Introduction
In today's world, the discovery of useful drugs is becoming increasingly challenging while the prevalence of untreatable diseases continues to rise. Developing new drugs is crucial for addressing diseases like antimicrobial resistance, tumors, and obesity, as well as improving the prediction of toxicity, a major factor in drug development failure. However, the current drug development process is highly inefficient, with a significant percentage of drugs failing in clinical trials. This inefficiency leads to high costs in the healthcare system and the availability of drugs with low efficacy and safety for the population.
An analysis of failed drugs in later stages of development revealed that the existing preliminary tests conducted on animals and 2D cell cultures accurately predicted human toxicity in only a portion of cases. This has raised concerns within the pharmaceutical industry about the reliability of current in vitro testing methods during the preclinical stage. Both animal models and 2D cell cultures lack the physiological resemblance to human tissue and are associated with time-consuming procedures, high costs, and ethical issues related to animal testing.
To overcome these limitations, recent advancements in 3D cell culture technology have introduced organoids, which are 3D humanized models that mimic the architecture and cell behavior of healthy and diseased organs. Organoids offer improved predictability for drug efficacy and safety, enhancing the quality of preclinical testing before proceeding to human clinical trials. Furthermore, when organoids are derived from patient cells, they hold the potential to contribute valuable insights to personalized medicine. However, organoid technology faces challenges such as limited production scalability, lack of automation, high costs, and issues with reproducibility, hindering its translation to the pharmaceutical industry.
A promising solution to address these technological challenges is the integration of organoids with microphysiological systems based on microfluidics technology, known as organ-on-a-chip. These microphysiological systems provide better control over physiological conditions, including perfusion, mechanical forces, and other parameters crucial for tissue and organ function. By combining the human cellular fidelity of organoids with the environmental control of microfluidic chips, a more accurate and efficient technology for drug discovery and development can be achieved. The pharmaceutical industry anticipates significant cost reductions in drug development by adopting microphysiological system technologies, which could potentially replace animal models. This mini-review aims to discuss recent advances in organoids and highlight the crucial technologies for achieving a synergistic strategy with microphysiological systems.
Drug development: Current scenario
The drug development process typically involves four main stages: discovery and development of promising compounds, preclinical research using in vitro and in vivo tests, clinical research, and application for regulatory approval. However, the high failure rate in drug development has prompted a comprehensive review of the preclinical stage, particularly concerning the use of non-human cells in tests, which often yield misleading results that are not replicated in clinical trials.
There is increasing pressure on governments and public administrations to find alternatives to animal testing. Legislative measures, such as the Humane Research and Testing Act, have been approved to allow drug manufacturers to seek market approval based on safety and effectiveness results obtained from alternative methods. These alternative methods include cell-based assays, organ chips, microphysiological systems, computer modeling, and other human biology-based test methods. The aim is to reduce, refine, and replace animal procedures while promoting animal welfare and technological innovation.
3D Cell culture
While 2D cell culture and animal models have contributed to our understanding of cellular and molecular biology, they fail to address important aspects of human cell physiology. In contrast, 3D cell culture models, specifically organoids, offer the potential to recreate the 3D tissue architecture of human organs and replicate morphogenetic events during stem cell differentiation. Animal models, on the other hand, do not accurately predict several human diseases and physiological responses due to their reliance on animal cells.
Complex 3D cell culture models generated from human cells have the potential to improve the prediction of drug development outcomes. Organoids bridge the gap between 2D cell culture and animal models, bringing cellular models closer in complexity to human tissues and organs. These models have become possible with the discovery of human adult stem cells and induced pluripotent stem cells, which can recapitulate tissue and organ development. Organoids, developed using non-adherent surfaces or matrigel, allow cell-cell and cell-extracellular matrix interactions to prevail, thereby simulating the tissue architecture and function of healthy and diseased organs.
Organoids can be derived from patient cells, including tumors, making them highly representative of specific disease states. Patient-derived organoids enable the discovery of new disease biomarkers and the testing of drugs in vitro, offering a personalized medicine approach. However, complex organoid models often lack reproducibility, which is crucial for the pharmaceutical industry. Additionally, organoids are not yet compatible with high-throughput screening methods. Overcoming these challenges requires addressing issues such as precise control of nutrient supply and the biochemical and biophysical microenvironments, reducing variability through automation, and improving the modeling of tissue-tissue and multi-organ interactions to better simulate body physiology.
A promising approach to tackle these challenges involves integrating organoids with microphysiological systems based on microfluidics technology, specifically organ-on-a-chip. Organ-on-a-chip devices provide better mimicry of physiological parameters crucial for tissue and organ function compared to 2D cell culture models. They can also be interconnected, creating multi-organ-on-a-chip systems that model whole-body physiology or pathology. These systems offer advantages such as reduced labor costs, automated operation, and minimized reagent usage. Notably, the FDA has already approved a clinical trial based on results from an organ-on-a-chip model, showcasing their potential to replace animal data.
Organ-on-a-chip
Organ-on-a-chip technologies rely on microfluidic devices seeded with cells and maintained under continuous fluid flow. The initial goal of these models was to replicate essential physiological parameters, primarily focusing on mechanical stimuli. For instance, the first published organ-on-a-chip model simulated the alveolar-capillary interface of the human lung using epithelial and endothelial cells. This device successfully replicated breathing-type movements and responded to pathogen stimuli.
Today, microphysiological systems come in various sizes and shapes, featuring small hollow channels with dimensions similar to blood capillaries to provide necessary nutrients and oxygen to the cultured tissues. These microfluidic devices recreate organ structures and mechanical forces necessary to mimic organ physiology. The biomechanical forces induced by microchannel flow mimic the pressures exerted by vascularization, promoting cell differentiation. Furthermore, these devices can be interconnected to model multi-organ systems, known as "body-on-a-chip" or "multi-organ-on-a-chip," which simulate whole-body physiology or pathology.
In recent years, conventional manufacturing techniques like photo-patterning, self-assembly, and soft lithography have been primarily used to develop microfluidic devices. However, 3D printing offers advantages such as unlimited design possibilities, complex geometries, and waste reduction. Researchers have successfully used 3D printing to fabricate inertial microfluidic devices with relevant geometries for cellular behavior. Another study employed 3D printing using extrusion fused deposition modeling technique and observed successful cell adhesion and viability on the surface of the devices during culture.
Despite advancements in organ-on-a-chip technologies, their cellular composition and tissue architecture remain relatively simple compared to organoids. Organ-on-a-chip models lack stem cell populations and tissue microenvironments, limiting their physiological relevance, especially in dynamic disease models like tumorigenesis with varying cell subpopulations.
Currently, it is possible to generate liver organoids composed not only of hepatocytes but also stromal cells like stellate and Kupffer-like cells. These advanced liver organoids can recapitulate the main steps of steatohepatitis, including steatosis, inflammation, and fibrosis phenotypes. They also exhibit a complex tissue architecture, such as a functional bile canaliculi system, capable of responding to drug stimuli.
The intestinal organoid was the first protocol described for organoids, using adult stem cells from the human intestine biopsy seeded on matrigel. These stem cells can mimic the epithelial polarity of the intestine without a mesenchymal niche. Over time, the complexity of intestinal organoids has been increased to simulate hyperplastic intestinal organoids under injury and to mimic different parts of the intestine, such as the small intestine and colon. However, the use of matrigel presents limitations for translation due to animal protein contamination and reproducibility issues. Researchers are working on developing alternative biomaterials, including synthetic polymers or biomaterials derived from decellularized extracellular matrix, to replace matrigel.
The cerebral organoid was the first organoid derived from induced pluripotent stem (iPS) cells and can replicate human cortical development in healthy and diseased states. It exhibits a diversity of cell types related to the human cerebral cortex, and functional aspects such as neuronal activity. Cerebral organoids have also been used as tumor models to study the invasion behavior of patient-derived stem cells from glioblastoma. Midbrain organoids have been developed as a model for Parkinson's disease.
The interest in lung organoid development has significantly increased in the last few years, especially due to the COVID-19 pandemic. Lung organoids derived from iPS cells show epithelial and mesenchymal compartments similar to native lung tissue. Recent advances include the use of extracellular matrix-free and chemically defined organoid culture derived from single adult human alveolar epithelial type II (AT2) cells, establishing a reproducible lung organoid model for studying distal lung infections, including COVID-19.
Significant progress has also been made in kidney organoids and tumor organoids as models of tumorigenesis.
Organoids have emerged as powerful tools for drug discovery, development, and testing. They can capture specific disease characteristics and individual drug responses, making them useful for personalized medicine. Tumor organoids, in particular, have been extensively used for precision cancer medicine, allowing the identification of resistant cell populations.
3D bioprinting has been used to engineer more complex and physiologically relevant tissue models. It provides higher resolution and the ability to organize cells, organoids, biomaterials, and growth factors in an automated and pre-designed manner. Bioprinted organoids have shown improved efficiency compared to non-bioprinted organoids in various applications.
The convergence of organoids and organ-on-a-chip technologies, often referred to as organoids-on-a-chip, holds great promise for advancing drug development, disease modeling, and personalized medicine. The use of microfluidic systems in combination with organoids can provide better control over cell functions, improved perfusion, and real-time monitoring of responses. Microfluidics can also enhance the vascularization and maturation of organoids, leading to more physiologically relevant models.
In summary, the convergence of organoids and microfluidic technologies offers several advantages for drug development and testing:
1. Reduced variability: Microfluidic devices provide better control over the culture environment, leading to decreased variability in organoid models. This is crucial for achieving reproducible results and improving the reliability of drug testing.
2. Automation and cost-effectiveness: Microfluidic systems can support automated operations, reducing labor costs and minimizing human error. They also require smaller volumes of reagents, which can be significant for organoid cultures due to the high cost of growth factors.
3. Accelerated maturation: The integration of microfluidics with organoids can enhance the maturation process, enabling faster and more efficient development of functional tissues. This is particularly important for organoids that require long-term culture to achieve maturity.
However, there are also some limitations and challenges associated with the convergence of organoids and microfluidics:
1. Design and fabrication of microfluidic devices: Developing scalable and versatile microfluidic devices that can accommodate various stages of organoid culture, from formation to maturation, remains a technological challenge. New designs and fabrication methodologies are needed to overcome these limitations.
2. Scalability and standardization: As an emerging field, organoid-on-a-chip technology lacks standardized protocols and scalability. Efforts are required to establish common standards and protocols to ensure reproducibility and compatibility with industrial manufacturing processes.
3. Alternative materials and manufacturing methods: Current microfabrication techniques often rely on materials that may not be scalable or compatible with long-term organoid culture. Exploring alternative materials and manufacturing methods is crucial for advancing organoid-on-a-chip technologies.
Despite these challenges, the integration of organoids and microfluidics has the potential to revolutionize drug development and testing. By addressing the limitations and leveraging the advantages, organoids-on-a-chip can provide more reliable and predictive models for personalized medicine, reduce the reliance on animal models, and contribute to more efficient and cost-effective drug discovery processes.
