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Department of Biomedical Engineering, Gachon University, Seongnam, 13120, Republic of KoreaCentral R & D Center, Medical & Bio Decision (MBD) Co., Ltd, Suwon, 16229, Republic of Korea
Three-dimensional (3D) cell culture technology has been steadily studied since the 1990′s due to its superior biocompatibility compared to the conventional two-dimensional (2D) cell culture technology, and has recently developed into an organoid culture technology that further improved biocompatibility. Since the 3D culture of human cell lines in artificial scaffolds was demonstrated in the early 90′s, 3D cell culture technology has been actively developed owing to various needs in the areas of disease research, precision medicine, new drug development, and some of these technologies have been commercialized. In particular, 3D cell culture technology is actively being applied and utilized in drug development and cancer-related precision medicine research. Drug development is a long and expensive process that involves multiple steps—from target identification to lead discovery and optimization, preclinical studies, and clinical trials for approval for clinical use. Cancer ranks first among life-threatening diseases owing to intra-tumoral heterogeneity associated with metastasis, recurrence, and treatment resistance, ultimately contributing to treatment failure and adverse prognoses. Therefore, there is an urgent need for the development of efficient drugs using 3D cell culture techniques that can closely mimic in vivo cellular environments and customized tumor models that faithfully represent the tumor heterogeneity of individual patients. This review discusses 3D cell culture technology focusing on research trends, commercialization status, and expected effects developed until recently. We aim to summarize the great potential of 3D cell culture technology and contribute to expanding the base of this technology.
The cultivation of animal cells began in the early 90′s through the cultivation of neuronal fibroblasts from the cochlea of the tadpole in the coagulated lymphatic fluid of the frog. An extracellular substrate (scaffold) was used to provide space for cells to grow and to construct a structure in vitro similar to the in vivo extracellular matrix (ECM) [
]. Thereafter, animal cells could be stably cultured in vitro with the development of cell lines with good reproducibility of proliferation, cell culture media, and apparatus and equipment. Animal cells were cultured by adhering and growing them on the surface of a plastic dish coated with a cell adhesion promoter. These animal cell cultures have been widely used in biotechnology and medicine for the discovery and production of physiologically active substances, the development of disease models, toxicity analysis, and drug development.
However, animal cells adhered to a plastic dish grow into a single layer, which is morphologically different from the 3D growth of animal cells in the living body. This environment also affects gene expression in cells. Animal cells exhibit differential gene expression in 2D cell culture models and biocompatible 3D cell culture models [
]. As a result, in vitro animal cell culture shows poor correspondence with animal cells in vivo. Therefore, cell-based experimental results should be cautiously applied to basic life science research and medical disease models. In particular, when animal cells are used for drug efficacy or toxicity analysis, drug reactivity in 3D cell culture models greatly differs from that in conventional 2D cell culture models [
]. When cells derived from cancer patients are cultured in 3D cell culture, the cell-cell interaction and ECM changes the morphology of the cells, as well as the type and expression of major genes [
]. In addition, there are many difficulties in deriving meaningful results at the clinical stage owing to the absence of disease-specific models in the human body during drug development. Human in vitro 3D cell cultures using stem cells from different organs has the potential to overcome these limitations. Recently, attempts have been made to use drug response data of organoids cultured in 3D in precision medicine for the personalized treatment of cancer patients as clinical data, as well as gene data [
]. Such an organoid model is composed of a combination of cells, an ECM (scaffold), a cell culture container, a cell growth factor, etc., and the model varies depending on the combinations thereof. The organoid model has been used to biologically model human organs in vitro and has recently developed into an organoid culture technology that further improved biocompatibility. [
] In particular, bioprinting technology using human-derived cells and organoid culture using a cell culture platform (organ-on-a-chip) has shown potential for drug screening and human disease research. Organoids are self-organizing 3D culture systems that closely resemble human organs [
]. Therefore, organoid formation and analysis studies using these complement the existing 2D cell culture model and are very useful in basic biological research, medical research, drug development, and drug research under conditions physiologically similar to the human environment. The potential of organoids is being increasingly recognized. However, compared to conventional cell lines and animal models, the development of organoid technology is still in its infancy, and there are still many challenges to overcome. During the last decades, organoid models have been variously developed, some of which have been commercialized but are still not standardized. In addition, attempts to increase the biocompatibility of existing models have been made. This review summarizes the latest technologies and commercialization trends of organoid models and discusses the future directions and applications of organoid models and technology.
2. Review of 3D cell culture
2.1 3D cell culture
A 3D cell culture model refers to a cell culture model in which an environment similar to a living body is artificially formed outside the body, allowing cells to grow in all dimensions and interact with the surrounding environment. In particular, since cells are implemented by 3D culture in an environment similar to the human body, they show similar characteristics to cells and organs of the human body [
]. In a 3D space composed of an ECM (scaffold), nutrients, oxygen, and drugs are supplied to the cells through a diffusion gradient and permeation to provide a biologically similar environment. Cell-to-cell and/or cell-to-ECM interactions and paracrine signaling by diffusion of cell secretions, that do not exist in conventional 2D cell culture, are the distinguishing features of the 3D cell culture model. Therefore, with advances in biotechnology, the 3D cell culture model is expected to become an optimized and efficient biotechnology platform for studying human physiology and disease.
2.2 Definition and difference of spheroid and organoid models
3D cell culture technology is a promising way to bridge the gap between conventional cell culture methods and animal models. 3D cell culture methods can be divided into spheroid and organoid models. Spheroids and organoids are 3D cell structures formed by gathering many cells. However, depending on the cell origin, type, and function of the cell structure implemented in 3D, the spheroids and organoids are classified as follows. Spheroids are simply the aggregation of cells into 3D structures, but organoids have self-differentiation capabilities, indicating structures and functions similar to those of human organs. To date, the terms—3D cell culture models, organoids, and spheroids—have been used interchangeably [
]. However, although these two models can both be applied to 3D cell-based research, they differ in their fabrication method and cell origin. Spheroids are 3D cell culture models having spherical cell units that are cultured as free-floating aggregates [
]. Compared to the existing 2D models, this method has the advantage of effectively implementing and analyzing cell-cell interactions. However, because spheroids are sometimes cultured without the ECM as a scaffold, they show relatively low structural complexity. Furthermore, spheroids are simple clusters of a wide range of cells, such as tumor tissues, embryonic bodies, hepatocytes, neural tissues, and mammary glands [
]. However, in cancer-related research, tumor spheroids are popular because they enable the culture of cancer cells in 3D and implement cell-cell and cell-ECM interactions. In particular, tumor spheroids are characterized by mimicking vascularized or poorly vascularized tumors. In particular, when cultured larger than 500 µm, a multilayered structure can be formed to achieve the outer layer, the middle layer of cells, and the inner layer structure of necrotic cells that show hypoxia [
]. In addition, tumor rotators generally exhibit anticancer drug resistance and radiation resistance + and are widely used in cancer cell migration and invasion and drug screening studies [
Organoids are complex clusters of organ-specific cells, such as those in the stomach, liver, or bladder. Organoids can be cultured from embryonic stem cells, induced pluripotent stem cells, and adult stem cells. They exhibit multi-stage organogenesis features that self-assemble and proliferate in a scaffolding extracellular environment such as Matrigel or collagen [
]. Organoids are structurally complex and recapitulate the development of organs in vitro, making them very useful in the study of organogenesis, heredity, and pathology, especially in organs with little or no regenerative capacity, such as the brain [
]. The term cancer organoids or tumoroids may not be appropriate because cancerous tissue is not an organ that originally exists within the body. However, since cancer organoids are implemented using cancer stem cells, the term organoid is commonly used depending on the origin of the cell. Unlike normal cells, cancer stem cells can produce cancer cells and indefinitely reproduce cancer progenitor cells. Therefore, cancer organoids exhibit self-renewal properties through cancer progression and re-proliferation, and their differentiation ability enables mimicking the unique characteristics of cancer tissues, such as intra-tumoral heterogeneity [
]. Cancer organoids are more complex than spheroids and have features and differences that better recapitulate the histological and genetic features of intrinsic malignant tissues [
2.3 Pros and cons of 2D cell culture and 3D cell culture
A 2D cell culture model in which cells are plated on a dish surface has been continuously developed over the past 100 years and is now standardized. This 2D cell culture model is easy to implement and commercially available products can be easily obtained, so users can conveniently perform experiments. However, a disadvantage of the 2D cell culture model is that it cannot accurately represent the response owing to its low biocompatibility. To solve this problem, 3D cell culture models have been developed. High Throughput Screening (HTS) assay was performed by uniformly culturing pancreatic organoids in 384 and 1536 well plates using a magnetic force. Cells were labeled with nanoparticles (NanoShuttle-PL) for 3D cell culture using the magnetic levitation method, and then the cells quickly formed 3D spheroids react with a magnetic plate. [
]. However, there are many variables, such as the type and shape of the ECM, cell culture factors, and shapes of cell culture containers, for imitating the living environment. The culture system is complicated for the user to perform the experiment easily. Despite these disadvantages, the 3D cell culture model has the advantage of providing a more accurate response owing to its high biocompatibility (Fig. 2). As shown in Table 1, 3D cell culture models have been reported to have higher biocompatibility than conventional 2D cell culture in terms of cell shape, cell differentiation, and drug metabolism. In addition, studies using 3D cell culture models have many potential advantages over animal-model-based experiments. 3D cell culture models can provide large amounts of information more quickly and efficiently. The 3D-cultured cells that are implemented using human-derived cells, can more accurately mimic human tissues compared to animal models, and the time and cost required to build a research model are less. In particular, there was a case in which the cytotoxicity efficacy of ~3300 approved drugs was evaluated using four patient-derived pancreatic cancer KRAS mutation-associated primary cells, including cancer-related fibroblasts [
]. Consequently, researchers can simultaneously conduct studies using a large number of 3D cell culture models. To meet this demand, 3D cell culture models have been built to gradually overcome the problems of complicated systems and difficulties of use, and are being commercialized. Corning, Insphero, 3D Biomatrix, and Trevigen are representative examples of commercialization of these 3D cell culture products, while SPL and MBD sell 3D cell culture models such as artificial ECM, culture plates, and culture equipment.
Fig. 2Comparative analysis of 2D and 3D cell culture models.
Because it is attached flat on the bottom of the culture dish, all the cells are uniformly exposed to the culture medium and the drug at the same time.
As the cells grow in many directions, the concentration gradient of the culture medium and drug permeates in the cell mass as in the in vivo environment, and the cells on the lump surface are more exposed than the cells on the inner surface.
It has a higher growth rate than 3D cell culture but is limited.
The rate of cell proliferation can be high or low and varies depending on the cell type and the 3D cell culture model, and it is possible to grow in a spheroid form.
Methods for implementing 3D cell culture models have not yet been standardized. The 3D cell culture model may be developed using a scaffold-free method that allows cells to grow together without an ECM, and a scaffolding method that cultivates cells in the ECM space (Fig. 3) [
]. The scaffold-free method is classified into four types depending on the method used to collect cells. Anti-adhesion methods prevent the adhesion of cells by coating the cell attachment material or flowing the culture media, including static suspension culture, spinner/rotational chamber culture, nano-pattern well culture, and magnetic levitation culture. The magnetic levitation method uses magnetic particles to aggregate cells. In this regard, there is the NanoShuttle™-PL product. Nanoshuttle™-PL is composed of gold, iron oxide, and poly-l-lysine, and magnetizes cells by electrostatically attaching to cell membranes during overnight incubation. NanoShuttle™-PL attached to the cell membrane in this way remains attached for up to 8 days and helps to form 3D spheroids by magnetic force. In addition, NanoShuttle™-PL is biocompatible, has no effect on metabolism, proliferation, and inflammatory stress, and does not interfere with experimental techniques such as fluorescence imaging and Western blotting [
]. In addition to adhesion prevention, hanging-drop cultures and U- or V-shaped well cultures are used where gravitational forces collect cells to form single spheroids; here, a 3D cell culture model is implemented by densely gathering cells in a narrow space (concave bottom of a water body, bottom surface of a U- or V-shaped well) to form a single cell mass (spheroid). The scaffold method can be divided into various types depending on the artificial polymers used, such as solid scaffolds or hydrogels, as an ECM. Structurally, they are divided into well, microchannel, and micropillar types, depending on the location of the ECM. As described, various models are being developed as 3D cell culture models to enable the application of various experimental parameters according to the purpose of the experiment. A description of each representative model is shown in Fig. 3, and comparisons of the 3D cell culture model of each model are shown in Table 2.
] is the most common 3D cell culture model. Here, cells are aggregated above the surface of an agar or anti-adhesion coating to prevent cell adhesion to the bottom of the dish or well plate. Floating cell lines, such as leukemia and murine ascites tumor cells, can be cultured with this model without bottom coating.
] is a 3D cell culture model that allows cells to float inside the media using a spinner or a rotational chamber to prevent the cells from adhering to the bottom. As the cells rotate in the culture media, cells with a high affinity adhere to each other through intercellular interactions. It is mainly used to induce cell metabolism or to mass-proliferate cells.
] is a 3D cell culture model in which a media droplet is hanging on the surface and cells in the media are aggregated in the lower part owing to gravity and cultured in a lump (spheroid).
] is a 3D cell culture model in which cells are collected using gravity or centrifugal force on the bottom of a well with a U- or V-shape to cultivate cells in a lump (spheroid).
] is a 3D cell culture model in which nanopatterns are formed on the bottom of a well, and cells are cultured in a lump-like 3D cell without adhering to the bottom of the well.
] is a 3D cell culture model in which cells are floated from the bottom of the well using magnet particles, and the cells are 3D cultured in a lump shape.
] is a 3D cell culture model in which hydrogel, which changes its state depending on temperature or a specific ion, is mixed with cells and is distributed at the bottom of the well. The cells proliferate while forming a mass in the hydrogel.
] is a recently introduced technique and 3D cell culture model in which hydrogel-containing cells are immobilized on pillar surfaces and combined with well chips containing culture media.
] is a 3D cell culture model in which hydrogels and cells are injected into a blood vessel-sized microchannel, and cells proliferate while forming a lump in the channel. In particular, this model shows excellent biocompatibility because it can flow a culture fluid or drug similar to blood vessels in vivo.
2.5 Hydrogel scaffold
The presence or absence of an ECM (scaffold) is a typical difference between 2D and 3D cell culture models. However, the effect of ECM composition on 3D cultured cells has not yet been standardized and defined. This uncertainty in ECM composition can affect the results of chemical and genetic screening studies using human 3D cultured cells [
]. Therefore, it is important to study and understand the ECM. Among them, hydrogel scaffolds have numerous endogenous factors that promote the survival, proliferation, function, development of cells, and efficient chemical transfer. The structure and configuration of hydrogels can be tailored to achieve appropriate chemical, biological, and physical properties that promote specific structures outside the body, and provide an environment of endogenous signaling that promotes cellular interactions that underlie tissue or 3D cell formation. Hydrogel Scaffold has a variety of endogenous factors to promote cell survival, proliferation, and differentiation. In addition, migration can be observed by inducing dynamic 3D phenotypic changes of cells through cell-hydrogel interaction [
]. Therefore, hydrogels are also used to 3D culture cells. The soft fibrous matrix provides appropriate physical parameters, and the Arg-Gly-Asp (RGD) adhesion domain is a key parameter for the formation of robust organisms [
]. Along with the development of a 3D cell culture model, the hydrogel scaffold is also being studied for use in vitro experiments. Efforts are being made to overcome existing obstacles to implement a more robust human 3D cell culture model. Table 3 shows the types and characteristics of representative hydrogels currently available in the market.
Table 3Types and properties of hydrogel scaffolds.
Invasion of interstitial matrix by a novel cell line from primary peritoneal carcinosarcoma, and by established ovarian carcinoma cell lines: role of cell-matrix adhesion molecules, proteinases, and E-cadherin expression.
It is refined in the rat tail tendon and is provided at a high concentration that can polymerize to form hydrogel, and is a major structural component of extracellular matrix (ECM) found in connective tissue and internal organs.
Cultrex BMEs are a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. Products include a full range of matrices and purified proteins including Basement Membrane Extracts, Laminin I, Collagen I, Collagen IV, Vitronectin, and Fibronectin.
Epicardial application of cardiac progenitor cells in a 3D-printed gelatin/hyaluronic acid patch preserves cardiac function after myocardial infarction.
Alginate Hydrogel is based on Alginate, an anionic polysaccharide derived from algae cell walls, and provides cells with growth hormone and other ECM proteins as well as cells lacking cell attachment sites. In addition, three-dimensional cell cultivation is convenient by gelling calcium ions and liquefaction in the presence of calcium-chelating agents.
Mebiol® Gel PNIPAAm-PEG 3D Thermoreversible Hydrogel
PEG hydrogels are copolymers of poly (N-isopropylacrylamide) and poly (ethylene glycol) (PNIPAAm-PEG) for research purposes in the early 2000s. It is characterized by temperature reversible sol-gel transition and has efficient gaps for transmission, gas and material exchange, and protection of cellular tissue from the outside.
Sigma F5386–1G Fibrin from Human Plasma, Insoluble Powder
A prospective, randomized, controlled trial of the efficacy and safety of fibrin pad as an adjunct to control soft tissue bleeding during abdominal, retroperitoneal, pelvic, and thoracic surgery.
The spheroid culture system is a very useful three-dimensional cell culture method that can be applied variously in life science research. Unlike the cells in the conventional two-dimensional monolayer culture model, the spheroid model exhibits cell-cell or cell-ECM interactions, maintaining intrinsic phenotypic characteristics [
]. The spheroid model was first developed by Sutherland et al. in 1970 to study the functional phenotype of human tumor cells and the effects of radiation therapy [
]. The spheroid model promotes the expression of stem cell markers, such as Oct-4 and Nanog, which influences cell proliferation, survival, and migration. It also secretes higher levels of cytokines and chemokines than those secreted by monolayer-cultured cells, leading to higher angiogenesis. The spheroid model can also better mimic the hypoxia model. In relation to hypoxia, it shows apoptotic resistance, improved viability, and secretion of angiogenic factors and chemokines [
]. The spheroid model can efficiently simulate the in vivo microenvironment of a tumor, and therefore, is a useful tool for researchers to predict drug efficacy in cancer research. Spheroids can also be used for stem cell research for developing embryonic bodies from induced pluripotent metaphase cells, as well as be used as high-purity neural stem cells for research on neurological diseases and related treatments. Recently, spheroid models have been used to study the cytotoxic effects of CAR-T cells, such as using the KILR® cytotoxicity assay developed by DiscoverX. Tumor spheroids transduced with CAR-T cells with KILR can be formed and cultured on the same microplate for spheroid culture, and antibody-mediated cytotoxicity (ADCC) effects can be analyzed with high uniformity and data reproducibility (Fig.4) [
Recently, organoid model studies have focused on the development of organotypic culture models. The organoid has structural and functional similarities to actual organs such as the stomach and liver. An organoid model with excellent biocompatibility was implemented in vitro for use in disease research and drug development (Fig. 5). When assessing whether the organoids have been successfully established, it is important to analyze whether the organoids consist of adequate cell types and whether the organoids accurately mimic the functions of the corresponding tissue in vivo [
]. Although it is not possible to make an organoid by simple lump formation of a cell, the organoid uses a hydrogel-in-well model, which is a conventional 3D cell culture method. However, cells are cultured in a suitable ECM for a long period with appropriate differentiation or growth factors to produce an artificial organ with functions similar to those of human organs. Because the ultimate goal of a 3D cell culture model is to create a highly biocompatible model in vitro, the final stage of development is considered to be an organoid. Organoid studies are led by the Hans Clevers Group [
] and have been actively pursuing research into application in precision medicine by developing a biocompatible model. Because the organoid model for producing organoids uses a highly viscous ECM, it is difficult to dispense a precise amount of cells and ECM using the conventional pipet used in biological experiments. High-throughput screening studies are performed using 384- or 1536- well plates. Therefore, since three-dimensional organoid samples and drugs are dispensed into numerous individual well plates, 3D bioprinting equipment capable of dispensing samples at high speed is required. The use of such 3D bioprinting equipment provides the researcher convenience, and a very small amount of liquid sample (less than a few nL) can be handled with very high uniformity and reproducibility. Therefore, it is possible to ensure high reproducibility of the experimental results. To solve this problem, the production of organoids now employs 3D bioprinting capable of precise liquid supply, as shown in Fig. 6 [
]. Recent clinical trials and studies have analyzed the efficacy of anticancer drugs in organoids developed from cancer patient-derived cells for precision treatment. A study aimed to measure the efficacy of anticancer drugs against patient-derived cancer cells and compare them with clinical data to select optimal anticancer drugs or to analyze the efficacy of new drugs (Fig. 7) [
]. In particular, when a patient-derived cell-based organoid model is used for research, it is possible to effectively represent the different genomic mutations, cancer cell characteristics, and tumor microenvironments of individual patients. Through the comparative analysis of patient-derived cancer organoid models and clinical data, cancer organoid models can represent actual clinical patients [
Personalized functional profiling using ex-vivo patient-derived spheroids points out the potential of an antiangiogenic treatment in a patient with a metastatic lung atypical carcinoid.
Fig. 5A schematic diagram used in the treatment of diseases after organogenesis using an organoid model developed by extracting patient-derived cells (PDCs) from patients
2.6 Commercialization trend of 3D cell culture market
Table 4 shows that the global 3D cell culture market is projected to grow from $1.3 billion in 2022 to $2.6 billion in 2027, with an average annual growth rate of 15.6% between 2022 and 2027 [
In particular, 3D cell culture models can be actively used to solve problems such as the increasing interest in personalized medicine and the increasing incidence of chronic diseases. However, there is still a lack of infrastructure for 3D cell culture model-based research, and the high cost of tumor biology research and clinical staging is expected to restrain the growth of this market. Currently, various 3D cell culture technologies from Corning, Insphero, and 3D Biomatrices are available in the market. Table 5 shows companies that sell 3D cell culture products.
Table 5Representative companies of 3D cell culture technology.
Corning sells spheroid microplates in either 96-, 384- or 1536- plate form (Fig. 8). Scaffold-free U-shaped wells use gravity to aggregate cells in one place for 3D cell culture. DU145 cell line, a human prostate cancer cell, was cultured in a corning spheroid 384-microplate, and then treated with doxorubicin for 24, 48, and 72 h to measure cell viability. In addition, drug efficacy can be quantitatively analyzed based on the dose-response curve (DRC) by treating the drug according to the concentration gradient. Although there is an advantage of enabling high-throughput screening (HTS) analysis using such 3D cultured cells, they are limited in that they cannot be cultured by mixing with the ECM (Fig. 9). In addition, corning sells various types of plate products such as Matrigel Matrix-3D Plates, Elplasia Plates, and Elplasia 12 K Flask for 3D cell culture experiments. By using Matrigel Matrix-3D Plates, polarized epithelial cells such as kidney, colon, lung, and mammary gland can create barriers separating the inside. Pre-coated Matrigel matrix-3D plates can be used to generate MDCK cysts for screening modulators of forskolininduced cyst swelling, which can be used to identify potential therapies for polycystic kidney disease (Fig. 10). [
] Corning Elplasia plates generate a high density of spheroids in a scaffold-free model using microcavity technology. The Elplasia plate is composed of individual U-bottom wells in the array well plate, so cells are cultured as 3D-spheroid without a scaffold (Fig. 11) [
]. Elplasia 12 K Flask consists of individual round wells with ultra-low attachment (ULA) surface on the bottom of the T-75 flask, so when cells are cultured, they form 3D-spheroids (Fig. 12) [
InSphero has commercialized a 3D cell culture container with a product called Akura™ PLUS Hanging Drop System. It features a 96- or 384-plate culture vessel designed for HTS analysis based on the hanging drop method. First, perform 3D spheroid culture in hanging drop plates to aggregate and grow single cells into spheroid shapes. The generated 3D cultured cells are again moved to the “V shape” well so that various tests can be performed. Fig. 13 shows the protocol for transferring the sphere to the V-shaped well after cell culture and the results of the uniform formation of the sphere in the 384-well plates. The product is limited in that it is not useful for culturing by mixing various ECM and cells.
Fig. 13Akura™ PLUS Hanging Drop System products and experimental results of cell clumps formed in 384 wells
EMD has commercialized a plate platform called CellASIC ONIX that can be used to 3D culture cells with ECMs using microfluidic technology. Fig. 14 illustrates the fluid control module that allows the culture and drug to flow through the plate. It is characterized by the use of microfluidic technology to provide biocompatibility by supplying continuous fluid to the cells. However, to continuously supply fluids, such as culture media and drugs, separate equipment, such as a fluid pump, is required. This has disadvantages in terms of cost and convenience during maintaining the culture.
Fig. 14Conceptual diagram of the CellASIC product and 3D cell culture image
MBD has commercialized a 3D cell culture plate and chip called Cellvitro. Fig. 15 shows the development of a 3D cell culture chip with immobilized organoids and ECM on the micropillar structure of the MBD product and immersion of the micropillar in microwells for 3D cell culture. This is advantageous in that the culture liquid and drug can be easily replaced by moving the micropillar. This structure overcomes the difficulty of replacing the culture media, drug, and staining reagent in 3D cell culture models using ECM. The corresponding 3D cell culture chip was miniaturized to the size of a glass slide. This has the advantage of minimizing the consumption of organoid samples, expensive staining reagents, and drug samples required for experiments. In the case of organoid samples derived from patients, the amount that can be obtained is limited, and intrinsic genetic characteristics are lost when cultured in vitro for a long time to obtain excess samples. Therefore, the 3D cell culture chip is advantageous as it uses a very small amount of patient-derived organoid samples. Fig. 16 shows the drug efficacy analysis and genomic data obtained using the developed pillar/well chips.
Fig. 15The concept of pillar/well and the organoid-based high-throughput screening product from the product of CellVitroTM
Fig. 16Analysis of drug efficacy and comparison with dielectric data of organoids Derived from Brain Tumor Patients Using Micro pillar/micro well chip, a product of CellVitroTM
SPL has commercialized a 3D cell culture method using a scaffold that can fit into a well, called SPL3DTM. The achievement of a 3D cell culture environment similar to the in vivo environment makes it possible to maintain the physiological characteristics of cells in vitro and maximize the cell culture surface area through scaffold construction. In addition, a spheroid forming unit (SFU) based on suspension cell culture was designed to facilitate the generation of spheroids and size up the generated spheroids. The SFU lid is an air-permeable cap with a hydrophobic filter membrane that is designed to optimize cell uptake by allowing the necessary gas supply during cell culture (Fig. 17). However, it is limited in that it cannot be directly utilized for large-scale data analysis such as high-throughput drug discovery based on the 3D cell culture vessel.
Fig. 17SPL3DTM product that enables 3D cell cultivation by inserting a scaffold into SPL's 12-well plate, and the Spheroid Forming Unit (SFU) 15 ml tube which can suspense cell culture
Organogenix provides a nano culture plate (NCP) in a well plate (24, 96, 384) or dish. NCP is a scaffold-type 3D cell culture system without hydrogel or chemical pretreatment. Cells cultured in this system exhibit high proliferation characteristics and high uniformity, making them suitable for conventional HTS analysis. In addition, low binding/high binding allows the formation of a 3D cell monolayer (Fig. 18). Nano-Culture Plate 96-well plates are intended for the primary culture of cancer in tumor tissue. They are characterized by a shorter incubation period compared to collagen gel, the ability to experiment with the media exchange without containing gel components in an exclusive media, and the convenience of the multi-plate type to test the susceptibility to different anticancer drugs.
Fig. 18Organogenix NanoCulture Spheroid product and 3D cell culture image
Trevigen commercialized the Cultrex® 3D culture spheroid cell invasion assay. The corresponding 3D cell culture model is a better representation of tumors in vivo. In particular, various physiological properties, such as similar morphology, cell-cell bond formation, decreased proliferation rate, increased tumor cell survival, tumor dormancy, and hypoxic core have been well implemented. By reflecting these characteristics in 3D culture cell invasion analysis, tumor invasion can be evaluated through a physiological approach. It also provides quantified visual analysis data through image-based analysis. The Cultrex® 3D spheroid cell invasion assay is performed using a special ECM that induces 3D cell aggregation and a 96-well plate for 3D cell culture. The use of a special ECM optimized to induce 3D cell invasion as a scaffold is an advantage of 3D cell culture model vessels. (Fig. 19).
Fig. 19Trevigen's Cultrex® 3D Culture Spheroid Cell Invasion Assay and 3D cell culture image
3D Biomatrix commercialized Perfecta3D® hanging drop plates (96, 384) easily aggregate cells into a single spoil using the hanging drop method. These products are sold commercially through Sigma-Aldrich product suppliers. The technique of making droplets by inserting a pipette tip into a hole and inserting cells and media is similar to Akura™ PLUS Hanging Drop System; however, the size and structure of the hole are different (Fig. 20). The product also has a limitation in that it cannot be cultured by mixing various ECM and 3D cultured cells.
Fig. 203D Biomatrix Perfecta3D hanging drop plates and graphs showing the change in spheroid size with cell number
Precision medicine refers to the analysis of multiple pieces of information, such as an individual's genomic and lifestyle information, to select patient-specific therapeutic agents. Previously, cell lines cultured in the form of a 2D monolayer were used to analyze the efficacy of cancer-related drugs [
]. However, recently, many studies have been conducted on sophisticated drug efficacy analysis systems that can better predict drug efficacy in clinical practice by simulating the in vivo tumor microenvironment based on a 3D cell culture system and modeling real cancer patients. In addition, analyzing drug efficacy using patient-derived cells is less expensive and time-consuming compared to animal models or clinical studies, facing fewer ethical issues [
]. By combining patient-derived cancer cells with a 3D cell culture model, molecular genomic profiling studies related to cancer drug discovery and tumor subtypes have been performed. In addition, these patient materials are accumulated along with data in the form of an organoid bank, which can ultimately open the door to preclinical screening of personalized drug candidate panels to improve low cancer treatment outcomes and by reducing side effects [
]. The 3D cell culture model is expected to provide a criterion for selecting the optimal therapeutic agent treatment by culturing patient-derived cells from the patient and analyzing the response to various drugs.
2.7.2 New drug development
Drug development involves the search for new drug candidates, non-clinical trials, and clinical trials for new drug candidates. Both non-clinical trials (animal trials) and clinical trials require considerable time and costs. For example, out of 50 million substances, approximately 250 new drug candidates enter non-clinical and clinical trials, from which only one new drug is considered to be at the market. The probability of new drug development has decreased recently. Drug development remains a time-consuming and expensive business because the success rate of drug development that has successfully reached the market through successful clinical trials is very low. Therefore, as the success rate of drug discovery remains low, the introduction of new 3D cell culture model technologies that can increase the accuracy of drug development and discovery is urgently needed. With low success rates in clinical trials, drug discovery remains a slow and expensive business. As attrition rates in drug discovery remain high, there is an urgent need for new technologies that provide better precision. Two of the most promising areas expected to improve the success rates of drug development are the advancement of precision medicine with the prospect of new biomarkers and more precise drug targets, and the availability of new preclinical models that better recapitulate in vivo microenvironmental factors. While traditional monolayer cultures are still predominant in cellular assays used for HTS, 3D cell culture model techniques for applications in drug discovery are making rapid progress [
]. One of the most promising areas that are expected to improve the success rate of drug development is a novel preclinical model that summarizes the in vivo and intra-tumoral microenvironment to precisely analyze target biomarkers in addition to drug efficacy. In drug efficacy analysis studies using HTS, the existing 2D cell culture method is still predominantly applied; however, recently, many efforts have been made to graft 3D cell culture models for HTS analysis as well as image-based high content screening (HCS) analysis [
]. Therefore, by changing the conventional 2D cell culture based HTS system to an HTS system using a 3D cell culture model, it can be more effective to identify potential drug candidates which are expected to dramatically reduce the astronomical costs and time for preclinical and clinical trials. There are examples of a unique ex vivo live tissue sensitivity assay (LTSA), in which precision-cut and uniform small tissue slices derived from pancreatic ductal adenocarcinoma PDX tumors were arrayed in a 96-well plate and screened against clinically relevant regimens within 3 to 5 days. The correlation between the sensitivity of the tissue section and the patient's clinical response and outcome was statistically analyzed. The LTSA assay results were further confirmed through in vitro biochemical methods and in vivo animal PDX models. The results of these PDX and LTSA methods reflect the response of clinical patients and can be used as a personalized strategy to improve the effectiveness of systemic treatment for pancreatic cancer patients, and can also be applied to the field of drug development to select efficacious anticancer drugs. [
Drug development has a very low success rate, and astronomical costs and time remain problematic. Therefore, efforts are being made to develop new drugs using drug-repurposing strategies. Drug repurposing (also called drug repositioning, reprofiling, or re-tasking) is a strategy for identifying new drug efficacy and use by expanding the range of medical indications for which a drug was originally approved [
]. This drug repurposing strategy offers several advantages over the development of an entirely new drug that targets a new indication. First, repurposed drugs are less likely to be discontinued owing to safety concerns in subsequent efficacy trials, as they have already been demonstrated to be sufficiently safe drugs in preclinical models and humans. Second, as the formulation for most preclinical and clinical trials in the safety evaluation stage has already been completed, it is possible to shorten the development period for further drug optimization and improvement to ensure safety. The time and cost required for phase 3 clinical trials are similar to those for drug development, but there may be significant cost and time savings in preclinical and phase 1 and phase 2 clinical trials [
]. The cost of bringing a repurposed drug to the market is estimated at $300 million on average, compared to an estimated $2–3 billion for new chemicals [
]. Therefore, if the 3D-HTS analysis method is applied to quickly and accurately discover new targets, routes, and indications of repurposed drugs, it is expected to increase the drug development success rate compared to new drug development and dramatically reduce cost and time.
2.7.4 Basic research
3D cell culture models mimicking the living body are expected to be applied to various basic biological studies, such as drug mechanisms and cell differentiation in the human body. But there is still remains to be done to develop systems that can accurately mimic the conditions and disease-related pathologies in vivo. [
] An ideal 3D cell culture model simulates a disease-associated tissue-specific physiological or pathophysiological disease microenvironment in which cells can proliferate, aggregate, and differentiate [
]. Reactivity to drugs may vary depending on the composition of the ECM, the interaction between the ECM and cells, immunomodulatory molecules, and the action of immune cells as well as disease-related target cells [
]. Therefore, it is very important to develop a 3D cell culture model considering that responsiveness to a wide range of drugs varies not only with specific cell lines or tumor types but also with the surrounding stroma. Thus, it may be possible to conduct research for efficient drug development and discovery, enabling a new pharmacological approach and development of a useful in vitro toxicity and efficacy testing platform.
3. Conclusions
In this paper, we summarized 3D cell culture-related technologies and commercially available products among these models and their application in preclinical and clinical research. To date, there is no standardized method for 3D cell culture model development because unlike in 2D cell culture model development, various methods can be implemented for 3D models depending on the ECM, structure of the culture vessel, characteristics of the cells used, and composition of the culture media. Researchers are still developing newer 3D cell culture models, and many attempts have been made to commercialize them. Although many products are on the market, there are still many obstacles to 3D cell culture models forming a market for consumption by life science researchers and healthcare workers. The obstacles that exist in research using 3D cell culture models can be categorized into the following two main categories.
1.
User-friendliness: The 3D cell culture model must be cultivated while forming a cell mass; therefore, care must be taken for the replacement of the culture media, maintenance of the ECM, or cross-contamination. These problems will require effort to simplify the experimental procedure by developing a culture container and a method that is easy to handle.
2.
Standardization of analysis: In the 2D cell culture, the activity of the cell is well standardized by the MTT assay or indirect measurement using the luminescence assay by measuring ATP. However, in 3D cell culture models, diffusion and permeability of existing MTT or ATP reagents differs for each cell mass. Currently, it is common to measure the size of a cell mass (spheroid) by staining. However, it is difficult to represent the activity of cells within a cell mass. Efforts to precisely measure the activity of cells in cell clusters using organization transparency (CLARITY) or confocal microscopes have been ongoing, but standardization of the technology has not yet been achieved. If these problems are resolved and the technology standardized, a biocompatible 3D cell culture model is expected to have a significant impact in the field of precision medicine, new drug bio-industry, and basic research.
The field of 3D cell culture-based HTS has grown exponentially over the past few years. In particular, 3D drug efficacy evaluation and toxicity tests are in progress targeting various organ diseases ranging from skin, digestive system, respiratory system, heart and central nervous system diseases. In this regard, there are efforts to develop an optimized 3D culture model that can mimic a microenvironment similar to in vivo tissue microenvironment and disease pathology. Considering that there are approximately 300 biochemical constituents that are not cellular components only in the ECM, this is still a difficult task. However, if various 3D cell culture methods that can be used are developed and knowledge of biomedical engineering is used and applied, it is expected that 3D biomimetic platform-based drug development, discovery, and precision medical research may be achieved.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Gachon University research fund of 2022.(GCU- 202205760001). This work also was supported by the Ministry of Science and ICT, (Project Number: 2023-22030007-11, NIDS:1711179109) and Commercialization Promotion Agency for R&D Outcomes (COMPA).
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