Covalent Immobilization of Collagen Type I to a Polydimethylsiloxane Surface for Preventing Cell Detachment by Retaining Collagen Molecules under Uniaxial Cyclic Mechanical Stretching Stress.

Surface modification of polydimethylsiloxane (PDMS) with an extracellular matrix (ECM) is useful for enhancing stable cell attachment. However, few studies have investigated the correlation between the stability of deposited ECM and cell behavior on the PDMS surfaces in external stretched cell culture systems. Herein, covalent collagen type I (Col)-immobilized PDMS surfaces were fabricated using 3-aminopropyl-trimethoxysilane, glutaraldehyde, and Col molecules. The immobilized collagen molecules on the PDMS surface were more stable and uniform than the physisorbed collagen. The cells stably adhered to the Col-immobilized surface and proliferated even under uniaxial cyclic mechanical stretching stress (UnCyMSt), whereas the cells gradually detached from the Col-physisorbed PDMS surface, accompanied by a decrease in the number of deposited collagen molecules. Moreover, the immobilization of collagen molecules enhanced cell alignment under the UnCyMSt. This study reveals that cell adhesion, proliferation, and alignment under the UnCyMSt can be attributed to the retention of collagen molecules on the PDMS surface.

Synthesis of Temperature-Responsive Polymers Containing Piperidine Carboxamide and N,N-diethylcarbamoly Piperidine Moiety via RAFT Polymerization.

In this study, poly(N-acryloyl-nipecotamide) (PNANAm), poly(N-acryloyl-isonipecotamide) (PNAiNAm), and poly(N-acryloyl-N,N-diethylnipecotamide) (PNADNAm) are synthesized as novel temperature-responsive polymers using reversible addition-fragmentation chain-transfer polymerization. Aqueous solutions of these three polymers are examined via temperature-dependent optical transmittance measurements. The PNANAm sample with a hydrophilic terminal group shows an upper critical solution temperature (UCST) in phosphate-buffered saline (PBS) when its molecular weight (Mn ) is 7600 or higher, whereas PNANAm (Mn < 7600) is soluble. The UCST is influenced by molecular weight and the polymer concentration. In contrast, PNANAm sample with nonionic terminal group shows UCST, when Mn is below 7600, suggesting that the terminal nonionic group possibly increases UCST of PNANAm. The urea addition experiment suggests that the driving force for expression of UCST of PNANAm is the formation of inter-and intramolecular hydrogen bonds among the polymer chains. PNAiNAm is soluble in PBS but exhibits an UCST in an appropriate concentration of ammonium sulfate. In contrast, PNADNAm exhibits a lower critical solution temperature. Comparing the chemical structure of these polymers and their phase transition behaviors suggests that the carboxamide group position in the piperidine ring could determine the UCST expression. These results could help design temperature-responsive polymers with a desired the cloud point temperature.

Poly( N-isopropylacrylamide)-Grafted Polydimethylsiloxane Substrate for Controlling Cell Adhesion and Detachment by Dual Stimulation of Temperature and Mechanical Stress.

Stretchable temperature-responsive cell culture surfaces composed of poly( N-isopropylacrylamide) (PIPAAm) gel-grafted polydimethylsiloxane (PIPAAm-PDMS) were prepared to demonstrate that dual stimulation of temperature and mechanical stress extensively altered graft polymer thickness, surface wettability, and cell detachment behavior. The PIPAAm-PDMS surface was hydrophilic and hydrophobic below and above the lower critical solution temperature, respectively, which was ascribed to the phase transition of PIPAAm chains. When uniaxial stretching was applied, the grafted PIPAAm gel surface was modulated to be more hydrophobic as shown by an increase in the contact angle. Atomic force microscopy observation revealed that uniaxial stretching made the grafted gel layer thinner and deformed the nanoscale aggregates of the grafted PIPAAm gel, implying extension of the PIPAAm chains. The stretched PIPAAm-PDMS became more cell adhesive than the unstretched PIPAAm-PDMS at 37 °C. Furthermore, dual stimulation, shrinking the already stretched PIPAAm-PDMS and decreasing the temperature, induced more rapid cell detachment than only a change in temperature did. Similarly, upon comparison with a single stimulation of a change in temperature or mechanical stress, dual stimulation accelerated cell sheet detachment and harvesting. This new stretchable and temperature-responsive culture surface can easily adjust the surface property to a different cell adhesiveness by appropriately combining each stimulus and enable the fabrication of cell sheets of various species.

Effect of Temperature Changes on Serum Protein Adsorption on Thermoresponsive Cell-Culture Surfaces Monitored by A Quartz Crystal Microbalance with Dissipation.

Thermoresponsive cell-culture polystyrene (PS) surfaces that are grafted with poly(N-isopropylacrylamide) (PIPAAm) facilitate the cultivation of cells at 37 °C and the detachment of cultured cells as a sheet with an underlying extracellular matrix (ECM) by reducing the temperature. However, the ECM and cell detachment mechanisms are still unclear because the detachment of cells from thermoresponsive surfaces is governed by complex interactions among the cells/ECM/surface. To explore the dynamic behavior of serum protein adsorption/desorption, thermoresponsive surfaces that correspond to thermoresponsive tissue-culture PS dishes were formed on sensor chips for quartz crystal microbalance with dissipation (QCM-D) measurements. X-ray photoelectron spectroscopy (XPS) measurements and temperature-dependent frequency and dissipation shifts, Δf and ΔD, using QCM-D revealed that the thermoresponsive polymers were successfully grafted onto oxidized, thin PS films on the surfaces of the sensor chips. Increased amounts of adsorbed bovine serum albumin (BSA) and fibronectin (FN) were observed on the thermoresponsive polymer-grafted surfaces at 37 °C when compared with those at 20 °C because of enhanced hydrophobic interactions with the hydrophobic, thermoresponsive surface. While the calculated masses of adsorbed BSA and FN using QCM-D were 3⁻5 times more than those that were obtained from radiolabeling, the values were utilized for relative comparisons among the same substrate. More importantly, the thermoresponsive, dynamic behavior of serum protein adsorption/desorption was monitored using the QCM-D technique. Observations of this dynamic behavior revealed that the BSA and FN that were adsorbed at 37 °C remained on both surfaces after decreasing the temperature to 20 °C.

Monolithic silica rods grafted with thermoresponsive anionic polymer brushes for high-speed separation of basic biomolecules and peptides.

Thermoresponsive anionic copolymer brushes, poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide) [P(IPAAm-co-AAc-co-tBAAm)], were grafted onto a monolithic silica rod column through surface-initiated atom-transfer radical polymerization (ATRP) to prepare an effective thermoresponsive anionic chromatography matrix. An ATRP initiator was attached to the rod surface. N-Isopropylacrylamide (IPAAm), tert-butyl acrylate (tBA), tert-butylacrylamide (tBAAm), and the ATRP catalyst CuCl/CuCl2/tris[2-(N,N-dimethylamino)ethyl]amine were dissolved in 2-propanol, and the reaction mixture was pumped into the initiator-modified column. After grafting P(IPAAm-co-tBA-co-tBAAm) on the monolithic silica surfaces, deprotection of the tert-butyl group of tBA was performed. Chromatographic analysis showed that the prepared column was able to separate catecholamine derivatives and angiotensin subtypes within a shorter analysis time (5 min) than a silica-bead-packed column modified with the same copolymer brush could. These results indicated that the prepared copolymer-modified monolithic silica rod column may be a promising bioanalytical and bioseparation tool for rapid analysis of basic bioactive compounds and peptides.

Thermoresponsive anionic copolymer brushes containing strong acid moieties for effective separation of basic biomolecules and proteins.

A thermoresponsive copolymer brush possessing the sulfonic acid group, poly(N-isopropylacrylamide (IPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-tert-butylacrylamide (tBAAm)), was grafted onto the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer and copolymer brushes on silica beads were characterized by observing the phase transition profile, CHNS elemental analysis, X-ray photoelectron spectroscopy, and gel permeation chromatography. The phase transition profile indicated that an appropriate AMPS composition for enabling thermally modulated property changes is 5 mol %, while excessive amounts of sulfonic acid groups prevented copolymer phase transition. Chromatographic elutions of catecholamine derivatives and basic proteins were observed, using the prepared copolymer brush-modified beads as chromatographic matrices, and the results suggest that the beads interact with these analytes through relatively strong electrostatic interactions. Thus, poly(IPAAm-co-AMPS-co-tBAAm) brush-modified beads will be useful for effective thermoresponsive chromatography matrices that separate basic biomolecules through strong electrostatic interactions.

Thermoresponsive copolymer brushes possessing quaternary amine groups for strong anion-exchange chromatographic matrices.

A thermoresponsive copolymer incorporating a quaternary amine group, poly(N-isopropylacrylamide-co-3-acrylamidopropyl trimethylammonium chloride (APTAC)-co-tert-butylacrylamide), was conjugated to the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer- and copolymer brush-modified beads were characterized by CHN elemental analysis, X-ray photoelectron spectroscopy, gel permeation chromatography, and observation of phase transition profiles. Phase transition profiles of the prepared copolymer indicated that 5 mol % APTAC is suitable for enabling thermally modulated property changes in the copolymer. Chromatographic elution behaviors of adenosine nucleotides and proteins were observed using prepared beads as chromatography matrices. Higher retention time of adenosine nucleotides and strong protein adsorption behavior were observed compared with those on beads with tertiary amine groups, because of the strong basic properties. Therefore, copolymer brush modified beads will be useful as thermoresponsive ion-exchange chromatographic matrices.

Thermoresponsive polymer brush on monolithic-silica-rod for the high-speed separation of bioactive compounds.

Poly(N-isopropylacrylamide), one of the most utilized thermoresponsive polymers, brush-grafted monolithic-silica columns were prepared through surface-initiated atom transfer radical polymerization (ATRP) for effective thermoresponsive-chromatography matrices. ATRP initiator was grafted on monolithic silica-rod surfaces by flowing a toluene solution containing ATRP initiator into monolithic silica-rod columns. N-Isopropylacrylamide (IPAAm) monomer and CuCl/CuCl(2)/Me(6)TREN, an ATRP catalytic system, were dissolved in 2-propanol, and the reaction solution was pumped into the preprepared initiator-modified columns at 25 °C for 16 h. The constructed PIPAAm-brush structure on the monolithic silica-rod surface was confirmed by XPS, elemental analysis, SEM observation, and GPC measurement of grafted PIPAAm. The prepared monolithic silica-rod columns were also characterized by chromatographic analysis. PIPAAm-brush-modified monolithic silica-rod columns were able to separate hydrophobic steroids with a short analysis time (10 min), compared to PIPAAm-brush-modified silica-beads-packed columns, because of the horizontally limited diffusion path length of monolithic supporting materials. Additionally, diluted PIPAAm-brush monolithic silica-rod column gave a further shorting analysis time (5 min). These results indicated (1) surface-initiated ATRP constructed PIPAAm-brush structures on monolithic silica-rod surfaces and (2) PIPAAm-brush grafted monolithic silica-rod column prepared by ATRP was a promising tool for analyzing hydrophobic-bioactive compounds with a short analysis time.

Preparation of thermoresponsive anionic copolymer brush surfaces for separating basic biomolecules.

Poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) (poly(IPAAm-co-AAc-co-tBAAm) brush grafted silica beads were prepared through a surface-initiated atom transfer radical polymerization (ATRP) with CuCl/CuCl(2)/Me(6)TREN catalytic system in 2-propanol at 25 degrees C for 4 h. The prepared beads were characterized by chromatographic analysis. Basic analytes, catecholamine derivatives, and angiotensin peptides could be separated by a short column length containing the beads because of its high densely grafted copolymer structure. Chromatograms for catecholamine derivatives were obtained with high resolution peaks due to their electrostatic and hydrophobic interactions to the densely grafted anionic copolymers on the beads. Effective separation of angiotensin peptides was performed near the lower critical solution temperature of copolymers, because the total electrostatic and hydrophobic interactions between the copolymer and the analytes become strong at the temperature. These results indicated that the copolymer brush grafted surfaces prepared by ATRP was an effective tool for separating basic biomolecules by modulating the electrostatic and hydrophobic interactions.

Preparation of thermoresponsive cationic copolymer brush surfaces and application of the surface to separation of biomolecules.

We have prepared poly( N-isopropylacrylamide (IPAAm)- co-2-(dimethylamino)ethylmethacrylate (DMAEMA)) brush-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) using the CuCl/CuCl 2/Me 6TREN catalytic system in 2-propanol at 25 degrees C for 16 h. The prepared temperature-responsive surfaces were characterized by chromatographic analysis using the modified silica beads as stationary phases. Chromatographic retention times for adenosine nucleotides in aqueous mobile phases were significantly increased compared to that previously reported for other cationic hydrogel surfaces, indicating that strong electrostatic cationic copolymer brush interactions occur between the surfaces and nucleotide analytes. Retention times for adenosine nucleotides significantly decreased with increasing column temperature, explained by the decreasing basicity in the copolymer with increasing temperature. Step-temperature gradients from 10 to 50 degrees C shorten ATP retention times. These results indicate that cationic copolymer brush surfaces prepared by ATRP can rapidly alter their electrostatic properties by changing aqueous temperature.

The use of biotin-avidin binding to facilitate biomodification of thermoresponsive culture surfaces.

Here, we report biomodification of temperature-responsive culture surfaces with biotinylated biomolecules utilizing streptavidin and biotinylation of the surfaces. Poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) was covalently grafted onto tissue culture polystyrene (TCPS) dishes. Biotinylated Arg-Gly-Asp-Ser (RGDS) peptides with different spacer lengths (biotin-conjugated G(n)RGDS (n=1,6,12,16)) were examined. Human umbilical vein endothelial cells (HUVECs) adhered and were well spread on G(12)RGDS-immobilized surfaces in the absence of serum at 37 degrees C, while much less cell adhesion was observed with the other peptides. Adhered HUVECs were detached on reducing temperature to 20 degrees C, or on adding free RGDS peptide. Interestingly, cell detachment was accelerated by applying both these techniques. Consequently, by optimizing the spacer length, biomolecules can be functionally immobilized onto thermoresponsive surfaces via the affinity binding between avidin and biotin.

Surface characterization of poly(N-isopropylacrylamide) grafted tissue culture polystyrene by electron beam irradiation, using atomic force microscopy, and X-ray photoelectron spectroscopy.

To understand features of polymers grafted by electron beam (EB) irradiation method, we investigated topology of poly(N-isopropylacrylamide) (PIPAAm) grafted tissue culture polystyrene (TCPS) (PIPAAm-TCPS) prepared by EB irradiation, using atomic force microscopy (AFM) in air and under aqueous conditions. Furthermore, surfaces properties of PIPAAm-TCPS surfaces before and after cell culture were also examined for evaluation of functionality of the surface as biomaterials, using XPS analysis. Three types of PIPAAm-TCPSs with different graft densities (1.0+/-0.1, 1.6+/-0.1, and 2.0+/-0.1 microg/cm2 of the grafted) were obtained (abbreviated as 11PIPAAm-, 16PIPAAm-, and 20PIPAAm-TCPS) by using different initial monomer concentration (20, 55, and 65 wt%). Contact angles (costheta value) of the surfaces increased with an increase in density of the grafted polymer. AFM observation in air clearly revealed that original TCPS surface possesses scratched and grooved topology (ca. 10 nm height of the scratch), while PIPAAm-TCPSs surfaces exhibited nanoordered PIPAAm particle-like domains. The size of the particles also increased proportionally initial IPAAm monomer concentration. The 11PIPAAm-and 16PIPAAm-TCPS surfaces having ca. 10-30 nm and ca. 40-50 nm size of the particles also displayed scratched and grooved topology featured in basal TCPS. However, the larger sizes of the particles (ca. 40-100 nm) formed on 20PIPAAm-TCPS surfaces adequately conceals the topological feature of the basal TCPS surfaces. The AFM images indicate that the graft polymer is as ultra thin as the scratch and grooves featured on basal TCPS are discernible, and the grafted PIPAAm layer become thicker with an increase of the monomer concentration. For 16PIPAAm-TCPS surfaces, the nanoordered particles were also observable in aqueous conditions at 20 degrees C and 37 degrees C. Comparison between the images obtained at 20 degrees C and 37 degrees C suggest that the domains are not likely to exhibit significant swelling and shrinking by temperature change, although the topology of PIPAAm grafted onto clover glass surface (50 microm thickness of the gel layer) were dramatically changed by temperature change in early reports. This difference should be due to ultra thin thickness of the grafted PIPAAm, which is subject to more restricted molecular motion by basal hydrophobic TCPS interfaces, as we reported previously. XPS C1s and N1s spectra of 16PIPAAm-TCPS surface after removal of cells suggest that proteins and/or peptides components possibly remained on the surfaces. Based on results from XPS analysis, we further discuss surface properties of 16PIPAAm-TCPS as biomaterials, comparing those of PIPAAm grafted polystyrene prepared by a radio frequency plasma method used in recent reports.

Fabrication of a cell array on ultrathin hydrophilic polymer gels utilising electron beam irradiation and UV excimer laser ablation.

Most of the surface patterning methods currently applied are based on lithography techniques and microfabrication onto silicon or glass substrates. Here we report a novel method to prepare patterned surfaces on polystyrene substrates by grafting ultrathin cell-repellent polymer layers utilising both electron beam (EB) polymerisation and local laser ablation techniques for microfabrication. Polyacrylamide was grafted onto tissue culture polystyrene (TCPS) dishes using EB irradiation. Water contact angles for these PAAm-grafted TCPS surfaces were less than 10 degrees (costheta = 0.99) with PAAm grafted amounts of 1.6 microg/cm(2) as determined by ATR/FT-IR. UV excimer laser (ArF: 193 nm) ablation resulted in the successful fabrication of micropatterned surfaces composed of hydrophilic PAAm and hydrophobic basal polystyrene layers. Bovine carotid artery endothelial cells adhered only to the ablated domains after pretreatment of the patterned surfaces with 15 microg/mL fibronectin at 37 degrees C. The ablated domain sizes significantly influenced the number of cells occupying each domain. Cell patterning functionality of the patterned surfaces was maintained for more than 2 months without loss of pattern fidelity, indicating that more durable cell arrays can be obtained compared to those prepared by self-assembled monolayers of alkanethiols, as described in previous reports. The surface fabrication techniques presented here can be utilised for the preparation of cell-based biosensors as well as tissue engineering constructs.

Accelerated cell-sheet recovery from a surface successively grafted with polyacrylamide and poly(N-isopropylacrylamide).

A double polymeric nanolayer consisting of poly(N-isopropylacrylamide) (PIPAAm) and hydrophilic polyacrylamide (PAAm) was deposited on tissue culture polystyrene (TCPS) surfaces using electron beam irradiation to form a new temperature-responsive cell culture surface in which the basal hydrophilic PAAm component in the double polymeric layer promotes the hydration of the upper PIPAAm layer and induces rapid cell detachment compared to a conventional temperature-responsive cell culture surface, PIPAAm-grafted TCPS (PIPAAm-TCPS). Take-off angle-dependent X-ray photoelectron spectroscopy spectral analysis demonstrated that the grafted PIPAAm and PAAm components were located in the upper and basal regions of the double polymeric layer, respectively, suggesting that the double polymeric layer forms an inter-penetrating-network-like structure with PAAm at the basal portion of the PIPAAm grafted chains. The wettability of the temperature-responsive cell culture surfaces with the double polymeric layer tended to be more hydrophilic, with an increase in the basal PAAm graft density at a constant PIPAAm graft density. However, when the graft densities of the upper PIPAAm and basal PAAm were optimized, the resulting temperature-responsive cell culture surface with the double polymeric layer exhibited rapid cell detachment while maintaining cell adhesive character comparable to that of PIPAAm-TCPS. The cell adhesive character was altered from cell-adhesive to cell-repellent with increasing PAAm or PIPAAm graft density. The cell adhesive character of the temperature-responsive cell culture surfaces was relatively consistent with their contact angles. These results strongly suggest that the basal PAAm surface properties affect the degree of hydration and dehydration of the subsequently grafted PIPAAm. In addition, the roles of the hydrophilic component in accelerating cell detachment are further discussed in terms of the mobility of the grafted PIPAAm chains. Applications of this insight might be useful for designing temperature-responsive cell culture surfaces for achieving efficient cell culture and quick target cell detachment.

Modulation of cell adhesion and detachment on thermo-responsive polymeric surfaces through the observation of surface dynamics.

Various thermo-responsive polymeric surfaces were evaluated in terms of cell adhesion/detachment and surface analysis. Three kinds of thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) surfaces were prepared by an electron beam irradiation (PIPAAm-EB), a reversible addition fragmentation polymerization (PIPAAm-RAFT), and a redox polymerization (PIPAAm-Redox). Although cell adhesion and detachment on surfaces of PIPAAm-EB and PIPAAm-RAFT were able to be modulated by altering their surface characters with changing the amounts of polymers, the adhesion and detachment were hardly controlled on PIPAAm-Redox surfaces, even though the amounts of polymers on the surface were able to be modulated. Atomic force microscopy (AFM) probed the interactions between AFM tip and the polymeric surface for further investigating a different conformation of polymeric surface. The modification of AFM tip surface coated with octadecyltrichlorosilane was found to change the interaction between the thermo-responsive surface and the tip. Adhesion force analysis clearly showed changes in the hydrophilic/hydrophobic characters of three kinds of thermo-responsive surfaces immediately after a change in temperature. From the kinetics study of AFM, PIPAAm-EB and PIPAAm-RAFT surfaces became hydrophilic less than 30 min after temperature decrease, but PIPAAm-Redox surfaces required 120 min to become hydrophilic after temperature reduction. These results indicated that a faster conformational change triggered cell detachment and a slow conformation change hardly affected cell detachment. Therefore, polymeric conformation on solid substrate was an important factor for modulating cell adhesion and detachment.

Thermally modulated cationic copolymer brush on monolithic silica rods for high-speed separation of acidic biomolecules.

Poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethylmethacrylate(DMAEMA)-co-tert-butylacrylamide (tBAAm)), a thermoresponsive-cationic-copolymer, brush-grafted monolithic-silica column was prepared through surface-initiated atom transfer radical polymerization (ATRP) for effective thermoresponsive anion-exchange chromatography matrices. ATRP-initiator was grafted on monolithic silica-rod surfaces by flowing a toluene solution containing ATRP initiator into monolithic silica-rod columns. IPAAm, DMAEMA, and tBAAm monomers and CuCl/CuCl2/Me6TREN, an ATRP catalytic system, were dissolved in 2-propanol, and the reaction solution was pumped into the preprepared initiator modified columns at 25 °C for 16 h. The constructed copolymer-brush structure on monolithic silica-rod surface was confirmed by X-ray photoelectron spectroscopy (XPS), elemental analysis, scanning electron microscopy (SEM) observation, and gel permeation chromatography (GPC) measurement of grafted copolymer. The prepared monolithic silica-rod columns were also characterized by chromatographic analysis. The cationic copolymer brush modified monolithic silica-rod columns were able to separate adenosine nucleotides with a shorter analysis time (4 min) than thermoresponsive copolymer brush-modified silica-bead-packed columns, because of the reduced diffusion path length of monolithic supporting materials. These results indicated that thermoresponsive cationic copolymer brush grafted monolithic silica-rod column prepared by ATRP was a promising tool for analyzing acidic-bioactive compounds with a remarkably short analysis time.

Shear stress-dependent cell detachment from temperature-responsive cell culture surfaces in a microfluidic device.

A new approach to quantitatively estimate the interaction between cells and material has been proposed by using a microfluidic system, which was made of poly(dimethylsiloxane) (PDMS) chip bonding on a temperature-responsive cell culture surface consisted of poly(N-isopropylacrylamide) (PIPAAm) grafted tissue culture polystyrene (TCPS) (PIPAAm-TCPS) having five parallel test channels for cell culture. This construction allows concurrently generating five different shear forces to apply to cells in individual microchannels having various resistance of each channel and simultaneously gives an identical cell incubation condition to all test channels. NIH/3T3 mouse fibroblast cells (MFCs) and bovine aortic endothelial cells (BAECs) were well adhered and spread on all channels of PIPAAm-TCPS at 37 °C. In our previous study, reducing culture temperature below the lower critical solution temperature (LCST) of PIPAAm (32 °C), cells detach themselves from hydrated PIPAAm grafted surfaces spontaneously. In this study, cell detachment process from hydrated PIPAAm-TCPS was promoted by shear forces applied to cells in microchannels. Shear stress-dependent cell detachment process from PIPAAm-TCPS was evaluated at various shear stresses. Either MFCs or BAECs in the microchannel with the strongest shear stress were found to be detached from the substrate more quickly than those in other microchannels. A cell transformation rate constant C(t) and an intrinsic cell detachment rate constant k(0) were obtained through studying the effect of shear stress on cell detachment with a peeling model. The proposed device and quantitative analysis could be used to assess the possible interaction between cells and PIPAAm layer with a potential application to design a cell sheet culture surface for tissue engineering.

High stability of thermoresponsive polymer-brush-grafted silica beads as chromatography matrices.

Thermo-responsive chromatography matrices with three types of graft architecture were prepared, and their separation performance and stability for continuous use were investigated. Poly(N-isopropylacrylamide)(PIPAAm) hydrogel-modified silica beads were prepared by a radical polymerization through modified 4,4'-azobis(4-cyanovaleric acid) and N,N'-methylenebisacrylamide. Dense PIPAAm brush-grafted silica beads and dense poly(N-tert-Butylacrylamide (tBAAm)-b-IPAAm) brush-grafted silica beads were prepared through a surface-initiated atom transfer radical polymerization (ATRP) using CuCl/CuCl(2)/ Tris(2-(N,N-dimethylamino)ethyl)amine (Me(6)TREN) as an ATRP catalytic system and 2-propanol as a reaction solvent. Dense PIPAAm brush-grafted silica beads exhibited the highest separation performance because of their strong hydrophobic interaction between the densely grafted well-defined PIPAAm brush on silica-bead surfaces and analytes. Using an alkaline mobile phase, dense themoresponsive polymer brushes, especially having a hydrophobic basal layer, exhibited a high stability for continuous use, because polymer brush on the silica bead surfaces prevented the access of water to silica surface, leading to the hydrolysis of silica and cleavage of grafted polymers. Thus, the precisely modulating graft configuration of thermoresponsive polymers provided chromatography matrices with a high separation efficiency and stability for continuous use, resulting in elongating the longevity of chromatographic column.

Thermally-modulated on/off-adsorption materials for pharmaceutical protein purification.

For the development of temperature-responsive adsorption materials for pharmaceutical protein purification, poly(N-isopropylacrylamide-co-N,N-dimethylaminopropylacrylamide-co-N-tert-butylacrylamide) (P(IPAAm-co-DMAPAAm-co-tBAAm) brush grafted silica beads were prepared through a surface-initiated atom transfer radical polymerization (ATRP). The prepared silica beads as a chromatographic stationary phase were evaluated by observing their thermo-responsive elution profiles of plasma proteins including human serum albumin (HSA) and γ-globulin. Chromatograms of two proteins indicated that negatively-charged HSA was adsorbed on the cationic copolymer brush modified silica beads at higher temperatures with low concentration of phosphate buffer (PB) (pH 7.0) as a mobile phase. The HSA adsorption was attributed to (1) an enhanced electrostatic interaction with the cationic copolymer brush at low concentration of PB and (2) an increased hydrophobic interaction from the dehydrated copolymer at high temperature. Step-temperature gradient enabled HSA and γ-globulin to be separated by the modulation of HSA adsorption/desorption onto the copolymer brush grafted silica beads. These results suggested that the prepared copolymer brush grafted silica beads adsorbed negatively-charged proteins both through electrostatic and hydrophobic interactions by the modulation of column temperature and gave attractive adsorption materials for protein purification process.

Effect of reaction solvent on the preparation of thermo-responsive stationary phase through a surface initiated atom transfer radical polymerization.

Poly(N-isopropylacrylamide) (PIPAAm) brush grafted silica beads, a thermo-responsive chromatographic stationary phase, were prepared through a surface-initiated atom transfer radical polymerization (ATRP) using 2-propanol, N,N-dimethylformamide (DMF), and water as reaction solvents. The rate of grafting PIPAAm on silica bead surfaces was different and found to be dependent on the reactivity of reaction solvent. Temperature-dependent elution profiles of hydrophobic steroids from the prepared-beads-packed columns were found to be different, although the graft amounts of PIPAAm were similar on silica bead surfaces. Especially, prepared beads using 2-propanol exhibited a higher resolution than those using DMF. Calibration curves using glucose and pullulan suggested that beads prepared using DMF prohibited analytes to diffuse into the pores. On the contrary, beads prepared using 2-propanol allowed analytes to diffuse into the pores. The pore diameter of the prepared beads, measured by N(2) adsorption-desorption measurement, suggested that beads using 2-propanol has relatively larger pore diameter than those using DMF. Thus, the reaction solvent in surfaces-initiated ATRP affected the grafting configuration of PIPAAm on porous silica-bead surfaces, leading to the different separation efficiency of stationary phase for bioactive compounds.