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. 2021 Oct 5;11(19):e4185.
doi: 10.21769/BioProtoc.4185.

Isolation of Primary Cytotrophoblasts From Human Placenta at Term

Arthur Colson  1   2 , Christophe Louis Depoix  1 , Corinne Hubinont  1   2 , Frédéric Debiève  1   2
Affiliations

Isolation of Primary Cytotrophoblasts From Human Placenta at Term

Arthur Colson et al. Bio Protoc. .

Abstract

The placenta is a multifaceted organ, fulfilling critical functions for the fetus and the mother. Therefore, it is a critical regulator of the pregnancy, and its dysfunction leads to diseases, including fetal growth restriction and preeclampsia. Studying the placenta is a difficult task since its existence is transient, and its structure is specific to our species. In vitro differentiation of primary cytotrophoblast isolated from term human placenta has been widely used in the placental research field as it represents a reliable model to study cellular differentiation and function. Direct alternatives include trophoblastic cell lines, explants, and organoids, but this protocol, based on the separation of the cells on a Percoll gradient, presents the advantage of being relatively cheap and easy to perform in every research laboratory. Furthermore, the 2D culture is a flexible method that can be adapted to various experimental conditions (transfection, drug exposure, metabolic study, observations, etc.), allowing mechanistic explorations of cellular processes.

Keywords: Fetal growth restriction; Placenta; Preeclampsia; Pregnancy; Syncytialization; Trophoblast.

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Figures

Video 1.
Video 1.. How to isolate of primary cytotrophoblasts from human placenta at term: an overview of the procedure accompanied by Summer vibes
Figure 1.
Figure 1.. Washing step.
A. After cutting the placenta into pieces, put them in the 2 L beaker and add 500 ml of NaCl 0.9%. B. Shake the beaker vigorously and drain the tissue in the sieve. Repeat this operation several times. After 4 to 8 L of NaCl 0.9%, the pieces must turn light red, and the wash solution must remain clear.
Figure 2.
Figure 2.. Cleaning step.
Prepare the pieces for digestion. A. After drying with paper towels, B. Remove big blood vessels and C-D. the chorionic plate. This step can be time-consuming, but reducing the amount of debris improves digestion and protocol yield.
Figure 3.
Figure 3.. Digestion step.
Dissolve 720 units of dispase I in 300 ml of preheated HBSS. A. Chop the pieces with the pair of big scissors and transfer the pulp to the baffled flask. B. Incubate the mixture 1 h in the water bath under strong shaking. Add predissolved DNAse I and incubate for another 15 min.
Figure 4.
Figure 4.. Filtration step.
A. After the digestion, the suspension is first prefiltered by using the little sieve. B. The suspension is then passed through the three test sieves, fixed on the stand by decreasing order. The filtrate is finally centrifuged; the pellet contains red blood cells, lymphocytes, debris, and cytotrophoblasts.
Figure 5.
Figure 5.. Gradient step.
The 6 ml contains a mix of debris, red blood cells, lymphocytes, and cytotrophoblasts. The discontinuous Percoll gradient allows separating the cell type by density. A. The gradient was freshly prepared on the day of isolation and stored at 4°C until use. B. After the filtration step, the cell suspension is meticulously deposited at the surface of the gradient. It is then centrifuged to separate the cells between the different layers.
Figure 6.
Figure 6.. The discontinuous Percoll gradient after centrifugation.
The cells form a compact cloud between 12.5 and 20 ml. The debris (red) are above, while the red blood cells form a pellet at the bottom of the tube. Some lymphocytes can be observed around the 12 ml graduation.
See this image and copyright information in PMC

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