Randomized clinical trial of an elastomeric sealant for hemostasis in thoracic aortic surgery.

OBJECTIVES: This study aimed to demonstrate the efficacy and safety of a newly developed elastomeric sealant, which does not require any blood coagulation system to exert its effect, during thoracic aortic surgery. METHODS: This is a multicenter, randomized study conducted in six hospitals in Japan. A total of 81 patients undergoing replacement surgery of a thoracic aortic aneurysm using cardiopulmonary bypass were randomized with a ratio of 2-:1 for those patients designated to receive the sealant (Group S, 54 patients) or those without the usage of the sealant (Group C, 27 patients). The primary endpoints were bleeding from each anastomosis at two time points: (1) immediately before applying protamine and (2) 15 min after applying protamine. The patients were followed for 6 months. RESULTS: The number of anastomoses checked for bleeding was 196 in Group S and 117 in Group C. Before protamine sulfate administration, complete hemostasis was obtained in 155 anastomoses (79%) in Group S compared to 45 anastomoses (38%) in Group C (p < 0.001). Fifteen minutes after the administration of protamine sulfate infusion, bleeding stopped completely in 173 anastomoses (88%) in Group S and in 71 anastomoses (61%, p < 0.001) in Group C. Between the two groups, there were no marked differences in the patient background or in the incidence of major adverse events. CONCLUSIONS: The sealant is effective in achieving hemostasis, even under fully heparinized conditions. The novel sealant is safe and effective in thoracic aortic surgery, one of the most demanding surgical situations for hemostasis.

Vascular endothelial growth factor-bound stents: application of in situ capture technology of circulating endothelial progenitor cells in porcine coronary model.

OBJECTIVES: We evaluated the in vivo performance of a newly devised vascular endothelial growth factor (VEGF)-bound stent in a porcine coronary model. BACKGROUND: An anti-CD34 antibody-bound stent, which captures endothelial progenitor cells (EPCs) to accelerate tissue formation, did not reduce intimal hyperplasia. By targeting the VEGF receptor, which is expressed on endothelial-lineage cells, we developed VEGF-bound stents that may enable selective capture of EPCs followed by rapid endothelialization. METHODS: Metallic stents were first coated with poly-(ethylene-co-vinyl alcohol), and then chemically bound with either VEGF or anti-CD34 antibody. These stents were placed in porcine coronary arteries for up to 14 days. Stent surface was evaluated by immunohistochemistry and by scanning electron microscope (SEM). RESULTS: After 2-day stenting with VEGF-bound stents, small populations of KDR (VEGF receptor-2)-positive cells adhered to the stent struts. After 7- and 14-day stenting, struts were fully covered with newly regenerated tissue. SEM images showed that the uniform tissue formed on struts was morphologically similar to native endothelium and was continuously connected with adjacent native endothelium. On the other hand, for the anti-CD34 antibody-bound stents, stent struts were rapidly covered by newly generated tissue that consisted of multicellular aggregates. CONCLUSIONS: Compared with anti-CD34 antibody-bound stents, VEGF-bound stents provide highly selective capture of EPCs, followed by rapid formation of intact endothelium tissue at an early period of stenting. These results suggest that VEGF-bound stents could represent a promising therapeutic option for cardiovascular stenting, although further long-term follow-up experiment with double-blinded fashion is needed prior to clinical application.

[Development of hemostatic sealant for arterial anastomosis; clinical application].

For the purpose of examining the clinical applicability of a newly developed surgical sealant, animal experiments were performed, and clinical trial was followed. In animal experiments, several animal models, including carotid artery anastomosis model and coronary artery bypass grafting model were undertaken. In each model, complete hemostasis of the anastomoses using four simple interrupted sutures, was obtained. In addition, elastomeric property of the sealant prevented thinning of the arterial wall. The clinical trial performed in patients with thoracic aortic surgery showed significantly better hemostasis even under heparinized condition. Based on these excellent results, clinical usage of the sealant was approved.

[Development of elastameric sealant designed for arterial field].

The development of a reliable surgical sealant specific for arterial tissues has been long awaited. In this article, first the "ideal" adhesion mechanism formulated from biomechanical concept is proposed for ensured hemostasis in dissected arteries with pulsatile flow. An urethane prepolymer prepared along the design criteria is viscous liquid. Due to its high water absorbility and high reactivity with water, the sealant applied to vascular tissues becomes an elastomer within several minutes. When the sealant was applied to dissected canine abdominal arteries with 3 stay sutures, followed by declamping 5 minutes, neither bleeding nor detrimental effect on tissue morphogenesis was observed. This sealant is being ready to the market.

Morphology and adhesion strength of myoblast cells on photocurable gelatin under native and non-native micromechanical environments.

We have quantitatively determined how the morphology and adhesion strength of myoblast cells can be regulated by photocurable gelatin gels, whose mechanical properties can be fine-tuned by a factor of 10(3) (0.1 kPa ≤ E ≤ 140 kPa). The use of such gels allows for the investigation of mechanosensing of cells not only near the natural mechanical microenvironments (E ~ 10 kPa) but also far below and beyond of the natural condition. Optical microscopy and statistical image analysis revealed that myoblast cells sensitively adopt their morphology in response to the substrate elasticity at E ~ 1-20 kPa, which can be characterized by the significant changes in the contact area and order parameters of actin cytoskeletons. In contrast, the cells in contact with the gels with lower elastic moduli remained almost round, and the increase in the elasticity beyond E ~ 20 kPa caused no distinct change in morphology. In addition to the morphological analysis, the adhesion strength was quantitatively evaluated by measuring the critical detachment pressure with an aid of intensive pressure waves induced by picosecond laser pulses. This noninvasive technique utilizing extremely short pressure waves (pulse time width ~100 ns) enables one to determine the critical pressure for cell detachment with reliable statistics while minimizing the artifacts arising from the inelastic deformation of cells. The adhesion strength also exhibited a transition from weak adhesion to strong adhesion within the same elasticity range (E ~ 1-20 kPa). A clear correlation between the cell morphology and adhesion strength suggests the coupling of the strain of the substrate and the mechanosensors near focal adhesion sites.

Surface design for in situ capture of endothelial progenitor cells: VEGF-bound surface architecture and behaviors of cultured mononuclear cells.

In situ capture of endothelial progenitor cells (EPCs) in the arterial bloodstream may allow the creation of a functioning endothelium on the luminal surfaces of implanted cardiovascular devices. Our strategy is to use highly biospecific interaction between the cell-surface marker and surface-bound protein. The target-cell marker defined is vascular endothelial growth factor (VEGF) receptor, which is exclusively expressed on endothelial lineage cells. The candidate surface-bound proteins are VEGF and anti-VEGF receptor (VEGF-R1 and VEGF-R2) antibodies, which were covalent-bound on poly(ethylene-co-vinyl alcohol) bearing a high-surface density of hydroxyl groups. Incubating human mononuclear cells on these substrates affected the histochemical expression of cell-surface markers specific for EPCs and endothelial cells (ECs). The VEGF-bound surface significantly increased the number of cells expressing both VEGF receptors after 1 or 2 weeks of culture, whereas both anti-VEGF receptor antibody-bound substrates did not affect the expression of the surface markers, and cells on these surfaces were eventually died. These results indicate that, among the three candidate molecules, VEGF is best able to capture EPCs and induces their differentiation. Additionally, a pilot study of surface architecture of stents and small-diameter artificial grafts was conducted for an ongoing implantation study in a porcine model.

Arterial shear stress augments the differentiation of endothelial progenitor cells adhered to VEGF-bound surfaces.

Our ongoing studies show that vascular endothelial cell growth factor (VEGF)-bound surfaces selectively capture endothelial progenitor cells (EPCs) in vitro and in vivo, and that surface-bound VEGF stimulates intracellular signal transduction pathways over prolonged culture periods, resulting in inductive differentiation of EPCs. In this article, we investigated whether simulated arterial shear stress augments the differentiation of EPCs adhered to a VEGF-bound surface. Human peripheral blood-derived mononuclear cells adhered to a VEGF-bound surface were exposed to 1 day of shear stress (15 dynes/cm(2), corresponding to shear load in arteries). Shear stress suppressed the expression of mRNAs encoding CD34 and CD133, which are markers for EPCs, and augmented the expression of mRNAs encoding CD31 and von Willebrand factor (vWF) as well as vWF protein, which are markers for endothelial cells (ECs). Shear stress enhanced expression of ephrinB2 mRNA, a marker for arterial ECs, but did not significantly change expression of EphB4 mRNA, a marker for venous ECs. Focused protein array analysis showed that mechanotransduction by shear stress activated the p38 and MAPK pathways in EPCs. Thus, arterial shear stress, in concert with surface-bound VEGF, augments the differentiation of EPCs. These results strongly support previous observation of rapid differentiation of EPCs captured on VEGF-bound stents in a porcine model.

Hydrodynamic shear-stress-dependent retention of endothelial and endothelial progenitor cells adhered to vascular endothelial growth factor-fixed surfaces.

The luminal surfaces of small-diameter artificial vascular grafts must be fully endothelialized to be nonthrombogenic following implantation. To achieve this goal, we have attempted to capture circulating endothelial progenitor cells (EPCs) in situ on the luminal surfaces of implanted grafts. We examined potential receptor-ligand pairs that promote selective and tight adhesion of EPCs using a radial flow chamber comprising three regions, each containing a specific protein-bound substrate: fibronectin (FN) for integrin, and vascular endothelial growth factor (VEGF) and anti-Flk-1 antibody for VEGF receptor. In the presence of shear stress, the greatest retention of endothelial cells and EPCs was observed with FN followed by VEGF and then anti-Flk-1 antibody. Regardless of the bound protein, cell adhesion increased with larger areas of cell adhesion and enhanced cell spreading; the latter was also greatest with FN followed by VEGF and then anti-Flk-1 antibody. The distribution of vinculin-a key protein in focal adhesion plaques-in adherent endothelial cells was examined using total internal reflection fluorescence microscopy; FN-bound surfaces resulted in larger areas of adhesion and more focal adhesion plaques compared with surfaces bound with VEGF. On the other hand, examining these parameters relative to the area of cell adhesion revealed that VEGF-bound surfaces resulted in larger focal adhesion areas and greater fluorescence signals, both of which indicate increased resistance to shear stress. We also discuss in situ capturing of EPCs on surfaces bound with VEGF or anti-Flk-1 antibody, with the goal of creating endothelialized small-diameter vascular grafts.

Electrospun Poly(γ-Benzyl-L-Glutamate) and Its Alkali-Treated Meshes: Their Water Wettability and Cell-Adhesion Potential.

The aim of this study was (1) to fabricate non-woven meshes from a biodegradable polymer, poly(γ-benzylL-glutamate), by electrospinning and subsequent hydrolysis of the ester bond on the polymer side-chain in an aqueous solution of NaOH, creating surface carboxyl groups on the fibers, and (2) to determine the effect of hydrolysis time on water wettability and cellular behaviors, in order to perform a preliminary evaluation for use of this polymer as a wound dressing matrix. A non-woven mesh composed of fibers, with minimal formation of 'bead' structures, was produced by electrospinning from tetrahydrofuran solution under optimally controlled conditions. The surface wettability largely depended on the hydrolysis time: an increase in hydrolysis time significantly reduced the advancing water contact angle. Instantaneous spreading of water droplets occurred at long hydrolysis times. An increase in hydrolysis time decreased adhesion of endothelial cells, but increased cell spreading. Neither proliferation nor invasion into the mesh interior was observed. We conclude by discussing the use of partially hydrolyzed non-woven mesh as a promising burn dressing.

Luminal surface design of electrospun small-diameter graft aiming at in situ capture of endothelial progenitor cell.

If endothelial progenitor cells (EPCs), which circulate in blood flow, are captured on the luminal surface of an implanted artificial graft and sooner or later proliferate to form a fully endothelialized surface, such a small-diameter artificial graft must exhibit a high patency rate. This study aimed at designing a luminal surface of elastomeric electrospun mesh graft, which is capable of selective capture of EPCs under arterial flow and has a high antithrombogenic potential until full endothelization is achieved. The designed luminal surface layer is composed of a photopolymerized gelatin gel layer that enables the release of impregnated heparin and selective adhesion of circulating EPCs via complexation between surface-fixed vascular endothelial growth factor (VEGF) and cellular VEGF receptor. Human mononuclear cells seeded and cultured on such a gel layer expressed endothelial cell surface markers. Confocal laser scanning microscopy observation revealed that VEGF is highly surface-enriched, and heparin is homogeneously distributed in the gel layer. A continuously slow release of heparin was observed. Thus, a prototype luminal surface was fabricated on electrospun segmented polyurethane tubes for in vivo study.

Articular cartilage tissue engineering based on a mechano-active scaffold made of poly(L-lactide-co-epsilon-caprolactone): In vivo performance in adult rabbits.

Our previous studies showed that a mechano-active scaffold made of poly(L-lactide-co-epsilon-caprolactone) (PLCL) exhibited a high potential to realize the formation of a functional, engineered cartilage in vitro. This animal study therefore was designed to investigate the feasibility of repairing on osteochondral defect with the use of bone marrow-derived mesenchymal stem cells (BMSCs) incorporated with a PLCL scaffold. Rabbit BMSCs, isolated and subsequently cultured in monolayer, were seeded into a porous PLCL scaffold sponge following an implantation onto a full-thickness osteochondral defect (diameter of 4.5 mm, depth of 5 mm) that was artificially created on the medial femoral condyles at a high load-bearing site on a rabbit's knee joint. Time-dependent healing of the defect was evaluated by macroscopic, histological examinations at both 3- and 6-month-implantations, respectively. A PLCL sponge incorporated with BMSCs exhibited sufficient structural support, resulting in new osteochondral tissue regeneration: a physiologically well-integrated subchondral bone formation, a hyaline cartilage-like morphology containing chondrocytes surrounded by abundant cartilaginous matrices. In addition, quantitative biochemical assays also demonstrated high potential for the synthesis of sulfated glycosaminoglycan and collagen, both of which are biomolecules essential to extracelluar matrix in normal cartilage tissue. In contrast, defects filled with cell-free PLCL scaffold or left empty showed a very limited potential for regeneration. Our findings suggest that a composite of PLCL-based sponge scaffold and BMSCs promote the repair of osteochondral defects at high load-bearing sites in adult rabbits.

Multiscale fabrication of a transparent circulation type blood vessel simulator.

We proposed and fabricated multiscale transparent arteriole and capillary vessel models with circular cross sections of 10-500 µm using photolithography. The circularities of the fabricated 10, 50, and 500 µm diameter microchannels were 84.0%, 61.5%, and 82.3%, respectively. Next, we connected these different models to realize a circulation type blood vessel model simulating arteriole networks. We proposed a novel connection method using an intermediate connector made of wax, which we used to connect these models to make a circulation model. In flow experiments, the fabricated models showed no leakage and circulation models with seamless connections were achieved.

Reversible hydrogel formation driven by protein-peptide-specific interaction and chondrocyte entrapment.

We developed a hydrogel self-assembling method driven by the interaction between recombinant tax-interactive protein-1 (TIP1) with the PDZ domain in a molecule, which is fused to each end of the triangular trimeric CutA protein (CutA-TIP1), and a PDZ domain-recognizable peptide which is covalently bound to each terminus of four-armed poly(ethylene glycol) (PDZ-peptide-PEG). Genetic manipulation based on molecular-dynamic simulation generated a cell-adhesive RGD tripeptidyl sequence in the CutA loop region [CutA(RGD)-TIP1]. Spontaneous viscoelastic hydrogel formation occurred when either CutA-TIP1- or CutA(RGD)-TIP1-containing buffer solution and PDZ-peptide-PEG-containing buffer solutions were stoichiometrically mixed. Dynamic viscoelasticity measurement revealed shear stress-dependent reversible-phase transformation: a spontaneous viscoelastic hydrogel was formed at low shear stress, but it was transformed into a sol at high shear stress. Upon the cessation of shear, hydrogel was restored. When chondrocytes were pre-mixed with one of these two components containing buffer solutions, the stoichiometric mixed solution was also spontaneously gelled. Individual rounded cells and multicellular aggregates were entrapped within both hydrogels without substantial cellular impairment regardless of the presence or absence of RGD motif in the CutA-TIP1 molecule. The potential use of such a shear-sensitive hydrogel for injectable cell delivery into diseased or lost cartilage tissue is discussed.

Experimental use of an elastomeric surgical sealant for arterial hemostasis and its long-term tissue response.

OBJECTIVES: Reliable suture line hemostasis should improve the outcome of aortic surgery. We examined the hemostatic effect and the tissue response of a novel elastomeric surgical sealant. METHODS: Using porcine internal carotid arteries, we performed 16 end-to-end anastomoses with four stitches of simple interrupted sutures under full heparinization. The anastomoses were divided into two groups (eight anastomoses per group). Either novel sealant or fibrin glue was applied. The amount of bleeding was measured during the 30 s period after removing the vascular clamp. In a separate experiment, we applied the novel sealant around the abdominal aorta of rabbits (n=6) to assess the effect of the elastomeric property of the sealant on arterial wall histology. For comparison, we applied cyanoacrylate, which has no elastomeric property (n=6). A histological study was performed three months after the operation. RESULTS: The novel sealant prevented arterial bleeding. The amount of bleeding from the anastomoses applied with novel sealant and fibrin glue was 0.12+/-0.03 g vs. 91.8+/-16.5 g, respectively (P<0.001). Thinning of the rabbit aortic wall was observed in the cyanoacrylate-treated abdominal aorta, whereas no thinning was observed in the novel sealant group. Histological examination revealed neither cell death nor necrosis in the novel sealant group. CONCLUSIONS: The novel sealant effectively prevented arterial bleeding from the anastomosis under full heparinization. In addition, the elastomeric property of the sealant prevented thinning of the aortic wall. The novel sealant may be a promising hemostatic agent for arterial anastomosis.

Nanoscale elongating control of the self-assembled protein filament with the cysteine-introduced building blocks.

Self-assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self-assembling proteins, named "Nanolego." Nanolego consists of "structural elements" of a structurally stable symmetrical homo-oligomeric protein and "binding elements," which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self-assembly process. The stabilization method is mediated by disulfide bonds between Cysteine-residues incorporated into the binding elements, and the termination method uses "capping Nanolegos," in which some of the binding elements in the Nanolego are absent for the self-assembled ends. With these technologies, we successfully constructed timing-controlled and size-regulated filament-shape complexes via Nanolego self-assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.

Lymphocyte adhesion and interactions with biomaterial adherent macrophages and foreign body giant cells.

To characterize the effects of adherent macrophages and biomaterial surface chemistries on lymphocyte adhesion and activation, lymphocytes were co-cultured with monocytes alone and together, directly and separated by a porous membrane transwell on hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic biomaterial surfaces. Surface adherent cells were quantitatively analyzed after 3 days utilizing immunofluorescence and phase contrast imaging. After periods of 3, 7, and 10 days, secreted interferon-gamma (IFN-gamma) was quantified by ELISA. Limited direct biomaterial-adherent lymphocytes were identified regardless of the presence of macrophages or foreign body giant cells (FBGC). The majority of adherent lymphocytes, which were T cells (>95%) rather than natural killer cells, predominantly interacted with adherent macrophages and FBGCs; greater than 90% were interacting on surfaces with higher levels of adherent macrophages and FBGCs and greater than 55% were interacting on surfaces with lower levels of macrophages and FBGCs. The hydrophilic/anionic surface promoted higher levels of macrophage- and FBGC-adherent lymphocytes but was nonselective for lymphocyte subtype interactions. The hydrophilic/neutral surface was selective for CD4+ T lymphocyte interactions while the hydrophobic surface was selective for CD8+ T lymphocyte interactions. IFN-gamma was produced in direct and indirect co-cultures but not in lymphocyte- and monocyte-only cultures suggesting that lymphocytes are activated via macrophage-derived cytokines rather than direct biomaterial contact. Direct lymphocyte interactions with adherent macrophages/FBGCs enhanced IFN-gamma production relative to indirect co-cultures. These results suggest that lymphocytes prefer interactions with adherent macrophages and FBGCs, resulting in lymphocyte activation, and these interactions can be influenced by biomaterial surface chemistries.

Antibody-bound cell microarray for immunophenotyping: surface modification and lymphocyte subpopulations.

Antibody-based cell microarrays may serve as a high-throughput diagnostic tool that requires precise surface design providing high biological specificity, high reliability, and high validity. A fundamental study on the quantitative evaluation of immunophenotyping using a cell microarray on which antibodies specific to cluster of differentiation (CD) antigens of blood cells are fixed was performed. The microarray, which was prepared by photomicropatterning self-assembled monolayers of alkanethiols, consisted of carboxyl group (3-mercaptopropionic acid)-packed domains regularly distributed in the methyl group (1-dodecanethiol)-packed matrix phase. This was verified by X-ray photoelectron spectroscopy and water wettability measurements. The patterned carboxylated domains of 1.0 mm or 0.2 mm diameter were covalently fixed with the anti-CD4 or anti-CD8 antibody by coupling reaction using a water-soluble carbodiimide and hydroxysuccinimide. Precision antibody fixation at almost complete conversion was verified by confocal laser scanning microscopy coupled with a specific dye staining technique. Peripheral blood mononuclear cells expressing CD4 or CD8 antigen, respectively, adhered on the anti-CD4 or anti-CD8 antibody-fixed domains of the microarray at a very high specificity, which was verified with flow cytometric analysis and an antibody-coated magnetic-bead-based cell isolation technique. The CD4/CD8 subset ratios determined using the antibody-fixed microarrays were very close to those obtained by flow cytometric analysis. These results indicate that microarrays, on which antibodies specific to cell surface antigens are covalently fixed, provide a solid basis of the high-throughput quantitative evaluation of immunophenotyping in medical diagnosis.

Electrospinning fabrication of high-trackable catheter tip with gradually graded or gradient flexibility.

Increasing demand for a flexible catheter tip exists in the rapidly growing fields of vascular and interventional radiology. This article describes a fabrication technology producing a catheter tip with gradually graded or gradient flexibility. Based on deflection mechanics of a tubular construct, three models were incorporated into structural designs. The models included graded wall thickness, tapered shape, or graded Young's moduli. Electrospinning using elastomeric polymer [poly(L-lactide-co-epsilon-caprolactone), polyurethane] solutions on a transversely and rotationally moving mandrel enabled preparation of three prototype tubular tips by either regio-specific control of wall thickness, tapered mandrel, or regiospecific deposition of polyurethanes with different Young's moduli. All prototype tips exhibited high flexibility at tip ends, as verified using a bifurcated vessel model.

Instability of self-assembled monolayers as a model material system for macrophage/FBGC cellular behavior.

Novel self-assembled monolayers (SAMs) designed to present homogenous surface chemistries were utilized to further investigate the material surface chemistry dependent macrophage and foreign-body giant cell (FBGC) behaviors, including macrophage adhesion, fusion, and apoptosis. Contact angle analysis revealed instabilities in the --CH(3) and --COOH terminated SAM surfaces upon incubation in serum-free media (SFM) at 37 degrees C or under dry, room temperature conditions. Further analysis indicated that the --CH(3) terminated SAM surface degraded rapidly within 2 h and loss of sufficient SAM units to be comparable to the gold (Au) control surface, within 24 h of incubation in SFM at 37 degrees C. After 5 days of incubation in SFM at 37 degrees C, the contact angles for the --COOH terminated SAM surfaces increased markedly. AFM analysis confirmed the desorption of --CH(3) terminated SAM molecules from the surface with increased roughness and marked appearance of peaks and valleys within 2 h. A decrease in the thickness of the --COOH terminated SAM surface also suggests molecular desorption over time. No significant changes in contact angle or AFM analyses were observed on the --OH terminated SAM surfaces. Cellular adhesion decreased more rapidly on the Au control and --CH(3) terminated SAM surfaces in comparison to the other surfaces. However by day 10, cellular adhesion, fusion, and apoptosis were comparable on all SAM surfaces and the Au control. These studies suggest that SAM surfaces may not be suitable for long-term studies where material dependent properties are investigated.

In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation.

A novel technique and instrumented device were developed to harvest target cells from multicellular mixture of different cell types under a microscope. The principle of the technique is that cells cultured on a thermoresponsive-substance-coated dish were detached by a region-specific cooling device and simultaneously harvested using a micropipette, both of which were assembled in an inverted microscope. Thermoresponsive coating consists of the mixture of poly(N-isopropylacrylamide) (PNIPAAm) and PNIPAAm-grafted gelatin. The former non-cell-adhesive polymer dissolves below at 32.1 degrees C in water and precipitates over that temperature (called lower critical solution temperature, LCST), and the latter cell-adhesive polymer has LCST of 34.1 degrees C. The appropriate mixing ratio of these thermoresponsive polymers exhibited high cell adhesion at physiological temperature and complete cell detachment at room temperature. A device developed as to cool at only a tiny area of the bottom of the dish, beneath which a cell that was targeted under a microscope, was assembled in a microscope. It was demonstrated that single cell or two cells that adhered to each other was detached from the surface and harvested by a micropipette within approximately 30s.