Considerations to Ensure Optimal Isolation of Single Cells

The applications of single cell biology range from the ability to isolate a clonal cell for recombinant protein production to characterization of a heterogenous population of cells to better understand the biology of a tissue or disease. The explosion of techniques that allow for the analysis of single cells has fundamentally changed modern life science research and development. It is important to consider the technique used to isolate single cells to preserve the original sample and obtain as unbiased results as possible. In cases where one wishes to clone the cell it is also important to ensure cell viability is maintained. Single Cell Applications The range of single cell applications is varied, it is important to consider the specific application requirements when deciding on the suitable method to use to isolate your cells to support the desired results of your downstream application. There are 2 broad categories of single cell applications, the first is the desire to analyze individual cells on a cellular level such as single cell RNA or DNA analysis to study heterogeneity or identify specific cells of interest. The second broad category is to isolate individual cells in order to clone them and grow them out into a clonal population to remove heterogeneity. A list of the various single cell applications along with reviews can be found in table 1. Another emerging trend is the topic of multi-omics combining several single cell analysis techniques allowing one to identify cell lineage with intracellular signatures for even more power analysis of cellular states (1). Table 1. List of Single Cell Applications Cell types Another obvious important consideration is the type of starting sample that you will be working with in order to isolate single cells. Table 2 lists the various types of starting samples and some consideration that one should make for isolating single cells. Table 2. Considerations of your cellular isolation method based on cell type There are various methods one may use in order to isolation single cells into plates. For the purposes of this article we are specifically considering techniques which allow for the physical separation and isolation of individual cells into plates or wells. The two most commonly used techniques for single cell separation have historically been manual pipetting and flow cytometry. Table 3 lists a number of technique’s and technology’s that have been developed for single cell isolation. Table 3. Comparison of Single Cell Isolation Methods With the advancement of single cell analysis techniques such as scRNA-seq and with the demands for cell cloning which requires the isolation of intact healthy cells for clonal selection the limitations of the traditional methods have driven the development of dedicated instruments for performing single cell isolation. Many of these have been developed with a specific application in mind while some have been developed as more general instruments for isolation of single cells as well as some traditional cell sorters which allow for isolation of single cells into microtiter plates. Table 4 lists some of the latest instruments for performing single cell isolation. Table 4. Single cell isolation instruments - dedicated single cell instruments and flow cytometers compared Tagging Cell Samples for Sorting and Isolation Using a fluorescence based method to isolate individual cells enables the isolation of cells of interest and helps avoid the isolation of dead cells or debris. In specific cases where viable cells are desired the use of a using viability dye can help enrich your isolation for viable cells. Table 5. Markers that may be used to identify / tag specific cell types of interest Selecting for cell viability If your downstream application includes growing cells after single cell isolation maintaining your cell viability through the isolation will obviously be critical. The other consideration one should make is if you want to specifically select for viable cells during the isolation phase or just select any cell. Selecting for viable cells should increase the percentage of positive clones that will grow downstream - increased outgrowth. There are some concerns around the dyes used for cell viability but in our experience these dyes do not affect the cell phenotype post isolation and they are generally used at low concentrations to limit any potential effects on the cells. Even if your application is analysis and does not require viable cells post isolation it is often useful to consider sorting for viable cells to enrich your cell population. Capturing healthy cells will help ensure better results in your analysis such as single cell genomics and transcription profiles will better reflect ‘normal’ cell population vs the dead or dying cells which likely have poor quality RNA or DNA. Table 6. Available Cell Viability Dyes Table 7. Suitable nucleic acid dyes useful for single cell sorting and isolation. References and Further Reading 1. Yuan et al. Challenges and emerging directions in single-cell analysis Genome Biology (2017) 18:84. 2. Hu et. al. Single Cell Isolation and Analysis Front. Cell Dev. Biol. (2016) 4:116. 3. Nguyen et al. Experimental Considerations for Single-Cell RNA Sequencing Front. Cell Dev. Biol. (2018) 6:108. 4. Ambriz-Aviña et. al. Applications of Flow Cytometry to Characterize Bacterial Physiological Responses Biomed Res Int. (2014) 2014:461941. 5. Yarrow, D. The Yeasts – A Taxonomic Study (1998).

Considerations to Ensure Optimal Isolation of Single Cells

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