1. Introduction

Currently, there are almost three general processes to produce organoids in vitro. The first very common and strikingly, the simplest of these approaches is based on extracellular matrix (ECM) (such as Matrigel or collagen-based) scaffolds . In 2009, Sato T. et al. demonstrated that Lgr5+ stem cells isolated from the intestinal crypt or crypt itself have the ability to form intestinal crypt-villus units, which are self-regulating organoid structures without a non-epithelial cellular niche by using Matrigel supplemented with growth factors. These organoids were cultured for more than eight months without losing their characteristics, and microarray analysis showed that they were highly similar to freshly isolated intestinal crypts ^[1]. Sachs et al. showed that the embedding of proliferating organoids in a contracting floating collagen gel allows them to line up in macroscopic tubes to form a simple intestinal epithelium structure that contains all major types of small intestinal cells, including epithelium and stem cells ^[2]. The collagen-based matrix, including type 1 collagen, Ham’s F12 nutrient medium, and bicarbonate, showed similar results to a matrigel-based niche in vitro. Since most of the ingredients in matrigel are not fully defined comparing to collagen, it is hard to predict its effect on the human body. Additionally, given the matrigel-based niche's ability to increase angiogenesis due to its ingredients such as cytokines and growth factors, for the in vivo organoid transplantation, the collagen base niche could be a valid alternative ^[3][4].

The second approach for generating organoids from the embryonic body (EB) or pluripotent stem cells is agitation using spinning-rotating bioreactors, which are often used to recapitulate human brain development in vitro. As demonstrated by Lancaster et al., by providing the environment essential for intrinsic cues together with improved growth conditions, EBs generate neuroectodermal tissues ^[5]. Neuroectodermal tissues were embedded in Matrigel droplets to create the 3D scaffold structure to provide more inclusive tissue growth. Later, Matrigel droplets were transferred to a spinning bioreactor designed to increase nutrient absorption. The rapid development of brain tissue has been made by this method requiring only 8–10 days for the appearance of neural identity and 20–30 days to form specific brain regions. This method was also used to produce retinal organoids. In 2018, Di Stefano et al. reported an approach to culture retinal organoids from mouse pluripotent stem cells using the NASA-designed rotating-wall vessel (RWV) bioreactor ^[6]. With the RWV bioreactor, organoids successfully demonstrated well-defined morphology and advanced neuronal differentiation by forming ganglion and S-cone photoreceptors cells and increased proliferation.

The last method for generating organoids was the air-liquid interface (ALI) method (Figure 1c). The ALI method is mainly used to produce gastrointestinal organoids ^[7]. In this method, the top layer of the cells is exposed to the air while the basal surface is inside the liquid matrix medium produced by Matrigel, collagen, or other biological matrices. Researchers also combine the organotypic slice culture method with the ALI method and generate air-liquid interface cerebral organoids, which exhibit improved survival and morphology with extensive axon enlargements reminiscent of nerve tracts ^[8].

2. Advantages of the Organoid Models

Organoid models have many advantages, as they form an almost physiological model system that starts from cells and creates an middle step for in vivo transition to studying tissues and diseases: (1) The ability of organoids to remain genetically and phenotypically stable while maintaining tumor heterogeneity and being extended over a long period of time without genetic changes have accelerated the application of organoid technology, thus creating a unique opportunity to promote and consolidate basic and clinical cancer research ^{[9][10][11][12][13]} (2) Organoids are comparably easy to grow and can be obtained from various sources such as adult cells, ESC, IPSC, cancer cells, and primary tissues with a limiting portion of starting materials. Moreover, high quality molecular and cellular imaging techniques can be easily applied for organoids ^{[12][13][14][15][16]}. Using gene editing technologies, normal organoids can be transformed into tumor organoids that mimic genetic changes during cancer initiation and progression, as well as cells can be selected or unselected to reflect the patient's heterogeneity and can be individually monitored ^[17]. For instance, by applying gene-editing technology, researchers have developed APC, TP53, KRAS, SMAD4 and/or PIK3CA mutated cancer driver pathway colorectal cancer (CRC) organoids, which were able to generate micrometastases when implanted into the mice. At the same time, organoids with both mutated cancer driver pathways and chromosomal instability were capable of creating large metastatic tumors when transplanted into the mice ^[18]. By using the patient-derived organoids, it is possible to develop biobanks and investigate diseases that are difficult to model in animals ^[19][20]. Recent studies have demonstrated that patient-derived tumor organoids can capture the cancer-specific genetic alterations and histopathology in individual patients that can be used for personal drug screenings, which make them suitable to correlate the genetic background of a tumor with drug response. Organoid technology has been utilized to discover cancer prognosis-related mechanisms. Broutier L et al. have shown 30 potential tumor biomarkers by systematically comparing transcriptional differences between healthy and primary liver cancer (PLC) organoid lines ^[21]. Among these 30 tumor biomarkers, 11 genes were reported for the first time, and 13 genes were associated with poor prognosis. (4) Organoids offer a cost-effective model for drug discovery and screening ^{[22][23][24][25]}. The production of cancer organoids even using simple cell culture setups makes them affordable and accessible and enables the culture to be accomplished in a short time ^[26] (5) Co-culturing of organoids with TME cells overcomes the inadequate inflammatory response due to the absence of immune, stromal, and blood cells and allows the study of complex interactions between TME and cancer^[27]. Fujii, M. and his colleagues have demonstrated that niche factors significantly impact colorectal cancer (CRC) organoids, which can support or inhibit organoids' growth ^[28].

3. Limitation of the Organoid Models

Compared to classical 2D culture models and spheroid cultures, organoids self-regulate themselves similar to in vivo; however, many existing organoid models are not subject to the influence of the inflammatory response as they are not supported by immune, stromal, and blood cells ^[29]

Besides, they are incapable to imitate biomechanical forces like shear stress that occurs with blood flow ^[30]. Due to the relatively rigid extra-cellular matrix used in organoid culture components, drug penetration may be limited. It is still difficult to culture organoids from tissues whose microenvironments and niche factors are poorly understood, like ovarian organoids. In addition to all these microenvironmental challenges, the same culture can be heterogeneous in terms of quantitative concepts such as cell viability, growth, and size due to the variability of organoid culture conditions ^[31]. Finally, in organoid models, as in many 3D models, as the organoids grow in size and volume, the core diverges from the surface and the simple diffusion process becomes insufficient to provide oxygen and nutrients. Given that the volume of waste removed from the growing cells will also become limited, only cells that come into contact with the fresh medium survive ^[32]. Overcoming all these limitations of existing organoid models, which are highly promising tools for understanding organ development, disease modeling and progression, can be accomplished by combining co-culture methodologies and microfluidic technologies that mimic in vivo conditions.