Hironori Yamazoe

Facile cell patterning on an albumin-coated surface.

Fabrication of micropatterned surfaces to organize and control cell adhesion and proliferation is an indispensable technique for cell-based technologies. Although several successful strategies for creating cellular micropatterns on substrates have been demonstrated, a complex multistep process and requirements for special and expensive equipment or materials limit their prevalence as a general experimental tool. To circumvent these problems, we describe here a novel facile fabrication method for a micropatterned surface for cell patterning by utilizing the UV-induced conversion of the cell adhesive property of albumin, which is the most abundant protein in blood plasma. An albumin-coated surface was prepared by cross-linking albumin with ethylene glycol diglycidyl ether and subsequent casting of the cross-linked albumin solution on the cell culture dish. While cells did not attach to the albumin surface prepared in this way, UV exposure renders the surface cell-adhesive. Thus, surface micropatterning was achieved simply by exposing the albumin-coated surface to UV light through a mask with the desired pattern. Mouse fibroblast L929 cells were inoculated on the patterned albumin substrates, and cells attached and spread in a highly selective manner according to the UV-irradiated pattern. Although detailed investigation of the molecular-level mechanism concerning the change in cell adhesiveness of the albumin-coated surface is required, the present results would give a novel facile method for the fabrication of cell micropatterned surfaces.

Cell micropatterning on an albumin‐based substrate using an inkjet printing technique

Positioning of cells in a desired pattern on a substrate is an important technique for cell-based technologies, including the fundamental investigation of cell functions, tissue-engineering applications, and the fabrication of cell-based biosensors and cell arrays. Recently, the inkjet printing technique was recognized as a promising approach to the creation of cellular patterns on substrates, and it has been achieved by the printing of living cells or cell adhesive proteins. In this article, we created complex cellular patterns by using an albumin-based substrate and inkjet printing technique. Albumin was cross-linked using ethylene glycol diglycidyl ether. Subsequent casting of the cross-linked albumin solution onto glass plates prevented cells from adhering to their surfaces. Through screening various chemical reagents, we found that these cross-linked albumin surfaces dramatically changed into cell adhesive surfaces after immersion in cationic polymer solutions. Based on this finding, cell adhesive regions were prepared with a desired pattern by printing the polyethyleneimine (PEI) solution onto a cross-linked albumin substrate using a modified commercial inkjet printer. Various cellular patterns including figures, letters, and gradients could be fabricated by seeding mouse L929 fibroblast cells or mouse Neuro-2a neuroblastoma cells onto the printed PEI-patterned substrate. Compared with the printing of fragile living cells or proteins, printing of stable PEI circumvents clogging of printer head nozzles and enables reproducible printing. Therefore, the present method will allow the creation of complex cell patterns.

Preparation of arrays of cell spheroids and spheroid-monolayer cocultures within a microfluidic device

This study describes a novel method for generation of an array of three-dimensional (3D) multicellular spheroids within a microchannel in patterned cultures containing one or multiple cell types. This method uses a unique property of a cross-linked albumin coated surface in which the surface can be switched from non-adhesive to cell adhesive upon electrostatic adsorption of a polycation. Introduction of a solution containing albumin and a cross-linking agent into a microchannel with an array of microwells caused the entire surface, with the exception of the interior of the microwells, to become coated with the cross-linked albumin layer. Cells that were seeded within the microchannel did not adhere to the surface of the microchannel and became entrapped in the microwells. HepG2 cells seeded in the microwells formed 3D spheroids with controlled sizes and shapes depending upon the dimensions of the microwells. When the albumin coated surface was subsequently exposed to an aqueous solution containing poly(ethyleneimine) (PEI), adhesion of secondary cells, fibroblasts, occurred in the regions surrounding the arrayed spheroids. This coculture system can be coupled with spatially controlled fluids such as gradients and focused flow generators for various biological and tissue engineering applications.

Preparation of coculture system with three extracellular matrices using capillary force lithography and layer-by-layer deposition.

Micropatterned cocultures were fabricated with 3 extracellular matrices, hyaluronic acid (HA), fibronectin, and collagen. The feature of the fabrication processes is to avoid the use of potentially cytotoxic materials and utilize capillary force of the solution and interactions between the extracellular matrix components. The coculture system can be used to investigate the effects of heterocellular interactions on cellular fate. Direct heterocellular connections between hepatocytes and fibroblasts were visualized by the transcellular diffusion of fluorescein in this coculture system. The interactions between hepatocytes and fibroblasts were crucial for the maintenance of albumin synthesis by hepatocytes. The coculture system was also beneficial for investigating the effects of cell-cell interactions on the induction of embryonic stem (ES) cell differentiation. In cocultures grown in a sea-island pattern, ES cells formed isolated colonies surrounded by PA6 cells and differentiated into neurons with branched neurites that extended from the colonies. This versatile and biocompatible coculture system could potentially be a powerful tool for investigating cell-cell interaction and for tissue engineering applications.

Fabrication of patterned cell co-cultures on albumin-based substrate: applications for microfluidic devices.

A surface coated with cross-linked albumin film resists the adhesion of cells, and subsequent exposure to UV irradiation or electrostatic adsorption of a cationic polymer switches the surface from non-adherent to adherent. Taking advantage of this unique property of cross-linked albumin, the authors fabricated patterned cell co-cultures with desired patterns and cell types. In this scheme, the cell-adherent region was initially created in the cell-non-adhesive albumin substrate, on which a first cell type was attached. Subsequently, the remaining region was also changed to adherent for the attachment of secondary cells in the same manner, thereby allowing distinctly localized co-cultures. As a proof of concept demonstration of the feasibility of this approach, a patterned co-culture of Neuro-2a cells with L929 cells was successfully prepared on the substrate. Furthermore, combining this technique with a microfluidic technique, a micropatterned co-culture of PA6 cells with 3T3 fibroblasts was created inside microfluidic devices. This approach could potentially be a useful tool for fundamental investigations of cell-cell interactions and for tissue engineering applications.

One-step induction of neurons from mouse embryonic stem cells in serum-free media containing vitamin B12 and heparin.

We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B12 and heparin, but without any expensive cytokines. After 14 days in culture, β-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B12/heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B12/heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B12/heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinsons disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.

Cell micropatterning inside a microchannel and assays under a stable concentration gradient

We describe the use of a microfluidic device to micropattern cells in a microchannel and investigated the behavior of these cells under a concentration gradient. The microfluidic device consisted of 3 parts: a branched channel for generating a stable concentration gradient, a main channel for culturing cells, and 2 side channels that flowed into the main channel. The main channel was coated with a cross-linked albumin that was initially cell-repellent but that could become cell-adherent by electrostatic adsorption of a polycation. A sheath flow stream, which was generated by introducing a polycation solution from the branched channel and a buffer solution from the 2 side channels, was used to change a specific region in the main channel from cell-repellent to cell-adhesive. In this way, cells attached to the central region along the main channel. The remaining surface was subsequently changed to cell-adhesive, thereby facilitating cell migration from a fixed location under a concentration gradient. We demonstrated that with this device, the gradient generator could be used to conduct simultaneous cytotoxic assays with anticancer agents; further, by combining this device with cell micropatterning, migration assays under a concentration gradient of biological factors could be conducted.

Drug-Carrying Albumin Film for Blood-Contacting Biomaterials

Surface-induced thrombosis is a major complication in the development of blood-contacting medical devices. Serum albumin has the ability to bind to a wide variety of compounds, including drugs, and neither cells nor proteins adsorb to an albumin-coated surface. These properties of albumin are useful for improving the blood compatibility of biomaterial surfaces. In the present study, we prepared a water-insoluble film by cross-linking pharmaceutical grade recombinant human serum albumin aiming to the clinical applications, and loaded the film with a synthetic antiplatelet drug, cilostazol. The resultant film possessed native albumin characteristics such as drug binding ability and resistance to cell adhesion. Mouse fibroblast L929 cells did not adhere on the albumin film, just as they did not adhere on native albumin-coated surfaces. Furthermore, when the albumin film carrying cilostazol was placed in PBS containing Tween-80, the release of cilostazol was sustained over 144 h. The results indicate that the surface coating with thus prepared albumin film can confer the biomaterials with antithrombogenic surface by virtue of its non-adhesiveness to cells and its release of cilostazol.

Generation of a patterned co-culture system composed of adherent cells and immobilized nonadherent cells.

UNLABELLED Patterned co-culture is a promising technique used for fundamental investigation of cell-cell communication and tissue engineering approaches. However, conventional methods are inapplicable to nonadherent cells. In this study, we aimed to establish a patterned co-culture system composed of adherent and nonadherent cells. Nonadherent cells were immobilized on a substrate using a cell membrane anchoring reagent conjugated to a protein, in order to incorporate them into the co-culture system. Cross-linked albumin film, which has unique surface properties capable of regulating protein adsorption, was used to control their spatial localization. The utility of our approach was demonstrated through the fabrication of a patterned co-culture consisting of micropatterned neuroblastoma cells surrounded by immobilized myeloid cells. Furthermore, we also created a co-culture system composed of cancer cells and immobilized monocytes. We observed that monocytes enhanced the drug sensitivity of cancer cells and its influence was limited to cancer cells located near the monocytes. Therefore, the incorporation of nonadherent cells into a patterned co-culture system is useful for creating culture systems containing immune cells, as well as investigating the influence of these immune cells on cancer drug sensitivity. STATEMENT OF SIGNIFICANCE Various methods have been proposed for creating patterned co-culture systems, in which multiple cell types are attached to a substrate with a desired pattern. However, conventional methods, including our previous report published in Acta Biomaterialia (2010, 6, 526-533), are unsuitable for nonadherent cells. Here, we developed a novel method that incorporates nonadherent cells into the co-culture system, which allows us to precisely manipulate and study microenvironments containing nonadherent and adherent cells. Using this technique, we demonstrated that monocytes (nonadherent cells) could enhance the drug sensitivity of cancer cells and that their influence had a limited effective range. Thus, our technique is useful for recreating complex tissues in order to investigate cellular interactions involving nonadherent cells.

Fabrication of protein micropatterns using a functional substrate with convertible protein-adsorption surface properties†

A functional substrate capable of regulating protein adsorption was prepared using a crosslinked albumin (cl-albumin) film for use in the fabrication of protein micropatterns. The adsorption of proteins with different characteristics onto cl-albumin film, including serum proteins, serum albumin, and lysozyme, was investigated using a quartz crystal microbalance. The results showed that surfaces coated with cl-albumin film are highly resistant to protein adsorption, regardless of protein charge and rigidity. In addition, this adsorption-resistance property can be easily converted to promote protein adsorption by exposing the cl-albumin film to a charged polymer solution. By combining the convertible surface property of cl-albumin film and inkjet printing techniques, a precise protein micropattern was successfully fabricated on the substrate. Protein adsorption onto the wall surface of microchannels could also be suppressed or promoted by coating the surface with cl-albumin film. This approach will aid in the development of biomaterials carrying protein micropatterns, such as biosensors, biochips, and cellular scaffolds.