette flush under stereo microscope.

b. Single cell can be isolated by aspiration and flushing.

c. Washing may be necessary depend on the analysis performed. Three washing steps in individual droplets of appropriate medium (often PBS) is recommended

d. Replace cell isolated in the container adapted for your downstream analysis.

Δ CRITICAL: Steps 1 to 6 are performed at 37°C; steps 7 to 8 - with accutase - were performed at room temperature.

## EXPECTED OUTCOMES

Upon laser opening of the zona pellucida, single cell / blastomeres dissociation at the cleavage or at the morula stage should be easily feasible. For example, out of 10 morula, we retrieved 85% of the cells for subsequent analysis (Methods video S5: Single cell dissociation at morula stage).

Upon dissection of B3/B4 blastocysts, you should first obtain a polar side and a mural side. Then, single cells can be dissociated. For example, out of 8 blastocysts, we retrieved 9 to 47 cells for subsequent analysis (average 18 cells per blastocyst).

The clumps that cannot be individualized after 30 min are processed as clumps.

## LIMITATIONS

Embryo culture setup should be appropriate in order to avoid poor embryo development, both in terms of developmental rate and morphology. When using frozen-thawed embryos for research, the thawing procedure needs to be perfectly tuned, strictly following manufacturer's instructions,
in order to maximize embryo survival rate and subsequent development. Please refer to IVF Guidelines (1) to get guidance towards high-standard human embryology practice.

Operator needs to be experienced with the use of microscope and micromanipulators in order to perform precise, stable and extremely careful manipulation of the embryos and isolated cells. Any suboptimal setup of the workstation, inadvertent movement or brutal manipulation will result in poor single cell recovery rate, or even embryo or cell lysis.

Of note, it is almost impossible to dissociate and collect all cells from each embryo. Part of cells can eventually be lysed during laser assisted-zona opening or lost during washing or enzymatic treatment steps.

## TROUBLESHOOTING

### Problem 1

Poor quality blastocysts (Figure 3A)

In the case of poor-quality blastocysts (grade C for ICM or trophectoderm), single cell picking can become particularly difficult and time-consuming.

#### Potential solution

Operators need to train with human embryos and become experts in embryo morphology classification to be able to identify good, medium and poor quality embryos. The use of Morphological atlas, reference morphological classifications and participation in external quality control program is strongly recommended (2,3).

### Problem 2

Retracted/hatched blastocysts (Figure 3B)

If expanded blastocysts are retracted/collapsed, it may become difficult to locate the ICM and therefore to separate accurately polar TE and ICM from mural TE. Similar issues can occur with hatching/hatched blastocysts which have a spontaneous tendency to collapse and retract during micromanipulation and laser shootings. As retracted blastocysts do not expand when zona pellucida is removed, it is important to avoid this situation and prefer (whenever possible) the use of expanded blastocyst when attempting to dissect mural and polar sides.

#### Potential solution

If the embryo is not or insufficiently expanded, it is advised to postpone experiment and prolong embryo culture for a few hours until proper expansion.

If the blastocyst is hatching (or has already hatched) or does not re-expand, it is however possible to perform laser dissection. After complete removing of the ZP with laser, the suspected polar side of the embryo (where ICM is located) should be aspirated in the biopsy pipette, while the rest of the blastocyst remains floating out of the pipette. The end of the biopsy pipette should then be put into direct contact with the end of the holding pipette and both should then be rubbed against each other, resulting in the mechanical dissection of the polar TE contained in the biopsy pipette on one side and mural TE released in the medium on the other side. This method is referred to as flicking technique and frequently used for trophectoderm biopsy in PGT-A program.

### Problem 3

Cell loss

Single cell handling requires isotonic medium in order to protect cells membrane integrity. This is often possible with the use of media containing HSA. Of note, HSA obviously interferes with single
cell proteomics, requiring multiple washing steps in ultrapure water to remove protein contaminants. However, cell handling in ultrapure water involves tricky and lengthy manipulation time and can result in an increased risk of cell loss.

**Potential solution**

The addition of Polyvinylpyrrolidone (PVP 7%) to ultrapure water could enable more comfortable manipulation and limit cell loss.

If cells are sticking to each other, it could also be due to poor Accutase quality. We recommend to aliquot Accutase medium and avoid multiple freeze / thaw cycles. Additional washes can also help to isolate cells.

**RESOURCE AVAILABILITY**

**Lead contact**

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Arnaud Reignier, arnaud.reignier@chu-nantes.fr.

**Materials availability**

No materials have been specifically designed for this protocol.

**Data and code availability**

Our original datasets and codes are available in our resource paper in Cell Stem Cell.⁴

**SUPPLEMENTAL INFORMATION**

Supplemental information can be found online at https://doi.org/10.1016/j.xpro.2023.102363.

**ACKNOWLEDGMENTS**

We thank Gedeon Richter, Theramex, Ferring, IBSA-Genévrier, MSD, MERck Serono for funding this work through donations to Nantes Université's Foundation. We thank the staff of the IVF unit of the CHU de Nantes for help and support.

**AUTHOR CONTRIBUTIONS**

Methodology, validation, investigation, writing, J.L., S.L.; supervision, review, editing, project administration, L.D., T.F.; methodology, validation, investigation, writing, project administration, A.R.

**DECLARATION OF INTERESTS**

The authors declare no competing interests.

**REFERENCES**

1. ESHRE Guideline Group on Good Practice in IVF Labs, De los Santos, M.J., Apter, S., Coticchio, G., Debrock, S., Lundin, K., Plancha, C.E., Prados, F., Rienzi, L., Verheyen, G., et al. (2016). Revised guidelines for good practice in IVF laboratories (2015). Hum. Reprod. 31, 685-686.

2. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology

in Reproductive Medicine and ESHRE Special Interest Group of Embryology (2011). The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum. Reprod. 26, 1270-1283.

3. Gardner, D.K., and Schoolcraft, W.B. (1999). Culture and transfer of human blastocysts. Curr. Opin. Obstet. Gynecol. 11, 307-311.

4. Meistermann, D., Bruneau, A., Loubersac, S., Reignier, A., Firmin, J., François-Campion, V., Kilens, S., Lelievre, Y., Lammers, J., Feyeux, M., et al. (2021). Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification. Cell Stem Cell 28, 1625-1640.e6.