Oxidative Stress Modulation by Carnosine in Scaffold Free Human Dermis Spheroids Model: A Proteomic Study.

Carnosine is an endogenous ß-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.

Mammalian Cell Spheroids on Mixed Organic-Inorganic Superhydrophobic Coating.

Three-dimensional cell culture has become a reliable method for reproducing in vitro cellular growth in more realistic physiological conditions. The surface hydrophobicity strongly influences the promotion of cell aggregate formation. In particular, for spheroid formation, highly water-repellent coatings seem to be required for the significant effects of the process. In this work, surfaces at different wettability have been compared to observe their influence on the growth and promotion of aggregates of representative mammalian cell lines, both tumoral and non-tumoral (3T3, HaCat and MCF-7 cell lines). The effect of increased hydrophobicity from TCPS to agarose hydrogel to mixed organic-inorganic superhydrophobic (SH) coating has been investigated by optical and fluorescence microscopy, and by 3D confocal profilometry, in a time scale of 24 h. The results show the role of less wettable substrates in inducing the formation of spheroid-like cell aggregates at a higher degree of sphericity for the studied cell lines.

3D hanging spheroid plate for high-throughput CAR T cell cytotoxicity assay.

BACKGROUND: Most high-throughput screening (HTS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor microenvironment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CAR T cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CAR T and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays. RESULTS: The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CAR T cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20°. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300-350 µm in diameter after 2 days in the 3DHSP. The cytotoxic effects of T cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR. CONCLUSIONS: The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CAR T cells on tumor spheroids.

Vascularization of iNSC spheroid in a 3D spheroid-on-a-chip platform enhances neural maturation.

In vitro platforms for studying the human brain have been developed, and brain organoids derived from stem cells have been studied. However, current organoid models lack three-dimensional (3D) vascular networks, limiting organoid proliferation, differentiation, and apoptosis. In this study, we created a 3D model of vascularized spheroid cells using an injection-molded microfluidic chip. We cocultured spheroids derived from induced neural stem cells (iNSCs) with perfusable blood vessels. Gene expression analysis and immunostaining revealed that the vascular network greatly enhanced spheroid differentiation and reduced apoptosis. This platform can be used to further study the functional and structural interactions between blood vessels and neural spheroids, and ultimately to simulate brain development and disease.

Stemness and immune evasion conferred by the TDO2-AHR pathway are associated with liver metastasis of colon cancer.

The aryl hydrocarbon receptor (AHR) pathway modulates the immune system in response to kynurenine, an endogenous tryptophan metabolite. IDO1 and TDO2 catalyze kynurenine production, which promotes cancer progression by compromising host immunosurveillance. However, it is unclear whether the AHR activation regulates the malignant traits of cancer such as metastatic capability or cancer stemness. Here, we carried out systematic analyses of metabolites in patient-derived colorectal cancer spheroids and identified high levels of kynurenine and TDO2 that were positively associated with liver metastasis. In a mouse colon cancer model, TDO2 expression substantially enhanced liver metastasis, induced AHR-mediated PD-L1 transactivation, and dampened immune responses; these changes were all abolished by PD-L1 knockout. In patient-derived cancer spheroids, TDO2 or AHR activity was required for not only the expression of PD-L1, but also for cancer stem cell (CSC)-related characteristics and Wnt signaling. TDO2 was coexpressed with both PD-L1 and nuclear ß-catenin in colon xenograft tumors, and the coexpression of TDO2 and PD-L1 was observed in clinical colon cancer specimens. Thus, our data indicate that the activation of the TDO2-kynurenine-AHR pathway facilitates liver metastasis of colon cancer via PD-L1-mediated immune evasion and maintenance of stemness.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air-liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

Stratified 3D Microtumors as Organotypic Testing Platforms for Screening Pancreatic Cancer Therapies.

Cancer-associated pancreatic stellate cells installed in periacinar/periductal regions are master players in generating the characteristic biophysical shield found in pancreatic ductal adenocarcinoma (PDAC). Recreating this unique PDAC stromal architecture and its desmoplastic microenvironment in vitro is key to discover innovative treatments. However, this still remains highly challenging to realize. Herein, organotypic 3D microtumors that recapitulate PDAC-stroma spatial bioarchitecture, as well as its biomolecular, metabolic, and desmoplastic signatures, are bioengineered. Such newly engineered platforms, termed stratified microenvironment spheroid models - STAMS - mimic the spatial stratification of cancer-stromal cells, exhibit a reproducible morphology and sub-millimeter size. In culture, 3D STAMS secrete the key molecular biomarkers found in human pancreatic cancer, namely TGF-ß, FGF-2, IL-1ß, and MMP-9, among others. This is accompanied by an extensive desmoplastic reaction where collagen and glycosaminoglycans (GAGs) de novo deposition is observed. These stratified models also recapitulate the resistance to various chemotherapeutics when compared to standard cancer-stroma random 3D models. Therapeutics resistance is further evidenced upon STAMS inclusion in a tumor extracellular matrix (ECM)-mimetic hydrogel matrix, reinforcing the importance of mimicking PDAC-stroma bioarchitectural features in vitro. The 3D STAMS technology represents a next generation of biomimetic testing platforms with improved potential for advancing high-throughput screening and preclinical validation of innovative pancreatic cancer therapies.

Isolation and in vitro cultivation of adrenal cells from mice.

The adrenal gland consists of two tissues, cortex and medulla, united under one capsule. Adrenal stem/progenitor cells play a key role in development and homeostasis. Here, we describe a protocol for generating primary cultures of adrenal cells from mice. We describe techniques for separating the cortex and medulla, generating spheroid cultures containing stem- and progenitor cells, and for the differentiation into steroidogenic and chromaffin cells, respectively. This protocol enables analysis of various treatments before, during, or after differentiation. For complete details on the use and execution of this protocol, please refer to Rubin de Celis et al. (2015), Steenblock et al. (2018), and Werdermann et al. (2021).

Stress generation, relaxation and size control in confined tumor growth.

Experiments on tumor spheroids have shown that compressive stress from their environment can reversibly decrease tumor expansion rates and final sizes. Stress release experiments show that nonuniform anisotropic elastic stresses can be distributed throughout. The elastic stresses are maintained by structural proteins and adhesive molecules, and can be actively relaxed by a variety of biophysical processes. In this paper, we present a new continuum model to investigate how the growth-induced elastic stresses and active stress relaxation, in conjunction with cell size control feedback machinery, regulate the cell density and stress distributions within growing tumors as well as the tumor sizes in the presence of external physical confinement and gradients of growth-promoting chemical fields. We introduce an adaptive reference map that relates the current position with the reference position but adapts to the current position in the Eulerian frame (lab coordinates) via relaxation. This type of stress relaxation is similar to but simpler than the classical Maxwell model of viscoelasticity in its formulation. By fitting the model to experimental data from two independent studies of tumor spheroid growth and their cell density distributions, treating the tumors as incompressible, neo-Hookean elastic materials, we find that the rates of stress relaxation of tumor tissues can be comparable to volumetric growth rates. Our study provides insight on how the biophysical properties of the tumor and host microenvironment, mechanical feedback control and diffusion-limited differential growth act in concert to regulate spatial patterns of stress and growth. When the tumor is stiffer than the host, our model predicts tumors are more able to change their size and mechanical state autonomously, which may help to explain why increased tumor stiffness is an established hallmark of malignant tumors.

Recapitulation of First Pass Metabolism Using 3D Printed Microfluidic Chip and Organoid.

The low bioavailability of oral drugs due to first pass metabolism is a major obstacle in drug development. With significant developments in the field of in vitro organ modeling and microfluidic chip three-dimensional (3D) printing, the challenge is to apply these for the production and evaluation of new drug candidates. This study aimed to produce a microfluidic chip to recapitulate and assess the feasibility of the first pass metabolism. The infill condition of the polycarbonate transparent filament and layer height was optimized to visualize and maintain the organoid or spheroid on the chip. Next, the chip was fabricated using a 3D printer after a computer-aided design (CAD). The chip consisted of three wells of different heights. The small intestinal (SI) organoid and colorectal adenocarcinoma spheroids were placed on the second and third wells, respectively. No additional equipment was assembled, and the tilted tunnel was connected to each well to transport the material by gradient force. The chip was fabricated using 50% and 0.1 um thickness. Among the three different prototypes of chip (chips 1, 2, and 3), the highest distribution of plasmids in the Matrigel of the second well was observed in Chip 2 at 48 h. The effect of first pass metabolism was analyzed using docetaxel. In the chip without an SI organoid, there was a marked decrease in the viability of colorectal adenocarcinoma spheroids due to drug efficacy. However, in the chip with the SI organoid, no significant change in viability was observed because of first pass metabolism. In conclusion, we presented a simple, fast, and low-cost microfluidic chip to analyze the efficacy change of candidate drug by the first pass metabolism.

From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish.

Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.

High-dose drug heat map analysis for drug safety and efficacy in multi-spheroid brain normal cells and GBM patient-derived cells.

To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In the chip, patient-derived cells were encapsulated in alginate and grown to maturity for more than 7 days to form cancer multi-spheroids. Multi-spheroids grown in conventional well plates require many cells and are easily damaged as a result of multiple pipetting during maintenance culture or experimental procedures. To address these issues, we applied a micropillar and microwell structure to the multi-spheroids array. Patient-derived cells from patients with Glioblastoma (GBM), the most common and lethal form of central nervous system cancer, were used to validate the array chip performance. After forming multi-spheroids with a diameter greater than 100µm in a 12×36 pillar array chip (25mm × 75mm), we tested 70 drug compounds (6 replicates) using a high-dose to determine safety and efficacy for drug candidates. Comparing the drug response of multi-spheroids derived from normal cells and cancer cells, we found that four compounds (Dacomitinib, Cediranib, LY2835219, BGJ398) did not show toxicity to astrocyte cell and were efficacious to patient-derived GBM cells.

A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation.

Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRASG12V exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRASG12V-expressing cells. This represents an applicable approach to explore protein-protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells.

Three-Dimensional Culture Systems for Dissecting Notch Signalling in Health and Disease.

Three-dimensional (3D) culture systems opened up new horizons in studying the biology of tissues and organs, modelling various diseases, and screening drugs. Producing accurate in vitro models increases the possibilities for studying molecular control of cell-cell and cell-microenvironment interactions in detail. The Notch signalling is linked to cell fate determination, tissue definition, and maintenance in both physiological and pathological conditions. Hence, 3D cultures provide new accessible platforms for studying activation and modulation of the Notch pathway. In this review, we provide an overview of the recent advances in different 3D culture systems, including spheroids, organoids, and "organ-on-a-chip" models, and their use in analysing the crucial role of Notch signalling in the maintenance of tissue homeostasis, pathology, and regeneration.

Bisphenol A Analogues Suppress Spheroid Attachment on Human Endometrial Epithelial Cells through Modulation of Steroid Hormone Receptors Signaling Pathway.

Bisphenol A (BPA) is a well-known endocrine disruptor, widely used in various consumer products and ubiquitously found in air, water, food, dust, and sewage leachates. Recently, several countries have restricted the use of BPA and replaced them with bisphenol S (BPS) and bisphenol F (BPF), which have a similar chemical structure to BPA. Compared to BPA, both BPS and BPF have weaker estrogenic effects, but their effects on human reproductive function including endometrial receptivity and embryo implantation still remain largely unknown. We used an in vitro spheroid (blastocyst surrogate) co-culture assay to investigate the effects of BPA, BPS, and BPF on spheroid attachment on human endometrial epithelial cells, and further delineated their role on steroid hormone receptor expression. We also used transcriptomics to investigate the effects of BPA, BPS, and BPF on the transcriptome of human endometrial cells. We found that bisphenol treatment in human endometrial Ishikawa cells altered estrogen receptor alpha (ERα) signaling and upregulated progesterone receptors (PR). Bisphenols suppressed spheroid attachment onto Ishikawa cells, which was reversed by the downregulation of PR through PR siRNA. Overall, we found that bisphenol compounds can affect human endometrial epithelial cell receptivity through the modulation of steroid hormone receptor function leading to impaired embryo implantation.

A Step-by-Step Refined Strategy for Highly Efficient Generation of Neural Progenitors and Motor Neurons from Human Pluripotent Stem Cells.

Limited access to human neurons, especially motor neurons (MNs), was a major challenge for studying neurobiology and neurological diseases. Human pluripotent stem cells (hPSCs) could be induced as neural progenitor cells (NPCs) and further multiple neural subtypes, which provide excellent cellular sources for studying neural development, cell therapy, disease modeling and drug screening. It is thus important to establish robust and highly efficient methods of neural differentiation. Enormous efforts have been dedicated to dissecting key signalings during neural commitment and accordingly establishing reliable differentiation protocols. In this study, we refined a step-by-step strategy for rapid differentiation of hPSCs towards NPCs within merely 18 days, combining the adherent and neurosphere-floating methods, as well as highly efficient generation (~90%) of MNs from NPCs by introducing refined sets of transcription factors for around 21 days. This strategy made use of, and compared, retinoic acid (RA) induction and dual-SMAD pathway inhibition, respectively, for neural induction. Both methods could give rise to highly efficient and complete generation of preservable NPCs, but with different regional identities. Given that the generated NPCs can be differentiated into the majority of excitatory and inhibitory neurons, but hardly MNs, we thus further differentiate NPCs towards MNs by overexpressing refined sets of transcription factors, especially by adding human SOX11, whilst improving a series of differentiation conditions to yield mature MNs for good modeling of motor neuron diseases. We thus refined a detailed step-by-step strategy for inducing hPSCs towards long-term preservable NPCs, and further specified MNs based on the NPC platform.

Spheroids of Bladder Smooth Muscle Cells for Bladder Tissue Engineering.

Cell-based tissue engineering (TE) has been proposed to improve treatment outcomes in end-stage bladder disease, but TE approaches with 2D smooth muscle cell (SMC) culture have so far been unsuccessful. Here, we report the development of primary bladder-derived 3D SMC spheroids that outperform 2D SMC cultures in differentiation, maturation, and extracellular matrix (ECM) production. Bladder SMC spheroids were compared with 2D cultures using live-dead staining, qRT-PCR, immunofluorescence, and immunoblotting to investigate culture conditions, contractile phenotype, and ECM deposition. The SMC spheroids were viable for up to 14 days and differentiated rather than proliferating. Spheroids predominantly expressed the late myogenic differentiation marker MyH11, whereas 2D SMC expressed more of the general SMC differentiation marker α-SMA and less MyH11. Furthermore, the expression of bladder wall-specific ECM proteins in SMC spheroids was markedly higher. This first establishment and analysis of primary bladder SMC spheroids are particularly promising for TE because differentiated SMCs and ECM deposition are a prerequisite to building a functional bladder wall substitute. We were able to confirm that SMC spheroids are promising building blocks for studying detrusor regeneration in detail and may provide improved function and regenerative potential, contributing to taking bladder TE a significant step forward.

Quantitative analysis of tumour spheroid structure.

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro.

Drug-induced liver injury (DILI) is the major reason for failures in drug development and withdrawal of approved drugs from the market. Two-dimensional cultures of hepatocytes often fail to reliably predict DILI: hepatoma cell lines such as HepG2 do not reflect important primary-like hepatic properties and primary human hepatocytes (pHHs) dedifferentiate quickly in vitro and are, therefore, not suitable for long-term toxicity studies. More predictive liver in vitro models are urgently required in drug development and compound safety evaluation. This review discusses available human hepatic cell types for in vitro toxicology analysis and their usage in established and emerging three-dimensional (3D) culture systems. Generally, 3D cultures maintain or improve primary hepatic functions (including expression of drug-metabolizing enzymes) of different liver cells for several weeks of culture, thus allowing long-term and repeated-dose toxicity studies. Spheroid cultures of pHHs have been comprehensively tested, but also other cell types such as HepaRG benefit from 3D culture systems. Emerging 3D culture techniques include usage of induced pluripotent stem-cell-derived hepatocytes and primary-like upcyte cells, as well as advanced culture techniques such as microfluidic liver-on-a-chip models. In-depth characterization of existing and emerging 3D hepatocyte technologies is indispensable for successful implementation of such systems in toxicological analysis.

Configurational fingerprints of multicellular living systems.

Cells cooperate as groups to achieve structure and function at the tissue level, during which specific material characteristics emerge. Analogous to phase transitions in classical physics, transformations in the material characteristics of multicellular assemblies are essential for a variety of vital processes including morphogenesis, wound healing, and cancer. In this work, we develop configurational fingerprints of particulate and multicellular assemblies and extract volumetric and shear order parameters based on this fingerprint to quantify the system disorder. Theoretically, these two parameters form a complete and unique pair of signatures for the structural disorder of a multicellular system. The evolution of these two order parameters offers a robust and experimentally accessible way to map the phase transitions in expanding cell monolayers and during embryogenesis and invasion of epithelial spheroids.