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Cell Prep Possibilities: Sample Prep and Cell Isolation Methods for Single-cell

This feature introduces the basic single-cell workflow, as well as best practices and the latest commercial methods in sample preparation and cell isolation, ready for single-cell sequencing. It is adapted from Chapter 1 of the Single-Cell And Spatial Buyer’s Guide, which can be downloaded for free here. All information contained in this feature was procured to the best of our ability in Q1 2024, we do not claim that the information is without error.

Single-Cell Sequencing

In some ways, single-cell sequencing can be seen as the natural progression from bulk tissue sequencing. Biological processes and diseases are complex and heterogenous, and the desire to understand this complexity arose at the same time that single-cell sequencing took off. Bulk sequencing still has its uses, but it can only take you so far. Single-cell sequencing was the tool, with the capacity to capture nuance, necessary for the job.

Primarily used for RNA sequencing as opposed to DNA sequencing, single-cell methods deconvolute bulk tissues into individual cells that can then be separately sequenced. Although this results in only a small amount of genetic material per cell, you gain an appreciation of each unit of the tissue, as opposed to an aggregate reading across it. Hence, single-cell RNA sequencing allows researchers to understand many aspects of a disease from the level of each individual cell, which is the level at which diseases tend to act. It allows us to ask how gene expression differs between healthy and diseased cell types and what might this mean for the disease profile and progression1.

Single-cell DNA sequencing is also valuable. While healthy cells in a tissue will have the same genome, single-cell DNA sequencing can identify somatic or germline mutations in specific cellular populations, which can help with investigations in cancer, ageing and neurodegeneration. The issue with this methodology arises from the fact that there are very small amounts of genomic material in a single cell. Whole genome DNA methods, such as multiple displacement amplifications (MDA)2, multiple annealing and looping-based amplification cycles (MALBAC)3 and degenerate oligonucleotide primed PCR (DOP-PCR)4, allow this analysis at genome-scale by amplifying low abundancies of DNA.

There are two main types of single-cell transcriptomic methods, based on whether they are sequencing mRNA through a primed tag or whether they use full-length transcriptomic methods. Both methods have seen the same overall advancement – an increase in cell throughput, alongside the subsequent decrease in cost per cell to run single-cell methodologies (Figure 1).

Figure 1. Development of single-cell RNA sequencing (scRNA-seq) technology. Timeline and throughput of various scRNA-seq methods. This scatterplot depicts the published date and throughput of sequencing for each technology. The colour indicates the different gene coverage. Size indicates the cost per sequenced cell of scRNA-seq methods. Source: Huang, et al. 12

This process has advanced, from the early days of manually isolating individual cells, to the first fluidic circuits that allowed the processing of 100s of cells5,6, and eventually to droplet methods (inDrop7 and Drop-seq8), allowing 10s of 1000s of cells to be processed. Finally, combinatorial indexing methods (sci-RNA-seq9,10 and SPLiT-seq11) have brought us to the current era, in which million-cell experiments are now a viable reality for most researchers.

Single-cell sequencing still relies on NGS sequencers, and single-cell research has benefited from the advancement in sequencing technology capabilities (see our Sequencing Buyer’s Guide for a full coverage of this).

The Single-Cell Workflow

A very simplistic single-cell sequencing workflow can be seen in Figure 2. Once tissue is procured, a single-cell suspension needs to be generated through gently breaking down the tissue. Individual cells need to then be isolated in well-plates or in contained reaction vesicles. Once individual cells are isolated, these cells are lysed so that the RNA is captured separately for each cell and then RNA is converted to cDNA to undergo standard NGS library prep, sequencing and analysis.

Figure 2. Basic workflow of single-cell sequencing. Source: Pan, et al. 13

Efficient sample prep is known for being crucial to performing an effective single-cell study. The common phrase in the field is – ‘crap in, crap out’. We direct readers to an excellent review paper from 2023 that covers a multitude of sample preparation techniques with advice and guidance14. The remainder of this feature will review the popular methods for the preparation of individual cells and review the commercial, automated instruments that could assist you to standardise the process.

Figure 3. Steps of the single-cell workflow with methods and key considerations. Source: Nguyen, et al. 15

Tissue Dissociation

Once the tissue samples are procured, the principal step in preparing the sample for a single-cell workflow is the tissue dissociation step. The goal is to convert a tissue sample into a suspension of single cells. This process is arguably the greatest source of unwanted technical variation and batch effects. A typical protocol for tissue dissociation involves (1) tissue dissection, (2) mechanical mincing, and (3) enzymatic breakdown.

Since tissue dissociation is such a critical step for ensuring experimental consistency, standardisation is essential. While there is little to replace the standardisation the comes from years of practiced wet lab work, commercial semi-automated platforms go a significant way to allow more reproducible, time-saving and efficient tissue dissection and single-cell preparation.

The benefits of automated tissue dissociation for single-cell sequencing can be seen as providing:

  1. Consistency: they allow for consistent processing of samples, reducing the variability between samples, and improving data accuracy.
  2. Speed: the dissociation process saves a lot of time, allowing researchers to process more samples in less time. This reduces labour costs and increases the number of samples that can be analysed.
  3. Quality: It provides more efficient dissociation of tissue, leading to higher yields of viable single cells. It also minimizes the risk of sample contamination.
  4. User-friendly: they are easy to use and require minimal training. They can be programmed to process various tissue types and sample sizes.
  5. Cost-effective: although automatic dissociation can be expensive, they can save money in the long term by reducing the need for multiple technicians and increasing the number of samples processed.

Below you will find a selection of the commercially-available dissociators on the marker. When choosing a tissue dissociator, it is important to consider the following factors:

  1. The tissue type. Check if there is already a program for it.
  2. The throughput requirements for your application.
  3. The cost (of the device and all specific materials, if required).
  4. The maintenance and service requirements.
  5. Other customers’ recommendations.

gentleMACS™ Dissociator – Miltenyi Biotec

The gentleMACS™ Dissociator is a benchtop instrument for semi-automatic or automatic tissue dissociation. The original dissociators is semi-automatic and can process 1 or 2 samples in parallel. The gentleMACS™ Octo Dissociator with Heaters is the larger, fully automated, counterpart with room to process 8 samples, and can also use heaters. By using one of the tissue-specific MACS Tissue Dissociation Kits with predefined enzyme mixes, and pre-defined programs for the gentleMACS Dissociators, there is no need to test multiple dissociation protocols to generate single cell suspensions with high viability.

Both dissociators use dedicated gentleMACS tubes (processing 20 mg – 4,000 mg of tissue per tube). The C tubes are for generating single-cell suspensions or single nuclei suspensions, while the M tubes are for further homogenization into subcellular material. The recently released gentleMACS Perfusers replaces tedious and complicated manual perfusion methods with automated ex vivo perfusion of liver to get reproducible results isolating viable hepatocytes. Over 40 preset programs exist for various human and mouse tissues plus user-defined programs can be created, saved, and shared. Click here for the user manuals for the original and Octo Dissociators.

PythoN® Tissue Dissociation System – Singleron

The PythoN® system integrates heating, mechanical and enzymatic dissociation in one 15 minute workflow. 8 samples can be processed in parallel with compatibility for 200+ tissue types. It also works with tissue weight from as little as 10mg to 4000mg. Up to 50 custom dissociation programs can be stored. The system uses disposable Dissociation Units and Singleron’s sCelLive® Tissue Dissociation Mix. Demo data shows this combination produces >85% cell viability across a range of tissues. The PythoN Junior™ is a compact and flexible version of the original. It works on the same principals and process with a 15 minute standard run time but with fewer samples (1-2) per run. This makes it ideal for clinical settings, since suspensions can still be generated from small amounts (~10mg).

Singulator™ – S2 Genomics

The Singulator™ Platform is a fully automated single cell and single nuclei isolation solution that is comprised of three components: Singulator instruments, single-use cartridge consumables, and cell or nuclei isolation kits. The reproducibility and precision of the Singulator platform removes a major bottleneck to conducting single cell genomics research, enabling more scientists to process more samples for single cell genomic applications. The Singulator Platform combines manual dissociation with enzymatic dissociation to isolate cells and manual extraction with chemical dissociation to isolate nuclei. Cells are dissociated from fresh tissue with a 20-60 minute run time achieving viability up to 90%. Nuclei are isolated from fresh, and flash frozen tissue with a 6-10 minute run time from as little as 2 mg of sample input. Finally, the Singulator 200+ automates deparaffinization and dissociation of nuclei from FFPE sections in as little as 40 minutes, enabling snRNA-Seq for FFPE samples.

VIA Extractor™ – Cytiva Life Sciences

The VIA Extractor™ works via single-use sample pouches that allow 3 samples to be processed in parallel. Run times can be as low as 10 minutes but are adjustable and pouches allow up to 1g of tissue. Viability scores tend to score highly (80%+) and yields for difficult tissues are high due to the VIA Freeze™ temperature control function, which can coordinate speed, temperature and time to maximize cell viability.

TissueGrinder – Fast Forward Discoveries

The FFX TissueGrinder is an enzyme free tissue dissociator for single-cell applications. This compact benchtop device has four grinding slots for mechanical dissociation and works in under 5 minutes to generate a suspension from tissue. Standard labware (Falcon Tubes) are fitted with the FFX grinder and strainer, which apply a combination of shearing and cutting to release viable cells into suspension. The pattern of mechanical processing is controlled by the instrument.

DSC-400/DSC-800 – RWD Life Science

The DSC-400 and DSC-800 are single cell suspension dissociators that use mechanical and enzymatic digestions. Runs typically take between 15 and 30 minutes. The 400 version has 4 independent working channels and the 800 has 8. Both systems can process samples from 20 mg to 4000 mg. Due to patent infringement, the tissue processing tubes of RWD are not available in the USA market.

Cell Sorting and Filtering

Filtering liquids or tissue dissociations to remove debris and dead cells is an important step in single-cell workflows. An associated optional step is single cell enrichment or cell sorting. In this step, a single cell suspension is filtered for debris and enriched for cell types of interest. This puts rare cell types to the forefront. There are several methods to accomplish this.

Filtering is a basic method of removing debris and clumps while retaining whole cells, and strainers are a good tool for the job. Commercial strainers include the pluriSelect strainers and the MACS® SmartStrainers. Different mesh sizes tend to be used, starting with large sizes (100 µm) and reducing as needed (e.g. to 70 µm and 40 µm). Every round of straining will result in unwanted cell loss, so a balance needs to be struck

When it comes to actually sorting those whole cells, the two most common high-throughput methods to achieve this, and enrich for rare cell types, are flow cytometry or fluorescence-activated cell sorting (FACS) and Magnetically Activated Cell Sorting (MACS) (see Figure 4). Manual sorting and Laser Capture Microdissection (LCM) can also be an option, but at a much lower throughput and require larger amounts of manual work.

Figure 4. Different methods of cell isolation and sorting. This includes FACS (A), MACS (B), LCM (C), Manual (D) and Microfluidic (E) strategies. Source: Hu, et al. 16

FACS works through fluorescently labelling cells via antibodies. The cells undergo flow cytometry but pass through a laser that allows cells to be sorted based on the colour emitted. This helps sort cells by proteins expressed on their exterior. Advantages of this approach include the extremely high throughput and the much higher cell purity levels that are achieved. However, FACS requires a large quantity of cells in suspension to sort, and the rapid flow can damage cells, impacting their viability downstream.

If the goal is to sort out one or two cell populations (for example T and myeloid cells), a benchtop model with two sort streams and 4-5 colour capabilities is sufficient. If, on the other hand, there is a need for multiple cell populations (for example, naïve, memory and effector subsets of T or B-cells), then a larger floor model with multiple sort streams and multiple fluorescent channels will be necessary (see below for a collection of options).

MACS, on the other hand, is simpler and more affordable and provides more opportunity to scale. It works using supermagnetic nanoparticles to tag target cells and then capture themthrough magnetically attaching to a column, while the remaining cells pass through.. The downsides are that the throughput and resulting purity are lower than FACS. Examples of MACS include MojoSort™ from BioLegend (see below), DynaCellect from Thermo Fisher Scientific and the MACS cell separators from Miltenyi Biotec. The latter works using MACS® Microbeads and includes a variety of column types. The automated instrument – autoMACS® NEO Separator (pictured) – automates the process, with capacity for 6 samples, for a high purity, reproducible and gentle cell isolation.

BioLegend’s MojoSort™ magnetic bead-based system is designed to reliably isolate desired populations of cells with high purity and yield. Each reagent is optimized for a sample for quick and easy isolation. The beads are small at 130 nm, and isolated cells have been tested in downstream in vivo and in vitro applications to demonstrate cell function, migration, and proliferation. MojoSort™ reagents are compatible with other magnets and column separation systems and have beads and kits designed to separate a wide variety of cell types from both mouse and human samples. They also offer MojoSort™ Human and Mouse Dead Cell Removal Kits, which does not require high calcium buffer exchange, resulting in higher live cell yield compared to Annexin-based kits.

Cell sorting is complicated to set up if high-throughput is required. The popular single-cell provider, 10x Genomics, has a set of best practices for sorting cells prior to their assays, which can be found here.  While MACS sorting is pioneered and led by Miltenyi Biotec, FACS sorting has a wide-variety of commercial options available for cell sorting in advance of single-cell sequencing, that we briefly review below.

FACSDiscover S8 – BD Biosciences

BD Biosciences has a selection of FACS based Cell Sorters with different specs for different applications. The FACSDiscover™ S8 (pictured) is the latest cell sorter with an impressive 86 detectors, 5 lasers and 3 different nozzle sizes. With BD CellView Image Technology and BD SpectralFX Technology, this spectral flow cytometer is combined with real-time spatial and morphological insights. A 96 well plate can be filled in less than 80 seconds with 80% cell viability.

Wolf & VERLO – Nanocellect

The WOLF G2 Cell Sorter uses up to 2 lasers and 9 colours for sorting. Cells are sorted into well plates or conical tubes and the back-to-back sorting speed is 200 events per second, with a sample pressure of <2psi for extremely gentle sorting. Sample volumes are a minimum of 150µl.

The VERLO Image Guided Sorter is the new instrument currently available for early access. This sorter uses image analysis alongside gentle microfluidic cell sorting. It captures actual images of each cells alongside morphology and marker localization.

Pala – Bio-Techne

The Pala™ benchtop Cell Sorter from Bio-Techne is a lightweight and portable instrument. It uses two lasers and up to 11 fluorescent channels with forward and side scatter. It can efficiently sort cells into well plates, at 1 minutes per plate. It also sorts at a low pressure of < 2 psi for gentle sorting. Input volume is between 100 µl – 600 µl. These single-cell dispensers use unique microfluidic cartridges, and these sorters have three different modes, capable of sorting cells at 2 cells per second up to 50,000 cells per second. The instrument is user-friendly, require no special training to operate, and need zero maintenance.

CellenONE – Cellenion

The CellenONE® X1 is Cellenion’s free standing single cell sorter and isolator using four fluorescent channels and two dispensing channels. Sample processing speed is < 3 mins for a 96-well plate. The imaging-based cell selection allows users to select cells based on morphology or fluorescent signature. Furthermore, it works on any sample volume, from as little as 1µl, meaning it excels at selecting out rare cell populations even in sparse samples.

CytoFlex – Beckman Coulter

The CytoFlex SRT Benchtop Cell Sorter comes in a variety of different models containing between 2-4 lasers and 5-15 colour filters to match different sorting applications. The most sensitive of these models can sort at <10,000 events per second sorting into plates, slides or conical tubes.

 F.SIGHT™ OMICS – Cytena

The SIGHT Dispenser range from Cytena have a variety of specialisation including the UP.SIGHT™ for colony tracking, the C.SIGHT™ for unlabelled cells and the F.SIGHT™ for dispensing of fluorescent cells. The F.SIGHT™ OMICS is their model specialised for single-cell genomics. This system uses brightfield and fluorescent imaging for precise cell identification and can isolate a 96 well plate in ~ 2 minutes. Single cells are dispensed in picolitre droplets at the centre of conical PCR plate wells. Cell size filters range from 10 μm – 40 μm.

DispenCell – MMI

The DispenCell Single-Cell Dispenser accurately dispenses individual cells into microplates, wells or slides using a disposable tip sorter rather than microfluidics. It operates under severely low pressure – <0.2 psi – similar to a manual pipette. This instrument is low throughput and takes 10-13 minutes to dispense a 96 well plate, but it is extremely gentle, perfect for low throughput delicate studies.

MACSQuant® Tyto® Cell Sorter – Miltenyi Biotech

With 3 lasers, 8 fluorescent channels and 2 scatter channels, the MACSQuant® Tyto® allows for complex sorting strategies with 10 parameters. Samples are kept sterile in the specially designed Tyto Cartridges, which allow micro-chip based fluorescent cell sorting. The cell flow rate is 55,000 cells per second for the normal cartridge and 110,000 cells for the HS cartridge. The operating pressure is 3 psi for a normal cartridge and <14 psi for the HS. The cartridge loads 100 µl up to 10ml.

Genesis Cell Isolation System – Bio Rad

The Genesis System with Celselect Slides™ utilize microfluidic channels and 140,000 individual microchambers to efficiently and gently capture 8 μm – 30 μm rare cells based on size. It can accommodate liquid biopsy sample inputs of <10 mL from two samples in parallel. After capture, enriched cells can be recovered for downstream processing (e.g., bulk and single-cell sequencing, digital PCR, etc.), or stained onslide for immunofluorescent applications such as enumeration and identification of various circulating tumor cells (CTCs) and other rare cell types using microscopy. For successful enumeration, Bio-Rad’s Celselect Slides Validated Antibodies for CTC enumeration can be used in tandem with the Celselect Slides Enumeration kit. For more information, visit the Genesis Cell Isolation System Knowledge Hub.

SH800S – Sony Biotechnology

The SH800S Cell Sorter uses 4 collinear excitation lasers and 6 fluorescent detectors and 2 scatter channels. It outputs into tubes, well plates and slides. The novel microfluidic sorting chip is available in three sizes including 70 μm, 100 μm, and 130 μm to permit sorting of a wide range of cell sizes. The 70 μm chip can sort 12,000 events per second with >98% purity. Up to 30,000 events per second can be achieved but it will impact purity.

Uno Single Cell Dispenser™ – Tecan

The Uno system is an automated benchtop instrument designed to improve single-cell workflows. This method does not use FACS but microfluidics to effectively dispense cells. It works with cells ranged 9 μm – 25 μm and dispenses them directly onto well plates. It can dispense a 384-well plate in ~5 minutes with 90% cell viability. The Uno dispenses both cells and reagents across the well plate.

On-chip® Sort – PHcbi

The On-chip® Sort is a disposable microfluidic chip cell sorter. This results in a damage-free, sterile system capable of sorting cells as large as 140 μm. It works at speeds of 1,000 targets a second using 3 lasers with six fluorescent detectors on top of the microfluidic chip.

Bigfoot Spectral Cell Sorter – Thermo Fisher Scientific

The Bigfoot system is fast, capable of sorting a 96-well pate in 11 seconds and 70,000 events per second. It has an impressive 9 lasers and 60 detectors capable of spectral sorting and spectral analysis. A six-position multi-sample loader allows six parallel samples. With configurable output holders and nozzles, this system can be adjusted to many applications, even dispensing directly onto a 10x Genomics chip.

Alerion™ – Akadeum Life Sciences

The Alerion™ is an interesting alternative to FACS and MACS, instead using BACS or Buoyancy Activated Cell Sorting. The system uses microbubbles that seek out and bind to target cells (based on a prechosen analyte) that then float to the surface of a suspension while the rest of cells remain untouched at the bottom. It’s an exceptionally gentle, targeted technique

Cell Counting and Quality Control

Following cell sorting, it is always recommended to perform separate cell counting with a reliable cell counter. This is to determine the effectiveness of the cell isolation to avoid wasting time, money and resources on poor quality samples that will produce misleading results. Cell counting also allows cell viability analysis and to determine whether cell clumps have been removed.

Cell counting can be performed manually with a hemocytometer. This is laborious but is preferable in certain situations since experienced cell biologists will be better at excluding debris and sometimes debris can be too overwhelming for automated counters.

However, manual cell counting is prone to error between individuals and between samples. Eliminating user-bias is a key reason why many people turn to automated cell counters. The automated instruments are also faster, a life-saver for when you have a large number of samples and tend to supply richer information on your cells.

Cell counters have a variety of uses in cellular biology. For single-cell sequencing applications, certain instrument features can offer particular benefits. First, it is worth checking the limit of quantification (LOQ) and CV for common sample types. It is also important to establish whether cell size information is generated. Cell size histograms allow one to readily determine the distribution of single cells in a sample and monitor the presence of clumps or debris. The ability to perform high-throughput cell counting can also be vital.

Finally, it is worth remembering that automated counters require proper size gating for your cell type, a sample concentration within the dynamic range of your instrument, and low sample debris for accurate and repeatable cell counts. Ensure your sample meets the specifications of your preferred automated cell counter. 

Examples of purchasable automated cell counters, using brightfield (BF) and fluorescent (FL) strategies include:

  • The Vi-CELL BLU from Beckman Coulter, which can hold 24 samples and 96-well plates, with reagent packs for trypan blue detection.
  • The LUNA-FL from Logos Biosystems for BF and FL cell counting and effective debris distinction, with reusable slides for affordable automated cell counting.
  • The CytoSMART Exact from Axion Biosystems for BF and FL counting and viability analysis
  • The Countess 3 model from Thermo Fisher Scientific for BF and FL counting with reusable slides.
  • The C100 from RWD for BF and FL counting taking information such as counts, viability and diameter measures.
  • The Cellaca MX, a high-throughput cell counter from Nexcelom. It can count 24 samples in under 3 minutes, with BF and 4 channel FL imaging and simultaneous imaging and analysis.
  • The TC20 Automated Cell Counter from Bio Rad performs counting and viability analysis using auto-focus technology and flexible cell size gating.

Additionally advice on counting cells from leading single-cell sequencing company, 10x Genomics, can be found here.

References

1.         Qu, H.-Q., Kao, C. & Hakonarson, H. Single-Cell RNA Sequencing Technology Landscape in 2023. Stem Cells 42, 1-12 (2023).

2.         Spits, C. et al. Whole-genome multiple displacement amplification from single cells. Nature Protocols 1, 1965-1970 (2006).

3.         Chapman, A.R. et al. Single cell transcriptome amplification with MALBAC. PLoS One 10, e0120889 (2015).

4.         Blagodatskikh, K.A. et al. Improved DOP-PCR (iDOP-PCR): A robust and simple WGA method for efficient amplification of low copy number genomic DNA. PLoS One 12, e0184507 (2017).

5.         DeLaughter, D.M. The use of the Fluidigm C1 for RNA expression analyses of single cells. Current protocols in molecular biology 122, e55 (2018).

6.         Brennecke, P. et al. Accounting for technical noise in single-cell RNA-seq experiments. Nature Methods 10, 1093-1095 (2013).

7.         Klein, A.M. et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187-1201 (2015).

8.         Macosko, E.Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202-1214 (2015).

9.         Cao, J. et al. Comprehensive single-cell transcriptional profiling of a multicellular organism. Science 357, 661-667 (2017).

10.       Domcke, S. et al. A human cell atlas of fetal chromatin accessibility. Science 370, eaba7612 (2020).

11.       Rosenberg, A.B. et al. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science 360, 176-182 (2018).

12.       Huang, D. et al. Advances in single-cell RNA sequencing and its applications in cancer research. Journal of Hematology & Oncology 16, 98 (2023).

13.       Pan, Y., Cao, W., Mu, Y. & Zhu, Q. Microfluidics Facilitates the Development of Single-Cell RNA Sequencing. Biosensors 12, 450 (2022).

14.       Sant, P., Rippe, K. & Mallm, J.-P. Approaches for single-cell RNA sequencing across tissues and cell types. Transcription 14, 127-145 (2023).

15.       Nguyen, Q.H., Pervolarakis, N., Nee, K. & Kessenbrock, K. Experimental Considerations for Single-Cell RNA Sequencing Approaches. Frontiers in Cell and Developmental Biology 6(2018).

16.       Hu, P., Zhang, W., Xin, H. & Deng, G. Single Cell Isolation and Analysis. Frontiers in Cell and Developmental Biology 4(2016).