Dynamic Micropatterning of Cells on Nanostructured Surfaces Using a Cell-friendly Photoresist.

Cellular dynamics under complex topographical microenvironments are important for many biological processes in development and diseases, but systematic investigation has been limited due to the lack of technology. Herein, we developed a new dynamic cell patterning method based on a cell-friendly photoresist polymer that allows in situ control of cell dynamics on nanostructured surfaces. Using this method, we quantitatively compared the spreading dynamics of cells on nanostructured surfaces to those on flat surfaces. Furthermore, we investigated how cells behaved when they simultaneously encountered two topographically distinct surfaces during spreading. This method will allow many exciting opportunities in the fundamental study of cellular dynamics.

Cholesterol selectively activates canonical Wnt signalling over non-canonical Wnt signalling.

Wnt proteins control diverse biological processes through ß-catenin-dependent canonical signalling and ß-catenin-independent non-canonical signalling. The mechanisms by which these signalling pathways are differentially triggered and controlled are not fully understood. Dishevelled (Dvl) is a scaffold protein that serves as the branch point of these pathways. Here, we show that cholesterol selectively activates canonical Wnt signalling over non-canonical signalling under physiological conditions by specifically facilitating the membrane recruitment of the PDZ domain of Dvl and its interaction with other proteins. Single-molecule imaging analysis shows that cholesterol is enriched around the Wnt-activated Frizzled and low-density lipoprotein receptor-related protein 5/6 receptors and plays an essential role for Dvl-mediated formation and maintenance of the canonical Wnt signalling complex. Collectively, our results suggest a new regulatory role of cholesterol in Wnt signalling and a potential link between cellular cholesterol levels and the balance between canonical and non-canonical Wnt signalling activities.

A comparative study on collagen type I and hyaluronic acid dependent cell behavior for osteochondral tissue bioprinting.

Bioprinting is a promising technique for engineering composite tissues, such as osteochondral tissues. In this study, as a first step toward bioprinting-based osteochondral tissue regeneration, we systematically examined the behavior of chondrocytes and osteoblasts to hyaluronic acid (HA) and type I collagen (Col-1) hydrogels. First, we demonstrated that cells on hydrogels that were comprised of major native tissue extracellular matrix (ECM) components (i.e. chondrocytes on HA hydrogels and osteoblasts on Col-1 hydrogels) exhibited better proliferation and cell function than cells on non-native ECM hydrogels (i.e., chondrocytes on Col-1 hydrogels and osteoblasts on HA hydrogels). In addition, cells located near their native ECM hydrogels migrated towards them. Finally, we bioprinted three-dimensional (3D) osteochondral tissue-mimetic structures composed of two compartments, osteoblast-encapsulated Col-1 hydrogels and chondrocyte-encapsulated HA hydrogels, and found viability and functions of each cell type were well maintained within the 3D structures up to 14 days in vitro. These results suggest that with proper choice of hydrogel materials, bioprinting-based approaches can be successfully applied for osteochondral tissue regeneration.

T cells sense biophysical cues using lamellipodia and filopodia to optimize intraluminal path finding.

Intraluminal crawling is considered to be important for extravasation of leukocytes in blood vessels, but biochemical/biophysical cues guiding the crawling of leukocytes have not been clearly understood. Here we provide evidence that T cells sense the topography of luminal surfaces and the nuclei of endothelial cells (ECs) using lamellipodia and filopodia, respectively, to optimize path finding during intraluminal crawling. Well-aligned EC layers or replicas of EC layers, which exhibit topography similar to that of EC layers, were fabricated, and flow was applied either parallel or perpendicular to the orientation of EC alignment. T cells crawled along the valleys of the topographical landscapes of the EC layers, while avoiding nuclei of ECs regardless of flow direction. Pharmacological inhibitor treatments revealed that sensing of topography and nuclei of EC layers was mediated by lamellipodia and filopodia, respectively. Lamellipodia or filopodia-inhibited T cells crawled significantly longer distances for extravasation than did normal T cells, indicating that sensing biophysical cues are critical for optimizing routes for extravasation.

Photopatterning with a printed transparency mask and a protein-friendly photoresist.

Microscope projection photolithography (MPP) based on a protein-friendly photoresist is a versatile tool for the fabrication of protein- and cell-micropatterned surfaces. Photomasks containing various features can be economically produced by printing features on transparency films. Features in photomasks are projected by the objective lens of a microscope, resulting in a significant reduction of the feature size to as small as ~1 µm, close to the practical limit of light-based microfabrication. A fluorescence microscope used in most biology labs can be used for the fabrication process with some modifications. Using such a microscope, multistep MPP can be readily performed with precise registration of each micropattern on transparency film masks. Here, we describe methods of the synthesis and characterization of a protein-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) and the setups of fluorescence microscopes and the MPP procedures. In addition, we describe the protocols used in the micropatterning of multiple lymphocytes and the dynamic micropatterning of adherent cells.

Negative role of inducible PD-1 on survival of activated dendritic cells.

PD-1 is a well-established negative regulator of T cell responses by inhibiting proliferation and cytokine production of T cells via interaction with its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), expressed on non-T cells. Recently, PD-1 was found to be expressed in innate cells, including activated DCs, and plays roles in suppressing production of inflammatory cytokines. In this study, we demonstrate that PD-1 KO DCs exhibited prolonged longevity compared with WT DCs in the dLNs after transfer of DCs into hind footpads. Interestingly, upon LPS stimulation, WT DCs increased the expression of PD-1 and started to undergo apoptosis. DCs, in spleen of LPS-injected PD-1 KO mice, were more resistant to LPS-mediated apoptosis in vivo than WT controls. Moreover, treatment of blocking anti-PD-1 mAb during DC maturation resulted in enhanced DC survival, suggesting that PD-1:PD-L interactions are involved in DC apoptosis. As a result, PD-1-deficient DCs augmented T cell responses in terms of antigen-specific IFN-γ production and proliferation of CD4 and CD8 T cells to a greater degree than WT DCs. Moreover, PD-1 KO DCs exhibited increased MAPK1 and CD40-CD40L signaling, suggesting a possible mechanism for enhanced DC survival in the absence of PD-1 expression. Taken together, our findings further extend the function of PD-1, which plays an important role in apoptosis of activated DCs and provides important implications for PD-1-mediated immune regulation.

Dynamic modulation of small-sized multicellular clusters using a cell-friendly photoresist.

Dynamics of small-sized multicellular clusters is important for many biological processes including embryonic development and cancer metastasis. Previous methods to fabricate multicellular clusters depended on stochastic adhesion and proliferation of cells on defined areas of cell-adhering islands. This made precise control over the number of cells within multicellular clusters impossible. Variation in numbers may have minimal effects on the behavior of multicellular clusters composed of tens of cells but would have profound effects on groups with fewer than ten cells. Herein, we report a new dynamic cell micropatterning method using a cell-friendly photoresist film by multistep microscope projection photolithography. We first fabricated single cell arrays of partially spread cells. Then, by merging neighboring cells, we successfully fabricated multicellular clusters with precisely controlled number, composition, and geometry. Using this method, we generated multicellular clusters of Madin-Darby canine kidney cells with various numbers and initial geometries. Then, we systematically investigated the effect of multicellular cluster sizes and geometries on their motility behaviors. We found that the behavior of small-sized multicellular clusters was not sensitive to initial configurations but instead was determined by dynamic force balances among the cells. Initially, the multicellular clusters exhibited a rounded morphology and minimal translocation, probably due to contractility at the periphery of the clusters. For 2-cell and 4-cell clusters, single leaders emerged over time and entire groups aligned and comigrated as single supercells. Such coherent behavior did not occur in 8-cell clusters, indicating a critical group size led by a single leader may exist. The method developed in the study will be useful for the study of collective migration and multicellular dynamics.

Microfabricated platforms to modulate and monitor T cell synapse assembly.

Immunological synapse (IS) is a complex supramolecular structure formed at the interface between T cells and antigen presenting cells (APCs) during T cell antigen recognition. Microfabricated platforms have made great contributions to our understanding of the assembly dynamics and functional roles of the T cell synapses over the last decade. Here, we review three different types of microfabricated platforms developed to modulate and monitor the T cell synapse assembly. Firstly, multi-protein micropatterned surfaces presenting key ligands for T cell activation that can modulate the spatial distribution of receptors/signaling molecules in T cell synapses are described. Secondly, micropatterned supported bilayers that can modulate the dynamics of T cell receptor (TCR) microclusters are introduced. Lastly, T-APC pair arrays that allow for improved fluorescence live cell imaging are discussed.

High-throughput quantitative imaging of cell spreading dynamics by multi-step microscopy projection photolithography based on a cell-friendly photoresist.

A new method for the high-throughput study of cell spreading dynamics is devised by multi-step microscopy projection photolithography based on a cell-friendly photoresist. By releasing a large number of rounded cells in single cell arrays and monitoring their spreading dynamics by interference reflection microscopy, a large number of cell spreading data can be acquired by a single experiment.

Nanotopography-guided migration of T cells.

T cells navigate a wide variety of tissues and organs for immune surveillance and effector functions. Although nanoscale topographical structures of extracellular matrices and stromal/endothelial cell surfaces in local tissues may guide the migration of T cells, there has been little opportunity to study how nanoscale topographical features affect T cell migration. In this study, we systematically investigated mechanisms of nanotopography-guided migration of T cells using nanoscale ridge/groove surfaces. The velocity and directionality of T cells on these nanostructured surfaces were quantitatively assessed with and without confinement, which is a key property of three-dimensional interstitial tissue spaces for leukocyte motility. Depending on the confinement, T cells exhibited different mechanisms for nanotopography-guided migration. Without confinement, actin polymerization-driven leading edge protrusion was guided toward the direction of nanogrooves via integrin-mediated adhesion. In contrast, T cells under confinement appeared to migrate along the direction of nanogrooves purely by mechanical effects, and integrin-mediated adhesion was dispensable. Therefore, surface nanotopography may play a prominent role in generating migratory patterns for T cells. Because the majority of cells in periphery migrate along the topography of extracellular matrices with much lower motility than T cells, nanotopography-guided migration of T cells would be an important strategy to efficiently perform cell-mediated immune responses by increasing chances of encountering other cells within a given amount of time.

Multiscale fabrication of multiple proteins and topographical structures by combining capillary force lithography and microscope projection photolithography.

We present new methods that enable the fabrication of multiscale, multicomponent protein-patterned surfaces and multiscale topologically structured surfaces by exploiting the merits of two well-established techniques: capillary force lithography (CFL) and microscope projection photolithography (MPP) based on a protein-friendly photoresist. We further demonstrate that, when hierarchically organized micro- and nanostructures were used as a cell culture platform, human colon cancer cells (cell line SW480) preferentially adhere and migrate onto the area with nanoscale topography over the one with microscale topography. These methods will provide many exciting opportunities for the study of cellular responses to multiscale physicochemical cues.

Addressable micropatterning of multiple proteins and cells by microscope projection photolithography based on a protein friendly photoresist.

We report a new method for the micropatterning of multiple proteins and cells with micrometer-scale precision. Microscope projection photolithography based on a new protein-friendly photoresist, poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP), was used for the fabrication of multicomponent protein/cell arrays. Microscope projection lithography allows precise registration between multiple patterns as well as facile fabrication of microscale features. Thin films of PDMP became soluble in near-neutral physiological buffer solutions upon UV exposure and exhibited excellent resistance to protein adsorption and cell adhesion. By harnessing advantages in microscope projection photolithography and properties of PDMP thin films, we could successfully fabricate protein arrays composed of multiple proteins. Furthermore, we could extend this method for the patterning of two different types of immune cells for the potential study of immune cell interactions. This technique will in general be useful for protein chip fabrication and high-throughput cell-cell communication study.

Radiofrequency ablation of renal VX2 tumors with and without renal artery occlusion in a rabbit model: feasibility, therapeutic efficacy, and safety.

The purpose of this study was to determine the therapeutic efficacy of radiofrequency ablation for treating renal VX2 tumors with and without renal artery occlusion in a rabbit model. Twenty-four New Zealand White rabbits were percutaneously implanted with renal VX2 tumors. Fifteen days after implantation, both kidneys were surgically exposed, and radiofrequency ablation was conducted. Group A (n=12) was treated with renal artery occlusion, and group B (n=12) was treated without occlusion. In each rabbit, the serum creatinine was measured to evaluate renal damage after arterial occlusion. Two days after radiofrequency ablation, computed tomography was performed to evaluate the difference in therapeutic results between the two groups. We also compared histopathologic findings after radiofrequency ablation. The mean tumor size was 2.4 cm (range, 1.2-3.1 cm). Radiofrequency ablation of renal tumors was technically feasible in all cases. Complete ablation was achieved in 11 of the 12 rabbits (92%) in group A but in only eight of the 12 rabbits (67%) in group B (P<0.05). The average time of radiofrequency application was shorter in group A (mean, 547 s) than in group B (mean, 826 s) (P<0.05). After radiofrequency ablation, the serum creatinine increased from 1.54 to 1.82 mg/dl in group A and from 1.46 to 1.78 mg/dl in group B. However, there was no significant difference between the two groups (P>0.05). In conclusion, radiofrequency ablation with renal artery occlusion can decrease the duration of treatment and increase the therapeutic efficacy for renal VX2 tumors.

Discrete hypoechoic ring in hepatic cavernous hemangioma resembling a malignant tumor: correlation with histologic features.

Differential diagnoses of hepatic nodules include hepatocellular carcinoma, focal nodular hyperplasia, hepatic adenoma, regenerative nodule, focal fatty changes, and hemangioma. However, differentiation of these nodules can often be difficult. Hemangiomas are frequently encountered during ultrasonogram incidentally and can be diagnosed easily because they have an almost distinctive sonographic appearance: a homogeneous hyperechogenicity and discrete posterior acoustic enhancement. They also sometimes have atypical findings, for example an internal echogenicity including hypoechogenicity, heterogeneous echogenicity, hyperechoic rim, central hypoechogenicity due to various changes (e.g., internal hemorrhage, necrosis, thrombosis, myxomatous change, and fibrosis), and (rarely) calcification. We report herein the case of an atypical hemangioma presenting with a hypoechoic peripheral ring, mimicking a hepatic malignancy. To our knowledge, there have been no other reports demonstrating a cavernous hemangioma with a discrete hypoechoic ring and without a pseudocapsule.

Benign submucosal lesions of the stomach and duodenum: imaging characteristics with endoscopic and pathologic correlation.

Benign submucosal lesions of the stomach and duodenum are occasionally encountered during endoscopy. But endoscopy has its limitations in the diagnosis and differentiation of these lesions, because submucosal lesions are often difficult to visualize at endoscopy due to minimal change of the overlying mucosa. Furthermore, endoscopic biopsy may not always yield adequate tissue for diagnosis due to the submucosal location of the lesions. For this reason, the role of radiologic imaging is important in the diagnosis of submucosal lesions of the stomach and duodenum. Recent advances in computed tomography (CT) and sonographic technology are helpful in narrowing the differential diagnosis of gastroduodenal submucosal lesions. In contrast to endoscopy and barium studies, CT or ultrasonography (US) provides information about both the gastric wall and the extragastric extent of the disease. Arterial phase contrast enhanced CT enables us to discriminate a mass of submucosal from that of a mucosal origin in the differential diagnosis of gastric or duodenal lesions. Although endoscopic sonography has been considered the better modality in the diagnosis of gastroduodenal submucosal lesions, transabdominal sonography can still be an alternative method to endoscopic sonography in assessing of the origin and character of the submucosal lesions. Some gastroduodenal submucosal lesions have similar radiologic findings that make differentiation difficult. But despite overlaps in radiologic findings, some lesions have characteristic radiologic features that may suggest a specific diagnosis. Knowledge of the differential diagnosis of benign submucosal lesions in the stomach and duodenum may promote correct diagnosis and appropriate treatment.

[Evaluation of predictive value of Okuda, TNM, CLIP and JIS staging systems for hepatocellular carcinoma patients].

BACKGROUND/AIMS: The aims of this study were to validate the prognostic value of the JIS score for HCC and to compare discriminatory ability and predictive power with other staging systems such as Okuda, TNM and CLIP. METHODS: We analyzed the clinical records of 210 patients who were diagnosed as HCC from 2000 to 2002. Univariate and multivariate survival analyses were done to find out factors to affect survival. To validate prognostic value of those staging systems, survival curve was obtained and analyzed by the Kaplan-Meier's method, and to compare discriminatory ability and predictive power, Homogeneity LR X(2) test and AIC score were used. RESULTS: The median survival was 19.5 months (19.1+/-14.9). The number of patients and 3-year survival rate for those staging systems were Okuda 1(126, 57.7%), 2(63, 9.0%) and 3(21, 0.0%) (p<0.001); TNM I (34, 63.1%), II (71, 59.4%), III (50, 22.4%), IV-A (6, 14.3%) and IV-B (1, 6.5%) (p<0.001); CLIP 0 (79, 68.5%), 1 (39, 34.2%), 2 (36, 16.7%), 3 (25, 20.0%), 4 (18, 5.1%), 5 (9, 11.1%) and 6 (4, 0.0%) (p<0.001) and JIS 0 (26, 78.9%), 1 (65, 65.3%), 2 (43, 21.9%), 3 (40, 25, 8.0%) and 5 (11, 2.0%)(p<0.001) in univariate analysis using Kaplan-Meier analysis. Homogeneity LR X(2) test showed more stratification power in JIS (Okuda, 102.8; TNM, 128.2; CLIP, 148.4 and JIS, 185.6) and AIC score showed superior predictive power in JIS system (Okuda, 1228.5; TNM, 1130.3; CLIP, 1117.1 and JIS, 1093.6). CONCLUSIONS: The proposed JIS system is useful system to predict survival of HCC patients. The discriminate ability of the JIS score is much better than other staging systems and has better prognostic predictive power compared to other staging systems.

[Prognostic factors for survival in patients with hepatocellular carcinoma after radiofrequency ablation].

BACKGROUND/AIMS: Currently there is no consensus on which staging system is the best in predicting the survival of patients with hepatocellular carcinoma (HCC). The aim of this study was to identify independent factors to predict survival and to compare 4 available prognostic staging systems in patients with early HCC after radiofrequency ablation. METHODS: We retrospectively studied 100 Korean patients with early HCC. Prognostic factors for survival were analysed by univariate and multivariate analysis using the Kaplan-Meier method and Cox proportional hazard regression models. Okuda, Cancer of the Liver Italian Program (CLIP), TNM and Japanese integrated staging score (JIS score) were evaluated before the treatments. RESULTS: Overall survival rates of 12, 24 and 36 months were 89%, 76%, and 64% respectively and the mean survival duration was 45 months. Multivariable analysis showed that albumin, total bilirubin and size of tumor were independent prognostic factors. Multivariate analysis showed that TNM and JIS score staging systems were significant staging systems for the prediction of prognosis. CONCLUSIONS: Both TNM and JIS score are more effective than the Okuda and CLIP staging systems in stratifying patients into different risk groups with early HCC. However, JIS score gives better prediction of prognosis in patients with HCC after radiofrequency ablation.