Single-cell RNA-sequencing (scRNA-seq) is a powerful technique for exploring the cellular composition of complex tissues. An essential prerequisite for performing scRNA-seq is the preparation of high-quality single-cell suspensions from the tissue of interest. Moreover, the use of cryopreservation coupled with batch sequencing (i.e., multiplexing) is essential for working with rare or difficult to obtain samples. Our line of research is focused on understanding the complex cellular interactions taking place in the placental tissues, and therefore a detailed protocol for the dissociation and cryopreservation of these tissues is necessary for our single-cell investigations. The placenta includes multiple distinct regions, which we have divided into the basal plate with placental villi (corresponding to the “body” of the placenta) and the extraplacental membranes, also known as chorioamniotic membranes. Importantly, these tissues include the “maternal-fetal interface”, the sites of contact between the mother and fetus. The maternal-fetal interface hosts important immunological, as well as other biological, interactions that are essential for the continuation of healthy pregnancy. Thus, the placenta continues to be a central target for investigating obstetrical syndromes such as preterm birth and preeclampsia.
In our publication, we describe in detail our method for the enzymatic dissociation of the placental tissues. The distinct composition of these placenta tissues precludes the use of a single digestion protocol for both, and thus we have validated two distinct methods tailored to the basal plate + placental villi and to the chorioamniotic membranes, which are presented in parallel in our protocol. For the basal plate with placental villi, we established that digestion using Collagenase A provided the best cell yields and viability, whereas a proprietary enzymatic cocktail was best suited for the chorioamniotic membranes. By combining these two enzymatic approaches together with the refinement of digestion times and sample handling, we demonstrate that high-viability single-cell suspensions can be obtained from the placental tissues. Importantly, the parallel design of our protocol makes it suitable for adaptation by researchers who may be interested in only the basal plate, placental villi, or chorioamniotic membranes.
A critical consideration for single-cell studies using clinical samples is the cryopreservation of single-cell suspensions for multiplexing, which can reduce the reagent and material costs associated with single-cell library preparation. To help researchers plan their single-cell experiments, we utilized flow cytometry to evaluate cellular viability and composition in fresh and cryopreserved single-cell suspensions of the placenta tissues. We found that neutrophils, which are short-lived and fragile cells, were negatively impacted by cryopreservation, and this factor should be taken into consideration by investigators. In addition, overall cell viability is reduced by cryopreservation; however, we show that the use of dead cell removal protocols can ensure a high proportion of viable cells prior to initiating single-cell experiments using cryopreserved samples.
We also validated the single-cell suspensions generated by this protocol for use with the droplet-based 10x Genomics Chromium system for scRNA-seq. By applying the quality control metrics recommended by 10x Genomics, we provide examples of high-quality and low-quality sample readouts before and after library preparation that can help researchers evaluate their sample quality at these critical points. We provide an overview of the typical analysis pipeline and tools that we use to interrogate our placental single-cell datasets, accompanied by examples of the cellular composition of the basal plate with placental villi and chorioamniotic membranes. A unique aspect of the placental tissues is the presence of cells from two genetically distinct individuals: the mother and the fetus. Therefore, we also describe the incorporation of maternal and fetal genotype information into our single-cell analysis, which allows for the distinction of cells according to their maternal and fetal origin. The latter step provides a key layer of information that we have utilized to evaluate the maternal and fetal contributions to pregnancy-specific processes.
Our protocol provides a useful systematic approach for the single-cell investigation of the human placenta (the basal plate, placental villi, and the chorioamniotic membranes), including the maternal-fetal interface. The presentation of parallel protocols for each tissue together with guidance on the use of fresh or cryopreserved single-cell suspensions for downstream applications makes the protocol easily adaptable for different research questions and study designs. An important consideration for investigators interested in this protocol is that we are primarily working with tissues collected in the third trimester; yet we consider that this protocol may also be useful for those interested in disorders of early pregnancy such as miscarriage or recurrent spontaneous abortion. We have outlined the advantages and disadvantages of our protocol to allow researchers to make the most informed decision regarding the use of their precious samples, and we hope that the accessibility and detail of our protocol can encourage other investigators to pursue novel research questions in the field of maternal-fetal biology.