Microfluidic organoids-on-a-chip: The future of human models

Abstract

Microfluidics opened the possibility to model the physiological environment by controlling fluids flows, and therefore nutrients supply. It allows to integrate external stimuli such as electricals or mechanicals and in situ monitoring important parameters such as pH, oxygen and metabolite concentrations. Organoids are self-organized 3D organ-like clusters, which allow to closely model original organ functionalities. Applying microfluidics to organoids allows to generate powerful human models for studying organ development, diseases, and drug testing. In this review, after a brief introduction on microfluidics, organoids and organoids-on-a-chip are described by organs (brain, heart, gastrointestinal tract, liver, pancreas) highlighting the microfluidic approaches since this point of view was overlooked in previously published reviews. Indeed, the review aims to discuss from a different point of view, primary microfluidics, the available literature on organoids-on-a-chip, standing out from the published literature by focusing on each specific organ.

Introduction

Microfluidics is a technology that allows to manipulate fluids by performing a high control on their flow. In microfluidics, at least one dimension of the components has an internal diameter inferior to 1 mm. Microfluidics is a powerful technology applied to different fields from fluid dynamics, synthetic and analytical chemistry to biology and medicine where it is exploited to assess drugs toxicity, develop and study drug delivery systems, regenerative medicine and single cell analyses. Microfluidics technology is such a powerful tool since not only it permits the high control on fluid flows but also allows to explore and exploit fluid features that are not present in the macroscopic systems. Indeed, decreasing the dimensions of the system, the forces acting on the fluid change shifting, for example, from gravity to surface tension. Furthermore, inertial, and viscous force actions on fluids, change in the micrometric scale with an increase of Reynolds number which is indeed given by the ratio among these forces:

Basing on Reynolds number, fluid flow is defined laminar when Re is inferior to 2000 or turbulent for Re higher than 4000. It can be understood from the Reynolds number formula that if the length scale i.e. the diameter of a circular cross-section microchannel decreases, Re decreases too. Indeed, in the microfluidic system, where the dimension is from tens to hundreds of micrometers, the Re is almost always in the laminar flow regime (usually in the range of 10⁻² −10⁻³) [1]. The laminar flow could be schematized as layers moving adjacent without mixing instead of chaotically mixing as it happens for turbulent flow. The characteristics of the laminar flow guarantee on one hand a high spatial control of the solute which can, on the other hand, moves in the flow only through a diffusion to the fluids nearby impeding a rapid and efficient mass transfer. To assess this issue, when required, recent microfluidic devices are equipped with mixing elements (later described).

Typically, microfluidics employs chips i.e. blocks of different materials, glass or polymers, where channels with micrometric dimensions are etched or molded. The chip design depends on the application. The term chip used for these microfluidic objects is because at first, they have been fabricated with the production techniques used for microchips such as photolithography and micro-matching. Photolithography is still widely used for producing microfluidic chips and it is based on photoactive materials for which light exposition changes their solubility by placing a mask (shaped basing on the desired pattern of the chip) between the light and the chip which is lit. Later, a photoreaction is generated in the exposed area of the chip allowing to obtain the channels molded into it. Thus, photolithography allows to obtain a mold chip that after could be obtained exploiting soft lithography, the process is depicted in Fig. 1 and it is widely used to obtain polydimethylsiloxane (PDMS) chips. Thermoplastic polymers are used to produce microfluidic chips by thermoforming, hot embossing or injection molding.

Furthermore, laser ablation could be also used to obtain chips. Among the several different materials that could be utilized for chip production, PDMS is the preferred one for most applications in cellular biology, thanks to its chemical and mechanical properties, biocompatibility, optical transparency, gas permeability and low cost [2]. In order to flow fluids into chip channels two different approaches have been developed, the active and passive flows. The active flow is generated by mechanical pumps, which could be syringe, peristaltic or pressure driven pumps. The latest ones are the most precise, stable, and versatile consequently are widely used for biomedical applications. The passive flow is indeed generated avoiding external actuators and exploiting forces acting on the flow such as gravity, surface tension, osmosis and hydrostatic pressure. These systems allow to create a space saving practical microfluidic devices but are less versatile and precise than active flow mechanisms.

For applications that require to increase fluid flow mixing, different strategies have been developed. Mixing could be achieved with passive methods, building up designated microstructures inside the channels or exploiting different channel geometries as happens in zigzag mixer, vortex mixer and staggered herringbone mixer. Moreover, active methods are available like magneto‐hydrodynamic mixers and acoustic mixers [3].

Since microfluidic systems can be designed basing on the requirements and being highly controlled, the technology can be particularly helpful in biomimetics, simulating the in vivo environment. Indeed, through microfluidics it is possible to simulate biomechanical forces such as electrical, shear stress and stretch [4] that are usefully used as stimuli for cell maturation and development [5]. Beside physical stimuli, microfluidic flows can be exploited to create perfusion-based culture systems where nutrients and factors in the media can be delivered in the chip controlling their concentration and mixing, supplying continuously fresh media to cells. Furthermore, it is possible to create gradients of solutes in the flow, which can be valuable to study and induce different cell behaviors such as proliferation, migration, differentiation, inflammation, and tumorigenesis. Together with media, the continuous perfusion provides to the cells also important gasses such as oxygen. Into microfluidic devices, it is possible to online monitoring different parameters important for cell culturing by the means of sensors which are fast, have small dimensions and therefore can be placed directly into the microfluidic systems.

The microfluidic simulation of in vivo conditions has brought to organ-on-a-chip: micro-scale systems replicating the human body for both drug testing and disease modeling with the ethical aim of progressively replace animal models. Organs-on-a-chip are made possible by the merging of the advanced knowledges in cell cocultures, stem cells, genome editing, sensors, 3D printing and microfluidics. Organs-on-a-chip simulate the function of tissues and organs; they are built integrating different tissues arranged into 3D systems on which bio and physical forces are applied in the context of simulate in vivo conditions. Moreover, different organs-on-a-chip can be combined creating a body-on-a-chip; valuable systems that permit to assess organ interactions, which determine the human body complexity. The body-on-a-chip is also a powerful strategy for the evaluation of absorption, distribution, metabolization, and elimination (ADME) of drugs under testing [6]. Others peculiar kind of organs-on-a-chip are barriers-on-a-chip, which replicate human body barriers to study their functional characteristics and the transport through them. In literature are reported numerous examples of barriers-on-a-chip among which the blood-brain-barrier [2], [7], [8], [9], [10], placental barrier [11], [12], [13], corneal barrier [14], [15], [16], blood-retinal barrier [17], glomerular filtration barrier [18] and skin barrier [19] on a chip; and the gut-brain axis on-a-chip from the interconnection of the gut epithelial barrier and the blood-brain barrier [20]. The union between biomimetic microfluidics and organoids has indeed brought to organoids-on-a-chip. Organoids are 3D cellular clusters derived from stem cells that self-organize creating self-renewing near physiological structures [3]. Organoids-on-a-chip differ from organs-on-a-chip since the last ones are created by an accurate system design that foreseen the choice of cell types and their seeding and growth, particular geometry of cell chamber on the chip and/or particular scaffolds while in the former, cells self-assemble stochastically and spontaneously. Organoids-on-a-chip, being based on microfluidic, are using the same technological tools of organs-on-a-chip and exploiting their same potentials. Organoids and organs-on-a-chip have some common requirements that can be satisfied by microfluidics such as an improved oxygenation of cells and similarly some capability of microfluidics can be useful for both of them such as the formation of gradients of solutes, which can be exploited for different purposes: from controlled differentiation of stem cells forming organoids, to intake study of a particular molecule from a specific organ simulated by an organ-on-a-chip.

Organoids have been chosen to simulate different organs since they can be cultivated for a long period of time maintaining their genetical and phenotypical features, starting from different possible sources of cells such as primary tissues, cancer cells, induced Pluripotent Stem (iPS) cells, and Embryonic Stem (ES) cells. The improvement of organoid technology into organoids-on-a-chip has been widely used by modeling different organs both healthy and pathological for drug testing; recent examples reported in literature are hereafter described sorted by organ. The scientific works reported in literature are described with a particular focus on the microfluidic devices since the microfluidic point of view is less detailed in others excellent reviews already published [21], [22], [23], [24].