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Drug-resistant cancer spheroids were fabricated by three-dimensional (3D) bioprinting for the quantitative evaluation of drug resistance of cancer cells, which is a very important issue in cancer treatment. Cancer spheroids have received great attention as a powerful in vitro model to replace animal experiments because of their ability to mimic the tumor microenvironment. In this work, the extrusion printing of gelatin-alginate hydrogel containing MCF-7 breast cancer stem cells successfully provided 3D growth of many single drug-resistant breast cancer spheroids in a cost-effective 3D-printed mini-well dish. The drug-resistant MCF-7 breast cancer spheroids were able to maintain their drug-resistant phenotype of CD44high/CD24low/ALDH1high in the gelatin-alginate media during 3D culture and exhibited higher expression levels of drug resistance markers, such as GRP78 chaperon and ABCG2 transporter, than bulk MCF-7 breast cancer spheroids. Furthermore, the effective concentration 50 (EC50) values for apoptotic and necrotic spheroid death could be directly determined from the 3D printed-gelatin-alginate gel matrix based on in situ 3D fluorescence imaging of cancer spheroids located out of the focal point and on the focal point. The EC50 values of anti-tumor agents (camptothecin and paclitaxel) for apoptotic and necrotic drug-resistant cancer spheroid death were higher than those for bulk cancer spheroid death, indicating a greater drug resistance. Statement of significance This study proposed a novel 3D bioprinting-based drug screening model, to quantitatively evaluate the efficacy of anticancer drugs using drug-resistant MCF-7 breast cancer spheroids formed within a 3D-printed hydrogel. Quantitative determination of anticancer drug efficacy using EC50, which is extremely important in drug discovery, was achieved by 3D printing that enables concurrent growth of many single spheroids efficiently. This study verified whether drug-resistant cancer spheroids grown within 3D-printed gelatin-alginate hydrogel could maintain and present drug resistance. Also, the EC50 values of the apoptotic and necrotic cell deaths were directly acquired in 3D-embedded spheroids based on in situ fluorescence imaging. This platform provides a single-step straightforward strategy to cultivate and characterize drug-resistant spheroids to facilitate anticancer drug screening.

Three-dimensional (3D) tissue-engineered in vitro models, particularly multicellular spheroids and organoids, have become important tools to explore disease progression and guide the development of novel therapeutic strategies. These avascular constructs are particularly powerful in oncological research due to their ability to mimic several key aspects of in vivo tumors, such as 3D structure and pathophysiologic gradients. Advancement of spheroid models requires characterization of critical features (i.e., size, shape, cellular density, and viability) during model development, and in response to treatment. However, evaluation of these characteristics longitudinally, quantitatively and non-invasively remains a challenge. Herein, Optical Coherence Tomography (OCT) is used as a label-free tool to assess 3D morphologies and cellular densities of tumor spheroids, generated via the liquid overlay technique. We utilize this quantitative tool to assess Matrigel’s influence on spheroid morphologic development, finding that the absence of Matrigel produces flattened, disk-like aggregates rather than 3D spheroids with physiologically-relevant features. Furthermore, this technology is adapted to quantify cell number within tumor spheroids, and to discern between live and dead cells, to non-destructively provide valuable information on tissue/construct viability, as well as a proof-of-concept for longitudinal drug efficacy studies. Together, these findings demonstrate OCT as a promising noninvasive, quantitative, label-free, longitudinal and cell-based method that can assess development and drug response in 3D cellular aggregates at a mesoscopic scale.

Cell spheroids as building blocks for engineering micro-tissue should be able to mimic the complex structure of natural tissue. However, control of the distribution of multiple cell populations within cell spheroids is difficult to achieve with current spheroid-harvest methods such as hanging-drop and with the use of microwell plates. In this study, we report the fabrication of core-shell spheroids with the ultimate goal to form 3D complex micro-tissue. We used endothelial cells and two types of stem cells (human turbinate mesenchymal stem cells (hTMSCs)/adipose-derived stem cells (ADSCs)). The stem cells and endothelial cells formed layered micro-sized cell sheets (µCSs) on polydopamine micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids by expansion of the hydrogels. The co-cultured spheroids formed a core-shell structure irrespective of stem cell type. In addition, the size of the core-shell spheroids was controlled from 90 ± 1 to 144 ± 3 µm by changing pattern sizes (200, 300, and 400 µm). The shell thickness gradually increased from 12 ± 3 to 30 ± 6 µm by adjusting the endothelial cell seeding density. Finally, we fabricated the micro-tissue by fusion of the co-cultured spheroids, and the spheroids with the core-shell structure rapidly induced in vitro vessel-like network in 3 days. Thus, the position of endothelial cells in co-cultured spheroids may be an important factor for the modulation of the vascularization process, which can be useful for the production of 3D complex micro-tissues using spheroids as building blocks. Statement of significance This manuscript describes our work on the fabrication of core-shell spheroids as building blocks to form 3D complex vascularized micro-tissue. Stem cells (human turbinate mesenchymal stem cells (hTMSCs) or adipose-derived stem cells (ADSCs)) and endothelial cells formed layered micro-sized cell sheets (µCSs) on micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids (core – stem cells, shell – endothelial cells), irrespective of stem cell type. In addition, the size and shell thickness of the core-shell spheroids were controlled by modifying pattern size and endothelial cell seeding density. We fabricated the vascularized micro-tissue by fusion of the spheroids and demonstrated that the spheroids with a core-shell structure rapidly induced vessel-like network.

Biologically inspired materials with tunable bio- and physicochemical properties provide an essential framework to actively control and support cellular behavior. Cell membrane remodeling approaches benefit from the advances in polymer science and bioconjugation methods, which allow for the installation of un-/natural molecules and particles on the cells’ surface. Synthetically remodeled cells have superior properties and are under intense investigation in various therapeutic scenarios as cell delivery systems, bio-sensing platforms, injectable biomaterials and bioinks for 3D bioprinting applications. In this review article, recent advances in the field of cell surface remodeling via bio-chemical means and the potential biomedical applications of these emerging cell hybrids are discussed. Statement of Significance Recent advances in bioconjugation methods, controlled/living polymerizations, microfabrication techniques and 3D printing technologies have enabled researchers to probe specific cellular functions and cues for therapeutic and research purposes through the formation of cell spheroids and polymer-cell chimeras. This review article highlights recent non-genetic cell membrane engineering strategies towards the fabrication of cellular ensembles and microtissues with interest in 3D in vitro modeling, cell therapeutics and tissue engineering. From a wider perspective, these approaches may provide a roadmap for future advances in cell therapies which will expedite the clinical use of cells, thereby improving the quality and accessibility of disease treatments.

Mechanical remodeling of stromal collagen, such as reorientation and deformation of collagen matrix, generated by invading cancer cells, plays an important role in the progression of cancer invasion and metastasis. In this study, we applied time-lapse microscopy in conjunction with particle displacement mapping to analyze time-dependent contraction and expansion deformations of collagen surrounding individual spheroids of head and neck squamous cell carcinoma cells (HNSCC), OECM-1 & SAS, as the cancer cells detached from the spheroid and invaded into the surrounding 3D collagen matrix. Our results revealed that highly-invasive HNSCC spheroids, stimulated by epidermal growth factor (EGF), generated a strong contraction deformation of the surrounding collagen in the very early stage, and aligned the collagen fibers radially with respect to the center of the spheroid. This initial collagen contraction deformation generated by the HNSCC spheroid bears a strong positive correlation with the overall extent of subsequent cancer cells invasion; hence, it may serve as an early indicator of the invasion capability of the HNSCC spheroids. STATEMENT OF SIGNIFICANCE: Mechanical remodeling of extracellular matrix (ECM) generated by cancer cells plays an important role in the progression of cancer invasion and metastasis. We observed that the extent of initial contraction deformation of collagen surrounding a head and neck squamous cell carcinoma cell (HNSCC) spheroid played an indispensable role in early stage to promote cancer cells invasion into the surrounding ECM. Our results revealed that more invasive HNSCC spheroids generated a larger extent of initial collagen contraction to align the surrounding collagen and to promote cancer cells invasion. This initial collagen contraction deformation generated by the HNSCC spheroids bears a strong positive correlation with the overall extent of cancer cells invasion; hence, it may serve as an early indicator of the invasion capability of the HNSCC spheroids.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with median patient survival of 12-15 months. To facilitate treatment development, bioengineered GBM models that adequately recapitulate the in vivo tumor microenvironment are needed. Matrix-encapsulated multicellular spheroids represent such model because they recapitulate solid tumor characteristics, such as dimensionality, cell-cell, and cell-matrix interactions. Yet, there is no consensus as to which matrix properties are key to improving the predictive capacity of spheroid-based drug screening platforms. We used a hydrogel-encapsulated GBM spheroid model, where matrix properties were independently altered to investigate their effect on GBM spheroid characteristics and drug responsiveness. We focused on hydrogel degradability, tuned via enzymatically degradable crosslinkers, and hydrogel adhesiveness, tuned via integrin ligands. We observed increased cellular infiltration of GBM spheroids and increased resistance to temozolomide in degradable, adhesive hydrogels compared to spheroids in non-degradable, non-adhesive hydrogels or to free-floating spheroids. Further, a higher infiltration index was noted for spheroids in adhesive compared to non-adhesive degradable hydrogels. For spheroids in degradable hydrogels, we determined that infiltrating cells were more susceptible to temozolomide compared to cells in the spheroid core. The temozolomide susceptibility of the infiltrating cells was independent of integrin adhesion. We could not attribute differential drug responses to differential cellular proliferation or to limited drug penetration into the hydrogel matrix. Our results suggest that cell-matrix interactions guide GBM spheroid drug responsiveness and that further elucidation of these interactions could enable the engineering of more predictive drug screening platforms. STATEMENT OF SIGNIFICANCE: Glioblastoma multiforme (GBM) multicellular spheroids hold promise for drug screening and development as they better mimic in vivo cellular responses to therapeutics compared to monolayer cultures. Traditional spheroid models lack an external extracellular matrix (ECM) and fail to mimic the mechanical, physical, and biochemical cues seen in the GBM microenvironment. While embedding spheroids in hydrogel matrices has been shown to better recapitulate the tumor microenvironment, there is still limited understanding as to the key matrix properties that govern spheroid responsiveness to drugs. Here we decoupled and independently altered matrix properties such as degradability, via an enzymatically degradable peptide crosslinker, and cell adhesion, via an adhesive ligand, giving further insight into what matrix properties contribute to GBM chemoresistance.

Cytoskeleton systems, actin microfilaments, microtubules (MTs) and intermediate filaments (IFs) provide the biomechanical stability and spatial organization in cells. To understand the specific contributions of each cytoskeleton systems to intrinsic properties of spheroids, we've scrutinized the effects of the cytoskeleton perturbants, cytochalasin D (Cyto D), nocodazole (Noc) and withaferin A (WFA) on fusion, spreading on adhesive surface, morphology and biomechanics of chondrospheres (CSs). We confirmed that treatment with Cyto D but not with Noc or WFA severely affected CSs fusion and spreading dynamics and significantly reduced biomechanical properties of cell aggregates. Noc treatment affected spheroids spreading but not the fusion and surprisingly enhanced their stiffness. Vimentin intermediate filaments (VIFs) reorganization affected CSs spreading only. The analysis of all three cytoskeleton systems contribution to spheroids intrinsic properties was performed for the first time.

The neurovascular unit (NVU) is a complex functional and anatomical structure composed of endothelial cells and their blood-brain barrier (BBB) forming tight junctions. It represents an efficient barrier for molecules and drugs. However, it also prevents a targeted transport for the treatment of cerebral diseases. The uptake of ultrasmall nanoparticles as potential drug delivery agents was studied in a three-dimensional co-culture cell model (3D spheroid) composed of primary human cells (astrocytes, pericytes, endothelial cells). Multicellular 3D spheroids show reproducible NVU features and functions. The spheroid core is composed mainly of astrocytes, covered with pericytes, while brain endothelial cells form the surface layer, establishing the NVU that regulates the transport of molecules. After 120 h cultivation, the cells self-assemble into a 350 µm spheroid as shown by confocal laser scanning microscopy. The passage of different types of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm) both into the spheroid and into three constituting cell types was studied by confocal laser scanning microscopy. Three kinds of covalently fluorophore-conjugated gold nanoparticles were used: One with fluorescein (FAM), one with Cy3, and one with the peptide CGGpTPAAK-5,6-FAM-NH2. In 2D cell co-culture experiments, it was found that all three kinds of nanoparticles readily entered all three cell types. FAM- and Cy3-labelled nanoparticles were able to enter the cell nucleus as well. The three dissolved dyes alone were not taken up by any cell type. A similar situation evolved with 3D spheroids: The three kinds of nanoparticles entered the spheroid, but the dissolved dyes did not. The presence of a functional blood-brain barrier was demonstrated by adding histamine to the spheroids. In that case, the blood-brain barrier opened, and dissolved dyes like a FITC-labelled antibody and FITC alone entered the spheroid. In summary, our results qualify ultrasmall gold nanoparticles as suitable carriers for imaging or drug delivery into brain cells (sometimes including the nucleus), brain cell spheroids, and probably also into the brain. Statement of significance 3D brain spheroid model and its permeability by ultrasmall gold nanoparticles. We demonstrate that ultrasmall gold nanoparticles can easily penetrate the constituting cells and sometimes even enter the cell nucleus. They can also enter the interior of the blood-brain barrier model. In contrast, small molecules like fluorescing dyes are not able to do that. Thus, ultrasmall gold nanoparticles can serve as carriers of drugs or for imaging inside the brain.

One of the fundamental steps needed to design functional tissues and, ultimately organs is the ability to fabricate thick and densely populated tissue constructs with controlled vasculature and microenvironment. To date, bioprinting methods have been employed to manufacture tissue constructs with open vasculature in a square-lattice geometry, where the majority lacks the ability to be directly perfused. Moreover, it appears to be difficult to fabricate vascular tissue constructs targeting the stiffness of soft tissues such as the liver. Here we present a method for the fabrication of thick (e.g. 1 cm) and densely populated (e.g. 10 million cells·mL−1) tissue constructs with a three-dimensional (3D) four arm branch network and stiffness in the range of soft tissues (1–10 kPa), which can be directly perfused on a fluidic platform for long time periods (>14 days). Specifically, we co-print a 3D four-arm branch using water-soluble Poly(vinyl alcohol) (PVA) as main material and Poly(lactic acid) (PLA) as the support structure. The PLA support structure was selectively removed, and the water soluble PVA structure was used for creating a 3D vascular network within a customized extracellular matrix (ECM) targeting the stiffness of the liver and with encapsulated hepatocellular carcinoma (HepG2) cells. These constructs were directly perfused with medium inducing the proliferation of HepG2 cells and the formation of spheroids. The highest spheroid density was obtained with perfusion, but overall the tissue construct displayed two distinct zones, one of rapid proliferation and one with almost no cell division and high cell death. The created model, therefore, simulate gradients in tissues of necrotic regions in tumors. This versatile method could represent a fundamental step in the fabrication of large functional and complex tissues and finally organs. Statement of Significance Vascularization within hydrogels with mechanical properties in the range of soft tissues remains a challenge. To date, bioprinting have been employed to manufacture tissue constructs with open vasculature in a square-lattice geometry that are most of the time not perfused. This study shows the creation of densely populated tissue constructs with a 3D four arm branch network and stiffness in the range of soft tissues, which can be directly perfused. The cells encapsulated within the construct showed proliferation as a function of the vasculature distance, and the control of the micro-environment induced the encapsulated cells to aggregate in spheroids in specific positions. This method could be used for modeling tumors and for fabricating more complex and densely populated tissue constructs with translational potential.

Understanding cellular behavior in response to microenvironmental stimuli is central to tissue engineering. An increasing number of reports emphasize the high sensitivity of cells to the physical characteristics of the surrounding milieu and in particular, topographical cues. In this work, we investigated the influence of dynamic topographic signal presentation on sprout formation and the possibility to obtain a space–time control over sprouting directionality without growth factors, in order to investigate the contribution of just topography in the angiogenic process. To test our hypothesis, we employed a 3D angiogenesis assay based on the use of spheroids derived from human umbilical vein endothelial cells (HUVECs). We then modulated the in situ presentation of topographical cues during early-stage angiogenesis through real-time photopatterning of an azobenzene-containing polymer, poly (Disperse Red 1 methacrylate) (pDR1m). Pattern inscription on the polymer surface was made using the focused laser of a confocal microscope. We demonstrate that during early-stage angiogenesis, sprouts followed the pattern direction, while spheroid cores acquired a polarized shape. These findings confirmed that sprout directionality was influenced by the photo-inscribed pattern, probably through contact guidance of leader cells, thus validating the proposed platform as a valuable tool for understanding complex processes involved in cell-topography interactions in multicellular systems. Statement of Significance The complex relationship between endothelial cells and the surrounding environment that leads to formation of a newly formed vascular network during tissue repair is currently unknown. We have developed an innovative in vitro platform to study these mechanisms in a space and time controlled fashion simulating what happens during regeneration. In particular, we combine a “smart” surface, namely a polymer film, with a three-dimensional living cell aggregate. The polymer is activated by light through which we can design a path to guide cells toward the formation of a new vessel. Our work lies at the intersection of stimuli-responsive biointerfaces and cell biology and may be particularly inspiring for those interested in designing biomaterial surface related to angiogenesis.

The proper functioning of the liver and tissues containing hepatocytes greatly depends upon the intricate organization of the cells. Consequently, controlling the shape of three-dimensional (3D) cellular constructs is an important issue for in vitro applications of fabricated artificial livers. However, the precise control of tissue shape at the microscale cannot be achieved with various commonly used 3D tissue-engineered building units, such as spheroids. Here, we present the fabrication of hepatic lobule-shaped microtissue (HLSM) containing rat liver (RLC-18) cells. By using cell-microcapsule technology, RLC-18 cells were encapsulated in the core region of poly-l-lysine-alginate microcapsules. After 14days of long-term cultivation, RLC-18 cells self-assembled into HLSM, and the cells fully occupied the microcapsule. By monitoring the cell number and albumin secretion during culture and characterizing the dimensions of the fabricated tissue, we demonstrated that the HLSM showed higher hepatic function as compared with normal cell spheroids. We also showcased the assembly of these microtissues into a 3D four-layered hepatic lobule model by a facile micromanipulation method. Our technology for fabricating 3D multilayer hepatic lobule-like, biofunctional tissue enables the precise control of tissue shape in three dimensions. Furthermore, these constructs can serve as tissue-engineered building blocks for larger organs and cellular implants in clinical treatment.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an unlimited cell source to treat cardiovascular diseases, the leading cause of death worldwide. However, current hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. Here, we examined two important factors that can affect functions of the nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. To examine the first factor, we prepared hiPSC cardiac spheroids with four different sizes by varying cell number per spheroid (∼0.5k, ∼1k, ∼3k, ∼7k cells/spheroid). Spheroids with ∼3k cells/spheroid was found to maximize the beneficial effects of the 3D spheroid microenvironment. This result was explained with a semi-quantitative theory that considers two competing factors: 1) the improved 3D cell-cell adhesion, and 2) the reduced oxygen supply to the center of spheroids with the increase of cell number. Also, the critical role of electrical conductivity of silicon nanowires has been confirmed in improving tissue function of hiPSC cardiac spheroids. These results lay down a solid foundation to develop suitable nanowired hiPSC cardiac spheroids as an innovative cell delivery system to treat cardiovascular diseases. Statement of Significance Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regenerative capacity of adult human hearts, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have received significant attention because they provide a patient specific cell source to regenerate damaged hearts. Despite the progress, current human hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. In this manuscript, we examined the effects of two important factors on the functions of nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. The results from these studies will allow for the development of suitable nanowired hiPSC cardiac spheroids to effectively deliver hiPSC-CMs for heart repair.

Previously we developed a fibrin hydrogel (FH) decorated with laminin-111 peptides (L1p-FH) and supports three-dimensional (3D) gland microstructures containing polarized acinar cells. Here we expand on these results and show that co-culture of rat parotid Par-C10 cells with mesenchymal stem cells produces migrating branches of gland cells into the L1p-FH and we identify FGF-7 as the principal morphogenetic signal responsible for branching. On the other hand, another FGF family member and gland morphogen, FGF-10 increased proliferation but did not promote migration and therefore, limited the number and length of branched structures grown into the gel. By controlling the mode of growth factor presentation and delivery, we can control the length and cellularity of branches as well as formation of new nodes/clusters within the hydrogel. Such spatial delivery of two or more morphogens may facilitate engineering of anatomically complex tissues/mini organs such as salivary glands that can be used to address developmental questions or as platforms for drug discovery. STATEMENT OF SIGNIFICANCE: Hyposalivation leads to the development of a host of oral diseases. Current treatments only provide temporary relief. Tissue engineering may provide promising permanent solutions. Yet current models are limited to salivary spheroids with no branching networks. Branching structures are vital to an effective functioning gland as they increase the surface area/glandular volume ratio of the tissue, allowing a higher output from the small-sized gland. We describe a strategy that controls branch network formation in salivary glands that is a key in advancing the field of salivary gland tissue engineering.

A new three-dimensional (3D) cell printing system was developed and investigated to organize multiple cells/biomaterials with a control precision within 100 μm. This system can be used for the in vitro construction of heterogeneous tissue models. The proposed printing system was achieved by the integration of extrusion printing and alternating viscous and inertial force jetting (AVIFJ) techniques using dual-nozzle switching. In this technique, hydrogels containing high cell densities were extruded using extrusion printing, while droplets containing single cells were precisely manipulated using AVIFJ. The droplets that contained single cells were at the scale of pico-liters and could be accurately positioned at the micron scale. Stable hydrogel structures with adjustable diameters were also printed, with cell viabilities exceeding 90% after printing. A heterogeneous tumor model that contained spheroids and human umbilical vein endothelial cells (HUVECs) was then constructed using the established integrated cell printing system in a stepwise or simultaneous fashion. HUVEC-loaded droplets were observed to locate around the preformed tumor spheroids as designed. Cells and spheroids in the model maintained high cell viability and sustained growth throughout the culture period. The ELISA results of albumin production also proved that the spheroids maintained increased cellular function during the culture. These results demonstrated the feasibility of this integrated 3D printing system for the engineering of in vitro heterogeneous tissue models for future biological and pathological studies. Statement of Significance Addressing the challenge of multi-scale printing in the construction of heterogeneous tissue models, a new 3D cell printing system was developed to organize cells/biomaterials of a control precision within 100 μm. AVIFJ was integrated with extrusion printing, thereby achieving the construction of cell interactions between single cells and spheroids, the manipulation of single cells in a 3D microenvironment with high accuracy, and the real-time on-demand printing. The printed heterogeneous tumor model maintained cell viability, sustained cell growth, and increased cell function during 7 days of culture. We believed that this work would benefit the production of functional artificial tissues, enabling the construction of more biomimetic cell arrangements and microenvironment to support cell functions.

The identification of a biologic substrate for maintaining hepatocyte functions is essential to provide reliable and predictable models for in vitro drug screening. In the current study, a three-dimensional culture of hepatocytes was established on highly porous fibrous scaffolds with grafted galactose and RGD to afford extensive cell-cell and cell-scaffold interactions spatially. The pore size and ligand densities indicated significant effects on the formation of hepatocyte spheroids in balancing the cell retention, adhesion, and migration on fibrous scaffolds. Fibrous scaffolds with an average pore size of 60 μm and surface grafting densities of galactose at 5.9 nmol/cm(2) and RGD at 6.9 pmol/cm(2) provided optimal microenvironments for hepatocyte infiltration and multicellular spheroid formation. Significant promotions were also demonstrated in the syntheses of albumin and urea and the activities of phase I (CYP 3A11 and CYP 2C9) and phase II enzymes. The in vitro metabolism tests on testosterone and acetaminophen by hepatocytes on the optimal scaffolds indicated the predicated clearance rates of 50.7 and 22.6 ml/min/kg, respectively, which were comparable to the in vivo values of rats. The in vitro hepatotoxicity tests on amiodarone hydrochloride and acetaminophen predicted the half maximal effective concentrations (EC50) to reflect the in vivo toxic plasma concentrations in human. In addition, the enzyme activities, predicted clearance rates and hepatotoxicity values of hepatocytes on the optimal scaffolds experienced sensitive responsiveness to specific inducers or inhibitors of CYP 3A11 and phase II enzymes, exhibiting in vivo-in vitro correlations to a certain extent. These results demonstrate the feasibility of hepatocyte spheroid culture on fibrous scaffolds as an potential in vitro testing model to predict the in vivo drug metabolism, hepatotoxicity, and drug-drug interactions.

Bioengineering approaches to regulate stem cell fates aim to recapitulate the in vivo microenvironment. In recent years, manipulating the micro- and nano-scale topography of the stem cell niche has gained considerable interest for the purposes of controlling extrinsic mechanical cues to regulate stem cell fate and behavior in vitro. Here, we established an optimal nanotopographical system to improve 3-dimensional (3D) differentiation of pancreatic cells from human pluripotent stem cells (hPSCs) by testing gradient-pattern chips of nano-scale polystyrene surface structures with varying sizes and shapes. The optimal conditions for 3D differentiation of pancreatic cells were identified by assessing the expression of developmental regulators that are required for pancreatic islet development and maturation. Our results showed that the gradient chip of pore-part 2 (Po-2, 200–300 nm diameter) pattern was the most efficient setting to generate clusters of pancreatic endocrine progenitors (PDX1+ and NGN3+) compared to those of other pore diameters (Po-1, 100–200 or Po-3, 300–400 nm) tested across a range of pillar patterns and flat surfaces. Furthermore, the Po-2 gradient pattern-derived clusters generated islet-like 3D spheroids and tested positive for the zinc-chelating dye dithizone. The spheroids consisted of more than 30% CD200 + endocrine cells and also expressed NKX6.1 and NKX2.2. In addition, pancreatic β- cells expressing insulin and polyhormonal cells expressing both insulin and glucagon were obtained at the final stage of pancreatic differentiation. In conclusion, our data suggest that an optimal topographical structure for differentiation to specific cell types from hPSCs can be tested efficiently by using gradient-pattern chips designed with varying sizes and surfaces. Statement of Significance Our study provides demonstrates of using gradient nanopatterned chips for differentiation of pancreatic islet-like clusters. Gradient nanopatterned chips are consisted of two different shapes (nanopillar and nanopore) in three different ranges of nano sizes (100–200, 200–300, 300–400 nm). We found that optimal nanostructures for differentiation of pancreatic islet-like clusters were 200–300 nm nano pores. Cell transplantation is one of the major therapeutic option for type 1 diabetes mellitus (DM) using stem cell-derived β-like cells. We generated 50 um pancreatic islet-like clusters in size, which would be an optimal size for cell transplantation. Futuremore, the small clusters provide a powerful source for cell therapy. Our findings suggest gradient nanopatterned chip provides a powerful tool to generate specific functional cell types of a high purity for potential uses in cell therapy development.

Development of a bone-like 3D microenvironment with stem cells has always been intriguing in bone tissue engineering. In this study, we fabricated composite spheroids by combining functionalized fibers and human adipose-derived stem cells (hADSCs), which were fused to form a 3D mineralized tissue construct. We prepared fragmented poly (ι-lactic acid) (PLLA) fibers approximately 100 μm long by partial aminolysis of electrospun fibrous mesh. PLLA fibers were then biomineralized with various concentrations of NaHCO3 (0.005, 0.01, and 0.04 M) to form mineralized fragmented fibers (mFF1, mFF2, and mFF3, respectively). SEM analysis showed that the minerals in mFF2 and mFF3 completely covered the fiber surface, and surface chemistry analysis confirmed the presence of hydroxyapatite peaks. Additionally, mFFs formed composite spheroids with hADSCs, demonstrating that the cells were strongly attached to mFFs and homogeneously distributed throughout the spheroid. In vitro culture of spheroids in the media without osteogenic supplements showed significantly enhanced expression of osteogenic genes including Runx2 (20.83 ± 2.83 and 22.36 ± 2.18 fold increase), OPN (14.24 ± 1.71 and 15.076 ± 1.38 fold increase), and OCN (4.36 ± 0.41 and 5.63 ± 0.51 fold increase) in mFF2 and mFF3, respectively, compared to the no mineral fiber group. In addition, mineral contents were significantly increased at day 7. Blocking the biomineral-mediated signaling by PSB 603 significantly down regulated the expression of these genes in mFF3 at day 7. Finally, we fused composite spheroids to form a mineralized 3D tissue construct, which maintained the viability of cells and showed pervasively distributed minerals within the structure. Our composite spheroids could be used as an alternative platform for the development of in vitro bone models, in vivo cell carriers, and as building blocks for bioprinting 3D bone tissue. Statement of Significance This manuscript described our recent work for the preparation of biomimeral-coated fibers that can be assembled with mesenchymal stem cells and provide bone-like environment for directed control over osteogenic differentiation. Biomineral coating onto synthetic, biodegradable single fibers was successfully carried out using multiple steps, combination of template protein coating inspired from mussel adhesion and charge-charge interactions between template proteins and mineral ions. The biomineral-coated single micro-scale fibers (1–2.5 μm in diameter) were then assembled with human adipose tissue derived stem cells (hADSCs). The assembled structure exhibited spheroidal architecture with few hundred micrometers. hADSCs within the spheroids were differentiated into osteogenic lineage in vitro and mineralized in the growth media. These spheroids were fused to form in vitro 3D mineralized tissue with larger size.

Extracellular matrix regulates hallmark features of cancer through biochemical and mechanical signals, although mechanistic understanding of these processes remains limited by lack of models that recreate physiology of tumors. To tissue-engineer models that recapitulate three-dimensional architecture and signaling in tumors, there is a pressing need for new materials permitting flexible control of mechanical and biophysical features. We developed a hybrid hydrogel system composed of collagen and alginate to model tumor environments in breast cancer and other malignancies. Material properties of the hydrogel, including stiffness, microstructure and porosimetry, encompass parameters present in normal organs and tumors. The hydrogel possesses a well-organized, homogenous microstructure with adjustable mechanical stiffness and excellent permeability. Upon embedding multicellular tumor spheroids, we constructed a 3D tumor invasion model showing follow-the-leader migration with fibroblasts leading invasion of cancer cells similar to in vivo. We also demonstrated effects of CXCL12-CXCR4 signaling, a pathway implicated in tumor progression and metastasis, in a dual-tumor spheroid invasion model in 3D hydrogels. These studies establish a new hydrogel platform with material properties that can be tuned to investigate effects of environmental conditions on tumor progression, which will advance future studies of cancer cell invasion and response to therapy. Statement of Significance Our manuscript describes a novel design of hybrid hydrogel system composed of collagen and alginate modeling 3D tumor environments in breast cancer. The hydrogel possesses a well-organized, homogenous microstructure with adjustable mechanical stiffness. Upon embedding tumor spheroids, we successfully showed a 3D tumor invasion model showing follow-the-leader migration with fibroblasts leading invasion of cancer cells similar to in vivo. To the best of our knowledge, this is the first study showing two spheroids invade simultaneously and forming bridge-like connection and effects of chemical gradients in 3D hydrogel environment. This research provides a new model for tumor-stromal interactions in cancer cell migration and establishes a novel hydrogel system for analyzing physical and biochemical signals regulating cancer progression and response to therapy.

Oxygen availability is a critical factor in regulating cell viability that ultimately contributes to the normal morphogenesis and functionality of human tissues. Among various cell culture platforms, construction of 3D multicellular spheroids based on microwell arrays has been extensively applied to reconstitute in vitro human tissue models due to its precise control of tissue culture conditions as well as simple fabrication processes. However, an adequate supply of oxygen into the spheroidal cellular aggregation still remains one of the main challenges to producing healthy in vitro spheroidal tissue models. Here, we present a novel design for controlling the oxygen distribution in concave microwell arrays. We show that oxygen permeability into the microwell is tightly regulated by varying the poly-dimethylsiloxane (PDMS) bottom thickness of the concave microwells. Moreover, we validate the enhanced performance of the engineered microwell arrays by culturing non-proliferated primary rat pancreatic islet spheroids on varying bottom thickness from 10 μm to 1050 μm. Morphological and functional analyses performed on the pancreatic islet spheroids grown for 14 days prove the long-term stability, enhanced viability, and increased hormone secretion under the sufficient oxygen delivery conditions. We expect our results could provide knowledge on oxygen distribution in 3-dimensional spheroidal cell structures and critical design concept for tissue engineering applications. Statement of Significance In this study, we present a noble design to control the oxygen distribution in concave microwell arrays for the formation of highly functional pancreatic islet spheroids by engineering the bottom of the microwells. Our new platform significantly enhanced oxygen permeability that turned out to improve cell viability and spheroidal functionality compared to the conventional thick-bottomed 3-D culture system. Therefore, we believe that this could be a promising medical biotechnology platform to further develop high-throughput tissue screening system as well as in vivo-mimicking customised 3-D tissue culture systems.

Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5-7% w/v) strongly influenced hydrogel stiffness (0.5±0.2kPa to 9.0±1.8kPa) but had little effect on solute diffusion. The diffusion coefficient of 70kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33-37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.

Various galactosylated scaffolds have been developed for hepatocyte culture because galactose ligands help maintain cell viability, facilitate the formation of multicellular spheroids and help maintain a high level of liver-specific functions. However, it is difficult to harvest the cell spheroids generated inside the three-dimensional scaffolds for their further biological analysis and applications. Here we developed a new galactosylated hydrogel scaffold which solidifies in situ upon heating to physiological temperature, but liquefies again upon cooling back to room temperature. The new scaffold is composed of poly(N-isopropylacrylamide) (PNIPAM) microgel and poly(ethylene glycol) (PEG). Because of the thermosensitivity of PNIPAM microgel, the mixed dispersions gel upon heating and liquefy upon cooling. PEG was added to reduce the shrinkage of the gels. Part of the PNIPAM microgel was replaced with a galactosylated one to provide a series of blend gels with various galactose ligand contents. HepG2 cells, a human hepatocarcinoma cell line, were encapsulated in the in situ-formed gels. As expected, the cell viability increases with increasing content of galactose ligands. In addition, compact multicellular spheroids were obtained in gels containing galactose ligands, while loose spheroids formed in gel without galactose ligands. The cells cultured in galactose-containing gels also exhibit a higher level of liver-specific functions, in terms of both albumin secretion and urea synthesis, than those cultured in gel without these ligands. The new galactosylated scaffold not only promotes the formation of hepatocyte spheroids, but also allows for their harvest. By cooling back to room temperature to liquefy the gel, the hepatocyte spheroids can be facilely harvested from the scaffold. The reversible galactosylated scaffold developed here may be used for large scale fabrication of hepatocyte spheroids.

Magnetic nanoparticles (MNPs), primarily iron oxide nanoparticles, have been incorporated into cellular spheroids to allow for magnetic manipulation into desired shapes, patterns and 3-D tissue constructs using magnetic forces. However, the direct and long-term interaction of iron oxide nanoparticles with cells and biological systems can induce adverse effects on cell viability, phenotype and function, and remain a critical concern. Here we report the preparation of biological magnetic cellular spheroids containing magnetoferritin, a biological MNP, capable of serving as a biological alternative to iron oxide magnetic cellular spheroids as tissue engineered building blocks. Magnetoferritin NPs were incorporated into 3-D cellular spheroids with no adverse effects on cell viability up to 1 week. Additionally, cellular spheroids containing magnetoferritin NPs were magnetically patterned and fused into a tissue ring to demonstrate its potential for tissue engineering applications. These results present a biological approach that can serve as an alternative to the commonly used iron oxide magnetic cellular spheroids, which often require complex surface modifications of iron oxide NPs to reduce the adverse effects on cells.

Bioactive and biodegradable hydrogels that mimic the extracellular matrix and regulate valve interstitial cells (VIC) behavior are of great interest as three-dimensional (3-D) model systems for understanding mechanisms of valvular heart disease pathogenesis in vitro and the basis for regenerative templates for tissue engineering. However, the role of stiffness and adhesivity of hydrogels in VIC behavior remains poorly understood. This study reports the synthesis of methacrylated hyaluronic acid (Me-HA) and oxidized and methacrylated hyaluronic acid, and the subsequent development of hybrid hydrogels based on modified HA and methacrylated gelatin (Me-Gel) for VIC encapsulation. The mechanical stiffness and swelling ratio of the hydrogels were tunable with the molecular weight of the HA and the concentration/composition of the precursor solution. The encapsulated VIC in pure HA hydrogels with lower mechanical stiffness showed a more spreading morphology compared to their stiffer counterparts and dramatically up-regulated alpha smooth muscle actin expression, indicating more activated myofibroblast properties. The addition of Me-Gel in Me-HA facilitated cell spreading, proliferation and VIC migration from encapsulated spheroids and better maintained the VIC fibroblastic phenotype. The VIC phenotype transition during migration from encapsulated spheroids in both Me-HA and Me-HA/Me-Gel hydrogel matrixes was also observed. These findings are important for the rational design of hydrogels for controlling the VIC morphology, and for regulating the VIC phenotype and function. The Me-HA/Me-Gel hybrid hydrogels accommodated with VIC are promising as valve tissue engineering scaffolds and 3-D models for understanding valvular pathobiology.

3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro , and typically require 3D culture systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) gel shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures. Statement of Significance Our paper combines an interesting new way for making capsules with cultivation of difficult-to-maintain primary epithelial cells (hepatocytes). The microcapsules described here will enable high density suspension culture of hepatocytes or other cells and may be used as building blocks for engineering tissues.

Tumor associated macrophages (TAMs) are critical stromal components intimately involved with the progression, invasion, and metastasis of cancer cells. To address the need for an in vitro system that mimics the clinical observations of TAM localizations and subsequent functional performance, a cancer cell/macrophage spheroid model is described. The central component of the model is a triple negative breast cancer spheroid embedded in a three-dimensional collagen gel. Macrophages are incorporated in two different ways. The first is a heterospheroid, a spheroid containing both tumor cells and macrophages. The heterospheroid mimics the population of TAMs infiltrated into the tumor mass, thus being exposed to hypoxia and metabolic gradients. In the second model, macrophages are diffusely seeded in the collagen surrounding the spheroid, thus modeling TAMs in the cancer stroma. The inclusion of macrophages as a heterospheroid changes the metabolic profile, indicative of synergistic growth. In contrast, macrophages diffusely seeded in the collagen bear the same profile regardless of the presence of a tumor cell spheroid. The macrophages in the heterospheroid secrete EGF, a cytokine critical to tumor/macrophage co-migration, and an EGF inhibitor decreases the metabolic activity of the heterospheroid, which is not observed in the other systems. The increased secretion of IL-10 indicates that the heterospheroid macrophages follow an M2/TAM differentiation pathway. Lastly, the heterospheroid exhibits resistance to paclitaxel. In summary, the collagen embedded heterospheroid model promotes TAM-like characteristics, and will be of utility in cancer biology and drug discovery.Two in vitro collagen-embedded multicellular spheroid models are described that mimic the clinical observations of macrophage localization within a tumor. Incorporation of macrophages within a breast cancer spheroid emphasizes cell-cell interactions with subsequent differentiation toward a tumor-promoting TAM phenotype. In contrast, macrophages seeded around the tumor spheroid display decreased interaction with cancer cells and no indication of a TAM phenotype. Finally, the presence of macrophages in the heterospheroid increases resistance to paclitaxel. This study demonstrates that cell-cell interactions and 3D collagen matrix direct macrophage activity, and, thus, highlights the important role the local environment itself plays in macrophage behavior.

Vascularization is of great importance to adipose tissue regeneration. Here we introduced a paradigm that using scaffold to induce ASC spheroids, so to promote vascularized adipose tissue regeneration. Poly ( l -glutamic acid) (PLGA) was activated by EDC, followed by being cross-linked by Adipic dihydrazide (ADH) to form a homogeneous hydrogel. Lyophilization was then carried out to create porous structure. The PLGA hydrogel scaffold possessed a significant swollen hydrophilic network to weaken cell-scaffold adhesion but drive ASCs to aggregate to form spheroids. Increase of seeding cell density was proved to result in the increase of spheroid size, upregulating angiogenic genes (VEGF and FGF-2) expression by enhancing the hypoxia-induced paracrine secretion. Also, the adipogenic differentiation of ASCs was achieved in spheroids in vitro. Moreover, the in vivo vascularized adipose tissue regeneration was evaluated in the dorsum of nude mice. After 12 weeks post-implantation, the significant angiogenesis was found in both adipogenic induced and non-induced engineered tissue. In adipogenic induced group, the clear ring-like morphology, the large vacuole in the middle of the cell and the Oil red O staining demonstrated adipose tissue formation. Statement of Significance Vascularization is of great importance to adipose tissue regeneration. Adipose derived stem cell (ASC) spheroids possessed not only the high efficiency of vascularization, but also the improved differentiation ability. Several research works have illustrated the advantage of ASC spheroids in vascularization. However, in adipose regeneration, ASC spheroid was rarely used. Even so, it is reasonable to believe that ASC spheroids hold a great promise in vascularized adipose tissue engineering. Thus in the present study, we introduced a method to create lots of ASC spheroids that acted as lots of individual adipogenesis and angiogenesis units inside of a porous hydrogel scaffold. Then, the scaffold carrying ASC spheroids was implanted subcutaneously in nude mice to preliminarily evaluate the adipose tissue generation and blood vessel formation.

Development of a tissue-engineered, salivary bio-gland will benefit patients suffering from xerostomia due to loss of fluid-secreting acinar cells. This study was conducted to develop a bioengineering system to induce self-assembly of human parotid epithelial cells (hPECs) cultured on poly ethylene glycol (PEG) hydrogel-micropatterned polycaprolactone (PCL) nanofibrous microwells. Microwells were fabricated by photopatterning of PEG hydrogel in the presence of an electrospun PCL nanofibrous scaffold. hPECs were plated on plastic dishes, Matrigel, PCL nanofibers, or PCL nanofibrous microwells. When the cells were plated onto plastic, they did not form spheres, but aggregated to form 3D acinar-like spheroids when cultured on Matrigel, PCL, and PCL microwells, with the greatest aggregating potency being observed on the PCL microwells. The 3D-assembled spheroids in the PCL microwells expressed higher levels of salivary epithelial markers (α-amylase and AQP5), tight junction proteins (ZO-1 and occludin), adherence protein (E-cadherin), and cytoskeletal protein (F-actin) than those on the Matrigel and PCL. Furthermore, the 3D-assembled spheroids in the PCL microwells showed higher levels of α-amylase secretion and intracellular calcium concentration ([Ca2+]i) than those on the Matrigel and PCL nanofibers, suggesting more functional organization of hPECs. We established a bioengineering 3D culture system to promote robust and functional acinar-like organoids from hPECs. PCL nanofibrous microwells can be applied in the future for bioengineering of an artificial bio-salivary gland for restoration of salivary function. Statement of Significance Three dimensional (3D) cultures of salivary glandular epithelial cells using nanofibrous bottom facilitate the formation of acinar-like organoids. In this study, we adapted a PEG hydrogel-micropatterned PCL nanofibrous microwell for the efficient bioengineering of human salivary gland organoids, in which we could easily produce uniform size of 3D organoids. This 3D culture system supports spherical organization, gene and protein expression of acinar markers, TJ proteins, adherence, and cytoskeletal proteins, as well as to promote epithelial structural integrity and acinar secretory functions, and results showed superior efficiency relative to Matrigel and nanofibrous scaffold culture. This 3D culture system has benefits in terms of inert, non-animal and serum-free culture conditions, as well as controllable spheroid size and scalable production of functional SG organoids and is applicable to bioengineering approaches for an artificial bio-gland, as well as to investigations of salivary gland physiology and regeneration.

In ophthalmology, hyaluronic acid (HA) is an important extracellular matrix (ECM) component and is appropriate for use in generating a microenvironment for cell cultivation. The aim of this work was to evaluate the rabbit corneal keratocyte (RCK) growth in response to HA coatings under serum-free conditions. After modification with HA of varying molecular weights (MWs: 35-1500kDa), the surfaces were characterized by atomic force microscopy and contact angle measurements, and were used for cell culture studies. Our data indicated that the substrates coated with higher negatively charged HA become rougher and are more hydrophilic, resulting in the decrease of cell adhesion and cell-matrix interaction. This early cellular event was likely responsible for the determination of keratocyte configuration. Additionally, for the growth of RCKs on dry HA coatings with surface roughness of 1.1-1.7 nm, a strong cell-cell interaction was observed, which may facilitate the formation of multicellular spheroid aggregates and maintenance of mitotically quiescent state. At each culture time point from 1 to 5 days, a better biosynthetic capacity associated with a higher prevalence of elevated ECM production was found for the cells in a spherical configuration. Irrespective of polysaccharide MW of surface coatings, the RCKs presented good viability without hypoxia-induced death. As compared with a monolayer of adherent keratocytes on tissue culture polystyrene plates and low MW HA-modified samples, the cell spheroids (76-110 μm in diameter) showed significantly higher expressions of keratocan and lumican and lower expressions of biglycan, similar to those of keratocytes in vivo. Moreover, the expression levels of corneal crystallin aldehyde dehydrogenase (7-9-fold increase) and nestin (10-16-fold increase) were greater in larger-sized spheroids, indicating higher ability to maintain cellular transparency and self-renewal potential. It is concluded that the cultured RCKs on surfaces coated with HA of different MWs can sense ECM cues, and the multicellular spheroids may potentially be used for corneal stromal tissue engineering applications.

Multicellular spheroids are important in biomedical applications, such as drug research and regenerative medicine. We developed microcapsules from sodium alginate and gelatin for mass production of size-controlled spheroids with diameters

A polymeric crosslinker was developed to promote the formation of cellular spheroids. Our approach was based on the crosslinking of cell membrane using a polymeric crosslinker that worked via hydrophobic interaction. The crosslinker, a poly(ethylene glycol) derivative with oleyl groups as a hydrophobic group at both ends, was synthesized and characterized by gel permeation chromatography and Fourier-transform infrared spectroscopy. Cell culture experiments were then performed to confirm spheroid formation. The rat pancreatic islet β-cell line RIN, which possesses the ability to secrete insulin, was cultured with the crosslinker in a round-bottomed 96-well plate. The formation of a spheroid was achieved when the crosslinker was added to the cell suspension, especially in the absence of serum. The size of the spheroid decreased with time and with increasing crosslinker concentration, and depended on the number of cells plated in each well. The number of cells cultured with crosslinker was almost constant during 7 days and hardly proliferated in crosslinker concentrations of 0–2.5 mg ml −1 , while the number of cells showed a decrease in the 25 mg ml −1 crosslinker concentration. It was shown that the insulin protein secretion in the spheroid cultured with crosslinker for 1 week was enhanced. The cell adhesion protein E-cadherin mRNA expression of the resulting spheroid was also enhanced. These results indicate that the promoted cell function was due to the cell–cell and cell–matrix interactions in the spheroid, suggesting that this polymeric crosslinker was useful for the formation of cell spheroids.

The purpose of this study was to evaluate the behaviors of rat tooth germ (TG) cells cultured on poly(vinyl alcohol) (PVA). It was found that TG cells suspended and aggregated to form three-dimensional spheroids on PVA. Compared with traditional monolayered cells on tissue culture polystyrene, TG cell spheroids on PVA obviously increased the alkaline phosphatase activity, the degree of mineralization, and upregulated both osteopontin and dentin matrix protein 1 genes, regardless of the seeding density. Surprisingly, PVA appears to activate the alkaline phosphatase activity and mineralization effects on TG cell spheroids in the absence of a differentiation medium. Furthermore, the present study indicates that integrins may play an important role in the mineralization on TG cell spheroids by adding Arg-Gly-Asp (RGD) peptides. Therefore, the information presented here should help to clarify the role of PVA in the regulation of the mineralization, differentiation and integrin-mediation of TG cells.

This study describes the morphological and functional behavior of rat hepatocytes on a polydimethylsiloxane (PDMS)-coated surface. Hepatocytes were cultured on hydrophobic or hydrophilic PDMS-coated surfaces in serum-free and serum-containing media. In the serum-free medium, almost all hepatocytes adhered onto the surface irrespective of the wettability, with a cell adhesion ratio of >90% at 24h. In the serum-containing medium, although they strongly adhered onto the hydrophilic surface (cell adhesion ratio >85%), the ratio on the hydrophobic surface was

Biochemical processes of tissue growth lead to production of new proteins, cells, and other material particles at the microscopic level. At the macroscopic level, growth is marked by the change of the tissue shape and mass. In addition, the appearance of the new material particles is generally accompanied by deformation and, consequently, stresses in the surrounding material. Built upon a microscopic toy-tissue model mimicking the mechanical processes of mass supply, a simple phenomenological theory of tissue growth is used in the present work for explaining residual stresses in arteries and studying stresses around growing solid tumors/multicell spheroids. It is shown, in particular, that the uniform volumetric growth can lead to accumulation of residual stresses in arteries because of the material anisotropy. This can be a complementary source of residual stresses in arteries as compared to the stresses induced by non-uniform tissue growth. It is argued that the quantitative assessment of the residual stresses based on in vitro experiments may not be reliable because of the essential stress redistribution in the tissue samples under the cutting process. Concerning the problem of tumor growth, it is shown that the multicell spheroid or tumor evolution depends on elastic properties of surrounding tissues. In good qualitative agreement with the experimental in vitro observations on growing multicell spheroids, numerical simulations confirm that stiff hosting tissues can inhibit tumor growth.

Adipose-derived mesenchymal stem cells (adMSCs) exhibit a high angiogenic activity. Accordingly, their incorporation into tissue constructs represents a promising vascularization strategy in tissue engineering. In the present study, we analyzed whether the efficacy of this approach can be improved by seeding adMSCs as three-dimensional spheroids onto porous scaffolds. Green fluorescent protein (GFP)-positive adMSCs expressing CD13, CD73, CD90 and CD117 were isolated from C57BL/6-TgN(ACTB-EGFP)1Osb/J mice for the generation of spheroids using the liquid overlay technique. Porous polyurethane scaffolds were seeded with these spheroids or a comparable number of individual adMSCs and implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice. The vascularization of the implants was analyzed and compared to non-seeded scaffolds by means of intravital fluorescence microscopy and immunohistochemistry. The adMSC spheroids exhibited a homogeneous diameter of ~270μm and could easily be incorporated into the scaffolds by dynamic seeding. After implantation, they induced a strong angiogenic host tissue response, resulting in an improved scaffold vascularization with a significantly higher functional microvessel density when compared to non-seeded scaffolds and scaffolds seeded with individual adMSCs. Immunohistochemical analyses revealed that a high fraction of ~40% of all microvessels within the center of spheroid-seeded scaffolds developed from GFP-positive adMSCs. These vessels inosculated with ingrowing GFP-negative vessels of the host. This indicates that adMSC spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside implanted tissue constructs. Thus, adMSC spheroids may be used to increase the efficacy of MSC-based vascularization strategies in future tissue engineering applications.

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.

It is known that cellular behavior is affected by nano-patterned topography. For example, many cell types tend to align and extend along the direction of nano-grooves/ridges structures. In this study, we investigated the impact of nano-grooves/ridges on hepatocyte morphology and functions. HepG2/C3A (C3A) cells were cultured on nano-grooved silicon or polystyrene substrata with various widths (from 100 to 500 nm) and depths (from 100 to 380 nm). Nano-grooved substrates induced dramatic changes in C3A cell morphology. The cells formed spheroids on the flat substrates, while C3A cells spread and grew confluently with elongated and aligned morphology along the nano-grooves/ridges. Albumin synthesis was enhanced on the nano-grooved silicon substrates compared to the flat surface, and was decreased with increasing groove depths. Urea conversion on the shallow grooves (400 nm wide and 100 nm deep) remained at the same level of that on the flat surfaces, but was decreased on the deeper grooves. We found that the functions of hepatocytes were enhanced on the substrates with shallow grooves. The nano-grooved substrates may be applied as in vitro culture systems of hepatocytes for both diagnostic and therapeutic applications.

This paper describes a new technique for the control of spheroid diameter in liver-derived cell lines using microfabricated chips that were prepared by combining microfabrication with chemical surface modification. The chip possesses multicavities in a triangular arrangement in the central region (10 mm x 10 mm) of a polymethylmethacrylate (PMMA) plate (24 mm x 24 mm), and the surface of the chip was modified with polyethylene glycol, thereby producing a surface that is non-adhesive to cells. HepG2 cells, a model liver-derived cell line, inoculated onto the chip were trapped within each cavity and proliferated to gradually form spheroids with smooth surfaces and high circularity. Although the spheroid diameters increased with cell proliferation during the initial 10 days of culture, they remained constant thereafter. The spheroid diameters were dependent on the scales of the multicavities on the chip, and the spheroid configuration with uniform diameter was maintained for at least 1 month. In particular, it was demonstrated using chips of various designs that the cavity diameter and the pitch between cavities were effective factors in controlling the spheroid diameter. Furthermore, the protein secretion activities of the spheroid formed on the chip were higher than those of the monolayers for at least 1 month of culture. These results indicate that this chip is a useful technique for the control of spheroid diameter and for the mass preparation of uniform spheroids.