Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

Microfluidic 3D Cytotoxic Assay.

Microfluidic-based cytotoxic assays provide high physiological relevance with the potential to replace conventional animal experiments and two-dimensional (2D) assays. Here, a 3D method utilizing a microfluidic platform for analysis of lymphocyte cytotoxicity is introduced in detail, including platform design, cell culture method, real-time cytotoxic assay setup, and image-based analysis. A 2D experimental method is used for comparison, which effectively demonstrates the advantages of 3D microfluidic platforms in closely recapitulating immune responses within the tumor microenvironment. Moreover, a wide range of experimental possibilities and applications using microfluidic 3D cytotoxic assays is introduced in this chapter, along with their capabilities, limitations, and future outlook.

Rocking- and diffusion-based culture of tumor spheroids-on-a-chip.

Tumor spheroids are now intensively investigated toward preclinical and clinical applications, necessitating the establishment of accessible and cost-effective methods for routine operations. Without losing the advantage of organ-chip technologies, we developed a rocking system for facile formation and culture of tumor spheroids in hydrogel microwells of a suspended membrane under microfluidic conditions. While the rocking is controlled with a step motor, the microfluidic device is made of two plastic plates, allowing plugging directly syringe tubes with Luer connectors. Upon injection of the culture medium into the tubes and subsequent rocking of the chip, the medium flows back and forth in the channel underneath the membrane, ensuring a diffusion-based culture. Our results showed that such a rocking- and diffusion-based culture method significantly improved the quality of the tumor spheroids when compared to the static culture, particularly in terms of growth rate, roundness, junction formation and compactness of the spheroids. Notably, dynamically cultured tumor spheroids showed increased drug resistance, suggesting alternative assay conditions. Overall, the present method is pumpless, connectionless, and user-friendly, thereby facilitating the advancement of tumor-spheroid-based applications.

Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads.

To investigate the role of mechanical constraints in morphogenesis and development, we have developed a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.

Phenotypic change of mesenchymal stem cells into smooth muscle cells regulated by dynamic cell-surface interactions on patterned arrays of ultrathin graphene oxide substrates.

The topographical interface of the extracellular environment has been appreciated as a principal biophysical regulator for modulating cell functions, such as adhesion, migration, proliferation, and differentiation. Despite the existed approaches that use two-dimensional nanomaterials to provide beneficial effects, opportunities evaluating their impact on stem cells remain open to elicit unprecedented cellular responses. Herein, we report an ultrathin cell-culture platform with potential-responsive nanoscale biointerfaces for monitoring mesenchymal stem cells (MSCs). We designed an intriguing nanostructured array through self-assembly of graphene oxide sheets and subsequent lithographical patterning method to produce chemophysically defined regions. MSCs cultured on anisotropic micro/nanoscale patterned substrate were spontaneously organized in a highly ordered configuration mainly due to the cell-repellent interactions. Moreover, the spatially aligned MSCs were spontaneously differentiated into smooth muscle cells upon the specific crosstalk between cells. This work provides a robust strategy for directing stem cells and differentiation, which can be utilized as a potential cell culture platform to understand cell-substrate or cell-cell interactions, further developing tissue repair and stem cell-based therapies.

Pillar-Based Mechanical Induction of an Aggressive Tumorigenic Lung Cancer Cell Model.

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.

Construction of a Fibroblast-Associated Tumor Spheroid Model Based on a Collagen Drop Array Chip.

Spheroid, a 3D aggregate of tumor cells in a spherical shape, has overcome the limitations of conventional 3D cell models to accurately mimic the in-vivo environment of a human body. The spheroids are cultured with other primary cells and embedded in collagen drops using hang drop plates and low-attachment well plates to construct a spheroid-hydrogel model that better mimics the cell-cell and cell-extracellular matrix (ECM) interactions. However, the conventional methods of culturing and embedding spheroids into ECM have several shortcomings. The procedure of transferring a single spheroid at a time by manual pipetting results in well-to-well variation and even loss or damage of the spheroid. Based on the previously introduced droplet contact-based spheroid transfer technique, we present a poly(dimethylsiloxane) and resin-based drop array chip and a pillar array chip with alignment stoppers, which enhances the alignment between the chips for uniform placement of spheroids. This method allows the facile and stable transfer of the spheroid array and even eliminates the need for a stereomicroscope while handling the cell models. The novel platform demonstrates a homogeneous and time-efficient construction and diverse analysis of an array of fibroblast-associated glioblastoma multiforme spheroids that are embedded in collagen.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Mammary Tumor Organoid Culture in Non-Adhesive Alginate for Luminal Mechanics and High-Throughput Drug Screening.

Mammary tumor organoids have become a promising in vitro model for drug screening and personalized medicine. However, the dependency on the basement membrane extract (BME) as the growth matrices limits their comprehensive application. In this work, mouse mammary tumor organoids are established by encapsulating tumor pieces in non-adhesive alginate. High-throughput generation of organoids in alginate microbeads is achieved utilizing microfluidic droplet technology. Tumor pieces within the alginate microbeads developed both luminal- and solid-like structures and displayed a high similarity to the original fresh tumor in cellular phenotypes and lineages. The mechanical forces of the luminal organoids in the alginate capsules are analyzed with the theory of the thick-wall pressure vessel (TWPV) model. The luminal pressure of the organoids increase with the lumen growth and can reach 2 kPa after two weeks' culture. Finally, the mammary tumor organoids are treated with doxorubicin and latrunculin A to evaluate their application as a drug screening platform. It is found that the drug response is related to the luminal size and pressures of organoids. This high-throughput culture for mammary tumor organoids may present a promising tool for preclinical drug target validation and personalized medicine.

Melanoma cells adopt features of both mesenchymal and amoeboid migration within confining channels.

For metastasis to occur, cancer cells must traverse a range of tissue environments. In part, this is accomplished by cells adjusting their migration mode to one that is best suited to the environment. Melanoma cells have been shown to be particularly plastic, frequently using both mesenchymal and amoeboid (bleb-based) modes of migration. It has been demonstrated that 2D confinement will promote the transition from mesenchymal to bleb-based migration. However, if melanoma cells similarly transition to bleb-based migration in response to 3D confinement, such as within narrow channels, is unknown. Here, using micro-fabricated channels, we demonstrate that metastatic, A375-M2, melanoma cells adopt features of both mesenchymal and bleb-based migration. In narrow (8 µm; height and width) channels coated with fibronectin, ~ 50% of melanoma cells were found to use either mesenchymal or bleb-based migration modes. In contrast, the inhibition of Src family kinases or coating channels with BSA, completely eliminated any features of mesenchymal migration. Detailed comparisons of migration parameters revealed that blebbing cells, particularly in the absence of adhesions, were faster than mesenchymal cells. In contrast to what has been previously shown under conditions of 2D confinement, pharmacologically inhibiting Arp2/3 promoted a fast filopodial-based mode of migration. Accordingly, we report that melanoma cells adopt a unique range of phenotypes under conditions of 3D confinement.

Rational design of a highly porous electronic scaffold with concurrent enhancement in cell behaviors and differentiation under electrical stimulation.

Conductive polymers (CPs) have received increasing attention as promising materials for studying electrophysiological signals in cell and tissue engineering. The combination of CPs with electrical stimulation (ES) could possibly enhance neurogenesis, osteogenesis, and myogenesis. To date, research has been prioritized on capitalizing CPs as two-dimensional (2D) structures for guiding the differentiation. In contrast, relatively little is conducted on the implementation of 3D conductive scaffolds. In this research, we report the synergic assembly of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) and multi-walled carbon nanotubes (MWCNTs) as a biocompatible, electrically conductive, mechanically robust and structurally porous 3D scaffold. To showcase the bioelectronic utilization, a proof-of-concept demonstration of electrically stimulated cell culture under ES is conducted. The ES effects coupled with the 3D scaffold are promising on pheochromocytoma 12 (PC12), a neuronal cell line, and the ES effect on osteogenesis of human adipose-derived stem cells (hASC) was further studied. PC12 cultured on this PEDOT:PSS/MWCNT 3D scaffolds was induced to differentiate toward a more mature neuronal phenotype with the ES treatment. Furthermore, hASC osteogenesis could be highly promoted in this conductive scaffold with ES. Calcium deposition concentration and osteo-differentiated gene markers were significantly higher with ES. The facile assembly of 3D conductive scaffolds sheds light on both platforms for investigating the 3D microenvironment for electrophysiological simulation of cells and tissues under the ES treatment of in vivo tissue engineering.

Advances in Smoking Related In Vitro Inhalation Toxicology: A Perspective Case of Challenges and Opportunities from Progresses in Lung-on-Chip Technologies.

The inhalation toxicology of multifaceted particulate matter from the environment, cigarette smoke, and e-cigarette liquid vapes is a major research topic concerning the adverse effect of these items on lung tissue. In vitro air-liquid interface (ALI) culture models hold more potential in an inhalation toxicity assessment. Apropos to e-cigarette toxicity, the multiflavor components of the vapes pose a complex experimental bottleneck. While an appropriate ALI setup has been one part of the focus to overcome this, parallel attention towards the development of an ideal exposure system has pushed the field forward. With the advent of microfluidic devices, lung-on-chip (LOC) technologies show enormous opportunities in in vitro smoke-related inhalation toxicity. In this review, we provide a framework, establish a paradigm about smoke-related inhalation toxicity testing in vitro, and give a brief overview of breathing LOC experimental design concepts. The capabilities with optimized bioengineering approaches and microfluidics and their fundamental pros and cons are presented with specific case studies. The LOC model can imitate the structural, functional, and mechanical properties of human alveolar-capillary interface and are more reliable than conventional in vitro models. Finally, we outline current perspective challenges as well as opportunities of future development to smoking lungs-on-chip technologies based on advances in soft robotics, machine learning, and bioengineering.

The biochemical composition of the actomyosin network sets the magnitude of cellular traction forces.

The regulation of cellular force production relies on the complex interplay between a well-conserved set of proteins of the cytoskeleton: actin, myosin, and α-actinin. Despite our deep knowledge of the role of these proteins in force production at the molecular scale, our understanding of the biochemical regulation of the magnitude of traction forces generated at the entire-cell level has been limited, notably by the technical challenge of measuring traction forces and the endogenous biochemical composition in the same cell. In this study, we developed an alternative Traction-Force Microscopy (TFM) assay, which used a combination of hydrogel micropatterning to define cell adhesion and shape and an intermediate fixation/immunolabeling step to characterize strain energies and the endogenous protein contents in single epithelial cells. Our results demonstrated that both the signal intensity and the area of the Focal Adhesion (FA)-associated protein vinculin showed a strong positive correlation with strain energy in mature FAs. Individual contents from actin filament and phospho-myosin displayed broader deviation in their linear relationship to strain energies. Instead, our quantitative analyzes demonstrated that their relative amount exhibited an optimum ratio of phospho-myosin to actin, allowing maximum force production by cells. By contrast, although no correlation was identified between individual α-actinin content and strain energy, the ratio of α-actinin to actin filaments was inversely related to strain energy. Hence, our results suggest that, in the cellular model studied, traction-force magnitude is dictated by the relative numbers of molecular motors and cross-linkers per actin filament, rather than the amounts of an individual component in the cytoskeletal network. This assay offers new perspectives to study in more detail the complex interplay between the endogenous biochemical composition of individual cells and the force they produce.

Drug Evaluation Based on a Multi-Channel Cell Chip with a Horizontal Co-Culture.

We developed a multi-channel cell chip containing a three-dimensional (3D) scaffold for horizontal co-culture and drug toxicity screening in multi-organ culture (human glioblastoma, cervical cancer, normal liver cells, and normal lung cells). The polydimethylsiloxane (PDMS) multi-channel cell chip (PMCCC) was based on fused deposition modeling (FDM) technology. The architecture of the PMCCC was an open-type cell chip and did not require a pump or syringe. We investigated cell proliferation and cytotoxicity by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-dphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and analysis of oleanolic acid (OA)-treated multi-channel cell chips. The results of the MTT and LDH assays showed that OA treatment in the multi-channel cell chip of four cell lines enhanced chemoresistance of cells compared with that in the 2D culture. Furthermore, we demonstrated the feasibility of the application of our multi-channel cell chip in various analysis methods through Annexin V-fluorescein isothiocyanate/propidium iodide staining, which is not used for conventional cell chips. Taken together, the results demonstrated that the PMCCC may be used as a new 3D platform because it enables simultaneous drug screening in multiple cells by single point injection and allows analysis of various biological processes.

Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To assist anchoring of spheroids onto skin wounds, we used a 120-nm-thick free-standing film (nanosheet) that has a highly adhesive property. Bioluminescence imaging showed that ASC spheroids carried by the nanosheet survived for 14 days, which is about two-times longer than that previously reported. Wounds treated with a nanosheet carrying ASC spheroids were 4-times smaller than untreated wounds on day 14. This method for transplantation of spheroids could be applied to cell therapy for various refractory skin wounds.

SpheroidPicker for automated 3D cell culture manipulation using deep learning.

Recent statistics report that more than 3.7 million new cases of cancer occur in Europe yearly, and the disease accounts for approximately 20% of all deaths. High-throughput screening of cancer cell cultures has dominated the search for novel, effective anticancer therapies in the past decades. Recently, functional assays with patient-derived ex vivo 3D cell culture have gained importance for drug discovery and precision medicine. We recently evaluated the major advancements and needs for the 3D cell culture screening, and concluded that strictly standardized and robust sample preparation is the most desired development. Here we propose an artificial intelligence-guided low-cost 3D cell culture delivery system. It consists of a light microscope, a micromanipulator, a syringe pump, and a controller computer. The system performs morphology-based feature analysis on spheroids and can select uniform sized or shaped spheroids to transfer them between various sample holders. It can select the samples from standard sample holders, including Petri dishes and microwell plates, and then transfer them to a variety of holders up to 384 well plates. The device performs reliable semi- and fully automated spheroid transfer. This results in highly controlled experimental conditions and eliminates non-trivial side effects of sample variability that is a key aspect towards next-generation precision medicine.

Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft.

Tissue engineered vascular grafts (TEVG) are potentially clear from ethical and epidemiological concerns sources for reconstructive surgery for small diameter blood vessels replacement. Here, we proposed a novel method to create three-layered TEVG on biocompatible glass fiber scaffolds starting from flat sheet state into tubular shape and to train the resulting tissue by our developed bioreactor system. Constructed tubular tissues were matured and trained under 3 types of individual flow programs, and their mechanical and biological properties were analyzed. Training in the bioreactor significantly increased the tissue burst pressure resistance (up to 18 kPa) comparing to untrained tissue. Fluorescent imaging and histological examination of trained vascular tissue revealed that each cell layer has its own individual response to training flow rates. Histological analysis suggested reverse relationship between tissue thickness and shear stress, and the thickness variation profiles were individual between all three types of cell layers. Concluding: a three-layered tissue structure similar to physiological can be assembled by seeding different cell types in succession; the following training of the formed tissue with increasing flow in a bioreactor is effective for promoting cell survival, improving pressure resistance, and cell layer formation of desired properties.

Novel low shear 3D bioreactor for high purity mesenchymal stem cell production.

Bone marrow derived human Mesenchymal Stem Cells (hMSCs) are an attractive candidate for regenerative medicine. However, their harvest can be invasive, painful, and expensive, making it difficult to supply the enormous amount of pure hMSCs needed for future allogeneic therapies. Because of this, a robust method of scaled bioreactor culture must be designed to supply the need for high purity, high density hMSC yields. Here we test a scaled down model of a novel bioreactor consisting of an unsubmerged 3D printed Polylactic Acid (PLA) lattice matrix wetted by culture media. The growth matrix is uniform, replicable, and biocompatible, enabling homogenous cell culture in three dimensions. The goal of this study was to prove that hMSCs would culture well in this novel bioreactor design. The system tested resulted in comparable stem cell yields to other cell culture systems using bone marrow derived hMSCs, while maintaining viability (96.54% ±2.82), high purity (>98% expression of combined positive markers), and differentiation potential.

Isolation of Mitochondria from Retinal Pigment Epithelial Cell Cultures and an Application of High-Resolution Respirometric Assay (XFe96 Seahorse Assay).

Intracellular Ca2+ is strictly regulated to maintain optimal levels for function of cellular organelles as well as mitochondrial respiratory signaling at the tricarboxylic acid cycle and electron transport chain level. Optimal Ca2+ concentration for these processes vary between cell types. Furthermore, exposure of mitochondria to sustained, elevated levels of Ca2+ induces mitochondrial Ca2+ overload and damage to mitochondrial oxidative phosphorylation and ATP production. Isolated mitochondria are widely used to study mitochondrial physiology and drug effects on mitochondrial metabolism and respiratory function. However, isolated mitochondria are easily damaged during the mitochondrial isolation process. The present article describes a mitochondrial isolation method using Ca2+-chelation to minimize mitochondrial damage. We follow up the isolation process with an application that requires an optimized buffer Ca2+ concentration: the characterization of their respiratory function using a high-resolution respirometric assay.

3D Engineering of Human Hematopoietic Niches in Perfusion Bioreactor.

The hematopoietic microenvironment, also referred to as hematopoietic niche, is a functional three-dimensional (3D) unit of the bone marrow (BM) that planar culture systems cannot recapitulate. Existing limitations of 2D protocols are driving the development of advanced 3D methodologies, capable of superior modeling of the native organization and interactions between hematopoietic cells and their niche.Hereafter we describe the use of a 3D perfusion bioreactor for in vitro generation of human hematopoietic niches. The approach enables the recapitulation of the interactions between hematopoietic stem and progenitor cells (HSPCs), mesenchymal cells (MSCs), and their extracellular matrix in a 3D relevant setting. This was shown to support the functional maintenance of blood populations, self-distributing in the system compartments depending on their differentiation status. Such 3D niche modeling represents an advanced tool toward uncovering human hematopoiesis in relation to its host microenvironment , for both fundamental hematopoiesis and personalized medicine applications.