Microfluidic cell culture integrates knowledge from biology, biochemistry, engineering, and physics to develop devices and techniques for culturing, maintaining, analyzing, and experimenting with cells at the microscale. It merges microfluidics, a set of technologies used for the manipulation of small fluid volumes (μL, nL, pL) within artificially fabricated microsystems, and cell culture, which involves the maintenance and growth of cells in a controlled laboratory environment. Microfluidics has been used for cell biology studies as the dimensions of the microfluidic channels are well suited for the physical scale of cells (in the order of magnitude of 10 micrometers). For example, eukaryotic cells have linear dimensions between 10 and 100 μm which falls within the range of microfluidic dimensions. A key component of microfluidic cell culture is being able to mimic the cell microenvironment which includes soluble factors that regulate cell structure, function, behavior, and growth. Another important component for the devices is the ability to produce stable gradients that are present in vivo as these gradients play a significant role in understanding chemotactic, durotactic, and haptotactic effects on cells.
Fabrication
Some considerations for microfluidic devices relating to cell culture include: fabrication material (e.g., polydimethylsiloxane (PDMS), polystyrene) culture region geometry control system for delivering and removing media when needed using either passive methods (e.g., gravity-driven flow, capillary pumps, or Laplace pressure based 'passive pumping') or a flow-rate controlled device (i.e., perfusion system) Fabrication material is crucial as not all polymers are biocompatible, with some materials such as PDMS causing undesirable adsorption or absorption of small molecules. Additionally, uncured PDMS oligomers can leach into the cell culture media, which can harm the microenvironment. As an alternative to commonly used PDMS, there have been advances in the use of thermoplastics (e.g., polystyrene) as a replacement material.
Spatial organization of cells in microscale devices largely depends on the culture region geometry for cells to perform functions in vivo. For example, long, narrow channels may be desired to culture neurons. The perfusion system chosen might also affect the geometry chosen. For example, in a system that incorporates syringe pumps, channels for perfusion inlet, perfusion outlet, waste, and cell loading would need to be added for the cell culture maintenance. Perfusion in microfluidic cell culture is important to enable long culture periods on-chip and cell differentiation.
Other critical aspects for controlling the microenvironment include: cell seeding density, reduction of air bubbles as they can rupture cell membranes, evaporation of media due to an insufficiently humid environment, and cell culture maintenance (i.e. regular, timely media changes).
Cell's health is defined as the collective equilibrium activities of essential and specialized cellular processes; while a cell stressor is defined as a stimulus that causes excursion from its equilibrium state. Hence, cell health may be perturbed within microsystems based on platform design or operating conditions. Exposure to stressors within microsystems can impact cells through direct and indirect ways. Therefore, it is important to design the microfluidics system for cell culture in a manner that minimizes cell stress situations. For example, by minimizing cell suspension, by avoiding abrupt geometries (which tend to favor bubble formation), designing higher and wider channels (to avoid shear stress), or avoiding thermosensitive hydrogels.
Advantages
Some of the major advantages of microfluidic cell culture include reduced sample volumes (especially important when using primary cells, which are often limited) and the flexibility to customize and study multiple microenvironments within the same device. A reduced cell population can also be used in a microscale system (e.g., a few hundred cells) in comparison to macroscale culture systems (which often require 105 – 107 cells); this can make studying certain cell-cell interactions more accessible. These reduced cell numbers make studying non-dividing or slow dividing cells (e.g., stem cells) easier than traditional culture methods (e.g., flasks, petri dishes, or well plates) due to the smaller sample volumes. Given the small dimensions in microfluidics, laminar flow can be achieved, allowing manipulations with the culture system to be done easily without affecting other culture chambers. Laminar flow is also useful as is it mimics in vivo fluid dynamics more accurately, often making microscale culture more relevant than traditional culture methods. Compartmentalized microfluidic cultures have also been combined with live cell calcium imaging, where depolarizing stimuli have been delivered to the peripheral terminals of neurons, and calcium responses recorded in the cell body. This technique has demonstrated a stark difference in the sensitivity of the peripheral terminals compared to the neuronal cell body to certain stimuli such as protons. This gives an excellent example as to why it is so important to study the peripheral terminals in isolation using microfluidic cell culture devices.
Culture platforms
Traditional cell culture Traditional two-dimensional (2D) cell culture is cell culture that takes place on a flat surface, e.g. the bottom of a well-plate, and is known as the conventional method. While these platforms are useful for growing and passaging cells to be used in subsequent experiments, they are not ideal environments to monitor cell responses to stimuli as cells cannot freely move or perform functions as observed in vivo that are dependent on cell-extracellular matrix material interactions. To address this issue many methods have been developed to create a three-dimensional (3D) native cell environment. One example of a 3D method is the hanging drop, where a droplet with growing cells is suspended and hangs downwards, which allows cells to grow around and atop of one another, forming a spheroid. The hanging drop method has been used to culture tumor cells but is limited to the geometry of a sphere. Since the advent of poly(dimethylsiloxane) (PDMS) microfluidic device fabrication through soft lithography microfluidic devices have progressed and have proven to be very beneficial for mimicking a natural 3D environment for cell culture.
Microfluidic cell culture Microfluidic devices make possible the study of a single cell to a few hundred cells in a 3D environment. Comparatively, macroscopic 2D cultures have 104 to 107 cells on a flat surface. Microfluidics also allow for chemical gradients, the continuous flow of fresh media, high through put testing, and direct output to analytical instruments. Additionally, open microfluidic cell cultures such as "microcanals" allow for direct physical manipulation of cells with micropipettes. Many microfluidic systems employ the use of hydrogels as the extracellular matrix (ECM) support which can be modulated for fiber thickness and pore size and have been demonstrated to allow the growth of cancer cells. Gel free 3D cell cultures have been developed to allow cells to grow in either a cell dense environment or an ECM poor environment. Although these devices have proven very useful, there are certain disadvantages such as cells sticking to the PDMS surface, small molecules diffusing into the PDMS, and unreacted PDMS polymers washing into cell culture media.
The use of 3D cell cultures in microfluidic devices has led to a field of study called organ-on-a-chip. The first report of these types of microfluidic cultures was used to study the toxicity of naphthalene metabolites on the liver and lung (Viravaidya et al.). These devices can grow a stripped-down version of an organ-like system that can be used to understand many biological processes. By adding an additional dimension, more advanced cell architectures can be achieved, and cell behavior is more representative of in vivo dynamics; cells can engage in enhanced communication with neighboring cells and cell-extracellular matrix interactions can be modeled. In these devices, chambers or collagen layers containing different cell types can interact with one another for multiple days while various channels deliver nutrients to the cells. An advantage of these devices is that tissue function can be characterized and observed under controlled conditions (e.g., effect of shear stress on cells, effect of cyclic strain or other forces) to better understand the overall function of the organ. While these 3D models offer better model organ function on a cellular level compared with 2D models, there are still challenges. Some of the challenges include: imaging of the cells, control of gradients in static models (i.e., without a perfusion system), and difficulty recreating vasculature. Despite these challenges, 3D models are still used as tools for studying and testing drug responses in pharmacological studies. In recent years, there are microfluidic devices reproducing the complex in vivo microvascular network. Organs-on-a-chip have also been used to replicate very complex systems like lung epithelial cells in an exposed airway and provides valuable insight for how multicellular systems and tissues function in vivo. These devices are able to create a physiologically realistic 3D environment, which is desirable as a tool for drug screening, drug delivery, cell-cell interactions, tumor metastasis etc. In one study, researchers grew tumor cells and tested the drug delivery of cis platin, resveratrol, tirapazamine (TPZ) and then measured the effects the drugs have on cell viability.
Applications of cells in microfluidic systems Microfluidic systems can be used to culture several cell types.
Culture of mammalian cells Mammalian cell cultures can be seeded, grown for several weeks, detached, and passaged to a fresh culture medium ad nauseam by digital microfluidic (DMF) devices on a macro-scale.
Culture of non-mammalian cells
Algae Algae can be incubated, and their growth rate and lipid production can be monitored in a hanging-drop microfluidic system. For example, Mishra et al. developed a 25 × 75 mm, easily accessible microfluidic device. This design is used to optimize the conditions by changing well diameters, UV light exposure (causing mutagenesis), and light/no light tests for culturing Botryococcus braunii, which is one of the most common freshwater microalgae for biofuel production.
Bacteria and yeast Microfluidic systems can be used to incubate high volumes of bacteria and yeast colonies. The two-layer microchemostat device is made to allow scientists to culture cells under chemostatic and thermostatic conditions without moving cells around and causing intercellular interaction. Yeast cell suspension droplets can be placed on a plate with patterned hydrophilic areas and incubated for 24 hours; then the droplets are split the produced proteins from yeast are analyzed by MALDI-MS without killing the cells in the original droplets.
Multi-culture in microfluidics