INTRODUCTION:

An ultimate quest of most biomedical research is to determine the basic communication between cells and molecules, which assemble to form human tissues and organs. The expectation is that with this knowledge in both health and disease, scientists can identify most potent targets, against which drugs can be discovered and tested. However, such knowledge cannot be attained directly from humans, and therefore models are used. Our understanding of the complex molecular singling in disease modalities, and subsequent development of therapeutic targets, has been heavily influenced by animal models. In the last4 decades, animal models have been extensively used, to develop our understanding several lethal diseases, such as, cancer progression and metastasis vascular disorders, diabetes mellitus, and cardiac complications.

Figure 1 Overview of the in vitro and in vivo models to study cellular, tissue-level, or organ-level functioning. As the complexity of the disease pathophysiology increases, the requirements for a modelling modality that can more faithfully recapitulate the exact mechanistic events increase.

Organ-on-a-chip:

Organ-on-a-Chip is a promising interdisciplinary technique emulating in vivo physiology and pathology for in vitro disease model, drug screening, and precision medicine. The future development of personalized Organ-on-a-Chip and continuous integration of novel engineering tools (e.g., automation handling, 3D printing. and in situ multi sensors).

3D structures:

The recent advancement in microfabrication and soft lithographic processes, researchers have engineered 3D in vitro systems that cannot only mimic the cellular/tissue level micro physiological environment but can also allow a systematic analysis of the key factors responsible for disease progression.

Why is organ-on-a-chip a hot topic?

We need real human organ–like devices that are superior to animal models. Organ-on-a-chip may be a good solution, which has minimal functional units that use primary human cells, rather than animal cells, like a real human organ. The ideal methodswillnotonlyusehumancellsbutalsomimic3D architecture and flow conditions with in real human organs. Microfluidic devices seeded with human cell sand perfused with cell culture media in a physiologically relevant manner have already been developed to provide a minimal functional unit to mimic real organs. The small size allows easy flow control and requires few cells and only small volumes of samples and reagents. Parallel experiments with large numbers of samples at the same time can also be realized. An additional advantage of the devices is optical transparency that allows visualization, at the cellular level, of the whole drug response process, something that is difficult to do in actual living organs.

Figure 2 Concept of organ chip

MATERIALS AND METHODS:

In vitro preclinical models: organ-on-a chip:

An organ-on-a-chip (OOC) is a multichannel, 3D microfluidic cell culture chip, that simulates the activities, mechanics, and physiological response of entire organs, or mechanics, or part of organ systems, representing a type of artificial organ.

The first pioneering design of organ-on-a-chip of the lung was by Huh et al. Their bio inspired microdevice reproduced complex integrated organ-level responses to bacteria an inflammatory cytokines, introduced into the alveolar space, by mimicking the cyclic mechanical strain caused by physiological breathing, using multichannel microfluidic separated by thin PDMS membranes. After this first demonstration, they further advanced the platform to model the alveolocapillary interface.

This system contained two microfluidic channels separated by a thin porous PDMS membrane, and revealed that cyclic mechanical strain expresses the inflammatory responses of the lung to nanoparticles more prominently. Later, the same platform was used to model small airways and viral infections. Other groups have shown that the functionality of the alveolar barrier could be restored by coculturing epithelial and endothelial cells. Moreover, they could successfully enhance the cell culture efficacy, in this dynamical environment Incorporating smooth muscle cells in the lung-on-a-chip, to mimic their interactions with epithelial cells was achieved by Youngs group, by culturing SMCs in collagen and Matrigel hydrogels that provide the necessary environment for cellular growth. Jain et al. have successfully model inflammation induced in their microfluidic alveolus thrombosis, in their microfluidic alveolus-on-a-chip, by coculturing human primary alveolar epithelium and endothelium, in blood flow conditions. Scientists could use the alveolus-on-a-chip to assess the therapeutic potentiality of a molecule, since it creates opportunities for future drug assays at lower cost, and extremely high speed. The door is also open for model of other pulmonary diseases by the way of the microchip technique.

Figure 3 In vivo and In vitro models study developing organ-on-a-chip

Organ-on-chip for human risk assessment:

Conventional systems for studying the toxicological responses of a toxicant generally focusing on a single cell type. However, in in vivo conditions, many cell types are involved in the toxicological responses. Due to the increasing accumulation of xenobiotics and toxicants in the human population and the environment, a study that determines their adverse effects needs to be carried out carefully to avoid potential harmful effects. The inherent advantages of OOC, including recapitulation of in vivo like conditions and dynamic responses, enables physiology-relevant toxicity assessment and allows the study of the fate of the toxicants and their metabolites on a specific target organ or multiple organs by interconnecting various single OOC platforms. For example, Theobald et al. demonstrated a liver-kidney-on-a-chip for studying the primary and secondary toxicity induced by toxins such as Aflatoxin B1 and Bezoalphapyrene. Compartmentalization in OOC platforms enable cellular crosstalk improve the efficiency of toxicity studies. The microfluidic systems also provide toxicity assessment based on flow-dependent manner also provide toxicity assessment based on flow-dependent manner Pulmonary exposure to pollutants such as aerosols can be modelled using lung-on-a-chip platforms. The lung-liver-on-a-chip modelled by D Bovard and colleagues manifested that the presence of HepaRGTM spheroids modulates the toxicity associated with the aflatoxin B1. Effective metabolism and detoxification of aflatoxin B1 due to the presence of the liver compartment in the liver-lung-on-a-chip protect the cells in the lung compartment from aflatoxin B1 mediated toxicity. Due to the ethical dilemma and lack of a suitable test group, assessing pathophysiology associated with environmental pollutants in humans is difficult. However, the biomimetic OOC platform can fill the gaps in the toxicological evaluation of environmental pollutants in the human body. The microfluidic pulmonary alveolus system developed by Zhang et al. revealed the lung inflammation and injury induced by environmental pollutants. The OOC, therefore, holds a great promise in the assessment of human risk associated with pollutants, environmental monitoring and drug testing.

Different types of organ-on-chip models:

1) Heart on-a chip

2) Kidney on a chip

3) Gut on a chip

4) Liver on a chip

5) Lung on a chip

6) Brain on a chip

1. Gut on a chip:

In order to effectively design gut-on-a-chip devices, one must understand the key structures and functions of the organ. The primary functions of the gut are to absorb and transport nutrients, electrolytes and drugs from the digestive system to the vasculature for distribution throughout the rest of the body The secondary involvement in both the immune and endocrine system arise from the presence of specialized human cells and the microbiome. The gut is characterized by its enormous surface area, achieved through the presence of folded micro structures known as the intestinal villi and microvilli. These finger-like protrusions of epithelial and specialized cells facilitate the multiple functions of the gut. In addition, the intestines have important microorganisms that aid in digestion, immune regulation, and protection from foreign pathogens]. These mutualistic microorganisms are able to survive due to the unique hypoxic environment found in the intestines]. In addition to the physical and chemical environment in the intestines, mechanical stress is applied by the characteristic peristaltic motion which stretches and squeezes the tissue to propel the contents of the bowel along the gastrointestinal tract (GIT).

Figure 4: Gut on a chip model

Applications:

Thus far, gut-on-a-chip development has been motivated by the need to understand the basic functions of the gut and how they are influenced by environmental conditions, drugs, and other cells. Absorption and barrier function have been extensively studied with these microfluidic in vitro models. One study by Pocock et al. characterized absorption of products of the chemotherapeutic agent SN38. When comparing the cells cultured under microfluidic conditions compared to the static Trans well model, the device demonstrated superior biological relevance as the cells self-assembled microvilli and produced significantly more Fractin. Investigating the permeability of the intestine was another early research objective of gut-on-a chip studies. The most common methods of testing barrier function of tissue within a device include tracking diffusion of marker molecules and TEER [18].

Kidney-on-a-chip:

The initial design of a published kidney-on-a-chip has two compartments. A top channel mimics the urinary lumen and has fluid flow, where as the bottom chamber mimic interstitial space and is filled with media.Kidney cells are under much lower shear stress than the endothelial or lung cells. This device used rat distal tubular cells or MDCK cells, and its shear stress was_1 dyne/cm2 A second report showed the same design, but human proximal tubular cells were used. In this model, the authors tried to reproduce cisplatin nephron toxicity.Proximal tubular cells have much lower shear stress 0.2 dyne /cm2 The foot processes of podocyte, a glomerular visceral epithelial cell, form a size-and charge-selective barrier to plasma protein, and derangement of the barrier causes podocyte injury and proteinuria. Some scientists have tried podocyte-on-a-chip, but It is still challenging. It may be because podocyte is exposed under a very slow shear stress in vivo and requires a sophisticated culturing condition.

Kidney-on-a-chip has various potential applications:

Kidney-on-a-chip has various potential applications. Cisplatin nephron toxicity was evaluated in the kidney-on-a-chip using human proximal tubular cells. Cisplatin was introduced into the bottom space, and cisplatin-induced cellular damage was monitored to the cells for 24 hours. Duringthefollowing72 hours, shear stress was helpful for facilitating recovery of the injured cells and associated biomarkers. Shear stress in the devices can facilitate translocation of aquaporin-2and relocation of actin Cito skeleton in the kidney-on-a-chip using primary cultured inner medullary collecting duct cells of rat kidneys which is a good example of a physiological experiment using kidney-on-a-chip. Moreover, renal tubular epithelial cells are continuously exposed to the changes of extracellular micro- environment, e.g.trans epithelial osmotic gradient, and changes of luminal or interstitial pH. The effect of these physiological factors on the functions of renal tubular cells could also be investigated by exploiting micro fluidics.

Heart-on-a-chip:

Cardiovascular disease is one of the leading causes of morbidity in the world. Furthermore, drug-induced cardiotoxicity is one of the main reasons for post-marketing drug withdrawal. Screening the drugs for their cardiotoxicity, optimizing drug efficacy during the drugs frothier cardiotoxicity, optimizing drug efficacy during the drug screening stage can ensure their safety. Traditionally animal models and static cell culture models are employed for the drug models and static cell culture models are employed for the drug models and static cell culture models are employed for the drug development process. However, these models are inapt for recapitulating human physiology relevant settings. The advent of OOC technology has imparted a great interest in designing and developing various heart-on-a-chip microfluidic devices. These heart-on-a-chip devices can mimic the heart's functional characteristics, thus an optimistic approach to developing drug screening platforms. Recapitulation of microenvironment through the introduction of cyclic stretch, electrical signals, an anisotropic arrangement of cells43 and fluidic flow, as well as the possibility of integration of sensors for the evaluation of cardiomyocytes contraction, calcium transients, electrical action potential and stress response in the heart-on-a-chip, is making them a superior platform for drug screening and much other application. Proper selection of cell types is necessary to develop a functionally relevant model.

Figure 5: Comparison of conventional and modern 3D printed scaffold-based tissue engineering techniques: (A) decellularization, (B) hydrogels, (C) nanofibers, (D) spheroids and hydrogel hybrid bioprinting, (E) 3D scaffold printing, and (F) 3D printed microfluidics chip.

Liver-on-a-Chip:

Early tissue/organoid-on-a-chip devices were geometrically designed to drive cell aggregation, thereby creating multicellular organoids. For example, devices were designed with microwells with a convergent geometry that terminated in a cell substrate of some type. Based on the microwell design, liver-derived cell lines could be formed into either spheroid or cylindrical constructs in a highly controlled manner. These 3D constructs maintained much better cellular function than did 2D controls . In another example, spheroids were created from a cell line using an array of channels connecting inverted, pyramid-shaped microwells, allowing for the delivery of cells and test compound to multiple chambers simultaneously. This integration of microfluidics with an array of microreactors greatly increased the throughput potential for drug screening.

Liver-on-a-chip devices have become much more complex. They often employ controlled fluid flow to address nutrient circulation, drug or toxin administration, sample collection, and the integration of liver organoids with other tissue types. The latter will be discussed in detail later in this chapter. In one such liver-on-a-chip, hydrogels were used to encapsulate HepG2 cells with National Institutes of Health (NIH)-3T3 fibroblasts. These arrays of 3D organoids had increased liver function compared with 2D controls and produced an appropriate toxic response to acetyl-para-aminophenol (acetaminophen [APAP]) in a drug screening experiment . Our group employed a versatile photopolymerizable hyaluronic acid biopolymer system for in situ photopatterning of HepG2 cells to generate 3D liver constructs. The constructs were formed in parallel channel fluidic devices that were fabricated by soft lithography and moulded polydimethylsiloxane. This system was used for toxicity screening by administering multiple alcohol concentrations within each chip. As expected, alcohol administration resulted in a dose-dependent decrease in viability and cellular function . Efforts within our group are focused on miniaturizing this and other systems to increase throughput further. Miniaturization and microfabrication approaches can be employed to generate more intricate biological microarchitecture such as liver sinusoids. Precise seeding and layering of hepatocytes and endothelial cells within microfluidic circuits can be used to generate structures with the resolution required to produce sinusoid-like models. Another approach to generating biologically relevant microarchitecture involves mating synthetic and biological components. As an example, semiporous membrane to separate two adjacent chambers may be used to partition human hepatocytes from sinusoidal endothelial cells. Such a design was shown to generate higher albumin and urea production compared with traditional hepatocyte cultures; it demonstrates another strategy for recapitulating normal microarchitecture to increase cell functioning

Lung-on-a-chip:

Lung-on-a-chip is a micro-engineered cell culture device that replicates the 3D microarchitecture and microenvironment, breathing movements as well as primary physiological functions of the human lung .Lung-on-a-chip models have shown potential in investigating the physiology and disease etiology of human lungs, toxicological studies and drug screening. These in the near future models are likely to decrease the need for both traditional 2D cell culture methods and animal studies. Therefore, the focus of this review is to describe and discuss current and future applications of lung-on-a-chip models in the field of respiratory diseases and pharmacological therapies.

New advances in diagnosis and therapy are urgently required. “Lung-on-a-chip” models provide fast, reliable and robust in vitro platforms to achieve these developments.

Figure 6: Lung on a chip preclinical model

Skin-on-a-Chip:

The biggest human organ is the skin, which protects the entire body from external conditions.

This is one of the most accessible cells for several stress factors causing several reactions. Experiments to test almost every imaginable condition have a great impact on the use of in vitro and in vivo models, and some of these are not efficient in humans. To reduce animal use and better approach the impact on human skin it is important to provide a better alternative. Skin-on-a-chip is becoming the main in vitro model. An example is a model with epidermal, dermal and endothelial layers developed to reproduce inflammation and edema treated with dexamethasone as a drug testing model (similar work was presented in Reference ). The skin is more complex than a general division of epidermis and dermis. Wrinkles are one of the phenomena that happen over time and through external stress. Ultraviolet light, chemicals, physical stimuli, and other processes cause wrinkles. Recent work was able to reproduce wrinkled skin-on-a-chip with the use of magnetic stretching. This work can help to test products for cosmetics and pharmaceutics with a more realistic approximation . The work done by Sriram et al. offers a full-thickness skin chip with novel fibrin-based dermal matrix support for 3-D culture. Additionally, they surpassed problems like inconsistent seeding, epithelial damage ,and contraction of the dermal matrix.

Table 1 A summary of organ‐on‐chip models to understand human diseases

Organ	Latest advances	Future directions
Heart	Ability to mimic Frank-staarling mechanics in cardiomyocytes as well as display propoer auxotonic contractions	Lack of well-developed models which utilize adult phenotype cardiomyocytes for drug studies.
Kidney	Recapituable mature and functional podocytes in glomerulus aiiows for more selective filtering in kidney models.Mimic diabetes melitus type 2.	Produce a more complete nephronmimetic device which incorporates glomerulus with podocytes and proximal convoluted tubule.
Liver	Bioprinting liver tissue that maintains function for longer term than before,so that they have a better opportunity to study drug metabolism	Bioprinting should combine with microfludics for multiorgan so better understand first pass metabolisam.
Intestine-GUT-Stomach	Many host-microbe interactions with intestine organoid(Salmonella, H.pylori)	Do not undrstand epithelial and mesenchymal interctions,lumen is an organoid issue

3D- Printing recently use COVID-19 Testing and Shortage Supply:

COVID-19 was first detected in China on December 19 causing an expanding and accelerating health crisis around the world. This pandemic has placed massive stress and a huge gap in demand and supply chains in terms of diagnostic kits,reagents, drugs, face masks, and PPE kits The 3D bioprinting-based additive manufacturing appears to be an. attractive solution and holds promise to combat the shortage of these supplies. Using 3D printing, the fabrication of complex structures can be done and the printing can be adjustable for the production of the respiratory mask, face shields, ventilator valves, testing kits, and other equipment that are desirable during this pandemic. However, standard safetyand quality measures of medical devices should be regulated and taken care of in 3D printing labs to ensure the goodness of fit of the bioprinted materials for human use which can be improved through education, monitoring, and quality testing of procedures. In the current pandemic, 3D printers have been used for rapid design and development of the equipment and testing kits which include a nasopharyngeal swab, personal protective equipment, and face shields. Moreover, an alternative approach for drug production and validation was done by multi-compartment and multi-layer 3D printing which was used for developing variable or fixed-dose combinations of two or more anti-viral therapeutics . The cost effective nasal swabs were designed and printed using polyethylene terephthalate glycol (PETG) filament by major type of filament-based printer at the Department of Pathology and Microbiology, University of Nebraska Medical Centre, Omaha, NE, USA. These swabs are durable, chemically inert, and well suited for structural applications . Similarly, an inkjet-powder bed printer was used for drug loading as it dissolves drugs quickly and easily therefore can lower the drug administration burden for critically ill patients. This enhancement of drug solubility using FDM filaments which are created with ASD formulations is done by locking the active drug compounds into a formulated filament. This reduces the potential misuse of drug treatment during the panic of an ongoing crisis.

DISCUSSION:

3D printing of in vitro models is an exciting research area in which some preliminary results have been obtained over the last few years. The range of available 3D printing techniques has the potential to facilitate the development of realistic in vitro models. For the successful application of 3D printed tissues as in vitro disease models, standardization and optimization of the printing process with respect to the final requirement are necessary in addition to complying with good manufacturing practice (GMP). Hence, there is a great need for studies targeted toward understanding the different stages of disease development within the 3D printed constructs.

FUTURE SCOPE:

The use of conventional models are restricted in terms of fabrication of multiple layers or complex and composite structural designs that require different materials to be piled up or aligned because these processes involve a manual layer-by-layer assembly process. In this regard, 3D printing technique will be a suitable choice of fabrication that has the significant potential for the generation of tissue-mimetic microenvironment and thus development of disease models. Using 3D printing, the cost of drug screening on disease models can be reduced substantially by miniaturization of the model from large size while maintaining all the parameters. The cost can further be reduced by sharing the digital data between the users. Nevertheless, these 3D printed in vitro disease or tissue models could be a powerful substitute for animal models or even human trials in drugs, cosmetics development, and toxicology testing.

CONCLUSION:

As the number of organs that have been successfully simulated increase, the recent focus is to use these technologies in translational research, perhaps for advanced point-of-care procedures. Organ-on-a-chip researchers have already begun incorporating patient-specific biomarkers, including blood and its flow rate, iPSCs derived from patients, and patient-specific atherosclerotic plaque geometry. Drug development and patient treatment expenses will eventually diminish. However, before implementing organ-on-a-chip and 3D bioprinting in precision medicine, it is crucial to verify that their readouts are able match in vivo data. Access of developers to patient-specific information is required, which is sensitive, and ethical concerns could arise. However, long-term studies that correlate organ-chip and 3D printing research labs, and clinics need to adopt innovations, and be willing to test these new models

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Received on 01.04.2022 Accepted on 24.06.2022

Int. J. Tech. 2022; 12(1):1-8.

DOI: 10.52711/2231-3915.2022.00001