Changes in Cell Adhesiveness and Physicochemical Properties of Cross-Linked Albumin Films after Ultraviolet Irradiation.

We discovered the unique cell adhesive properties of ultraviolet (UV)-irradiated albumin films. Albumin films prepared using a cross-linking reagent with epoxy groups maintained native albumin properties, such as resistance to cell adhesion. Interestingly, the cell adhesive properties of films varied depending upon the UV irradiation time; specifically, cell adhesiveness increased until 2 h of UV irradiation, when the cell number attached to the film was similar to that of culture dishes, and then cell adhesiveness decreased until 20 h of UV irradiation, after which the surface returned to the initial non-adhesive state. To elucidate the molecular mechanisms underlying this phenomenon, we examined the effect of UV irradiation on albumin film properties. The following changes occurred in response to UV irradiation: decreased α-helical structure, cleavage of albumin peptide bonds, and increased hydrophilicity and oxygen content of the albumin film surface. In addition, we found a positive correlation between the degree of cell adhesion and the amount of fibronectin adsorbed on the film. Taken together, UV-induced changes in films highly affect the amount of cell adhesion proteins adsorbed on the films depending upon the irradiation time, which determines cell adhesion behavior.

Multifunctional Micromachines Constructed by Combining Multiple Protein-Based Components with Different Functions.

Untethered mobile micromachines have considerable potential to realize more effective and minimally invasive medicine. Although diverse medical micromachines have been reported over the past few decades, these machines were developed for performing only specific tasks and the functions imparted to them were limited to a few. Hence, the methodologies for imparting a wide variety of functions to machines have not been fully explored. In this study, a novel construction strategy for the multifunctional micromachines is presented, where a specific function can be added to the machine in one step by directly combining the protein-based component, possessing the biological function of constituent proteins, to an arbitrary position of the machine by using an inkjet printing technique. As a proof-of-concept demonstration, various types of machines were constructed by combining multiple components with different functions. These constructed machines successfully performed functions as diverse as enzyme-powered self-propulsion, collection of target objects, including the bilirubin and living cells, enzyme-mediated conversion of substrate molecules to different ones, magnetic guidance, and release of anti-inflammatory drug diapocynin. The study's progressive approach as well as multifunctional and biocompatible machines composed of proteins will profoundly impact the development of intelligent machines equipped with multiplex sophisticated functionalities.

Superior Adhesion of a Multifunctional Protein-Based Micropatch to Intestinal Tissue by Harnessing the Hydrophobic Effect.

Drug delivery systems comprising drug carriers capable of adhering to intestinal tissue have considerable potential to realize more sophisticated systemic drug delivery and topical drug treatments in the intestinal tract. The development of innovative strategies for improving the adhesion efficiency of carriers is of high importance for the advancement of this field. Herein, a novel approach to achieving high adhesion efficiency of drug carriers is presented, where the accessibility of the carrier to the intestinal surface and its subsequent adhesion to the intestinal tissue are promoted by utilizing the thermodynamic tendency of the hydrophobic carrier and its dispersion solvent, triacetin, to be excluded from the aqueous environment. Drug carriers are fabricated using proteins, imparting multiple functions, including drug release and the removal of reactive oxygen species (ROS). Results of ex vivo studies indicate that this multifunctional protein-based carrier, "protein micropatch," adheres to various mouse intestinal tissues, including the small intestine, colon, and inflamed colon, with high efficiency. Furthermore, protein micropatches, administered to mice via oral or rectal routes, successfully adhere to the intestinal tract. This approach and the highly functionalized carrier described in the study have the potential to significantly contribute to the development of bioadhesive carrier-based drug delivery systems.

Potential of the Coordinated Actions of Multiple Protein-Based Micromachines for Medical Applications.

Untethered mobile micromachines hold great promise in the development of effective and minimally invasive therapies. Although diverse medical micromachines for specific applications have been developed over the past few decades, the coordinated action of multiple machines with different functions remains largely unexplored. In this study, we created three types of biocompatible micromachines using proteins and demonstrated the potential of their coordinated action for medical applications. As a proof of concept, we demonstrated neural replacement therapy, in which neuroblastomas were killed by using an anticancer prodrug and the first machine that contains enzymes, enabling the conversion of the prodrug into a cytotoxic drug. Subsequently, a second machine composed of extracellular matrix was placed on the dead cancer cells to provide a suitable environment for cell adhesion, on which embryonic stem (ES) cells and stromal cells that promote neural differentiation of stem cells were attached by using third machines capable of delivering cells to target positions with desired patterns. As a result, neuroblastomas were replaced with novel healthy neurons derived from ES cells by teaming multiple protein-based machines. We believe that this work highlights the potential of heterogeneous machine groups for medical treatment and the utility of highly biocompatible and functional micromachines made from proteins, representing an important step forward in building more sophisticated micromachine-based therapies.

Flow analysis on microcasting with degassed polydimethylsiloxane micro-channels for cell patterning with cross-linked albumin.

Patterned cell culturing is one of the most useful techniques for understanding the interaction between geometric conditions surrounding cells and their behaviors. The authors previously proposed a simple method for cell patterning with an agarose gel microstructure fabricated by microcasting with a degassed polydimethylsiloxane (PDMS) mold. Although the vacuum pressure produced from the degassed PDMS can drive a highly viscous agarose solution, the influence of solution viscosity on the casting process is unknown. This study investigated the influences of micro-channel dimensions or solution viscosity on the flow of the solution in a micro-channel of a PDMS mold by both experiments and numerical simulation. It was found experimentally that the degassed PDMS mold was able to drive a solution with a viscosity under 575 mPa·s. A simulation model was developed which can well estimate the flow rate in various dimensions of micro-channels. Cross-linked albumin has low viscosity (1 mPa·s) in aqueous solution and can undergo a one-way dehydration process from solution to solid that produces cellular repellency after dehydration. A microstructure of cross-linked albumin was fabricated on a cell culture dish by the microcasting method. After cells were seeded and cultivated on the cell culture dish with the microstructure for 7 days, the cellular pattern of mouse skeletal myoblast cell line C2C12 was observed. The microcasting with cross-linked albumin solution enables preparation of patterned cell culture systems more quickly in comparison with the previous agarose gel casting, which requires a gelation process before the dehydration process.

Antibody immobilization technique using protein film for high stability and orientation control of the immobilized antibody.

The development of antibody immobilization techniques is essential for creating antibody-based biomaterials. Although numerous methods for antibody immobilization have been demonstrated, low stability and disordered orientation of the immobilized antibody remain an important problem. In this work, an original antibody immobilization technique using a protein film, which achieved a high stability and orientation control of the immobilized antibody, has been described. In this method, an antibody-immobilized albumin film was prepared by adding the cross-linked albumin solution to the substrate, where antibodies were attached in uniform orientation, followed by subsequent drying, and detaching the formed film from the substrate by heating at 120 °C in a dry state. Antibodies in the film showed high antigen-binding capacity, at a level comparable to the oriented immobilized antibody using protein G. The stability of antibodies in the film was found to be significantly high; their antigen-binding capacity was completely retained even after storage at 40 °C in a dry state for one month. Thus, this approach provides useful information to immobilize the antibody on solid surfaces while controlling its orientation and increasing its stability.

Spectroscopic study on the conformation of serum albumin in film state.

Protein is a promising material for fabricating the biocompatible films used in the biomedical fields and food industry. Previously, we successfully prepared a water-insoluble albumin film possessing native albumin properties such as resistance to cell adhesion and drug-binding ability. Here, I quantitatively investigated the conformation of albumin in a film state using circular dichroism (CD) spectroscopy. The albumin film was prepared by crosslinking albumin with ethylene glycol diglycidyl ether (EGDE). CD measurements of albumin films revealed that approximately 70% of the α-helical structure was retained after film formation. Albumin molecules in the films acquired high stability. The conformation of albumin was completely retained even after heating at 100 °C for 1 h. For comparison, crosslinked albumin film was also prepared using glutaraldehyde (GA). Unlike EGDE-crosslinking, GA-crosslinking induced significant conformational changes in albumin; 46% of the α-helical structure was destroyed in GA-crosslinked albumin films. Cell adhesion studies showed that EGDE-crosslinked albumin film maintained the cell-nonadhesive property inherent in native albumin. This property was lost in GA-crosslinked albumin film, and cells adhesion occurred at a level comparable to that of cell culture dishes. These results indicate that EGDE-crosslinking is a useful method for preparing albumin films in which the native albumin structure and property are retained. The approach described here provides valuable information for creating protein films possessing high functionality.

Multifunctional protein microparticles for medical applications.

Micro- and nano-scale intelligent devices can revolutionize the medical field. Although proteins are promising materials for creating biocompatible miniature medical devices with biological functions, construction of complicated solid-state architectures, using inherently vulnerable proteins, remains challenging. Here, I present a sophisticated strategy for constructing a multifunctional microparticle for medical applications using multiple proteins; this strategy achieved the retention of function, increased stability, and orientation control of the proteins in the fabricated particle. As proof-of-concept, the particle, designed to cope with excess reactive oxygen species (ROS) involved in many diseases, was constructed by combining three proteins with different functions. The body of the particle was fabricated using albumin and superoxide dismutase (SOD), and the antibody was incorporated into the surface of the particle in an orientation-controlled manner. The constructed protein microparticle exhibited coordinated activities for coping with ROS, such as capture of the ROS-secreting cells by the incorporated antibody, followed by the elimination of 70% ROS, secreted from the captured cells, by the SOD in the particle. Additionally, diapocynin, loaded to the particle via the drug-binding ability of albumin, was released from the particle, preventing ROS production in the cells. This multifunctional microparticle, constructed from proteins, will profoundly impact the development of intelligent protein-based miniature devices used in medical fields.

Induction of dopamine-releasing cells from primate embryonic stem cells enclosed in agarose microcapsules.

Dopamine-releasing cells derived from embryonic stem cells (ESCs) are potentially valuable in cell transplantation therapy for Parkinson's disease. There have been many recent investigations of the induction of dopamine-releasing cells from mouse and primate ESCs. However, there are major obstacles to application of dopamine-releasing ESC progeny to cell transplantation therapy, including host immune responses to transplanted cells and the difficulty of collecting dopamine-releasing cells from culture dishes undamaged. To overcome these obstacles, in the present study, cynomolgus monkey ES cell (cESC) aggregates enclosed in agarose microcapsules were cultured in 3 kinds of media: Glasgow minimum essential medium-based medium (GBM); GBM-containing conditioned medium of PA6 cells; and GBM supplemented with fibroblast growth factor (FGF)8, sonic hedgehog, and ascorbic acid (GBM(+)) under free-floating culture conditions. Of these 3 culture media, GBM(+) most efficiently induced dopamine-releasing cells. Addition of FGF8, sonic hedgehog, and ascorbic acid to the culture medium during culture days 10 to 15, days 12 to 15, and days 16 to 20, respectively, facilitated the generation of dopamine-releasing cells. Because various characteristics of cESCs are reported to be similar to those of human ESCs, we expect that the study using cESCs will provide useful information for cell transplantation therapy of Parkinson's disease.

Efficient generation of dopaminergic neurons from mouse embryonic stem cells enclosed in hollow fibers.

Transplantation of dopamine neurons is a promising approach to treat Parkinson's disease. Embryonic stem (ES) cells are expected to be a cell source of the dopaminergic neurons. Various difficulties, however, need to be overcome to realize cell therapy of Parkinson's disease using dopaminergic neurons from ES cells. For example, they are highly sensitive to enzymatic treatment and physical dissociation, and the patient's immune system may attack the transplanted cells. In this study, we attempted to induce dopaminergic neurons from mouse ES cells enclosed in hollow fibers using conditioning medium from PA6 cells, the stromal cells derived from skull bone marrow. beta-tubulin type III positive cells and tyrosine hydroxylase positive cells were efficiently derived in hollow fibers after 16 days in culture, and dopamine release was observed when the hollow fibers containing cells were exposed to 56mm KCl for 15min to induce dopamine release through depolarization of the neurons. By our procedure, enclosure of dopaminergic neurons in hollow fibers was easily performed without loss of cells, and the hollow fiber membrane is expected to efficiently protect dopaminergic neurons from mechanical disturbances and attacks by the host immune system. Although there are many issues, especially related to immuno-isolation, that still remain to be addressed, we believe that differentiation of ES cells within hollow fibers is one of the crucial procedures so that cell therapy of Parkinson's disease can be realized.

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, beta-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 1.5-fold 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 Parkinson's 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.

Induction dopamine releasing cells from mouse embryonic stem cells and their long-term culture.

Cell transplantation therapy using dopaminergic neurons derived from embryonic stem (ES) cells for the treatment of Parkinson's disease has been proposed as one of the major applications for stem cell-based therapy. However, the low collection efficiency of neurons from a culture dish and the rejection of cells after transplantation are expected to limit their future clinical applications. To overcome these problems, we examined the induction of neurogenesis of ES cells under free-floating conditions and microencapsulation of the obtained cell aggregates into an agarose hydrogel. Cell aggregates from ES cells were cultured in various media under the free-floating condition. Immunohistochemical staining for tyrosine hydroxylase (TH) and RT-PCR analyses for TH and Nurr1 showed that dopaminergic neurons were induced in ES cell aggregates cultured in a 1:2 mixture of conditioned medium of PA6 stromal cells and Glasgow minimum essential medium (GMEM) after 16 days in culture. The cell aggregates could be collected and were encased within agarose microcapsules without loss of dopaminergic neurons. The cell aggregates with/without microencapsulation were maintained in CM/GMEM for an additional period. KCl stimulation assays were done at day 23, 30, 37, 44, 51, and 58 to examine dopamine release. Dopamine release abilities were well maintained during 58 days of observation. Amounts of dopamine release from encapsulated cell aggregates were slightly higher than those of unencapsulated cell aggregates from day 16 to 58. Although efficacy for immunoisolation of the agarose microcapsules still remains for future in vivo studies, microencapsulation did not adversely affect viability and functions of the dopamine releasing ES cell progeny.

Multicomponent Coculture System of Cancer Cells and Two Types of Stromal Cells for In Vitro Evaluation of Anticancer Drugs.

In vitro evaluation of anticancer drugs using cancer cells has long been performed for the development of novel drugs and the selection of effective drugs for different patients. Recent studies have suggested that tumor stromal cells affect the drug sensitivity of cancer cells; however, most conventional culture systems for drug evaluation lack stromal cells. In this study, we fabricated a multicomponent coculture system that takes account of cancer-stroma interactions for drug evaluation. In this system, small-cell and nonsmall-cell lung cancer cells embedded in collagen gel were cocultured with two types of stromal cells, including stromal fibroblasts and proinflammatory cytokine-secreting monocytes, thus recreating the in vivo cancer microenvironment. Cancer drug sensitivity was significantly altered by the presence of stromal cells. Fibroblasts induced resistance of cancer cells to anticancer drugs. Monocytes induced the upregulation of thymidine phosphorylase in cancer cells, promoting the conversion of an anticancer prodrug to a cytotoxic drug, and consequently enhanced the sensitivity of cancer cells to the anticancer prodrug. These results clearly show the importance of incorporating stromal cells into culture systems for drug evaluation. Our system will help to improve the accuracy of in vitro drug evaluation and provide useful information for the in vitro recreation of cancer microenvironments.

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

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.

Collection of neural inducing factors from PA6 cells using heparin solution and their immobilization on plastic culture dishes for the induction of neurons from embryonic stem cells.

Embryonic stem (ES) cells have the ability to replicate themselves and differentiate into various mature cells. Recently, dopaminergic neurons were efficiently induced from ES cells using mouse stromal cells (PA6 cells) as a feeder cell layer. This simple procedure seems to be very efficient to obtain dopamine-releasing cells for future clinical cell transplantation treatment of Parkinson's disease. In this study, we prepared stock solutions containing neural inducing factors (NIFs) by washing PA6 cells with phosphate-buffered saline containing heparin. ES cells grew successfully in culture media supplemented with 33 v/v% NIFs stock solution, and the rate of neural differentiation of ES cell progeny increased with increasing heparin concentration in the culture media. In addition, NIFs-immobilized surfaces were prepared by exposing polyethyleneimine-modified surfaces to NIFs stock solutions. The NIFs-immobilized culture dish effectively supported cell growth as the culture medium supplemented with NIFs stock did, but its induction effect to dopaminergic neurons from ES cells was much smaller than free NIFs. NIFs stock solutions have two different activities. One can stimulate cell growth and the other induces differentiation of ES cells to the neural fate when heparin existed. The former factors were effectively immobilized on the culture dish, but those that induce differentiation may not be. Further optimization is required.

Cell microarray for screening feeder cells for differentiation of embryonic stem cells.

Microarrays are currently recognized as one of major tools in the assessment of gene expression via cDNA or RNA analysis and are now accepted as a powerful experimental tool for high-throughput screening of a large number of samples, such as cDNA and siRNAs. In this study, we examined the potential of the microarray methodology for high-throughput screening of candidate cells as feeder cells which effectively differentiate embryonic stem (ES) cells to the specific lineage. Cell arrays were prepared by applying three kinds of cells, PA6, human umbilical vein endothelial, and COS-1 cells, to circular spots, 2 mm in diameter, on a glass plate, followed by the application of mouse ES cells to the cell microarray. After 8 d in culture, TuJ1 (neuron-specific class III beta-tubulin) immunocytochemical staining clearly demonstrated that only PA6 cell spots had the capability to induce ES cells to neuronal differentiation. Although this is a model experiment, these findings clearly indicate that the cell microarray will become a powerful tool for high-throughput screening large numbers of candidate feeder cells for specific differentiation.

Facile immunostaining and labeling of nonadherent cells using a microfluidic device to entrap the cells.

We fabricated a microfluidic device for the entrapment of nonadherent cells. Solution exchange was easily performed by introducing the solution into the cell-trapping microchannel. Immunostaining and labeling of the cell membrane of THP-1 cells were demonstrated using this device, which does not require cumbersome repetition of centrifugation and resuspension steps.

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.

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.

Combining the cell-encapsulation technique and axon guidance for cell transplantation therapy.

In cell transplantation therapy for the treatment of neurodegenerative disorders, encapsulation of implanted cells in a semipermeable membrane is a promising approach to protect the implanted cells from host immune rejection and inhibit the invasion of tumor into surrounding tissue if the implanted cells form a tumor after transplantation. However, implanted neurons isolated by capsules could not build connections with host neurons, preventing the implanted neurons from responding to stimuli from host neurons. In the present study, we focused on the passage of neurites and axons navigated by axon guidance molecules through membrane pores to enable encapsulated neurons and host neurons to form connections. The type of matrix coated on membranes and the pore size of the membranes greatly affected the successful passage of PC12 neurites through membrane pores. PC12 neurites preferably passed through collagen-coated membranes with pores greater than 0.8 µm in diameter, but the neurites did not pass through albumin- or fibronectin-coated membranes or membranes with pores less than 0.1 µm in diameter. We could navigate the direction of commissural neural axon extensions by utilizing the axon guidance molecules secreted from floor plate and make guided axons pass through the membrane pores. These results suggest the feasibility of building connections between encapsulated neurons and host neurons by encapsulating the implanted neurons and axon guidance molecules, which attract the axons of host neurons into the capsule, in the porous membranes with suitable pore size and matrix coating.