Applications of Tumor Cells in an In Vitro 3D Environment

: Spherical, multicellular aggregates of tumor cells, or three-dimensional (3D) tumor models, can be grown from established cell lines or dissociated cells from tissues in a serum-free medium containing appropriate growth factors. Air–liquid interfaces (ALIs) represent a 3D approach that mimics and supports the differentiation of respiratory tract and skin 3D models in vitro . Many 3D tumor cell models are cultured in conjunction with supporting cell types, such as ﬁbroblasts, endothelial cells, or immune cells. To further mimic the in vivo situation, several extracellular matrix models are utilized to support tumor cell growth. Scaffolds used for 3D tumor cell culture growth include both natural and synthetic hydrogels. Three-dimensional cell culture experiments in vitro provide more accurate data on cell-to-cell interactions


Introduction
Cell-based in vitro assays are complex and require control of the three-dimensional (3D) environment to obtain reliable precision data on, for example, therapeutic candidates in humans before predicting drug cytotoxicity and entering clinical trials [1].Spherical, multicellular aggregates, or spheres, are grown from established cell lines or dissociated cells from tissues in a serum-free medium containing appropriate growth factors.When performing 3D cell culture experiments in vitro, the environment can be adjusted to mimic the in vivo environment to provide more accurate data on cell-to-cell interactions and aid in tumor characterization, drug discovery, metabolic profiling, and stem cell research, as well as being useful for studying other types of disease [2].The tumor microenvironment (TME) requires various chemical, physical, and biological factors that shape and support the proliferation, evolution, invasion, and metastatic properties of cancer cells [3,4].In addition, tumor growth and metastasis are continuously in need of oxygen supply, nutrients, and growth factors provided by the vasculature.More complex 3D models are under development to enable not only cell-cell interactions but also nutrient/waste transport, vascular perfusion, and microfluidics [4,5].The organ-on-a-chip model utilizes microfluidic technology and tissue engineering to mimic and monitor dynamic 3D tissue microenvironments.It should be noted that animal models are capable of simulating the TME, but animal modeling is expensive, difficult to manipulate, and limited in its ability to recreate human immune biology [5].
Structural proteins, such as collagen, laminin, fibronectin, proteoglycans, and carbohydrate hyaluronic acid (HA) provide the cells with support [6][7][8].Interstitial collagen is secreted by fibroblasts, organizing the collagen fibrils I and III and promoting cell differentiation, growth, and migration [9].The basement membrane provides an additional physical barrier between the epithelial cells and the stroma, allowing gas diffusion.The ECM can, therefore, play a critical role in mimicking the 3D culture environment since its structural and biochemical support are essential for many basic cellular processes [9].Li et al. showed that the structure and composition of the ECM change during the process of oncogenesis and the development of CRC tumors [10].There are different collagens and matrix metalloproteinases (MMPs) that can change in expression during tumor development.Moreover, CRC liver and lung metastases have been investigated for how the TME influences tumor progression and invasion, which is important to understand to be able to develop TME-targeted treatments [11].

2D vs. 3D Cell Cultures
Standard protocols for 2D cell cultures are well established and frequently used in life science.The maintenance of 2D cell cultures is simple and cost-efficient, but the possibilities for the cells to differentiate and communicate with other cells and matrices (if added) are limited [9, [12][13][14].Cells grown in vitro in 2D form flat cells adhering to the plastic surface.The morphology of the cells is altered with lost polarity and phenotype, where both the proliferation rate and sensitivity to chemotherapeutic drugs have been shown to be higher for cells grown in 2D compared to the natural 3D environment (reviewed in [9, 12,13]).One of the reasons for this is that the cells can detect the stiffness of the substrate to which they attach, leading to cytoskeleton remodeling [14].Three-dimensional scaffolds provide a natural environment for studying cell migration and may provide information for further understanding cancer metastasis [15].Matrix topography, including porosity and microarchitectures in 3D models, is required for regulating cell mobility and retaining ECM components produced upon cell differentiation.Moreover, gene and protein expression differ between 2D and 3D cultured cells.Due to the lack of physiologically relevant 2D cell cultures for preclinical models, many researchers are instead exploring 3D cell cultures that better mimic the tissue or organ.It is now well established that 3D culture systems mimic tissue factors and are the best representatives of in vivo cellular phenomena in comparison to 2D models [1].Cells grown in a 3D environment resemble physiological conditions more closely in terms of cell morphology, protein expression, biomarker expression, and gene expression [16].Still, protocols and procedures for more complex cell growth in 3D need to be further developed and optimized.

Scaffolds and Matrices
There are several commonly used methods for 3D cultures, including scaffold-free spheroid formation, such as the hanging drop method, non-adherent surface culture, suspension culture, and nanoimprinted scaffolds [9, 16,17].Scaffold-free 3D cell cultures are preferred when high numbers of spheroids are needed.Hanging drop plates allow the formation of spheroids via self-aggregation by relying on gravity.The spheroids hang in open, bottomless wells in order to regulate the environmental humidity of the cells.Hanging drop plate methods have a wide range of uses due to their replicability [2].The hanging drop method can be easily applied to a wide range of cell lines, and the efficiency of spheroid formation relies on the inherent ability of the cells to self-aggregate.Indeed, the advantages of scaffold-free 3D cell cultures lie in the simplicity of the models, the reproducibility of results, the ease of preparation, scalability, and applicability to various cell lines [18].
The scaffolds used for 3D cultures are based on either natural or synthetic hydrogels, or hybrids of both, and are of animal or plant origin.The particular type is chosen depending on the biocompatibility advantage or the physicochemical nature, respectively [9].Natural gels (natural polymers) are, for example, fibrinogen, hyaluronic acid (HA), collagen, Matrigel ® (Corning, Corning, NY, USA), gelatin, chitosan, and alginate [19,20], whereas most synthetic hydrogels are synthesized by modification of synthetic polymers, which exhibit versatile biophysical, mechanical, and biological properties [9].Polymer scaffolding offers a favorable 3D setting for cellular performance.Collagen-based hydrogels are the most often used scaffolding models since collagen is the major constituent of the basement membrane [9].
Hydrogels are cross-linked networks formed of hydrophilic polymers attached through physical, electrostatic, or covalent interactions.Indeed, hydrogels are an attractive scaffolding material because of their mechanical properties that can mimic those of natural tissues [19].Collagen-based hydrogels are expensive, difficult to prepare reproducibly, and also require extensive handling and specific equipment [9].Nonetheless, they offer excellent support regarding cellular heterogeneity and spontaneous cell organization.The stiffness of the 3D culture can be controlled by adjusting the hydrogel concentration.This system can also be applied to study the interaction between any type of neoplastic cell, even using more than two different cell types [21].Cellular adhesion directly to the gel can be favored over suspension within the scaffold by the incorporation of various peptide domains into the hydrogel structure, thus dramatically increasing the tendency for cellular attachment [19].A successful strategy to mediate cellular attachment is the inclusion of an RGD adhesion peptide sequence (arginine-glycine-aspartic acid).Fibroblasts, endothelial cells, smooth muscle cells, osteoblasts, and chondrocytes are cell types that have been shown to bind to RGD.This way, cellular migration, proliferation, growth, and tissue regeneration were enhanced.Hydrogels are unique because of their ability to mimic the ECM while allowing soluble factors, such as cytokines and growth factors, to migrate through the tissue-like gel.The ECM is rich in growth factors and metabolic precursors [9].In addition, the ECM plays a basic role in the development and maintenance of epithelial tissues.For example, the ECM of human breast tissue is composed of protein fibril complexes intertwined in a network of glycosaminoglycan (GAG) chains.
Natural polymers include the commercially available "golden standard" Matrigel ® , which is a mixture of gelatinous proteins derived from the basement membrane isolated from the mouse sarcoma Engelberth-Holm-Swarm [9].The biological activity of Matrigel ® includes the production of a large amount of ECM-related components, such as type I collagen, laminin-111, heparin sulfate proteoglycans (perlecan), and nidogen [22], as well as soluble growth factors, such as fibroblast growth factor (FGF), epidermal growth factor (EGF), transformative growth factor beta (TGF-beta), and MMPs, including MMP-2 and -9.Although easy to use and very popular, the presence of these growth factors in unknown and uncontrollable quantities can have both positive and negative impacts on research [9].Another issue is the lack of control over its exact composition and content-wise batch-to-batch variability since Matrigel ® is produced and purified from an animal [23].
Johansson et al. developed methods for using the self-assembly of recombinant silk into fiber networks with concurrent integration [24].Silk proteins functionalized with a cell adhesion motif from the ECM protein fibronectin (FN) were used to create BioSilk [25].With the FN motifs, endothelial cells were coated and seeded, which enabled the work of engineered vascular constructs or synthetic grafts [26].Next, FN-silk together with recombinant human laminin 521 (LN 521) created a 3D culture system suitable for the expansion of human pluripotent stem cells (hPSCs) and supported neural differentiation [27].The FN-silk furnishes a unique 3D culture framework with an ECM-like network that allows both cell-cell and cell-matrix interactions, which are lacking in commonly used scaffold-free spheroid cultures [28].Moreover, in a recent report on recombinant spider silk nanomembranes, it was shown that those nanomembranes were able to support a cell co-culture into an in vitro blood vessel wall model that can be used as substrates in drug delivery systems and in organs-on-a-chip [29].

Organoids
Tissue fragments from the stroma were originally separated, and cells were cultured within 3D gels to form organoids [30,31].Today, organoid cultures include techniques that result in self-organizing, self-renewing 3D cultures derived from a variety of tissues, including primary tissues, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) (reviewed in [30]).ESC-or iPSC-derived organoids make it possible to generate the complex structure of adult organs in which all cells are fully differentiated [14].They may contain mesenchymal, epithelial, or endothelial components but tend to also constitute other types of cells.Adult stem cell (ASC)-derived organoids can mimic both physiological and pathological adult tissues, including cancer.
Organoids can be produced from individual patient tumor samples, meaning that the biodiversity is similar to that of in vivo tumors [16].With more experience and knowledge of the precise culture techniques of organoids, screening platforms for drug discovery that are more cost-effective than animal models will be developed.Today, transcriptome profiling is one of the most common downstream applications of organoids, together with applications in drug discovery and precision therapy [30].
The selection of appropriate media, growth factors, and morphogens is also fundamental to preserving the functional characteristics of cells and obtaining the desired 3D construct [23].Many different technologies have promoted the development of specific 3D models applicable in various biomedical and medical fields.According to Marchini et al., the definition of organoids can be summarized in the following four points: (i) two or more interacting cell types; (ii) 3D multicellular structure; (iii) self-organization of multiple distinct cell phenotypes into specific supracellular structures found in organs; and (iv) functional properties resembling the corresponding native tissue [23].Moreover, self-organization is a key feature of organoids due to multicellular structures with similar architecture as the in vivo organs evolving from stem and progenitor cells [23,32].
Mitrakas et al. discussed the challenges of 3D cell cultures versus in vivo tumors in a recent review [33].The spheroids are designed to more accurately reproduce the gradients of nutrients and oxygenation found in the tumor microenvironment.The added complexity also requires consideration of the experimental design as well as monitoring and acquisition.For example, some reagents will reach the center of a larger 3D cell culture, whereas for others, lytic assays disrupting the cells within these structures are necessary [33].
Imaging spheroids is challenging due to light scattering, and fluorescent imaging of fixed spheroids is limited to the outer layers of cells.Live imaging requires compatible in vitro culture chambers attached to the microscope.A solution for imaging 3D cultures could be using digital holographic (DH) microscopy, a convenient noninvasive method to analyze living cells [34][35][36][37].With this technique, in vitro cell cultures can be imaged in 2D and pseudo-3D, and measurements of the size and morphology of the cells will be provided.Recently, novel methodologies utilizing DH microscopy for the analysis of spheroids were presented [34,38].In addition, light-sheet-based fluorescence microscopes can be used to study spheroid development over longer time scales [33].
Three-dimensional cell culture systems and organoids both provide excellent artificial microenvironments in which cells grow, differentiate, and interact with each other and with the encapsulating biomaterial in 3D, mimicking the in vivo microenvironment.

Co-Culture Systems
Over the past ten years, the field has made progress in incorporating multiple elements of the TME by using co-culturing models [39].Moreover, CAFs have a prominent role in cancer development, from initiation to primary and metastatic progression, and in drug resistance [40].There has also been a growing appreciation of the ability of cancerassociated fibroblasts (CAFs) to modulate the immune system [41].CAFs modulate cancer metastasis through the synthesis and remodeling of the ECM, the production of growth factors, and their influence on angiogenesis, tumor mechanics, drug access, and therapy responses.Targeting CAFs by altering their numbers, subtypes, or functionality is a very intriguing potential approach to improving cancer therapies.CAFs are perhaps the most effective cells within the TME in depositing on and remodeling the ECM [41].The alterations in matrix production and tumor mechanics largely result from the action of CAFs and have complex consequences for tumors.In a recent review, Mao et al. describe that most CAF subpopulations usually exhibit cancer-promoting effects, while the discovery of cancer-restraining CAFs (rCAFs) was reported to instead exert inhibitory effects on tumor progression [42].Many studies have now shown that interactions between CAFs and immune cells, or other immune components, are capable of inhibiting the anti-tumor immune response.Shiga et al. showed that CAFs are an important IL-6 source and that anti-IL-6 receptor antibodies suppressed angiogenesis and inhibited tumor-stroma interactions.Furthermore, it was demonstrated that CAFs contribute to drug resistance acquisition in cancer cells [43].
Moreover, 3D multicellular tumor spheroid (MCTS) models were developed by combining three different cell types: epithelial colon cancer cells (HCT116), human intestinal fibroblasts, and monocytes [44].These CRC models mimicked ECM production, spatial organization, and the formation of a necrotic core and proved to be adequate for drug screening.

Microfluidic Organ-on-a-Chip-Based Cell Culture Devices
Advances in material fabrication techniques (e.g., microfluidic devices, 3D bioprinting, and high-throughput robotic systems) are supporting the development of sophisticated 3D cancer invasion models [45].Microfluidic organ-on-a-chip-based cell culture devices are valuable tools that enable co-culturing of 3D cell culture systems in a spatially controlled manner (e.g., controlled perfusion flow and gradient control of cytokines) to better simulate tissue and organ physiologies [23].Microfluidic technology, with its unique characteristics, has become a promising tool in cancer research, especially for studying the metastasis process [46].The details of each step in the metastasis process are not yet fully understood, and appropriate models are, therefore, needed to shed light on the complex multistep process.For example, mouse models have been previously exploited to study the mechanism of metastasis, but animal models have also been challenged as they possess some disadvantages [46].By mimicking the TME and angiogenesis, microfluidic platforms are used to study in vitro tumor growth, and the platforms are utilized for the detection and identification of CTCs with different phenotypes [46].

Colorectal Cancer
CRC is the third most common cause of cancer-related deaths worldwide [20,47].Due to the anatomical proximity, CRC most often metastasizes in the liver.Up to 25% of patients present with CRC liver metastases at the time of diagnosis, and 50-60% of CRC patients will develop liver metastases at some point later on [5].Unfortunately, all patients are not eligible for hepatectomy at the time of diagnosis and will, therefore, suffer from progressive metastatic CRC.
The incidents of CRC have been increasing, especially in younger patients [16].Despite significant improvements in treatment, the prognosis for patients with metastatic CRC (mCRC) is still poor [48].Molecular profiling consists of a pathologic assessment of the tumor tissue to test for microsatellite instability-high (MSI-H) and mismatch repair-deficient (MMR-D), as well as for sequential variants in KRAS, NRAS, and BRAF genes that have been applied for the prognosis of mCRC and the prediction of response to adjuvant therapy in some local clinical practices [49,50].
Novel and more effective therapeutic treatments are, therefore, necessary.Twodimensional (2D) cell cultures are not accurately representing the interactions between the CRC cells and other components of the TME, including immune cells, cancer-associated fibroblasts (CAFs), and stromal cells, in vivo.It has been shown that the stromal compartment of CRC cells can be involved in alterations in disease progression and therapeutic response [51].In addition, cytokines and growth factors that are present can affect CRC initiation and progression [16].
The CRC HCT116 cell line is frequently used for culturing 3D spheroids.The cell line has a mutation in codon 13 of the ras proto-oncogene [52].Feist et al. used HCT116 cells to detect over 1350 proteins with very little variation between replicate spheroids [53].Moreover, cellular populations of the CRC cell line HT29 showed differences between the outer proliferating layer, the senescent middle layer, and the necrotic core, which is in line with comparable in vivo models [54].The HCT116 and HT29 cancer cell lines belong to the most often utilized CRC model cells in 2D cultures and have, therefore, frequently been investigated in 3D spheroid cultures [55,56].
Ivanov et al. published one of the first devices that allowed the building of tissue microarrays of spheroids cultured under various conditions or from different cell lines straight from the culture plate, suited to spheroids grown in high-throughput plate-based formats [57].Eleven different cell lines were cultured and analyzed, including HCT116.The HCT116 cell line did not require addition or Cultrex basement membrane extract and formed compact spheroids.Moreover, Ivanov et al. used immunohistochemistry to detect protein markers, but the method could equally be applied to detect RNA or DNA markers, e.g., using in situ hybridization methods [57].
Reidy et al. described the time line for the first organoid studies on CRC cells performed less than fifteen years ago [16].Indeed, the patient material of tumor tissue was more comparable with CRC cells than spheroids based on cell lines [58].Organoids are being used to examine the molecular and genetic bases of CRC initiation and progression [59].
Patient-derived organoids (PDOs) are robust 3D in vitro models that retain the histopathological features of in vivo tumors, including patient-specific drug responses [60].Ponsioen et al. analyzed the interplay between EGF signaling and the activation of MAPK/ERK using PDOs from KRAS and BRAF mutant CRCs.Without activity through the EGF receptor (EGF-R), signaling by these two oncogenes is insufficient to sustain the full cell proliferative potential.The study provided data that revealed that EGFR signaling actively amplifies the signaling output of oncogenic MAPK effectors.Individual treatment of CRC is preferred since the disease is very heterogeneous.Today, first-line treatment consists of resection of the primary tumor and, for patients with invasive tumors and a risk of relapse, adjuvant treatment with 5-fluorouracil (5-FU) together with other chemotherapeutics [61].The addition of targeted therapies includes molecules targeting growth factor receptors.The combination of folinic acid, 5-FU, oxaliplatin, and/or irinotecan (FOLFOXIRI) is the standard of care for mCRC.This strategy inhibits tumor growth but provokes drug resistance and serious side effects [62,63].These 3D systems have been performed with spheroids and organoids, with or without co-cultures of stroma cells, and/or with endothelial cells.Since the development of CRC organoids, many research groups have also used high-throughput analyses or biobanks to identify potential inhibitors of KRAS and EGF-R to study the regenerative responses to a large quantity of compounds or to identify small-molecule drugs [59].

Prostate Cancer
Prostate cancer (PCa) is the second most frequent malignancy in men and accounts for about one-fourth of all cancer cases amongst them worldwide [47].Even though it is not the most fatal cancer type and its course is often indolent, the mortality rate is still relatively high, and every fourth PCa patient eventually dies.The occurrence and mortality rates of PCa exhibit a significant correlation with factors such as high age, ethnicity, or race, as well as environmental and geographic variables [64,65].PCa develops from normal prostate epithelium through a multistep histological transformation process governed by various underlying molecular changes [66,67].Most patients can benefit from treatment at an early stage through a reduction in tumor volume and a rapid decrease in prostate-specific antigens (PSAs) [68].
PSA screening and its enhancements have long been considered "the golden standard" in PCa detection and have significantly lowered the PCa mortality rate.Despite the controversy caused by the US Preventive Services Task Force reports in 2008 and 2012 referring to overdiagnosis of insignificant cancers and unnecessary prostate biopsies, it still has an important role in PCa screening and early detection [69].Novel biomarkers found in both serum and urine, along with their various combinations, are becoming prevalent and have been extensively reviewed [70].Androgen deprivation therapy (ADT) is an effective therapeutic strategy for PCa.Since androgens have important roles in the body, their deprivation, especially in terms of continuous ADT, can lead to numerous unfavorable effects, such as cardiovascular maladies, bone density loss, metabolic changes, sexual dysfunction, hot flushes, and, last but not least, adverse psychological reactions [71].Despite the initial response to ADT, patients develop castration resistance, leading to recurrence and/or castration-resistant PCa [72].Moreover, metastatic PCa that progresses after castration resistance remains incurable [73].
Various clinical PCa models have been developed to clarify the complex underlying mechanisms regarding the treatment resistance of PCa [74,75].The existing in vitro 3D cell culture methods for PCa cells have been reviewed, with advantages and disadvantages discussed [17,75].The 3D cell culture methods of PCa include suspension cell cultures, hanging drop methods, microfluidic devices, gel-embedded scaffolds, and patient-derived xenografts or explants.As for all tumor types investigated, cell lines are widely used in in vitro 3D cell culture methods.The three classic PCa cell lines, DU145, PC-3, and LNCaP, are from clinical metastatic lesions and the most widely used cell lines in PCa research [74].At present, several PCa cell lines have been established from primary PCa tumors, PCa metastases, and PCa xenograft models.In addition, many sublines have been developed from these three classic PCa cell lines, especially the LNCaP cell line [74].The parental LNCaP cells are androgen-sensitive, but sublines of these cells, such as LNCaP-abl and LNCaP-LTAD, are androgen-insensitive due to their establishment through the depletion of androgen in culture media.However, the utilization of cell lines comes with limitations due to their lack of resemblance to the tumors of origin [76].
Petric et al. discussed the development of organoid cultures of PCa tumor cells [75].The origin of the organ-specific cells is often stem cells or progenitor cells.Together with a scaffolding ECM, they often grow to resemble the original tumor, both in structure and function [77].Moreover, advantages with organoids include the ability to keep them in long-term culture, perform genetic manipulations, and cryopreserve them [75,78].Of importance is also maintaining the TME in vitro with the addition of supporting cells, such as CAFs, immune cells, and endothelial cells.
Several examples of the successful establishment of organoids from patient biopsies have been discussed by Saranyutanon et al. [66].In addition, efforts are made to develop organoid models for more rare PCa forms, such as castration-resistant neuroendocrine PCa [79].The organoids were established from metastatic lesions and could demonstrate genomic, transcriptomic, and epigenomic concordance with their corresponding tumors.Organoids and patient-derived xenograft (PDX) models are essential tools for the development of useful PCa models, giving results that can more accurately predict clinical responses in patients [66,80].PCa PDXs and patient-derived organoids (PDOs) from both primary and metastatic lesions have also been developed and can lead to new strategies for cancer precision medicine [73,74,80,81].Moreover, several biobank collections have been established for PCa PDXs and PDOs, giving valuable possibilities for sharing samples and data [80,82,83].
Osteoblasts are vital contributors to PCa metastasis, but it has been reported that bone marrow adipocytes could also play a role.This is exemplified in the work undertaken by Bessot et al., in which a gelatine methacrylamide-based hydrogel was used as a 3D platform for human BM adipocytes and PCa cell investigation in both in vitro and in vivo models.In this study, PCa cells co-cultured with adipocytes in a 3D environment not only had a significantly higher proliferation rate and more heterogeneous morphology but also altered metabolism compared to tumor cells co-cultured with adipocytes in a 2D environment [84].

ALI Systems of the Skin
Historically, the ALI technique was primarily used to mimic respiratory tract epithelia in vitro [23].The first ALI method was developed in the 1980s, which introduced a model of epidermal and respiratory tract epithelial tissues in vitro.Since then, routine approaches to growing fully differentiated in vitro skin models have been established, even for clinical applications of wound healing [85,86].Chen et al. illustrated this point clearly by elegantly reviewing different ALI systems, including skin [87].
To establish and propagate cells in an ALI culture, epithelial cells are seeded in compartmentalized systems on porous filter supports under submerged conditions for 3-7 days [87].After an initial attachment, proliferation phase, and formation of a confluent monolayer, the apical medium of the epithelial cells is removed.The cells "interface" with the surrounding air, differentiating and generating an apical microenvironment [87].Since the basal surface of the cells has access to the culture medium, its nutrients and other additives can nourish cells via diffusion through the porous membrane.The full differentiation of the in vitro structure is completed in approximately 2 weeks [88,89] (Figure 1A).The different barrier functions of human skin and their role in maintaining tissue homeostasis are important to understand when designing 3D reconstructed skin models [90,91].The epidermis is the outer compartment of the skin, and it contains keratinocytes, which comprise around 95% of all the cells in the epidermis [92].These cells are essential for epidermis development as they differentiate and migrate upward to create four strata that facilitate skin barrier function [93,94].The stratum basale is the bottom layer of the epidermis and is responsible for constantly renewing the cells of the epidermis.This layer contains just one row of undifferentiated epidermal cells, known as basal keratinocytes, that divide frequently [95].
The skin protects against chemical exposure, ultraviolet (UV) radiation, and penetration of pathogens and also regulates the diffusion of molecules.In cases of any barrier defect, Langerhans cells, keratinocytes, and skin-resident immune cells provide a secondary immunological barrier [91,95].In both the epidermis and the dermis, cells and molecules from the immune system are present to protect against pathogens and induce an immune response.Langerhans cells are a type of dendritic cell in the epidermal layer of the skin [95].Those cells, which are the main skin-resident immune cells, patrol and protect the epidermis together with T cells.Keratinocytes are also producers of cytokines and chemokines, contributing to the immune defense.There are twice as many T cells present in the skin compared to in the bloodstream, indicating the importance of the skin as an immunological reservoir [91].The epidermis also harbors the melanocytes, which produce the pigment melanin [95].Examples of in vitro 3D models to study different aspects of the skin and its function in a controlled laboratory environment are shown in Figure 1.
Melanoma is the most aggressive form of skin cancer and is caused by a malignant transformation of melanocytes [96].While melanoma has a positive outlook if it does not metastasize, it is associated with the majority of skin-cancer-related deaths.However, stage IV malignant melanoma carries a worse prognosis [97].Multicellular tumor spheroids (MCTSs) are composed of tumor cells forming sphere-like 3D structures.The MCTS structures of melanoma cells can feature in vivo-like cell-cell adhesion, barriers to mass transport, extracellular matrix deposition, as well as cell-matrix adhesion [40].The development of engineered human skin equivalents (hSEs) has recently progressed significantly and serves as an important link between animal models, conventional 2D cell culture systems, and human skin biopsies [98][99][100].

Figure 1.
In vitro 3D models of skin.(A) Epidermis model.A thin layer of keratinocytes is grown on a porous filter support.After culture at the air-liquid interface (ALI), the keratinocytes are arranged in a multi-layered structure, with the outermost layer consisting of flattened cells resembling the stratum corneum.Below this layer, several layers of keratinocytes are present, forming the stratum granulosum, stratum spinosum, and stratum basale, mimicking the epidermis layer of the skin.(B) Epidermis/dermis model (full-thickness model).The top layer is the epidermis, similar to (A), and the bottom layer is the dermis.The dermis layer consists of fibroblasts cultured in collagen or a polymeric scaffold for support.(C) A full-thickness model with melanocytes and immune cells.In addition to the keratinocytes and fibroblasts, melanocytes and/or immune cells can also be added to the epidermal layer.These cells are important for studying the immune response of the skin and how it reacts to different stimuli.Created with BioRender.com.

Melanocytes
The vital role of melanocytes in the skin comes not only from their ability to synthesize melanin, which allows skin pigmentation.More importantly, pigment absorbs the harmful In vitro 3D models of skin.(A) Epidermis model.A thin layer of keratinocytes is grown on a porous filter support.After culture at the air-liquid interface (ALI), the keratinocytes are arranged in a multi-layered structure, with the outermost layer consisting of flattened cells resembling the stratum corneum.Below this layer, several layers of keratinocytes are present, forming the stratum granulosum, stratum spinosum, and stratum basale, mimicking the epidermis layer of the skin.(B) Epidermis/dermis model (full-thickness model).The top layer is the epidermis, similar to (A), and the bottom layer is the dermis.The dermis layer consists of fibroblasts cultured in collagen or a polymeric scaffold for support.(C) A full-thickness model with melanocytes and immune cells.In addition to the keratinocytes and fibroblasts, melanocytes and/or immune cells can also be added to the epidermal layer.These cells are important for studying the immune response of the skin and how it reacts to different stimuli.Created with BioRender.com.
Moreover, more and more companies are commercializing skin analogues composed of fibroblasts and keratinocytes that replicate the skin anatomy to some extent.Phenion ® (Düsseldorf, Germany) FT, StrataTest ® (Stratatech, Madison, WI, USA), VUmc skin equivalent, EpidermFT™ (MatTek Life Sciences, Ashland, MA, USA), and organotypic skin cultures (ORGs) are a few examples of reconstructed bi-layered hSEs that are commercially available to date [101][102][103][104][105]. Those models are a quick, direct, and cheaper alternative to animal testing since they are considered safe and provide a human system, although in vitro [100].Further advantages with hSE systems are that cellular components can be added and evaluated to better mimic human skin.Currently, two main types of engineered 3D tissue models exist, viz., scaffold-free and scaffold-based models [98].The division dictates how the model's dermal part is reconstructed, and it has far-reaching consequences that determine further advantages and disadvantages of the models [92,93].Hence, scaffold-free 3D skin models are based on the idea of stimulating native ECM production, which is advantageous because of the low cost and high reproducibility owing to the batch-to-batch independence of the applied scaffold.Moreover, such models are instrumental in high-throughput cellular function and cytotoxicity studies or biochemical analyses.However, a full-thickness stratified epidermis is required to obtain proper tissue architecture.Scaffold models of 3D tissues are cells grown in the presence of support scaffolds, either hydrogel-based or polymeric fiber-based support [98].Such scaffold-based models exhibit superior biocompatibility and cell adhesion but are limited by their short lifespan and weakened mechanical strength.In addition, 3D hSEs can also be assembled using de-epidermized dermis (DED), where the donated human samples undergo the process of de-cellularization.This allows for the creation of a dermal substitute that is rich in ECM scaffolds, facilitating keratinocyte attachment and proliferation [93].

Melanocytes
The vital role of melanocytes in the skin comes not only from their ability to synthesize melanin, which allows skin pigmentation.More importantly, pigment absorbs the harmful UV radiation that penetrates the skin and protects the cells from DNA damage.The elongated dendritic extensions of melanocytes that allow melanin transfer from the stratum basale to more distant epidermal layers [92] also allow for the transfer of growth factors from keratinocytes, stimulating melanocyte proliferation and differentiation [106].Moreover, when melanocytes are co-cultured with keratinocytes and fibroblasts, the latter cells secrete various factors that support melanocyte migration and survival and allow melanocytes to return to their typical phenotypic characteristics, such as high proliferation rates and active expression of various melanoma-associated antigens [107].Interestingly, in some hSEs, melanocytes have also been reported to mimic naïve melanocytes by protecting the basal keratinocytes against UVB-induced apoptosis [108].
For a long time, 2D monolayer cultures were regarded as the standard models for studying melanocyte or melanoma biology but are now considered quite inferior.This is caused by culture-induced changes in cell morphology and gene expression patterns that alter the phenotype and behavior of the cells in situ.Indeed, the intricate network of cell-cell interactions in native skin requires more advanced systems mimicking cellular communication in a more realistic way.The development of 3D hSEs allows for complex interactions between different cells and between cells and matrix to occur, minimizing the emergence of culture artifacts [109].
One of the most common ways to construct 3D hSEs involves using a collagen-based dermal matrix.A better alternative could be a broadly emerging technology known as 3D bioprinting, which allows for the production of full-thickness hSEs by sequentially printing single melanocytes and keratinocytes on top of dermal equivalents.This not only resolves the problem of drug absorption in the collagen but also allows a more precise allocation of the cells in the epidermis.
The neural crest stem cell (NCSC) origin of melanocytes underlies the ability of metastasizing melanoma cells to both migrate to and thrive in the brain and other major organs, including the lungs [97].Common mutations in genes involved in melanomas include BRAF, NRAS, and NF-1.Significant treatment efficacy has been observed with some regimens, including targeted-and immune-based therapeutic strategies.The understanding of the genetic and non-genetic mechanisms in melanomas has increased, which has set the scene for further technological developments to achieve more advanced in vitro cell culture models.Another significant aspect of melanocytes grown in 3D skin reconstruction is that they exhibit homeostasis and progression similar to what is observed in the skin of patients [97].Moreover, when incorporating melanoma cells into 3D skin reconstructions, they exhibit aggressiveness analogous to their action in patients [97].Recently, Dror et al. demonstrated that melanoma cells use melanosomes to communicate with fibroblasts at the early stages of development, resulting in CAF formation [110].

Immune Cells
Given that the skin's immune component is the body's primary immune barrier and a fundamental source of virtually all skin diseases, considerable interest in incorporating it into 3D skin culture systems is highly anticipated [93,111].Undeniably, for a greater understanding of skin pathophysiology, the successful recreation of skin immunological functions is integral for mimicking native immune reactions, investigating key sensitization events, and modeling skin diseases [93,94].The skin's resident immune cells include dermal dendritic cells (DCs), Langerhans cells (LCs), macrophages, T cells, mast cells, and innate lymphoid cells (ILCs), and each skin layer possesses its own characteristic contingent of cells responsible for regulating immune responses [93,94].However, given the immense complexity of the skin as a biological barrier, comprised of diverse immune cell types involved in immunological responses, it is incredibly challenging to incorporate the entire immune component in hSEs.Thus, to date, most commercially available hSEs either lack the integrated immune component of the skin or focus on incorporating only a single immunological aspect of it [93,94].
To date, various approaches have been employed to integrate immune cells into 3D skin reconstruction models.The incorporation of LCs, which are antigen-presenting cells located in the stratum spinosum of the epidermis, was the primary focus of the first skin immune-competent models that were developed [93,111].In 1997 and 2005, the Schmidt group published two independent studies in which they reported a successful incorporation of cord-blood-derived LCs into a reconstructed human skin epidermis [112,113].However, given the lack of the dermal equivalent in their model as well as logistical limitations regarding the fresh cord blood supply and donor variation, the extensive application of the model was significantly restricted [111].
With this in mind, Ouwehand et al. pushed ahead by incorporating MUTZ-3 cellderived LCs, establishing the hSE VUmc model [103,114].MUTZ-3 cells are a human acute myeloid leukemia (AML) cell line that can be differentiated into LCs, known as MUTZ-LCs, through stimulation with the transforming growth factor β (TGF-β).This triggers MUTZ-3 cell pre-differentiation in MUTZ-LCs, indicated by the emergence of langerin, HLA-DR, and CD1a expression on the cell surface as well as the intracellular presence of Birbeck granules.Moreover, in response to stimulation by an allergen or irritant, the cells begin to resemble their native immune equivalents in the skin by expressing characteristic maturation markers, such as CCR7, CXCR4, and CD83, and acquiring the ability for CXCL12-induced migration [103,111].This model was then successfully employed as a standardized assay for irritant and sensitizer discrimination by investigating MUTZ-LCs' phenotypic plasticity and chemotactic migration into the dermis [111].
Other 3D immune skin reconstruction models have also been developed, which contain melanoma cells, autologous T-cells, and fibroblasts that can be allogeneic [115].This approach allows for the visualization and quantitation of T-cell-mediated tumor cell killing.

The Vascular System
To improve the physiological relevance of 3D skin models, an appropriate vasculature with a capillary structure is necessary.This allows for the creation of an in vitro environment that supplies oxygen and nutrients and eliminates waste products in a physiological manner, mimicking in vivo circulation at the organ level [93,98].Organ-on-chips constitute perfusable culture devices that simulate the structure and function of the vascular system, connecting the cells or tissues via vascular flow [93,116].Such physiological communication between the cells facilitates greater control and maintenance of homeostasis within the system, improving the model's viability [94].Since it creates a potential route for immune cell migration, it also constitutes a functional tool for immunological research, specifically in mediating inflammatory responses [93,94].To date, many skin-based organ-on-a-chip models have been developed and described in detail [117][118][119].Some researchers utilized the fast-emerging and revolutionary technology known as 3D printing [120], while others, such as Ronaldson-Bouchard et al. ( 2022), combined the recent advances in tissue engineering technology to design more complex multi-organ tissue chips.However, in ex vivo conditions, vascular systems could enhance skin barrier function [94].Finally, system perfusion allows for the creation of a biomolecular gradient that can serve as a signaling route guiding the growth, differentiation, and migration of cells in their 3D environment [94,121].

Conclusions
The limitations of 2D cell cultures have provided an excellent new technique for 3D in vitro models.Recent advances in 3D cell culture technology and applications have improved the success rate for the establishment of spheroids and organoids in vitro and provided more accurate data on drug discovery, metabolic profiling, and stem cell research.Here, we provide characteristics and advancements for 3D cancer cell cultures of CRC, prostate, and ALI of the skin.We have discussed different types of natural and synthetic scaffolds, supporting cell types such as fibroblasts, endothelial cells, or immune cells, and the importance of modeling the TME in vitro.With the future expansion of different 3D culture models, a major breakthrough can be foreseen in drug development and disease mechanisms.

Figure 1 .
Figure 1.In vitro 3D models of skin.(A) Epidermis model.A thin layer of keratinocytes is grown on a porous filter support.After culture at the air-liquid interface (ALI), the keratinocytes are arranged in a multi-layered structure, with the outermost layer consisting of flattened cells resembling the stratum corneum.Below this layer, several layers of keratinocytes are present, forming the stratum granulosum, stratum spinosum, and stratum basale, mimicking the epidermis layer of the skin.(B)Epidermis/dermis model (full-thickness model).The top layer is the epidermis, similar to (A), and the bottom layer is the dermis.The dermis layer consists of fibroblasts cultured in collagen or a polymeric scaffold for support.(C) A full-thickness model with melanocytes and immune cells.In addition to the keratinocytes and fibroblasts, melanocytes and/or immune cells can also be added to the epidermal layer.These cells are important for studying the immune response of the skin and how it reacts to different stimuli.Created with BioRender.com.