3D Cell Culture - McLean Taggart, Emma Villares, and Maximillian Marek

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CHEM-ENG 535: Microfluidics and Microscale Analysis in Materials and Biology

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While most microfluidic devices incorporate a 2D cell culture design, in which a single layer of cells is grown on the bottom of the 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, devices are being made that support 3D cell culture - in which cells are suspended in a three-dimensional matrix or seeded on a three-dimensional scaffold, each allowing the cells to grow at varying height.

Background

Figure 1. Standard microfluidic cell culture chip design, containing well for cell growth and parallel channels for media delivery; specifically, this chip is used for tumor microenvironment biomarker profiling.[5] Reproduced from Enabling cell recovery from 3D cell culture microfluidic devices for tumor microenvironment biomarker profiling by Munoz, M. V. et al, under Creative Commons Attribution 4.0 License.

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).[17] 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.[4]

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.[6] Despite its benefits, the usage of 2D cell culture can lead to dubious 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.[7] 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.[8]

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.

Device Materials

There are several different materials that are used for microfluidic devices that impact overall growth and behavior of 3D cell culture.[10]

PDMS

Poly(dimethylsiloxane) (PDMS) is one of the most commonly used materials in microfluidics due to its wide range of properties. PDMS is an elastomer that has a high surface area to its bound substrate which is important in its soft lithography process in order to hold the desired device template. Specific to 3D cell culture, PDMS is chemically inert, isotropic, and can allow for observation of light down to 300 nm. Since the device is chemically inert, there are no negative reactions that could occur when trying to apply different types of treatments on the actual device or that would alter the device to impact the cells.[10] Since the device is isotropic, there would be no difference in cellular conditions throughout the device such that one side does not favor growth over the other. With visibility of substances at 300 nm or above, this is critically important in cell culture for observing the fluorescence of novel treatments. Since green and red fluorescence are above 300 nm, proper visualization can occur in order to determine if the treatment properly works.

Polystyrene

There are some instances where PDMS cannot be used due to potential impacts on cell proliferation due to the absorption of growth factors from the scaffold and interference with the cell culture media. In addition, in the case of therapeutics testing, this can skew the results such that the therapeutic can be absorbed into the surrounding material rather than target the cells. From a design standpoint, a polystyrene based microfluidic does not require micropillars as PDMS does since polystyrene is hydrophobic to the point where interactions between wells would not occur. Micropillars are often used in PDMS devices in order to make sure that there is no mixing between different wells or culture media with cells if needed. Polystyrene however is a commonly used material in standard 2D cell culture and as such can be seen as a recommended substitute for a microfluidic device if there are experimental cytotoxicity materials. However 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. But it does provide the possibility that polystyrene would be able to determine different gas testing abilities, such as with cigarette smoke.[11]

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 with an increase of visibility and imaging quality of the 3D cell culture systems based on fluorescence tagging. There have also been studies using silica for 3D cell cultures, mostly for nutrient delivery to different cells.[10],[12] However, due to the fragility of the glass devices and the cost of the silica devices, the use of glass or silica is relatively less common relative to polymeric devices, which are typically cheap and easy to make.

Paper-Based

Figure 2: Diagram showing the construction of a diffusion-based paper microfluidic cell culture device.[13] Reproduced from Paper-supported 3D cell culture for tissue-based bioassays by Derda, R. et al. under Creative Commons Attribution 4.0 License.

Considered a much cheaper option for microfluidic devices to sustain 3D cell culture, paper microfluidics was first established by the Whitesides group.[13] 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.

Hydrogel

The most common scaffolding technique in microfluidic devices mixes a hydrogel substance with cells before 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 manner including physical structures such as posts and ridges; molding through pressure force; and post-injection external force. Due to differences in environmental preference between cell types and cell lines, different types of hydrogels are used depending of the cell lines being seeded. Endothelial cells and tumor cells are typically cultured in collaged; stem cells are commonly cultured in fibrin; embryonic stem cells are typically cultured in matrigel.[9] While most cell lines are compatible with most gels, these combinations have been found to best facilitate adhesion, migration, and proliferation.

Applications

The usage of three-dimensional (3D) cell culture in microfluidic devices was first published in 2009 in a collaborative study from MIT. It had already been established that 3D cell cultures were considerably better than 2D cell cultures due to their inclusion of the extracellular matrix (ECM) and other growth factors. Upon this initial study, it was found that the formation of these 3D spheroids showed more accurate growth pattern and treatment results compared to their 2D counterpart. However, a different set of considerations must occur for 3D cell culture at the microfluidic scale, such as the materials used and unintentional oxidation.[14]

These devices are typically made with PDMS consisting of several different micropillars. These micropillars are evenly spaced such that there is an increase of cell-to-cell interactions and a decrease in physical shear stress.[15] PDMS is a material that has always been considered the norm for microfluidic devices and 2D cell culture. As such, to prove the 3D model, PDMS devices were also fabricated with these microarrays. These devices consist of several inlet and outlet streams for the injection of cells and removal of byproducts, respectively. In addition, an agarose gel or some other scaffold must be coated on the device where the cells would be present in order for proper growth/behavior to occur. Otherwise a 3D cell model could become deformed that can skew experimental results. As such, the cells must have a longer doubling time in order for proper experiments can occur for adding any form of therapeutic. Another method for loading cells onto the microfluidic device involves directed scaffolds with growth factors, such as collagen IV attached. This would allow for continuous cell growth after several passages.[16]

Osteogenesis on a Chip

One common application of 3D cell culture, is organ-on-a-chip studies. A study performed by Bahmaee et al., recently designed and evaluated a microfluidic device as an osteogenesis-on-a-chip model.[17] In simple terms, osteogenesis essentially means the formation of bone. This model combined both the importance of a 3D environment and fluid shear stress. The chip utilizes a polymerized High Internal Phase Emulsion (polyHIPE) and modeled the flow dynamics computationally using an intermittent flow profile with rest periods to more strongly promote differentiation and matrix formation. In addition, this intermittent flow profile also enhances mineralized matrix deposition when tested. Further results showed that higher flow rates compromise the initial attachment of the cells, but not growth in the long term. Overall, the cells were able to penetrate the polyHIPE network and successfully filled the channels within the chip.

Due to the predicted potency of the intermittent flow profile being investigated, fluid shear stress alone was tested and found to be insufficient to induce the osteogenic differentiation. Instead, media containing specific factors such as dexamethasone were necessary, but shear stress increases the overall differentiation compared to the components alone. Further exciting results indicated that primary cilia were present with the device channels. In the bone, primary cilia are rigid structures that extend from the cell into the extracellular space to allow for mechanosensing in bone, critical for development and bone formation.[18] This shows the cilia can be exposed to the fluid shear stress and are a potential mechanism by which the stress could be mechano-transduced within the device. Overall, in this study, a successful microfluidic device was developed to establish an osteogenesis-on-a-chip model.

Chemotherapeutic Drug Cytotoxic Activity

Figure 3. Microfluidic chip incorporating tumor spheroids for screening of chemotherapeutic drug cytotoxic drug activity.[13] Reproduced from Characterising a PDMS based 3D cell culturing microfluidic platform for screening chemotherapeutic drug cytotoxic activity by Khot, N. I. et al, under Creative Commons Attribution 4.0 License.

Since the first three-dimensional culture was made in the 1970s, similarities between the morphology and behavior of cells in 3D culture conditions and tumor cells have been observed. This type of 3D culture, a spheroid, formed of cells making various layers, mimics both the physical and biochemical features characteristic of a solid tumor mass. This allows for a platform to be used for various cancer-biology studies involving cancer-initiating cells, invasion processes, drug testing, cell response to irradiation, and metastasis.[1]

Studies comparing drug sensitivity in 2D versus 3D cultures found that tumor cells were less sensitive to chemotherapeutic drugs in 3D cultures. There are a few explanations for this such as diffusion limitations leading to reduced access of compounds in the medium, hypoxia-induced pathophysiological differences, or the cell cycle. Therefore, 3D cultures may prove to be more efficient models for understanding tumor responses to drugs by introducing necessary complications more relevant to the actual physiology. Among all 2D and 3D models, 3D cell culture with the same cell density as natural tissue shows a drug response that is relevant and comparable to a solid tumor. Microfluidic approaches to establishing spheriod cultures can be broken down into three categories: (i) microwell, (ii) microtrap, and (iii) droplet-based approaches.[19] For all approaches, working on the microscale utilizes micrometer-sized channels to allow for spatial control over co-cultures, perfusion flow, and signaling gradients.[20]

Furthermore, Khot et al. developed a PDMS-based microfluidic platform for evaluating chemotherapeutic drug cytotoxic activity (Figure 3).[21] Microscopic wells were created that allowed for HT29 cancer cells to coagulate into tumor spheroids. Over multiple days, with constant fluid flow, the spheroids continuously gained volume as the cells agglomerated to form the structure. After full development, chemotherapeutic drugs - including 5-fluorouracil - were added to the transient fluid to measure their anti-tumor efficacy. Using stained cells, the group could measure the drug ability by measuring well fluorescence, showing decreased cell viability over time after treatment. This model offers a high-throughput, cost-effective method for evaluating the efficiency by which such drugs operate.

Difficulties

Flowing Media

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. In microfluidic 3D cell culture, 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

One of the current challenges with 3D spheroid formation 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 Nitrogen environment. Different culture flows were tested at media concentrations ranging from 10% to 40% with these endpoints providing the lowest amount of bubble formation. This is a major consideration with 3D cell culture since proper growth needs to occur for accurate results.[22]

References

[1] Kapałczyńska, M.; Kolenda, T.; Przybyła, W.; Zajączkowska, M.; Teresiak, A.; Filas, V.; Ibbs, M.; Bliźniak, R.; Łuczewski, Ł.; Lamperska, K. 2D And 3D Cell Cultures – a Comparison of Different Types of Cancer Cell Cultures. Archives of Medical Science 2016.

[2] Segeritz, C.-P.; Vallier, L. Cell Culture. Basic Science Methods for Clinical Researchers 2017, 151–172.

[3] Young, E. W.; Beebe, D. J. Fundamentals of Microfluidic Cell Culture in Controlled Microenvironments. Chemical Society Reviews 2010, 39 (3), 1036.

[4] Kim, L.; Toh, Y.-C.; Voldman, J.; Yu, H. A Practical Guide to Microfluidic Perfusion Culture of Adherent Mammalian Cells. Lab on a Chip 2007, 7 (6), 681.

[5] Virumbrales-Muñoz, M.; Ayuso, J. M.; Lacueva, A.; Randelovic, T.; Livingston, M. K.; Beebe, D. J.; Oliván, S.; Pereboom, D.; Doblare, M.; Fernández, L.; Ochoa, I. Enabling Cell Recovery from 3D Cell Culture Microfluidic Devices for Tumour Microenvironment Biomarker Profiling. Scientific Reports 2019, 9 (1).

[6] Coluccio, M. L.; Perozziello, G.; Malara, N.; Parrotta, E.; Zhang, P.; Gentile, F.; Limongi, T.; Raj, P. M.; Cuda, G.; Candeloro, P.; Di Fabrizio, E. Microfluidic Platforms for Cell Cultures and Investigations. Microelectronic Engineering 2019, 208, 14–28.

[7] Tehranirokh, M.; Kouzani, A. Z.; Francis, P. S.; Kanwar, J. R. Microfluidic Devices for Cell Cultivation and Proliferation. Biomicrofluidics 2013, 7 (5), 051502.

[8] Kapałczyńska, M.; Kolenda, T.; Przybyła, W.; Zajączkowska, M.; Teresiak, A.; Filas, V.; Ibbs, M.; Bliźniak, R.; Łuczewski, Ł.; Lamperska, K. 2D And 3D Cell Cultures – a Comparison of Different Types of Cancer Cell Cultures. Archives of Medical Science 2016, 14 (4), 910-919.

[9] Li, X. J. (J.; Valadez, A. V.; Zuo, P.; Nie, Z. Microfluidic 3D Cell Culture: Potential Application for Tissue-Based Bioassays. Bioanalysis 2012, 4 (12), 1509–1525.

[10] Xia, Y. & Whitesides, G. M. Soft Lithography. Angewandte Chemistry International Edition 1998, 37, 550-575.

[11] Chan, C. Y.; Goral, V. N.; DeRosa, M. E.; Huang, T. J.; Yuen, P. K. A Polystyrene-Based Microfluidic Device with Three-Dimensional Interconnected Microporous Walls for Perfusion Cell Culture. Biomicrofluidics 2014, 8 (4), 046505.

[12] Li, X. J. (J.; Valadez, A. V.; Zuo, P.; Nie, Z. Microfluidic 3D Cell Culture: Potential Application for Tissue-Based Bioassays. Bioanalysis 2012, 4 (12), 1509–1525.

[13] Derda, R.; Laromaine, A.; Mammoto, A.; Tang, S. K.; Mammoto, T.; Ingber, D. E.; Whitesides, G. M. Paper-Supported 3D Cell Culture for Tissue-Based Bioassays. Proceedings of the National Academy of Sciences 2009, 106 (44), 18457–18462.

[14] Sudo, R.; Chung, S.; Zervantonakis, I. K.; Vickerman, V.; Toshimitsu, Y.; Griffith, L. G.; Kamm, R. D. Transport‐Mediated Angiogenesis in 3D Epithelial Coculture. The FASEB Journal 2009, 23 (7), 2155–2164.

[15] Kapałczyńska, M.; Kolenda, T.; Przybyła, W.; Zajączkowska, M.; Teresiak, A.; Filas, V.; Ibbs, M.; Bliźniak, R.; Łuczewski, Ł.; Lamperska, K. 2D And 3D Cell Cultures – a Comparison of Different Types of Cancer Cell Cultures. Archives of Medical Science 2016.

[16] Kim, M. S.; Hwang, H.; Choi, Y.-S.; Park, J.-K. Microfluidic Micropillar Arrays for 3D Cell Culture. The Open Biotechnology Journal 2008, 2 (1), 224–228.

[17] Bahmaee, H.; Owen, R.; Boyle, L.; Perrault, C. M.; Garcia-Granada, A. A.; Reilly, G. C.; Claeyssens, F. Design and Evaluation of an Osteogenesis-on-a-Chip Microfluidic Device Incorporating 3D Cell Culture. Frontiers in Bioengineering and Biotechnology 2020, 8.

[18] Temiyasathit, S. & Jacobs, C. R. Osteocyte primary cilium and its role in Bone Mechanotransduction. Annals of the New York Academy of Sciences 2010, 422–428 (1192).

[19] Lee, J. M. et al. Generation of tumor spheroids using a droplet-based microfluidic device for photothermal therapy. Microsystems & Nanoengineering 2020, (6).

[20] van Duinen, V., Trietsch, S. J., Joore, J., Vulto, P. & Hankemeier, T. Microfluidic 3D cell culture: From tools to tissue models. Current Opinion in Biotechnology , 2015, 118–126 (35).

[21] Khot, M. I.; Levenstein, M. A.; de Boer, G. N.; Armstrong, G.; Maisey, T.; Svavarsdottir, H. S.; Andrew, H.; Perry, S. L.; Kapur, N.; Jayne, D. G. Characterising a PDMS Based 3D Cell Culturing Microfluidic Platform for Screening Chemotherapeutic Drug Cytotoxic Activity. Scientific Reports 2020, 10 (1).

[22] Park, D. H.; Jeon, H. J.; Kim, M. J.; Nguyen, X. D.; Morten, K.; Go, J. S. Development of a Microfluidic Perfusion 3D Cell Culture System. Journal of Micromechanics and Microengineering 2018, 28 (4), 045001.