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.

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial–mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

Similar to stem cells, naïve T cells undergo asymmetric division following activation. While asymmetric division of T cells has been shown to be an important mechanism for the generation of lymphocyte fate diversity during immune responses, key factors that influence whether T cells will undergo symmetric or asymmetric divisions are not completely understood. Here, we utilized immunological synapse arrays (ISAs) to begin to dissect mechanisms of asymmetric T lymphocyte division. ISAs are protein micropatterned surfaces composed of two segregated regions, activation sites and adhesion fields. Activation sites are small spots presenting activation signals such as anti-CD3 and anti-CD28, and adhesion fields are the remaining regions surrounding activation sites immobilized with interintercel adhesion molecule 1 (ICAM-1). By varying the size and the distance between the activation sites and measuring the incidence of asymmetric cell divisions, we found that the distance between activation sites is an important regulator of asymmetric division. Further analysis revealed that more symmetric divisions occurred when two nascent daughter cells stably interacted with two distinct activation sites throughout and following cytokinesis. In contrast, more asymmetric divisions occurred when only one daughter cell remained anchored on an activation site while the other daughter became motile and moved away following cytokinesis. Together, these results indicate that TCR signaling events during cytokinesis may repolarize key molecules for asymmetric partitioning, suggesting the possibility that the density of antigen presenting cells that interact with T cells as they undergo cytokinesis may be a critical factor regulating asymmetric division in T cells.

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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.