Cell Spheroids Research Articles

Quantitative image analysis of intracellular protein translocation in 3-dimensional tissues for pharmacodynamic studies of immunogenic cell death

A recent development in cancer chemotherapy is to use cytotoxics to induce tumor-specific immune response through immunogenic cell death (ICD). In ICD, calreticulin is translocated from endoplasmic reticulum to cell membrane (ecto-CRT) which serves as the ‘eat-me-signal’ to antigen-presenting cells. Ecto-CRT measurements, e.g., by ecto-CRT immunostaining plus flow cytometry, can be used to study the pharmacodynamics of ICD in single cells, whereas ICD studies in intact 3-dimensional tissues such as human tumors require different approaches. The present study described a method that used (a) immunostaining with fluorescent antibodies followed by confocal microscopy to obtain the spatial locations of two molecules-of-interest (CRT and a marker protein WGA), and (b) machine-learning (trainable WEKA segmentation) and additional image processing tools to locate the target molecules, remove the interfering signals in the nucleus, cytosol and extracellular space, enable the distinction of the inner and outer edges of the cell membrane and thereby identify the cells with ecto-CRT. This method, when applied to 3-dimensional human bladder cancer cell spheroids, yielded drug-induced ecto-CRT measurements that were qualitatively comparable to the flow cytometry results obtained with single cells disaggregated from spheroids. This new method was applied to study drug-induced ICD in short-term cultures of surgical specimens of human patient bladder tumors.

A Synergic Strategy: Adipose-Derived Stem Cell Spheroids Seeded on 3D-Printed PLA/CHA Scaffolds Implanted in a Bone Critical-Size Defect Model.

Bone critical-size defects and non-union fractures have no intrinsic capacity for self-healing. In this context, the emergence of bone engineering has allowed the development of functional alternatives. The aim of this study was to evaluate the capacity of ASC spheroids in bone regeneration using a synergic strategy with 3D-printed scaffolds made from poly (lactic acid) (PLA) and nanostructured hydroxyapatite doped with carbonate ions (CHA) in a rat model of cranial critical-size defect. In summary, a set of results suggests that ASC spheroidal constructs promoted bone regeneration. In vitro results showed that ASC spheroids were able to spread and interact with the 3D-printed scaffold, synthesizing crucial growth factors and cytokines for bone regeneration, such as VEGF. Histological results after 3 and 6 months of implantation showed the formation of new bone tissue in the PLA/CHA scaffolds that were seeded with ASC spheroids. In conclusion, the presence of ASC spheroids in the PLA/CHA 3D-printed scaffolds seems to successfully promote bone formation, which can be crucial for a significant clinical improvement in critical bone defect regeneration.

Bond-selective full-field optical coherence tomography.

Optical coherence tomography (OCT) is a label-free, non-invasive 3D imaging tool widely used in both biological research and clinical diagnosis. Conventional OCT modalities can only visualize specimen tomography without chemical information. Here, we report a bond-selective full-field OCT (BS-FF-OCT), in which a pulsed mid-infrared laser is used to modulate the OCT signal through the photothermal effect, achieving label-free bond-selective 3D sectioned imaging of highly scattering samples. We first demonstrate BS-FF-OCT imaging of 1 µm PMMA beads embedded in agarose gel. Next, we show 3D hyperspectral imaging of up to 75 µm of polypropylene fiber mattress from a standard surgical mask. We then demonstrate BS-FF-OCT imaging on biological samples, including cancer cell spheroids and C. elegans. Using an alternative pulse timing configuration, we finally demonstrate the capability of BS-FF-OCT on imaging a highly scattering myelinated axons region in a mouse brain tissue slice.

Macrophages as a photosensitizer delivery system for photodynamic therapy: Potential for the local treatment of resected glioblastoma

BackgroundPhotodynamic therapy (PDT) efficacy is determined in part by the concentration of photosensitizer (PS) at the treatment site. The blood-brain barrier (BBB) poses a significant limitation on the transport of PS into the post-operative resection region where brain tumors most often recur. Macrophages (Ma), as opposed to free or nanoparticle bound agents, are known to actively migrate to and around tumors, and can therefore be used as delivery vectors for both drugs and photosensitizers. MethodsMouse Ma (RAW264.7) and F98 rat glioma cells were used in all experiments along with the photosensitizer AlPcS2a. Mitomycin-treated Ma were loaded with photosensitizer (PS) and mixed with glioma cells, forming hybrid spheroids. F98 spheroids were incubated with supernatants derived from PS-loaded Ma (MaPS). Light treatment (PDT) was administered at various radiant exposures from a 670 nm diode laser. The growth of both types of spheroids was evaluated by measurement of spheroid volume after 14 days in culture. ResultsPDT on F98 cell spheroid cultures, mediated by either free or PS-released from Ma, demonstrated a significant growth inhibition with supernatants harvested after 4 and 24 h. A significant PDT-induced growth inhibition was demonstrated in the MaPS/F98 hybrid spheroid experiments. ConclusionSince the efficacy of PDT, mediated by either free or released photosensitizer was comparable, the uptake and released photosensitizer was not degraded. MaPS, incorporated in hybrid tumor spheroids also mediated effective PDT. These results indicate that Ma have potential as an effective vector for photosensitizer delivery to resected brain tumors.

Endochondral Repair of Jawbone Defects Using Periosteal Cell Spheroids

Recapitulation of the natural healing process is receiving increasing recognition as a strategy to induce robust tissue regeneration. Endochondral ossification has been recognized as an essential reparative approach in natural jawbone defect healing. However, such an approach has been overlooked in the recent development of cell-based therapeutics for jawbone repair. Therefore, this study aimed to explore a bioinspired stem cell–based strategy for jawbone repair by mimicking the mesenchymal condensation of progenitor cells during the early endochondral ossification process. For this purpose, passage 3 of jawbone periosteum-derived cells (jb-PDCs) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 96.4 ± 5.8 µm after 48 h. Compared to monolayer culture, the jb-PDC spheroids showed a significant reduction of stemness marker expression evidenced by flow cytometry. Furthermore, a significant upregulation of chondrogenic transcription factor SOX9 in both gene and protein levels was observed in the jb-PDC spheroids after 48 h of chondrogenic induction. RNA sequencing and Western blotting analysis further suggested that the enhanced SOX9-mediated chondrogenic differentiation in jb-PDC spheroids was attributed to the activation of the p38 MAPK pathway. Impressively, inhibition of p38 kinase activity significantly attenuated chondrogenic differentiation jb-PDC spheroids, evidenced by a significant decline of SOX9 in both gene and protein levels. Strikingly, the jb-PDC spheroids implanted in 6- to 8-wk-old male C57BL/6 mice with critical-size jawbone defects (1.8 mm in diameter) showed an evident contribution to cartilaginous callus formation after 1 wk, evidenced by histological analysis. Furthermore, micro–computed tomography analysis showed that the jb-PDC spheroids significantly accelerated bone healing after 2 wk in the absence of exogenous growth factors. In sum, the presented findings represent the successful development of cell-based therapeutics to reengineer the endochondral bone repair process and illustrate the potential application to improve bone repair and regeneration in the craniofacial skeleton.

Dinuclear gold(I) complexes based on carbene and diphosphane ligands: bis[2-(dicyclohexylphosphano)ethyl]amine complex inhibits the proteasome activity, decreases stem cell markers and spheroid viability in lung cancer cells.

Three new dinuclear gold(I) complexes (1-3) containing a carbene (1,3-Bis(2,6-di-isopropylphenyl)imidazol-2-ylidene (IPr)) and diphosphane ligands [bis(1,2-diphenylphosphano)ethane (Dppe), bis(1,3-diphenylphosphano)propane (Dppp) and bis[2-(dicyclohexylphosphano)ethyl]amine (DCyPA)], were synthesized and characterized by elemental analysis and, ESI-MS, mid FT-IR and NMR spectroscopic methods. The structures of complexes 2 and 3 were determined by X-ray crystallography, which revealed that the complexes are dinuclear having gold(I) ions linearly coordinated. The anticancer activities of the complexes (1-3) were evaluated in lung (A549), breast (MC-F7), prostate (PC-3), osteosarcoma (MG-63) and ovarian (A2780 and A2780cis) cancer models. Growth inhibition by the new complexes was higher than cisplatin in all cell lines tested. The mechanism of action of complex 3 was investigated in A549 cells using 2-dimensional (2D) models and 3D-multicellular tumor spheroids. Treatment of A549 cells with complex 3 caused: the induction of apoptosis and the generation of reactive oxygen species; the cell cycle arrest in the G0/G1 phase; the inhibition of both the proteasome and the NF-kB activity; the down-regulation of lung cancer stem cell markers (NOTCH1, CD133, ALDH1 and CD44). Complex 3 was more active than cisplatin also in 3D models of A549 lung cancer cells.

3D bioprinting of dECM-incorporated hepatocyte spheroid for simultaneous promotion of cell-cell and -ECM interactions.

The cell spheroid technology, which greatly enhances cell-cell interactions, has gained significant attention in the development of in vitro liver models. However, existing cell spheroid technologies still have limitations in improving hepatocyte-extracellular matrix (ECM) interaction, which have a significant impact on hepatic function. In this study, we have developed a novel bioprinting technology for decellularized ECM (dECM)-incorporated hepatocyte spheroids that could enhance both cell-cell and -ECM interactions simultaneously. To provide a biomimetic environment, a porcine liver dECM-based cell bio-ink was developed, and a spheroid printing process using this bio-ink was established. As a result, we precisely printed the dECM-incorporated hepatocyte spheroids with a diameter of approximately 160-220μm using primary mouse hepatocyte (PMHs). The dECM materials were uniformly distributed within the bio-printed spheroids, and even after more than 2weeks of culture, the spheroids maintained their spherical shape and high viability. The incorporation of dECM also significantly improved the hepatic function of hepatocyte spheroids. Compared to hepatocyte-only spheroids, dECM-incorporated hepatocyte spheroids showed approximately 4.3- and 2.5-fold increased levels of albumin and urea secretion, respectively, and a 2.0-fold increase in CYP enzyme activity. These characteristics were also reflected in the hepatic gene expression levels of ALB, HNF4A, CPS1, and others. Furthermore, the dECM-incorporated hepatocyte spheroids exhibited up to a 1.8-fold enhanced drug responsiveness to representative hepatotoxic drugs such as acetaminophen, celecoxib, and amiodarone. Based on these results, it can be concluded that the dECM-incorporated spheroid printing technology has great potential for the development of highly functional in vitro liver tissue models for drug toxicity assessment.

CCRG-04. AUTOMATED REAL TIME TRACKING OF GLIOBLASTOMA CELL CYCLE TRANSITIONS IN ASSEMBLOIDS LINKS G2 CELLS TO INFILTRATION

Abstract Glioblastoma (GB) is the most aggressive primary brain tumour in adults associated with survival rates below 5% despite treatment. Tumour repopulating residual cells in the surgical cavity resist treatment due to their molecular heterogeneity. Single-cell transcriptomic studies have linked GB heterogeneity to the cell cycle; however, it remains unclear to what extent cell cycle states affect the infiltrative capacity of GB. The “go or grow” paradigm predicts that migration and proliferation are separate spatiotemporal events. We used a brain tumour-cerebral organoid (assembloid) approach to test the migratory potential of GB cell cycle phases during assembloid infiltration. Human induced pluripotent cells were differentiated into cerebral organoids that self-assemble with spheroids of patient-derived GB cells, expressing a fluorescent cell cycle reporter (FUCCI). Utilising live-cell confocal imaging, the process of GB assembloid infiltration was captured. Resultant videos were analysed with automated image analysis capable of tracking cell cycle transitions and cellular migration in confocal microscopy planes, enabling the longitudinal quantification of GB migration and invasion. Our analysis revealed a subset of highly infiltrative GB cells, containing a significant number of cycling G2 phase cells that demonstrated a ~1.4-fold increase in directional infiltrative capacity compared to their non cycling G1 phase counterparts. To isolate and characterize the G2 migratory GB cell state, we developed a phenotypic separation assay using microenvironmental cues. Specifically, we found that these cues markedly affect GB migration routes enabling the isolation of the highly migratory GB cells for further investigation. Our results question the “go or grow” hypothesis at the level of GB cellular subpopulations. We hypothesise that the observed G2 migratory capacity of GB cells could be critical to tumour recurrence following surgery. Further research is underway to define the underlying mechanisms of G2 GB cell migration.

DDEL-12. A CONFORMABLE IMPLANTABLE DEVICE - MICROMESH - FOR THE SUSTAINED DELIVERY OF CHEMOTHERAPEUTIC MOLECULES AND IMMUNE CHECK POINT INHIBITORS AGAINST HIGH-GRADE GLIOMAS

Abstract High-grade gliomas, like glioblastoma (GBM), continue to have a poor prognosis with therapeutic options limited to maximal safe resection and adjuvant radiochemotherapy. Effective treatment is hampered by the blood-brain barrier, limiting the intratumor permeation of systemically administered agents; and biological heterogeneity of malignant cells, requiring complex therapeutic regimens. A conformable, compartmentalized polymeric implant – microMESH – has been engineered to overcome these obstacles by realizing a localized, sustained release of drugs, small inhibitors, antibodies, and nanomedicines. In the present configuration, microMESH comprises a water-soluble polyvinyl alcohol microlayer, loaded with the immune checkpoint aCD47, intercalated with a microfabricated network of poly lactic-co-glycolic acid, carrying docetaxel (DTXL). Upon deposition over the tumor margins, microMESH adheres and conforms to the local tissue releasing thereof its therapeutic cargo for several weeks, circumventing possible systemic toxicity. Gel permeation chromatography, nuclear magnetic resonance spectroscopy, weight and pH variations were measured to assess the microMESH biodegradation over time. microMESH biocompatibility and cytotoxicity were assessed on cancer cell monolayers and spheroids. The preclinical performance of microMESH was evaluated in two syngeneic mouse models of high-grade gliomas obtained via the intracranial inoculation of murine cancer cells – CT-2A, cold tumor; GL261, hot tumor. Flow cytometry was used to map the immune component in the tumor and assess the expression of immune checkpoints. Bioluminescence imaging was used to monitor tumor proliferation longitudinally. microMESH biodegraded in ~2 months with no documented toxicity. Preliminary experiments with an aCD47/DTXL–microMESH applied over the unresected margins of an orthotopic CT-2A model documented a median overall survival of 33.5 days against 18 days for untreated mice, 22 days for intravenously injected DTXL, 24 days for intraperitoneally administered aCD47 (statistically not significant difference among the control groups). microMESH allowed the sustained delivery of complex combination therapies directly at the tumor bed demonstrating superior efficacy over systemic therapies.

Circular RNA ZNF800 (hsa_circ_0082096) regulates cancer stem cell properties and tumor growth in colorectal cancer

BackgroundCancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines.MethodsSpheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.ResultsCircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.ConclusionsCircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.

EXTH-61. A MULTI-TARGETED APPROACH TO EVALUATE THERAPEUTIC SELECTION AND EFFICACY IN PRECLINICAL GBM MODELS

Abstract Glioblastoma (GBM) is a devastating primary brain cancer with approximately 10,000 new US diagnoses annually. The current standard of care for GBM includes surgical resection followed by radiation therapy (RT) and temozolomide (TMZ), however there is near universal recurrence and development of resistance after treatment. Relapse in disease is tightly linked with dynamic changes in gene expression during tumor evolution, highlighting the need for stronger preclinical GBM models. Here we report developing a pair of patient-derived xenograft (PDX) models from surgical resections obtained from initial GBM incidence in a patient (CRT00433) and subsequent recurrence following treatment (CRT00435) in order to study disease progression and novel treatment strategies. Recent studies have suggested the utilization of combination therapy approaches for GBM patients to address TMZ resistance. To this end, Certis has developed a personalized, AI-based approach to predict and test combination therapies for CRT00433/435. Accuracy of the predicted sensitivities to treatment was then evaluated in vivo using both subcutaneously and orthotopically engrafted mouse modeling. Luciferase-tagged CRT00433 and CRT00435 spheroid cell lines were transduced with firefly luciferase and implanted intracranially by stereotactic surgery for orthotopic monitoring. Optical bioluminescence imaging (BLI) and MRI were used to assess therapeutic response of AI-predicted combination therapies and standard of care (RT and/or TMZ). Both in-life imaging and terminal histologic analysis of the tumors confirmed high fidelity to several of the therapies tested. In all, these results demonstrate a novel, combinatorial platform with which to assess therapeutic efficacy in GBM and exploit the overall ability of this deadly disease to adapt to an ever-changing molecular and genetic landscape.

OptoDyCE-plate as an affordable high throughput imager for all optical cardiac electrophysiology

We present a simple low-cost system for comprehensive functional characterization of cardiac function under spontaneous and paced conditions, in standard 96 and 384-well plates. This full-plate actuator/imager, OptoDyCE-plate, uses optogenetic stimulation and optical readouts of voltage and calcium (parallel recordings from up to 100 wells in 384-well plates are demonstrated). The system is validated with syncytia of human induced pluripotent stem cell derived cardiomyocytes, iPSC-CMs, grown as monolayers, or in quasi-3D isotropic and anisotropic constructs using electrospun matrices, in 96 and 384-well format. Genetic modifications, e.g. interference CRISPR (CRISPRi), and nine compounds of acute and chronic action were tested, including five histone deacetylase inhibitors (HDACis). Their effects on voltage and calcium were compared across growth conditions and pacing rates. We also demonstrated optogenetic point pacing via cell spheroids to study conduction in 96-well format, as well as temporal multiplexing to register voltage and calcium simultaneously on a single camera. Opto-DyCE-plate showed excellent performance even in the small samples in 384-well plates. Anisotropic structured constructs may provide some benefits in drug testing, although drug responses were consistent across tested configurations. Differential voltage vs. calcium responses were seen for some drugs, especially for non-traditional modulators of cardiac function, e.g. HDACi, and pacing rate was a powerful modulator of drug response, highlighting the need for comprehensive multiparametric assessment, as offered by OptoDyCE-plate. Increasing throughput and speed and reducing cost of screening can help stratify potential compounds early in the drug development process and accelerate the development of safer drugs.

An in vitro model system based on calcium- and phosphate ion-induced hMSC spheroid mineralization

A challenge in regenerative medicine is creating the three-dimensional organic and inorganic in vitro microenvironment of bone, which would allow the study of musculoskeletal disorders and the generation of building blocks for bone regeneration. This study presents a microwell-based platform for creating spheroids of human mesenchymal stromal cells, which are then mineralized using ionic calcium and phosphate supplementation. The resulting mineralized spheroids promote an osteogenic gene expression profile through the influence of the spheroids’ biophysical environment and inorganic signaling and require less calcium or phosphate to achieve mineralization compared to a monolayer culture. We found that mineralized spheroids represent an in vitro model for studying small molecule perturbations and extracellular mediated calcification. Furthermore, we demonstrate that understanding pathway signaling elicited by the spheroid environment allows mimicking these pathways in traditional monolayer culture, enabling similar rapid mineralization events. In sum, this study demonstrates the rapid generation and employment of a mineralized cell model system for regenerative medicine applications.

In Vitro Models of Head and Neck Cancer: From Primitive to Most Advanced.

For several decades now, researchers have been trying to answer the demand of clinical oncologists to create an ideal preclinical model of head and neck squamous cell carcinoma (HNSCC) that is accessible, reproducible, and relevant. Over the past years, the development of cellular technologies has naturally allowed us to move from primitive short-lived primary 2D cell cultures to complex patient-derived 3D models that reproduce the cellular composition, architecture, mutational, or viral load of native tumor tissue. Depending on the tasks and capabilities, a scientific laboratory can choose from several types of models: primary cell cultures, immortalized cell lines, spheroids or heterospheroids, tissue engineering models, bioprinted models, organoids, tumor explants, and histocultures. HNSCC in vitro models make it possible to screen agents with potential antitumor activity, study the contribution of the tumor microenvironment to its progression and metastasis, determine the prognostic significance of individual biomarkers (including using genetic engineering methods), study the effect of viral infection on the pathogenesis of the disease, and adjust treatment tactics for a specific patient or groups of patients. Promising experimental results have created a scientific basis for the registration of several clinical studies using HNSCC in vitro models.

Comparative clinical significance and biological roles of PFKFB family members in oral squamous cell carcinoma

BackgroundCancer cells promote glycolysis, which supports rapid cell growth and proliferation. Phosphofructokinase-fructose bisphosphatases (PFKFBs), a family of bidirectional glycolytic enzymes, play key roles in the regulation of glycolysis in many types of cancer. However, their roles in oral squamous cell carcinoma (OSCC), the most common type of oral cancer, are still unknown.MethodsWe compared the gene expression levels of PFKFB family members and analyzed their clinical significance in oral cancer patients, whose clinical data were obtained the Cancer Genome Atlas database. Moreover, real-time quantitative polymerase chain reaction, western blotting, assays for cell viability, cell cycle, cell migration and viability of cell spheroid were performed in scramble and PFKFB-silenced cells.ResultsWe discovered that PFKFB3 expression in tumor tissues was slightly higher than that in tumor adjacent normal tissues but that PFKFB4 expression was significantly higher in the tumor tissues of oral cancer patients. High PFKFB3 and PFKFB4 expression had different effects on the prognosis of oral cancer patients with different clinicopathological outcomes. Our data showed that PFKFB3 and PFKFB4 play different roles; PFKFB3 is involved in cell viability, G2/M cell cycle progression, invasion, and migration, whereas PFKFB4 is involved in the drug resistance and cancer stemness of OSCC cells. Furthermore, oral cancer patients with co-expressions of PFKFB3/cell cycle or EMT markers and PFKFB4/stemness markers had poor prognosis.ConclusionsPFKFB3 and PFKFB4 play different biological roles in OSCC cells, which implying that they might be potential prognostic biomarkers for OSCC patients with certain clinicopathological outcomes.

Cortex-driven cytoplasmic flows in elongated cells: fluid mechanics and application to nuclear transport in Drosophila embryos.

The Drosophila melanogaster embryo, an elongated multi-nucleated cell, is a classical model system for eukaryotic development and morphogenesis. Recent work has shown that bulk cytoplasmic flows, driven by cortical contractions along the walls of the embryo, enable the uniform spreading of nuclei along the anterior-posterior axis necessary for proper embryonic development. Here, we propose two mathematical models to characterize cytoplasmic flows driven by tangential cortical contractions in elongated cells. Assuming Newtonian fluid flow at low Reynolds number in a spheroidal cell, we first compute the flow field exactly, thereby bypassing the need for numerical computations. We then apply our results to recent experiments on nuclear transport in cell cycles 4-6 of Drosophila embryo development. By fitting the cortical contractions in our model to measurements, we reveal that experimental cortical flows enable near-optimal axial spreading of nuclei. A second mathematical approach, applicable to general elongated cell geometries, exploits a long-wavelength approximation to produce an even simpler solution, with errors below [Formula: see text] compared with the full model. An application of this long-wavelength result to transport leads to fully analytical solutions for the nuclear concentration that capture the essential physics of the system, including optimal axial spreading of nuclei.

3D microperfusion of mesoscale human microphysiological liver models improves functionality and recapitulates hepatic zonation.

Hepatic in vitro models that accurately replicate phenotypes and functionality of the human liver are needed for applications in toxicology, pharmacology and biomedicine. Notably, it has become clear that liver function can only be sustained in 3D culture systems at physiologically relevant cell densities. Additionally, drug metabolism and drug-induced cellular toxicity often follow distinct spatial micropatterns of the metabolic zones in the liver acinus, calling for models that capture this zonation. We demonstrate the manufacture of accurate liver microphysiological systems (MPS) via engineering of 3D stereolithography printed hydrogel chips with arrays of diffusion open synthetic vasculature channels at spacings approaching in vivo capillary distances. Chip designs are compatible with seeding of cell suspensions or preformed liver cell spheroids. Importantly, primary human hepatocytes (PHH) and hiPSC-derived hepatocyte-like cells remain viable, exhibit improved molecular phenotypes compared to isogenic monolayer and static spheroid cultures and form interconnected tissue structures over the course of multiple weeks in perfused culture. 3D optical oxygen mapping of embedded sensor beads shows that the liver MPS recapitulates oxygen gradients found in the acini, which translates into zone-specific acet-ami-no-phen toxicity patterns. Zonation, here naturally generated by high cell densities and associated oxygen and nutrient utilization along the flow path, is also documented by spatial proteomics showing increased concentration of periportal- versus perivenous-associated proteins at the inlet region and vice versa at the outlet region. The presented microperfused liver MPS provides a promising platform for the mesoscale culture of human liver cells at phenotypically relevant densities and oxygen exposures. STATEMENT OF SIGNIFICANCE: A full 3D tissue culture platform is presented, enabled by massively parallel arrays of high-resolution 3D printed microperfusion hydrogel channels that functionally mimics tissue vasculature. The platform supports long-term culture of liver models with dimensions of several millimeters at physiologically relevant cell densities, which is difficult to achieve with other methods. Human liver models are generated from seeded primary human hepatocytes (PHHs) cultured for two weeks, and from seeded spheroids of hiPSC-derived human liver-like cells cultured for two months. Both model types show improved functionality over state-of-the-art 3D spheroid suspensions cultured in parallel. The platform can generate physiologically relevant oxygen gradients driven by consumption rather than supply, which was validated by visualization of embedded oxygen-sensitive microbeads, which is exploited to demonstrate zonation-specific toxicity in PHH liver models.

Light-responsive smart nanocarriers for wirelessly controlled photodynamic therapy for prostate cancers.

Photodynamic therapy (PDT) is an effective non-invasive or minimally invasive treatment method against different tumors. Loading photosensitizers in nanocarriers can potentially increase their accumulation in tumor sites. However, the PDT efficacy may be hindered because of self-quenching of the encapsulated photosensitizer and the small diffusion radii of the generated reactive oxygen species (ROS). Herein, light responsive nano assemblies composed of (Polyethylene glycol)-block-poly(4,5-dimethoxy-2-nitrobenzylmethacrylate) (PEG-b-PNBMA) were designed and loaded with the photosensitizer, Rose Bengal lactone (RB), to act as a smart nanocarrier (RB-M) for the delivery of the photosensitizer. A wirelessly activated light-emitting diode (LED) implant was designed to programmatically induce the release of the loaded RB and activate PDT after diffusion of RB into the cytoplasm. The results showed that sequential '405-580 nm' irradiation of the RB-M treated 22RV1 cells resulted in the highest PDT outcome among different irradiation protocols. The combination of this smart nanocarrier and sequential '405-580 nm' irradiation strategy exhibited good PDT efficacy against 2D 22RV1 prostate cancer cells as well as 3D cancer cell spheroids. This platform overcome the light penetration limitations in PDT, and can potentially be applied in cancer bearing patients who are unfit for chemotherapy. STATEMENT OF SIGNIFICANCE: Nanocarriers for the delivery of photosensitizer in photodynamic therapy may result in relatively low therapeutic efficacy because of self-quenching of the encapsulated photosensitizer and the small diffusion radii of the generated reactive oxygen species (ROS). Light responsive smart nanocarriers can potentially overcome this challenge. In this study, a light responsive polymer (Polyethylene glycol)-block-poly(4,5-dimethoxy-2-nitrobenzylmethacrylate) (PEG-b-PNBMA) was synthesized and utilized to fabricate the smart nanocarrier. A wirelessly activated light-emitting diode (LED) implant was designed for light delivery in deep tissue. This new approach permits wirelessly and programmatically control of photosensitizer release and PDT activation under deep tissue, thus significantly enhancing PDT efficacy against prostate cancer cells as well as 3D cancer cell spheroids. This design should have a significant impact on controllable PDT under deep tissue.

Tissue-specific micropattern array chips fabricated via decellularized ECM for 3D cell culture

Multicellular three-dimensional (3D) in vitro models, such as cell spheroids and organoids, can significantly improve the viability, histomorphology, genotype stability, function and drug metabolism of cells [1–3]. In general, several culture methods of 3D models, including the hanging drop, microwell-mesh and hydrogel encapsulating methods, have difficulty building a standard mode and controlling the size and arrangement of cell spheroids or organoids, which could severely affect the authenticity and repeatability of experimental results [4]. Another key factor in 3D in vitro models is the extracellular matrix (ECM), which can determine cell viability, proliferation, differentiation, function, migration and organization [5]. In this study, micropattern array chips combined with decellularized ECM (dECM) not only provide tissue-specific ECM but also control the size and arrangement of 3D models.•Methods have been established to demonstrate the use of dECM as a bioink to generate dECM-coated micropattern array chips by microcontact printing.•The micropattern can limit cell growth and migration, and cells spontaneously assemble into cell spheroids with uniform size and orderly arrangement.

IL-6 Reduces Spheroid Sizes of Osteophytic Cells Derived from Osteoarthritis Knee Joint via Induction of Apoptosis

Osteophytes in osteoarthritis (OA) joints contribute to restriction of joint movement, joint pain, and OA progression, but little is known about osteophyte regulators. Examination of gene expression related to cartilage extracellular matrix, endochondral ossification, and growth factor signaling in articular cartilage and osteophytes obtained from OA knee joints showed that several genes such as COL1A1, VCAN, BGLAP, BMP8B, RUNX2, and SOST were overexpressed in osteophytes compared with articular cartilage. Ratios of mesenchymal stem/progenitor cells, which were characterized by co-expression of CD105 and CD166, were significantly higher in osteophytic cells than articular cells. A three-dimensional culture method for cartilage and osteophyte cells was developed by modification of cultures of self-assembled spheroid cell organoids (spheroids). These spheroids cultured in the media for mesenchymal stem cells containing transforming growth factor-β3 showed characteristic morphologies and gene expression profiles of articular cartilage and osteophytes, respectively. The effects of IL-1β, tumor necrosis factor-α, and IL-6 on the spheroids of articular and osteophytic cells were studied. To the best of our knowledge, they provide the first evidence that IL-6 suppresses the spheroid size of osteophytic cells by inducing apoptosis and reducing extracellular matrix molecules. These data show that IL-6 is the suppressor of osteophyte growth and suggest that IL-6 expression and/or activity are implicated in the regulation of osteophyte formation in pathologic joints.