3D Cell Culture & Cell Confinement
This review outlines traditional and developing techniques of biological cell culture, focusing on the advantages that 3D cell culture techniques have over their 2D counterparts. These advantages can be further achieved through the integration of microfluidic technologies with cellular research, giving unprecedented control over key cell culture parameters, such as the spatial and temporal gradients of the cell microenvironment. Cell confinement, targeted isolation and deformation of small numbers of cells, is a novel technique that microfluidics has enabled, further expanding the applications and potential of microfluidic technologies, which have yet to be fully realised in this field of research.
In order to fully understand the complex form and function of living cells, consideration of a cell’s highly dynamic characteristics, such as its 3D structure, mechanical properties, and biochemical environment, is essential . In vitro cell culture techniques have long been vital research and industrial tools  used to gain a better understanding of cellular behaviour through numerous studies, ranging from cancer drug screening to developmental biology . Cell cultures have traditionally been based on two-dimensional glass, polystyrene, or hard plastic substrates; the cellular mono-layers, which these “petri dish”-based cell cultures generate, are severely limited by their failure to reconstruct the in vivo cellular microenvironment, and thus are not representative of those found in organisms , . As a result, biological responses measured in 2D cell culture studies, such as receptor expression, cell migration, and apoptosis, can differ significantly from those of the original tissue or environment in which the cells are found .
The advent of three-dimensional cell culture was an attempt to address the limitations of 2D cell culture  (see figure 1). These 3D cell cultures serve to imitate the natural extra-cellular matrix (ECM), a highly hydrated network of collagen and elastic fibers embedded in a gel-like material of glycosaminoglycans, proteoglycans, and glycoproteins . They can be fabricated using natural and synthetic materials in the form of matrices, or scaffolds, to better represent the spatial organisation of the cell environment , on which the behaviour of many cells are reliant, thus reducing the gap between cell cultures and native biological environments , . 3D cell culture has consequently become an essential means of increasing the relevance of cellular studies .
The emergence of microfluidics technology in recent decades has further expanded our ability to refine cell cultures, with more precise control of local cellular microenvironments , . Microfluidics, a field involving the manipulation of small volumes of fluid in geometries of micrometer scale, often on silicon-based chips, has allowed for a multitude of benefits applicable to cellular research, such as greater spatial and temporal control of the microenvironment , the ability to work with smaller reagent volumes, have shorter reaction times , and have greater potential for cell handling integration , . Microfluidics technology also gives rise to cell confinement potential, whereby small numbers of cells, or even a single cell, can be mechanically deformed  or simply isolated , allowing research into the effects of cell shape and mechanical influences, as well as more targeted insight into cell behaviour .
This review aims to provide a summary of conventional and microfluidic approaches to 3D cell culture and cell confinement, and their implications in the progression of cellular research.
The problems of dissimilarity between 2D cell culture and a cell’s natural environment have become evident after numerous studies demonstrated that the cellular microenvironment contributes to the complex spatial and temporal signaling domain that directs cell phenotype, whereby a cell is not solely defined by its genome, but also by a multitude of other influences, including the ECM, growth factors, hormones, and other small molecules .
3D cell matrices, or scaffolds, are porous substrates designed to support cell growth, proliferation, and differentiation on or within their structure (see figure 2), with the goal of mimicking natural ECM features such that cells in the matrix behave as they would in vivo . Cell matrices applications can be divided into either clinical modeling or in vitro approaches. Clinical methods, such as tissue engineering or regenerative medicine, are aimed at repairing, maintaining, or improving defective human tissue using artificial 3D matrices . In such cases, mechanical and biodegradability properties must be taken into consideration, allowing for the shape of the structure and seeded cells to be sufficiently protected from loads once implanted, and to insure the scaffold materials can be metabolised into the body without serious toxic effects . Scaffolds designed for 3D in vitro modeling are aimed at allowing systemic analysis of cell biology in order to contribute to the understanding of cell physiology and pathophysiology . In this experimental context, the 3D matrix should be designed to mimic the 3D organisation and function of whatever cellular microenvironment the experimenter is trying to simulate, with further attention being paid to accessibility of the matrix to imaging and analysis tools .
A key design aspect that is common to both clinical and in vitro cell matrix applications is the matrix macro and micro architecture, encompassing matrix pore size and geometry, porosity, interconnectedness of pores, and surface topology . The high surface areas of these porous structures allow cell ingrowth and anchorage, as well as transport of fluids and nutrients. Depending on the type of cell or tissue being cultured, the microporosity of a scaffold can be important for capillary growth and interactions between cell and matrix, while the macroporosity of the structure is significant for nutrient and cell metabolism waste transport .
Cell matrices can be fabricated using both natural and synthetic materials, commonly taking the form of hydrogels, crosslinked networks that possess high water content, which have shown high efficacy in this application . Naturally derived polymer materials, such as collagen, fibroin, chitosan, alginates, and starch, were the first biomaterials used for cell culture , and often exhibit good biocompatibility and biodegradability properties, though variability between batches and difficulty of processing some polymers are notable drawbacks , . Synthetic polymers, such as polyglycolic acid (PLG) and polylactic acid (PLA), while generally less biocompatible than their natural counterparts, provide opportunity for better control of key characteristics, such as surface to volume ratio, porosity, and mechanical properties , . Despite the distinct advantages that 3D cell matrices have introduced to cell culture techniques, they still cannot fully attain the spatiotemporal gradients of chemicals and oxygen or the mechanically active microenvironments that are among key characteristics of cellular environments .
The integration of microfluidics technology with cell biology has allowed for unprecedented control over the spatial and temporal gradients of chemicals and oxygen in the cell microenvironment that determine a cell’s behaviour – an ability to tailor variables on a micro-scale that cannot be achieved with conventional cell culture models , . These microfluidic devices designed for cell biology applications integrate 3D cell culture analysis with micro-fabrication techniques from the microchip industry . Poly(dimethylsiloxane) (PDMS), a silicon-based material, is the most popular medium for microfluidic cell culture research, often fabricated into small chips with micro-scale channels using soft lithography . The precise control of the cell microenvironment can be achieved by using microfluidic networks to transport chemicals, nutrients, and oxygen at user-determined flow rates, and by manipulation of the microfabrication and micropatterning processes of the chip to control the cell-ECM interactions and shear forces imposed on the cells . The microfluidic approach also brings with it the practical benefits of being able to work with significantly smaller samples, potentially reducing the cost of cellular studies, while allowing for easy integration of analytical standard operations into the system , .
While much of the progress in this field has been proof-of-concept demonstrations relating to different microfluidic geometries and platforms , , a number of microfluidic systems have been developed, including those with applications in biosensor development, drug-screening, stem cell research, and genetic analysis . Progress in microfluidic 3D cell culture has also been demonstrated in the modeling of ECM for supporting 3D cell growth and examining cell migration, an important property of living cells affecting embryogenesis, wound healing, immune response, tissue development, and disease processes, such as cancer metastasis and inflammation –. These microfluidic cell migration assays have been shown to produce accurate cell migration behaviour with limited reagents, while overcoming some of the drawbacks of traditional 2D cell migration assays, such as the potential damage to cells via cell scraping methods, and the unsuitability of scaling such models towards high throughput screening .
A novel capability brought about by microfluidic integration with cellular biology is what is known as cell confinement, encompassing both mechanical deformation of individual cells and cell isolation. Microfluidic devices have been used to isolate individual cells in nanoliter and picoliter sized droplets, thus greatly increasing cell concentration. This allows for faster accumulation of molecules released by the cell without the need for a pre-incubation stage, leading to the rapid detection of bacteria , .
Cell confinement can also be used to impose a desired shape onto a cell by forcing it into a specific geometry (i.e., a curved microfluidic channel), allowing study of the effects of cell shape and mechanical stress on a number of properties . Cell confinement has been used for a number of different cellular applications, including, for example, studies into the relationship between cell cytoskeleton and cell polarity , cell migration behaviour under confinement , cancer cell mechanical properties , cell separation , and label-free recognition of specific cells based on their deformability . The cell confinement techniques used in these studies, varying from utilising unique microfluidic channel geometries   to optical stretchers  and inertial forces , demonstrate the versatility of microfluidics technologies when applied to cellular biology.
In summary, cell culture in three dimensions has extensive benefits when compared to conventional two-dimensional cell culture. The in vivo spatial domain, in which cells are naturally found and which is vital to the biological signaling that dictates cell behaviour, cannot be reliably achieved on the cellular monolayers that are generated in 2D cell culture models. While current 3D cell culture techniques, namely those incorporating cell matrices, or scaffolds, greatly improve upon 2D cell culture techniques, they still lack the ability to reproduce key aspects of the cellular microenvironment. Some of these aspects, including the spatial and temporal gradients of chemicals and oxygen and mechanical stimuli, can be addressed through the use of microfluidics technology, which provides a cell culture platform with highly versatile applications when integrated with cellular biology. This includes the novel capability of cell confinement, giving targeted insight into the effects of cell deformation and mechanical stresses. Though many cell biology microfluidics studies have been only demonstrations , it is clear that there is great potential for increasingly more impactful and practical cell behaviour studies through the utilisation of microfluidics.
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