3D Cell Culture - McLean Taggart, Emma Villares, Maximillian Marek, Scott LeBlanc, and Adam Lyons
While most microfluidic devices incorporate a 2D cell culture design, in which a single layer of cells is grown on the bottom of a device, these systems suffer from poor in vivo mimicry, as, in the human body, most cells grow in all directions. To address this limitation, 3D cell culture devices have been developed - in which cells are suspended in a 3D matrix or seeded on a 3D scaffold, allowing the cells to grow in more directions than in a 2D culture.
In life sciences research, cell culture is a ubiquitous technique that allows researchers to study biological systems in an easy, reproducible manner. The necessity for cell culture derives from the need to isolate systems, cell lines, and physical regions to study their properties, reducing a complex organism to its constituent parts. Within cell culture, a strain of cells is grown in an ex vivo, highly controlled manner in which the culture can remain uncontaminated. Due to the high variability in growth conditions across the many, widely available cell lines, there are numerous different manners by which cell culture can be performed. A recently popularized method of cell culture is microfluidic cell culture, where populations of cells are introduced to and grown in a micro-scale system. There are many advantages to this manner of cell culture, however, one of the largest benefits is the complete control over microenvironment conditions, currently impossible at the macroscale. If the cells are seeded in a manner where they clump up to form small colonies, additional vasculature may be required to enable internal nutrient delivery, which can be enabled through microtubing in the device (Figure 1). In Figure 1a,c, the red coloration denotes cells that have died due to limited access to the growth medium. Using microfluidic techniques, additional vasculature within this portion will enable media flow to the obstructed cells. Due to the intrinsic physical properties of the microscale, these systems allow for unprecedented control over cell seeding, physicochemical gradients, heat and mass transport, shear stress, and bubble formation, all of which are pivotal to providing sustained, deterministic cell growth.
2D Cell Culture
2D cell culture occurs when the culture is grown on a flat surface, on which the cells adhere and spread out, eventually covering it once confluent. This method of cell culture is most commonly used due to its ease of access and lower material cost because all that is required is a flat, sterile plate with wells for the cells to rest in. Due to its ease of access, this method is universally known across the biological scientific community and allows for experiments to easily be reproduced. Despite its benefits, the usage of 2D cell culture can lead to unrepresentative results due to the lack of mimicry when compared to the biological systems in which cells typically grow. One large difference is that of morphology, as since the cells are flat on a plate, they will not grow to be spherical as they do when suspended in the body, rather, they will end up more disk-like. There are specific cell lines that can be grown in such a manner, however, such as epithelial cells, which show similar responses to drugs and toxins across assays. Further limitations to 2D culturing arise when taking into account the 3D environment in vivo, in which cells are bound to the extracellular matrix. Further complicating the usage of such systems, is the fact that cells grown on a monolayer have unlimited access to constituents of the media in which they are grown, such as oxygen, nutrients, metabolites, and signaling proteins. In the body, the architecture of cells can lead to this access being limited, as cells swarm each other and cut off the availability of external supplies.
3D Cell Culture
Recent advances in 3D cell culture technology have allowed for widespread adoption of the technique. With this increase in accessibility, many new findings have been published showing the advantages 3D cultures display over 2D. One such advantage is the better capacity of 3D culturing to mimic the diffusion-limited access to media nutrients found in in vivo models (Figure 1). Alongside the more biologically relevant media consumption, cultures incorporating 3D cell growth allow for deeper complexity of intercellular communication, such as signal transduction, extracellular matrix, and biologically relevant morphology, increasing the capacity for phenotypic matching. The increase in accessibility in 3D culture has been driven by the usage of microfluidic devices, which save on material costs that can cripple new labs, especially when learning more advanced, contemporary techniques. Microfluidic 3D culturing is performed similarly to 2D culturing, in which a chip supports a well channel for cellular growth and media delivery channel. The novel aspect implemented in 3D culture is the usage of a matrix that can support the growth of cells along varying heights while allowing for adequate media delivery to internal cells in cultures through artificial vasculature.
There are several different materials that can be used for the construction of microfluidic devices. These materials can impact the overall growth and behavior of 3D cell cultures.
The most common microfluidic scaffolding technique begins by mixing a hydrogel substance with cells prior injection into the growth well. This hydrogel allows for the cells to be suspended height wise while also allowing media and signaling molecules to diffuse, similar to biological systems. The geometry of such gels can be modulated in several manners including physical structures such as posts and ridges, molding through pressure, and post-injection external forces. Due to differences in environmental preference between cell types and cell lines, different types of hydrogels are used depending on the cell lines being seeded. Endothelial cells and tumor cells are typically cultured in collagen, stem cells in fibrin, and embryonic stem cells in matrigel. While most cell lines are compatible with most gels, these combinations have been found to best facilitate adhesion, migration, and proliferation.
Poly(dimethylsiloxane) (PDMS) is a commonly used material that can be fabricated with a design of interest through soft lithography to influence growth in a cell culture. PDMS can be used to imprint a hydrogel during its solidification which may influence media access and overall growth. A PDMS stamp can be fabricated with a vascular design which is done to improve cellular access to nutrients within the media. The thickness of the design can be determined during photolithography and during soft lithography, the PDMS itself is fabricated and and optionally plasma treated alongside glass to enable adhesion between the two materials.
PDMS devices are benefitted by their inert and isotropic properties, alongside its transparency down to 300 nm. Furthermore, its permeability to gas presents advantages and disadvantages. Its inert nature prevents any undesired interactions between the PDMS and other materials or chemicals used. Because devices made entirely of PDMS are isotropic, there would be no difference in cellular conditions throughout such that one side does not favor growth over the other. This isotropic nature may be impacted by the inclusion of other materials such as glass. With visibility of substances at 300 nm or above, this is critically important in cell culture for observing the fluorescence of novel treatments. Since fluorescence measurements and imaging is typically performed in the visible range of the spectrum (i.e., above 300 nm) proper visualization can occur in order to determine if the treatment properly works.
Polystyrene is frequently used in standard 2D cell culture, meaning that it can be a desirable substitute for microfluidic devices if there are experimental cytotoxic materials. Polystyrene use is highly-studied and is able to be molded using standard industrial techniques. In some instances, PDMS can diminish cell proliferation if it absorbs growth factors or an experimental therapeutic from the scaffold. From a design standpoint, polystyrene can be a desirable material because of its affordability, its impermeability to gas which limits contaminants from the air, and its ease of use. Its impermeability to gas enables studies conducted to determine the impact of gaseous solutions on cell growth, such as with cigarette smoke. Though glass can also be used for studies where gas impermeability is important, polystyrene is a convenient alternative due to easier fabrication. At this time, it is unknown how long a 3D cell culture environment can survive in a polystyrene device as the current study only shows a 4-day long experiment.
Glass is often used now as a base for polymeric microfluidic devices, such as PDMS. However, glass devices in the past have shown an increase of visibility and imaging quality of the 3D cell culture systems based on fluorescence tagging. Glass is impermeable to oxygen, making it a good material to monitor cell growth in low-oxygen environments, similar to some tumor microenvironments. One example of a glass-based device is used by Jang et al., to culture osteoblasts for drug screening. The device was fabricated using a combination of photolithography and wet etching. After coating the inside of the device, cells were seeded and media was fed continuously through the channel. However, due to the fragility of the glass devices, they are not as commonly used as polymeric devices.
Considered a much cheaper option for microfluidic devices to sustain 3D cell culture, paper microfluidics was first established by the Whitesides group. This method includes chromatography paper that is stacked and contains suspended cells (Figure 2). Cells are grown in a growth media before being spotted onto the paper, which is then stacked. This best modifies the oxygen gradient that occurs with 3D cell cultures where there is a higher concentration at the surface level than there is at the core of the 3D structure.
Difficulties with 3D Cell Culture
Although 3D cell cultures provide a variety of benefits over 2D cell cultures, they have their own set of limitations. One current challenge includes flowing media through the 3D cell cultures. Different hydrogels expand at different rates during solidification, and this expansion may cause a vascular design to close in on itself. Media flow would be blocked, leading to a pressure build-up and damage to the system. Another common issue is preventing air bubbles from entering the system. This affects the concentration of oxygen available to cells, which impacts cell growth.
Using flowing media in cell culture allows for growth conditions more reminiscent of those found natively in the body, as blood is always pumping through the heart. Some microfluidic cell cultures rely on a one time injection of nutrient-rich media, but this leads to shorter lifespans of the cells and therefore the device since the source of nutrients is finite. Introducing vasculature not only improves this shortcoming, but it also provides access to more growth medium over time and can prevent waste from building up in the culture environment. However, using non-stationary media poses an issue due to pressure build up. Since the cells must be seeded in a matrix, this matrix blocks the pathway for fluid flow, acting as a wall, which leads to pressure increasing at the matrix interface which can lead to backflow or leakage. To avoid this, some microfluidic devices incorporate flow through a mass or concentration gradient, however this decreases the flow rates which can be accessed and leads to unsteady flow as the flow rate will constantly decrease as the gradient decreases.
A current challenge of 3D cell cultures is air bubble formation. This can develop due to poor sealing and constant circulation of fluids. One of the problems with air bubbles is the increased amount of oxygen within the cell culture which will lead to reduction in stability and flow resistance in the normally nitrogenous environment. To prevent the buoyancy-driven entrapment of air bubbles, the velocity of the bubbles surfacing must be less than the media flow velocity. This can be controlled by increasing the media flow rate or by using a more viscous growth medium with a closer density to the bubble. 
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