Open access peer-reviewed chapter
Abstract
An organoid is an in-vitro platform that recreates 3D multicellular aggerates to form tissues that fabricate the human cellular environment in the lab and imitate the functionalities of the specific organ or disease. Organoids effectively overcomes the gaps in research between 2D cell line and in-vivo models. For organoid development, both pluripotent stem cells and embryonic stem cells can be utilized, and recently Patient-Derived Organoids (PDO) was developed that overcome the limitations caused by using other cell lines. With the development of many advanced technologies in the field of research, the organoid evolution also progressed slowly into the development of patient-specific organ structures. Since tumor organoids were heterogeneous as well as patient-specific, it has many advantages that aid cancer therapy effectively. Apart from cancer treatment, organoids have a variety of applications in cancer research, the study of tissue-specific models, and also in the analysis of the relationship between tissue-specific cancer with various pathogens. Thus, the development of organoids in an effective way can pave the way for various biomedical applications. This chapter focuses on the trends in the journey of organoid research and the latest technologies developed specifically for organoids.
Keywords
- organoid
- 3D tissues
- cancer research
- cancer therapy
1. Introduction
Cancer is a heterogeneous disease that is caused by the progress of somatic mutations in normal cells [1]. Based on studies it was confirmed that continuous exposure to physical agents such as X-rays, gamma rays, UV rays, and genotoxic factors may end up in the progress of cancer cells from normal cells [2]. Carcinogenesis is a complex process that involves various pathways which need to be studied to understand the response and treatment method required for targeted therapy which is a complicated mechanism [3].
The development of drugs by using medicinal plants as bio source has also been studied extensively in various research [4]. It is essential to understand the biological interaction among immune cells among tumor immune microenvironment, stroma, and tumor for the success of cancer clinical treatments [5]. The overall cancer therapy may vary with multiple metastatic sites which in turn depend on the tumor heterogeneity [6]. The major biological complexities make the treatment methods difficult which in turn varies with the metastatic sites among individual patients [7]. The microenvironment of tumor cells includes normal stroma, malignant cells, and immune response based on every individual [8]. The durability and patient-to-patient response extremely varies based on each patient and hence very difficult to predict the consequences [9].
An organoid is an emerging technology with various applications in biomedical applications such as biobanking, disease modeling, regenerative medicine, and precision medicine [10]. An organoid is a technology used to fabricate 3D tissues in the laboratory that resembles parent tissue in function and structure and hence bridges the gap between 2D
Figure 1.
Flowchart of use of normal organoids in making tumor organoids and utilization of tumor organoids in a biobank and its applications [
2. The use of humanized models for cancer treatment
In the initial phase, the genetically engineered mouse models (GEMMs) provided a better appreciation for cancer treatment [19]. But later for practical applications, GEMMs technologies were identified to be expensive, laborious, and fetch up into complications when transformed into therapeutic applications [20]. One of the methods of generation of organoids is the utilization of Pluripotent stem cells (iPSCs), but the tissues generated from these embryonic stem cells were found to be phenotypically unstable and hence have the same limitations [21]. Later, Patient-derived xenografts (PDXs) were contemplated as a better replacement to conquer these constraints since, in this technology, models were provoked from an enormous pool of patients [22]. Unfortunately, these techniques also ended up in complications as the cancer cell lines generated
Patient-derived tumor xenografts (PDTXs) is the recently available technology in cancer research that can maintain genomic stability and tumor heterogeneity but the major drawback of PDXs is that it is expensive and time-consuming and hence treatment gets delayed [13]. Later PDTXs technologies were upgraded by transplanting these cell lines into mouse models, but PDTXs failed to replicate the human-specific immune systems and were also ended up as a laborious and expensive process [14]. The clinical response of cancer treatment depends on the clinical model used for the study [24].
3. Organoids and their types
The unique way to improve cancer research has emerged with the help of organoid technology that accompanies tumor heterogeneity at low cost and with less time [25]. Cancer organoids serve as an effective tool to understand the interaction between tumor environment and genetic alterations [15]. The organoids derived from postnatal or adult tissue were termed ASC-derived organoids. In the case of ESCs and iPSCs derived organoids, the generation of organoids takes place from all three germ layers. For all these types of organoids, the growth of cells was carried out by using a series of differentiation protocols by utilizing growth factors and inhibitors in the process of organogenesis [11]. Recently many patient-derived organoids (PDTOs) have been developed that include liver, prostate cancer, and pancreatic cancer organoids [26]. A snapshot of types is shown in Figure 2.
Figure 2.
Development of 3D organoids culture [
To make more clarity to organoids, CRISPER gene-editing technology is being implemented to organoid to convert normal organoids into tumor organoids [25]. Upon various research, mutations have been induced in normal organoids like intestinal organoids [40], colon organoids [41], pancreatic organoids [42] to make them into tumor organoids that paves the way for flexible
The brief details about various types of organoids have been discussed below:
3.1 Intestinal organoids
The pluripotent stem cells (iPSCs) were used to derive intestinal organoids that contain both mesenchymal and epithelial cells [27]. The basic intestinal crypt cells were allowed to grow in an appropriate medium containing a matrix that tends to organize into 3D epithelial cells that contain both physical and genetic resemblance of their parent organ [28].
3.2 Colon organoids
The colon organoids were originated from intestinal crypts in the presence of appropriate growth factors [29]. The stem cells of intestinal crypts differentiate into an epithelial complex that contains all types of intestinal cells. The isolated crypts develop into three-dimensional epithelial cells that form a sphere towards the lumen that further develops into colon organoids [30].
3.3 Pancreatic organoids
The capability of pancreatic ducts in in-vitro expansion and development of three-dimensional hollow structures in the presence of collagen as the suitable medium is utilized in the development of pancreatic organoids [31]. Many recent developments were carried out in establishing three-dimensional adult pancreatic organoids as well as fetal pancreatic organoids and both types of organoids were found to differ in morphology similar to that of parent cells [32].
3.4 Endometrial organoids
To investigate the biological processes involved in disease modeling, endometrial organoids were developed from primary endometrial cells of the human being [33]. For the development of three-dimensional endometrial organoids, the primary endometrial cells were dissociated, suspended in a Matrigel medium that promotes endometrial organ formation [34].
3.5 Lung organoids
The lung organoids were developed from stem cells of the lungs through a self-organization process [35, 36, 37]. The lung organoid technology has also been extended to develop various structures of lungs such as lung buds, airways, and alveolar cells that can be used to treat pulmonary diseases [38].
3.6 Ovarian organoids
Ovarian cancer is spread among a wide range of populations and the development of patient-specific ovarian organoids was found to be one of the precise approaches to enhance the treatment of ovarian cancer [39]. The ovarian organoid can enumerate the morphological heterogeneity of parent tissue and can aid in personalized therapy to enhance the treatment efficiency [46].
3.7 Breast organoids
Breast cancer is considered to be one of the most widely spread cancers among women with various subtypes and more heterogeneity [47]. Many studies have been performed on breast organoids and breast organoids can be grown either from epithelial cells [48] or from adult stem cells (ASCs) [27].
3.8 Retinal organoids
Retinal organoids were developed from Human Pluripotent Stem Cells (hPSCs) that is considered a labor-intensive and simple method of organoid development [49]. The retinal organoids also developed a physiological response to light to a certain extent that can be used in certain therapies [50].
3.9 Heart organoids
The heart organoids have proven to exhibit structural and functional features of developing human hearts [51]. The human Pluripotent Stem Cells (hPSCs) were used to develop in-vitro cardiac cell types that help in understanding and treatment of various cardiac diseases [52].
3.10 Brain organoids
The summons besotted in understanding the diseases caused in the central nervous system has been overcome by the development of brain organoids from either human Pluripotent Stem Cells (hPSCs) or epithelial cells [53].
4. Advantages of organoids over 2D techniques for cancer treatment
The development of organoid technology has overcome the limitations caused by 2D cell cultures in the following ways: 2D cultures were not repetitive of parent cultures while organoids were a portrait of their parent cells, the lack of predictivity faced in the use of 2D cultures was not visible while using organoid technology, and in 2D cell, culture models waste were being generated from growth medium which was overcome by organoid technology [54]. In organoid technology, multiple cell types were developed
To overcome the technological limitations of cancer treatment, organoids were developed in which ASCs were proliferated in-vitro and self-organized into 3D structures by utilizing extracellular matrix proteins like BME and Matrigel as medium [60]. The main advantage of organoid technology is tissue-specific growth factors can be utilized based on the epithelial cells used for analysis [61]. Based on numerous studies, the ASCs derived organoids were found to be genetically and phenotypically stable and permit the expansion of stem cells in a required path which allows them to differentiate into tissue-specific cells [62]. Apart from tumor heterogeneity, more focus is required on lymphocyte infiltration trafficking along with the clinical response of individual patients to cancer treatment [63].
5. Stages of organoid development
Generally, the 3D culture of organoids was performed by using Matrigel derived from EHS (Engelbreth-Holam-Swarm) mouse sarcoma cells which were rich in adhesive proteins such as laminin, entactin, collagen, and proteoglycans. The Matrigel structural support, ECM signals along with the extracellular environment that supports the growth of cells [64]. In other techniques, Matrigel or fibroblasts were submerged in a medium to expose the upper layers of cells in the air. Later, the air-liquid interface will be utilized to culture the cells for better polarization and differentiation [65]. The organoid technology developed drastically after the successful generation of in-vitro organ generation from sponge cells [66], amphibians pronephros [67], chick embryos [68] followed by surface adhesion of cells using thermodynamic differentiation [69]. After the invention of pluripotent stem cells from mouse embryos, there was a significant impact on organoid development by utilizing stem cells [70].
The use of animal models has been considered in the initial days to predict the efficacy of drugs to tumor cells, later cell lines cultured in-vitro were used. But both these studies did not represent the actual model of tumor cells for which treatment is required [19]. A report by Radhakrishnan et al., 2017 [71] was used to analyze the immune target therapy for the treatment of cancer was carried out by utilizing the CANscript exvivo platform technology.
The various stages of organoid development were explained below [72] and the overview showed in Figure 3.
Figure 3.
Flowchart of various stages of organoid development [
1900–1950: In the early 1900s hanging drop culture method was employed in organ development followed by tube cultures. Subsequently in 1907 organoids were developed from sponge cells and later in 1944 organoid development was carried out from amphibian pronephros.
1950–1980: In 1952 organoids were developed from chick embryos. In 1975 floating collagen cells were developed followed by characterization of laminin using organoid technology in 1979.
1980–2000: The isolation of pluripotent stem cells from mouse tails was successfully performed in 1981 which paved the way for great progress in organoid technology in subsequent years. The development of human blastocytes was performed in 1998.
2000–2020: The development of the first organoid from pluripotent stem cells was performed in 2006 from mouse fibroblast, followed by the development of organoids from cortex tissue, adult stem cells, development of gastric organoids, development of retinal, kidney, pancreas, lungs, fallopian tubes and snake venom were performed successfully in subsequent years.
6. Role of organoids in drug development and cancer research
Apart from therapeutic applications, organoids can also be employed in cancer research to study the mechanism of cancer progression and the interaction between pathogens and organs in the development of cancer cells. In a study conducted by Scanu et al., 2016 [73] the interaction of pathogen
Organoid technology has also been implemented to study the genetic interaction between mutation and progression of various types of cancer [75]. Organoids guide in the study of the initiation of tumors with the progression of cancer at various genetic levels [76].
Organoids can be used to study the response of drugs to each patient so can be utilized in drug development for cancer research [77]. Though many drugs were working on cancer models they get eliminated in final clinical trials due to unbearable side effects or lack of efficiency compared to clinical models [78]. Many biobanks of organoids were used to study the efficiency of novel drugs [16]. By utilizing organoids in drug screening, it will be feasible to predict the patient’s response and value of Patient-Derived Organoids in chemotherapy. The data obtained from PDOs can be used to predict the outcome of individual patients which was not possible by using the cell line technique [17].
7. The use of biobanks for organoid research
The progression on the development of organoids from ASCs paved the way for the establishment of the biobank for cancer [18]. The cancer biobanks provide cancer organoid cultures of numerous cancer subtypes [79]. Based on an investigation of numerous organoids using biobanks it was concluded that the dependency of organoids on ligands varies from one type of organoids to other types of organoids [59]. The inception of biobanks of organoids for cancer paved the way for the up-gradation of anti-cancer drugs with a wide range of testing based on requirements by cancer patients [21]. In a study done by Smalley and coworkers in 2018 [80], CD 34 biomarker has been utilized effectively in cancer treatment and the outcome has been studied in detail.
8. Technological developments of organoids
The existing
The development of organoids paved the way for 3D imaging technology which can be used to visualize complex organizations that were not possible using 2D imaging technology [90]. The 3D imaging technology helped scientists to visualize cellular components, intracellular processes, and architecture of cells in a detailed manner [91]. Lungs were considered as one of the most complex organs in the human body with numerous types of cells and the development of lung organoids had a significant impact on the treatment of lung cancer, asthma, cystic fibrosis, and pulmonary fibrosis [92]. Based on 3D imaging technology major therapeutic drugs were developed that can be used successfully in lung cancer treatment [93]. The 3D models increased the predictability and reliability of preclinical assays and decreased the use of animal models [94]. The quantitative analysis of the organization of various cell types in aggregates uses 3D imaging technology for detailed analysis [95].
Another technological up-gradation seen in organoid technology is Organs-on-a-chip which is used to model various functional groups of organs for detailed study [96]. Initially, the anatomy of the particular organ was studied, and then its basic elements were investigated that can be used to study organ-specific physical and biochemical applications [97]. Retinal diseases were one of the major causes of vision loss in humans worldwide while the complexity of neuro-retinal organization and complex blood supply causes side effects by the use of therapeutic drugs in the treatment of retinal diseases [98]. The development of retinal organoids improved the treatment methods of retinal diseases and also organs-on-a-chip technology paved the way to study cell types in detail that can further enhance the treatment methodology of retinal disease [99]. In case the focus of cancer research is based on a particular cell type or organ miniature types of organoids can be developed by utilizing organ-on-a-chip technology [100]. The organoid development and organoid-on-a-chip technology has their unique characteristics and limitations and based on research requirement the suitable technology is to be utilized [101]. The usage of organoid-on-a-chip technology on brain organoids paved the way to cure many neurological diseases [102].
In earlier days, tumor spheroids were developed to analyze the capability of antitumor therapeutic drugs, and recently tissue-specific organoids were utilized effectively to model various organs that can be used in cancer treatment by overcoming the limitations of ethical concerns caused by tumor spheroids [103]. The use of brain organoids to study neural diseases has proven that the brain organoids developed in 3D cultures express a large number of genes entangled in neurological problems and hence make the study process feasible compared to 2D technology [104]. In recent areas of research organoid technology is being implemented extensively in image-based phenotypic high throughput screening [105].
9. Limitations of organoids
Generation of organoids from epithelial cells may lead to contamination or overgrowth of normal cells [58]. Generally, the organoids developed from cancer cells will have less growth than organoids developed from normal cells which result in overgrowth of normal cells in the medium which can be overcome by limiting the use of growth factors in the medium [106]. Upon analyzing the efficiency of various organoids on drug development in-vitro, a positive predictive value of 88% and a negative predictive value of 100% were endorsed upon numerous studies [107, 108]. The organoids developed
10. Conclusions
Cancer is a heterogeneous disease caused by a mutation in normal cells that results in the abnormal growth of cells. Though many treatment methods and drugs were available in cancer treatment, there is no proper cure, and the drugs used ended up in major side effects in patients. The development of cancer cells varies with the patient and also based on the type of cancer. Thus, there was a need to derive a patient-specific treatment method is cancer treatment. But the limitation of these 2D cell line models where it was a time-consuming process, expensive, and sometimes cannot meet the requirement of tumor heterogeneity to large extent. To overcome this many technologies such as GEMMs and PDTXs were developed and they have been proved to be effective techniques. To overcome these limitations organoid technology has emerged in which tissues were grown in
Acknowledgments
We would like to thank Ms. Amudha Madhavan for her critical review and proof reading of the chapter.
Acronyms and abbreviations
Genetically Engineered Mouse Models Patient-derived xenografts patient-derived organoids pluripotent stem cells hPSCs ASCs
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