The need to recapitulate both healthy and pathological states of human tissues in vitro is not unique to the field of female reproduction. Throughout history, many researchers focused their efforts on developing techniques to maintain cells in vitro (Figure 2). It was not until 1885 that Wilhelm Roux succeeded for the first time at maintaining frog cells in the laboratory. More than 20 years later (1912), Alexis Carrel defined a specific culture medium that grew and maintained somatic mammalian cells in vitro for several months (Carrel, 1912; Carrel and Baker, 1926). Shortly thereafter (1916), cell culture specialists discovered that trypsin could be used to isolate individual cells from subcultures (Rous and Jones, 1916).

More than 60 years later, in the 1980s, the scientific interest shifted beyond the ability to simply grow cells in vitro, as the role of the cell’s native microenvironment was revealed and bioengineering techniques were being developed. This environment was considered essential for good cell growth and development. Alongside emerging bioengineering strategies to improve in vitro cultures, in 1980 the first ex vivo extracellular matrix (ECM), derived from a rat liver, demonstrated high adhesion capacity, survival efficiency and cell culture growth (Rojkind et al., 1980).

Bioengineering is a multidisciplinary field combining principles and methods from engineering, biology, and medicine to develop technologies and devices that improve healthcare and solve biological problems as tissue regeneration or development of medical devices (Francés-Herrero et al., 2022). The use of bioengineering was a turning point in the approach of cell cultures to the native environment. In that sense, in 1986, the description of the use of gel-like extracellular matrix components as a suitable microenvironment for cell growth was published for the first time (Kleinman et al., 1986). The subsequent commercialization of this protein-rich matrix, coined Matrigel (BD Biosciences, formerly BD Pharmingen), was a significant research milestone because it supported three-dimensional (3D) in vitro models that reliably mimicked the native conditions that promote cell differentiation and growth, among other advantages.

Advanced in vitro bioengineering approaches became popular at the end of the 20^th century. Microfluidic devices were designed to mimic the endocrine and paracrine signalling between different tissues of human organs. Microfluidic devices combine principles from engineering, physics, chemistry, biology, and nanotechnology for highly specialized and efficient applications. For example, the first organ-on-a-chip dates back to 1997 and it involved chondrocytes on a polyglycolic acid polymer scaffold. They introduced these combination and helped to form cartilage 12 weeks post-implantation in mice (Cao et al., 1997). Finally, microfluidic platforms for in vitro cell cultures were first reported in 1998. Folch and Toner injected collagen- or fibronectin-rich protein solutions through microchannels to create a biocompatible scaffold to seed different cell populations such as fibroblasts (Folch and Toner, 1998).

The most revolutionary milestone in endometrial (and other tissues) cell culture was the establishment of continuous cell lines. This was important because healthy animal-derived somatic cells have a limited proliferative capacity. An important paradigm shift in in vitro culture practices began in 1951, when continuous human cell lines were established. The laboratory of George and Margaret O. Gey established a patient-derived monolayer cell culture from immortalized cervical cancer cells. The resulting HeLa cell line, named after the original patient from whom the primary cells were derived, Henrietta Lacks (Scherer et al., 1953), has become one of the oldest and most commonly used cell lines across the globe. The development of stable cell lines was crucial, as it allowed for homogenizing the cell population and did not require sacrificing any animals. HeLa cell cultures paved the way for more sophisticated monolayer cell culture techniques and helped characterize additional cell lines derived from reproductive tissues. Specifically, the human endometrial adenocarcinoma cell line (HEC-1) was characterized and immortalized in 1968 (Kuramoto et al., 1972). The HEC-1 cell line was mainly used to study the endometrium and endometrial carcinoma in a simplified cellular system (Kurarmoto et al., 2002). Following, in 1973, for the first time, viable uterine cells were directly obtained from a rat uterus. The authors defined the metabolic characterization of these cells and revealed the equilibrium dissociation constant for estrogen binding (Williams and Gorski, 1973), the guidelines for isolating and maintaining human endometrial epithelial glands in vitro were established in 1979 (Satyaswaroop et al., 1979). For the first time, these guidelines recommended that endometrial glands be isolated by enzymatic digestion with collagenase and cultured with a nutrient-rich media (Satyaswaroop et al., 1979). This approach led to the co-culture of primary human endometrial epithelial glands with stromal cells to study estradiol metabolism during the menstrual cycle (Liu and Tseng, 1979).

Five years later, in 1985, the Ishikawa cell line was derived from a stage II human endometrial adenocarcinoma (Nishida et al., 1985). Clones of these cells are still available to researchers across the globe, with varying expression of estrogen receptors (ERs) and progesterone receptors (PRs) depending on the clone’s degree of differentiation (Nishida, 2002). Nevertheless, it remains difficult to study human endometrial epithelial cells’ hormonal responses in monolayer cultures, as they do not accurately reflect the complexity of the uterine microenvironment. A 3D cellular arrangement was found to be necessary to maintain the cells’ differentiation state which is related to hormone sensitivity.

In 1986, Kirk and Alvarez developed a new method for in vitro culture of reproductive tissues using Matrigel, in which cells maintain their 3D morphology (Kirk and Alvarez, 1986). This technique is considered to have generated the first epithelial organoids from human endometrial biopsies. Small fragments of endometrial tissue were processed through gravity sedimentation to isolate the smallest glandular elements, which, in turn, were cultured with a defined media supplemented with a collagen gel (Kirk and Alvarez, 1986). Alternatively, in 1988, biopsy-derived endometrial gland elements that were cultured on a Matrigel coating for several weeks produced larger colonies, with 50% of the elements assembling into large organoid structures (Rinehart et al., 1988). Notably, epithelial cells grown on traditional plastic culture plates remained flat and squamoid whereas the cells grown on Matrigel adopted their natural columnar shape. Further, the ECM support from the Matrigel consistently provided longer periods of cell growth and maintenance compared to traditional plastic plates (3 months vs. 3 weeks, respectively). Finally, it was also in 1988 when another important event in the field of endometrial modelling took place. The hormone-responsive endometrial cell line (ECC1) was cloned from human endometrial adenocarcinoma cells (EnCa101AE) transplanted into mice (Tabibzadeh et al., 1988). To date, these cells have been widely used to study the endometrium’s hormone response, as they express ER and PR.

In the early 1990s, researchers focused on co-cultures to elucidate the interactions between different cell populations in the female reproductive system. The first experimental article combining endometrial epithelial glands cultured over a Matrigel coating with stromal cell monolayers was published in 1992. This co-culture modelled the native paracrine interactions, improving the understanding of endometrial function (Mahfoudi et al., 1992). The following year (1993), Tabibzadeh’s group demonstrated that the immune system (T cells) regulates the activity of endometrial epithelial cells. The presence of T cells in ECC1 cultures altered antigen expression and proliferation rates (Tabibzadeh et al., 1993), highlighting the role of the microenvironment. Next, to add another layer of complexity to endometrial models, in 1994, Bertin-Ley’s group co-cultured endometrial epithelial cells grown on a Matrigel membrane with endometrial stromal cells embedded in a collagen matrix. This 3D model faithfully recapitulated the natural interactions between the stromal compartment (composed of ECM and stromal cells) and epithelial compartment, providing a reliable model of the human endometrium in vivo (Bentin-Ley et al., 1994).

In the 2000s, independent research groups corroborated the existence of human endometrial stem cells. A cohort of epithelial and stromal cells with clonogenic activity was first observed in the human endometrium in 2004 (Chan et al., 2004). Three years later, Schwab and Gargett published a methodology for isolating the subpopulation of human endometrial stromal cells with mesenchymal stem cell colony-forming and multipotent properties (Schwab and Gargett, 2007). Finally, in 2007, Kato et al. published the isolation of the Side Population (endometrial stem cells that can participate in the regeneration of the tissue and have self-renewal potential), confirmed 3 years later by Kato et al. (2007), Cervelló et al. (2010). These findings have had wider implications in the field of reproduction such as the understanding of the mechanisms of endometrial regeneration and their role in this process.

Around 15 years later, Turco et al. established endometrial epithelial organoid models in 2017. In contrast to the endometrial organoids from 1986, these novel structures could be used for long-term cultures and preserved their genetic stability plus high proliferation rates even after freeze-thaw cycles. Turco’s group defined a culture medium for maintaining and expanding endometrial epithelial organoids as well as a method for differentiating these organoids into a secretory or gestational state (Turco et al., 2017). Meanwhile, Boretto et al. characterized expandable healthy and pathological (endometrial cancer) human endometrial organoid models (Boretto et al., 2017). Together, these advances created the foundation for patient-specific biobanks that could be used for personalized drug testing, among other applications.

The first approach of microfluidic models for the human endometrium emerged in 2017. Gnecco et al. developed a dual-chamber microfluidic platform co-culturing primary human endothelial and endometrial stromal cells to mimic the physiological changes occurring throughout the 28-day human menstrual cycle. The device consisted of two orthogonal microfluidic chambers, made with polydimethylsiloxane, divided by a biocompatible resin-based membrane permitting cell communication via soluble factors. This system allowed the simultaneous analysis of stromal decidualization and endometrial vascular function under controlled physiomimetic conditions, such as endothelial cells remodelling and vascular barrier formation (Gnecco et al., 2017). In 2017, Xiao et al. developed the EVATAR system to co-culture murine ovaries with human fallopian tube, endometrium, ectocervix and liver tissues for 28 days. Each tissue was cultured in its own chamber and connected by microfluidic channels with dynamic media flow to effectively recapitulate pituitary hormone circulation and regulation (Xiao et al., 2017). Next year, Edington et al. described a 10-way multi-microphysiological system platform including the liver, gut, lung, heart, pancreas, brain, skin, kidney, skeletal muscle and endometrium. This system replicates the functional interactions between multiple human tissues in a laboratory setting and offers a more precise approach to studying human biology and pharmacology (Edington et al., 2018).

Finally, in 2021, Ahn et al. recently developed a micro-engineered vascularized endometrium-on-a-chip (MVEOC) which reconstituted the physiologically relevant endometrial environment, containing the three main cell populations of the human endometrium. By combining endothelial, epithelial and stromal cells, this device serves as a promising model to study endometrial angiogenesis and vasculogenesis, among other biological processes. Notably, the MVEOC can improve efficiency of high-throughput drug screening and identify molecular pathways involved in the process of embryo implantation (Ahn et al., 2021).

In the near future, novel techniques have emerged such as synthetic or artificial organ development and bioprinting, which allow the creation of lab-grown tissues for regenerative medicine applications. Generating personalized models is also an alternative to test treatments in an individual’s specific biology. Tissue-specific ECMs are used in tissue regeneration and are designed to mimick the natural cellular environment. Finally, single-cell technologies and CRISPR genome editing are also emerging as new strategies, among others.

Cell culture techniques have been instrumental for studying the physiology of the healthy endometrium. Primary endometrial cells, stem cells and cell lines provide valuable models for investigating endometrial function, hormone responses, and cell interactions (Pijnenborg et al., 2023). These models are used to explore the endometrium’s cyclical changes and response to hormone therapy. Although 2D cell cultures have dominated the field for a long time, recent research has shifted toward culturing 3D structures as they provide enhanced biomimicry in terms of structure and physiology. Specifically, endometrial organoid models closely mimic the structural and functional characteristics of the native endometrial tissue, accurately recapitulating the conditions of the in vivo microenvironment (Boretto et al., 2017). Among many other applications, organoids are used to study endometrial regeneration, hormone regulation, and the interactions between endometrial and immune cells (Dai et al., 2024). Finally, the microfluidic devices and lab-on-a-chip technologies represent dynamic platforms to model the endometrium. These systems can simulate the menstrual cycle, among other physiological processes, by controlling the microenvironment and fluid flow around endometrial cells (De Bem et al., 2021). Such models are particularly useful for studying cell signaling, tissue dynamics, and the effects of pharmacological agents in a controlled setting (Murphy et al., 2022).

Models of endometrial diseases, particularly endometriosis or adenomyosis, are generated through various in vitro and in vivo approaches. In vivo rodent models are particularly valuable for studying the pathogenesis of endometriosis and testing new therapeutic approaches (Burns et al., 2021). Alternatively, in vitro endometrial organoid models provide insights into the molecular mechanisms driving disease initiation and progression (Luddi et al., 2020; Esfandiari et al., 2021a; Esfandiari et al., 2021b). Adenomyosis, has also been studied using diverse in vitro models. Notably, 3D cultures and organoid models were used to study the invasive behavior of endometrial cells along with their interactions with adjacent myometrial tissue (Mehasseb et al., 2010; Taylor et al., 2014). Endometrial cells co-cultured with immune cells or bone marrow-derived stem cells helped elucidate the complex cell interactions in endometriosis (Chan et al., 2017; Chen et al., 2021). Finally, microfluidic devices were used to mimic the excessively active uterine peristalsis (hyperperistalsis) driving development of adenomyosis or endometriosis. By simulating the dynamic microenvironment of the uterus, researchers were able to study the kinetics of cellular migration and invasion (Elad et al., 2020).

Endometrial cancer research has significantly benefited from biotechnological advancements. Cancer cell lines, patient-derived xenografts, and genetically engineered mouse models are used to study tumor biology and progression, identify genetic mutations, and evaluate potential treatments (Dai et al., 2024). These models have been instrumental in understanding the role of specific oncogenes and tumor suppressors in endometrial cancer development (Hibaoui and Feki, 2020). Specifically, organoids derived from endometrial cancer patients were developed to study the tumor microenvironment and test the efficacy of targeted and personalized therapies (Berg et al., 2021).

In summary, recent biotechnological approaches facilitated the development of high-throughput drug screening as well as monitoring of endometrial disease models. Automated systems accelerate evaluation of drug efficacy and toxicity in endometrial organoids (Girda et al., 2017), streamlining identification of new therapeutic compounds. Further, multiple drug candidates can simultaneously be tested on patient-derived organoids, providing personalized insights into how particular patient could respond to treatment, thereby helping clinicians optimize treatment regimens (Boretto et al., 2019). Further, live-cell imaging and biosensors can be used to monitor the dynamics of cellular responses to treatments in real-time allowing early detection of a treatment’s efficacy or toxicity before adverse effects occur (Peel and Jackman, 2021).