TRUE CELL Review written with the support of the Individual Fellowship Marie Curie #753785 obtained by A. Boussommier that funds her project “TrueCell”.Written by Alexandra Boussommier: Alexandra.boussommier@elvesys.com 3D cell culture _ Alexandra Boussommier

3D cell culture: market and industrial needs

All living tissues are made of cells. To study cells, scientists isolate them from their original tissues and grow them in controlled and artificial environments: this is what is called cell culture. It was first used in 1907 [1], and has since become ubiquitous in biomedical laboratories. Cell studies have evolved from being performed on 2D surfaces to a 3D configuration to mimic more closely their natural 3D habitat in the body: this is what we refer to as 3D cell culture.

Keywords:

In vitro: means “in the glass”, it refers to the cells being cultured outside their normal habitat.

In vivo: means in a living organism.

ECM: extra-cellular matrix, made of proteins that cells secrete and interact with.

1. The purpose of 3D cell culture

In this report, we identify the following two main purposes for the 3D cell market:

1. Cell therapy, or regenerative medicine, which consists in growing cells that will be transplanted inside the patient to repair damaged tissue. This field consists in several research areas [2], listed in Fig. 1.

3D cell culture _ Celltherapy_Elvesys

2.  3D in vitro modeling for drug discovery, which aims at creating 3D cellular models to mimic living tissues, so that the bulk of research and drug discovery can be done in vitro instead of on animals (or humans). 3D in vitro models are meant to be used at different stages of drug discovery [3] prior to clinical trials, as listed in Fig. 2.

3D cell culture _ Drugdiscovery_Elvesys

 It should be noted that one other fundamental goal of cell culture has conventionally been to use cells to produce viruses or antibodies. Yet, this has been traditionally accomplished with 2D cell culture, with little incentive to move towards a true 3D cell culture format, other than to increase the yield; as a consequence, it is only briefly mentioned in this report.

3D cell culture _ ResearchAreas_Elvesys

Allied Market research has estimated that as of 2015, the global 3D cell market was $765 million and expected to grow by 30% to $4691 million in 2022 [4]. They divide the market in 4 categories: drug discovery, cancer research, regenerative medicine and stem cell research. Per our broader categorization, we consider that part of stem cell research is involved in cell therapy along with regenerative medicine. That is because stem cells can be differentiated in vitro into the desired cell type and re-implanted to repair tissue. On the other hand, we consider that, while cancer and stem cell research are major actors of their own, they essentially play a part in the effort of developing 3D in vitro models. Therefore, we find that the bulk of the market contributes to 3D in vitro modeling for drug discovery (Fig. 3). This is largely driven by a major push in cancer research, which is itself forced by the rising incidence of cancer around the world.

2. The types of 3D cell culture

According to Allied Market Research [4], 3D cell culture products exist in the following different formats (that can sometimes be combined):

  1. 3D porous scaffolds: Cells grow inside the pores of 1- engineered scaffolds, or 2- naturally derived fibrous material such as collagen or laminin.
  2. Scaffold-free platforms: Cells form multi-cellular structures without the support of a scaffold, either on their own (e.g. spheroids, organoids) or using cell-sheet technology.
  3. Microchips: Cells are placed inside micro-chip compartments to form cellular micro-environments of higher structural complexity resembling organs (i.e. organs on chips).
  4. Hydrogels: Cells are embedded in 3D gels of ECM such as Matrigel.
  5. Bioreactors: Bioreactors are hollow cylindrical chambers that locally control factors such as perfusion, temperature, humidity, and gas exchange. Cells are placed in scaffolds inside those bioreactors to facilitate 3D cell culture.
  6. Customized service: Companies directly customize the product for researchers.
3D cell culture _ MarketProducts_Elvesys

In addition, 3D bio-printing can be identified as another emerging format for 3D cell culture, a technology that enables the “printing”, or the layer-by-layer positioning of cells and biological materials into the desired configuration [3]. Of all products, scaffold-based platforms and microchips were identified as the top investment pockets in the field [4] (Fig. 4). Scaffolds generate the most revenue because they are becoming key both for cell therapy and drug discovery and because of an increasing number of materials being characterized. Microchips will undergo significant growth because of the gap organ-on-a-chips will fill in creating powerful pre-clinical models [4]. Note that the growth of scaffolds and microchips might be synergistic, as often organs on chips rely on the use of scaffolds, such as hydrogels.

3. Who is interested in 3D cell culture?

 Geographically, Allied Market Research estimates that the USA is the largest actor in the 3D cell market in 2015 representing 41% of the market, while Europe contributes 29%, Asia-Pacific 19% and Brazil, South Africa and Saudi Arabia 11% (Fig. 5). In 2022, they estimate that the gap between the USA and Europe’s importance in the market will be reduced, with the USA contributing at 35.3%, more closely followed by Europe at 31.5% [4].

  Three main types of end users are participating in the 3D cell culture market: biotechnology and pharmaceutical companies, academic laboratories and contract research laboratories. Their contribution will evolve at similar rates until 2022 [4]. Often, the basic science is initiated in academia, but pharmaceutical companies are the ones who link scientific findings with medicinal and process chemistry, pharmacokinetics and safety sciences. It is estimated that between 67-97% of drug development is performed by private companies [5].

3D cell culture _ Worldmap_Elvesys

4. Why bother with an extra third dimension?

 The answer is easy: because in their normal environment, cells evolve in three dimensions! It is now established that cells behave drastically differently in 3D compared to 2D. A turning point in the field came when cells in 3D were shown to respond to drugs differently than cells in 2D [6]- [7], proving that it is vital to culture cells in 3D for drug testing.

   Briefly, culturing cells in 3D allows the cells to adopt a morphology and a migration mode similar to that found in vivo. Recapitulating the normal morphology of cells is important as their shape can directly affect their biological activity [8]. It is also fundamental to replicate migration modes faithfully in vitro, as cell migration is a central process in many diseases (such as cancer) that could be targeted pharmacologically. More generally, placing cells in 3D effectively increases their degree of interaction with their immediate surroundings. It forces them to interact more heavily through their entire surface area with their micro-environment (whether it is with a scaffold, a hydrogel or other cells), as they do in tissue. This is of utmost importance, as it is now known that the interactions of cells with their ECM and micro-environment are essential to many cellular functions[7], [9]-[10]. In addition, the 3D support system provides physical and biochemical anchorage that can be engineered to replicate in vivo conditions. Both the stiffness, pore size, or ligands that make up the extracellular matrix can be fine-tuned to resemble the tissue of interest. Finally, 3D cell culture allows the formation and study of more complex multi-cellular structures, such as spheroids, organoids or a microvasculature.

3D cell culture _ 2Dvs3D_Elvesys

While these are all much needed improvements, moving to the third dimension has in reality opened up many other dimensions to explore. In the remainder of this review, we summarize the 3D cell culture landscape and expose the needs that will need to be met for 3D cell culture to become a higher throughput, safer and better standardized technique in the biomedical field.

5. 3D in vitro models for drug discovery

  1. The problem: A real bottleneck in the drug discovery process

Congratulations, you found a drug that was successful in treating a disease in the lab! That is quite a milestone. This typically means you have studied the drug in mice, if not in cell studies too. Now, you need to decide whether you want to spend an incredible amount of time and money to go through heavy regulations and more pre-clinical testing, until your drug can be tested in a human clinical trial. Only to find out that … it does nothing, or worse, it has unforeseen side-effects and will not be prescribed to human patients.

3D cell culture _ drugdiscoverybottleneck_Elvesys

 This is the sad reality of the current drug discovery process: approximately 9 out of 10 drugs that work in mice, do not in humans [11] (Fig. 7). It has been suggested that badly designed human clinical trials may be at fault in some cases. However, these statistics suggest that a problem lies most likely in the predictive power of the pre-clinical models: we need models that better recapitulate the human response to drugs. In particular, it is thought that the major problem lies with the failure to predict efficacy of the drugs, and its toxicity [12]. This is the huge bottleneck that the current pharmaceutical industry faces. Imagine spending on average 10 years and 2.6 billion $ dollars for nothing [5]. Still wondering why we haven’t easily cured all major diseases to this day?

   Something needs to be done; we need to re-think the format of the drug discovery system so as to more selectively filter the drugs that will eventually work on humans. The aim is three-fold: to increase the success rate of the drug discovery process, increase the throughput and reduce the associated costs.

2. Pre-clinical models: of mice and men… and cell culture?

It goes without saying that new reagents cannot be directly tested on humans, without prior evidence of their potential toxicity.  Instead, scientists have traditionally been using surrogate models to predict what the drugs will do in humans, such as animal or cellular models, each with their pros and cons (Fig. 8). Note that most of the pros and cons listed in Fig. 8 apply to in vitro models as a whole, that is, both 2D and 3D cellular systems.

3D cell culture _ invivovsinvitro_Elvesys

The main advantage of using animal models is unrivaled: it is after all a whole living organism. This means one can assess not only the local effects of the drug on the cells of interest (as you would in typical simplistic cell studies), but also its systemic effect on the entire body (in other words, on numerous organs simultaneously). Similarly, it is now becoming evident that the micro-environment of cells affects not only the cell’s physiology but their response to drugs [42]: hence, it is an advantage to be able to study the effects of a drug in an animal where all cell types are interacting in real time.

While numerous powerful mouse models have been characterized, some of their intrinsic limitations will be hard to overcome. For example, results in animals are inherently based on non-human cells and pathogens, which might not faithfully mimic the human pharmacological response. While studies have introduced human cells into mice, this typically must be done in mice that have a compromised immune system to prevent the rejection of human cells. This in turn means the animal model does not properly reflect the role of immune cells, which should be taken into account, as it has become clear that immune cells are pivotal actors in many diseases [13]. In any case, the human cells that are introduced in the mouse are interacting with a cellular micro-environment of murine origin, which again might not properly reflect the all-human cellular interactions that happen in patients. In contrast, cell studies can be exclusively made of human cells. This avoids discrepancies caused by the extrapolation of results from animals to humans, which can be particularly stark for the immune system [14]. Even though cell models are still failing to always predict the human response to drugs correctly, the 3D cell culture field is still in its infancy and is evolving rapidly; it might therefore be more amenable to fill the current gap in preclinical predictive models than animal models. In fact, many of the emerging 3D cell culture products have already been implemented in the drug discovery process. Traditionally, spheroids, organoids and organs on chips have been extensively used for disease modeling in an academic setting. They are now also being translated towards more pharmacological applications, such as drug screening, toxicity and pharmacokinetics testing (as detailed in an exhaustive review by Fang & Eglen [3]). You’d be right to think this is already a lot that 3D cell culture can do for drug discovery. If you think this is all, think again: somehow, 3D cell culture can also greatly advance the field of cell therapy.

6. Cell therapy

Cell therapy’s goal is to replace the tissues of patients that have damaged or failing organs. To reach this goal, a huge effort has been made in performing basic in vitro studies and developing protocols to grow cells for eventual implantation inside the patient, using a variety of 3D cell culture products.

    Scaffolds are loaded with cells and eventually implanted inside the patient to regenerate locally the tissue of interest.

   Three-dimensional scaffolds can be placed inside bioreactors to facilitate the study of cells in 3D, to optimize tissue growth and predict the mechanical response of the construct after their implantation. This is uniquely facilitated in bioreactors because they provide such a tight control of the culture environment and enable physical conditioning of the tissue to mimic the forces the cells would experience post transplantation [2]. Sensors are routinely integrated in the bioreactors, which helps to build computation models and obtain precise experimental data for predicting the cell response to their implantation in the body.

   Stem cells can be injected in patients for promoting tissue-specific regeneration. Interestingly, it has been shown that growing stem cells in 3D has clinical benefits as compared to their culture in 2D. For example, expanding mesenchymal stem cells in spheroids in 3D enhanced their anti-inflammatory response post-transplantation in mice [15]. Spheroids of stem cells have also been used to increase the yield of stem cell production for clinical trials.

   Organoids are developed in vitro from stem cells or organ progenitors which self-organize to resemble the basic micro-anatomy of organs [16]. Therefore, it is thought they could one day become a source for transplantation to replace some organs. As of today, organoids replicating kidneys or colons have been transplanted in mice, with successful long-term engraftment.

   Instead of using self-assembled organoids, one could 3D bio-print an organ for transplantation. While we are far from being able to print exact replicas of entire organs, there have been successful bio-printings of bladder, tracheal, bone and cartilage that have been implanted in animals and humans[17].

   Recently, a new procedure called cell-sheet engineering was also developed [18], which consists in layering confluent monolayers of cells along with the underlying ECM they secreted. This has been used to create tissues rich in ECM resembling the cornea, the kidney or liver.

   Finally, while microfluidic chips tend to be more relevant for creating 3D in vitro models, some microfluidic tools have been developed to encapsulate cells in a 3D ECM, which could be implanted into patients. A study used microfluidic devices to create hydrogel microfibers, which were then used to encapsulate and implant pancreatic islets in mice [19].

7. Commercial state of the art in 3D cell culture

Here we describe representative examples of commercial 3D cell culture methods.

Commercialized scaffold-free based products consist essentially in platforms that facilitate the formation and screening of spheroids. Spheroids are self-organized clusters of cells that recapitulate important aspects of the tumor, such as a necrotic and hypoxic core and reduced drug diffusion. The aggregation of cells into spheroids can be promoted either 1- by using non adherent surfaces (e.g. Happy Cell or Sumitomo Bakelite) 2- by suspending cells in a drop of fluid that hangs from an upside down well plate (e.g.: InSphero, 3D Biomatrix), 3- by agitating the cells, with spinner flasks or rotary systems, thereby preventing their adherence (e.g.: Wheaton, Synthecon).  Spheroids can also be placed into a 3D matrix like Matrigel (BD biosciences) [20].

 Commercialized scaffold-based products that facilitate 3D cell culture consist either in selling 1- matrices/hydrogels that are extracted directly from animal tissues (e.g. Geltrex by Invitrogen) or secreted by cultured cells (e.g. Matrigel, by BD biosciences), 2- well-defined scaffolds made of different pore sizes in polystyrene (i.e. Alvetex by Amsbio, or 3D biotek insert by Sigma), or 3- engineered carrier beads, where cells grow either as a monolayer on the outside (e.g. GEM from Global Cell Solutions, ProNectin F beads from Solohill, Cytodex from GE healthcare) or on the inside pores in a more three-dimensional configuration (Cytopore from GE Healthcare). Note that these beads increase the yield of cell culture because of the large culture surface area to volume ratio, and are therefore also used to increase the production yield of antibodies or viruses by cells.

Bioreactors have become a staple product of many laboratories dedicated to 3D cell culture and in particular regenerative medicine[2]. While often the bioreactors have been engineered and customized in the lab, several scaled up products exist commercially, such as the 3D Culture Pro from Bose Corporation, a portable bioreactor that contains up to 6 chambers which can accommodate a variety of samples, including cellular constructs. Another example is the XRS20 from Pall Corporation, a bioreactor with integrated sensors that can agitate the cells in a controlled fashion with a single-use 3D bio-container which reduces risk of contamination.

There are fewer off-the-shelf products for cell therapy purposes, as the process of implanting the resulting tissues inside patients’ adds a significant need for safety. To the best of our knowledge, only a few companies provide services to manufacture grafts. Autologous Clinical Tissue Engineering Systems (ACTES) by Octane Medical Group is trying to automate entire the process by creating a bioreactor system that would digest a patient’s cartilage biopsy, expand the chondrocytes and directly generate either a cell suspension or an osteochondral graft. Automation of such protocols require a huge up-front investment but could help the field with increasing the standards of safety and traceability [2].

Finally, microfluidic chips are slowly seeping into the 3D cell market. Sackmann et al., in 2015 have described the technology as still being composed principally of “proof-of-principle” studies published in engineering journals, that describe microfluidic prototypes without a clear biomedical need [21]. Slowly, the biological relevance of these platforms is growing stronger, and there are now many microfluidic platforms in the academic community that have been created to mimic and investigate a wide range of biological processes, ranging from cancer cell migration through hydrogels or the vasculature, to neuronal communication or organoid development. There is a particular effort in designing organs on chips [22] so as to interconnect organs, ranging from heart, brain, liver to lung-on-a-chip which is giving rise to several companies. Emulate Inc is pioneering the commercial development of such organs-on-a-chip. Otherwise, there are commercialized arrays of basic microfluidic units that allow the introduction of cells in a 3D extracellular matrix between two reservoirs that can be continuously perfused (Bell Brooks lab and Cell Asics). The microfluidic technology is now ripe for further development, and in particular for translation from the academic to the industrial stage.

8. Microfluidics: a macro-step towards increased physiological relevance

Microfluidics rely on the use of small channels that range from 10-100 µm in height or width for handling small volumes of fluids. Their key attribute over other more conventional 3D cellular models is that they enable a higher control of the cellular, physical and biochemical micro-environment. This enables researchers to recreate complex cellular models that mimic more faithfully human tissues [23].

   The design of microfluidic chips can consist of many different channels or compartments, that can be easily accommodated to the researcher’s needs. This compartmentalization allows a unique spatial control of cell distribution at physiological length scales, especially for co-cultures of different cell types. Many microchips have now integrated electronic and mechanical actuators such as valves or switches onto microchips that facilitate the realization of several sequential steps onto the same chip. This concept is what is often referred to as lab-on-a-chip: the idea that many of the time-consuming laboratory tasks can be miniaturized and automated onto a microfluidic chip. This miniaturization also diminishes the volumes of reagents and cells needed as compared to conventional macroscopic assays, which reduces costs.

  The presence of channels allows the control of flow at physiological length scales. This facilitates the formation of chemical or physical gradients across the channels (e.g. cytokine gradients, or interstitial pressure), which are important in many biological processes. The establishment of flow in the assay facilitates perfusion of media, recreates important biomechanical processes such as shear stress, or simply allows to recreate the fluidic environment of the cells in the blood flow.

   Last but not least, the short distance between the cells in the microfluidic chip and the microscope objective enables imaging of the entire specimen at higher resolution than other conventional macroscopic in vitro systems.

  There are a few important limitations associated with microchips: the main one is that cells are hard to retrieve, if subsequent tests need to be done. In addition, even if they were retrieved the volumes would potentially not be large enough for subsequent testing. Yet even these challenges are gradually being overcome along with others, to adapt to the needs of the 3D cell culture market.

9. The future of 3D cell culture under a microscope

To keep up with the growing demand of the drug discovery and cell therapy market, 3D cell culture is facing many challenges.

The first one is reproducibility: often, 3D cell culture results are too variable. The source of this variability is three-fold. The first one is biological. Cells and cell-derived products (such as Matrigel, or serum) are known widely vary amongst batches of production. The second source of variability is user-based: many of the procedures associated with 3D cell culture such as pipetting are manually done, and thus are prone to user-dependent variability. Lastly, many protocols can be affected by environmental factors that are often not locally controlled in the assay, such as temperature or humidity, which can very much affect crucial steps such as 3D gel polymerization.    At the same time, 3D cell culture procedures are far from being easy. They require specialized and highly trained staff, the lack of which is hampering the market growth. In addition, these tasks are time-consuming and expensive.

These problems all point to two major needs of the 3D cell culture field: automation and standardization. Ideally, new products are needed to facilitate these 3D cell culture procedures and reduce human intervention so as to minimize human errors, accelerate protocols and increase throughput and reproducibility. Already, the automation of tasks like cell passaging or cell expansion are under way [2].

Another major bottleneck in the field of 3D cell culture is the lack of easy readouts. Too many studies are heavily relying on 3D imaging to analyze cell morphology, migration or viability. This requires a lot of time and the use of expensive and specialized equipment such as confocal microscopy. While this will remain necessary seeing as these variables are very informative, there also needs to be an effort in developing easier readouts. In parallel, there also needs to be an effort in standardizing analysis methods for quantifying these results: reconstructing 3D images is far from being easy. While several 3D reconstruction software packages are available commercially, it is still hard to define, extract and quantify new variables from these complex datasets.

3D cell culture _ FutureDirections_Elvesys

   All in all, we foresee that these methodological challenges will need to be solved in order to facilitate the stream-lining of 3D cell culture in the clinical context. These technological advances are expected to help the field steer towards the following directions: vascularization, organoids/iPS cells, organ-on-a-chip, personalized medicine, and immunocompetent models.

  There is an acute interest in vascularizing cellular models [24]. First, this drastically increases their physiological relevance for drug discovery purposes. Indeed, many diseases and biological processes are heavily linked to the presence, disruption or formation of vasculature. Cancer cells for example heavily interact with the vasculature as they migrate from the primary tumor and form a secondary tumor in a distant organ [23]. Modelling a vasculature in vitro would also help recreate drug delivery through the blood circulation. Second, vascularized tissue will have a much higher chance of long-term engraftments in patients for cell therapy purpose.

  As previously discussed, another area of growing interest is the use of organoids. Current efforts consist in standardization of the protocols for their fabrication, but there is also a need to vascularize them, for the reasons previously described.

  Organ-on-a-chips are generating a lot of interest in the field of drug discovery[25], and have been pioneered by many academic groups in the last decades. Slowly but surely, prototypes of many different organs are emerging, and the technology is now moving towards chips that integrate several interconnected organs at once to mimic a more systemic and complete model. These models could become surrogates to test cytotoxicity both of drugs and of the biomaterials that make up cellular implants.

  All of these developments are emerging under the more general paradigm shift of personalized medicine. Personalized medicine tends towards developing therapies and diagnostic tools that are customized to the patient’s own cells. In other words, it is now thought that in the close future cells from each patient could be extracted and integrated in one of any of the 3D cell culture assays described in this review, so as to recreate the patient’s very own tissues in vitro.

  Finally, we expect immunocompetent models to become a key development in the 3D cell culture field. It is now becoming clear that immune cells play a pivotal role in many diseases, and can actually be directly targeted as a means to help patients. This is evidenced by the success of immunotherapy in cancer, a treatment that re-educates the immune cell to better recognize and fight cancer. It is therefore going to become essential to include immune cells in 3D vitro cellular models  [23], to better understand their role and screen for drugs that can target them. Interestingly, a study has shown that many drugs that were shown to work in mice had unforeseen side effects in humans because they were immunosuppressive [26]. Because they debilitated the immune system, opportunistic pathogens became the cause for infections in patients. Therefore, pre-clinical models should include immune cells to predict the risk for immune suppression in patients. The immune system is also the culprit of the inflammation that is often triggered after the implantation of biomaterials. Including immune cells to in vitro models would help to better predict the chance of biomaterials causing a harmful reaction.

Interestingly, the study that revealed that immune-suppressive drugs were often associated with toxic side effects in humans sheds the light on the immune cells but also on the pathogens [26]. In other words, while those drugs also suppressed the immune system of mice, the pathogens in mice are not the same as those in humans. This is especially true of patients that can have unique microbiomes depending on their conditions or treatments. Therefore, an ideal in vitro model for drug testing could also contain human pathogens to predict infections associated with drug treatments.

  For all of these 3D cell culture developments, the biggest challenge will consist in assay validation [27]. Indeed, one needs to verify the relevance and validity of in vitro data, and compare them to in vivo results. First, this implies that we need to identify relevant biomarkers for a wide range of diseases that we can compare between in vitro models and human clinical data. Second, there needs to be an effort in searching for human clinical data that can be compared to in vitro models.  While so many challenges will most likely be solved by a myriad of societal and technological improvements, we propose that microfluidic chips might be one promising avenue for addressing several of the issues we raised.

9. What can be micro-done?

  First microfluidics have already shown great potential for automating and accelerating cumbersome bench-top procedures. For example, some microfluidic chips have automated ELISAs protocols[28], and a lot of work is going into combining mass spectrometry onto microfluidic chips. This minimizes cost and time, but also helps standardize results by minimizing human intervention. However, moving towards the manipulation of cells in 3D might prove harder. While it is routine to manipulate fluids in microfluidic tools, it is more challenging to manipulate and mix solutions of different viscosities such as cell suspensions and 3D hydrogels, because it requires a set-up to overcome laminar flow.

    Groups have also found ways to combine microfluidic chips with easy readouts. For example, the Whitesides group has developed colorimetric paper-based microfluidics, such that the result of the assay is simply revealed by a color change [29]. Microfluidic chips also present an obvious advantage for facilitating personalized medicine, in the sense that they require small volumes of cells, which could therefore all come from the patient.

  In parallel, efforts have been made towards developing microfluidic chips with controlled environments, much like bioreactors. For example, microfluidic tools that re-create a hypoxic environment have been created to mimic hypoxia in cancerous tissues [30]. In another study, a microfluidic chip was connected to a pressure controller so as to exert cyclical pressure and strain on cells, as they would experience in the lung [31]. In other words, microfluidic chips could become micro-bioreactors of their own, thereby reaping many of the benefits we have discussed bioreactors offer for 3D cell culture. At the same time, microfluidics with integrated sensors are being developed [32] and will most likely become more ubiquitous in the near future. The presence of sensors, much like in bioreactors, enables the monitoring of important variables and facilitates automation and traceability.

  The automation, miniaturization and precision enabled by microfluidics will also help standardize sensitive procedures such as cell differentiation. Indeed, cells that need to be differentiated such as stem cells or monocytes, are often differentiated using conventional macroscopic in vitro protocols, that can be prone to large variabilities if chemical and environmental stimuli are not controlled for. Microfluidic chips in contrast can precisely control the delivery of nutrients in media, or oxygen concentration or fluidic properties, all of which have been shown to affect stem cell differentiation [33]. In parallel, the combination of these chips with sensors such as protein analytics enables the unraveling of the role of specific cues and signaling pathways in the differentiation process of cells [34].

  The miniaturization and automation facilitated by microfluidic offers the eventual possibility of decentralizing cumbersome procedures from scientific hubs to more remote areas. This has already been used with microfluidics used for testing liver function[35]. In contrast, microfluidic applications for 3D cell culture currently requires numerous steps that can only be performed in laboratories. However, it is not impossible that with the developments under way 3D cell culture microfluidics might also become point-of-care diagnostics used, if not in remote areas, directly in the clinic.

    Uniquely, microfluidic chips enable the perfusion of vascular structures. Briefly, when endothelial cells are mixed in the appropriate 3D gel (such as fibrin) they form vessels. While this can happen in any format, the vessels in the microfluidic chip form openings that directly connect to the microfluidic channel [36], [37]. This means that one has direct access to the intravascular compartment of the vessels through the microfluidic channel, such that vessels can be perfused with particles, cells or media. This is of utmost importance to recreate many biological processes that involve the transport of cells or molecules inside the vasculature, or simply to be able to perfuse drugs inside the vasculature, as they would be delivered in patients. On the other hand, organoids could also be grown in microfluidic chips for higher control of their development. That is, the microfluidic compartments would limit and thus govern the organoids’ shape and size to some extent. Ultimately, organoids could be mixed with vascular networks inside microfluidic chips to obtain perfusable organotypic models, and achieve ever more complex tissue models. Ultimately, combining stem cells/organoids in microfluidic chips is meant to enable the use of patient-derived cells to create personalized cellular models[27].

In parallel, microfluidic systems are uniquely amenable to the study of immune cells, as extensively reviewed elsewhere [23]. This is because they allow the establishment of cytokine gradients, which are important guiding cues for immune cells. The flow control that microfluidic models provide is essential to re-creating immune cell transport in the blood circulation. Along the same lines, the previously discussed ability to perfuse vessels on microfluidic chips is also uniquely helpful to recreating immune cell transport inside and across the vasculature, as they do in vivo. Finally, immune cells are highly mobile and heterogeneous cells, which must often be visualized as a means to investigate them. Microchips greatly facilitate their high resolution imaging for detailed analysis.

  Lastly, microchips have been the sole technology behind the soaring organs on chips effort [25]. As we have previously discussed, the increased control of the micro-environment provided by microchips has rendered this technology unique to developing organotypic models. In addition, the facilitation of flow enables the connection and interaction between said organs, and the transport of molecules from one organ to the other. These interactions have been shown to be key for drug testing. For example, gut and liver have been combined on chip because drugs are first absorbed through the gut and then metabolized by the liver [38]. Their combination has been shown to affect drug fate differently than chips where liver of guts are cultured separately [39]. Adding a stomach on a chip might also be important to mimic gastric emptying, all of which can affect drug pharmacokinetics [40]. Ultimately, one could imagine a body-on-a-chip product where basic organotypic tissues are interconnected by a perfusable vasculature so as to test the systemic human response to drugs.

  Recently, researchers have bio-printed in 3D for the first time a heart on a chip: this shows great promise for facilitating and standardizing organs on chip fabrication. In addition, soft strain sensors were integrated within the microarchitecture of the tissue, paving the way for instrumented organs on chips [41].

  All in all, we believe microfluidics have much to offer for the field of 3D cell culture, if there is a real effort to translate its potential from the academic setting to the clinical world. Most likely, solutions will borrow aspects not just from microfluidics but also from advances in hydrogel and bioreactor technology to eventually merge into a synergistic field. Yet all of this will require a combined and collaborative effort between engineers, biologists, clinicians, health regulators… off to work!

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