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Will Microfluidic Cell Culture Fulfill its Long-awaited Potential?

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Will Microfluidic Cell Culture Fulfill its Long-awaited Potential?

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The first paper ever published on microfluidic cell culture is 19 years old1. Microfluidic cell culture has now outgrown its infancy and is about to survive its teenage years. It has matured considerably but still needs to transition from academia into clinics and industry. Will it come of age?

The birth of microfluidic cell culture


By the late 1980s, biologists started benefiting from the progress achieved in micro-electronics and microfabrication by placing cells on micro-patterns and studying the effect of micro-architecture on cell growth in 2D2. A decade later, an increasing number of groups had understood the potential offered by microfabrication techniques for controlling cell distribution in vitro. These in vitro models were however only composed of one cell type. The first turning point came when scientists concentrated their efforts on facilitating co-culture of different cell types1,3, realizing that microfabrication “will allow us to precisely manipulate cell–cell interactions of interest”. Scientists realized that these micro-systems could not only “pave the way for studying the functional significance of tissue architecture at the resolution of individual cells”3, but also help in developing tissue substitutes for tissue engineering or for screening toxic compounds4.

Microfluidic cell culture came to life when cells were first manipulated and cultured via microfluidic channels1; at first, this was used to micro-pattern more than one cell type, each perfused in separate channels. This also materialized the second turning point, when it was realized that “The handling and delivery of small quantities of liquid is a critical part of many physical, chemical, and biological processes”1. Scientists recognized that the ability to recreate and control flow at the micro-scale was something that could be exploited for more than facilitating cellular micropatterning. Within a few years, groups used flow in microfluidic systems to study the effects of complex gradients on cells5, cell transport and adhesion under dynamic flow6, the transport of molecules across monolayers7 or to deliver small molecules with sub-cellular precision8. In parallel, microfluidic actuators, valves and sensors were integrated to miniaturize and accelerate experiments in the effort of developing lab-on-chips. Microfluidics had made its first promising steps into the field of biology9.

Microfluidic cell culture has entered the big league


Since then, microfluidic models have become a predominant tool for biology research for tissue modeling. “We have made incredibly good advances on the scientific front; we can create complicated tissue-like structures.” says Prof. Mehmet Toner from Massachusetts General Hospital at the Harvard Medical School, one of the pioneers of microfluidic cell culture. Indeed, microfluidic models have allowed scientists to create tissue-like models with unrivalled physiological relevance. In particular, “The relative ease in manufacture of these devices has led to the burgeoning field of organ-on-chip studies” says Dr. Kristina Haase, a post-doctoral researcher studying in vitro vasculogenesis at Massachusetts Institute of Technology, referring to the use of PDMS as an inexpensive and easy to use material for fabricating microfluidic devices.

Organs-on-chips are cellular models composed of different cell types cultured in microfluidic chips that mimic the organ micro-architecture and recreate key functions of organs in vitro. This makes them unique surrogates for humans, and unique candidates to replace the use of animal models. In addition to minimizing the use of animals for ethical reasons, these organs-on-chips are envisioned to help with the current bottleneck in drug discovery. Currently, 9 out of 10 drugs that work in mice do not work in humans10, chiefly because current pre-clinical models fail to recapitulate faithfully the human response to drugs11. The hope is that the improved physiological relevance of organs-on-chips will increase their predictive power, accelerate drug discovery and decrease the massive costs currently associated with developing new drugs, estimated at $2.6 billion12.  As a result, numerous publications have now characterized microfluidic models for the lungs, heart, kidney and liver amongst others13.

Organs-on-chips are being used for numerous applications: the most exploited have been to unravel mechanisms underlying diseases such as cancer14, or to test drugs. Recently, groups have even gone so far as to combine several organs on the same chip15 to mimic a more systemic and faithful drug response16,17. Note that organs-on-chips are not the only microfluidic cell culture application, but it is one of the applications in cell culture for which microfluidics have demonstrable advantages over traditional methods18. It is also one of the applications generating the most interest because it is believed to fill an important gap in pre-clinical models to increase drug testing efficiency. Other interesting applications of microfluidic cell culture are being explored, such as finding biomarkers for diseases19, clinical diagnosis, studying developmental biology20 or regenerative medicine21 or encapsulating cells in 3D for transplantation22. There are also studies interested in performing 2D cell culture in microfluidic chips to accelerate and standardize benchtop procedures, such as stem cell differentiation23.

Microfluidic cell culture publications have steadily been increasing for the last 20 years, betraying an avid academic interest into exploring as many applications as can be offered by this technology. The question remains: will these applications move out of the academic realm and become implemented clinically, or is this a hype?

Could it be a hype?


Microfluidic technology has in fact gone through a hype cycle like other emerging technologies24, named after the company Gartner that introduced the model in 1995. In this model a trough of disillusionment follows the peak of inflated expectations, which for microfluidic technology happened in the 1990’s. In fact, microfluidics should not be considered as the solution to everything. But in 2009, Becker said that microfluidics was going through the slope of enlightenment because people were “finally realizing that the technology can solve certain problems that conventional technologies can’t”, as reported by Dr. Mukhopadhyay25.

When it comes to cell culture, it is indeed now established that microfluidic models hold advantages over both conventional macroscopic in vitro models and animal models. Most notably, microfluidic models enable a more precise control over the cellular and biochemical microenvironment than conventional in vitro models26. This is because cells can be compartmentalized in a range of user-defined microscopic configurations, thus allowing for the distribution of cells to be controlled at physiological length scales. The microenvironment control is also facilitated by the presence of channels: these allow the establishment of complex and persistent biochemical or physical gradients27, the control of biomechanical factors such as shear stress and the precise delivery of cells or reagents inside the assay. In addition, microfluidic models enable high resolution imaging, which can be difficult in animals and macroscopic in vitro systems. Imaging complex cellular microfluidic models has enabled researchers to observe in great details rare events that are hard to capture in vivo, such as specific steps of cancer metastasis28. Finally, microfluidic models require less reagents and cells than conventional macroscopic in vitro systems. While microfluidic technology has some inherent limitations, these numerous benefits are now clear to the scientific community. What is, then, holding it up from bridging the gap with industry?

Time to leave the academic nest?


By now, it was predicted that microfluidic technology would have reached its plateau of productivity25, where mainstream adoption starts to take off. As far as microfluidic cell culture is concerned, academic adoption has certainly become wider but industrial translation has been slow.

Not surprisingly, one of the main sources of resistance stems from the fact that microfluidic cell culture is pitted against conventional cell culture which, as Prof. Toner points out, “has been optimized for a century; it is a gold standard that is outstanding and hard to overcome”. Indeed, it is hard for people to adopt change, especially when the new solution is perceived as more expensive and cumbersome, as microfluidics can be thought of compared to conventional cell culture. While microfluidics technology has the potential to be higher throughput and save money in the long run compared to conventional cell culture or animal models, the upfront cost in setting it up can be a scary investment. Manufacturing microfluidic chips can now be relatively cheap; it is often the fact that it entails switching from 2D to 3D culture that increases the associated costs. Indeed, microfluidic cell culture can be done in 2D, but one of its main attractions lies in creating complex 3D models, and as Prof. Toner says, “it is very expensive to switch to 3D”. According to him, to motivate this investment, “there needs to be more data to prove that drugs should be tested in 3D vs. 2D”. While some studies have been published to that effect29,30, more could be done to convince a larger audience of the need to switch to 3D cell culture, and with it to using microfluidic devices. On the other hand, he says “people are not going to use more complicated devices unless it’s data they couldn’t get before”. This echoes previous warnings18,25, stating that the field of microfluidic cell culture needs to focus on applications that cannot be done in conventional cell culture to become more widely adopted. As Dr. Haase points out, “engineers need to collaborate further with biologists in order to exploit the microfluidic models to their maximum extent. “

Yet, even for “killer applications” such as organs-on-chips there seems to be some delay in adoption. Some limitations are clearly technical and logistical: for example, Dr. Haase emphasizes that “one hurdle preventing clinical adaptation of microfluidic technologies is that highly skilled personnel are often required to develop and operate the systems.” She also points out that PDMS can be problematic as “it absorbs small molecules and cannot be used to study any long-term (days) drug interactions”. Yet these issues will and are slowly being worked around, for example with new polymers31 being developed or thermoplastic devices being used instead of PDMS chips. A separate issue exists, related to the business model associated with microfluidic cell culture ventures. After all, “it is complicated to maintain living things, let alone selling living products” as Prof. Toner points out. Some companies like Emulate Inc. are taking on the challenge through academic and industrial collaborations; others like Aim Biotech Inc. sell ready-made modular chips that scientists fill with cells in their laboratories.

Even though one might dwell on these limitations, ongoing academic breakthroughs promise to provide enough incentives to overcome them. Currently, the field is focusing much of its energy on developing 3D personalized cellular models using cells derived from patients32,33. These could become miniaturized avatars to test the unique effect of drugs on individual patients and are generating great interest in facilitating personalized medicine34. This is proving hard, but according to Dr. Haase, chiefly because “it is a challenge harvesting patient cells and obtaining large enough cell volumes to work with”. Therefore, some of the limitations in implementing microfluidic cell culture in the clinic are not inherent to microfluidic technology and will require progress in other areas. Many groups are also concentrating on improving on-chip vascularization35, to improve physiological relevance and long-term tissue survival in vitro. According to Dr. Haase, mastering in vitro vascularization will also “undoubtedly influence drug testing, considering that the circulatory system is the primary transport mechanism”. Finally, some effort is being dedicated towards implementing on-chip bio-sensors36 so that data can be gathered and analyzed reproducibly and fast from the microfluidic chips. This needs to be more actively pursued so that microfluidic technology can become truly high throughput and stream-lined in the drug discovery process.

Conclusion


We can be sure of one thing: microfluidic systems have already accomplished a great deal for cell culture, especially for improving the physiological relevance and giving unprecedented momentum to 3D cell culture. Microfluidic cell culture has proved its worth but needs to make more of an effort to leave the comfortable academic home that has done so much to nurture it. Indeed,  there seems to be a disconnect between academia and industry18,25, typical of other fields too, whereby the pressure to publish drives “certain academics to try to do the next big fancy thing; there is a need to listen to the voice of the customers”, as Prof. Toner points out. In other words, much of academic research in microfluidic cell culture is pushing the technology; it might be up to companies to instead find the market that pulls the technology. Companies might also be better incentivized to focus energy on improving the practical aspects of device design and fabrication that might have fallen by the wayside in academia,  and adapt microfluidic cell culture to robust industrial workflows which cannot be as finnicky as that often found in academic labs25.  At any rate, while the field of microfluidics is ready to leave the academic nest it will not cut ties: it might just be time to learn to be marketable outside of academia while keeping strong links with it. After all, who says adulthood should be entered alone?

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