Potential Use of Organoids in Regenerative Medicine

Abstract

Background:

In vitro cell culture is crucial for studying human diseases and development. Compared to traditional monolayer cultures, 3D culturing with organoids offers significant advantages by more accurately replicating natural tissues' structural and functional features. This advancement enhances disease modeling, drug testing, and regenerative medicine applications. Organoids, derived from stem cells, mimic tissue physiology in a more relevant manner. Despite their promise, the clinical use of regenerative medicine currently needs to be improved by reproducibility, scalability, and maturation issues.

Methods:

This article overviews recent organoid research, focusing on their types, sources, 3D culturing methods, and applications in regenerative medicine. A literature review of "organoid" and "regenerative medicine" in PubMed/MEDLINE highlighted relevant studies published over the past decade, emphasizing human-sourced organoids and their regenerative benefits, as well as the availability of free full-text articles. The review uses descriptive data, including tables and text, to illustrate the challenges and potential of organoids in regenerative medicine.

Results:

The transition from 2D to 3D models, particularly organoids, has significantly advanced in vitro research. This review covers a decade of progress in various organoid types—such as liver, cholangiocyte, intestinal, pancreatic, cardiac, brain, thymus, and mammary organoids—and their 3D culture methods and applications. It addresses critical issues of maturity, scalability, and reproducibility and underscores the need for standardization and improved production techniques to facilitate broader clinical applications in regenerative medicine.

Conclusions:

Successful therapy requires increased scalability and standardization. Organoids have enormous potential in biological research, notwithstanding obstacles.

Background

Modeling human development and disease via in vitro cell culture is a critical research component. Even though traditional monolayer cell culture has been in use for a while, it lacks the complexity and tissue architecture necessary to represent real biological processes that occur in vivo accurately. The 3D culturing technique avoids direct contact between the cells and the plastic. As a deviation from their usual environment, this lack of direct contact with the culture surface with the plastic can be viewed as a stress for the cells. It could result in alterations to their physiology or behavior. This method can be done with or without using a scaffold. The scaffold is usually made of artificial or biological hydrogels that resemble the extracellular matrix (ECM) that is present in nature. The main part of a developmental model is to mimic the physiological orchestration conditions that generally exist in the human body, where different cellular and ECM components come together to generate a particular microenvironment for each kind of tissue [1, 2].

In 2016, Hans Clever utilized a term that has undergone a rebirth and taken on new connotations. The term' Organoid' has been utilized extensively in developmental biology experiments over the past forty years to understand organogenesis by cell. The term organoid describes 3D aggregates of cells made from tissue-specific adult stem cells, induced pluripotent stem cells (iPSCs), or embryonic stem cells (ESCs). Further, with a consistent pattern, differentiation develops in the organoids. Yoshiki Sasai was the first to propose this novel idea [3].

The concept that pluripotent cells can differentiate and generate intricate, well-organized structures approximating typical organ or tissue development when cultured in three dimensions was first proposed by the Sasai group. In contrast to conventional cell cultures, which confine cells to a flat, two-dimensional surface, cells can freely move around in a three-dimensional environment such as a culture plate. Instead, individuals are free to engage with their surroundings in various aspects. Different physical and chemical characteristics of the culture plate's surface, such as texture, stiffness, or the presence of particular chemicals, might affect how cells behave. Organoids are cells that have been cultivated in an in vitro three-dimensional (3D) environment to generate small cell clusters that can organize and develop into specific functional cells, which mimic the shape and function of organs in vivo (so they are often called "mini-organs") [1, 3, 4].

Organoid technology represents a groundbreaking approach to therapy, and while research is ongoing, there have been some recent trials, such as intestinal organoid transplantation in humans. However, further development is needed to address challenges related to repeatability and scalability [5–7]. Organoid technology is promising for biomedical research and treatment, but some challenges still need to be addressed before it can be used on humans. Organoids must overcome several obstacles before being successfully applied in therapeutic settings. The absence of uniform and repeatable findings across several organoid batches is a significant concern. Each organoid culture may differ in size, cell makeup, functionality, and general excellence. This variation hampers the trustworthiness of experimental results, and uniform techniques for therapeutic applications are difficult to create [8, 9]. Manufacturing organoids at a large enough scale is another formidable obstacle. Organoids are now mainly produced in small amounts and need a lot of labor to culture. It is challenging to scale up the production process to produce large numbers of standardized organoids appropriate for therapeutic application. Researchers are looking into several strategies to increase scalability, including automation and bioreactors. To produce organoids at a low cost and with high throughput, considerable improvements are still needed [8]. Further research is required in organoids' long-term stability and maturation domains. Organoids can mimic some characteristics of human organ development, although they frequently lack real organs' full maturity and functionality. It is still unclear how successfully organoids can simulate human organ complexity and long-term dynamics in a controlled laboratory environment [10].

This article will give a brief overview of numerous organoids that have been investigated for a considerable amount of time from various cell sources, the 3D methods applied, and the research findings. Organoids vs. spheroids, organoid production techniques, liver, cholangiocyte, intestinal, pancreatic, cardiac, brain, thymus, and mammary organoids are all topics that will be covered in this review of the potential use of organoids in the field of regenerative medicine over the past ten years.

Methods

The terms "organoid" and "regenerative medicine" were used in literature searches on PubMed/MEDLINE on 15 December 2022. Articles in English that discussed organoids, human sources, and their advantages in regenerative medicine, which free full text were available, and published within the last 10 years (2012–2022), were qualified as inclusion criteria. We checked the abstracts for studies on the use of organoids for precision medicine, 3D culture organoid models, and stem-cell-derived organoids in regenerative medicine, which were included in this review. Articles with no available full texts or content were irrelevant to the aim of this review; letters, commentaries, conference, congress, and meetings abstracts were excluded. Data were examined and descriptively presented using text, and tables (Tables 1, 2).

Table 1.

Comparison of spheroid and organoid

	Spheroid	Organoid	References
Aggregates	Homogenous or multi cell type, any type of cells	Heterogenous (mini organs), usually contains stem cells	[11, 12]
Physiological relevance	Less	More/mimic organ	[1]
Exogenous genes expression	Easy	Difficult	[13]
Cost	Cheaper	Expensive	[14]

Table 2.

A description of the several types of organoids

No.	Type of organoid	Cell Source	Requirements for support of 3D culture	Main findings	References
1	Liver organoid	Isogenic adult primary non-parenchymal cells, iPSCs	Matrigel	While displaying decreased TGF- and Wnt signaling activity, liver organoids significantly enhanced albumin expression, generated more of it, and showed higher expression of TDO2, CYP1A1, andCYP1A2	[15]
2	Liver organoid	Hepatic progenitor cells	Polylactic acid fabric mesh using a bioreactor	A unique bioreactor system was used to create liver organoid tissue. Fibroblasts were inserted in collagen fibril networks to form an artificial tissue, which was comparable to liver lobules. It can be used to recreate the hepatic interstitial structure	[16]
3	Liver Organoid	Human iPSCs	Inverted colloid crystal (ICC)	Compared to 2D and 3D controls, the produced organoids were more similar to adult tissue	[17]
4	Hepatic organoids (ARPKD organoids)	Human iPSC lines	Matrigel scaffold	ARPKD organoids could be utilized to examine the pathophysiology of congenital types of liver fibrosis and to assess the effectiveness of proposed anti-fibrotic treatments	[15]
5	Liver and kidney organoid-based multi-organ-on-a-chip model	Kidney tubuloids, MSCs	TissUse HUMIMIC Chip2 microfluidic model	The therapeutic effectiveness and biodistribution of Mesenchymal Stromal Cell (MSC)-derived small extracellular vesicles (sEVs) as seen in animal models are replicated by multi-organ-on-a-chip (MOC) models	[5]
6	Cholangiocyte organoids	Cholangiocytes	Droplet encapsulation single-cell RNA sequencing (scRNA-seq)	Proof-of-concept that the human biliary epithelium can be repaired using cholangiocyte organoids. The interchangeability of intra- and extra-hepatic cholangiocytes for regenerative medicine applications is revealed by single-cell RNA sequencing analysis in conjunction with a novel model for cell transplantation in human livers	[19]
7	Bile-cholangiocyte organoid	Cholangiocyte-like cells	Basement membrane extract-BME-/matrigel domes	It is now possible to design bile duct tissue and replicate specific diseases by employing bile as a novel source for extrahepatic cholangiocyte organoids	[20]
8	Colonic Organoids	Human colonic biopsy crypt-derived single cells	QGel CN99	Displaying prolonged genetic stability as well as de novo establishment, growth, and organoid maintenance	[21]
9	Intestinal organoids	Human iPSC	Matrigel	(1) To quickly create and perfect conditioned media for human intestine organoid culture, they used a lentiviral vector. (2) They supplemented WNT3A and fibroblast growth factor 2 to stimulate differentiation into definitive endoderm, which improved the efficiency with which they produced intestinal organoids from human iPSCs. (3) They successfully transduced exogenous genes into organoids using 2D culture and subsequent re-establishment of organoids. (4) To facilitate harvesting and tests, they looked at suspension organoid culture without scaffolds	[13]
10	Cardiomyocytes, liver organoids, pancreatic endocrine cells, and dopaminergic neurons	Human pluripotent stem cells (hPSCs)	Matrigel	For mimicking COVID-19 disease as well as understanding the biological responses of human tissues to SARS-CoV-2 infection, hPSC-derived cells and organoids are invaluable models	[22]
11	Pancreatic organoids	MODY3 patient and CRISPR/Cas9-engineered human induced pluripotent stem cells (hiPSCs)	Matrigel	This study emphasizes how pancreas progenitor-derived organoids can be used as in vitro disease models	[23]
12	Cardiac organoid	hPSC-derived ventricular cardiomyocytes	Collagen-based hydrogel	In this study, a small human ventricle-like cardiac organoid chamber with fluid ejection in three dimensions (3D) was created	[24]
13	Cardiac organoids	HCMECs, HCFs, Cor.4U-CMs, Human iPSC Lines	Hanging drop and Poly-L-Lysine coated	The appropriateness of organoids as a 3D cellular model to depict the phenotypic characteristics of normal and cardiomyopathic hearts. Organoids could thus be a useful tool for developing and testing patient-specific therapies and serving as a base for more secure and effective medication development	[25]
14	Cardiac organoids	Human cardiac fibroblasts, cardiomyocytes, stromal, and endothelial cells	Agarose microwells	When subjected to hypoxic and ischemic conditions in vitro, cardiac organoids treated with molidusat increase their lifespan	[26]
15	Kidney organoids	hiPSCs	Low adhesion spindle-shaped bottom 96-well plates	Ureteric bud and metanephric progenitors that are produced from hiPSC recapitulate nephrogenic niches. Interconnected kidney organoids are produced by ureteric buds and hiPSC-derived progenitors	[27]
16	Midbrain organoids	Small molecule neural precursor cells (smNPCs), originate from pluripotent stem cells (PSCs)	Standardized mechanical stresses and matrigel embedding by using an ALHS	Organoids are characterized by relatively homogenous appearance, size, cellular makeup, and structure	[28]
17	Cortical brain blood–brain barrier and liver spheroid model	hiPSC	Matrigel and Microfluidic systems	The cutting-edge multi-organ system allowed for the determination of metabolite distribution at the blood–brain barrier as well as parent compound concentrations	[29]
18	Neural tube organoids	hPSC	PEG hydrogels	The methods shown here have broad applicability and should make it possible to produce more programmable and reproducible organoid shape, patterns, and identity, opening up possibilities for their usage in disease modelling and regenerative medicine applications	[30]
19	Cerebral organoids	Induced pluripotent stem cells (iPSCs) with APOE ε3/ε3 or ε4/ε4 genotype	U-bottom ultra-low-attachment and matrigel	APOE4 may be a prospective therapeutic target for Alzheimer disease (AD) since isogenic conversion of APOE4 into APOE3 reverses several of the AD-related symptoms in cerebral organoids from AD	[31]
20	Cerebral organoids	Human iPSC	U-bottom ultra-low-attachment, Matrigel and orbital shaker	Findings provide APOE4 mechanism of action in raising the risk for synucleinopathies by demonstrating the predominance of apoE in lipid metabolism and Syn disease in cerebral organoids produced from iPSCs	[32]
21	Cortical organoids (human cortex)	Human induced pluripotent stem cell lines and embryonic stem cells line	v-bottom low adhesion plate, orbital shaker and matrigel	Organoids have fewer cell subtypes than original tissue, and they frequently co-express marker genes, leading to broad type designations like pan-radial glia or pan-neuron	[33]
22	Thymic organoids	Hematopoietic stem and progenitor cells (HSPCs), human thymocyte, and MS5-mDLL4 and MS5-hDLL4 cell lines	Transwell insert	HSPCs from all sources can be used in an artificial thymic organoid (ATO) system to support very effective and repeatable positive selection and in vitro differentiation of human T cells	[34]
23	Thymic organoid	Murine Bone Marrow HSPCs, murine and human thymocyte, and MS5-mDLL4	Transwell insert	Highly conserved metabolic changes are essential for the formation of thymic T cells	[35]
24	Thymic organoid	IPSC from Fibroblasts were cultured from a skin biopsy specimen obtained from the RAG2 deficient patient and MS5-hDLL4 cells	Transwell Insert	A promising therapeutic strategy for treating this immunodeficiency may be targeted gene editing	[36]
25	Artificial Thymic organoid (ATO)	MS5-hDLL4 with CD34 cells from patients carrying RAG mutations	Aggregating	The ATO system may be used to pinpoint the precise stage at which T-cell development is inhibited and establish whether T-cell shortage is caused by hematopoietic or thymic intrinsic defects	[37]
26	Mammary Organoids	Non-tumorigenic human breast epithelial cell lines MCF10A and MCF12A	Rat tail collagen I, LDEV-free Matrigel, growth factor–reduced hydrogels	To create all-human 3D culture systems, human collagen hydrogels can be used for organoid 3D printing	[38]
27	Mammary organoids	Human IPSC	3D floating mixed gel culture	It is possible to in vitro drive human iPSCs toward mammary lineage development. This study findings offer an iPSC-based model for investigating the control of breast disease development as well as modulation of normal mammary cell destiny and function	[39]
28	Retinal Organoid	Human embryonic stem cell (hESC)	Low attachment and orbital shaker	encourages the creation of regenerative medicine treatments that use hESC-derived retinal organoids (hESC-retinal implants) as a means of preventing vision loss	[40]
29	Retinal organoids	Embryonic stem cell	Low attachment v bottom	A method for effectively producing big, 3D-stratified retinal organoids without the need to remove features resembling optic vesicles, which had previously reduced the organoid yield	[41]
30	Retinal organoids	Human Pluripotent Stem Cell Human fetal retinal tissue	Poly-L-lysine (Sigma-Aldrich) and laminin	In order to enrich for human photoreceptor cells, a combination of a fluorescence-activated cell sorting method and cell surface marker monoclonal antibodies can be applied. This method isolates photoreceptors without genetic alteration or insertion of reporter genes, which is a crucial prerequisite to create clinical medicines	[42]
31	Retinal organoids	Embrionic stem cells and fetal retina	3-D free floating serum-free	Human fetal retinal chromatin accessibility shows transcription factor cascades. Organoids generated from hESCs with accessible chromatin exhibit preserved development. Notch signaling differs between fetal retina and retinal organoids	[43]
32	Adipose organoid model	Adipose stem/progenitor cells (ASCs) isolated from white adipose tissue (WAT)	Hanging drop technique and transferred into agar-coated cell cultur	Strong elevation of fatty acid-binding protein 4 (FABP4), adiponectin, peroxisome proliferator-activated receptor (PPAR) and CCAAT/enhancer-binding protein (C/EBP) expression during adipose organoid development	[44]

iPSCs = induced pluripotent stem cells, hPSCs = human pluripotent stem cells, Cor.4U-CMs = Cor.4U Cardiomyocytes, TDO2: Tryptophan 2,3-Dioxygenase, CYP1A1: Cytochrome P450 1A1, CYP1A2: Cytochrome P450 1A2, MSCs = mesenchymal stem cells, 3D: Three-Dimensional, AD: Alzheimer's disease, ALHS: automated liquid handling system, APOE: Apolipoprotein E, ARPKD: Autosomal Recessive Polycystic Kidney Disease, ASC: adipose stem/progenitor cells, ATO: artificial thymic organoid, BME: basement membrane extract, C/EBP: CCAAT/enhancer-binding protein, CN99: QGel CN99 (a type of gel used in research), Cor.4U, CMs: Cardiomyocytes Derived from Human Pluripotent Stem Cells, COVID-19: Coronavirus Disease 2019, CRISPR/Cas9: Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9, FABP4: fatty acid-binding protein 4, HCFs: Human Cardiac Fibroblasts, HCMECs: Human Cardiac Microvascular Endothelial Cells, hESC: human embryonic stem cell, hiPSCs: Human Induced Pluripotent Stem Cells, hPSC: Human Pluripotent Stem Cell, hPSCs: Human Pluripotent Stem Cells, HSPCs: hematopoietic stem and progenitor MS5-mDLL4, HUMIMIC: Human-Microbial Cross-Talk Model, hyCM: Human Cardiomyocyte, ICC: inverted colloid crystal, iPS: induced pluripotent stem cells, iPSC: induced pluripotent stem cell, ALHS = automated liquid handling system, LDEV: Large DNA Virus, MCF10A: Mammary Epithelial Cell Line 10A, MCF12A: Mammary Epithelial Cell Line 12A, MOC: multi-organ-on-a-chip, MODY3: Maturity-Onset Diabetes of the Young 3, MS5-hDLL4 cell lines: Human Stromal Cell Line Expressing Delta-Like Protein, MS5-mDLL4: Mouse Stromal Cell Line Expressing Delta-Like Protein 4, MSC: mesenchymal stem cells, PPAR: peroxisome proliferator-activated receptor, PSCs: pluripotent stem cells, RAG2: Recombination Activating Gene 2, RNA: Ribonucleic Acid, SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2, scRNAseq: single-cell RNA sequencing, sEVs: small extracellular vesicles, smNPCs: small molecule neural precursor cells, TGF: Transforming Growth Factor, WAT: white adipose tissue, Wnt: Wingless/Integrated, WNT3A: Wingless-Type MMTV Integration Site Family Member 3A

Result and discussion

We got 32 articles that were summarized in Table 2.

Organoid versus spheroid

Two-dimensional cell culture, which has long been the gold standard in in vitro biology, has been used in practically every significant discovery in cell biology. However, as our knowledge of biological complexity grows, so does our appreciation of the value of model systems that more accurately reflect tissues' genuine three-dimensional (3D) environment [45]. Organoids and human-induced pluripotent stem cells (hiPSCs) are crucial for in vitro human development and disease modeling. Organoids are three-dimensional cell clusters that are generated in vitro and replicate essential characteristics of real organs. Organoid technology in regenerative medicine is a promising method for replacing severely damaged organs [46].

Single-cell and multi-cell type spheroids are effective for enhancing and getting around the drawbacks of in vitro conventional systems. Spheroid culture is an easy-to-use and frequently used method for 3D cell culture that enables cellular interactions and more closely resembles physiological circumstances than conventional 2D cultures. By promoting cell–cell and cell–matrix interaction, which are important in many cellular mechanisms that consequently retain cellular features, a spheroid culture system offers a comparable physicochemical structure that closely resembles the in vivo tissue counterparts. Spheroids in long-term suspension culture frequently aggregate, which result in necrotic centers because there is insufficient movement of waste out of and nutrients into the aggregate [12]. Despite being frequently used in many biological researches, 2D cell line cultures are typically regarded as non-physiological since they lack tissue architecture and complexity and are typically immortalized. Suspension culture is used to create the 3D culture system in order to prevent direct physical contact with the plastic dish. Scaffolding or approaches without scaffolding can be used to accomplish this. Organoids are characterized as an organized 3D structure that is formed from various types of stem cells, in which cells spontaneously self-organize into a variety of functional cell types or progenitors, to obtain some of the properties of native tissue. Organoids imitate an organ's in vivo structure and complexity (Table 2) [1].

Advantages of organoids over spheroids for broader clinical applications in regenerative medicine

Since organoids mimic the complexity of native tissues, including the multicellular identity of an in vivo tumor, they are more medically relevant. Therefore, they are a superior choice for researching the causes of diseases and creating customized therapies [47]. Spheroids can generate data with low clinical translational potential, even if they are helpful for drug screening and toxicity assessment because they lack the multicellular identity of an in vivo tumor [48]. Because of their ability to self-renew, organoids can be cultivated for several months. This characteristic allows for repeated testing and long-term investigations, which is essential for regenerative medicine applications [49, 50]. Genetic modifications can be applied to organoids, which is crucial for comprehending the mechanisms behind disease and creating specialized treatments. The versatility of genetic modification increases its usefulness in regenerative medicine [51]. From patient cells, organoids can be made, offering models relevant to the patient for examining the course of the disease and evaluating treatment outcomes. A personalized strategy is essential for regenerative medicine to customize treatments for each patient effectively [52]. Organoids have demonstrated therapeutic solid use, mainly searching for new cancer treatments. Compared to tumor spheroids, they indicate a patient's reaction to medication because they may be created directly from primary tumor tissue or patient-derived xenografts [53, 54].

Organoids frequently need particular cofactors to cause differentiation and particular medium conditions to ensure the survival of their cultures. Because of their complexity, producing them may be costly and impracticable [55]. Production has a great deal of batch-to-batch variability since the matrices used to generate organoids are frequently sourced from animal tissue. This variability may impact the accuracy and repeatability of these models [56].

Methods of organoid production

The 3-dimensional culture system is constructed to prevent direct physical contact with the plastic. This can be done with or without the use of a scaffold. The scaffold is made of artificial or biological hydrogels that resemble the extracellular matrix (ECM) present in nature. The central part of the tissue/organ developmental model is to mimic the physiological orchestration conditions that generally exist in the human body, where different cellular and ECM components come together to generate a particular microenvironment for each kind of tissue/organ [2].

Adult stem cells and pluripotent stem cells are the two primary biological sources from which organoids can be produced. Since pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), may develop into any cell, they are perfect for producing a variety of organoids. These cells are grown in culture, ESCs or iPSCs are then differentiated into distinct cell types utilizing growth factors and medium conditions, and lastly, the cells are sown into a matrix, such as Matrigel or synthetic extracellular matrix, to produce organoid structures. [8, 57, 58]

Tissue-specific organoids may be produced using adult stem cells (ASCs), obtained from particular tissues and can develop into multiple cell types within their original tissue. To create organoids, adult tissues are separated, ASCs are grown in culture, differentiated using tissue-specific factors, and then seeded into a matrix. Specific extracellular matrices (ECMs) and microenvironmental cues are necessary to form organoids to promote growth and maturation. The creation of artificial extracellular matrix (ECM) and sophisticated culture systems is the primary goal of current research to enhance reproducibility and consistency [8, 58–60].

Currently, researchers have access to a variety of 3D culture systems, including spheroids, more complicated organoids, on-chip organs, 3D single-cell types, co-cultures with microfluidic flow control, and hybrid 2- and 3-dimensional culture systems using biomedical microelectronic devices. Organoids can be created using a variety of techniques, including mechanically assisted culture for primary tissue differentiation, self-assembly on ECM-coated surfaces, by floating culture to form embryoid body-like aggregates using ultra low-adhesion vessels, or using hanging drop culture method in serum-free conditions. Organoids developed from tissue-specific stem cells are now more effective than cell lines as in vitro models for studying physiological and pathological processes. As a result, the development and testing of drugs and customized treatment are huge prospective applications of organoid technology [61–63].

The broader subject of tissue engineering and regenerative medicine encompasses human organoid biology and microfluidics technologies, which are used to develop in vitro chip-based models for investigating the (patho-) physiology of human organs. Regenerative medicine uses various techniques, such as cell-based therapies, tissue engineering, and the creation of biomaterials, to replace or repair diseased or damaged tissues and organs.

Liver organoid

With a high rate of morbidity and mortality, liver failure is one of the most serious health issues in the world. The gold standard treatment for liver failure, such as in end-stage liver disease is still allogeneic liver transplantation, which offers a better quality of life while also being cost-effective. However, a lack of donor organs has led to longer transplant waiting lists. By creating bioengineered organs that can support hepatic physiological processes like detoxification, protein synthesis, and the creation of bile, which is essential for digestion, to support regenerative medicine seeks to solve this problem. Consequently, this strategy aims to finish the so-called “halfway technology, which means an approach or tactic intended to close the gap between the present state of technology or medical care and the ideal or desired state or to offer an alternative.

Recently, a liver regenerative medicine-based strategy has been investigated to support the liver till transplantation using bio-artificial devices (BALs) and a liver-on-a-chip platform. BALs could remove toxins that build up in liver failure and provide liver cell function to help the liver in terms of its regulatory and synthetic activities [62].

Future approaches to regenerative medicine show considerable promise for liver organoid technology. According to recent studies, it is possible to reliably create isogenic (genetically identical) liver organoids using isogenic adult primary non-parenchymal cells or liver parenchymal and non-parenchymal cells derived from iPSCs. This can be accomplished by employing either genetically modified or produced from the same source isogenic adult primary non-parenchymal cells or liver parenchymal and non-parenchymal cells from induced pluripotent stem cells (iPSCs). Researchers can limit genetic diversity and provide more dependable and consistent models for studying liver biology and disease by ensuring the cells used to construct organoids are isogenic. However, using entire iPSC-derived cells may cause significant translational problems, i.e., immunogenicity, tumorigenicity, heterogeneity, and safety concerns. Organoid technology advancements have increased the range of conditions and diseases that can be treated using human iPSC-based regenerative medicine; however, organoid mass production and the creation of supplements and animal origin-free chemically defined (AOF-CD) media are still open problems that limit the clinical viability of these strategies. AOF-CD medium and supplements guarantee the reproducibility and quality of culture systems by reducing variance from lot to lot and the danger of virus or toxin contamination [15, 64].

A large-scale organoid production platform was constructed using endoderm, endothelial, and mesenchymal progenitor populations that were derived completely from human iPSCs, and was reproducibly proven to be functioning both in vitro and in vivo. The production of human organoids from iPSCs opens up promising opportunities for cell treatment. Significant progress toward accomplishing those aims has been hampered by the reliance on animal-derived matrices like Matrigel, immortalized cell lines, and the difficulties involved with managing and scaling up the resulting structures [6, 17].

Cholangiocyte organoids

Organoids that are made of cholangiocytes have the potential to treat cholangiopathies and are capable of regeneration of the cholangiocytes. Organoids that are made from cholangiocytes are regarded as promising cell sources for this disease. An innovative method of cell engraftment in ex vivo normothermic perfused human livers is used to show how extrahepatic cholangiocyte organoids can repair human intrahepatic ducts after transplantation. Cholangiocyte-derived organoids offer a potent tool for bile duct epithelial characterization and cholangiocyte expansion for tissue engineering applications.. However, the initial development of organoids required invasive tissue biopsies through high-risk surgical procedures [19, 20].

Sampaziotis et al. established a proof-of-concept that human biliary epithelium could be repaired using cholangiocyte-derived organoids. According to single-cell RNA sequencing analysis, intra- and extra-hepatic cholangiocytes are similar, and thus they are interchangeable for regenerative medicine applications, and provide a new model for cell transplantation in human livers [19]. Recent studies showed that it was possible to culture cholangiocyte organoids from bile that was collected by minimally invasive method via endoscopic retrograde pancreaticography (ERCP). The cellular location of origin of these bile cholangiocyte organoids (BCOs) and a thorough examination of them have not yet been proven. BCOs are described by Roos et al., who compare them to the established organoids derived from intra- and extrahepatic biliary duct tissue. They showed that extrahepatic bile taken from the gallbladder after surgery and percutaneous transhepatic cholangiography from a range of individuals can successfully initiate cholangiocyte organoid. These three bile sources all produced BCOs, and all of them exhibited characteristics of in vivo cholangiocytes. The exclusive expression of regional common bile duct genes (HOXB2 and HOXB3) by gallbladder-derived and ERCP-derived BCOs, which expressed gallbladder-specific genes represented the regionally distinct properties of the BCOs. Furthermore, compared to intra-hepatic cholangiocyte-derived organoids, BCOs exhibited less potential for hepatocyte-fate differentiation. These findings suggest that the organoid-initiating cells in bile are probably extrahepatic in origin rather than intrahepatic [20].

Intestinal organoid

The modeling of both healthy and pathological tissues has greatly advanced with intestinal organoid technology. These systems, however, typically lack genuine tissue architectures and ECM, and as a result, they are still far from accurately simulating physiological circumstances. Recent technological developments have made it possible to separate natural cellular matrix that has retained ECM and regular three-dimensional (3D) tissue architecture, which offers a potential new method for creating more physiologically accurate intestinal organoid. Basic intestinal physiology and pathophysiological mechanisms, such as TNF-induced epithelial cell death during inflammatory bowel disease, are being studied using intestinal organoids. Organoids, which are aggregates of multiple tissue-specific cell types containing 3D structures that mimic native organs, can be formed by direct differentiation of human pluripotent stem cells due to temporal manipulation of growth factors and in vitro environment. Therefore, understanding of early organoid formation mechanism is essential to improve the potency of these methods, before using them in regenerative medicine [63, 65, 66].

According to Arora et al., 13% of early-stage spheroids with varying size, shape, and cell density were transformed into intestine organoids. They sorted the spheroids using an automated micropipette system and discovered that a diameter greater than 75 µm and a distinct core mass were crucial elements to enhance pre-organoid populations by 1.5 and 3.8-fold, respectively. These results provide crucial criteria for developing a reliable procedure to create high-quality intestinal organoids, notably by engineering process to increase pre-organoid yield from hindgut cultures that were produced from human pluripotent stem cells (hPSCs) [66].

A crucial step toward translational applications is the deployment of a fully defined ECM for the development and growth of intestinal organoids using cells that are taken directly from fresh biopsies. Numerous studies, therapeutic uses, and regenerative medicine use intestinal organoids. The use of animal tumor-derived basement membrane extracts (BMEs) in intestinal organoid research has slowed down the development of clinical applications due to their inadequate characterization and ineligibility for regulatory approval, primarily due to their origin and the variation seen in different batches. The move to clinical use has been partially hampered by this fact [21, 65].

Pancreatic organoid

A significant milestone in this endeavor would involve isolating human pancreas organoids (hPOs) in a chemically specified, serum-free culture medium. In the realm of pancreatic tissue engineering, the difficulty in producing human pancreatic islet organoids from stem cells has largely been attributed to our limited understanding of the essential microenvironments (niches) that are required for multicellular tissue self-assembly in vitro. Several studies aimed to address this knowledge gap and enable the successful generation of hPOs [46, 67, 68].

A methodology for differentiating hiPSC into beta-like cells and pancreatic progenitor (PP) organoids was published by Pedraza et al. The seeding and expansion of hiPSC, PP differentiation, organoid expansion, and PP differentiation into beta cells were all covered in depth. Organoids produced beta, delta, and alpha cells after differentiating. Dossena et al. created a large-scale method for producing therapeutically significant amounts of undifferentiated organoids using discarded pancreatic tissues, which avoided the need of operator-dependent pancreatic duct plucking and enzymatic processing. They implemented a freezing process that complied with GMP. The diameter and area of hPOs were expanded three- and eight-fold in 7 days, respectively, which demonstrated exponential growth. This discovery marks a turning point in the development of GMP-compliant hPOs and, ultimately, their clinical use in type 1 diabetes treatment [46, 67].

Cardiac organoid

A deeper understanding of the mechanisms behind heart development and disease has been made possible by stem cell-derived in vitro cardiac models. One of the key breakthroughs that permitted customized cardiovascular disease modeling methodologies was the efficient differentiation of particular cardiac cell types from human pluripotent stem cells utilizing a three-step Wnt signaling regulation. The formation of different cardiac cell types that coincides with important developmental phases during embryogenesis, has made them as good models for studying the patterning of heart tissue in early cardiac structures. Exciting progress was achieved by stem cell biologists and tissue engineers in developing aligned monolayers or simplified models of human heart muscles to close the gap between clinical trials and experimental animals [24, 69].

There are currently no in vitro methods available in regenerative medicine that can evaluate the efficiency of the heart's pumping. Li et al. invented an advanced biomimetic model by using human ventricular cardiomyocytes (hvCM) that were derived from human pluripotent stem cells, which were embedded in a collagen-based extracellular matrix hydrogel, to build a three-dimensional (3D) electro-mechanically connected cardiac organoid chamber (COC). This human ventricular cardiac organoid chamber (hvCOC) resembled the human ventricle in important physiological and molecular aspects and responded properly to diverse pharmacological interventions. They contend that by giving early-stage drug development human-specific preclinical data, this "human heart in a jar" technique has the potential to hasten medication discovery in the area of regenerative medicine [24].

It has been demonstrated that pre-vascularized 3D micro-tissues are a useful cell delivery platform for heart repair. In agarose microwells, Coyle et al. co-seeded human cardiomyocytes with cardiac fibroblasts, endothelial cells, and stromal cells to study the creation of self-organizing, pre-vascularized human cardiac organoids. They proposed that the endogenous stimulation of the hypoxia-inducible factor (HIF) pathway in the avascular 3D micro-tissues facilitated this pre-vascularization process. They showed that targeted HIF-stabilization offered a reliable method for enhancing endothelial expression and lumen creation in cardiac microtissues, which would offer a strong foundation for pre-vascularization of different micro-tissues in the development of effective cell transplantation therapies [26].

Kidney organoid

The kidneys are crucial organs that maintain fluid and electrolyte homeostasis while filtering the blood and eliminating urine waste. Animal models and static cell cultures that were used in conventional research today were either inadequate to understand the complicated human in vivo condition or have no practical application. It is crucial to investigate the molecular and morphological aspects of nephron patterning in mice and humans, looking at both their similarities and differences. The kidney's functional unit is called a nephron. A fundamental framework for nephron creation has been established in a mouse research however, it remains uncertain how nephrons arise in the human kidney [4, 24].

The pronephros, mesonephros, and metanephros are the three nephric stages in the development of the mammalian kidney; they are all derived from the mesoderm. According to studies that have been done on mice, the kidney organoids that are known as the ureteric bud (UB) and metanephric mesenchyme (MM), which are produced from IM (Intermediate Mesoderm), are reciprocally induced to become the adult metanephros. All of the expected components of the fetal human kidney, including patterned and segmented nephrons, have been seen in these organoids [4].

In their analysis of human embryonic kidney development, Lindström et al. found that, despite species-specific dynamics, similar inductive processes were active in mouse and human kidney development. In order to compare the formation of cellular variety during mouse and human nephrogenesis, they used high-resolution transcriptional factor mapping. Deep conservation was seen in the emerging patterns, probably due to shared regulating mechanisms between man and mice. These findings benchmark human nephron development and address the first appearance of mature cell markers within developing nephrons. They will also guide developing in vitro model systems replicating typical human nephrogenesis [70].

Koning et al. initiated and studied vasculogenesis in kidney organoids via intracoelomic transplantation in chicken embryos, followed by single-cell RNA sequencing and cutting-edge imaging techniques. Their study showed that human organoid-derived endothelial cells (ECs) divide and rearrange to produce functional capillaries, integrating with the host's blood vessels to generate a hybrid vascular network. After transplantation, they discovered a variety of possible interactions between human ECs and perivascular cells using ligand-receptor analysis, which supports the stabilization of the arterial walls. Perfused glomeruli advanced to the capillary loop stage and began to exhibit symptoms of maturation. Their findings demonstrate that vascularization has a beneficial effect on the mesenchymal compartment as well as the epithelial cells. It also encourages the growth of specific perivascular stromal cells, which are necessary for the continued development and stabilization of the newly established vasculature. The ability of kidney organoids to form blood vessels is essential for obtaining notable glomerular maturation and kidney morphogenesis in vitro, as explained in this article [71].

Kidneys are capable of healing themselves after damage, but severe or recurrent damage causes partial healing and fibrosis, which exacerbates the condition and makes chronic kidney disease (CKD) more likely. Gupta et al. showed that the transition from intrinsic to partial repair could be modeled using kidney organoids. They found differently expressed genes and pathways in tubular cells undergoing repair following cisplatin exposure using single-nuclear RNA sequencing. FANCD2 and RAD51, two genes implicated in homology-directed repair, were up-regulated during repair but down-regulated in incomplete repair. SCR7 was found through drug screening to be a viable treatment to stop the progression of CKD by re-establishing FANCD2/RAD51-mediated repair. Their research emphasizes the potential of renal organoids in CKD modeling and treatment discovery [72].

Brain organoid

An innovative new technology to develop brain organoids has the potential to fundamentally alter how disorders of the brain are recognized and treated. These three-dimensional neural tissues, which mimic the development of the human brain down to the earliest cortical layers and progenitor zones, are produced when pluripotent stem cells self-organize. Investigating various facets of comparative biology and developmental neurobiology have benefited from the use of brain organoids. Organoids are appealing as models for brain illnesses due to a number of properties. Tools for neural regenerative medicine and tissue engineering research include lab-on-a-chip devices and ex vivo models for researching the regeneration of peripheral nerve, spinal cord, and brain tissues. For brain tissue engineering applications, the need for ex vivo systems, lab-on-a-chip technologies, and disease models is rising to overcome the limitations and shortcomings of conventional in vitro systems and animal models [73, 74].

Ex vivo models have experienced substantial progress, ranging from conventional 2D cell culture models to more sophisticated 3D tissue-engineered scaffold systems, bioreactors, and the more recent invention of organoid test beds. Through the combination of gene regulatory networks, cell–cell communication, and physical interactions mediated by mechanical forces, tissues in the body develop their sophisticated spatial organization. However, rather than actively applying mechanical stresses, most current techniques for creating in-vitro tissues predominantly rely on extracellular matrices. As a result, these techniques typically need help simulating the dynamic and coordinated mechanical manipulations in real tissues [30, 74].

The development of the human brain is a complex process that requires the carefully scheduled coordination of cell division, differentiation, migration, and integration of various cell types. The inability to prospectively research human neurodevelopment at the molecular, cellular, and system levels, as well as the limited availability of fetal brain tissue, have severely restricted our understanding of these fundamental processes. Although non-human model species have offered significant insights into the mechanisms underpinning brain development, these systems do not completely recapitulate many human-specific traits that are frequently associated with disease [75].

Thymic organoid

Strong model systems that replicate the entire course of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells, are necessary for studies of human T cell development. Human HSPCs are committed to T cells in existing in vitro models; however there are a few differentiations into mature CD3+ TCRab+ single positive (SP) CD8+ or CD4+ cells. In vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs are supported by a serum-free, artificial thymic organoid (ATO) system that Seet et al. described. ATOs offer a powerful tool for researching tailored T cell therapeutics that are based on stem cells and human T cell development [34].

The limitations of in vitro T-cell differentiation tests and the lack of thymic samples make it difficult to research early T-cell development in humans. Bosticardo used an artificial thymic organoid (ATO) platform to study T-cell development from CD341 cells of patients who carried hematopoietic intrinsic or thymic defects that resulted in T-cell lymphopenia. The platform was created by combining CD341 cells that were isolated from mobilized peripheral blood or bone marrow with a DLL4-expressing stromal cell line (MS5-hDLL4). The ATO system may be used to pinpoint the precise stage at which T-cell development is inhibited and establish whether T-cell shortage is caused by hematopoietic or thymic intrinsic defects [37].

Despite the fact that the impact of metabolic pathways on the differentiation and activation of peripheral T cells has been demonstrated, little is known about the metabolic mechanisms that control the development of thymic T cells, particularly in human tissues. Transcriptomics and extracellular flow analysis were used in a study by Sun et al. to look at the metabolic characteristics of primary thymic and in vitro-derived mouse and human thymocytes. Their findings showed that the decrease in metabolic activity that was seen at the double-positive (DP) stage of T cell development is not caused by a requirement for the rearrangement of the T-cell receptor (TCR). They conclude that highly conserved metabolic changes are essential for the development of thymic T cells [35].

Mammary organoid

Human iPSCs show considerable potential in regenerative medicine and disease-modeling applications since they can give rise to various cell types. Mammary epithelial cells are commonly cultured using conventional three-dimensional (3D) in vitro procedures, which rely on the random distribution of cells within hydrogels. Despite these systems' advantages over traditional 2D models, there are still certain restrictions because it is impossible to precisely control where the cells are placed within the hydrogel. This leads to inconsistent experimental outcomes and variable organoid shape. This results in varied experimental outcomes and organoid shapes that are not controlled or predictable. More standardized 3D epithelial culture methods are required for robust, high-throughput experiments [38, 39].

Qu et al. have created a trustworthy two-step technique to produce human mammary-like organoids from iPSCs. These organoids could be stimulated to make milk protein and displayed common luminal, basal, and breast tissue markers, including estrogen receptors. These findings show that human iPSCs can be stimulated in vitro to differentiate into mammary lineages. Their findings offer a model for investigating how normal mammary cell destiny and function are regulated and how breast illness develops [39].

The use of a 3D bioprinting platform as a research tool to direct the 3D production of organoids by the "self-assembly" of human mammary epithelial cells was described in detail by Reid et al. Experimental bioprinting techniques enabled the controlled production of arrays of distinct mammary organoids. They specified the distance and cell number parameters that were required to manufacture both big continuous organoids, which were connected across many print locations, as well as individual organoids that did not interact between print places. According to this study, cells from different prints might combine to create structures with continuous lumens [38].

Retinal organoid

Pluripotent stem cells' adaptability opens up new avenues for research into growth, aging, and regeneration. Protocols for developing retinal organoids from embryonic stem cells have been created to increase photoreceptor generation or mimic the eyecup's complete morphogenesis. Millions of people worldwide have progressive vision loss due to retinal degenerative (RD) illnesses like age-related macular degeneration, retinitis pigmentosa, and Leber congenital amaurosis, which negatively influences the quality of life. One of the most significant unmet clinical needs is finding effective treatments for disorders that cause blindness. One of the primary causes of blindness in the industrialized world is the loss of photoreceptor cells due to retinal degeneration. Although there is no cure, cell replacement therapy employing photoreceptor cells that are produced from stem cells may offer a viable alternative [40–42].

Multiple cell types that are crucial for vision can be found in the complex tissue that is known as the retina. Potential uses for this knowledge in regenerative medicine include understanding of gene expression patterns of different retinal cell types. Through single-cell RNA-sequencing research, retinal organoids (optic vesicles) that are produced from pluripotent stem cells have started to shed light on the transcriptomics of developing retinal cell types in humans. Human pluripotent stem cells (hPSCs) can create an endless supply of three-dimensional retinal tissue using retinal organoid technology for regenerative medicine applications. The high similarity between the transplantable human fetal retina and organoid-derived retinal tissue offers a chance to test and model retinal tissue replacement strategies in certain animal models to create a functional retinal patch to restore vision in patients with severe retinal degeneration-related blindness. The chromatin landscape, as well as differences between hESC-derived retinal organoids and human fetal retina in term of Notch responses were compared by Finkbeiner et al. to profile accessible chromatin using single-cell ATAC-seq, and the results revealed transcription factor cascades that led to specific cell fates [43, 76, 77].

Organoid biobank

Organoid biobanks have already been created. Their emergence opens up new avenues for tailored drug screening and holds great promise for personalized regenerative medicine. Therefore, a large-scale, reproducible organoid synthesis with little variability is required, which renders manual procedures useless. Organoid biobanking is expanding quickly. Thus, it should be carefully examined to see if and how much organoids alter the ethical dilemmas in stem-cell research and related fields [14, 78].

European Biobanks often encounter several challenges, including establishing quality standards and standard operating procedures (SOPs) for material collection and storage, efficiently distributing collected materials, determining suitable governance models for the European context, ensuring long-term sustainability, and addressing cultural and legal differences in ethical, legal, and social implications (ELSI) [79]. The unprecedented potential to create self-organizing, three-dimensional tiny organs that closely resemble in vivo settings has been made possible by the invention of human organoid culture models. Such organoids can now be expanded, cultured, and banked, giving researchers a chance to create next-generation living biobanks that will significantly advance translational research in a variety of fields, such as drug discovery and testing, regenerative medicine, as well as the creation of a personalized treatment strategy. However, creating a living organoid biobank necessitates a far higher degree of coordination, additional resources, and scientific know-how than typical tissue repositories [80].

Conclusions

This review highlights the substantial advancements in organoid technology for regenerative medicine. Organoids accurately represent the human body's physiological circumstances and cellular interactions by cultivating cells in a three-dimensional environment. Using the organoid for regenerative study enables more precise and trustworthy experimental results. However, various issues still need to be solved before organoid technology can be successfully used in therapeutic settings. Problems with repeatability, scalability, and maturity hamper organoids' broad use in clinical applications.

Standardizing methods, utilizing bioengineering techniques to regulate microenvironments, and combining organoids with microfluidic devices have been the main focuses of current work to address organoids' reproducibility, scalability, and maturity barriers. Vascularization and tissue-tissue interaction engineering have been used to try and improve organoid maturity. Furthermore, the creation of complex functional readouts has attempted to enhance the evaluation of organoid functionality, increasing their dependability for scientific investigations.

Further studies and developments are needed to standardize organoid production methods, enhance scalability, and ensure long-term stability and maturation of organoids. Organoid technology, despite these difficulties, has enormous potential for biomedical research and regenerative medicine. Overall, in the last ten years, substantial progress has been made in organoid research. Still, further investigation and improvement of organoid models are required to fully realize their promise for clinical applications and transform the regenerative medicine industry.

Author contribution

WLS: Performed the experiment; Analyzed and interpreted the data; Wrote the paper, JAP: Analyzed and interpreted the data, wrote the paper, reviewed the manuscript.