PIPAAm-grafted Surfaces Research Articles

Design of Temperature-Responsive Polymer-Grafted Surfaces for Cell Sheet Preparation and Manipulation

Abstract This review focuses on the design of poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces for cell sheet preparation and manipulation, which are revolutionary tools for the creation of transplantable two-dimensional (2D) and engineered three-dimensional (3D) cellular tissues. Particularly, the thickness of grafted PIPPAm chains in the perpendicular direction is regulated to achieve temperature-dependent alteration of cell sheet preparation/harvesting. The 2D positioning of grafted PIPAAm in a direction parallel to the material surfaces facilitates spatially controlled micropatterns containing heterotypic cells. This review also describes the 2D manipulation of cell sheets and the creation of cell sheet-layered 3D tissue using the PIPAAm-grafted surface. With the aid of supporting materials such as membranes and gelatin hydrogels, cell sheets on PIPAAm-grafted surfaces can be manipulated and applied for transplantation in clinical settings and for the formation of 3D tissues in vitro. For the next generation of cell sheet-based tissue engineering, a challenging issue is the creation of large, thick tissues/organs such as cardiac and hepatic tissues/organs. The integration of various technologies including bioreactors and micropatterning is essential to achieve the creation of functional engineered 3D organs.

Cell sheet tissue engineering: Cell sheet preparation, harvesting/manipulation, and transplantation.

Cell sheet tissue engineering is a concept for creating transplantable two-dimensional (2D) and three-dimensional (3D) tissues and organs. This review describes three elements of cell sheet tissue engineering in terms of the chemical and physical effects of material surfaces and the interfacial properties of cell sheets: preparation, harvesting/manipulation and transplantation of cell sheets. An essential technology for the preparation of cell sheets is the use of a temperature-responsive cell culture surface, where the surface of tissue culture polystyrene (TCPS) dish is modified with thin layer of temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). PIPAAm-immobilized TCPS allows cultured cells to be harvested as a contiguous cell sheet with extracellular matrices (ECMs) by reducing the temperature, while chemical and physical disruption impair ECMs, cell-cell junction, and membrane proteins. Ligand-immobilized and porous hydrophilic PIPAAm-grafted surfaces are able to accelerate cell sheet preparation and harvesting, respectively. In addition, the manipulation of harvested cell sheets with the aid of cell sheet manipulator facilitates the formation of 3D tissues. Cell sheet-based tissues and their transplantation are in seven clinical settings such as heart, cornea, esophagus, periodontal, middle chamber of ear, knee cartilage, and lung. In order to create thick and large 3D tissues and organs, large production of differentiated parenchymal cells from induced pluripotent stem (iPS) cells and vascularization within 3D tissues are key issues. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 955-967, 2019.

Switching of growth factor binding on heparin-functionalized thermoresponsive surface for hepatocyte sheet manipulation with maintenance of hepatic functions

Event Abstract Back to Event Switching of growth factor binding on heparin-functionalized thermoresponsive surface for hepatocyte sheet manipulation with maintenance of hepatic functions Jun Kobayashi1, Yoshikatsu Akiyama1*, Masayuki Yamato1* and Teruo Okano1* 1 Tokyo Women's Medical University, Institute of Advanced Biomedical Engineering and Science, Japan Introduction: Liver tissue engineering using primary hepatocytes is an attractive method for the treatment of liver diseases[1]. Engineered Hepatocyte sheets were effectively engrafted in pre-vascularized subcutaneous site and exhibited liver-specific functionalities. By contrast, cultured hepatocytes rapidly lose their viability and phenotypic functions on isolation from the native in vivo microenvironment of the liver. To overcome this problem, heparin-functionalized poly(N-isopropylacrylamide) (PIPAAm)-grafted cell culture surface[2],[3], which interacts with heparin-binding proteins such as heparin-binding EGF-like growth factor (HB-EGF), was been designed for maintaining hepatic functions during the cultivation (Figure 1, left). In addition, the detachment of the cultured hepatocytes as a sheet was examined when lowering temperature to 20°C (Figure 1, right). Figure 1. Schematic of affinity regulation between heparin-binding protein and immobilized heparin by conformational change of PIPAAm. Materials and Methods: Heparin-functionalized thermoresponsive surfaces were prepared as described previously[2]. Briefly, poly(IPAAm-co-2-carboxyisopropylacrylamide) (poly(IPAAm-co-CIPAAm))-grafted surfaces on tissue culture polystyrene (TCPS) dishes were prepared by electron beam irradiation. Heparin was covalently immobilized on the poly(IPAAm-co-CIPAAm)-grafted dishes by condensing reaction. Then, affinity binding of HB-EGF was performed on the heparinized thermoresponsive surfaces by incubation of HB-EGF solution at 37°C for 24 h. Amounts of bound HB-EGF on the heparinized surface were quantified using [125I]-labeled HB-EGF. Primary rat hepatocytes were seeded on the dishes in DMEM with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2. Results: The addition of soluble HB-EGF in the cell culture media was essential for the survival of hepatocytes. When the medium contained less than 10 ng/cm2 of soluble HB-EGF, the hepatocytes were not able to adhere and form their cell sheets. By contrast, hepatocytes adhered and formed their sheets on heparin-functionalized thermoresponsive surface with 10 ng/cm2 of bound HB-EGF. In addition, the secretion of albumin on bound HB-EGF was maintained and higher than that on PIPAAm-grafted surfaces with soluble HB-EGF. Therefore, bound HB-EGF gave a high activity of maintenance of hepatocyte adhesion and function compared with soluble HB-EGF. Finally, when lowering temperature to 20°C, the cultured hepatocyte sheets were detached from the surface through the reduction of affinity binding between HE-EGF and immobilized heparin with increasing the mobility of heparin and steric hindrance of the swollen PIPAAm chains (Figure 1, right). Conclusions: Heparin-functionalized thermoresponsive cell culture surfaces facilitated the manipulation of hepatic cell sheets with maintaining hepatic functions by changing temperature. Creation of transferable and functional liver tissues is considered to have a potential to treat liver disease. Part of this work was financially supported by JSPS KAKENHI Grant Numbers 23106009 and 15K01317

Fabrication and Characterization of Thermoresponsive Polystyrene Nanofibrous Mats for Cultured Cell Recovery

Rapid cell growth and rapid recovery of intact cultured cells are an invaluable technique to maintain the biological functions and viability of cells. To achieve this goal, thermoresponsive polystyrene (PS) nanofibrous mat was fabricated by electrospinning of PS solution, followed by the graft polymerization of thermoresponsive poly(N-isopropylacrylamide)(PIPAAm) on PS nanofibrous mats. Image analysis of the PS nanofiber revealed a unimodal distribution pattern with 400 nm average fiber diameter. Graft polymerization of PIPAAm on PS nanofibrous mats was confirmed by spectroscopic methods such as ATR-FTIR, ESCA, and AFM. Human fibroblasts were cultured on four different surfaces, PIPAAm-grafted and ungrafted PS dishes and PIPAAm-grafted and ungrafted PS nanofibrous mats, respectively. Cells on PIPAAm-grafted PS nanofibrous mats were well attached, spread, and proliferated significantly much more than those on other surfaces. Cultured cells were easily detached from the PIPAAm-grafted surfaces by decreasing culture temperature to 20°C, while negligible cells were detached from ungrafted surfaces. Moreover, cells on PIPAAm-grafted PS nanofibrous mats were detached more rapidly than those on PIPAAm-grafted PS dishes. These results suggest that thermoresponsive nanofibrous mats are attractive cell culture substrates which enable rapid cell growth and recovery from the culture surface for application to tissue engineering and regenerative medicine.

Surface design of antibody-immobilized thermoresponsive cell culture dishes for recovering intact cells by low-temperature treatment

Antibody-immobilized thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [poly(IPAAm-co-CIPAAm)]-grafted cell culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces.

Switching of cell growth/detachment on heparin-functionalized thermoresponsive surface for rapid cell sheet fabrication and manipulation

Heparin-functionalized poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [P(IPAAm-co-CIPAAm)] grafted surface was designed for the switching of cell growth/detachment, achieved by the regulation of affinity binding between basic fibroblast growth factor (bFGF) and immobilized heparin through the temperature-dependent conformational change of grafted P(IPAAm-co-CIPAAm) chains. At 37 °C, bFGF-bound heparin-thermoresponsive surfaces were able to hold the two- to three-fold number of mouse fibroblast (NIH/3T3) cells than both bFGF-physisorbed surface and PIPAAm surface with soluble bFGF after a 3-day cultivation. Bound bFGF via heparin on shrunken grafted P(IPAAm-co-CIPAAm) chains at 37 °C was able to reinforce the formation and stabilization of bFGF-FGF receptor complex, although the activity of physisorbed bFGF on PIPAAm-grafted surfaces was decreased by non-specific and randomly oriented adsorption. At 20 °C, the cultured NIH/3T3 cell sheet with bFGF detached from heparin-functionalized thermoresponsive surface. The release of bFGF from the surfaces was induced by reducing the affinity binding between bFGF and immobilized-heparin due to increasing the mobility of the swollen grafted P(IPAAm-co-CIPAAm) chains. Therefore, heparin-functionalized thermoresponsive surface was able to enhance cell proliferation, and confluent cells detached themselves as a contiguous cell sheet due to switching cell growth by changing temperature. A cell culture system using this surface is useful for rapid cell sheet fabrication and manipulation.

Stereoregulation of Thermoresponsive Polymer Brushes by Surface-Initiated Living Radical Polymerization and the Effect of Tacticity on Surface Wettability.

In this study, stereocontrolled poly(N-isopropylacrylamide) (PIPAAm) brushes were grafted from surfaces by atom transfer radical polymerization (ATRP) in the presence of a Lewis acid, and the effect of PIPAAm brush tacticity on the thermoresponsive wettabiliy was investigated. PIPAAm grafted by ATRP in the presence of Y(OTf)(3) showed high isotacticity, while the control brush polymerized in the absence of Y(OTf)(3) was clearly atactic. The isotacticity and molecular weight of PIPAAm brushes were controlled by polymerization conditions. The wettability of isotactic PIPAAm-grafted surfaces decreased slightly below 10 °C, although the phase transition temperature of atactic surface was 30 °C, and the bulk isotactic polymer was water-insoluble between 5 and 45 °C.

Effects of Graft Densities and Chain Lengths on Separation of Bioactive Compounds by Nanolayered Thermoresponsive Polymer Brush Surfaces

We have prepared various poly(N-isopropylacrylamide) (PIPAAm)-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) by changing graft densities and brush chain lengths. The prepared surfaces were characterized by chromatographic analysis using the modified silica beads as chromatographic stationary phases. ATRP initiator (2-(m,p-chloromethylphenyl)ethyltrichlorosilane) density on silica bead surfaces was modulated by changing the feed composition of the self-assembled monolayers (SAMs) of mixed silane coupling agents consisting of ATRP initiator and phenethyltrichlorosilane on the surfaces. IPAAm was then polymerized on SAM-modified silica bead surfaces by ATRP in 2-propanol at 25 degrees C. The chain length of the grafted PIPAAm was controlled by simply changing the ATRP reaction time at constant catalyst concentration. The thermoresponsive surface properties of the PIPAAm-grafted silica beads were investigated by temperature-dependent elution behavior of hydrophobic steroids from the surfaces using Milli-Q water as a mobile phase. On the surfaces grafted with shorter PIPAAm chains, longer retention times for steroids were observed on sparsely grafted PIPAAm surfaces compared to dense PIPAAm brushes at low temperature, because of hydrophobic interactions between the exposed phenethyl groups of SAMs on silica surfaces and steroid molecules. Retention times for steroids on dilute PIPAAm chain columns decreased with temperature similarly to conventional reverse-phase chromatographic modes on octadecyl columns. This effect was due to limited interaction of solutes with the PIPAAm-grafted surfaces. Retention times for steroids on dilute PIPAAm brush surfaces with longer PIPAAm chains became greater above the PIPAAm transition temperature. At low-temperature regions, hydrated and expanded PIPAAm at low temperatures prevented hydrophobic interactions between the phenethyl group of SAMs on the silica bead surfaces and steroid molecules. Retention times for steroids on a dense PIPAAm brush column increased with temperature since solvated polymer segments within the dense brush layer undergo dehydration over a broad range of temperatures. In conclusion, PIPAAm graft density has a crucial influence on the elution behavior of steroids because of the interaction of analytes with silica bead interfaces, and because of the characteristic dehydration of PIPAAm in dense-pack brush surfaces.

Interfacial Property Modulation of Thermoresponsive Polymer Brush Surfaces and Their Interaction with Biomolecules

Dense poly(N-isopropylacrylamide) (PIPAAm) brushes were created on silica bead surfaces by surface-initiated atom transfer radical polymerization (ATRP). Interfacial properties of PIPAAm brushes were characterized by thermoresponisve interaction with biomolecules. The grafted amounts of PIPAAm on silica bead surfaces exceeded that from previously reported polymer-hydrogel-modified silica beads prepared by conventional radical polymerization by nearly 1 order of magnitude. Temperature-dependent chromatographic interactions with soluble analytes were modulated by changing the grafted PIPAAm chain lengths. Short PIPAAm-grafted silica beads produce insufficient dehydration and chain aggregation to separate steroids using weak hydrophobic interactions. In contrast, broad unresolved peaks were observed on silica beads column grafted with long PIPAAm chains due to steroid partitioning into thick, densely grafted PIPAAm brush layers. Thus, silica beads column grafted with PIPAAm chains of proper length can demonstrate baseline separation of steroids with relatively high resolution among the tested columns. Relatively longer retention times for steroid analytes were observed on all columns compared to those previously reported for other PIPAAm-grafted silica beads. This indicates that densely PIPAAm-grafted chains enable control of strong hydrophobic interactions with steroids by changing the column temperature. Densely grafted PIPAAm columns were also successful in separating two peptides into two peaks as the column temperature was increased to 40 degrees C. This provides an effective separation alternative for peptides using substantial hydrophobicity without modification of hydrophobic surfaces and/or low mobile phase pH. In conclusion, densely PIPAAm-grafted surfaces exhibit strong, reversible temperature-modulated hydrophobic interactions, facilitating baseline separations of steroids and peptides in aqueous milieu without changes in the mobile phase pH and high ionic strength.

Development of Rapid Cell Recovery System Using Temperature-Responsive Nanofiber Surfaces

PHBV ultrafine fibers were fabricated by electrospinning process. Electrospun PHBV fiber structures revealed randomly aligned fibers with average diameter of 400 nm. PIPAAm was grafted on the surface of PHBV nanofibrous mat by electron beam irradiation. PIPAAm-grafted PHBV mats were determined by ATR-FTIR and ESCA. Water contact angles were determined by a sessile drop method at 20 and 37. To examine the tissue compatibility, human fibroblasts were evenly seeded onto PIPAAm-grafted PHBV mat and cast film, ungrafted PHBV mat and film. Attached and spread fibroblasts on nanofibrous mat were proliferated more rapidly than that of flat film surface. Initial cell attachment on PIPAAm-grafted surfaces was higher than ungrafted surfaces. The surface property changed to hydrophilic by PIPAAm graft, which increased initial cell attachment. Detachment of single cells from PIPAAm-grafted PHBV matrixes was measured by low temperature treatment after incubation at 37. Cultured cells were rapidly detached from PIPAAm-grafted PHBV mat compared with film. With porous mats, the water molecules easily reach to grafted PIPAAm from underneath and peripheral to the attached cells, resulting in rapid hydration of grafted PIPAAm molecules and detachment of the cells.

Thermal Modulated Interaction of Aqueous Steroids Using Polymer-Grafted Capillaries

Poly(N-isopropylacrylamide) (PIPAAm) of controlled molecular weight was densely grafted onto glass capillary lumenal surfaces using surface-initiated atom transfer radical polymerization (ATRP). Temperature-dependent changes of these thermoresponsive brush surfaces with hydrophobic steroids were investigated by exploiting thermoresponsive aqueous wettability changes of the polymer-modified surfaces in microfluidic systems. IPAAm was polymerized on ATRP initiator-immobilized glass surfaces using CuCl/CuCl(2)/tris(dimethylaminoethyl)amine (Me(6)TREN) as an ATRP catalyst in water at 25 degrees C. PIPAAm graft layer thickness and its homogeneity on glass surfaces are controlled by changing ATRP reaction time. Aqueous wettability changes of PIPAAm-grafted surfaces responses drastically changed to both grafted polymer layer thickness and temperature, especially at lower temperatures. Temperature-responsive surface properties of these PIPAAm brushes within capillary inner wall surfaces were then investigated using capillary chromatography. Effective interaction of hydrophobic steroids with dehydrated, hydrophobized PIPAAm-grafted capillary surfaces was observed above 30 degrees C without any column packing materials. Steroid elution behavior from PIPAAm-grafted capillaries contrasted sharply with that from PIPAAm hydrogel-grafted porous monolithic silica capillaries prepared by electron beam (EB) irradiation wherein significant peak broadening was observed at high-temperature regardless of sample hydrophobicity factors (log P values), indicating multistep separation modes in coated monolithic silica capillaries. In conclusion, thermoresponsive polymer-grafted capillary inner wall surfaces prepared by ATRP exhibit useful temperature-dependent surface property alterations effective to regulate interactions with biomolecules without requirements for separation bed packing materials within the capillary lumen.

Nanostructured designs of biomedical materials: applications of cell sheet engineering to functional regenerative tissues and organs

Biomaterials surface design is critical for control of cell–materials interactions. Materials surface characteristics important to cell–materials interactions are the following: (a) nonfouling surfaces where cells cannot interact; (b) surfaces that interact with cells but do not alter cell morphology or metabolism (passive adhesion processes); and (c) surfaces that strongly interact with cells and cell–surface receptors to alter cell shape after metabolic interactions (active adhesion). In this paper, we briefly discuss the relationship between materials surface characteristics and cells for biomaterials designs in these categories. We have extensively investigated the thermoresponsive polymer, poly( N-isopropylacrylamide) (PIPAAm), as grafted surfaces allowing recovery of confluent cell monolayers as contiguous living cell sheets for tissue engineering applications. Cellular interactions with PIPAAm-grafted surfaces can be regulated vertically using the thickness of the PIPAAm-grafted layers in nanometer-scale levels, as well as laterally (spatially) using nano-patterned PIPAAm chemistry on various other surface chemistries. PIPAAm-grafted surfaces with 15–20-nm thick layers exhibit temperature-dependent cell adhesion/detachment control, while surfaces with PIPAAm layer thicknesses of more than 30 nm do not support cell adhesion. These changes in cell adhesion are explained by the limited mobility of the surface grafted polymer chains as a function of grafting, hydration, and temperature.

Incorporation of new carboxylate functionalized co-monomers to temperature-responsive polymer-grafted cell culture surfaces

Several cultured cell types are easily detached from poly( N-isopropylacrylamide) (PIPAAm)-grafted surfaces only by reducing culture temperature without traditional proteolytic treatments that might damage certain cell functions. We have exploited these novel surfaces for tissue engineering applications where harvested intact cell sheets are useful for fabricating tissue-like constructs. We now extend the polymer chemistry of such grafted surfaces with new charged co-monomers. Functional carboxylate groups are incorporated into temperature-responsive surfaces with newly designed analogous carboxylate co-monomers, 2-carboxyisopropylacrylamide (CIPAAm) and 3-carboxy- n-propylacrylamide (CNPAAm), which have a small structural difference in the placement of the carboxylate group ( iso or normal to the monomer propyl group). P(IPAAm- co-CIPAAm) demonstrates a similar phase transition behavior to that of pure PIPAAm, and the ionic dissociation of carboxyl groups is suppressed (elevated p K′a) even under physiological conditions. By contrast, P(IPAAm- co-CNPAAm) exhibits a higher charge density (lower p K′a), higher hydration, and reduced temperature-sensitivity under identical conditions. Introduction of 5 mol% CNPAAm into PIPAAm grafted surfaces produces no cell attachment under typical cell culture conditions, while identical introductions of CIPAAm into grafted copolymers functions well for cell attachment. Cultured cell spreading efficiency was essentially similar on PIPAAm-grafted surfaces as on copolymer-grafted surfaces with 1 mol% introduction of either carboxylate co-monomer. Accelerated cell detachment upon reducing culture temperature was observed for the 1 mol% these copolymer-grafted surfaces since polymer hydration and swelling kinetics are enhanced by the increased ionizable moiety in these grafted surfaces.

Ultrathin Poly(N-isopropylacrylamide) Grafted Layer on Polystyrene Surfaces for Cell Adhesion/Detachment Control

We investigated physicochemical properties of two types of poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture polystyrene (TCPS) surfaces, to elucidate the influential factors for thermally regulated cell adhesion and detachment to PIPAAm-grafted surfaces. The two types of PIPAAm-grafted surfaces were prepared by the electron beam polymerization method. Attenuated total reflection Fourier transform infrared spectroscopy revealed that amounts of the grafted polymers were 1.4 +/- 0.1 microg/cm2 for PIPAAm-1.4 and 2.9 +/- 0.1 microg/cm2 for PIPAAm-2.9. Both PIPAAm-grafted surfaces showed hydrophobic/hydrophilic property alterations in response to temperature. However, PIPAAm-1.4 surfaces were more hydrophobic (cos theta = 0.21 at 37 degrees C and cos theta = 0.35 at 20 degrees C) than PIPAAm-2.9 (cos theta = 0.42 at 37 degrees C and cos theta = 0.50 at 20 degrees C) both above and below the PIPAAm's transition temperature. Thicknesses of the grafted PIPAAm layers were estimated to be 15.5 +/- 7.2 nm for PIPAAm-1.4 and 29.5 +/- 8.4 nm for PIPAAm-2.9, by the use of UV excimer laser and atomic force microscope. Bovine carotid artery endothelial cells (ECs) adhere to the surfaces of PIPAAm-1.4 and proliferate to form confluent cell monolayers. The cell monolayers were harvested as single cell sheets by temperature decrease from 37 to 20 degrees C. On the contrary, ECs did not adhere to the surfaces of PIPAAm-2.9. This phenomenon was correlated with an adsorption of cell adhesion protein, fibronectin, onto surfaces ofPIPAAm-1.4 and -2.9. In the case of nano-ordered thin grafted surfaces, the surface chain mobility is strongly influenced by the thickness of PIPAAm grafted layers because dehydration of PIPAAm chains should be enhanced by the hydrophobic TCPS surfaces. PIPAAm graft amounts, that is, thickness of the PIPAAm grafted layers, play a crucial role in temperature-induced hydrophilic/hydrophobic property alterations and cell adhesion/detachment behavior.

Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces.

The hydrophobic monomer, n-butyl methacrylate (BMA) has been incorporated into thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) to lower PIPAAm phase transition temperatures necessary for systematically regulating cell adhesion on and detachment from culture dishes at controlled temperatures. Poly(IPAAm-co-BMA)-grafted dishes were prepared by electron beam irradiation methods, systematically changing BMA content in the feed. Copolymer-grafted surfaces decreased grafted polymer transition temperatures with increasing BMA content as shown by water wettabilities compared to homopolymer PIPAAm-grafted surfaces. Bovine endothelial cells readily adhered and proliferated on copolymer-grafted surfaces above collapse temperature at 37 degrees C, finally reaching confluence. Cell sheet detachment behavior from copolymer-grafted surfaces depended on the culture temperature and BMA content. In conclusion, cell attachment/detachment can be controlled to an arbitrary temperature by varying the content of hydrophobic monomer incorporated into PIPAAm grafted to culture surfaces.

Copolymerization of 2-carboxyisopropylacrylamide with N-isopropylacrylamide accelerates cell detachment from grafted surfaces by reducing temperature.

Acrylic acid (AAc) has been utilized to introduce reactive carboxyl groups to a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). However, AAc introduction shifts the copolymer phase transition temperatures higher and dampens the steep homopolymer phase transition with increasing AAc content. We previously synthesized 2-carboxyisopropylacrylamide (CIPAAm) having both a similar side chain structure to IPAAm and a functional carboxylate group in order to overcome these shortcomings. In the present study, these copolymers, grafted onto cell culture plastic, were assessed for cell adhesion control using their phase transition. AAc introduction to PIPAAm-grafted surfaces resulted in excessive surface hydration and hindered cell spreading in culture at 37 degrees C. In contrast, CIPAAm-containing copolymer-grafted surfaces exhibited relatively weak hydrophobicity similar to both homopolymer PIPAAm-grafted surfaces as well as commercial ungrafted tissue culture polystyrene dish surfaces. Cells adhered and spread well on these surfaces at 37 degrees C in culture. As observed previously on PIPAAm-grafted surfaces, cells were spontaneously detached from the copolymer-grafted surfaces by reducing culture temperature. Cell detachment was accelerated on the CIPAAm copolymer-grafted surfaces compared to pure IPAAm surfaces, suggesting that hydrophilic carboxyl group microenvironment in the monomer and polymer is important to accelerate grafted surface hydration below the lower critical solution temperature, detaching cells.

Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Cell Culture Surfaces

Recent progress in cell transplantation therapy to repair impaired hearts has encouraged further attempts to bioengineer 3-dimensional (3-D) heart tissue from cultured cardiomyocytes. Cardiac tissue engineering is currently pursued utilizing conventional technology to fabricate 3-D biodegradable scaffolds as a temporary extracellular matrix. By contrast, new methods are now described to fabricate pulsatile cardiac grafts using new technology that layers cell sheets 3-dimensionally. We apply novel cell culture surfaces grafted with temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), from which confluent cells detach as a cell sheet simply by reducing temperature without any enzymatic treatments. Neonatal rat cardiomyocyte sheets detached from PIPAAm-grafted surfaces were overlaid to construct cardiac grafts. Layered cell sheets began to pulse simultaneously and morphological communication via connexin43 was established between the sheets. When 4 sheets were layered, engineered constructs were macroscopically observed to pulse spontaneously. In vivo, layered cardiomyocyte sheets were transplanted into subcutaneous tissues of nude rats. Three weeks after transplantation, surface electrograms originating from transplanted grafts were detected and spontaneous beating was macroscopically observed. Histological studies showed characteristic structures of heart tissue and multiple neovascularization within contractile tissues. Constructs transplanted into 3-week-old rats exhibited more cardiomyocyte hypertrophy and less connective tissue than those placed into 8-week-old rats. Long-term survival of pulsatile cardiac grafts was confirmed up to 12 weeks. These results demonstrate that electrically communicative pulsatile 3-D cardiac constructs were achieved both in vitro and in vivo by layering cardiomyocyte sheets. Cardiac tissue engineering based on this technology may prove useful for heart model fabrication and cardiovascular tissue repair. The full text of this article is available at http://www.circresaha.org.

Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured cardiomyocyte sheets.

Recent progress in stem cell biology is likely to provide implantable sources of human cardiomyocytes in the near future. This possibility has encouraged cardiac tissue engineering. To construct heart-like tissue, we have exploited the capabilities of novel cell culture surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), to produce intact viable monolayer cell sheets simply by reducing culture temperature. Cultured chick embryonic cardiomyocyte sheets detached from PIPAAm-grafted surfaces were layered into tissue-like laminate stacks using hydrophilic support and transfer membranes. The layered cell sheets rapidly adhered to each other, establishing cell-to-cell connections characteristic of heart tissue, including desmosomes and intercalated disks. Bilayer cell sheets pulsed spontaneously and synchronously, altering their characteristic pulsing frequency with applied electric stimulation transmitted across the sheets. These results demonstrate that electrically communicative three-dimensional cardiac constructs can be achieved by stacking monolayer cardiomyocyte sheets. Cardiac tissue engineering based on this technology will facilitate new in vitro heart models and may prove useful for in vivo cardiovascular tissue repair.

Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface.

We developed a novel method to obtain designed shape cell sheets for tissue engineering. Shaping of cell sheets were achieved by the use of poly(N-isopropylacrylamide) (PIPAAm) and poly(N,N'-dimethylacrylamide) (PDMAAm) for temperature-responsive cell adhesive and cell nonadhesive domains, respectively. These polymers were covalently grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation with mask patterns. At 37 degrees C, human aortic endothelial cells (HAECs) attached, spread, and proliferated to make a monolayer only on PIPAAm-grafted domains. HAECs did not adhere on PDMAAm-grafted domains for more than 1 month even under the serum-supplemented condition. By reducing the culture temperature below 32 degrees C, PIPAAm changed to hydrophilic and HAEC sheets were detached from PIPAAm-grafted surfaces without any need of an enzyme such as trypsin. Cell-cell junctions were retained in the recovered cell sheets and easily moved to virgin TCPS dishes with the aid of hydrophilically modified polyvinylidenefluoride membranes as a supporter during the transfer. Moved cell sheets rapidly adhered onto the dish surfaces, and the supporter was easily peeled off from the cell layers. HAEC sheets transferred to new dishes revealed the identical shape and size to those before transfer. This novel technique is the only way to create, harvest, and transfer designed shape cell sheets and would have promising applications in tissue engineering.

Temperature-dependent modulation of blood platelet movement and morphology on poly( N-isopropylacrylamide)-grafted surfaces

Poly( N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32°C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32°C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ.