A high-throughput 3D kinetic killing assay.

Our work presents a high-throughput kinetic killing assay in the 3D matrix using high-content imaging that is a robust and powerful cytotoxicity assay for evaluating the killing efficiency of immune killer cells or conducting drug screening under physiologically and pathologically relevant scenarios, particularly in the context of solid tumors.

Full maturation of in vitro Plasmodium falciparum oocysts using the AlgiMatrix 3D culture system.

BACKGROUND: Plasmodium falciparum oocysts undergo growth and maturation in a unique setting within the mosquito midgut, firmly situated between the epithelium and the basal lamina. This location exposes them to specific nutrient exchange and metabolic processes while in direct contact with the mosquito haemolymph. The limited availability of in vitro culture systems for growth of the various P. falciparum mosquito stages hampers study of their biology and impedes progress in combatting malaria. METHODS: An artificial in vitro environment was established to mimic this distinctive setting, transitioning from a 2D culture system to a 3D model capable of generating fully mature oocysts that give rise to in vitro sporozoites. RESULTS: A two-dimensional (2D) chamber slide was employed along with an extracellular matrix composed of type IV collagen, entactin, and gamma laminin. This matrix facilitated development of the optimal medium composition for cultivating mature P. falciparum oocysts in vitro. However, the limitations of this 2D culture system in replicating the in vivo oocyst environment prompted a refinement of the approach by optimizing a three-dimensional (3D) alginate matrix culture system. This new system offered improved attachment, structural support, and nutrient exchange for the developing oocysts, leading to their maturation and the generation of sporozoites. CONCLUSIONS: This technique enables the in vitro growth of P. falciparum oocysts and sporozoites.

Bioengineered 3D microfibrous-matrix modulates osteopontin release from MSCs and facilitates the expansion of hematopoietic stem cells.

The osteopontin (OPN) released from mesenchymal stem cells (MSCs) undergoing lineage differentiation can negatively influence the expansion of hematopoietic stem cells (HSCs) in coculture systems developed for expanding HSCs. Therefore, minimizing the amount of OPN in the coculture system is important for the successful ex vivo expansion of HSCs. Toward this goal, a bioengineered three dimensional (3D) microfibrous-matrix that can maintain MSCs in less OPN-releasing conditions has been developed, and its influence on the expansion of HSCs has been studied. The newly developed 3D matrix significantly decreased the release of OPN, depending on the MSC culture conditions used during the priming period before HSC seeding. The culture system with the lowest amount of OPN facilitated a more than 24-fold increase in HSC number in 1 week time period. Interestingly, the viability of expanded cells and the CD34+   pure population of HSCs were found to be the highest in the low OPN-containing system. Therefore, bioengineered microfibrous 3D matrices seeded with MSCs, primed under suitable culture conditions, can be an improved ex vivo expansion system for HSC culture.

Dissecting Alzheimer's disease pathogenesis in human 2D and 3D models.

The incidence of Alzheimer's disease is increasing with the aging population, and it has become one of the main health concerns of modern society. The dissection of the underlying pathogenic mechanisms and the development of effective therapies remain extremely challenging, also because available animal and cell culture models do not fully recapitulate the whole spectrum of pathological changes. The advent of human pluripotent stem cells and cell reprogramming has provided new prospects for tackling these challenges in a human and even patient-specific setting. Yet, experimental modeling of non-cell autonomous and extracellular disease-related alterations has remained largely inaccessible. These limitations are about to be overcome by advances in the development of 3D cell culture systems including organoids, neurospheroids and matrix-embedded 3D cultures, which have been shown to recapitulate extracellular pathologies such as plaque formation in vitro. Recent xenograft studies have even taken human stem cell-based disease modeling to an in vivo scenario where grafted neurons are probed in a disease background in the context of a rodent brain. Here, we review the latest developments in this emerging field along with their advantages, challenges, and future prospects.

WDR5 modulates cell motility and morphology and controls nuclear changes induced by a 3D environment.

Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability, and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting nongenomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5 and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking, and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared with cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn up-regulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a nongenomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity, and cell migratory capacity.

Human elastin-like polypeptides as a versatile platform for exploitation of ultrasensitive bilirubin detection by UnaG.

A new, bifunctional recombinant protein was expressed as the fusion product of human elastin-like polypeptide (HELP) and the bilirubin-binding protein UnaG. The engineered product displays both the HELP-specific property of forming a functional hydrogel matrix and the UnaG-specific capacity of emitting green fluorescence upon ligand binding. The new fusion protein has been proven to be effective at detecting bilirubin in complex environments with high background noise. A cell culture model of the stress response, consisting of bilirubin released in the cell culture medium, was set up to assess the bilirubin-sensing properties of the functional matrix obtained by cross-linking the HELP moiety. Our engineered protein allowed us to monitor cell induction by the release of bilirubin in the culture medium on a nanomolar scale. This study shows that elastin-like protein fusion represents a versatile platform for the development of novel and commercially viable analytical and biosensing devices.

Microtubules in 3D cell motility.

Three-dimensional (3D) cell motility underlies essential processes, such as embryonic development, tissue repair and immune surveillance, and is involved in cancer progression. Although the cytoskeleton is a well-studied regulator of cell migration, most of what we know about its functions originates from studies conducted in two-dimensional (2D) cultures. This research established that the microtubule network mediates polarized trafficking and signaling that are crucial for cell shape and movement in 2D. In parallel, developments in light microscopy and 3D cell culture systems progressively allowed to investigate cytoskeletal functions in more physiologically relevant settings. Interestingly, several studies have demonstrated that microtubule involvement in cell morphogenesis and motility can differ in 2D and 3D environments. In this Commentary, we discuss these differences and their relevance for the understanding the role of microtubules in cell migration in vivo We also provide an overview of microtubule functions that were shown to control cell shape and motility in 3D matrices and discuss how they can be investigated further by using physiologically relevant models.

A novel method to understand tumor cell invasion: integrating extracellular matrix mimicking layers in microfluidic chips by "selective curing".

A major challenge in studying tumor cell invasion into its surrounding tissue is to identify the contribution of individual factors in the tumor microenvironment (TME) to the process. One of the important elements of the TME is the fibrous extracellular matrix (ECM) which is known to influence cancer cell invasion, but exactly how remains unclear. Therefore, there is a need for new models to unravel mechanisms behind the tumor-ECM interaction. In this article, we present a new microfabrication method, called selective curing, to integrate ECM-mimicking layers between two microfluidic channels. This method enables us to study the effect of 3D matrices with controlled architecture, beyond the conventionally used hydrogels, on cancer invasion in a controlled environment. As a proof of principle, we have integrated two electrospun Polycaprolactone (PCL) matrices with different fiber diameters in one chip. We then studied the 3D migration of MDA-MB-231 breast cancer cells into the matrices under the influence of a chemotactic gradient. The results show that neither the invasion distance nor the general cell morphology is affected significantly by the difference in fiber size of these matrices. The cells however do produce longer and more protrusions in the matrix with smaller fiber size. This microfluidic system enables us to study the influence of other factors in the TME on cancer development as well as other biological applications as it provides a controlled compartmentalized environment compatible with cell culturing.

Heterogeneity of Retinal Pigment Epithelial Cells from Adult Human Eye in Different Culturing Systems.

We studied the behavior of retinal pigment epithelial cells from adult human eye derived from different donors in different culturing systems: on plastic, in collagen gel, and on decellularized neural retina substrate. The cells diverge into two subpopulations similar by their morphology and behavior: one subpopulation migrated to the surface of the dense substrate and the other formed spheroid structures consisting of aggregated cells. This fact confirms the data on genetically-predetermined phenotypic heterogeneity of retinal pigment epithelium cells that should be taken into account when using these cells in tissue engineering.

Role of dynamin in elongated cell migration in a 3D matrix.

The use of 3-dimensional (3D) collagen gels has yielded new insights into the migratory behaviour of cancer cells. While the large GTPase dynamin has emerged as an important regulator of cancer cell migration and invasion under 2D conditions, its role in 3D migration is unclear. We have used a potent dynamin modulator, a bis-tyrphostin derivative, Ryngo® 1-23, to investigate the role of dynamin in 3D migration in 3 different cell lines. The compound specifically inhibits persistent, elongated 3D migration in U87MG and SMA-560 cells. Treated U87MG cells adopt a rounded morphology that is not due to apoptosis, loss of matrix metalloprotease activity or inhibition of clathrin-mediated endocytosis. Given that Ryngo 1-23 is known to regulate dynamin oligomerisation and actin dynamics at the leading edge, we analysed actin filament distribution. Ryngo 1-23 induced a switch in actin filament organization in 3D cultures resulting in the generation of multiple short actin-rich microspikes. Correlated with the change in actin filament distribution, cells displayed reduced collagen gel contraction. Since acto-myosin force transmission to the extra-cellular matrix underpins persistent, elongated migration, our results suggest that Ryngo 1-23 modulates this process in 3D migration via dynamin-mediated regulation of acto-myosin force transmission to the extra-cellular matrix.

Two-way regulation between cells and aligned collagen fibrils: local 3D matrix formation and accelerated neural differentiation of human decidua parietalis placental stem cells.

It has been well established that an aligned matrix provides structural and signaling cues to guide cell polarization and cell fate decision. However, the modulation role of cells in matrix remodeling and the feedforward effect on stem cell differentiation have not been studied extensively. In this study, we report on the concerted changes of human decidua parietalis placental stem cells (hdpPSCs) and the highly ordered collagen fibril matrix in response to cell-matrix interaction. With high-resolution imaging, we found the hdpPSCs interacted with the matrix by deforming the cell shape, harvesting the nearby collagen fibrils, and reorganizing the fibrils around the cell body to transform a 2D matrix to a localized 3D matrix. Such a unique 3D matrix prompted high expression of ß-1 integrin around the cell body that mediates and facilitates the stem cell differentiation toward neural cells. The study offers insights into the coordinated, dynamic changes at the cell-matrix interface and elucidates cell modulation of its matrix to establish structural and biochemical cues for effective cell growth and differentiation.

Mechanosensing of cells in 3D gel matrices based on natural and synthetic materials.

Cells in vivo typically are found in 3D matrices, the mechanical stiffness of which is important to the cell and tissue-scale biological processes. Although it is well characterized that as to how cells sense matrix stiffness in 2D substrates, the scenario in 3D matrices needs to be explored. Thus, materials that can mimic native 3D environments and possess wide, physiologically relevant elasticity are highly desirable. Natural polymer-based materials and synthetic hydrogels could provide an better 3D platforms to investigate the mechano-response of cells with stiffness comparable to their native environments. However, the limited stiffness range together with interdependence of matrix stiffness and adhesive ligand density are inherent in many kinds of materials, and hinder efforts to demonstrate the true effects contributed by matrix stiffness. These problems have been addressed by the recently emerging exquisitely designed materials based on native matrix components, designer matrices, and synthetic polymers. In this review, a variety of materials with a wide stiffness range that mimic the mechanical environment of native 3D matrices and the independent affection of stiffness for cellular behavior and tissue-level processes are discussed.

An integrative alginate-based 3D in vitro model to explore epithelial-stromal cell dynamics in the breast tumor microenvironment.

The tumor microenvironment (TME) orchestrates cellular and extracellular matrix (ECM) interactions, playing a key role in tumorigenesis, tumor growth, and metastization. Investigating the interplay between stromal-epithelial cells within the TME is paramount for understanding cancer mechanisms but demands reliable biological models. 3D-models have emerged as powerful in vitro tools, but many fall short in replicating cell-cell/cell-matrix interactions. This study introduces a novel hybrid 3D-model of the breast TME, combining epithelial cells, cancer-associated fibroblasts (CAFs), and their ECM. To build the stromal compartment, porous 3D-printed alginate scaffolds were seeded with CAFs, which proliferated and produced ECM. The pores were infused with oxidized peptide-modified alginate hydrogel laden with MCF10A cells, forming the parenchymal compartment. The hybrid system supported epithelial morphogenesis into acini surrounded by fibroblasts and ECM, and could be readily solubilized to recover cells, their matrix, and sequestered soluble factors. Proteome profiling of the retrieved ECM showed upregulation of proteins associated with matrix assembly/remodeling, epithelial-to-mesenchymal transition (EMT), and cancer. The TME-like microenvironment induced a partial EMT in MCF10A cells, generating a hybrid population with epithelial and mesenchymal features, characteristic of aggressive phenotypes. Our model provided new insights into epithelial-stromal interactions within the TME, offering a valuable tool for cancer research in a physiologically-relevant 3D setting.

Macrophage Mechano-Responsiveness Within Three-Dimensional Tissue Matrix upon Mechanotherapy-Associated Strains.

Mechano-rehabilitation, also known as mechanotherapy, represents the forefront of noninvasive treatment for musculoskeletal (MSK) tissue disorders, encompassing conditions affecting tendons, cartilage, ligaments, and muscles. Recent emphasis has underscored the significance of macrophage presence in the healing of MSK tissues. However, a considerable gap still exists in comprehending how mechanical strains associated with mechanotherapy impact both the naïve and pro-inflammatory macrophage phenotypes within the three-dimensional (3D) tissue matrix, as well as whether the shift in macrophage phenotype is contingent on the mechanical strains inherent to mechanotherapy. In this study, we delineated alterations in mechano-adaptation and polarization of both naive and M1 macrophages within 3D matrices, elucidating their response to varying degrees of mechanical strain exposure (3%, 6%, and 12%). To evaluate macrophage mechano-adaptation and mechano-sensitivity within 3D collagen matrices under mechanical loading, we employed structural techniques (scanning electron microscopy, histology), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time polymerase chain reaction. Our data reveal that the response of macrophages to mechanical loading is not only contingent on their specific sub-phenotype but also varies with the amplitude of mechanical strain. Notably, although supra-mechanical loading (12% strain) was requisite to induce a phenotypic shift in naive (M0) macrophages, as little as 3% mechanical strain proved sufficient to prompt phenotypic alterations in pro-inflammatory (M1) macrophages. These findings pave the way for leveraging the macrophage mechanome in customized and targeted applications of mechanical strain within the mechano-therapeutic framework. Considering the prevalence of MSK tissue injuries and their profound societal and economic implications, the development of well-informed and effective clinical mechanotherapy modalities for MSK tissue healing becomes an imperative endeavor. Impact statement Mechanotherapy is a primary noninvasive treatment for musculoskeletal (MSK) tissue injuries, but the effect of mechanical strain on macrophage phenotypes is not fully understood. A recent study found that macrophage response to mechanical loading is both sub-phenotype specific and amplitude-dependent, with even small strains enough to induce phenotypic changes in pro-inflammatory macrophages. These findings could pave the way for using macrophage mechanome in targeted mechanotherapy applications for better MSK tissue healing.

Organic nano carbon source inducing 3D silica nanoparticles-graphene nanosheet layer on Cu current collector for high-performance anode-free lithium metal batteries.

Anode-free lithium metal batteries (AFLBs) have attracted considerable attention due to their high theoretical specific capacity and absence of Li. However, the heterogeneous Li deposition and stripping on the lithiophobic Cu collector hamper AFLBs in practice. To achieve a uniform and reversible Li deposition, a carbon-based layer on the Cu collector has attracted intense interest due to its high conductivity. However, the 2D single-component carbon-based interface is inadequate lithiophilic for obtaining the homogeneous Li deposition and preventing the lithium dendrite from piercing the separator. Herein, we present a 3D embedded lithiophilic SiO2 nanoparticles-graphene nanosheet matrix (SiO2@G-M) on the Cu collector by organic nano carbon source. In this structure, the lithiophilic SiO2 nanoparticles as active points promote the homogeneous lithium nucleation and the 3D graphene nanosheet matrix offers homogenous electron distribution and voids to prevent the piercing. Finally, SiO2@G-M/Li cell shows a high coulombic efficiency of 98.62 % after 100 cycles at a high current density of 2 mA cm-2 with an areal capacity of 1 mAh cm-2.

Advancing Bioactive Material for Mandibular Bone Regeneration: Transformation of Fibrous Mat into 3D Matrix Cotton for Enhanced Shape Retention and Rapid Hemostasis.

Transformation of a fibrous mat into a three-dimensional (3D) scaffold opens up abundant innovative prospects in biomedical research, particularly for studying both soft as well as hard tissues. Electrospun nanofibers, which mimic the extracellular matrix have attracted significant attention in various studies. This research focuses on rapidly converting a fibrous mat made of polycaprolactone (PCL)/pluronic F-127 (PF-127) with different percentages of monetite calcium phosphate (MCP) into desirable 3D matrix cotton using a unique gas foaming technology. These matrix cottons possess biomimetic properties and have oriented porous structures. Using this innovative technique, various shapes of 3D matrix cotton, such as squares, hollow tubes, and other customizable forms, were successfully produced. Importantly, these 3D matrix cottons showed a consistent distribution of monetite particles with total porosity ranging from 90% to 98%. The structure of the 3D matrix cotton, its water/blood absorption capacity, the potential for causing non-hemolysis, and rapid hemostatic properties were thoroughly investigated. Additionally, periodontal cells were cultured on the 3D matrix cotton to assess their viability and morphology, revealing promising results. Furthermore, a coculture study involving NIH-3T3 and MG-63 cells on the 3D matrix cotton showed spheroidal formation within 24 h. Notably, in vitro assessments indicated that the matrix cotton containing 15% monetite (PCL-MMC15%) exhibited superior absorbent capabilities, excellent cell viability, and rapid hemostatic characteristics. Subsequently, the effectiveness of PCL-MMC15% in promoting mandibular bone regeneration was evaluated through an in vivo study on rabbits using a mandibular injury model. The results demonstrated that PCL-MMC15% facilitated the resolution of defects in the mandibular region by initiating new bone formation. Therefore, the presented 3D matrix cotton (PCL-MMC15%) shows significant promise for applications in both mandibular bone regeneration and hemostasis.

The role of cell-matrix adhesion and cell migration in breast tumor growth and progression.

During breast cancer progression, there is typically increased collagen deposition resulting in elevated extracellular matrix rigidity. This results in changes to cell-matrix adhesion and cell migration, impacting processes such as the epithelial-mesenchymal transition (EMT) and metastasis. We aim to investigate the roles of cell-matrix adhesion and cell migration on breast tumor growth and progression by studying the impacts of different types of extracellular matrices and their rigidities. We embedded MCF7 spheroids within three-dimensional (3D) collagen matrices and agarose matrices. MCF7 cells adhere to collagen but not agarose. Contrasting the results between these two matrices allows us to infer the role of cell-matrix adhesion. We found that MCF7 spheroids exhibited the fastest growth rate when embedded in a collagen matrix with a rigidity of 5.1 kPa (0.5 mg/mL collagen), whereas, for the agarose matrix, the rigidity for the fastest growth rate is 15 kPa (1.0% agarose) instead. This discrepancy is attributable to the presence of cell adhesion molecules in the collagen matrix, which initiates collagen matrix remodeling and facilitates cell migration from the tumor through the EMT. As breast tumors do not adhere to agarose matrices, it is suitable to simulate the cell-cell interactions during the early stage of breast tumor growth. We conducted further analysis to characterize the stresses exerted by the expanding spheroid on the agarose matrix. We identified two distinct MCF7 cell populations, namely, those that are non-dividing and those that are dividing, which exerted low and high expansion stresses on the agarose matrix, respectively. We confirmed this using Western blot which showed the upregulation of proliferating cell nuclear antigen, a proliferation marker, in spheroids grown in the 1.0% agarose (≈13 kPa). By treating the embedded MCF7 spheroids with an inhibitor or activator of myosin contractility, we showed that the optimum spheroids' growth can be increased or decreased, respectively. This finding suggests that tumor growth in the early stage, where cell-cell interaction is more prominent, is determined by actomyosin tension, which alters cell rounding pressure during cell division. However, when breast tumors begin generating collagen into the surrounding matrix, collagen remodeling triggers EMT to promote cell migration and invasion, ultimately leading to metastasis.

A Finite Element Analysis of Single, Double & Matrix Miniplate in Fracture of the Mandibular ANGLE Region: An In Vitro Study.

Purpose: To evaluate and compare the efficacy of three osteosynthesis systems in fixation of mandibular angle fractures using Finite Element Analysis. Materials and Methods: In this study, we used a three-dimensional finite element analysis to assess the stress, deformation and strain in three different groups with bite force loads. A three-dimensional finite element model of the mandible with three different plating techniques using modelling software 'Solidworks2018' and was analysed for stress, deformation and strain produced in the bone following biting loads of different magnitude using analysing software 'ANSYS Workbench'. Results: In this study, we found out that the tensile forces in the matrix miniplate with vertical struts were well distributed in the cortical and cancellous bone on comparison with other two fixation systems in fixation of the mandibular angle fracture and therefore prevents lateral displacement, torsion and bending. The matrix miniplate system revealed less displacement of the fracture segments as compared to the other two plating systems. Conclusion: The use of matrix miniplate for the treatment of mandibular angle fractures can be considered efficacious. The stress transferred onto the cortical & cancellous bone is least in the matrix plate leading to better stability of the fixation system.

Graph theoretical descriptors differentiate d-Mannose isomers in the principal component proposed feature space: A computational approach.

The intricate nature of carbohydrates, particularly monosaccharides, stems from the existence of several chiral centers within their tertiary structures. Predicting and characterizing the molecular geometries and electrostatic landscapes of these substances is difficult due to their complex electrical properties. Moreover, these structures can display a substantial degree of conformational flexibility due to the presence of many rotatable bonds. Moreover, identifying and distinguishing between D and L enantiomers of monosaccharides presents a significant analytical obstacle since there is a need for empirically measurable properties that can distinguish them. This work uses Principal Component Analysis (PCA) to explore the chemical information included in 3D descriptors in order to comprehend the conformational space of d-Mannose stereoisomers. The isomers may be discriminated by utilizing 3D matrix-based indices, geometrical descriptors, and RDF descriptors. The isomers can be distinguished by descriptors, such as the Harary-like index from the reciprocal squared geometrical matrix (H_RG), Harary-like index from Coulomb matrix (H_Coulomb), Wiener-like index from Coulomb matrix (Wi_Coulomb), Wiener-like index from geometrical matrix (Wi_G), Graph energy from Coulomb matrix (SpAbs_Coulomb), Spectral absolute deviation from Coulomb matrix (SpAD_Coulomb), and Spectral positive sum from Coulomb matrix (SpPos_Coulomb). Among these descriptors, the first two, H_RG and H_Coulomb, perform the best in differentiation among the 3D-Matrix-Based Descriptors (3D-MBD) class. The results obtained from this study provide a significant chemical insight into the structural characteristics of the compounds inside the graph theoretical framework. These findings are likely to serve as the basis for developing new methods for analytical experiments.

Dorsal and ventral stimuli in sandwich-like microenvironments. Effect on cell differentiation.

While most of the in vivo extracellular matrices are 3D, most of the in vitro cultures are 2D--where only ventral adhesion is permitted--thus modifying cell behavior as a way to self-adaptation to this unnatural environment. We hypothesize that the excitation of dorsal receptors in cells already attached on a 2D surface (sandwich culture) could cover the gap between 2D and 3D cell-material interactions and result in a more physiological cell behavior. In this study we investigate the role of dorsal stimulation on myoblast differentiation within different poly(L-lactic acid) (PLLA) sandwich-like microenvironments, including plain material and aligned fibers. Enhanced cell differentiation levels were found for cells cultured with dorsal fibronectin-coated films. Seeking to understand the underlying mechanisms, experiments were carried out with (i) different types of dorsal stimuli (FN, albumin, FN after blocking the RGD integrin-binding site and activating dorsal cell integrin receptors), (ii) in the presence of an inhibitor of cell contractility, and (iii) increasing the frequency of culture medium changes to assess the effect of paracrine factors. Furthermore, FAK and integrin expressions, determined by Western blotting, revealed differences between cell sandwiches and 2D controls. Results show a stimuli-dependent response to dorsal excitation, proving that integrin outside-in signaling is involved in the enhanced cell differentiation. Due to their easiness and versatility, these sandwich-like systems are excellent candidates to get deeper insights into the study of 3D cell behavior and to direct cell fate within multilayer constructs.