3D Microenvironment Research Articles

Abstract A19: Immunologic dysfunctions of NSG mice confer higher engraftment levels of xenograft Ewing sarcoma metastasis in the PuMA model compared to NOD-SCID mice

Abstract Introduction: Ewing sarcoma is characterized by expression of EWS-ETS fusion proteins generated by chromosomal translocations and comprises bone and/or soft tissue tumors affecting mainly children and young adults. The majority of patients respond well to conventional therapy, but 5-year overall survival of patients with pulmonary metastasis at diagnosis or patients with recurrence remains dismal. The pulmonary metastasis assay (PuMA) is an ex vivo lung explant system developed to study the biology of lung colonization in osteosarcoma (OS), using fluorescence microscopy. The most significant among several advantage of the PuMA model over conventional assays for metastasis is that it recapitulates the initial stages of lung colonization (detachment from primary tumor, intravasation to blood circulation, and arrest to the secondary site) and allows the assessment of antimetastatic effects of a given intervention (candidate gene knockdown or drug treatment) in a 3D microenvironment. For unknown reasons, Ewing sarcoma cell lines do not survive lung colonization using NOD-SCID mice in the PuMA model. NSG mice are generated by crossing NOD-SCID with IL2γ receptor null mice (NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ). These further immunocompromised mice have emerged as successful models to study breast cancer and melanoma metastasis. We herein compared metastatic engraftment of Ewing sarcoma A673 cells in NOD-SCID versus NSG mice in the PUMA model. Methods: Metastatic tumor growth was accessed using PuMA. Briefly, 1 × 106 viable A673 dTomato labeled tumor cells were injected into NOD-SCID or NSG mice via tail veins. Mice were then euthanized, chest cavities exposed, and tracheas cannulated, and PneumaCult-ALI medium (Stem Cell Tech) mixed with 1.2% agarose was infused into the lung. Lung slices of ~1-2mm from each lung lobe were incubated in 6 well plates with PneumaCult-ALI (day 0). Lung slice images were captured on days 0, 3, 7, and 14 using a Leica Stereo Microscope to evaluate tumor growth and H&E staining. Results: In NOD-SCID mice, A673 cell did not form metastatic lesions 3 days after successful injection. In NSG mice, tumor cells were able to successfully colonize the lungs (fold change of tumor burden = 1, 1.5, and 3.2 on day 0, 3, and 7, respectively). Conclusion: Our results show that NSG mice confer higher engraftment levels of xenografted A673 cells in PuMA assays compared to NOD-SCID. NSG and NOD-SCID mice share T- and B-cell depletion and loss of C5 complement, but NSG mice present extremely low NK activity and innate immunity. NOD-SCID may rely on functionally immature macrophages and residual NK activity to target Ewing sarcoma cells. Better understanding specific immunologic response in Ewing sarcoma patients and the mechanisms by which Ewing tumor cells evade immune system may help the development of novel therapy able to prevent advanced disease. Citation Format: Renata Scopim-Ribeiro, Michael M. Lizardo, Anne-Chloe Dhez, Amal M. El-Naggar, Poul H. Sorensen. Immunologic dysfunctions of NSG mice confer higher engraftment levels of xenograft Ewing sarcoma metastasis in the PuMA model compared to NOD-SCID mice [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A19.

Confocal scanning of intervertebral disc cells in 3D: Inside alginate beads and in native microenvironment.

The interaction between cells and their extracellular matrix (ECM) is crucial to maintain both tissue and cellular homeostasis. Indeed, cell phenotype is significantly affected by the 3D microenvironment. Although highly convenient, isolating cells from the intervertebral disc (IVD) and growing them in 2D on plastic or glass substrates, causes them to rapidly lose their phenotype and consequently alter their gene and protein expression. While characterization of cells in their native or simulated 3D environment is preferred, such approaches are complexed by limitations in phenotypic readouts. In the current article, we describe a detailed protocol to study nucleus pulposus cells in 3D—embedded in alginate as a permeable cell‐staining reservoir, as well as adaptation for cell staining and imaging in their native ECM. This method allows for detection of phenotypical and cytoskeletal changes in cells within native tissue or 3D alginate beads using confocal microscopy, without the need for histological processing.

Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive-Manufactured Scaffolds.

Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules-based optical sensors in cell-seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules-based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.

Three-dimensional environment and vascularization induce osteogenic maturation of human adipose-derived stem cells comparable to that of bone-derived progenitors.

While human adipose‐derived stem cells (hADSCs) are known to possess osteogenic differentiation potential, the bone tissues formed are generally considered rudimentary and immature compared with those made by bone‐derived precursor cells such as human bone marrow‐derived mesenchymal stem cells (hBMSCs) and less commonly studied human calvarium osteoprogenitor cells (hOPs). Traditional differentiation protocols have tended to focus on osteoinduction of hADSCs through the addition of osteogenic differentiation media or use of stimulatory bioactive scaffolds which have not resulted in mature bone formation. Here, we tested the hypothesis that by reproducing the physical as well as biochemical bone microenvironment through the use of three‐dimensional (3D) culture and vascularization we could enhance osteogenic maturation in hADSCs. In addition to biomolecular characterization, we performed structural analysis through extracellular collagen alignment and mineral density in our bone tissue engineered samples to evaluate osteogenic maturation. We further compared bone formed by hADSCs, hBMSCs, and hOPs against mature human pediatric calvarial bone, yet not extensively investigated. Although bone generated by all three cell types was still less mature than native pediatric bone, a fibrin‐based 3D microenvironment together with vascularization boosted osteogenic maturation of hADSC making it similar to that of bone‐derived osteoprogenitors. This demonstrates the important role of vascularization and 3D culture in driving osteogenic maturation of cells easily available but constitutively less committed to this lineage and suggests a crucial avenue for recreating the bone microenvironment for tissue engineering of mature craniofacial bone tissues from pediatric hADSCs, as well as hBMSCs and hOPs.

Real-time monitoring of oncolytic VSV properties in a novel in vitro microphysiological system containing 3D multicellular tumor spheroids.

As a new class of cancer therapeutic agents, oncolytic viruses (OVs) have gained much attention not only due to their ability to selectively replicate in and lyse tumor cells, but also for their potential to stimulate antitumor immune responses. As a result, there is an increasing need for in vitro modeling systems capable of recapitulating the 3D physiological tumor microenvironment. Here, we investigated the potential of our recently developed microphysiological system (MPS), featuring a vessel-like channel to reflect the in vivo tumor microenvironment and serving as culture spaces for 3D multicellular tumor spheroids (MCTSs). The MCTSs consist of cancer A549 cells, stromal MRC5 cells, endothelial HUVECs, as well as the extracellular matrix. 3D MCTSs residing in the MPS were infected with oncolytic VSV expressing GFP (oVSV-GFP). Post-infection, GFP signal intensity increased only in A549 cells of the MPS. On the other hand, HUVECs were susceptible to virus infection under 2D culture and IFN-β secretion was quite delayed in HUVECs. These results thus demonstrate that OV antitumoral characteristics can be readily monitored in the MPS and that its behavior therein somewhat differs compared to its activity in 2D system. In conclusion, we present the first application of the MPS, an in vitro model that was developed to better reflect in vivo conditions. Its various advantages suggest the 3D MCTS-integrated MPS can serve as a first line monitoring system to validate oncolytic virus efficacy.

Engineering the cellular mechanical microenvironment to regulate stem cell chondrogenesis: Insights from a microgel model

Biophysical cues (especially mechanical cues) embedded in cellular microenvironments show a critical impact on stem cell fate. Despite the capability of traditional hydrogels to mimic the feature of extracellular matrix (ECM) and tune their physicochemical properties via diverse approaches, their relatively large size not only induces biased results, but also hinders high-throughput screening and analysis. In this paper, a microgel model is proposed to recapitulate the role of 3D mechanical microenvironment on stem cell behaviors especially chondrogenesis in vitro. The small diameter of microgels brings the high surface area to volume ratio and then the enlarged diffusion area and shortened diffusion distance of soluble molecules, leading to uniform distribution of nutrients and negligible biochemical gradient inside microgels. To construct ECM-like microenvironment with tunable mechanical strength, three gelatin/hyaluronic acid hybrid microgels with low, medium and high crosslinking densities, i.e., Gel-HA(L), Gel-HA(M) and Gel-HA(H), are fabricated in microfluidic devices by Michael addition reaction between thiolated gelatin (Gel-SH) and ethylsulfated hyaluronic acid (HA-VS) with different substitution degrees of vinyl sulfone groups. Our results show that mouse bone marrow mesenchymal stem cell (BMSC) proliferation, distribution and chondrogenesis are all closely dependent on mechanical microenvironments in microgels. Noteworthily, BMSCs show a clear trend of differentiating into hyaline cartilage in Gel-HA(L) and fibrocartilage in Gel-HA(M) and Gel-HA(H). Whole transcriptome RNA sequencing reveals that mechanical microenvironment of microgels affects BMSC differentiation via TGF-β/Smad signaling pathway, Hippo signaling pathway and Integrin/YAP/TAZ signaling pathway. We believe this microgel model provides a new way to further explore the interaction between cells and 3D microenvironment. Statement of SignificanceIn recent years, hydrogels have been frequently used to construct 3D microenvironment for cells. However, their relatively large size not only brings biased experimental results, but also limits high-throughput screening and analysis. Herein we propose a gelatin/hyaluronic acid microgel model to explore the effects of 3D cellular mechanical microenvironment (biophysical cues) on BMSC behaviors especially chondrogenesis, which can minimize the interference of biochemical gradients. Our results reveal that BMSC differentiation into either hyaline cartilage or fibrocartilage can be regulated via tailoring the mechanical properties of microgels. Whole transcriptome RNA sequencing proves that “TGF-β/Smad signaling pathway”, “Hippo signaling pathway” and “Integrins/YAP/ TAZ signaling pathway” are activated or inhibited in this process.

Modulating Alginate Hydrogels for Improved Biological Performance as Cellular 3D Microenvironments.

The rational choice and design of biomaterials for biomedical applications is crucial for successful in vitro and in vivo strategies, ultimately dictating their performance and potential clinical applications. Alginate, a marine-derived polysaccharide obtained from seaweeds, is one of the most widely used polymers in the biomedical field, particularly to build three dimensional (3D) systems for in vitro culture and in vivo delivery of cells. Despite their biocompatibility, alginate hydrogels often require modifications to improve their biological activity, namely via inclusion of mammalian cell-interactive domains and fine-tuning of mechanical properties. These modifications enable the addition of new features for greater versatility and control over alginate-based systems, extending the plethora of applications and procedures where they can be used. Additionally, hybrid systems based on alginate combination with other components can also be explored to improve the mimicry of extracellular microenvironments and their dynamics. This review provides an overview on alginate properties and current clinical applications, along with different strategies that have been reported to improve alginate hydrogels performance as 3D matrices and 4D dynamic systems.

Three-Dimensional Microtumors for Probing Heterogeneity of Invasive Bladder Cancer.

Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.

Nanogrooves for 2D and 3D Microenvironments of SH-SY5Y Cultures in Brain-on-Chip Technology.

Brain-on-chip (BOC) technology such as nanogrooves and microtunnel structures can advance in vitro neuronal models by providing a platform with better means to maintain, manipulate and analyze neuronal cell cultures. Specifically, nanogrooves have been shown to influence neuronal differentiation, notably the neurite length and neurite direction. Here, we have drawn new results from our experiments using both 2D and 3D neuronal cell culture implementing both flat and nanogrooved substrates. These are used to show a comparison between the number of cells and neurite length as a first indicator for valuable insights into baseline values and expectations that can be generated from these experiments toward design optimization and predictive value of the technology in our BOC toolbox. Also, as a new step toward neuronal cell models with multiple compartmentalized neuronal cell type regions, we fabricated microtunnel devices bonded to both flat and nanogrooved substrates to assess their compatibility with neuronal cell culture. Our results show that with the current experimental protocols using SH-SY5Y cells, we can expect 200 – 400 cells with a total neurite length of approximately 4,000–5,000 μm per 1 mm2 within our BOC devices, with a lower total neurite length for 3D neuronal cell cultures on flat substrates only. There is a statistically significant difference in total neurite length between 2D cell culture on nanogrooved substrates versus 3D cell culture on flat substrates. As extension of our current BOC toolbox for which these indicative parameters would be used, the microtunnel devices show that culture of SH-SY5Y was feasible, though a limited number of neurites extended into microtunnels away from the cell bodies, regardless of using nanogrooved or flat substrates. This shows that the novel combination of microtunnel devices with nanogrooves can be implemented toward neuronal cell cultures, with future improvements to be performed to ensure neurites extend beyond the confines of the wells between the microtunnels. Overall, these results will aid toward creating more robust BOC platforms with improved predictive value. In turn, this can be used to better understand the brain and brain diseases.

Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy

Recent advances in the field of in-cell NMR spectroscopy have made it possible to study proteins in the context of bacterial or mammalian cell extracts or even entire cells. As most mammalian cells are part of a multi-cellular complex, there is a need to develop novel NMR approaches enabling the study of proteins within the complexity of a 3D cellular environment. Here we investigate the use of the hanging drop method to grow spheroids which are homogenous in size and shape as a model system to study solid tumors using solid-state NMR (ssNMR) spectroscopy. We find that these spheroids are stable under magic-angle-spinning conditions and show a clear change in metabolic profile as compared to single cell preparations. Finally, we utilize dynamic nuclear polarization (DNP)-supported ssNMR measurements to show that low concentrations of labelled nanobodies targeting EGFR (7D12) can be detected inside the spheroids. These findings suggest that solid-state NMR can be used to directly examine proteins or other biomolecules in a 3D cellular microenvironment with potential applications in pharmacological research.

Mechanics and Mechanisms of 3D Mesenchymal Cell Migration

Cell migration has classically been characterized on two-dimensional (2D) surfaces as a series of distinct spatiotemporal steps. However, it remains unclear whether cells in a three-dimensional (3D) microenvironment use cell migration cycles or other mechanisms for efficient migration. Here, we describe a unique contractile mechanism employed by fibroblasts and mesenchymal cancer cells (HT-1080s) to migrate in 3D collagen gels. During 3D cell spreading, fibroblasts strongly deform the matrix. They protrude, polarize, and initiate migration in the direction of highest ECM deformation (prestrain). This prestrain is maintained through a novel uniaxial cellular contraction behind the leading edge prior to protrusion that coordinates a distinct 3D migration cycle that varies between cell types. We have found that myosin IIA is required for 1) polarizing force during spreading, 2) generating local uniaxial contraction, and 3) maintaining the matrix prestrain for efficient directional cell migration. Local matrix severing disrupts the matrix prestrain and migration, suggesting force transmission precedes migration. We find that epithelial cancer cells (MDA-MB-231) rarely demonstrate a sustained prestrain or uniaxial contraction seen in mesenchymal cells. We propose that mesenchymal cells can sense ECM stiffness in 3D and generate their own matrix prestrain using myosin IIA to migrate efficiently using polarized periodic uniaxial contractions for maintaining a unique 3D migration cycle.

3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber.

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.

Effect of three-dimensional ECM stiffness on cancer cell migration through regulating cell volume homeostasis

The extracellular matrix (ECM) stiffness has direct effect on cancer cells homeostasis (e.g., cell volume), which is critical for regulation of their migration. However, the relationship among ECM stiffness, cell volume and cancer cell migration in three-dimensional (3D) microenvironment remains elusive. In this work, we prepared the collagen-alginate hydrogels with tunable stiffness to study how the 3D ECM stiffness influences cell volume and their migration. We found the cell volume homeostasis and migration speed of the MDA-MB-231 cells are both regulated by 3D ECM stiffness, while cell migration speed shows the same stiffness-dependent trend with cell volume. Deviating the cell volume from its homeostasis state can cause a significant decrease in its migration ability, which can be recovered through recovering the cell volume to its homeostasis state. This work reveals for the first time that 3D ECM stiffness regulates cell migration behavior through regulating cell volume homeostasis, which may provide a novel view in the exploration of the underlying mechanisms of cancer metastasis and cellular mechanotransduction.

Screening of integrins localized on the surface of human epidermal melanocytes.

In vivo, melanocytes occupy three-dimensional (3D) space. Nevertheless, most experiments involving melanocytes are performed in a two-dimensional microenvironment, resulting in difficulty obtaining accurate results. Therefore, it is necessary to construct an artificial in vivo-like 3D microenvironment. Here, as a step towards engineering a precisely defined acellular 3D microenvironment supporting the maintenance of human epidermal melanocytes (HEMs), we examined the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in HEMs. Real-time PCR and fluorescent immunoassay analyses were used to elucidate the expression of integrin α and β subunit genes at the transcriptional and translational levels, respectively. The functionality of the presumed integrin heterodimers was confirmed using attachment and antibody-inhibition assays. Among the genes encoding 12 integrin subunits (α1, α2, α3, α4, α5, α6, α7, αV, β1, β3, β5, and β8) showing significantly higher transcription levels, proteins translated from the integrin α2, α4, α5, β1, β3, and β5 subunit genes were detected on the surface of HEMs. These HEMs showed significantly increased adhesion to collagen I, fibronectin, laminin, and vitronectin, and functional blockade of the integrin α2 subunits significantly inhibited adhesion to collagen I, fibronectin, and laminin. In addition, there was no significant inhibition of the adhesion to fibronectin or vitronectin in HEMs with functional blockade of the integrin α4, α5, or αV subunits. These results indicate that the active integrin α2β1 heterodimer and the inactive integrin α4, α5, αV, β3, and β5 subunits are all localized on the surface of HEMs.

Epidermal growth factor and three-dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte-like cells.

Three-dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold-based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte-like cells using embryoid body protocol in the two-dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte-like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or -EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate-based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene-expression patterns, we can conclude that alginate-based 3D coculture system provided a highly efficient protocol for oocyte-like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte-like cell differentiation.

Continuous microfluidic encapsulation of single mesenchymal stem cells using alginate microgels as injectable fillers for bone regeneration

The encapsulation of cells in microscale hydrogels can provide a mimic of a three-dimensional (3D) microenvironment to support cell viability and functions and to protect cells from the environmental stress, which have been widely used in tissue regeneration and cell therapies. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. This microfluidic technique integrated on-chip encapsulation, gelation, and de-emulsification into a one-step fabrication process, which enables scalable cell encapsulation while retaining the viability and functionality of loaded cells. Remarkably, we observed MSCs encapsulated in Ca-alginate microgels at the single-cell level showed significantly enhanced osteogenesis and accelerated mineralization of the microgels which occurred only after 7 days of induction. Furthermore, MSCs laden in alginate microgels displayed significantly enhanced bone formation compared to MSCs mixed with microgels and acellular microgels in a rat tibial ablation model. To conclude, the current microfluidic technique represents a significant step toward continuous single cell encapsulation, fabrication, and purification. These microgels can boost bone regeneration by providing a controlled osteogenic microenvironment for encapsulated MSCs and facilitate stem cell therapy in the treatment of bone defects in a minimally invasive delivery way. Statement of SignificanceThe biological functions and therapeutic activities of single cells laden in microgels for tissue engineering remains less investigated. Here, we reported a microfluidic-based method for continuous encapsulation of single MSCs with high viability and functionality by integrating on-chip encapsulation, gelation, and de-emulsification into a one-step fabrication process. More importantly, MSCs encapsulated in alginate microgels at the single-cell level showed significantly enhanced osteogenesis, remarkably accelerated mineralization in vitro and bone formation capacity in vivo. Therefore, this single-cell encapsulation technique can facilitate stem cell therapy for bone regeneration and be potentially used in a variety of tissue engineering applications.

Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.

Transmigration of Trypanosoma cruzi trypomastigotes through 3D cultures resembling a physiological environment.

To disseminate and colonise tissues in the mammalian host, Trypanosoma cruzi trypomastogotes should cross several biological barriers. How this process occurs or its impact in the outcome of the disease is largely speculative. We examined the in vitro transmigration of trypomastigotes through three-dimensional cultures (spheroids) to understand the tissular dissemination of different T. cruzi strains. Virulent strains were highly invasive: trypomastigotes deeply transmigrate up to 50 μm inside spheroids and were evenly distributed at the spheroid surface. Parasites inside spheroids were systematically observed in the space between cells suggesting a paracellular route of transmigration. On the contrary, poorly virulent strains presented a weak migratory capacity and remained in the external layers of spheroids with a patch-like distribution pattern. The invasiveness-understood as the ability to transmigrate deep into spheroids-was not a transferable feature between strains, neither by soluble or secreted factors nor by co-cultivation of trypomastigotes from invasive and non-invasive strains. Besides, we demonstrated that T. cruzi isolates from children that were born congenitally infected presented a highly migrant phenotype while an isolate from an infected mother (that never transmitted the infection to any of her children) presented significantly less migration. In brief, we demonstrated that in a 3D microenvironment each strain presents a characteristic migration pattern that can be associated to their in vivo behaviour. Altogether, data presented here repositionate spheroids as a valuable tool to study host-pathogen interactions.

Engineering of brain-like tissue constructs via 3D Cell-printing technology

The development of 3D Cell-printing technology contributes to the application of tissue constructs in vitro in neuroscience. Collecting neural cells from patients is an efficient way of monitoring health of an individual target, which, in turn, benefits the enhancement of medicines. The fabricated sample of neural cells is exposed to potential drugs for the analysis of neuron responses. 3D cell-printing as an emerging biofabrication technology has been widely used to mimic natural 3D models in in vitro tissue research, especially in vitro brain-like tissue constructs in neuroscience. Fabricated brain-like tissue constructs provide a 3D microenvironment for primary neural cells to grow within. After more than several weeks of in vitro culturing, the formation of neural circuits in structures equips them with the capability of sensitively responding to a stimulus. In this study, an in vitro layered brain-like tissue construct is first proposed and later developed by 3D cell-printing technology. The layered structure is systematically analyzed, starting from printing parameter optimization to biological functionality performance. The optimized diameter of printing nozzle and printing speed are 0.51 mm and 5 μl s−1, respectively, and the elastic modulus is approximately 6 kPa. Live/dead and immunostaining imaging is used to verify the growth of neural cells in the printed structure. The survival rate of neural cells in 2D and 3D samples is compared, and the results demonstrate that the 3D-printed structures exhibit a better artificial culturing environment and a higher survival rate. Both 2D and 3D samples are directly cultured in a 4 × 4 multiple electrode array. Local field potentials are collected and validated by the Med64 recording system. Tetrodotoxin is used to test the drug sensitivity of the printed structure, and the excitatory postsynaptic potential signals of the physiological performance indicate that the 3D-printed structure has great potential as a drug testing model in the pharmaypeceutical study.

Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments.

Extracellular vesicles (EVs) showed therapeutic properties in several applications, many in regenerative medicine. A clear example is in the treatment of osteoarthritis (OA), where adipose-derived mesenchymal stem cells (ASCs)-EVs were able to promote regeneration and reduce inflammation in both synovia and cartilage. A still obscure issue is the effective ability of EVs to be internalized by target cells, rather than simply bound to the extracellular matrix (ECM) or plasma membrane, since the current detection or imaging technologies cannot fully decipher it due to technical limitations. In the present study, human articular chondrocytes (ACHs) and fibroblast-like synoviocytes (FLSs) isolated from the same OA patients were cocultured in 2D as well as in 3D conditions with fluorescently labeled ASC-EVs, and analyzed by flow cytometry or confocal microscopy, respectively. In contrast with conventional 2D, in 3D cultures, confocal microscopy allowed a clear detection of the tridimensional morphology of the cells and thus an accurate discrimination of EV interaction with the external and/or internal cell environment. In both 2D and 3D conditions, FLSs were more efficient in interacting with ASC-EVs and 3D imaging demonstrated a faster uptake process. The removal of the hyaluronic acid component from the ECM of both cell types reduced their interaction with ASC-EVs only in the 2D system, showing that 2D and 3D conditions can yield different outcomes when investigating events where ECM plays a key role. These results indicate that studying EVs binding and uptake both in 2D and 3D guarantees a more precise and complementary characterization of the molecular mechanisms involved in the process. The implementation of this strategy can become a valuable tool not only for basic research, but also for release assays and potency prediction for clinical EV batches.