Stem Cell Research

Consistent Cell Count for Differentiation Success

Stem cells have gained significant attention in academic and industry sectors over the past two decades. This is due to these cells showing potential to alleviate or even cure many diseases. However, prior to utilization in advanced therapy medicinal products (ATMPs), much research and developmental work goes into technologies specific to the therapy. Human pluripotent stem cells (hPSCs) are derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) and are traditionally cultured on feeder cells in a 2D setting (a tissue culture dish/flask). To create a controlled stem cell niche, culture settings have moved towards more restricted conditions, where all components are serum-free, or even xeno-free, and using various plastic or microcarrier coatings, instead of attaching feeder cells in the dish. A restricted environment allows for better cell-to-cell signaling and control of factors that cause, and should be used, for the type of directed differentiation you are interested in.

Recent developments have seen stem cell culturing shift from traditional 2D methods to more physiologically relevant 3D methods, such as spheroid and organoid cultures, allowing for proper cell-to-cell signaling and interactions during cell growth and differentiation. These 3D structures have great promise in the fields of drug discovery and regenerative medicine, as they allow researchers to better simulate how their treatments affect tissues in a physiologically/biologically relevant setting, rather than experiencing their effect in isolated layers of cells. Because they are so physiologically relevant, organoid cultures can also reduce the use of certain animal experiments without compromising the scientific significance of the results obtained.

What’s the Challenge?

Apart from culture media, surface coatings, and pluripotency level of the cells you work with, the cell density is the fourth major contributor to experimental success. Intercellular signaling is difficult to control, but seeding cells at the right concentration can significantly contribute towards getting the directed differentiation of interest. For instance, differentiation protocols designed for mesoderm and endoderm cell types (e.g. pancreatic progenitors) have shown that low cell densities positively affect percentages of the cells of interest1,2. As an added benefit, low cell densities mean lower cytokine concentrations are required to obtain the same effect, and in turn saving considerable costs at the cell culture step.

Conversely, neural differentiation protocols often require high cell densities, as cell-to-cell signaling is indispensable and cell density at protocol onset determines the ratio of neuronal cells and cerebral organoids3,4.

Maintenance cultures of hPSCs are usually carried out by passaging dense cultures as medium-sized aggregates (clumps) of cells instead of dissociating them to single cells before re-plating. This ensures that the hPSC niche is not disrupted too much during passage and over multiple passages, which could lead to genetic drift and a decrease of pluripotency.

Improving Cell Counting & Viability Analysis in iPSC and ESC Work

To ensure consistency and reproducibility in culturing stem cells, one needs to have precise methods to estimate cell concentration and viability. The NucleoCounter® NC-202™ automated cell counter offers a wide range of specialized assays for hPSCs in single cell suspension, in aggregates, or grown on microcarriers. It is easy to use, eliminates human interference and variation from the counting process and takes only 30 seconds per cell count.

If you want to combine cell counting and viability analysis along with customized cellular stains of your choice, the NucleoCounter® NC-3000™ comes with a flexible protocol providing up to five channels of analysis of up to eight samples per slide. The instrument’s software (NucleoView™) produces full fluorescent image data and histograms with modifiable gate settings to give you full control over your data analysis.

For more information on how you can improve your analysis of hPSCs for stem cell therapies, please visit the following pages:

Mesenchymal stem cells (MSCs)
Induced pluripotent stem cells (iPSCs)
Hematopoietic stem (HSCs)

References

  1. AE Chen, M Borowiak, RI Sherwood et al.: Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation. Development. 2013; 140(3):675-86. 
  2. M Hansson, DR Olesen, JML Peterslund et al.: A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells. Dev Biol. 2009; 330(2):286-304. 
  3. Srimasorn, M Kirsch, S Hallmeyer-Ellgner et al.: Increased Neuronal Differentiation Efficiency in High Cell Density-Derived Induced Pluripotent Stem Cells. Stem Cells Int. 2019; 2019:2018784. 
  4. MA Lancaster and JA KnoblichGeneration of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014; 9(10):2329-40. 

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