INTRODUCTION:

An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidiccell culturechip that simulates the activities, mechanics and physiological response of entire organs and organ systems, a type of artificial organ. Organ-on-a-chip devices aim to duplicate the function and microstructure of multicellular human organs in vitro on a microfluidics chip. Such devices include the biological functions of organs and the biochemical, bioelectrical, and biomechanical properties of cellular microenvironments and extracellular matrixes (ECMs). Recent studies have demonstrated the feasibility of organ-on-a-chip systems, which can closely mimic in vivo tissues and provide platforms for drug delivery test and biological cell characterization. It constitutes the subject matter of significant biomedical engineering research, more precisely in bio-MEMS.

The convergence of labs-on-chips (LOCs) and cell biology has permitted the study of human physiology in an organ-specific context, introducing a novel model of in vitro multicellular human organisms. One day, they will perhaps abolish the need for animals in drug development and toxin testing. Although multiple publications claim to have translated organ functions onto this interface, the movement towards this microfluidic application is still in its infancy. Organs-on-chips will vary in design and approach between different researchers. As such, validation and optimization of these systems will likely be a long process. Organs that have been simulated by microfluidic devices include the heart, the lung, kidney, artery, bone, cartilage, skin and more.^[1]

ORGANS:

Human-on-a-chip:

Researchers are working towards building a multi-channel 3D microfluidic cell culture system that compartmentalizes microenvironments in which 3D cellular aggregates are cultured to mimic multiple organs in the body. Most organ-on-a-chip models today only culture one cell type, so even though they may be valid models for studying whole organ functions, the systemic effect of a drug on the human body is not verified.^[2]

Conceptual schematic of a human-on-a-chip – Designing a whole body biomimetic device will potentially correct one of the most significant limitations on organs-on-chips: the isolation of organs. In particular, an integrated cell culture analog (µCCA) was developed and included lung cells, drug-metabolizing liver and fat cells. The cells were linked in a 2D fluidic network with culture medium circulating as a blood surrogate, thus efficiently providing a nutritional delivery transport system, while simultaneously removing wastes from the cells culturing. Four different cell types to mimic four human organs: liver, lung, kidney and fat. They focused on developing a standard serum-free culture media that would be valuable to all cell types included in the device. Optimized standard media are generally targeted to one specific cell-type, whereas a human-on-a-chip will evidently require a common medium (CM). In fact, they claim to have identified a cell culture CM that, when used to perfuse all cell cultures in the microfluidic device, maintains the cells functional levels.^[4]

Fig 1: Conceptual schematic of a human-on-a-chip

Lung-on-a-chip:

In human body, liver is a major metabolism organ, and its functions include regulation of glycogen storage, decomposition of red blood cells, plasma protein synthesis, hormone production, and detoxification. Ample vascular and bile network provides supplement of nutrient–oxygen and elimination of metabolites and waste products. The hepatocyte makes up to 70%–85% of the liver’s mass and is the main functional parenchymal cell of the liver. Lung-on-a-chips are being designed in an effort to improve the physiological relevance of existing in vitro alveolar-capillary interface models.^[5]Such a multifunctional micro device can reproduce key structural, functional and mechanical properties of the human alveolar-capillary interface (i.e., the fundamental functional unit of the living lung). Their fabrication of a system containing two closely apposed micro channels separated by a thin (10µm) porous flexible membrane made of PDMS. The device largely comprises three microfluidic channels, and only the middle one holds the porous membrane. Culture cells were grown on either side of the membrane: human alveolar epithelial cells on one side, and human pulmonary microvascular endothelial cells on the other. The compartmentalization of the channels facilitates not only the flow of air as a fluid which delivers cells and nutrients to the apical surface of the epithelium, but also allows for pressure differences to exist between the middle and side channels. During normal inspiration in a human’s respiratory cycle, intrapleural pressure decreases, triggering an expansion of the alveoli. As air is pulled into the lungs, alveolar epithelium and the coupled endothelium in the capillaries are stretched. Since a vacuum is connected to the side channels, a decrease in pressure will cause the middle channel to expand, thus stretching the porous membrane and subsequently, the entire alveolar-capillary interface. The pressure-driven dynamic motion behind the stretching of the membrane, also described as a cyclic mechanical strain (valued at approximately 10%), significantly increases the rate of nanoparticle translocation across the porous membrane, when compared to a static version of this device, and to a Transwell culture system. ^[6]

Fig. 2: Schematic of lung-on-a-chip

The device consists of three hollow micro channels, and only the middle channel contains a horizontal porous membrane, coated on either side by either an endothelium or an epithelium tissue. The side channels are connected to a vacuum and can therefore simulate the stretching of the membrane. The contraction of the diaphragm triggers the intrapleural pressure to decrease, leading to an expansion of alveoli. This is the phenomenon essentially mimicked by this lung-on-a-chip. In order to fully validate the biological accuracy of a device, its whole-organ responses must be evaluated. In this instance, researchers inflicted injuries to the cells: Pulmonary inflammatory responses entail a multistep strategy, but alongside an increased production of epithelial cells and an early response release of cytokines, the interface should undergo an increased number of leukocyte adhesion molecules. The pulmonary inflammation wassimulated by introducing medium containing a potent proinflammatory mediator. Only hours after the injury was caused, the cells in the microfluidic device subjected to a cyclic strain reacted in accordance with the previously mentioned biological response. Pulmonary infection.

Living E-coli bacteria was used to demonstrate how the system can even mimic the innate cellular response to a bacterial pulmonary infection. The bacteria were introduced onto the apical surface of the alveolar epithelium. Within hours, neutrophils were detected in the alveolar compartment, meaning they had transmigrated from the vascular microchannel where the porous membrane had phagocytized the bacteria.^[7]

Heart-on-a-chip:

Past efforts to the development of heart-on-a-chip platforms is one of the most challenging areas in organ-on-a-chip research. Microfluidics has already contributed to in vitro experiments on cardio myocytes, which generate the electrical impulses that control the heart rate. For instance, researchers have built an array of PDMS micro chambers, aligned with sensors and stimulating electrodes as a tool that will electrochemically and optically monitor the cardiomyocytes’ metabolism.^[8]

Fig. 3: Schematic of heart-on-a-chip

Preparation of the Heart-on-a-chip substrate and contractility test samples- After applying a stimulating the contraction of the myocytes via the field electrodes, strips/teeth in the MTF start to curl. Researchers have developed a correlation between tissue stress and the radius of curvature of the MTF strips during the contractile cycle, validating the demonstrated chip as a heart-on-a-chip (in the realm of their respective needs). This chip determines that the alignment of the myocytes in the contractile apparatus made of cardiac tissue and the gene expression profile (affected by shape and cell structure deformation) contributes to the force produced in cardiac contractility. This heart-on-a-chip is a biohybrid construct: an engineered anisotropic ventricular myocardium is an elastomeric thin film.^[9]The final steps involve the spin coating of protective surface of PDMS over the cover slip and curing. Muscular thin films (MTF) enable cardiac muscle monolayers to be engineered on a thin flexible substrate of PDMS. In order to properly seed the 2D cell culture, a microcontact printing technique was used to lay out a fibronectin "brick wall" pattern on the PDMS surface. Once the ventricular myocytes were seeded on the functionalized substrate, the fibronectin pattern oriented them to generate an anisotropic monolayer. After the cutting of the thin films into two rows with rectangular teeth, and subsequent placement of the whole device in a bath, electrodes stimulate the contraction of the myocytes via a field-stimulation thus curving the strips/teeth in the MTF."^[10]

Kidney-on-a-chip:

Renal cells and nephrons have already been simulated by microfluidic devices. "Such cell cultures can lead to new insights into cell and organ function and be used for drug screening".^[11] A kidney-on-a-chip device has the potential to accelerate research encompassing artificial replacement for lost kidney function. Nowadays, dialysis requires patients to go to a clinic up to three times per week. A more transportable and accessible form of treatment would not only increase the patient’s overall health (by increasing frequency of treatment), but the whole process would become more efficient and tolerable.^[12] Artificial kidney research is striving to bring transportability, wearability and perhaps implantation capability to the devices through innovative disciplines: microfluidics, miniaturization and nanotechnology.^[13]

Fig. 4: Schematic of kidneys-on-a-chip

Nephron-on-a-chip:

The nephron is the functional unit of the kidney and is composed of a glomerulus and a tubular component. Researchers at MIT claim to have designed a bioartificial device that replicates the function of the nephron’s glomerulus, proximal convoluted tubule and loop of Henle. Each part of the device has its unique design, generally consisting of two microfabricated layers separated by a membrane. The only inlet to the microfluidic device is designed for the entering blood sample. In the glomerulus’ section of the nephron, the membrane allows certain blood particles through its wall of capillary cells, composed by the endothelium, basement membrane and the epithelial podocytes. The fluid that is filtered from the capillary blood into Bowman’s space is called filtrate or primary urine.^[14]

Fig. 5: Schematic of a nephron-on-a-chip

Schematic of a nephron-on-a-chip device with cross-sections of 3 functional units- C- Connector; G- Glomerulus; T- Tubule; L – Henle's loop Black arrows: passive transport.

White arrows: cell-mediated active transport.

In the tubules, some substances are added to the filtrate as part of the urine formation, and some substances reabsorbed out of the filtrate and back into the blood. The first segment of these tubules is the proximal convoluted tubule. This is where the almost complete absorption of nutritionally important substances takes place. In the device, this section is merely a straight channel, but blood particles going to the filtrate have to cross the previously mentioned membrane and a layer of renal proximal tubule cells. The second segment of the tubules is the loop of Henle where the reabsorption of water and ions from the urine takes place. The device’s looping channels strives to simulate the countercurrent mechanism of the loop of Henle. Likewise, the loop of Henle requires a number of different cell types because each cell type has distinct transport properties and characteristics.^[15]

These include the descending limb cells, thin ascending limb cells, thick ascending limb cells, cortical collecting duct cells and medullary collecting duct cells. One step towards validating the microfluidic device’s simulation of the full filtration and reabsorption behavior of a physiological nephron would include demonstrating that the transport properties between blood and filtrate are identical with regards to where they occur and what is being let in by the membrane.^[16] One recent report illustrates a biomimic nephron on hydrogel microfluidic devices with establishing the function of passive diffusion. The complex physiological function of nephron is achieved on the basis of interactions between vessels and tubules (both are hollow channels). However, conventional laboratory techniques usually focus on 2D structures, such as petri-dish that lacks capability to recapitulate real physiology that occurs in 3D.^[17] Therefore, the authors developed a new method to fabricate functional, cell-lining and perfusable micro channel inside 3D hydrogel. The vessel endothelial and renal epithelial cells are cultured inside hydrogel microchannel and form cellular coverage to mimic vessels and tubules, respectively. They employed confocal microscope to examine the passive diffusion of one small organic molecule (usually drugs) between the vessels and tubules in hydrogel. The study demonstrates the beneficial potential to mimic renal physiology for regenerative medicine and drug screening.^[18]

Artery-on-a-chip:

Cardiovascular diseases are often caused by changes in structure and function of small blood vessels. A microfluidic platform simulating the biological response of an artery could not only enable organ-based screens to occur more frequently throughout a drug development trial, but also yield a comprehensive understanding of the underlying mechanisms behind pathologic changes in small arteries and develop better treatment strategies. Conventional methods used to examine intrinsic properties of isolated resistance vessels (arterioles and small arteries with diameters varying between 30µm and 300µm) include the pressure myography technique. An artery-on-a-chip could overcome several of these limitations by accommodating an artery onto a platform which would be scalable, inexpensive and possibly automated in its manufacturing. An organ-based microfluidic platform has been developed as a lab-on-a-chip onto which a fragile blood vessel can be fixed, allowing for determinants of resistance artery malfunctions to be studied.^[19] The artery microenvironment is characterized by surrounding temperature, transmural pressureand luminal &abluminal drug concentrations. The multiple inputs from a microenvironment cause a wide range of mechanical or chemical stimuli on the smooth muscle cells (SMCs) and endothelial cells (ECs) that line the vessel’s outer and luminal walls, respectively. Endothelial cells are responsible for releasing vasoconstriction and vasodilator factors, thus modifying tone. Vascular tone is defined as the degree of constriction inside a blood vessel relative to its maximum diameter. Pathogenic concepts currently believe that subtle changes to this microenvironment have pronounced effects on arterial tone and can severely alter peripheral vascular resistance. The engineers behind this design believe that a specific strength lies in its ability to control and simulate heterogeneous spatiotemporal influences found within the microenvironment, whereas myography protocols have, by virtue of their design, only established homogeneous microenvironments. They proved that by delivering phenylephrine through only one of the two channels providing superfusion to the outer walls, the drug-facing side constricted much more than the drug opposing side.^[20]

Fig. 6: Schematic of artery-on-a-chip

Artery-on-a-chip and detail of inspection area- The green microchannel is used for loading the artery segment, and perfusion (delivery of nutrients to the luminal walls); the fixation channels in yellow are used to adjust the positioning of the organ in the inspection zone by applying sub-atmospheric pressures at each end; in red is the super fusion channel, used to deliver nutrients to the abluminal wall of the artery.

The artery-on-a-chip is designed for reversible implantation of the sample. There is a microchannel used for loading the artery segment, and when the loading well is sealed, it is also used as a perfusion channel, to replicate the process of nutritive delivery of arterial blood to a capillary bed in the biological tissue.^[2] Another pair of micro channels serves to fix the two ends of the arterial segment. Finally, the last pair of micro channels is used to provide super fusion flow rates, in order to maintain the physiological and metabolic activity of the organ by delivering a constant sustaining medium over the abluminal wall. A thermoelectric heater and a thermo resistor are connected to the chip and maintain physiological temperatures at the artery inspection area. The protocol of loading and securing the tissue sample into the inspection zone helps understand how this approach acknowledges whole organ functions.^[3] After immersing the tissue segment into the loading well, the loading process is driven by a syringe withdrawing a constant flow rate of buffer solution at the far end of the loading channel. This causes the transport of the artery towards its dedicated position. This is done with closed fixation and super fusion in/outlet lines. After stopping the pump, sub-atmospheric pressure is applied through one of the fixation channels. Then after sealing the loading well shut, the second fixation channel is subjected to a sub-atmospheric pressure. Now the artery is symmetrically established in the inspection area, and a transmural pressure is felt by the segment. The remaining channels are opened and constant perfusion and super fusion are adjusted using separate syringe pumps.^[22]

Animal-on-a-Chip:

The ultimate extension of multi-compartment organ mimics is a system that combines multiple organ compartments together to construct an animal -or human-on a-chip. In an eﬀort to move toward models of the body, which account for such systemic interaction between diﬀerent organs that occur naturally in vivo, researchers have included multiple cell types in microﬂuidic chips. Animal-on-chip systems that are a ﬁrst step toward realistic artiﬁcial models of interactions between physiologically complex organ chambers can be used for fundamental and applied research.^[23]

CONCLUSION:

Organ-on-a-chip technology holds great promise and offers the prospect of a wide range of applications in a variety of industries. The most striking application is the pharmaceutical industry where there is a high demand for better predictive and translational models to study human physiological and pathophysiological processes related to toxicity, If this promising technological advance is to achieve its full potential, biologists and engineers must continue to collaborate on building models and on gaining a better understanding of what the results from these models have to say about the processes we can expect to find in humans.

ACKNOWLEDGEMENT:

Authors wish to express their sincere thanks to Hon. Smt. Padmatai Shinde madam, President, Shriram Dnyan Shikshan Prasarak Mandal, Vaijapur, Dist-Aurangabad, MS, India-423701 and Hon. Shri. Rajesh Shinde sir, President, Shriram Dnyan Shikshan Prasarak Mandal, Vaijapur, Dist-Aurangabad, MS, India-423701 for their constant encouragement and support.

REFERENCES:

1. C. Moraes, G. Mehta, S. C. Lesher-Perez and S. Takayama: Ann. Biomed. Eng. 40 (2012) 1211

2. C. Luni, E. Serena, N. Elvassore (2014), "Human-on-chip for therapy development and fundamental science", CurrOpin Biotech 25, 45-50

3. K. Viravaidya and M. L. Shuler (2004), Biotechnol. Prog.,20, 590-597

4. C. Zhang, Z. Zhao, N. Rahim, D. Noort, H. Yu (2009), "Towards a human-on-chip: Culturing multiple cell types on a chip with compartmentalized microenvironments"

5. Maton A, Jean H, Charles W, et al. Human Biology and Health. Englewood Cliffs, NJ, USA: Prentice Hall; 1993.

6. D. Huh, B. D. Matthews, A. Mammoto, M. Montoya-Zavala, H. Y. Hsin, D. E. Ingber (2010), "Reconstituting Organ-Level Lung Functions on a Chip

7. Franke, W. W., Borrmann, C. M., Grund, C. and Pieperhoff, S. (2006). The area composite of adhering junctions connecting heart muscle cells of vertebrates: Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins. Eur J Cell Biol 85, 69-82.

8. S. R. Shin, S. M. Jung, M. Zalabany, K. Kim, P. Zorlutuna, S. Kim, M. Nikkhah, M. Khabiry, M. Azize, J. Kong, K. Wan and T. Palacios: ACS Nano 7 (2013) 2369.

9. A. Werdich, E. A. Lima, B. Ivanov, I. Ges, M. E. Anderson, J. P. Wikswo and F. J. Baudenbacher, Lab on a Chip, 2004, 4, 357–362

10. Patrick W. Alford, Adam W. Feinberg, Sean P. Sheehy, Kevin K. Parker (2009). Biohybrid thin films for measuring contractility in engineered cardiovascular muscle

11. Kidney on a Chip, Highlights in Chemical Biology, RSC Publishing

12. Cruz D, Bellomo R, Kellum JA, De Cal M, Ronco C. The future of extracorporeal support. Crit Care Med 2008;36(4 Suppl):S243-52.

13. C. Ronco, A. Davenport, V. Gura (2011), The future of the artificial kidney: moving towards wearable and miniaturized devices

14. Maton, Anthea; Jean Hopkins, Charles William McLaughlin, Susan Johnson, MaryannaQuon Warner, David LaHart, Jill D. Wright (1993). Human Biology and Health. Englewood Cliffs, New Jersey, USA

15. Koeppen B, Stanton B. Renal physiology; 3rd ed. St Louis, MO: Mosby, 2001

16. E. Weinberg, M. Kaazempur-Mofrad, J. Borenstein (2008), "Concept and computational design for a bioartificial nephron-on-a-chip"

17. X. Mu, W. Zheng, L. Xiao, W. Zhang, X. Jiang, Engineering a 3D vascular network in hydrogel for mimicking a nephron. Lab Chip 13, 1612 (2013)

18. D. C. Eaton and J. P. Pooler, Vander’s Renal Physiology, McGraw-Hill, 2009 (2013)

19. Lippincott, Williams & Wilkins (2011), Cardiovascular Physiology Concepts 2nd Ed.

20. N. Marieb, K. Hoehn (2006), Human Anatomy & Physiology 7th Ed.

21. C. Zhang, Z. Zhao, N. Rahim, D. Noort, H. Yu (2009), "Towards a human-on-chip: Culturing multiple cell types on a chip with compartmentalized microenvironments"

22. Li, A. P. In vitro evaluation of human xenobiotic toxicity: scientiﬁc concepts and the novel integrated discrete multiple cell co-culture (IdMOC) technology. ALTEX 25(1):43–49, 2008.

Received on 08.09.2020 Modified on 05.10.2020

Asian J. Pharm. Tech. 2021; 11(1):66-71.

DOI: 10.5958/2231-5713.2021.00011.8