An out-and-out solution for Agarose Encapsulation
The ideal habitat for cells
The holy grail of cell culture methods would combine the high throughput encapsulation of cells in a 3D scaffold (spheroid) with the free perfusion of molecules whilst also compartmentalizing the cells to maintain their monoclonal identity. At the single cell level, cell biology focuses on understanding the behavior and interactions of individual cells. This is useful to, for example, discern the heterogeneity within a tumor. So, what is the ideal workflow that involves capturing single cells and allowing for growth and proliferation at the same time?
- Microfluidic approach to hydrogel projects
- What is a hydrogel scaffold?
- Use of agarose scaffolds in research
- The nadAROSE kit
- Overcoming obstacles
- Downstream applications
- Find out more
The Microfluidic route
After decades worth of work, microfluidics has emerged as a game changer in Life Science research. With devices like the Nadia Instrument, we now have a way to carry out thousands of independent, localised reactions on single cells at the same time. Some breakthrough methodologies that have emerged include continuous-flow microfluidics for a simultaneous test of compounds on different cell types, 3D cell culture, and droplet microfluidics for fast generation of millions of individual microreactors with volume in the picolitre range (10).
As far as cell screening is concerned, FACS (fluorescence-activated cell sorting) and FADS (fluorescence-activated droplet sorting) methods have quite often been used to screen droplet emulsions. FADS devices require advanced operational skills since they are often performed on chips, deliver low sorting rates, and have poor viability making them an unpopular choice. FACS cytometers boast high-quality signal detection and sensitivity. FACS instruments are also easy to operate and are ubiquitously available at institutions. As a result, FACS remains the predominant choice. However, there is a technical challenge associated with sorting droplets with FACS. Water-in-oil emulsions are incompatible with the aqueous ‘sheath fluid’ that allows the processing of samples with FACS cytometers. One strategy to overcome this is the generation of double (water-in-oil-in-water) emulsions. But the downstream cell recovery is vulnerable with this method.
An alternative approach is the addition of hydrogel-forming polymers like that of agarose to the cell-containing aqueous solution prior to encapsulation. In doing so, cell viability and growth is retained due to hydrogels providing a ‘scaffold’ which mimics functions of the extracellular matrix. Moreover, this approach does not impair the droplet generation process.
What is a hydrogel scaffold?
Hydrogels are generally understood as crosslinked scaffolds of long protein strands which freely allow the movement of aqueous solutions through their structures. Most of a hydrogel’s volume is made up by empty space, whilst still giving ample support to encased cells.
A key hydrogel material within the field of single cell microdroplet encapsulation is agarose due to its inherent biocompatibility. Agarose droplets are picolitre-volume spherical scaffolds which remain stable in an aqueous solution and represent a potent solution for many single cell applications (1). Agarose encapsulation allows cells to be grown in a supported and individual microenvironment for extended periods of time (2).
Use of agarose scaffolds in research
The properties of agarose scaffolds are several-fold and are applicable in several scientific fields, such as, studying tumour development (3), drug screening and delivery (4,5), bacteriology (6), assessing plant development (7), and many more. With these applications in mind, the development of the protocols and instruments enabling single cell agarose encapsulation has great potential for advancing several fields of science. To make this technique an advancement from well plate-based methods, droplet-based hydrogel capture must achieve high-throughput via automation. Generating these droplets in a quick and stable microfluidic manner introduces an astounding level of scalability to this cellular technique, the only factor limiting cell number being time and reagent availability.
Our newest reagent kit and what does it offer?
With the use of the Nadia instrument and the newest nadAROSE kit, cells can be continuously captured in a high throughput manner within droplets containing hydrogel using droplet microfluidics, which surrounds tiny pockets of molten agarose containing cells in cell buffer with an immiscible oil shell. The hydrogel droplets, once hardened and removed from their oil shells, are spherical scaffolds which remain stable in an aqueous solution such as cell buffer.
Beyond single cell encapsulation, the nadAROSE kit enables the incorporation of more than one cell type within a droplet. Co-encapsulation of two distinct cell types can create precise niches for some cell types to develop in. Such setups can be feasible to study single cell-cell signalling or cell-pathogen interactions in normal and diseased states over biologically relevant timeframes in a scalable and high throughput manner. Furthermore, both single encapsulation and co-encapsulation of cells in agarose provide a platform for assaying single molecule-cell interactions through the addition of pico-litre volumes of substrate, such as active drugs, stressors or growth factors in hydrogel bead-based cell delivery systems (7).
If you would like to know more about the nadAROSE kit, please refer to our application note
Overcoming obstacles with the nadAROSE kit – Dropletize and Immobilize
Unlike preceding cell culture techniques, hydrogel microdroplets on the Nadia achieve high throughput, naturalistic 3D culture and maintenance of single-cell identity when desired. It is a laborious task to perfuse individual wells of cell culture on a plate. Hydrogel scaffolds containing cells can be continuously perfused with nutrients or drug molecules alleviating the cell culturing burden. Culturing cells in picolitre volumes also means using smaller volumes of media and other reagents, therefore reducing the amount of reagent expenditure. Techniques like colony picking are often time consuming. With droplet-based methods, colonies are physically separated and require less growth time before picking. Time is money and you don’t have to waste it.
Immobilized cell growth is pertinent from both a laboratory as well as an industrial perspective. Agarose is identified as a suitable solid support for bioligands and enzymes and has gained widespread use in the Biotechnology Industry.
Beyond cell encapsulation – downstream applications
The capacity to easily capture, culture and image does not stop at single cells. The precision of modern microfluidic system allows smaller molecules to be partitioned into single droplets. Molecules and drugs can be captured, alongside single or multiple cells, to assess their downstream impact (5). Agarose encapsulation also holds potential for bacterial isolation and analysis, a key area that previously was hard to tap into within single cell research (8).
The nature, in terms of both size and properties, of the agarose droplets enables them to be sorted using flow cytometry (9). FACS can be performed for single cells, co-encapsulated cells, and to gain a better understanding of the local microenvironments. Particularly, it enables us to dive into the heterogenic world of biology and knit pick the different cells as individual entities and their surrounding environments.
Moving away from mammalian cells, the study of plant cells is also an important field of study. Research in plant biology often focuses on cellular interactions with either abiotic molecules (such as pesticides) or pathogens (such as viruses or bacteria). Being able to co-encapsulate all these different cell-like molecules within a single droplet can allow vast tracts of plant and crop research to branch into single cell analyses. Many areas of plant research stand to advance rapidly by joining the single cell revolution bolstered by techniques such as agarose droplets.
Find out more about the nadAROSE kit
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2. Joseph JS, Malindisa ST, Ntwasa M. Two-Dimensional (2D) and Three-Dimensional (3D) Cell Culturing in Drug Discovery. In: Cell Culture [Internet]. 2018 [cited 2019 Sep 19]. Available from: https://www.intechopen.com/books/cell-culture/two-dimensional-2d-and-three-dimensional-3d-cell-culturing-in-drug-discovery
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