3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research in Regenerative Medicine

Abstract

Regenerative medicine has been coming into spotlight ever since the realisation that conventional treatments are not enough, and the need for specific therapies has emerged. This, however, has paved way for cell-free therapy using extracellular vesicles. A two-dimensional (2D) cell culture model is widely recognised as the “gold standard” for researching cellular communications ex vivo. Although the 2D culture technique is straightforward and easy to use, it cannot replicate the in vivo ECM interactions & microenvironment. On the contrary, 3D culture culturing technology has emerged which include structures such as spheroids and organoids. Organoids are small replicas of in vivo tissues and organs, which faithfully recreate their structures and functions. These could be used as models to derive stem cells based EVs for manufacturing purposes. The linkages between infection and cancer growth, as well as mutation and carcinogenesis, may be modelled using this bioengineered platform. All in all, 3D culturing derived EVs serves as a novel platform for diagnostics, drug discovery & delivery, and therapy.

1. Introduction

Cell culture has become an indispensable tool for elucidating fundamental biophysical and biomolecular mechanisms that govern how cells construct into tissues and organs, how well these tissues function, and how that function is disrupted in disease. Cell culture is now used extensively in biomedical research, biomedical engineering, stem cell therapy, and commercial applications. In regenerative medicine, stem cells are the central players, however the shortcomings and risks associated with cellular therapy are higher as compared to non-cellular therapy thereby EVs are better. Although adherent, two-dimensional (2D) cell culture has long been the norm, recent research has shifted toward three-dimensional (3D) structures and more feasible biochemical and biomechanical microenvironments. Understanding the in vivo processes that leads to the formation and purpose of tissues and organs requires deciphering the mechanisms underlying these behaviours. Laboratory experiments should ideally be carried out using a user-defined three-dimensional (3D) model that closely mimics the cell’s microenvironment [1, 2, 3]. However, challenges in developing such a model include the building of the tissue-tissue interface, control of the spatiotemporal distributions of oxygen, carbon dioxide, nutrients or waste, and further followed by the customization of other microenvironmental factors known to regulate activities in vivo. It is well understood that cells adapt to their surroundings by reacting to local signals and cues, which has implications for cell proliferation, differentiation, and function [4, 5]. Traditional culture methods for growing mammalian cells in vitro are far removed from the complexities that cells encounter in real-life tissues. One of the most noticeable physical differences is the shape and geometry that cells acquire when grown on a flat substrate, such as a cell culture plate or flask. When cells grow on two-dimensional (2D) surfaces, they flatten and remodel their internal cytoskeletons. Lacking the ability to form more natural tissue-like structures, existing in vitro 2D cell culture models are frequently a poor substitute when used to study cell growth and various associated aspects [6]. This has a substantial impact on cell performance, as well as, the outcomes of biological assays. Monolayers of cultured cells, for example, are assumed to be more sensitive to therapeutics. Moreover, due to the limited cell interactions, culturing cells on rigid surfaces may increase cell proliferation, but adversely impact cell differentiation. A more adequately engineered cell culture environment might enhance drug discovery predictive accuracy and aid in the interpretation of tissue morphogenesis [4, 5, 6].

Some significant aspects of cancer cells, for example, cannot be adequately modelled in 2D cultures. To overcome the limitations, novel 3D cell culture platforms that better mimic in vivo conditions are now being developed, which are sometimes referred to as spheroid or organoid culture. In many cases, these new platforms have shown to be more capable of stimulating in vivo-like cell fates for the processes under investigation. 3D research shows that increasing the dimensionality of the extracellular matrix (ECM) surrounding cells from 2D to 3D has a significant impact on cell proliferation, differentiation, mechano-responses, and cell survival [5, 7, 8].

For example, Extracellular vesicles (EVs) are membrane-enclosed structures that are released by almost all cell types. They transport biologically active molecules such as RNAs, lipids, and proteins from the delivering cell to the target cell, allowing for a novel mode of intercellular communication. The use of EVs as diagnostic tools is highly influenced not only by the molecular cargo but also by the quantity of EVs derived from various cell subpopulations in tissues and body fluids. Moreover, overall mechanisms and factors influencing EV release are still unknown [9, 10]. Organoids are obtained from animal or patient samples, cultured in 3D matrices like Matrigel under well-defined conditions, and retain the cellular heterogeneity found in in vivo epithelial tissues. As a result, they depict one of the most cutting-edge technologies for studying human diseases, allowing for the investigation of pathways and factors that influence EV release.

Although these findings suggest that 3D systems should be used anytime feasible, the system of choice is often governed by the specific process of interest, and there is currently no universal 3D platform; additionally, 2D cell culture approaches can still recapitulate in vivo behaviour for many bioactivities, and new advances in substrate configuration continue to offer new capabilities for this platform [11]. All in all, 3D platforms are probable to become a more appealing alternative to 2D cell culture as technology advances to enable a broader range of processes. Technological advances have opened up new avenues for cell culture and the formation of 3D tissue-like frameworks. This is primarily due to research activities between cell biology and biophysical sciences, which has introduced new materials and manufacturing techniques to produce technologies tailored to support 3D cell growth in vitro [12]. The culture of cells in 3D is rapidly progressing, as evidenced by the increasing number of publications in the scientific literature. The adoption, validation, and implementation of these novel strategies will guarantee the effectiveness of this technology. This will most likely take time as the scientific community recognises the limitations of traditional 2D cell culture and recognises the value of new methods to reliably culture cells in 3D.

2. Technologies for 2D and 3D cell culture

2.1 2D cell culture techniques

Traditional 2D cell culture relies on the cells adhering to a flat surface, typically a glass or polystyrene petri dish, to provide mechanical support. Culturing in 2D monolayers allows the cell access to a significant amount of growth factors and nutrients in the form of media, resulting in homogeneous proliferation and expansion [13, 14]. However, a majority of these 2D approaches do not allow for regulation of cell shape, which influences biophysical cues that affect cell biological properties in vivo. Micro-patterned surfaces, such as cell-adhesive islands, microwells, and micropillars, have indeed been developed to control cell shape in 2D culture and aid in the investigation of the effects of cell shape on bioactive components [15]. This induced polarity may alternate cell functions such as expansion and migration for perceiving soluble components and other microenvironmental signals. One of the strategies to completely eradicate apical-basal directionality is by the sandwich culture procedure, which adds a layer of ECM atop the cells and provides a mimic of 3D ECM that can be used to mitigate the effect of cell polarisation in 2D cell culture (Figure 1). The sandwich culture method, which involves placing cells between two layers of ECM, polyacrylamide, collagen or any type of suitable ECM, has long been shown to produce cell cultures with morphology and function that more closely resemble the in vivo behaviour. This is especially important in drug discovery, in which scientists aim to understand pharmacokinetic profile in relation to the organs [15]. The sandwich method was discovered to reduce oxygen diffusion sufficiently, resulting in an 80% survival rate over 5 days, compared to a 32% success rate over 2 days in a mixing process population culture. Many researchers have been able to examine the consequences of pharmacokinetics, which is crucial to consider when modelling physiological and pathological events, thanks to the sandwich culture [16, 17]. Another strategy could including micropatterning, which is a designed 2D surface that allows imprinting and alteration to create a 2D microenvironment for cell culture that contains distinct physiochemical factors, topography, stiffness, and mechanical load. In a typical 2D cell culture, cells are subjected to a homogeneous surface free of defects that could interfere with their development [18, 19]. Moreover, cells cultured on patterned and un-patterned PLLA surfaces differentiated at a slower rate than cells cultured on tissue culture-treated polystyrene (PS), the experiment’s control. In terms of lipid production, it was discovered that a later time points, shaped PLLA surfaces produced the most lipids, followed by PS, and then non-patterned PLLA [20, 21]. The findings revealed that the micro-patterning and surface type both influenced the rate of cell differentiation. Overall, since the early 1900s, two-dimensional (2D) cell culture has been the technique used to culture cells, which plays an important role in research but has many drawbacks due to 2D models imprecisely representing tissue cells in vitro [22, 23, 24].

Figure 1.

The representative diagram showing different kinds of cell culture techniques and their impact on cells fate.

2.2 3D cell culture techniques

2.2.1 Aggregate cultures and the formation of spheroids

A 3D culture model is supposed to provide a tissue-like microenvironment in which cells can proliferate, aggregate, and differentiate. This could have an application in predicting the effect of a drug on cells. For several reasons, cells cultured in 3D respond differently to drugs than cells cultured in 2D [25, 26]. Changes in physical and biological features between 2D and 3D cultures make 2D cultured cells more susceptible to drug effects than 3D cultured cells because 2D cultured cells cannot maintain constant morphology like 3D cells. Since 3D cultured cells have greater depth than 2D cells, the variation in shape between 2D and 3D cultured cells creates a change in local pH levels within the cells. Lower intracellular pH levels have been shown to reduce drug efficacy, giving back to drug resistance. Further, Microfluidics, microchips, embryoid bodies (EBs), collagen gels (GELs), and hanging-drop culture are all methods for spheroid cultures [26, 27]. Various studies have established the experiments in two different 3D culture methods based on the differentiation and proliferation of embryonic stem cells (ESCs). To promote adherence, cells were cultured as embryoid bodies (EBs) in either a collagen type I gel (GEL) or in non-tissue culture-treated dishes. GEL and unattached EB cultures produced cluster morphology that was similar, with defined boundaries and the occasional cavity. The existence of enlarged masses along the edges of the gel form presented a marked difference in the morphology of GEL cultures. Over the same 12-day period, free EBs had more continuous change in the genotypic expression profiles of cytoskeletal genes than GEL cultures [28, 29]. These findings suggest that, despite having similar morphologies, the gene expression of each kind of 3D culture is unique in its adaptation to its microenvironment. Similarly, hanging drop method is another technique to establish spheroids but because of the difficulty in maintenance measures, like changing of media, the traditional hanging-drop method does not allow for extended cell culture.

All such 3D scaffolds and associated cell-encapsulation techniques provide valuable tools for understanding how the ECM influences cell fate. During the last decade, major advances have been made in the techniques for encapsulating cells in 3D using tissue engineering scaffolds with customised biochemical and biophysical components. Majorly, biopolymers derived from animal tissues are especially popular because they contain similar biochemical components to those found in cells’ native tissue and may encourage tissue regeneration. However, one of the most pressing issues is the inability to individually control the key elements required in modulating cell bioactivities, such as biochemical properties, matrix elasticity, and macro-porosity [26, 28, 29]. Therefore, a prefabricated scaffolds has the advantage of a configurable biochemical composition, matrix elasticity, and micro-architectures. These scaffolds can be made using polymer phase separation, 3D printing, lyophilizing, gas foaming, stereo-lithography and porogen leaching with soluble templates to form pores or channels. However, current methods for creating prefabricated scaffolds frequently involve procedures that create circumstances which are too severe for cells to survive, such as extreme pressure, non-physiological osmotic pressure, and the use of organic solvents [28, 29]. As a result, cell diffusion is primarily used to deliver cells into scaffolds, and this method is frequently associated with low cell penetration rates and poor scaffold cellularization. Hydrogels made up of various types of biopolymers have been broadly used as scaffolds in contrast to prefabricated scaffolds because of their ease of cell encapsulation. They have tissue-like water content as well as effortlessly controllable biochemical and mechanical characteristics. Most hydrogels, on the other hand, are composed of micron/nanometre-sized mesh that is frequently too small to facilitate post fabrication cellularization and lack the microtopography required for influencing cell shape and supporting cell mobility, proliferation, and matrix production [30, 31, 32]. The main disadvantage of hydrogels in tissue regeneration is that matrix degradation simultaneously changes biochemical elements and matrix elasticity, both of which require careful control. Furthermore, matching the rate of hydrogel degradation with the rate of tissue formation is extremely difficult, which is essential for maintaining the shape and structural stability of tissue engineering.

Making multiple layers of cell sheets is another method for engineering organs and tissues without relying on constructed scaffolds. A plethora of studies have successfully replicated cardiomyocyte pulsatile function and functional dopaminergic neurons in a 3D construct by stacking multiple cell sheets or on the single cell sheet by twitching the mechanical properties of scaffold [29, 31, 32, 33].

Apart from this, bioreactors are designed to study cell behaviour during the development of micro-tissues or organs and to generate more cells for clinical use or laboratory research. Large-scale bioreactors involve simple systems like spinner flasks and rotating wall bioreactors, which enable for semi-adherent cell growth, in addition to more complicated systems like gravimetric bioreactors. The impacts of fluid transport on a cell membrane scale have been investigated using bioreactors with cell-sized conduits. Micro-bioreactors have also shown promise in drug screening and controlling the cell microenvironment.

2.2.2 Organoid formation from diseased microenvironment and microfluidic 3D cell culture

The shift from 2D to 3D culture techniques is a significant step toward more biologically relevant tissue models. However, 3D culture techniques do not yet capture the multicellular intricacies of tissues, they lack vasculature, do not provide precise control over gradients, and exchange medium at discrete time points rather than continuously. Microfluidic techniques enable spatial control of fluids in micrometre-sized channels, which can be used to investigate the biological significance of 3D culture models (Figure 1) [29]. Early examples of spatial patterning of adhesion molecules and hydrogels, which are still employed in microfluidic 3D cell culture, are depicted in Figure 1. The three foremost drivers for using microfluidic methodologies in 3D cell culture today are as follows:

1. The ability to co-culture cells in a controlled manner.

2. Generating and controlling (signalling) gradients.

3. Perfusion/flow integration.

An even more functional aspect that can be introduced using microfabrication techniques is mechanobiological aspects such as active stretch and tension. Microfluidic devices can conquer the drawbacks of traditional cell/stem cell culture techniques and tissue engineering approaches by better simulating in vivo interplay between ECM and cells thereby enabling high-resolution in situ imaging [25]. The combination of the unique benefits of microfluidics and the breadth of possibilities offered by stem cell technologies can also provide alternatives for the management of neurodegenerative diseases such as Alzheimer’s and Parkinson’s, as well as other disorders or injuries of the central or peripheral nervous system. This method has even progressed so far as to suggest the development of devices known as “brain-on-a-chip” [34, 36]. In neuro-regeneration, for example, these systems enable the development of uniform populations of neuronal and glial cells. The potential to co-culture cells in a 3D arrangement, better controlled signalling, and the ability to combine diffusion and laminar flow are the most significant advantages of microfluidic cell culture. Microfluidics allows researchers to precisely control stem cell/cell numbers and growth conditions, as well as arrange or design cells in spatially controlled positions and track cell responses to various internal/external mechanical, chemical, and optical stimuli [35, 36, 37]. Furthermore, microfluidics techniques enable high-throughput studies of single cells in microenvironments closely mimicking biologically relevant conditions by creating gradients of mechanical forces and different chemical agents. These benefits are classified into four categories: (a) fabrication characteristics, (b) Biomaterial ingredients, (c) Biochemical properties, and (d) bio-physico-mechanical (Table 1) [37, 38, 39].

S. no.	Properties	Benefits
1	Fabrication characteristics	In tissue engineering and biological research, soft lithography is the most commonly used method for fabricating microfluidic devices. This technique entails replica moulding, embossing, and printing. In this regard, following two topics should be cited: large scale assays and sensor integration. Microfluidic large scale integration can be achieved by parallelizing assays with valves or droplet-based microfluidics and designing a single platform to achieve a series of successive steps. This strategy can be used to investigate the consequences of various biochemical factors on stem cell behaviour and fate, as well as to develop controlled gene/drug delivery systems and nanotoxicological assays [40, 41].
2	Biomaterial ingredients	The biomaterial properties of microfluidics-based tissue engineering devices are composed of various factors; microfabrication, numerous biomaterial methods to ECM and biological materials have been used. The extracellular matrix (ECM) of cells and stem cells is made up of various biological molecules such as proteins, proteoglycans, and soluble factors. Distinct natural or synthetic materials were used to make these microdevices, which can then be modified to mimic the ECM [43, 44].
3	Biochemical properties	Various biochemical molecules have been employed in microdevices to better mimic the physiological properties of the target tissue. In their in vivo microenvironment, stem cells are exposed to a variety of soluble signalling cues, including extracellular calcium ions, various growth factors, nutrients, and oxygen. The most studied conventional and microfluidics strategies for this purpose are automated culture systems, soluble gradients, and temporal exposure regimens. Microfluidic systems can also be used to achieve automated temporal control of the delivery of soluble factors. A gradient generating microfluidic device, for example, was used to create a continuous growth factor gradient containing a mixture of PDGF, EGF, and FGF2 [45].
4	Bio-physico-mechanical	Controlling physical and mechanical factors such as spatial confinement, biomolecular tensions, shear stress, surface rigidity, and topography is possible with microfluidics. Other benefits of microfluidics in stem cell research include control over fluidic flow, which affects dynamic cell culture situations, the availability of shear stress as found naturally in different organs, and the availability of different medium compositions [46, 47, 48].

Table 1.

Benefits of different aspects of 3D cell culture.

Overall, the fabrication of an artificial human organ (even going as far as a brain) is beginning to be considered possible due to the multidisciplinary overlap of biology and engineering combined with emerging new trends such as microfluidics, stem cells, and nanotechnology; this has been imagined as a “Organ-on-a-chip.” Organs on a chip will provide much better mimicking of real human physiology and will be beneficial for tissue engineering, disease modelling, and drug screening; however, much more well-designed research in this field is still required [37, 38, 39, 49].

3. Impact of culture strategy on the secretion and content of extracellular vesicles

Extracellular vesicles are a novel modality in the scope of diagnosis, drug delivery & regenerative medicine. These are nanoscale vesicles which can be sub divided based upon their mode of synthesis, size and content. The sub category among them which has recently gained popularity is the small vesicles identified in the range of 30–150 nm & found to be synthesised via the endosomal route, also commonly known as exosomes. These vesicles are naturally found to be involved in mediating inter-cellular communication. They carry a diverse compass of functional molecules including DNA, RNA, miRNA, Proteins, Enzymes & many other are yet under discovery. The release & content of these vesicles are largely influenced by the cell microenvironment & the extracellular cues which are received in a mechanosensitive manner [9]. These vesicles happen to package the content from inside the cell & deliver them to the cell-in-need. These are primarily known to fuse with the cells & release their content in order to facilitate the functioning, however, at certain instances they might as well be phagocytosed. Recent gain of limelight have brought up diverse and divergent functions & applications of these small vesicles, for e.g. Tian et al., suggested the role of these vesicles in the detection of breast cancer. They suggested 8 EV proteins that could serve as a biomarker for diagnosing and differentiation between non metastatic and metastatic breast cancer [50]. Being membrane bound structures, these circulating EVs succeed in preserving their content & can be easily captured by any kind of normal cells. When released from the cancer cells, EVs have been evidenced to enhance the malignancy by causing malignant transformations in the recipient normal cells. Tumorigenicity and cancer spread are highly attributable to the intercellular communication in the tumour microenvironment & in the blood stream via the release of EVs. It was stated by Bebelman et al., that cancer cells are found to exhibit higher EV secretion as compared to the non-cancerous cells [51]. This could be attributed to the fact that cancer cells hold a diminish property of contact inhibition, therefore forming a tightly packed 3D layered & inculcated mass of cells which advances into a tumour. In order to study the biology of this deadly near-pandemic disease, it is essential that the conditions subjected to the tumour in vivo be replicated in in vitro set ups so that the exact nature & habits of a tumour could be elucidated. This has led to invoking the question regarding the culture conditions in which cancer cells & their fate are studied. The most popular choice of cell culture is based upon a 2D sub culturing method wherein the cells are allowed to form a sheet like structure and their propensities are studied [25, 27]. A 2D culture set up is popular due to its ease of handling and other properties as discussed in the previous section, however it does not necessarily confirm that the results being obtained from such a set up are actually a simulation of the in vivo scenario. On that account, 3D culture is the recent technique of choice of researchers as it is expected & anticipated to model the tumour microenvironment in vivo. Pertaining the same, the EVs which are released from the 3D kind of culture have also been found to mimic the in vivo secretions in a more identical manner. Upon comparison of EVs from 2D and 3D culture of the same cell types, it was also found that there were differences in both EV secretion and EV content.

3.1 Effect of 3D culture on EV secretion

Extracellular vesicles have been the modality of interest for diagnostic & therapeutic purposes. However, their less yield hinders their successful commercialization. Thereby in order to enhance the yield of EVs for commercial purposes, many strategies have been explored. One of these strategies is the culturing of cells in a 3D manner [46]. Kim et al., compared the secretion of mesenchymal stem cell derived EVs in a 2D monolayer culture format vs. a 3D culture format via spheroid formation using the hanging drop method & the poly-HEMA coating. From this study, they found that exosome secretion was significantly enhances upon culturing in a 3D format. This led them to sought the cause of increase in EV production, which was found to be the creation of hypoxic niche in a 3D culture format, along with the increased cell density and circular cellular morphology [52]. This finding has also been evidenced by many other studies, for e.g. Yan et al., cultured umbilical cord derived MSCs in a hollow fibre bioreactor & found that the EV secretion was increased by 7.5 folds as compared to the EV release in monolayer culturing [53]. Similar finding was also reported by Haraszti et al., wherein 3D culturing of MSCs resulted in 20 fold increase in exosomes concentration when combining 3D culturing of MSCs along with isolation via differential ultracentrifugation. They also established another technique wherein they combined 3D culturing with Tangential flow filtration which thereby enhanced the yield up to 27 folds. Patel et al., developed a culture system by combining a tubular perfusion bioreactor system & a 3D printed scaffold, wherein they found a 100-fold increase in EV production by endothelial cells [54]. Not just in primary cells, but these results are also observed in cancer cells based 3D Culturing [55]. Hwang et al. suggested that the EV release was increased upon 3D culturing in colorectal cancer [56].

3.2 Effect of 3D culture on EV content

The effect of 3D culturing of cells on EV content is exceptionally significant. 3D culturing has proven time and again that it is a better model to study the cell-ECM & cell-cell interactions as the dynamics of 2D and 3D culturing are poles apart. As a means of cell-cell communication, EVs regulate vibrant interplay mechanisms, and thereby 3D culturing leads to modifications in the cargo of EVs as the stimuli perceived by the cells is varied as compared to 2D culture. It was concluded in one of the studies that 3D culturing leads to an overall depression of protein expression while upregulation of miRNA cargo in EVs due to the downregulation of ARF6 pathway influencing the cell arrangement & secretion profile thereof [57]. Many studies have presented varied views on this matter. It was found that culturing of HeLa cell line in a 3D manner results in the secretion of EVs which were up to 96% similar in their RNA profile with the circulating EVs collected from the plasma of a cervical cancer patient, however the genetic profile of EVs i.e. DNA was unaltered [58]. It is also speculated that EV release in 3D culture systems is aided by the higher expression of transporters [59]. Due to the mechano-sensing based activities in cells upon culturing in the 3D microenvironment, the gene and protein expression of the same cells are differential. For e.g., Eguchi et al. observed that upon 3D culturing, the neuroendocrine adenocarcinoma cells formed large organoids in a steady growing pattern which further expressed numerous stem cell specific markers, neuroendocrine markers and intercellular adhesion molecules. While in case of 2D culturing, it was found that cells had a faster growth rate, while intercellular adhesion molecules were decreased and mesenchymal transition was increased. It was deduced thereby that the 3D culturing of cells leads to the formation of more realistic tumoroids in terms of morphology & gene expression [60]. Furthermore due to enhanced intercellular communication in 3D culturing, EVs which are involved in transcellular transport are more in number when compared to the 2D culture system. Tu et al., realised 3D culturing as a better model for tumour progression, as they estimated the miRNA content of exosomes and protein expression of GPC-1, and found that the trend observed in EVs derived from spheroids presented higher relevance to the progression of pancreatic cancer [61]. Not just in cancer, but 3D culturing has also been tested for primary cells like Mesenchymal Stem Cells (MSCs). It was found the culturing of MSCs in a 3D manner leads to multi-fold increase in the exosome concentration & enrichment of cargo such that they were more efficient in their uptake capabilities and improved the viability of the recipient cells [62]. Furthermore, it was deduced that the cargo content of MSCs-EVs was vividly distinct when derived from a 3D culture microenvironment. The results of a microarray suggested that expression of 193 miRs were varied wherein 68 miRs were up regulated and 125 miRs were downregulated [63]. Yu et al., also explored the EV dynamics in 3D vs. 2D culture of mesenchymal stem cells & observed that there was a 2.5 fold increase in exosome production upon 3D culturing along with 2.9 fold increase in the enrichment of proteins. Furthermore, they also suggested that exosomes derived from 3D culturing of MSCs had heightened expression of osteo-inductive genes and proteins, which could be attributed to the upregulation of YAP pathway [64]. 3D culture system derived exosomes were also proven to possess extended anti-inflammatory effects and were able to restore the homeostatic balance of Th17 and Treg cells in a model of periodontal inflammation. This was suggested to be happening as a result of enhanced expression of miR1246 in the 3D derived exosomes, thereby affecting the Nfat5 expression which plays a role in Th17 polarity [65]. However, there have also been studies that suggest that EVs isolated from MSCs cultured in a 3D manner did not sufficiently execute the properties which are a characteristic of their parent cells like immunomodulation & anti-fibrotic activity. It was observed that the level of IDO was significantly downregulated when the cells were cultured in 3D & there was a rise in pro-inflammatory capacity of macrophages upon culturing of EVs derived from 3D culture as compared to 2D culture. This could be due to the extensive networking and interactions between the cells itself during 3D culturing & the decline in cell volume thereby affecting the packaging of EVs so released [66].

3D culturing for EV derivation is a budding area of research, and so there is yet nothing conclusive about the possible effects of 3D culturing on exosome release & cargo profile. There have been many contrasting views that support or reject the hypothesis of culturing cells in a 3D manner. It can be accepted that 3D culture model might best be able to replicate the cancer biology due to its ability to replicate tumour like interactions & features in vitro, and concurrent release of in vivo like EVs. Such a culture model could aid the advancements in identification of cancer biomarkers which may be specifically analysed in a simulated manner. While 3D culturing is being preferred in carcinoma-based studies, culturing of primary cells in a 3D microenvironment is still a topic of debate. There have been divergent perspectives of researchers regarding the derivation of EVs from 3D culture of primary cells however, it still needs to be developed further to enhance its benefits, more than its shortcomings. EVs are a recent popular candidate for therapeutics, drug loading & delivery, and 3D culture has shown tremendous potential in enhancing their yield, therefore it could be an interesting application & strategy to exploit these modalities for commercial manufacturing of the EVs.

4. Future prospects of using organoid as drug screening and EVs as biomarker analysis

Research in cancer has relied heavily for a considerable amount of time on cancer cell lines as a model system. Recent research has made use of high-throughput screening of broad panels of cancer cell lines to detect patterns of drug sensitivity and to correlate drug sensitivity to genetic changes [67]. These high-throughput cell-line-based research paint a picture of a complex network of biological variables that affect sensitivity to most cancer medicines. It is possible, for example, that there is no direct connection between sensitivity to a particular medicine and individual genetic changes. Instead, the outcome of drug sensitivity may be determined by the complicated interactions that occur between several genetic changes, which are difficult to find. Therefore, despite the new information that has recently become available, it is still difficult to develop algorithms that can accurately predict the drug sensitivity of a patient’s tumour based on the spectrum of genomic alterations that are present, in the context of the individual’s specific genetic background [68]. Even though there is a great deal of information accessible on the biology of cancer, there are still a great deal of questions regarding this international health issue [69]. There is a clear and pressing requirement to keep researching and developing improved therapies for cancer patients. Incomplete or inaccurate modelling of cancer is one of the primary roadblocks in the way of the development of additional treatment regimens. This is because, at times, cancer models can only poorly recapitulate clinical conditions. Because of this, a significant number of medications that produce encouraging outcomes in cancer models fail when tested in humans. Therefore, despite the fact that animal models appear to provide useful insights into the fundamentals of cancer biology, it is vital to keep in mind that these models frequently fail to faithfully recreate the pathogenic processes that take place in patients [70]. As a result, the field of oncology requires the development of new methodologies and approaches to create fresh targeted medicines and to continue lowering the number of fatalities caused by cancer.

An important advance in scientific methodology over the course of time has led to the development of three-dimensional (3D) organoid culture as well as 3D printed scaffolds, both of which are able to simulate human biology as well as diseases more accurately. In 1946, Smith and Cochrae were the first people to use the term “organoid,” which means “resembling an organ,” to describe a case of cystic teratoma. This term was used to describe the growth of a cystic teratoma [71]. However, the term “organoid” now has a more restricted definition. This definition states that organoids are self-assembled in vitro 3D structures, which are primarily generated from primary tissues or stem cells such as adult stem cells, induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs). The production of organoids is dependent on the self-assembly and differentiation of cells, as well as the signalling signals from the extracellular matrix (ECM) and the conditioned medium. This is true regardless of the circumstances. When the three-dimensional constructions are complete, they are able to replicate the intricate features of their real-life organ equivalents, as well as undergo genetic engineering, long-term expansion, and cryopreservation [72]. Organoids and 3D cultures have emerged because of several attempts to replicate the biology of human organs, such as stem cell development in 2D cultures with or without a 3D matrix, cell culture on a microfluidic device (organ-on-a-chip), and bio-printing of cells. Opportunities for medication discovery and human disease study have been expanded because of these modelling initiatives [73]. The term “organoids” refers to three-dimensional structures that can self-organise through the processes of self-renewal and tenogenic differentiation. These structures are formed from pluripotent stem cells that have been cultivated from organ-specific tissues. Organoids have a distinct organisation that places them in the category of micro physiological systems. This is because they are capable of both self-renewal and self-organisation, and, more importantly, that they display organ functionalities that are analogous to those of the tissue(s) from which they originated. Therefore, it is essential to establish cultures of functional tissues, but these cultures should be devoid of the mesenchymal, stromal, immune, and neuronal cells that interspace tissues in vivo. This will allow for the development and maintenance of optimal conditions for organoid design. In fact, this process is dependent on the construction of artificial extracellular matrices in order to allow organoid self-organisation into structures that are analogous to the architecture of the native tissue [69, 74]. To this day, organoids have been successfully created from the intestine, liver, pancreas, colon, and prostate of murine animals, as well as from the small intestine, colon, stomach, and prostate of human beings. The fact that these organoids can be grown for an extended period and, according to whole-genome sequencing, match the patient tissue from whence they originated suggests that their phenotypic and genetic traits are consistent [75].

Patient derived organoids (PDOs) have recently proven valuable in translational research because these models can be maintained for an extended period and cryopreserved. In addition, PDOs are genetically stable, which makes them a perfect choice for modelling diseases. In addition, PDO models are helpful because they enable the expansion of normal cells as well as tumour cells in parallel, which contributes to the formation of a living tumour organoid biobank. PDO models, on the other hand, solely represent the epithelial tissues of organs; they do not include the stroma, nerves, or vasculature that are seen in real organs, which is an essential distinction to make. When organoids are generated from different types of tissues, different types of growth components are required (Figure 2) [74].

Figure 2.

Culture additives/growth factors used for generation of different organoid models.

The use of murine and human embryonic stem cell lines and induced pluripotent stem cell lines to generate organoids gets around the limited availability of high-quality human primary material. However, in order to perform directed differentiation, in-depth knowledge of the factors involved in germ layer formation and subsequent lineage specification is required. In contrast to the employment of ESCs, the utilisation of iPSC lines necessitates the performance of an additional step. Specifically, the expression of OCT4, KLF4, SOX2, and MYC29 is required in order to transform somatic cells into iPSCs. Following this step, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are subjected to germ layer and tissue-specific patterning factors. Next, the cells are embedded in Matrigel in order to facilitate the development of 3D architecture. Finally, the cells are treated with differentiation factors in order to produce the desired organoids.

The first successful mesoderm-derived organoids were reported not too long ago. Renal organoids were formed by manipulating GSK3 and FGF signalling pathways in human iPSCs. This was done while the cells were in an intermediate mesodermal state. The architecture and segmentation of human foetal nephrons into ducts, tubules, and glomeruli is replicated in these organoids, which have the same name [76]. The human renal organoids provide a 3D model to study human renal development and disease under well-defined conditions, thus overcoming various limitations of previous models such as 2D monolayers, short-term 3D aggregates, and co-cultures with mouse fibroblasts [77, 78].

The existence of cell types other than those intended for lineage in ESCs and iPSCs is one of the most distinguishing characteristics of organoids created from primary tissue as opposed to those generated from ESCs and iPSCs. This is due to the fact that the factors that are used for the directed differentiation of ESCs and iPSCs are not completely efficient in driving all of the cells toward the lineage of choice. As a result, many ectodermal and endodermal organoids, such as those of the intestine, stomach, and kidney, have reported the limited presence of mesenchymal cell types [79, 80]. Despite these developments, some tissues remain resistant to organoid culture but have been successfully cultivated in 3D as whole-tissue explants or organotypic/mechanically supported cultures (for example, skin or ovary) [81]. Understanding the endogenous stem cell microenvironment and signalling pathways driving lineage specification in organoid cultures is crucial. Our limited knowledge in these areas for certain tissues prevents us from logically designing niche parameters for organoid formation. Identifying stem cells is not necessary for growing primary tissue units but understanding the stem cell niche is essential for long-term culture sustainability [82]. Small-molecule modulators of critical signalling pathways and organ-specific hormones could facilitate organoid growth from organs like the ovaries. A tight dependence on growth factor/signalling gradients for stem cell renewal and lineage specification may also complicate organoid formation from tissues. Microfluidics could be utilised to create in-vivo-like concentration gradients [83]. In vivo stem cell behaviour and differentiation are also highly impacted by local biomechanical factors, such as interactions with the extracellular matrix49. In order to create more robust organoid culture models for a larger spectrum of tissues, researchers are screening for substrates and ECM factors that influence cell behaviour in vitro [84].

In the field of cancer research, the improvement of culture techniques has been applied to the study of EVs, which models the environment and physiological conditions that are present in the area surrounding tumours. The role of EVs in tumour physiology is not limited to cell-to-cell communication; rather, they are also a promising source of biomarkers, a tool to deliver drugs, and a mechanism to induce antitumor activity [10, 85]. Extracellular vesicles, also known as EVs, were discovered in the 1980s and were initially thought to be carriers of waste products that were produced inside of cells [86]. After it was discovered that RNA could be found enclosed within the lipid membrane of EVs, approximately 20 years later, EVs began to be seen in a different light; specifically, as crucial mediators in the process of intercellular communication [87]. The nucleic acids contained in EV RNAs were found to be distinct from those found in the cell from which they originated, displaying distinct sequences and even concentration profiles. In the course of time, research has demonstrated that EVs are responsible for transporting nucleic acids, such as RNA and DNA, as well as a wide variety of biomolecules, which includes proteins and lipids, into and out of cells [88]. For instance, in cancer, the EV content of the tumour is tumour-like, and a class of EVs known as exosomes help the progression of the tumour by signalling to the tumour cells that they should establish the pre-metastatic niche. In another scenario, EVs that are released from cells that have been infected with a virus such as HIV can contain fragments of viral RNA as well as viral proteins; consequently, the function of EVs in HIV is unclear at the present time. In addition, extracellular vesicles released by breast cancer cells have been shown to contribute to the spread of the disease to the brain and to have triggered the breakdown of the barrier that separates the blood and the brain. In general, EVs can break through natural barriers such as the blood-brain barrier and others. These mechanisms can be exploited to deliver therapeutic agents to parts of the body that are difficult to reach. EVs have also been shown to play a role in reproductive biology, the differentiation of stem cells, angiogenesis, and a variety of other biological processes [89]. Various clinical trials are ongoing and have been completed where they have used EVs for therapy of various cancer types [90]. EVs are critically important for tumour communication with their intended target cells. Therefore, the study and modification of EVs have opened so many doors for diagnosis and therapy. It is well-established that elevated levels of circulating EVs are linked to the development of most cancers. Blood EV concentration has also been shown to correlate with tumour volume in several tumour types. These EVs have become the substrate for biomarker mining in a variety of cancers, including prostate cancer, due to the valuable information they transport about the tumour (Table 2) [91, 92, 93].

Id/reference	Disease	EV source	Stage	Goal	Status
NCT01294072	Colon cancer	Plant	Phase 1	To see if curcumin could be delivered using plant EVs	Active
NCT03608631	Pancreatic cancer	Mesenchymal stromal cells	Phase 1	To evaluate the side effects of mesenchymal stromal EVs on pancreatic cancer cells	Not recruiting
NCT01159288	Lung cancer	Dendritic cells	Phase 2	To determine if patients’ conditions improve after treatment with EVs	Completed
Dai et al.	Colorectal cancer	Ascites	Phase 1	To determine the role of ascites EVs in immunotherapy	Completed
Morse et al.	Lung cancer	Dendritic cells	Phase 1	Role of dendritic EVs in immunotherapy	Completed
Besse et al.	Non-small cell lung cancer	Dendritic cells	Phase 2	To assess the role of dendritic EVs on NSCLC patients	Complete

Table 2.

Human clinical trials using EVs for therapeutic purposes.

Three-dimensional (3D) culture enables cell growth in a physiological topology, and organoids and spheroids continue to release EVs, which are essential for tumour communication with targeted cells, and the released EVs are functional (Figure 3). The extracellular vesicles that are released by pancreatic cancer organoids have the ability to activate p38 MAPK and increase the expression of F-box protein 32 and UBR2 in myotubes. In the case of colorectal cancer stem cells, 3D cultures exhibit a higher level of EVs release in comparison to 2D conformations. The presence of APC mutations in colon cancer organoids that activated the WNT pathway resulted in an increase in the amount of EVs released in cultures based on Matrigel. This release was presumably likely helped along by the presence of collagen, which is a component of the extracellular matrix and is present in this sort of gel [61]. Collagen is a component of the gel. Additionally, a further potential hypothesis is that the greater expression of transporters in 3D cultures may be partially responsible for the release of EVs [94, 95, 96]. It was shown that tumoroids of colon cancer cells with improved stemness had significant levels of expression of the ATP-binding cassette transporter G1, which is a cholesterol lipid efflux pump. Similarly, inhibiting this transporter prevents the release of EVs and leads to an increase in the number of vesicles found inside the cell [97].

Figure 3.

The schematic diagram showing the establishment of patient derived organoid cell culture depicting the organoid microenvironment and respective EV distribution.

Research has been done to investigate the spontaneous effect of EVs derived from normal cells in order to use them as natural antitumor agents. For instance, extracellular vesicles produced from glia have been shown to have an anticancer effect in spheroids composed of glioma cells. This effect was demonstrated by a gradual reduction in the tumour potential to invade surrounding tissue. Another example is the EVs that are produced by mesenchymal stem cells (MSCs). These EVs could initiate angiogenesis and preserve vascular homeostasis in activated endothelial cells [98, 99]. On the other hand, most of the publications centre their attention on the prospect of loading EVs with anticancer medicines and biomolecules such as amino acids, lipoproteins, or nucleic acids. In a microfluidic system that contained a variety of cell types, an anticancer effect of EVs that were loaded with a particular miRNA (miR-497) was evaluated [100]. These kinds of devices are helpful when used in conjunction with an extracellular matrix because doing so makes it possible to investigate migration in response to a factor that is controlled via microfluidic channels. In this experiment, the non-small cell lung cancer cell (NSCLC) line A549 was cultured alongside human umbilical vein endothelial cells (HUVEC). Both cell types were grown in a dish (HUVEC). When the experiment was run under these conditions, the production of tubes by endothelial cells was prevented, and the amount of tumour migration was significantly reduced in comparison to the control. Both types of cells were separated in the microfluidic devices using the matrigel component. This was done so that the limitations of cocultures that are related with cell separation after analysis could be avoided [101, 102]. This is a fascinating illustration of how 3D culture may be used to recreate the physiological intricacy of tumours (Figure 3).

One of the most fascinating uses of 3D cultures is the large-scale and standardizable production of EVs. This is one of the most exciting applications of 3D cultures because till yet there is no established biomanufacturing platform for EVs, which poses restriction for clinical translation (Figure 4). The utilisation of bioreactor flasks is a straightforward method that can be utilised because these flasks boost the creation of EVs that are discharged by tumour cells. Utilising cell cultures on microfluidic substrates is a more interesting application of this technology [103, 104]. These automated systems can manufacture therapeutic exosomes, which could also be modified, and harvest them in real-time from the cultures that are performed on the chip. As a device of this kind has been utilised in the process of isolating leukocytes from human blood. An alternative method that has been utilised is a 3D-printed scaffold-perfusion bioreactor system to investigate the impact that dynamic cultures have on the production of EVs from endothelial cells. Because of this method, the cells were able to keep up their level of functionality (i.e., pro-vascularization bioactivity or pro-angiogenic gene expression) [95, 101].

Figure 4.

The schematic diagram shows the isolation of EVs and modification of EVs for targeted delivery.

5. Conclusion

Three-dimensional (3D) cell culture models are more functionally important than two-dimensional (2D) cell cultures and include a broad range of structures such as embryoid bodies, spheroids, patches, and scaffolds. Whereas, organoids and 3D culture systems are becoming being recognised as a tool for advancing medical research without relying on animal models. Improvements to these methods may even result in new methods for creating 3D models. Understanding the limitations of the systems is critical for their improvement and determining the model’s suitability for investigating EVs. Overall, cellular architecture influences the concentration and cargo profile of EVs. Many studies, for example, found that 3D in vitro systems secreted more EVs than their 2D culture counterparts. Moreover, the possibility of a necrotic core developing in multicellular cultures poses a unique challenge to isolating EVs from 3D in vitro systems. The necrotic core can generate EVs composed primarily of apoptotic bodies rather than small vesicles or large vesicles. To address the challenges, developing cell viability criteria and measures to normalise the outcomes compared against controls such as 2D cell monolayer cultures are required. With the start of human clinical trials for EV therapeutics, these challenges become even more important. Despite the widespread use of organoids in biology, the technology is still in its infancy for certain disorders. Most neurodevelopmental or neuropsychiatric disorders, such schizophrenia, Parkinson, or autism, are examined using animal models. Autism spectrum diseases or Parkinson’s have clinical heterogeneity (epilepsy, sleep disruptions, motor difficulties), making organoid culture challenging to use. Researchers have been developing techniques to create more mature and complicated brain organoids. Organoids can be utilised to explore developmental brain injuries and disorders (DBD) Stem cells are linked to several disorders. Scientists do not know how stem cells develop abnormalities or which type of specialised cell to generate. The organoids method can answer questions about stem cells in diseases like emphysema, when lung stem cells fail to heal damage. Further, scientists have suggested utilising organoids to screen medications that can produce specialised cell types for hereditary illnesses like cystic fibrosis, where ciliated cells that remove mucus from the lung do not work properly. Generate organoids from cystic fibrosis patient tissues, then design a medication to make ciliated cells operate better in organoid cultivation. Since scientists can co-culture organoids with immune cells, the approach can be used to investigate autoimmune disease mechanisms and screen medications.

Acknowledgments

The authors would like to acknowledge the Biorender software for figures.

Conflict of interest

The authors declare that they have no competing interest.

Abbreviations

2D	two-dimensional
3D	three-dimensional
APC	adenomatous polyposis coli
ATP	adenosine triphosphate
DNA	deoxyribonucleic acid
EBs	embryoid bodies
ECM	extracellular matrix
EGF	epidermal growth factor
ESCs	embryonic stem cells
EVs	extracellular vesicles
FGF	fibroblast growth factor
GELs	collagen gels
GPC-1	Glypican 1
HIV	human immunodeficiency virus
HUVEC	human umbilical vein endothelial cell
IDO	indoleamine 2,3-dioxygenase
MAPK	mitogen-activated protein kinase
miR	micro RNA
MSCs	mesenchymal stem/stroma cells
Nfat5	nuclear factor of activated T cells
NSCLC	non-small cell lung cancer
PDGF	platelet derived growth factor
PDOs	patient derived organoids
PLLA	ploy (l-lactic acid)
PS	polystyrene
RNA	ribonucleic acid
UBR2	ubiquitin protein ligase E3 component N-recognin 2