Microfluidic pressure control for organ-on-a-chip applications: A comprehensive guide

Overview of microfluidic pressure control for organ-on-a-chip applications 

Definition of microfluidics  

Microfluidics is the science that studies the behavior, manipulation, and control of small volumes of fluids (picolitres-microliters) at the micrometer scale. It involves channels and structures with dimensions ranging from micrometers to tens of micrometers (1). It offers advantages such as reduced sample and reagent consumption, rapid analysis and processing times, high sensitivity, and the ability to perform complex and multiplexed assays in a miniaturized format. It has emerged as a powerful technology for various applications, including organ-on-chip systems.  

Definition of Organ-on-chip  

Organ-on-chip (OOC) is a rapidly evolving field that aims to mimic the structure and function of human organs in a microscale device (2). OOC devices typically consist of microfluidic channels lined with cells that are organized to replicate the cell types found in a specific organ. By recreating the microenvironment and cellular interactions of an organ, OOC technology provides a more physiologically relevant model for studying organ function, disease mechanisms, and drug responses compared to traditional two-dimensional cell cultures or animal models.  

Figure 1: Example of an OOC: Brain organoid on chip combining biology, microfluidics, and recreation of microenvironment.   

Common microfluidic technologies used for OOC applications   

Mimicking the physiological microenvironment of living organs employing OOC devices is essentially achieved by implementing controlled liquid perfusion systems to simulate the flow of nutrients, drugs, and other substances through the microfluidic system. Several microfluidic flow control technologies are commonly used by researchers, each having their advantages and their disadvantages (Figure 2).   

Peristaltic Pumps  

Peristaltic pumps use positive displacement to squeeze a leak-proof flexible tube to create a pulsatile flow of liquid through the tube. It is an accessible and easy-to-set-up system used in biological fields, as it is compatible with various types of fluids and presents a low risk of contamination since the liquid only contacts the tubing. However, its flow rate control and pressure capabilities are relatively low, and its pulsatile aspect may not be suitable for certain applications.   

Syringe Pumps  

A syringe pump is a motor-driven precision pump that uses one or more syringes to deliver precise and accurate amounts of fluid. They are generally user-friendly, with intuitive control interfaces for programming and adjusting parameters such as flow rate, volume, and infusion rates. They can provide both continuous and intermittent flow, depending on the programming settings. This feature is useful for experiments or procedures that require specific flow patterns. Despite their advantages, syringe pumps have a few limitations such as low flow rate control and responsiveness, limited volume of the syringe which will require periodic refilling steps, and an increased risk of contamination as the fluid is in direct contact with the syringe.   

Pressure generators  

Pressure generators utilize external pneumatic or hydraulic systems to generate pressure and drive the liquid flow through the microfluidic system. Among multiple advantages, high flow rate stability and fast responsiveness are the most pertinent features needed for microfluidic pressure control for OOC applications. They can also provide continuous and pulsatile flow options, depending on the application, and can be easily integrated into multi-organ systems. This technology is more complex than the latter ones, as it requires additional equipment and a sophisticated setup.    

It’s important to note that the choice of liquid perfusion technology depends on the specific requirements of the organ-on-chip application, such as the desired flow rate, pressure, pulsatility, and compatibility with the cells or tissues being studied. Researchers often select the most appropriate technology based on the advantages and disadvantages mentioned above and tailor it to their experimental needs. 

Figure 2: Advantages and disadvantages of different microfluidic technologies used for OOC.  

Why use microfluidic pressure control for Organ-on-Chip applications?  

Microfluidic pressure control plays a crucial role in the success of organ-on-chip applications. It enables researchers to precisely control and manipulate the flow rates, shear stresses, and mechanical forces experienced by cells and tissues within the microfluidic device. By mimicking the in-vivo physiological conditions, microfluidic pressure control enhances the accuracy and relevance of organ-on-a-chip experiments. Pressure control is closely related to flow control, so by modulating the pressure, flow rates can be accurately adjusted, enabling precise control over fluid dynamics and directing the flow to specific regions of the chip. Accurate flow control allows for recreating complex fluidic environments and mimicking microenvironmental cues (such as shear stress and pressure gradients), affecting the behavior and function of cells. Moreover, precise pressure control is essential for controlled drug delivery and perfusion within the organ-on-a-chip device. It allows for controlled exposure of cells and tissues to drugs, toxins, or other substances, enabling the assessment of their effects on organ function and response. 

Understanding microfluidic pressure control 

Components and Mechanisms

Within microfluidics systems, various components play crucial roles in ensuring precise fluid control and pressure management. 

• Pressure sources: employed to generate controlled fluid flow and pressure within microfluidic systems. Common types of pumps used in microfluidics include syringe pumps, peristaltic pumps, and pressure-driven pumps. They enable the precise delivery of fluids and the generation of desired flow rates and pressures. 

• Pressure regulators: they ensure that the pressure remains constant or follows a predefined profile by using feedback from pressure sensors to adjust the flow rate and maintain the desired pressure level.  
• Pressure Sensors: used to measure the pressure within microfluidic channels. These sensors can be integrated into the chip or connected externally to monitor and provide feedback on the pressure conditions in real-time. They convert the pressure exerted by the fluid into electrical signals, which are then used by the pressure regulators to make necessary adjustments. Common types of pressure sensors used in microfluidics include piezoresistive and capacitive sensors.  
• Valves: crucial components of a microfluidic system that regulate fluid flow within microfluidic channels. They can be mechanical, pneumatic, or electrostatic, and they control the opening and closing of specific channels to direct the flow of fluids as required. Valves allow researchers to create complex fluidic networks and dynamic flow patterns within OOC devices. 
• Feedback Control Systems: use information from pressure sensors or other sensors in the systems to adjust the pressure or flow rates in real-time. These systems provide a closed-loop control mechanism, enabling the device to maintain desired pressure conditions. 

By integrating  these components, researchers can effectively use microfluidic pressure control (Figure 3) via Fluigent systems to create accurate fluidic environments necessary for organ-on-chip applications. 

Figure 3: Example of Fluigent’s recirculation system using microfluidic pressure controllers. Two Flow EZ are connected to two reservoirs. Tubing passes through the L-SWITCH (allowing media recirculation), a flow unit, and the microfluidic device. The software used to control the system is OxyGEN.   

Applications in Organ-on-Chip   

In recent years, researchers have made significant progress in developing organ-on-a-chip models for various organs. Examples include kidney on a chip (4), lung on a chip (5), heart on a chip (6), skin on a chip (7), pancreas on a chip (8), brain on a chip, gut on chip, and blood-brain barrier on a chip (9). The use of microfluidic technologies has been crucial in overcoming several limitations associated with these models, such as: 

• Replicating the organ’s exposure to fluids in a controlled and directional manner 

• Facilitating the flow of nutrients into the chip and the removal of waste products 
• Establishing gradient flows and allowing for more precise dosing studies 
• Enabling the layering of multiple cell types to mimic the complex structure of organs 
• Using human-derived cells in organ-on-a-chip studies thus providing a more physiologically relevant model compared to animal models 

In summary, organ-on-a-chip microfluidic devices have revolutionized research in several areas, including high throughput drug screening, single-cell analysis, cell-cell interactions, and the study of various biological processes and diseases. These advancements have opened new possibilities for understanding human physiology and developing more effective therapies. 

Successful Organ-on-chip examples using Fluigent’s systems  

Fluigent’s microfluidic pressure control systems have been  used by many researchers in their investigations on novel Organ-on-a-chip models. 

• Kidney-on-chip 

MJ Hoogduijn’s team (10) performed the first vascularization of kidney organoids in HUVEC co-culture conditions using a microfuidic organ-on chip system. They first cultured kidney organoids (Figure 4) that presented nephron structures on chip and demonstrated an increased maturation of endothelial populations based on a colocalization analysis of endothelial markers (MCAM & PECAM) compared to transwell organoid cultures (Figure 5). They also performed co-cultures of these organoids along with human umbilical vein endothelial cells (HUVECs). These cells were able to migrate and proliferate inside the organoid tissue, and to interconnect with endogenous endothelial cells, which led to the formation of structures presenting an open lumen resembling vessels (Figure 6). They used Fluigent’s microfluidic pressure controller, FlowEZ, at a constant rate of 3 µL/min for 48 h after static culture.  

Figure 4: Kidney organoid-on-a-chip 

Figure 5: Kidney organoids cultured on a chip show expanded EC populations and directed endothelial maturation patterns 

Figure 6: Vascular structures derived from the GFP+ HUVECs integrated in the organoid tissue. 

• Cancer-on-chip  

Van Gent’s team developed a Cancer-on-a-Chip microfluidic platform (Figure 9) for the assessment of patient treatment responses using tumor tissue slices under controlled growth conditions (12). Their microfluidic pressure control system consists of Fluigent’s MFCS-EZ (Figure 10). They were able to maintain cell viability and sustain proliferation of breast and prostate cancer PDX tumor slices for  up to 14 days. They also studied the tumor slices response to cisplatin and antidrogen chemotherapeutic treatment. Their platform could be used for future studies including biopsies of patient tumors that will be treated with the same chemotherapy.   

Figure 9: (A) Cross-section of the Cancer-on-a-Chip system illustrating the diffusion and perfusion toward the tissue slice. 

Figure 10: (A’) The Cancer-on-a-Chip platform connected to a Fluigent Microfluidic flow control system  further connected to flowrate sensors (Fluigent FLOW UNIT-S) using Fluigent’s software throughout the culture period. 

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References 

1. Whitesides, G.M. The origins and the future of microfluidics. Nature 2006, 442, 368–373. 

2. Leung, C.M., de Haan, P., Ronaldson-Bouchard, K. et al. A guide to the organ-on-a-chip. Nat Rev Methods Primers 2, 33 (2022). https://doi.org/10.1038/s43586-022-00118-6. 

3. Leung, C.M., de Haan, P., Ronaldson Bouchard, K. et al. A guide to the organ-on-a-chip. Nat Rev Methods Primers 2, 33 (2022). https://doi.org/10.1038/s43586-022-00118-6. 

4. S.K. Jeonghwan Lee, Kidney-on-a-Chip: a new technology for predicting drug efficacy, interactions, and drug-induced nephrotoxicity, Curr. Drug Metab. 19 (2018) 577–583. 

5. P. Zamprogno, S. Wüthrich, S. Achenbach, Second-generation lung-on-a-chip with an array of stretchable alveoli made with a biological membrane, Commun. Biol. 4 (2021) 168.  

6. H. Liu, O.A. Bolonduro, J.J. Ning Hu, A.A. Rao, B.M. Duffy, Z. Huang, L.D. Black, B.P. Timko, Heart-on-a-Chip model with integrated extra- and intracellular bioelectronics for monitoring cardiac electrophysiology under acute hypoxia, Nano Lett. 20 (2020) 2585–2593.  

7. B. Luk´ acs, A. ´ Bajza, D. Kocsis, A. Csorba, I. Antal, K. Ivan, ´ A.J. Laki, F. Erdo, Skinon-a-Chip device for ex vivo monitoring of transdermal delivery of drugs—design, fabrication, and testing, Pharmaceutics 19 (2019) 445.  

8. K. Shik Mun, K. Arora, Y. Huang, Patient-derived pancreas-on-a-chip to model cystic fibrosis-related disorders, Nat. Commun. 10 (2019) 3124. 

9. L.I. Ilaria Raimondi, Marta Tunesi, Manola Comar, Diego Albani, Carmen Giordano, Organ-On-A-Chip in vitro models of the brain and the bloodbrain barrier and their value to study the microbiota-gut-brain axis in neurodegeneration, Front. Bioeng. Biotechnol. (2020).  

10. Creating a kidney organoid‑vasculature interaction model using a novel organ‑on‑chip system Amanda Bas‑Cristóbal Menéndez 1,2*, Z. Du1 , T. P. P. van den Bosch3 , A. Othman4 , N.Gaio4 , C. Silvestri4 , W. Quirós4 , H. Lin5 , S. Korevaar1 , A. Merino1 , J. Mulder2,6 & M. J. Hoogduijn1.  

11. Multiplexed fluidic circuit board for controlled perfusion of 3D blood vessels-on-a-chip† Mees N. S. de Graaf, a Aisen Vivas, bc Dhanesh G. Kasi,ade Francijna E. van den Hil,a Albert van den Berg, c Andries D. van der Meer, b Christine L. Mummery ab and Valeria V. Orlova *a. 

12. (A Microfluidic Cancer-on-Chip Platform Predicts Drug Response Using Organotypic Tumor Slice Culture Sanjiban Chakrabarty1,2, William F. Quiros-Solano3,4, Maayke M.P. Kuijten1,5, Ben Haspels1 , Sandeep Mallya6 , Calvin Shun Yu Lo1 , Amr Othman4 , Cinzia Silvestri4 , Anja van de Stolpe7 , Nikolas Gaio4 , Hanny Odijk1 , Marieke van de Ven8 , Corrina M.A. de Ridder9 , Wytske M. van Weerden9 , Jos Jonkers8 , Ronald Dekker3,7, Nitika Taneja1 , Roland Kanaar1,5, and Dik C. van Gent1. 

13. Emulating the chondrocyte microenvironment using multi-directional mechanical stimulation in a cartilage-on-chip† Carlo Alberto Paggi,ab Jan Hendriks,a Marcel Karperien *a and Séverine Le Gac *b