Microfluidic cell culture – a beginner friendly review

Microfluidic cell culture for beginners

Microfluidics is an advanced technique based on multiple disciplines. Hence, getting behind the technique and its underlying principles can be very challenging. Cell biologists have already discovered the potential of microfluidics and are working on cell in microfluidic chambers [1]. Why this field is so promising to them and which hurdles they should expect when joining the microfluidic community and getting started with their first microfluidic experiments will be discussed in this review.

Limits of classical cell culture

Since it first appeared in the early 1980s much has changed in the knowledge about and the usage of microfluidics. Originally developed in the domain of physics, meanwhile most biologists may have heard about the stunning diversity of new possibilities which microfluidics offers, especially in cell biology with microfluidic cell culture. And they are completely right. Classical cell culture experiments and in vivo studies offer a variety of possibilities to analyze cell interactions, but they have certain shortcomings. Both human and animal in vivo models offer most realistic results. Logically, human in vivo studies can be conducted only at very high safety standards that have been proven in animal studies. For example, in drug development, this approval of harmlessness is a very time-consuming and expensive process, that also imposes major ethical concerns. Furthermore, the results of animal studies cannot be translated to humans unscrupulously, as there are relevant differences in enzyme activities and cellular behavior between the different species. Nonetheless, in vivo models represent most reliably the interaction of different tissues and their physiological activity.

Cell-culture however, is afflicted with less ethical concerns and allows the investigation of human derived cells and tissues. In general, such experiments are less sumptuous to conduct as well. Since the establishment of induced pluripotent stem cells (iPSCs) in 2006 it is now possible to culture diverse human-derived cell types. This has been an enormous advance in individualized medical research. As a result there have been efforts to develop stem cell culture further. While those cells and their induced differentiated descendants had once only been grown in two-dimensional cultures, it is nowadays possible to grow them as spheroids or in three-dimensional scaffolds as a novel approach to an in-vitro-model of whole organs [2]. This also includes the co-culture of different cell types, which has – in the exemplary case of neuron-astrocyte-co-culture – facilitated the culture of iNeurons over the long-term and rendered this cellular model more realistic as it includes the neuronal interactions with other naturally occurring cell types in the brain. Nevertheless this is only a first step in the direction of in-vitro-cultivation of integrated organic systems. After all, 3D-organoids do not grow in a perfectly controlled way and the cultivation on a petri-dish or in suspension is hardly comparable to the physiological situation in vivo. New approaches had to be developed to combine the advantages of low-cost, ethically immaculate and eventually even patient-specific cell culture with physiological architecture and interactions which had so far not been reproduced in vitro satisfyingly.

How microfluidic cell culture has become indispensable

Microfluidic cell culture is this next step towards the in-vitro modeling of interconnected organoids, sometimes also referred to as “organs-on-chip” [3]. It can give new insights in the interaction of different tissue types in an environment which is more similar to their natural context, especially including physical shear stress and exposure to fluid flow [4].

Microfluidic cell culture enables the modeling of tissue-tissue-interfaces and can provide further findings about physiological barriers. Furthermore it offers a straight-forward approach to investigate both signaling between different organs and cooperative function of different organs regarding endogenous synthesis of molecules [5] and drug-metabolism [6]. Different organs-on-chip can be therefore connected in a microfluidic cell culture system using the same flow source to allow diverse cell types to communicate via the tubing [7].

Technically, it features high spatio-temporal control over the cellular microenvironment and allows different ways of analyzing the microfluidic cell culture, including microscopy and electrophysiology of the cells themselves and analyses of the outlet medium. It works with less medium changes, with fewer medium evaporation thanks to the low surface and can be run automatically. Thanks to the reduced cell number, the general usage of media and supplements can be reduced as well. Microfluidic cell culture experiments are not only used to model physiological states and to gain basic insights into intercellular processes. They are also an attractive means to understand pathologies since they allow well-controlled manipulation of the modeled processes.

Watch the webinar by Lisa Muiznieks, PhD about Microfluidic 3-Dimensional cell culture!

Hurdles to expect during the first microfluidic cell culture experiment

As microfluidic cell culture aims to model complex processes, its handling might occur complex to some first-time-users. Although easy once understood, microfluidic experiments have to be well-prepared. This might appear obvious, but there is an immeasurable diversity of different commercial microfluidic chips for different applications. More advanced users may even need to design their own chips for specific experiments. This diversity is needed to offer the cells some kind of scaffold to grow on. Furthermore you will need different chip architectures depending on how you like to apply flow on the cells. There are different types of experiments you have to think about before choosing a defined setup:

As microfluidic cell culture aims to model complex processes, its handling might occur complex to some first-time-users. Although easy once understood, microfluidic experiments have to be well-prepared. This might appear obvious, but there is an immeasurable diversity of different commercial microfluidic chips for different applications. More advanced users may even need to design their own chips for specific experiments. This diversity is needed to offer the cells some kind of scaffold to grow on. Furthermore you will need different chip architectures depending on how you like to apply flow on the cells. There are different types of experiments you have to think about before choosing a defined setup:

• simple flow experiments to investigate the effects of shear stress on the cells, e.g. endothelial cells, gut tissue and more [8]
• serial co-culture of different cell types, e.g. for connecting gut, liver and kidney derived cells to investigate the metabolism of orally applied drugs [9, 10]
• membrane-separated parallel chambers to model physiological barriers, e.g. blood-brain-barrier using endothelial cells, pericytes and astrocytes [11, 12]
• hollow fibers, e.g. for co-culturing endothelial cells and podocytes for modeling the kidney’s glomerular filtration barrier [13]
• integration for electrophysiological recordings [14]
• microchannels to guide dendritic growth [Fig. 1] [15]
• open channels for easily manipulating supplements in the medium [16]
• complex individualized chips for modeling anatomical microstructures with spheroids, e.g. liver lobules [Fig. 2] [17]

Figure 1. Image source [15]

Figure 2. Image source [17]

Apart from the chip architecture, its material has to be chosen carefully as well. Many chips are made of PDMS, as it shows fine bio-compatibility and transparency for microscopy applications. As it reportedly absorbs small molecules there are attempts to use other materials such as polystyrene or polylactic acid [18]. As it has not yet been fully clarified whether the properties of PDMS really influence the culture in a negative way, it is so far the most-commonly used substance thanks to its convenient prototyping suitability.

The mentioned components of a microfluidic cell culture experiment are quite static and can be easily standardized. The most variable element indeed is the flow. To maintain stable flow rates and to adapt them when necessary requires additional gadgets which should ideally be easy and intuitive to operate. In any case these systems will require some practice, but in the end they help to regulate the flow in a desired range and to increase the reproducibility.

Beside these preparatory considerations, first-time-users should be aware of some practical application-hurdles as well. The major nuisances in flow-experiments are air bubbles and leakage, both of which can hardly be over-estimated, as they can lead to serious malfunction of the experiment. Fortunately there are commercially acquirable solutions for these practical problems like bubble traps and tubing adapters which nonetheless require prudent application.

Figure 3. Image source [7]

For the microfluidic cell culture with several cell types (co-culture), there is always a common medium to be found which all the different cell types can utilize [Fig. 3]. It is recommended to search literature for other research groups who may have already established a certain combination of cells and are thereby experienced with the experiment. Non-microfluidic co-cultures may serve as a model as well in this case.

First-time users might also be surprised by the space occupied by one single microfluidic cell culture experiment, which is not comparable to the common static well plates and flasks, as the tubing system, the adapters and the eventually involved regulatory equipment require quite extensive and large setups.

In any case, newcomers have to consider that microfluidic cell culture is not always intuitive, even with theoretical background knowledge. It is recommended to always stay in touch with your supplier and to learn from experienced users by taking opportunities for hands-on-training. After all it is a completely unfamiliar setup for most cell biologists which has to be explored step-by-step. With some experience the user will notice that in spite of the elaborate installation of an experiment, the handling of microfluidic experiments can get far more convenient as soon as the experiment is running.

Future approaches

Microfluidic cell culture has to become more accessible for first-time-users who are not familiar with the physical details behind the technology. Plug-and-play-installations have to be a major aim for reaching more potential users and to provide higher levels of standardization and reproducibility. It will be inevitable to get the laborious experimental setups smarter and to integrate more functions in less devices. ELVEFLOW is working on such smart plug-and-play solutions for microfluidic cell culture and will gladly support newcomers during their first steps towards microfluidic expertise.

References