In recent decades, cell culture has emerged as one of the most extensively employed techniques for substituting, preceding, or complementing the use of animals in research. In the 20th century, cell culture was developed to study animal cell behavior outside the body in a controlled environment. Initially, it employed crude extracts and animal lymph as culture mediums. However, it has since evolved into an indispensable tool for investigating degenerative diseases, cell therapy, stem cell research, testing the toxicity and efficacy of novel compounds, and understanding pathogenesis, among other applications ^[1][2][16,17].

The predominant cell culture model employed in examining fungal pathogenesis is the monolayer (2D) culture due to its convenient alteration of culture mediums, subculturing, analysis, and standardization. Through phagocytosis assays involving macrophages ^[3][18], multiple researchers have discovered that phagocytic cells are more inclined to engulf planktonic cells as opposed to cells originating from biofilms, such as Cryptococcus neoformans and Candida albicans ^[3][4][18,19]. Some investigations have centered around elucidating the behavior of exocytosed macromolecules from fungi during cellular infection. For instance, exoantigens extracted from the fungus P. brasiliensis stimulated the proliferation of pulmonary fibroblasts in vitro. In contrast, a mannoprotein extracted from the cell wall of C. albicans induced alterations in the cell cycle and inhibited apoptosis in human keratinocyte cells (HaCat) ^[5][20]. Cell cultures also offer insights into interaction studies. For example, some researchers have demonstrated the relevance of the Gmp1 binding protein in immune evasion, cell adhesion, invasion, and colonization of C. albicans in human umbilical vein endothelial (HUVEC) and HaCat host cells. The utilization of primary or lineage-specific cells from human and mammalian hosts (macrophages, neutrophils, dendritic, endothelial, epithelial, and stem cells, among others) has facilitated comprehension of the fungus–host relationship and the pursuit of prospective therapeutic targets ^[6][7][8][21,22,23].

Nevertheless, this kind of culture model has limitations due to its simplistic architecture, leading to diminished physiological functionality caused by the decline of characteristics during culturing. Despite its close resemblance to natural conditions, the in vitro process still presents challenges for cell development. Its cell–cell and cell–matrix adhesion is diminished, and it lacks the attributes (such as heterogeneity and three-dimensional architecture) of an in vivo tissue since its nutritional and hormonal environment undergo modification ^[2][9][17,24].

Thus, three-dimensional (3D) cell cultures are extensively employed for novel drug screening of antifungals. Several advantages are tied to obtaining more precise toxicity and efficacy results than in vivo models, along with host–pathogen interactions closely mimicking reality ^[10][11][25,26]. Recently, pulmonary spheroids have been employed to screen broad-spectrum antifungal drugs, yielding outcomes that indicate three-dimensional cell cultures are less susceptible to the toxic effects of drug treatment compared to monolayer cultures. Furthermore, it was observed that monolayer cell cultures are approximately ten times more sensitive than their 3D counterparts, underscoring that 3D cell cultures better resemble in vivo models. Consequently, three-dimensional (3D) cultures have emerged as a viable alternative for replicating the in vivo environment ^[11][26].

Three-dimensional (3D) cultures are pivotal in vitro studies as an alternative model that curtails research expenses ^[9][12][24,27]. Although exploration of 3D culture began around the 1970s, historical records indicate that early 20th-century approaches to cell differentiation and aggregate models were already underway ^[12][13][14][27,28,29]. Nonetheless, during the late 20th and early 21st centuries, 3D cultures gained prominence, notably following an article by Weaver et al. in 1997 ^[15][30], followed by subsequent publications that underscored the significance of 3D culture, particularly in advancing tumor research ^[15][16][17][30,31,32].

Numerous researchers demonstrated that specific antibodies could hinder a surface receptor for β1-integrin, causing cancer cells to mimic normal cells, a phenomenon not observed in a 2D environment. This event marked the continued attention on 3D cultures, initially for tumor studies and subsequently in fields such as toxicology, pathogenesis, disease physiology, and other applications ^[18][19][33,34].

Evidence indicates that 3D cultures can create a microenvironment more akin to that of animals than 2D cultures: cells in 3D settings exhibit distinct morphology and physiology from their 2D counterparts. Moreover, cellular responses, proliferation rates, gene and protein expression levels, and susceptibility to molecules render 3D cultures more akin to responses obtained within an in vivo environment, differing from those witnessed in 2D models ^{[9][18][20][21]}[24,33,35,36].

Diverse methodologies exist for creating 3D cultures, although not all techniques have been validated for studying fungal pathogenesis. The principal methodologies include scaffold-free spheroids; spheroids formed using scaffolds, organoids, and skin/mucosa models. The scaffold-free spheroid approach requires no material to form 3D cultures; cells autonomously organize and naturally connect ^[22][23][24][38,39,40]. The established methodologies include suspended droplet microplates, magnetic levitation, and spheroid microplates with ultra-low fixation coating ^[25][41]. This approach is straightforward, demanding less economic investment and time than animal experiments and obviating the need for ethical committee approval. Consequently, spheroids facilitate cell interaction, aggregation, and the establishment of 3D cultures ^[26][27][42,43].

Conversely, spheroids formed through scaffolds necessitate hydrogels, polymeric materials characterized by varying physical, chemical, and biological attributes ^[28][29][44,45]. Prominent hydrogels in 3D cultures include alginate, collagen, agarose, and natural polymers. However, some scaffolds can be fashioned from synthetic hydrogels. These hydrogels enclose cells in the culture medium, forming miniature pearls through their pores. This arrangement permits the entry of nutrients and waste products exit alongside favorable biocompatibility with the culture medium ^[18][19][28][33,34,44].

Organoids entail multilayered cell structures enveloped by an extracellular matrix that safeguards cellular connections and delineates tissue functions and structures. Organoids are self-arranging cells that closely mimic human organs. For sustained organoid function and differentiation, a culture medium is necessary. Two types of stem cells—embryonic pluripotent stem cells (ESCs), governing embryonic organ development, and adult somatic stem cells (ASCs), specific to maintaining homeostasis and regenerating mature organs—give rise to organoids. The culture platform can reside on a biopolymer surface where cells are distributed in culture flasks coated with extracellular matrix molecules like collagen I and Matrigel. Alternatively, cells are encapsulated within a natural or synthetic biopolymer gel to facilitate more physiological 3D organization. Culture on mechanical supports, organs, or organ fragments is maintained at the liquid–air interface to facilitate gas exchange, and cells are grown on a porous membrane at this interface ^[30][31][46,47].

Skin 3D models, also called artificial skin or reconstructed skin, replicate the layers constituting human skin—a complex structure encompassing three layers: the epidermis, dermis, and hypodermis ^[31][32][33][47,48,49]. The epidermis predominantly comprises keratinocytes, Langerhans cells, and melanocytes that contribute to microbial defense, melanin synthesis for radiation protection, and endocrine functions. The dermis houses fibroblasts, synthesizing the extracellular matrix, in addition to endothelial cells, macrophages, and various components such as blood vessels, sebaceous glands, sweat glands, hair follicles, and nerves, collectively upholding physiological activities. The hypodermis, rich in adipocytes, offers mechanical protection and energy storage alongside playing a pivotal role in temperature regulation ^[33][34][35][49,50,51].

Skin 3D cultures effectively recreate the epidermal and dermal components via cells and biopolymers for skin reconstruction. A frequently utilized methodology involves employing biopolymers to construct skin layers, enabling the incorporation of fibroblasts, keratinocytes, stem/progenitor cells, or reprogrammed cells, thereby mimicking the tissue’s morphological and biological characteristics. Alternatively, 3D printers can assemble the cellular layers ^{[31][33][34][36]}[47,49,50,52]. In recent years, the burgeoning demand for skin models has propelled the rapid expansion of the market, resulting in commercially available skin models such as EpiDermTM (MatTek, Ashland, MA, USA) and EpiskinTM (formerly by Episkin, Chaponost, France; now by L’Oreal, SkinEthic, Nice, France), among others ^{[37][38][39][40][41]}[12,53,54,55,56].

Aside from skin models, 3D mucosal cultures have also been reported, particularly in models concerning oral mucosa and other mucous membranes encompassing the organism ^[42][57]. The oral mucosa comprises stratified epithelium that safeguards against physical and chemical harm, microorganisms, and dehydration. It also encompasses a basement membrane composed of thin layers of extracellular matrix, type IV collagen, and laminin, contributing to protection and homeostasis ^[43][44][45][58,59,60]. Analogous to skin models, these cultures exhibit organizational complexity, effectively simulating an in vivo environment ^[42][57].

3D mucosal cultures, similar to skin models, can also be created through scaffolds to generate cellular layers or procured commercially. At least two available models include one featuring oral squamous cell carcinoma (Skinethic Laboratory, Nice, France) and another comprising primary oral keratinocytes (MaTek Corporation, Ashland, MA, USA). These models are cultured in chemically defined mediums in an air–liquid interface configuration to mimic oral epithelial tissue, with or without keratinization ^[42][57]. Each 3D cultivation method presents advantages and disadvantages, contingent on the desired experimental outcomes. The employment of 3D cultures fosters a highly physiological environment characterized by direct cell–cell contact and interactions with extracellular matrix components, including collagen I, collagen III, and fibronectin, which are principal constituents ^[1][20][16,35].

One of the primary attributes of organoid models is their capacity to replicate pathogen–host interactions, with current studies primarily centered on virology, particularly in the context of brain organoid models and the Zika virus ^[46][47][48][61,62,63]. Moreover, recent research has explored the application of organoid brain technology to develop drugs for preventing or treating Zika infections ^{[49][50][51][52]}[64,65,66,67]. Organoids derived from intestinal and gastrointestinal tissues have also found utility in investigating pathogen–host interactions for various pathogens, including rotavirus, enteric bacteria, Clostridium difficile, Helicobacter pylori ^[53][54][68,69], and norovirus ^[55][56][57][70,71,72].

However, a paucity of literature addresses the utilization of organoid models for studying fungal–host interactions. Demonstrations have been made that the pulmonary and intestinal organoid model exhibited cytokine release evaluation after infection with heat-inactivated A. fumigatus and other strains. Moreover, a notable rise in the expression of biologically significant Toll-like receptors elicited a range of pro-inflammatory mediators, signifying the model’s efficacy in scrutinizing and quantifying immune responses ^[58][73].

Employing organoids derived from esophageal and gastric cancer cells to explore the interaction of fungal plaques revealed that the microenvironment favored the growth and invasive fungal infection by Candida spp. ^[58][73]. The use of explant culture from murine intestinal mucosa exhibited a highly prismatic epithelium with specific cell-to-cell epithelial connections, a basal lamina, and diverse types of connective tissue cells. This approach evaluated the infection process with C. albicans, revealing its capacity to adhere, penetrate, and infiltrate the epithelial barrier ^[59][74].

Lung organoids have been harnessed to probe Pneumocystis spp. infections; organisms predominantly confined to the alveolar lumen. This study extracted type 1 and 2 lung epithelial cells through lung digestion, followed by organoid development. The insights garnered facilitated establishing a mean organoid size standard for introducing target microorganisms; smaller organoids tended to collapse during the infection ^[60][75].

However, akin to any model, organoids possess limitations. Notably, they lack innervation, blood vessels, and immune system cells, and the disease processes they replicate might not fully parallel those observed in the human body ^[61][76]. The self-organizing characteristics of organoids present the prospect of incorporating additional cellular or microbial elements, and although their exploration has increased due to their human-organ-like resemblance, there are still gaps in understanding many approaches. Particularly in studies employing organoids as models for fungal infections, substantial avenues remain unexplored ^[60][61][62][75,76,77].

Comprehending the conduct and adaptation of fungal pathogens within the human body has surged in importance, given the escalating prevalence of fungal infections. Recent investigations have led to the development of 3D models representing epithelial tissues designed to scrutinize fungal infections of the respiratory and oral mucosal regions.

A 3D epithelial model using a transwell scaffold was deployed to investigate upper and lower respiratory tract infections. Infection with A. fumigatus within this model revealed the detection of pathogens, phagocytosis, and migratory characteristics, illustrating the functionality of this in vitro system in simulating the in vivo environment ^[63][78].

Creating a reconstituted oral mucosa model utilizing fibroblasts, type IV collagen, and keratinocytes extracted from gingival biopsies facilitated infections with C. albicans and S. aureus, simulating co-infections within the oral mucosa. The model exhibited surface damage upon C. albicans infection alone and invasion of the subepithelial collagen matrix in the case of co-infection ^[45][60].

This reconstituted oral mucosa model further highlighted fungal infection behavior, both with and without the addition of polymorphonuclear cells. Including immune cells exhibited protective effects and reduced epithelial damage, implying a Th1-linked immune response ^[64][79].

A recent study featuring a 3D co-culture model of oral mucosa infected with Candida spp. investigated the efficacy of silver microcrystals. This model demonstrated satisfactory biocompatibility, and the tested compound exhibited promising effectiveness in treating fungal infections ^[64][79].

These investigations are pivotal for comprehending the host’s defense mechanisms against pathogens. Nonetheless, the validation of 3D models as reliable predictors of biological responses is necessary, and a demand persists for more robust models capable of mimicking the in vivo environment while diminishing the need for mammalian usage in research, aligning with the principles of the 3Rs.