1,3-Disubstituted Thiourea Derivatives: Promising Candidates for Medicinal Applications with Enhanced Cytotoxic Effects on Cancer Cells.

The distinct chemical structure of thiourea derivatives provides them with an advantage in selectively targeting cancer cells. In our previous study, we selected the most potent compounds, 2 and 8, with 3,4-dichloro- and 3-trifluoromethylphenyl substituents, respectively, across colorectal (SW480 and SW620), prostate (PC3), and leukemia (K-562) cancer cell lines, as well as non-tumor HaCaT cells. Our research has demonstrated their anticancer potential by targeting key molecular pathways involved in cancer progression, including caspase 3/7 activation, NF-κB (Nuclear Factor Kappa-light-chain-enhancer of activated B cells) activation decrease, VEGF (Vascular Endothelial Growth Factor) secretion, ROS (Reactive Oxygen Species) production, and metabolite profile alterations. Notably, these processes exhibited no significant alterations in HaCaT cells. The effectiveness of the studied compounds was also tested on spheroids (3D culture). Both derivatives 2 and 8 increased caspase activity, decreased ROS production and NF-κB activation, and suppressed the release of VEGF in cancer cells. Metabolomic analysis revealed intriguing shifts in cancer cell metabolic profiles, particularly in lipids and pyrimidines metabolism. Assessment of cell viability in 3D spheroids showed that SW620 cells exhibited better sensitivity to compound 2 than 8. In summary, structural modifications of the thiourea terminal components, particularly dihalogenophenyl derivative 2 and para-substituted analog 8, demonstrate their potential as anticancer agents while preserving safety for normal cells.

Culture and Immunomodulation of Equine Muscle-Derived Mesenchymal Stromal Cells: A Comparative Study of Innovative 2D versus 3D Models Using Equine Platelet Lysate.

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.

Non-Invasive Quality Control of Organoid Cultures Using Mesofluidic CSTR Bioreactors and High-Content Imaging.

Human brain organoids produce anatomically relevant cellular structures and recapitulate key aspects of in vivo brain function, which holds great potential to model neurological diseases and screen therapeutics. However, the long growth time of 3D systems complicates the culturing of brain organoids and results in heterogeneity across samples hampering their applications. We developed an integrated platform to enable robust and long-term culturing of 3D brain organoids. We designed a mesofluidic bioreactor device based on a reaction-diffusion scaling theory, which achieves robust media exchange for sufficient nutrient delivery in long-term culture. We integrated this device with longitudinal tracking and machine learning-based classification tools to enable non-invasive quality control of live organoids. This integrated platform allows for sample pre-selection for downstream molecular analysis. Transcriptome analyses of organoids revealed that our mesofluidic bioreactor promoted organoid development while reducing cell death. Our platform thus offers a generalizable tool to establish reproducible culture standards for 3D cellular systems for a variety of applications beyond brain organoids.

A Three-Step Protocol to Differentiate iPSC into Colon Organoids.

Organoids, three-dimensional, stem cell-based structures that mimic the cellular and functional architecture of tissues, have emerged as an innovative in vitro tool. They offer highly efficient models for studying both embryonic development and disease progression processes. Colon organoids can also be generated from biopsies obtained during a colonoscopy. However, the invasive nature of biopsy collection poses practical challenges and introduces biases when studying patients who are already afflicted. Therefore, the use of iPSC-derived colon organoids can be considered a more practical approach for researchers and patients alike. Numerous protocols have been published for generating colon organoids from iPSCs. While most of these protocols share a common developmental process, some are labor-intensive or require additional equipment. Taking these considerations into account, we present a cost-effective and straightforward yet functionally robust colon organoid protocol: (1) definitive endoderm differentiation, (2) hindgut endoderm differentiation, and (3) maturation of colon spheroids into mature organoids.

An induced pluripotent stem cell-based approach for hair follicle development and regeneration.

Because hair loss is a common concern for many individuals, potential regenerative therapies of hair follicles have been extensively researched. Induced pluripotent stem cells (iPSCs) are a promising avenue for hair follicle regeneration. This review explores current iPSC-based approaches and highlights their potential applications and challenges in hair restoration. The principles of iPSC technology, iPSC differentiation into hair follicle precursor cells, and potential clinical implications for hair follicle regeneration are also discussed. This overview of iPSCs and their applications aims to contribute to our understanding of their role in hair restoration and potential future therapeutic applications.

Targeted Cancer Therapy-on-A-Chip.

Targeted cancer therapy (TCT) is gaining increased interest because it reduces the risks of adverse side effects by specifically treating tumor cells. TCT testing has traditionally been performed using two-dimensional (2D) cell culture and animal studies. Organ-on-a-chip (OoC) platforms have been developed to recapitulate cancer in vitro, as cancer-on-a-chip (CoC), and used for chemotherapeutics development and testing. This review explores the use of CoCs to both develop and test TCTs, with a focus on three main aspects, the use of CoCs to identify target biomarkers for TCT development, the use of CoCs to test free, un-encapsulated TCTs, and the use of CoCs to test encapsulated TCTs. Despite current challenges such as system scaling, and testing externally triggered TCTs, TCToC shows a promising future to serve as a supportive, pre-clinical platform to expedite TCT development and bench-to-bedside translation.

The application of retinal organoids in ophthalmic regenerative medicine: A mini-review.

Retinal organoids are three-dimensional (3D) microscopic tissues that are induced and differentiated from stem cells or progenitor cells in vitro and have a highly similar structure to the retina. With the optimization and development of 3D retinal culture system and the improvement of induced differentiation technology, retinal organoids have broad application prospects in retinal development, regenerative medicine, biomaterial evaluation, disease mechanism investigation, and drug screening. In this review we summarize recent development of retinal organoids and their applications in ophthalmic regenerative medicine. In particular, we highlight the promise and challenges in the use of retinal organoids in disease modeling and drug discovery.

Developing Device of Death Operation (DODO) to Detect Apoptosis in 2D and 3D Cultures.

The real-time detection of intracellular biological processes by encoded sensors has broad application prospects. Here, we developed a degron-based modular reporting system, the Device of Death Operation (DODO), that can monitor various biological processes. The DODO system consists of a "reporter", an "inductor", and a "degron". After zymogen activation and cleavage, the degron will be released from the "reporter", which eventually leads to the stabilization of the "reporter", and can be detected. By replacing different "inductors" and "reporters", a series of biological processes can be reported through various signals. The system can effectively report the existence of TEV protease. To prove this concept, we successfully applied the DODO system to report apoptosis in 2D and 3D cultures. In addition, the reporter based on degron will help to design protease reporters other than caspase.

Advanced Hydrogels in Breast Cancer Therapy.

Breast cancer is the most common malignancy among women and is the second leading cause of cancer-related death for women. Depending on the tumor grade and stage, breast cancer is primarily treated with surgery and antineoplastic therapy. Direct or indirect side effects, emotional trauma, and unpredictable outcomes accompany these traditional therapies, calling for therapies that could improve the overall treatment and recovery experiences of patients. Hydrogels, biomimetic materials with 3D network structures, have shown great promise for augmenting breast cancer therapy. Hydrogel implants can be made with adipogenic and angiogenic properties for tissue integration. 3D organoids of malignant breast tumors grown in hydrogels retain the physical and genetic characteristics of the native tumors, allowing for post-surgery recapitulation of the diseased tissues for precision medicine assessment of the responsiveness of patient-specific cancers to antineoplastic treatment. Hydrogels can also be used as carrier matrices for delivering chemotherapeutics and immunotherapeutics or as post-surgery prosthetic scaffolds. The hydrogel delivery systems could achieve localized and controlled medication release targeting the tumor site, enhancing efficacy and minimizing the adverse effects of therapeutic agents delivered by traditional procedures. This review aims to summarize the most recent advancements in hydrogel utilization for breast cancer post-surgery tissue reconstruction, tumor modeling, and therapy and discuss their limitations in clinical translation.

In Vitro Generation of Haploid Germ Cells from Human XY and XXY Immature Testes in a 3D Organoid System.

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

An Overview of Multiple Sclerosis In Vitro Models.

Multiple sclerosis (MS) still poses a challenge in terms of complex etiology, not fully effective methods of treatment, and lack of healing agents. This neurodegenerative condition considerably affects the comfort of life by causing difficulties with movement and worsening cognition. Neuron, astrocyte, microglia, and oligodendrocyte activity is engaged in multiple pathogenic processes associated with MS. These cells are also utilized in creating in vitro cellular models for investigations focusing on MS. In this article, we present and discuss a summary of different in vitro models useful for MS research and describe their development. We discuss cellular models derived from animals or humans and present in the form of primary cell lines or immortalized cell lines. In addition, we characterize cell cultures developed from induced pluripotent stem cells (iPSCs). Culture conditions (2D and 3D cultures) are also discussed.

Long-termin vitromaintenance of plasma cells in a hydrogel-enclosed human bone marrow microphysiological 3D model system.

Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived fromin vivostudies in mice, asin vitrostudies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survivalin vitrofor up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCsin vitro. Our innovative model system is suitable for in-depthin vitrostudies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics.

Lung decellularized matrix-derived 3D spheroids: Exploring silicosis through the impact of the Nrf2/Bax pathway on myofibroblast dynamics.

Silicosis is an occupational respiratory disease caused by long-term inhalation of high concentrations of free silica particles. Studies suggest that oxidative stress is a crucial initiator of silicosis fibrosis, and previous studies have linked the antioxidative stress transcription factor known as Nrf2 to fibrosis antagonism. Myofibroblasts play a pivotal role in tissue damage repair due to oxidative stress. Unlike physiological repair, myofibroblasts in fibrosis exhibit an apoptosis-resistant phenotype, continuously synthesising and secreting significant amounts of collagen and other extracellular matrices, which could be a direct cause of silicosis fibrosis. However, the relationship and mechanism of action between oxidative stress and myofibroblast apoptosis resistance remain unclear. In this study, a new 3D cell culture model using mice lung decellularised matrix particles and fibroblasts was developed, simulating the changes in myofibroblasts during the development of silicotic nodules. Western Blot results indicate that silica stimulation leads to increased collagen deposition and decreased apoptosis-related protein Bax and oxidative stress-related protein Nrf2 in the 3D spheroid model. Immunofluorescence experiments reveal co-localisation in their expression. In Nrf2 overexpressing spheroids, Bax exhibits significant upregulation. In the Nrf2 knockout spheroids, Bax is also significantly downregulated; after intervention with Bax inhibitors, a significant downregulation of Bax-induced apoptosis was also detected in the Nrf2-overexpressed spheroids. In contrast, Bax-induced apoptosis showed a significant upregulation trend in Nrf2-overexpressed spheroids after intervention with Bax agonists. The results demonstrate that the spheroid model can mimic the development process of silicotic nodules, and silica stimulation leads to an apoptosis-resistant phenotype in myofibroblasts in the model, acting through the Nrf2/Bax pathway. This research establishes a new methodology for silicosis study, identifies therapeutic targets for silicosis, and opens new avenues for studying the mechanisms of silicosis fibrosis.

High-Throughput Assembly of Compositionally Controlled 3D Cell Communities for Developmental Engineering.

Cell patterning for 3D culture has increased our understanding of how cells interact among themselves and with their environment during tissue morphogenesis. Building cell communities from the bottom up with size and compositional control is invaluable for studies of morphological transitions. Here, we detail Photolithographic DNA-programmed Assembly of Cells (pDPAC). pDPAC uses a photoactive polyacrylamide gel substrate to capture single-stranded DNA on a 2D surface in large-scale, highly resolved patterns using the photomask technology. Cells are then functionalized with a complementary DNA strand, enabling cells to be temporarily adhered to distinct locations only where their complementary strand is patterned. These temporary 2D patterns can be transferred to extracellular matrix hydrogels for 3D culture of cells in biomimetic microenvironments. Use of a polyacrylamide substrate has advantages, including a simpler photolithography workflow, lower non-specific cell adhesion, and lower stiction to ECM hydrogels during release of patterned hydrogels. The protocol is equally applicable to large (cm)-scale patterns and repetitive arrays of smaller-scale cell interaction or migration experiments.

The generation of islet-like insulin-producing cells from Wharton's jelly-derived mesenchymal stem cells on the PES/fish gelatin scaffold.

Diabetes Mellitus (DM) disrupts the body's capability to control blood glucose statuses. Type 1 diabetes mellitus (T1DM) arises from inadequate insulin production and is treated with insulin replacement therapy. Stem cell therapy is a hopeful treatment for T1DM that involves using adult stem cells to generate insulin-producing cells (IPCs). Mesenchymal stem cells (MSCs) are particularly advantageous for generating IPCs. The islet cells require interactions with the extracellular matrix for survival, which is lacking in conventional 2D culture systems. Natural or synthetic polymers create a supportive 3D microenvironment in tissue engineering. We aim to construct superior differentiation conditions employing polyethersulfone (PES)/Fish gelatin scaffolds to differentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) to IPCs. In this study, the PES/fish gelatin scaffold (3D) was manufactured by electrospinning, and then its biocompatibility and non-toxicity were investigated by MTT assay. After that, scaffold-supportive effects on WJ-MSCs differentiation to IPCs were studied at the gene and protein levels. After exposure to the differentiation media, 2D and 3D (PES/Fish gelatin) cultured cells were slowly aggregated and developed spherical-shaped clusters. The viability of cells was found to be comparable in both 2D and 3D cultures. The gene expression analysis showed that efficiency of differentiation was more elevated in 3D culture. Additionally, ELISA results indicated that C-peptide and insulin release were more significant in 3D than in 2D culture. In conclusion, the PES/fish gelatin scaffold is highly promising for pancreatic tissue engineering because it supports the viability, growth, and differentiation of WJ-MSCs into IPCs.

Generation of functionally active resident macrophages from adipose tissue by 3D cultures.

Introduction: Within adipose tissue (AT), different macrophage subsets have been described, which played pivotal and specific roles in upholding tissue homeostasis under both physiological and pathological conditions. Nonetheless, studying resident macrophages in-vitro poses challenges, as the isolation process and the culture for extended periods can alter their inherent properties. Methods: Stroma-vascular cells isolated from murine subcutaneous AT were seeded on ultra-low adherent plates in the presence of macrophage colony-stimulating factor. After 4 days of culture, the cells spontaneously aggregate to form spheroids. A week later, macrophages begin to spread out of the spheroid and adhere to the culture plate. Results: This innovative three-dimensional (3D) culture method enables the generation of functional mature macrophages that present distinct genic and phenotypic characteristics compared to bone marrow-derived macrophages. They also show specific metabolic activity and polarization in response to stimulation, but similar phagocytic capacity. Additionally, based on single-cell analysis, AT-macrophages generated in 3D culture mirror the phenotypic and functional traits of in-vivo AT resident macrophages. Discussion: Our study describes a 3D in-vitro system for generating and culturing functional AT-resident macrophages, without the need for cell sorting. This system thus stands as a valuable resource for exploring the differentiation and function of AT-macrophages in vitro in diverse physiological and pathological contexts.

Hypoxia-preconditioned WJ-MSC spheroid-derived exosomes delivering miR-210 for renal cell restoration in hypoxia-reoxygenation injury.

BACKGROUND: Recent advancements in mesenchymal stem cell (MSC) technology have paved the way for innovative treatment options for various diseases. These stem cells play a crucial role in tissue regeneration and repair, releasing local anti-inflammatory and healing signals. However, challenges such as homing issues and tumorigenicity have led to exploring MSC-exosomes as a promising alternative. MSC-exosomes have shown therapeutic potential in conditions like renal ischemia-reperfusion injury, but low production yields hinder their clinical use. METHODS: To address this limitation, we examined hypoxic preconditioning of Wharton jelly-derived MSCs (WJ-MSCs) 3D-cultured in spheroids on isolated exosome yields and miR-21 expression. We then evaluated their capacity to load miR-210 into HEK-293 cells and mitigate ROS production, consequently enhancing their survival and migration under hypoxia-reoxygenation conditions. RESULTS: MiR-210 overexpression was significantly induced by optimized culture and preconditioning conditions, which also improved the production yield of exosomes from grown MSCs. The exosomes enriched with miR-210 demonstrated a protective effect by improving survival, reducing apoptosis and ROS accumulation in damaged renal cells, and ultimately promoting cell migration. CONCLUSION: The present study underscores the possibility of employing advanced techniques to maximize the therapeutic attributes of exosomes produced from WJ-MSC spheroid for improved recovery outcomes in ischemia-reperfusion injuries.

Variations in fluid chemical potential induce fibroblast mechano-response in 3D hydrogels.

Mechanical deformation of skin creates variations in fluid chemical potential, leading to local changes in hydrostatic and osmotic pressure, whose effects on mechanobiology remain poorly understood. To study these effects, we investigate the specific influences of hydrostatic and osmotic pressure on primary human dermal fibroblasts in three-dimensional hydrogel culture models. Cyclic hydrostatic pressure and hyperosmotic stress enhanced the percentage of cells expressing the proliferation marker Ki67 in both collagen and PEG-based hydrogels. Osmotic pressure also activated the p38 MAPK stress response pathway and increased the expression of the osmoresponsive genes PRSS35 and NFAT5. When cells were cultured in two-dimension (2D), no change in proliferation was observed with either hydrostatic or osmotic pressure. Furthermore, basal, and osmotic pressure-induced expression of osmoresponsive genes differed in 2D culture versus 3D hydrogels, highlighting the role of dimensionality in skin cell mechanotransduction and stressing the importance of 3D tissue-like models that better replicate in vivo conditions. Overall, these results indicate that fluid chemical potential changes affect dermal fibroblast mechanobiology, which has implications for skin function and for tissue regeneration strategies.

In Vitro and In Vivo Systems to Study Tumor Dormancy and the Transition to Overt Metastases Induced by the Fibrotic Milieu.

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.

Three-Dimensional Rotary Culture to Model Mycobacterial Biofilms in Low-Shear Detergent-Free Liquid Suspension.

In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.