Why consistent cell counting is key to understanding early embryonic development

Dr. Naomi Moris and her team from the Francis Crick Institute are using embryonic stem cell systems to model the early stages of human embryonic development. Their processes rely on highly accurate and repeatable cell counting. As a result, they recently invested in a NucleoCounter® NC-202™. In this post, we talked to Dr. Peter Baillie-Benson, Senior Laboratory Research Scientist at the Moris group, to find out more about their work and why cell counting is so important to them.

Studying developing embryos can help us understand the mechanisms behind human development and what happens when those mechanisms go wrong. However, studying human embryos comes with ethical considerations and regulatory restrictions, which limit how long human embryos can be used for research.

The Moris laboratory is interested in the process of gastrulation. Gastrulation is a step in early embryonic development where the embryo transforms from a single layer of epithelial cells into a multilayered, multidimensional structure. Gastrulation forms three embryonic germ layers, the endoderm, ectoderm, and mesoderm, which later develop into different body parts. In short, gastrulation provides the first step of forming a body plan.

Studying gastrulation with stem cell systems

Gastruloid viewed through a microscope

“UK regulations only allow you to study human embryos up to 14 days or the appearance of the primitive streak, whichever is sooner. So, we can’t study anything beyond the very early stages of gastrulation using embryos,” explains Peter. Instead, he and the team are using human gastruloids, three-dimensional structures of embryonic stem cells, to model gastrulation.

“Our work centers around a key assay where we’re growing human stem cells under self-renewing conditions. Then we aggregate them in very defined numbers to form very small clusters of cells like a 3D spheroid.

Over time those aggregates start to polarize their gene expression and develop the rudiments of a body plan. They gain head-to-tail polarity, and they start to elongate in that direction and express some of the genes you might expect to see expressed in a developing human embryo,” he says.

“We study them for about 3 days, so quite a short period of time. But in that time, we can learn quite a lot about their gene expression.”

Peter and the team hope that interrogating human gastruloids will give them a better understanding of human development principles and developmental diseases like congenital malformations.

“We can also use gastruloids to screen for potential drug teratogenicity, so looking at whether a particular compound might dysregulate development in a way that might be damaging to the embryo, which could help us understand drug safety while reducing the use of animal models in research” he adds.

Why is consistent cell counting so important?

“The reason that cell counting has been so important for us is because our human gastruloid assay only works if you culture a very specific number of cells in the aggregate to begin with,” explains Peter. “They have to be at a particular density to reach a state that they can self-organize into aggregates.”

“With stem cell systems, there’s often quite a lot of variability, especially between lines, experiments, and users, so we’re always trying to drive out that variability and get more consistent results. When this assay works, it works very well, but it relies on everything being as consistent as possible,” he explains.

“As part of our protocol, we start plating the cells at a particular density, and then we leave them for about 5 days before we make the aggregates. But when you plate the cells at a particular density and then expand them, any error in that first measurement gets magnified,” Peter explains. “Consistency in our cell counting is really important. We want to know when we are plating 40,000 cells that we’ve really got that number in the dish and that there’s not going to be great variation”.

From manual to automated cell counting

“In the very beginning, we were doing all manual counting using hemocytometers, which limits throughput because it relies on having people trained to do it quickly and accurately,” says Peter. The first automated cell counter they tried relied on disposable slides and preparing suspensions with acridine orange and propidium iodide to count the live and dead cells. This system was inconsistent, and Peter suspects much of the variation came from how different users prepared their suspensions and adjusted the setting on the cell counter.

Peter knew he needed a more consistent instrument but also a counter that could work across a large dynamic range. “How many cells we are working with varies quite a lot. We’re plating tens of thousands of cells into wells, but when we’re preparing the cells, we can work with a million or even ten million cells. So, we’re trying to work on two orders of magnitude: from tens of thousands to millions. Our cell counter needed to keep up with that”.

“Another important thing to us was that the counter could count cells within clumps or clusters. This is important because when you’ve got new users in the lab, they might not be as effective at making single-cell suspensions that are very easy to count, so you need to have a counter that will work out how many cells there actually are before you plate them,” he says.

Aggregated stem cells viewed through a microscope

Testing the NC-202™

“We did quite a long demo with the NC-202™, and we tested it really extensively,” Peter explains.

“I made a lot of cell suspensions at different dilutions and densities and put them through all three counters. I was looking at the variability of repeated counts of the same suspension, so I could find the most consistent counter,” he says.

The NucleoCounter® NC-202™ is designed to eliminate the variation between users. It uses our individually volume-calibrated Via2-Cassette™, which contains immobilized, pre-portioned dyes to eliminate variation from sample preparation. The NC-202™ also uses fixed focus and standardized protocols, so there is no variation caused by how operators use the counter.

“I also checked that the counts were good across a range from around 1 in 1000 to an undiluted suspension, just to make sure there was a good dynamic range of the count.” The NC-202™ also provided the large dynamic range Peter and the team needed, providing reliable cell counting from 5×104 to 1×107 cells/ml. It also features our powerful declustering algorithm for accurately counting aggregated cells.

Now that Peter has found a cell counter that meets their needs, he and the team can focus on the important work of understanding gastrulation.

To learn more about the Moris lab research, you can visit their website or read a recent publication from the group.

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