Applied Materials Today
Recent advances in organoid engineering: A comprehensive review
Graphical abstract
Introduction
The three-dimensional (3D) cell culture in the form of cell aggregation was attained its first popularity in 1965-1985, according to the number of published materials [1,2]. Despite the name “organoids” was introduced to the scientific community half a century ago, this name was used to describe classical biological experiments that sought to define organogenesis by cell dissociation and reaggregation experiments [1]. Later, the emergence of the 3D cell cultures has fueled the knowledge on the effectiveness of the 3D cell cultures compared to the conventional two-dimensional (2D) cell cultures. The 2D cell cultures do not provide the conditions required for cellular organization and cell interactions in vivo. Also, the cell signaling networks are altered in 2D cell culture versus 3D cell cultures. In contrast to the 2D cell culture, 3D cell culture models exhibit very close properties to the in vivo conditions. The 3D culture models provide more realistic findings similar to in vivo conditions in translational research [3]. The 3D culture models are generally either two models: scaffold-based models or scaffold-free models. In scaffold-based 3D culture, the cells are grown in the substrate, which mimics the extracellular matrix (ECM) properties in either hydrogel or solid scaffolds; while cells are unable to attach to the substrate and force to make cell aggregates or cell spheroids in scaffold-free method [4,5]. The past decade again witnessed the organoid's resurgence, yet in a somewhat different form due to the parallel development of 3D culture techniques. The breakthrough of the current organoid resurgence was made by Hans Clevers and his colleagues in 2007 after discovering a new type of stem cells [6]. Organoid technology is now on the verge of emerging as an independent research field.
The term organoid has lost accuracy in recent years and has been defined broadly according to the different cell culture techniques and their respective applications. However, this broad applications from small tissue explants to clonally expanding cells that self organize have made it difficult to define or otherwise, have made its meaning ambiguous. Recently, experts from different part of the world came together and defined the organoid as a “3D structure derived from (pluripotent) stem cells, progenitor, and/or differentiated cells that self-organize through cell-cell and cell-matrix interactions to recapitulate aspects of the native tissue architecture and function in vitro” [7]. However, according to the early definitons and studies of the organoid, it has been established that organoids are exclusively derived from the stem cells [1,[8], [9], [10], [11]]. It is now clear that organoid can generate from differentiated cells such as cholangiocytes [12,13]. Due to this recent expert meeting, previously believed [11] organoid definition and characteristics of the organoid was changed to a newer version and in this review we focused on the latest definition of the organoid. So, recently new catergorization for the organoid was proposed. Organoids cen be devided into three distict groups based on their characteristics; (i) epithelial organoids, (ii) multi-tissue organoids, and (iii) multi-organ organoids [7].
The first category, epithelial organoids are derived from a single germ layer (endoderm, mesoderm, or ectoderm). Epithelial organoid can self-renew under appropriate culture conditions and also represents the most widely studied organoid type. Since epithelial organoids do not contain mesodermal component, in some cases, supporting cells are co-cultured with epithelial organoids [14,15]. Multi-tissue organoids at least have two germ layer cells or co-differentiation of PSCs, which are established through co-culture. Interestingly, current studies do not support the self-renewal of multi-tissue organoids in normal pathways, but cells interact to achieve a stable level of maturity and function [16,17]. The most complex and the least studied organoid type is multi-organ organoids, where these organoid can make a significant contribution for study of organogenesis.
Organoids can be derived from different types of cells: (i) pluripotent embryonic stem cells (PESCs) and their synthetic counterparts, induced pluripotent stem cells (iPSCs); (ii) organ restricted adult stem cells (aSCs) [1]; and (iii) differentiated cell types such as cholangiocytes and hepatocytes, as described in the recent literature [18]. PESCs and iPSCs are simply known as pluripotent stem cells (PSCs) in most literature. Both these PSCs and aSCs have shown an unprecedented capacity to self-organize into structures that mimic the fundamental properties of the tissues which they are supposed to form. As mentioned above, human iPSCs (hiPSCs) play a significant role in the recent development of novel organoid models [10,[19], [20], [21]]. The discovery of the iPSCs has resulted in the development of multiple differentiation protocols in vitro using various endoderm-derived tissue types, including stomach [22], kidney [23], liver [24], [25], [26], lung [27], [28], [29], pancreas [30], and intestine [31]. The invention of directed differentiation of tissue-specific cell types from iPSCs has resulted in the investigation of different organoids, including the brain [32], kidney, liver, lung [33], pancreas, and stomach. A more detailed review of some organoid types is discussed in the latter part of this paper.
As previously mentioned above, unlike the traditional in vitro cell cultures, organoids have complex cellular composition and architecture, making them a physiologically complex model to study tissue development process, tissue homeostasis, and cellular functions and signaling pathways present in tissues in vitro [10,34]. Theoretically, the organoids made from human stem cells should mimic the actual human organ functions, especially conditions that do not replicate well in experimental animals [6,34]. However, most organoid models do not replicate the actual human organ models for various reasons, including the difficulty of generating full tissue components, difficulty controlling cell types, and complexity in cell-cell and cell-matrix interactions in these systems. The dependence on the limited animal-derived hydrogels, including Matrigel and collagen as an ECM, makes organoids unsuitable for expansion and downstream clinical applications due to their uncontrolled modifications and risk of pathogen and immunogen transfer [35]. Due to these reasons, despite the extensive use of organoids in basic and translational research, organoids are currently restricted to drug screening and initial cell replacement strategies [8].
In this review, we discuss the recent advancement and trends in organoid technology, including different organoid models and their significance to the development of current therapeutic studies. We critically evaluate the use of synthetic or natural polymer as an ECM for organoid formation. Spheroids as an intermediate stage for organoid development are also be addressed by emphasizing recent discoveries. We also highlight the key challenges and limitations of the current organoid technologies, which remain to be addressed in the future.
Section snippets
The formation of organoids: self-organization
The formation of an organoid from iPSCs or aSCs or differentiated cells is a controlled process, uses biochemical and physical cues for tissue development and homeostasis. The development of the human body from the zygote is a precisely controlled process, which uses stepwise differentiation and self-organization of the cells. Understanding the self-assembly of organoids solely depends on the classic developmental biology theories. The tissue patterning is essential to understand the
Organoid systems
Organoid systems offer promising platforms for future therapeutic studies and stem cell applications focusing on tissue and organ regeneration. However, similar to most model systems, there is a significant difference between in vitro and in vivo models, which can be addressed through various bioengineering techniques. Bioengineering approaches can be applied to develop bottom-up synthetic organoid constructs, and facilitate controlled organoid formation through different cell cultures. The
Brain organoids
The development of human brain embodies involves a high degree of coordination between the neural stem cells (NSCs) and the dynamic microenvironment in which they exist [34]. The expansion of the neuroepithelium to generate radial glial stem cells (RGS) is the beginning of human brain development [32]. The human brain is a complex structure consisting of different region-specific cell types and complex cellular arrangements. In most situations, part of the specific interest region of the brain
Drug development and toxicity screening
The current drug toxicity screening methods are heavily dependent on the traditional 2D cell cultures (monolayer) on plastic, which results in unacceptable failure rates [152,187,188]. The new investigational drugs that have gone through phase II and phase III clinical trials were failed to proceed into the next stage of development 66% and 30%, respectively [152]. The current toxicity assays are the main reason for this high failure rate and hence highlight the need for reliable toxicity
Future perspectives and limitations
Organoids are one of the most versatile and physiologically relevant models to study human diseases and drug toxicity screening. So far, the traditional cell biology principles, together with the novel bioengineering approaches, have evolved organoid into a new era of translational research platform instead of being a basic research tool. Most importantly, the development of miniature organ-like structures using available microengineering techniques eventually curtail the time to develop
Conclusion
Organoids have the capacity to mimic the specific cellular functions, 3D architecture, and cell-type compositions similar to the actual organ and thereby hold great promise for a range of biological and biomedical applications. In the study of drug discovery, organoid cultures may reduce millions of dollars spent on unnecessary drug developments due to the variability in animal models and humans. Since the organoids can resemble actual human organs, the number of animal models and clinical
Declaration of Competing Interest
The authors declare no conflict of interest
Acknowledgments
The authors acknowledge funding support from the National Institutes of Health (R01DE023356) and the University of Toledo.
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