3D Cell Culture - McLean Taggart, Emma Villares, Maximillian Marek, Scott LeBlanc, Adam Lyons and Jacob Belden
Introduction
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.[1] 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.
Background
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.[2] 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.[3] 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).[4] 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.[6]
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.[7] 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.[8] 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.[1]
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.[9] 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 multiple ways to create a 3D cell culture. Methods include scaffold; which creates a 3D matrix for cells to grow off of, non-scaffold; which uses micropatterned wells to induce growth in a desired manner which better mimics real life applications, and droplet microfluidics; which is the process of creating droplets with desired concentrations of cells.
Device Materials
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.[10]. It is important to know the most used materials before deciding what design for a 3D cell culture to follow.
Hydrogel
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.[9] While most cell lines are compatible with most gels, these combinations have been found to best facilitate adhesion, migration, and proliferation. This material can be used for setting the stage for unsupported cell cultures such as non-scaffold cell cultures which are reliant on a stable base to develop. It has also been found that hydrogel is easily manipulated to create complex networks which can be found in scaffold cell culture designs.
A problem however are 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.
PDMS
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. Its inert nature prevents any undesired interactions between the PDMS and other materials or chemicals used.[<550::AID-ANIE550>3.0.CO;2-G 10] 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. Furthermore, its permeability to gas presents advantages and disadvantages. 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.
PDMS is an advantageous material because it is cheap and easily moldable to whichever design is desired. While the material itself is hydrophobic, hydrophilic treatments can be layered into a device for initial cell adhesion. Reactive coatings are a novel innovation which has been found to support the self-assembly of proteins, antibodies, and mammalian cells [18]. The reactive coating, poly[para-xylylene carboxylic acid pentafluorophenolester-co-para-xylylene], was added to the device by the use of chemical vapor deposition (CVD) showed great adhesion when deposited in films at or under 100 nm according to Jörg Lahann et al. surface treatments like these are great for non-scaffold or scaffold based cultures.
Polystyrene
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.[11] 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/Silica
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.[12] 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.
Paper-Based
Considered a much cheaper option for microfluidic devices to sustain 3D cell culture, paper microfluidics. The benefits of paper is that it is cheap and it already has a premade structure which can be used to create scaffolded 3D cell cultures.
first established by the Whitesides group.[13] This method includes paper (chromatography paper was used by the Whiteside group) 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.
Techniques and Applications
The main purpose of 3D cell cultures is to synthesize a system that is representative of real life. While monolayer (2D) cell culture has been effective in research, cells have been proven to lose their specific properties over time in a dish. There are many effective techniques currently that do this such as 3D non-scaffold based and scaffold based cellular aggregates (including hydrogel-based cell cultures), and droplet microfluidics.[17] The techniques have been most effectively used in drug screening and cell therapies along with cancer research.
Non-scaffold based cell aggregates
Also known as scaffold free 3D cell aggregates, This is the cell culture technique that does not require a support system used in scaffold based cell cultures. The reasons for this technique are favorable due to a better replication of in vivo conditions such as cell-cell interactions and scalability. A popular technique for scaffold free cell culture development are using micropatterned wells via a forced aggregation technique [15]. This technique uses the pre-patterned wells which control cell-cell interactions by determining the special arrangement of cells. The growth of the culture is determined by this and can influence cell behavior in a system which can lead to tissue like structures. Another strategy is referred to as the cell sheet technique [19]. In this scaffold-free method, 2D cell cultures are created on a surface that is hydrophilic only at a certain temperature. By then changing the temperature, surface becomes hydrophobic and the cells are joined together creating a detachable “sheet” of cells which can later be stacked on each other to create a 3D culture as was performed by Ayidah Alghywainem Et al.
An example of an application of the non-scaffold technique was used to develop Mesenchymal stem cells (MSCs) by Rettinger et al. [15], which have applications in a wide range of therapies. The cells were grown using micropatterned wells via a forced aggregation, as stated above. To test the viability of the proliferative capability, the aggregates were tested via live/dead staining. The results were positive with cells in smaller cell aggregates but at around 4000 cells/aggregate, many cells became non-viable.
The materials for non-scaffold based 3D cell cultures are important to take note of. A specific material is not recommended rather each culture requires a different base. Due to the structures being independent from support it is important to have a foundational material that both mimics the native tissue environment and cues specific cell behavior and one that allows for optimal flow for nutrients and waste. Using surface treatments that fulfil the criteria can open opportunity for more dependable material such as PDMS to be used.
Droplet Microfluidics
Stemming off of Non-scaffold cell cultures are droplets. Encapsulating biological and chemical assays can be done via droplet microfluidics. Developed off of single cell screenings, cell culturing within droplets provides many benefits. One of which is the ability to create 3D cultures of various sizes and concentrations using any droplet microfluidic device which can quickly be developed. Once developed, the droplets provide effective long term culturing abilities due to the lower diffusion distance for nutrient and waste products from the feed stream. [18]. The droplets also provide quick production rates which can be benefitial in research that needs rapid testing. Additionally, droplets are effective at analyzing co-culture interactions which best mimic real life applications. This method of culturing also provides the ability to have multiple different types of cells in each droplet and can be effective at seeing how they interact.
Currently droplet microfluidics have been used for observing mammalian cells such as stem cells, or cancer cells in a desired medium. In most cases hydrogel was used, but water can also be used for this process. While hydrogel may be more stable, water can be as effective in droplet microfluidics because surface tension will maintain the droplet structure. The droplets are created with the desired concentration of cells and once formed, the droplets are left to sit in a liquid media or the hanging drop method is used. As mentioned above, the individual droplets provide great opportunity for 3D cell culture while maintaining the easy access to add and remove waste.
The creation of droplets in question here can be made in many ways. In many cases droplets are formed by a method known as the hanging drop technique 20. Cells are culture in a drop of media suspended on the lid of a cell culture dish as performed by Eleanor Knight Et al. Some other methods include capillary microfluidic devices, or flow focusing microfluidics devices which can be found in more detail under Emulsion Droplets.
Scaffold based cell cultures
Scaffold based cell cultures are 3D cultured created with the use of an external matrix composed of a firm material. These cultures can be advantageous due to their predictable results and design that caters to any desired outcome. These devices can be designed by 3D printing, photolithography, or any other method that properly creates an area for cells to grow and spread in multiple directions. Scaffold based cell cultures want to mimic environments that already have these supports naturally and are required for a cells proper function.
Scaffolds are entirely dependent on the material used. One popular material here are polymers. Polymer scaffolds have improved 3D cell culture by providing a biomimic environment that imitates extracellular matrix components (ECM). Examples of ECM’s are collagens, elastin, proteoglycans, etc. These are essential to some cell growth and survival due to the ability to provide structure which can improve nutrition and waste flow in the system. They can be placed into two systems. Protein based scaffolds and polysaccharide-based scaffolds [16]. Protein based scaffolds are created by molecules composed of amino acids which mimics in vivo interactions, while polysaccharide-based scaffolds are built from long chains of sugar molecules, these have been more effective at mimicking tumors grown in vivo as shown by Abuwatfa et al. Other types of scaffold materials are hydrogel scaffolds and paper-based scaffolds. The properties of hydrogel or paper are ideal for creating a system of 3D hydrophilic networks that provide a cheap yet effective way to mimic an in vivo system. PDMS can be used for devices that desire flow through them to mimic flow through the body.
The applications of scaffold-based cell cultures can vary. In all cases the strategy is used to have the results most related to real life. Cancer research is the major goal for most research studies. The applications in cancer research include drug testing, angiogenesis dynamics, or malignancy mechanisms as shown by Abuwatfa et al.
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.
Media Flow
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.
Air Bubbles
A current challenge of 3D cell cultures is air bubble formation. While air bubbles are generally associated with flow, they can also disrupt cell cultures by interfering with their structure. 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. [14].
References
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