Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3D models. Using muscle microbiopsies as the primary cell source in both models showed promising results. Initial investigations indicated that small variations in heparin concentration in 2D cultures strongly influenced medium coagulation with an optimal proliferation observed at final heparin concentrations of 1.44 IU/mL. The two novel models investigated showed that expansion of mdMSCs is achievable. At the end of expansion, the 3D model revealed a higher total number of cells harvested (64.60 ± 5.32 million) compared to the 2D culture (57.20 ± 7.66 million). Trilineage differentiation assays confirmed the multipotency (osteoblasts, chondroblasts and adipocytes) of the mdMSCs generated in both models with no significant difference observed. Immunophenotyping confirmed the expression of the mesenchymal stem cell (MSC) markers CD-90 and CD-44, with low expression of CD-45 and MHCII markers for mdMSCs derived from the two models. The generated mdMSCs also had great immunomodulatory properties. Specific immunological extraction followed by enzymatic detection (SIEFED) analysis demonstrated that mdMSCs from both models inhibited myeloperoxidase (MPO) activity in a strong dose-dependent manner. Moreover, they were also able to reduce reactive oxygen species (ROS) activity, with mdMSCs from the 3D model showing significantly higher dose-dependent inhibition compared to the 2D model. These results highlighted for the first time the feasibility and efficacy of using 10% ePL for mdMSC expansion in novel 2D and 3D approaches and also that mdMSCs have strong immunomodulatory properties that can be exploited to advance the field of regenerative medicine and cell therapy instead of using FBS with all its drawbacks.

Metastatic breast cancer is a major cause of mortality among breast cancer patients (Sauer et al. Front Oncol: 11:659963, 2021). It may emerge years or even decades after the initial treatment of the primary tumor. This latency in the manifestation of the disease is attributed to the presence of early disseminated tumor cells (DTCs) that lay quiescent (dormant) for years until they emerge as clinically overt metastases. Given that to date we have no treatment to cure metastatic disease, it is vital to investigate ways to eradicate dormant DTCs and/or prevent their emergence to overt metastases. Here, we present a modified 3-dimensional in vitro system to model the in vivo growth characteristics of several tumor cell lines that exhibit either dormant behavior (D2.0R, MCF7) or transient dormant metastatic behavior (D2A1) at a metastatic secondary site. Additionally, we present an in vitro and complementary in vivo system to study the switch from dormancy to metastatic growth driven by a fibrotic-like milieu enriched with the deposition of type I collagen.

Breast cancer, a leading cause of cancer-related deaths globally, exhibits distinct subtypes with varying pathological, genetic, and clinical characteristics. Despite advancements in breast cancer treatments, its histological and molecular heterogeneity pose a significant clinical challenge. Triple-negative breast cancer (TNBC), a highly aggressive subtype lacking targeted therapeutics, adds to the complexity of breast cancer treatment. Recent years have witnessed the development of advanced 3D culture technologies, such as organoids and spheroids, providing more representative models of healthy human tissue and various malignancies. These structures, resembling organs in structure and function, are generated from stem cells or organ-specific progenitor cells via self-organizing processes. Notably, 3D culture systems bridge the gap between 2D cultures and in vivo studies, offering a more accurate representation of in vivo tumors\' characteristics. Exosomes, small nano-sized molecules secreted by breast cancer and stromal/cancer-associated fibroblast cells, have garnered significant attention. They play a crucial role in cell-to-cell communication, influencing tumor progression, invasion, and metastasis. The 3D culture environment enhances exosome efficiency compared to traditional 2D cultures, impacting the transfer of specific cargoes and therapeutic effects. Furthermore, 3D exosomes have shown promise in improving therapeutic outcomes, acting as potential vehicles for cancer treatment administration. Studies have demonstrated their role in pro-angiogenesis and their innate therapeutic potential in mimicking cellular therapies without side effects. The 3D exosome model holds potential for addressing challenges associated with drug resistance, offering insights into the mechanisms underlying multidrug resistance and serving as a platform for drug screening. This review seeks to emphasize the crucial role of 3D culture systems in studying breast cancer, especially in understanding the involvement of exosomes in cancer pathology.

Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+ activated Cl- channel Anoctamin 1 (ANO1) and the epithelial Na+ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl- measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.

A three-dimensional (3D) lung aggregate model based on sodium alginate scaffolds was developed to study the interactions between Paracoccidioides brasiliensis (Pb) and lung epithelial cells. The suitability of the 3D aggregate as an infection model was examined using cell viability (cytotoxicity), metabolic activity, and proliferation assays. Several studies exemplify the similarity between 3D cell cultures and living organisms, which can generate complementary data due to the greater complexity observed in these designed models, compared to 2D cell cultures. A 3D cell culture system of human A549 lung cell line plus sodium alginate was used to create the scaffolds that were infected with Pb18. Our results showed low cytotoxicity, evidence of increased cell density (indicative of cell proliferation), and the maintenance of cell viability for seven days. The confocal analysis revealed viable yeast within the 3D scaffold, as demonstrated in the solid BHI Agar medium cultivation. Moreover, when ECM proteins were added to the alginate scaffolds, the number of retrieved fungi was significantly higher. Our results highlight that this 3D model may be promising for in vitro studies of host-pathogen interactions.

Cholesterol remains a vital molecule required for life; however, increasing evidence exists implicating cholesterol in cancer development and progression. Numerous studies investigating the relationship between cholesterol and cancer in 2-dimensional (2D) culture settings exist, however these models display inherent limitations highlighting the incipient need to develop better models to study disease pathogenesis. Due to the multifaceted role cholesterol plays in the cell, researchers have begun utilizing 3-dimensional (3D) culture systems, namely, spheroids and organoids to recapitulate cellular architecture and function. This review aims to describe current studies exploring the relationship between cancer and cholesterol in a variety of cancer types using 3D culture systems. We briefly discuss cholesterol dyshomeostasis in cancer and introduce 3D in-vitro culture systems. Following this, we discuss studies performed in cancerous spheroid and organoid models that focused on cholesterol, highlighting the dynamic role cholesterol plays in various cancer types. Finally, we attempt to provide potential gaps in research that should be explored in this rapidly evolving field of study.

Microgravity has been shown to induce many changes in cell growth and differentiation due to offloading the gravitational strain normally exerted on cells. Although many studies have used two-dimensional (2D) cell culture systems to investigate the effects of microgravity on cell growth, three-dimensional (3D) culture scaffolds can offer more direct indications of the modified cell response to microgravity-related dysregulations compared to 2D culture methods. Thus, knowledge of 3D cell culture is essential for better understanding the in vivo tissue function and physiological response under microgravity conditions. This review discusses the advances in 2D and 3D cell culture studies, particularly emphasizing the role of hydrogels, which can provide cells with a mimic in vivo environment to collect a more natural response. We also summarized recent studies about cell growth and differentiation under real microgravity or simulated microgravity conditions using ground-based equipment. Finally, we anticipate that hydrogel-based 3D culture models will play an essential role in constructing organoids, discovering the causes of microgravity-dependent molecular and cellular changes, improving space tissue regeneration, and developing innovative therapeutic strategies. Future research into the 3D culture in microgravity conditions could lead to valuable therapeutic applications in health and pharmaceuticals.

Liver cancer remains one of the most common cancers worldwide with limited therapy options. The main risk factors for hepatocellular carcinoma (HCC), the most common form of liver cancer, include chronic infection with hepatitis B or hepatitis C viruses, alcohol abuse, and metabolic disease. Current systemic therapies for advanced HCCs have greatly improved in the last decade, but there is still a need to develop more targeted drug therapy for HCCs. The development of liver organoids, a self-organising and self-renewal three-dimensional cell culture model, has greatly improved cancer research, including liver cancer. The generation of liver organoids provides a physiologically relevant model to study cancer drug screening and development, personalized medicine, liver disease modeling, and liver regeneration. However, the advent of organoid development also comes with few shortcomings that must be overcome, including the high cost of the model, the availability of origin tissues, and the need for multilineage liver organoids to replicate the true cellular heterogeneity of the liver. Despite all the limitations, liver organoids provide a reliable in vitro model for translational applications to develop more effective HCC therapy and to understand the underlying pathogenic mechanism in various liver diseases.

Background: Patients with obstructive sleep apnea (OSA) experience recurrent hypoxemic events with a frequency sometimes exceeding 60 events/h. These episodic events induce downstream transient hypoxia in the parenchymal tissue of all organs, thereby eliciting the pathological consequences of OSA. Whereas experimental models currently apply intermittent hypoxia to cells conventionally cultured in 2D plates, there is no well-characterized setting that will subject cells to well-controlled intermittent hypoxia in a 3D environment and enable the study of the effects of OSA on the cells of interest while preserving the underlying tissue environment. Aim: To design and characterize an experimental approach that exposes cells to high-frequency intermittent hypoxia mimicking OSA in 3D (hydrogels or tissue slices). Methods: Hydrogels made from lung extracellular matrix (L-ECM) or brain tissue slices (300-800-μm thickness) were placed on a well whose bottom consisted of a permeable silicone membrane. The chamber beneath the membrane was subjected to a square wave of hypoxic/normoxic air. The oxygen concentration at different depths within the hydrogel/tissue slice was measured with an oxygen microsensor. Results: 3D-seeded cells could be subjected to well-controlled and realistic intermittent hypoxia patterns mimicking 60 apneas/h when cultured in L-ECM hydrogels ≈500 μm-thick or ex-vivo in brain slices 300-500 μm-thick. Conclusion: This novel approach will facilitate the investigation of the effects of intermittent hypoxia simulating OSA in 3D-residing cells within the parenchyma of different tissues/organs.

BACKGROUND: In vitro culture of preantral follicles is a promising technology for fertility preservation.
OBJECTIVE: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation.
METHODS: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E2) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization.
RESULTS: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro.
CONCLUSIONS: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.