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.     

Surface-active and stimuli-responsive polymer--Si(100) hybrids from surface-initiated atom transfer radical polymerization for control of cell adhesion

A simple two-step method was developed for the covalent immobilization of atom-transfer radical polymerization (ATRP) initiators on the hydrogen-terminated Si(100) (Si-H) surface. Well-defined functional polymer-Si hybrids, consisting of covalently tethered brushes of poly(ethylene glycol) monomethacrylate (PEGMA) polymer, N-isopropylacrylamide (NIPAAm) polymer, and NIPAAm-PEGMA copolymers and block copolymers on Si-H surfaces, were prepared via surface-initiated ATRP. Kinetics study revealed that the chain growth from the silicon surface was consistent with a "controlled" process. Surface cultures of the cell line 3T3-Swiss albino on the hybrids were evaluated. The PEGMA graft-polymerized silicon [Si-g-P(PEGMA)] surface is very effective in preventing cell attachment and growth. At 37 degrees C [above the lower critical solution temperature (LCST, approximately 32 degrees C) of NIPAAm], the seeded cells adhered, spread, and proliferated on the NIPAAm graft polymerized silicon [Si-g-P(NIPAAm)] surface. Below the LCST, the cells detached from the Si-g-P(NIPAAm) surface spontaneously. Incorporation of PEGMA units into the NIPAAm chains of the Si-g-P(NIPAAm) surface via copolymerization resulted in more rapid cell detachment during the temperature transition. The "active" chain ends on the Si-g-P(PEGMA) and Si-g-P(NIPAAm) hybrids were also used as the macroinitiators for the synthesis of diblock copolymer brushes. Thus, not only are the hybrids potentially useful as stimuli-responsive adhesion modifiers for cells in silicon-based biomedical microdevices but also the active chain ends on the hybrid surfaces offer opportunities for further surface functionalization and molecular design.     

Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers

We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339-344, 1997.     

Swelling induced detachment of chondrocytes using RGD-modified poly(N-isopropylacrylamide) hydrogel beads

Thermally sensitive poly(N-isopropylacrylamide, NIPAAm) hydrogel beads conjugated with a cell adhesive motif, GRGDY, were prepared and utilized as cell culture substrate for chondrocytes. They were produced to be uniform in size and distribution by using calcium alginate as a temporal mold. The RGD moieties were introduced, in a spatially selective manner, to the surface of the beads by conjugating GRGDY under the precollapsed state at a higher temperature above the lower critical solution temperature (LCST). These RGD-conjugated polyNIPAAm beads demonstrated a reversible swelling and deswelling behavior around the LCST, which enabled the chondrocytes attached on the surface of collapsed beads at 37 degrees C to readily detach when the temperature was shifted below 37 degrees C. The cell detachment percentage was largely affected by the temperature-dependent reswelling extent of the collapsed RGD-modified beads.     

RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.     

Regulation of cell adhesion using a signal-responsive membrane substrate

We have developed a novel cell culture material that regulates cell adhesion by changes in potassium ion concentration. The material is a polyethylene substrate grafted to a copolymer of the thermoresponsive polymer N-isopropylacrylamide (NIPAM) and benzo-18-crown- 6-acrylamide (BCAm), with a pendant crown ether as sensor. The crown ether recognizes potassium ion concentrations and NIPAM conformational changes lead to changes in the hydrophobicity/hydrophilicity balance of the entire polymer at constant cell culture temperatures. Although cells were successfully cultured on the ion recognition material in normal culture medium at 37 degrees C, the cells could be detached from the material surface by adding potassium ions alone, without proteolytic enzymes, because the surface to which the cells were attached altered its surface characteristics to a more hydrophilic state. Therefore, cell layers with intact cell-to-cell junctions and high activities were successfully recovered. Furthermore, by changing the target sensors, this material will be able to control cell adhesion through various cellular signals.     

A novel method to prepare multicellular spheroids from varied cell types

A simple method for preparing multicellular spheroids from varied cell types has been successfully developed by using a stepwise gradient surface in cell attachability or detachability. The surface was composed of poly-N-isopropylacrylamide (PNIPAAm), a temperature responsive polymer, as a cell detaching component, and collagen as a cell attaching component. The surface functions as a culture substratum at 37 degrees C; then, when lowering the temperature of culture medium, the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature (LCST; about 30 degrees C), but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. The stepwise gradient surface which consisted of six different sectors was prepared by exposing a surface of the PNIPAAm-collagen mixture to ultraviolet (UV) irradiation six times using a photomask, sliding the hole position in the photomask, and changing the energy of UV irradiation. This was because crosslinking of collagen depended on the energy of UV irradiation, then, cell attachability to and detachability from the surface were tightly controlled by changing the energy.The stepwise gradient surface allowed us to easily determine optimal surface conditions to obtain good cell attachment and detachment as a self-supporting sheet from the surface to prepare multicellular spheroids. According to the evaluation of the attachability and detachability of 23 cell types, the optimal surface condition remarkably depended on each cell type. The detached cells under optimal surface conditions, including fibroblasts, osteoblastic cells, smooth muscle cells, and measangial cells, which were very difficult to form spherioids using conventional methods, were able to form multicellular spheroids. The results clearly demonstrate that the above-described method for preparing multicellular spheroids can be applied to varied cell types. (c) 1995 John Wiley & Sons, Inc.     

A novel method to prepare size-regulated spheroids composed of human dermal fibroblasts

A simple method to prepare size-regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly-N-isopropyl-acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37 degrees C, then when lowering the temperature of culture medium the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30 degrees C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV-irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 mug/cm(2) (collagen ratio, 4.5%), UV-irradiation energy of 2000 J/m(2) was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV-irradiation, it was possible to obtain cell-adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 mum in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 mum in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size-regulated spheroids for different applications; for example, in toxicology tests. (c) 1994 John Wiley & Sons, Inc.     

Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels

In this study, a novel yet simple method, using a thermoreversible hydrogel system coated on tissue culture polystyrene (TCPS) dishes, was developed for harvesting living cell sheets. The hydrogel system was prepared by simply pouring aqueous methylcellulose (MC) solutions blended with distinct salts on TCPS dishes at 20 degrees C. For the applications to cell culture, only those aqueous MC compositions that may form a gel at 37 degrees C were chosen for the study. It was found that the hydrogel coating composed of 8% MC blended with 10 g/L PBS (phosphate buffered saline) (the MC/PBS hydrogel, with a gelation temperature of approximately 25 degrees C) stayed intact throughout the entire course of cell culture. To improve cell attachments, the MC/PBS hydrogel at 37 degrees C was evenly spread with a neutral aqueous collagen at 4 degrees C. The spread aqueous collagen gradually reconstituted with time and thus formed a thin layer of collagen (the MC/PBS/collagen hydrogel). After cells reached confluence, a continuous monolayer cell sheet formed on the surface of the MC/PBS/collagen hydrogel. When the grown cell sheet was placed outside of the incubator at 20 degrees C, it detached gradually from the surface of the thermoreversible hydrogel spontaneously, without treating with any enzymes. The results obtained in the MTT assay demonstrated that the cells cultured on the surface of the MC/PBS/collagen hydrogel had an even better activity than those cultured on an uncoated TCPS dish. After harvesting the detached cell sheet, the remaining viscous hydrogel system is reusable. Additionally, the developed hydrogel system can be used for culturing a multilayer cell sheet. The obtained living cell sheets may be used for tissue reconstructions.     

Artificial cell adhesive proteins engineered by grafting the Arg-Gly-Asp cell recognition signal: factors modulating the cell adhesive activity of the grafted signal.

An artificial cell adhesive protein could be engineered by grafting the RGDS tetrapeptide, the core sequence of the major cell adhesive site of fibronectin, to a truncated form of Staphylococcal protein A (tSPA) via cassette mutagenesis of the tSPA expression vector pRIT2T [T. Maeda et al. (1989) J. Biol. Chem. 264, 15165-15168]. We synthesized a panel of tSPA derivatives grafted with various RGDS-containing oligopeptides to address the problem of how the cell adhesive activity of the resulting tSPA derivatives was affected by the length and amino acid sequence of the grafted oligopeptides and by the sites on tSPA where the extra oligopeptides were inserted. The results showed that (i) the amino acid residues flanking the RGDS core sequence played a key role in modulating the cell adhesive activity of the grafted RGDS signal; (ii) at least two sites on tSPA, each corresponding to on e of the two HindIII sites of pRIT2T, were competent in sustaining the cell adhesive activity of the grafted signal; and (iii) the divalent tSPA containing the RGDS signal at both sites was more active than monovalent derivatives containing only one signal at either site. These results provide a strategic basis for engineering of artificial cell adhesive proteins by grafting the RGDS signal.     

Simultaneous enhancement of cell proliferation and thermally induced harvest efficiency based on temperature-responsive cationic copolymer-grafted microcarriers

The development of large-scale suspension cell cultures using microcarriers has long been a focus of attention in the fields of pharmacy and biotechnology. Previously, we developed cell-detachable microcarriers based on temperature-responsive poly(N-isopropylacrylamide) (PIPAAm)-grafted beads, on which adhering cells can be noninvasively harvested by only reducing the temperature without the need for proteolytic enzyme treatment. In this study, to improve the cell harvest efficiency from bead surfaces while maintaining cell adhesion and proliferation properties, we prepared temperature-responsive cationic copolymer-grafted beads bearing a copolymer brush consisting of IPAAm, positively charged quaternary amine monomer (3-acrylamidopropyl trimethylammonium chloride; APTAC), and hydrophobic monomer (N-tert-butylacrylamide; tBAAm). The incorporation of positively charged APTAC into the grafted copolymer brush facilitated bead dispersibility in a cell culture system containing Chinese hamster ovary (CHO-K1) cells and consequently allowed for enhanced cell proliferation in the system compared to that of unmodified CMPS and conventional PIPAAm homopolymer-grafted beads. Additionally, P(IPAAm-co-APTAC-co-tBAAm) terpolymer-grafted beads exhibited the most rapid and efficient cell detachment behavior after the temperature was reduced to 20 °C, presumably because the highly hydrated APTAC promoted the overall hydration of the P(IPAAm-co-APTAC-co-tBAAm) chains. Therefore, P(IPAAm-co-APTAC-co-tBAAm) terpolymer-grafted microcarriers are effective in facilitating both cell proliferation and thermally induced cell detachment in a suspension culture system.     

The cell surface glycoprotein Mac-1 (CD11b/CD18) mediates neutrophil adhesion and modulates degranulation independently of its quantitative cell surface expression

It has previously been shown that during degranulation Mac-1 (CD11b/CD18)--a glycoprotein that plays a central role in neutrophil adhesion-is up-regulated on PMN surfaces. It has been assumed that this quantitative change in adhesion Ag expression on the cell surface would in turn lead to increased cellular adhesiveness. In contrast, we found that at an incubation temperature of 16 degrees C, stimulated neutrophil adhesion to plastic tissue culture dishes in the presence of FMLP (2.5 x 10(-6) M), TNF (10 ng/ml), or PAF (1 x 10(-4) M) occurred without cellular degranulation or Mac-1 surface up-regulation as measured cytofluorometrically. As shown by functional inhibition studies employing monoclonal antibodies 60.3 (anti-CD18) and 60.1 (anti-CD11b), adhesion at 16 degrees C, where no CD11b/CD18 up-regulation was seen, is mediated by CD11b/CD18 just as it is at 37 degrees C, where degranulation and CD11b/CD18 up-regulation could be demonstrated. The physiologic importance of these findings was underscored by experiments done on endothelial monolayers, which showed that PMN association with endothelial cells is absolutely independent from the quantitative up-regulation of Mac-1 on PMN surfaces. When neutrophils were stimulated at 37 degrees C by endotoxin, an agent that does not induce aggregation (a form of intercellular adhesion), Mac-1 surface expression increased only after cells had become adherent, whereas cells held in suspension to prevent cell-substrate adhesion neither degranulated nor up-regulated their Mac-1 surface expression. Thus, not only is adherence independent of degranulation and Mac-1 cell surface up-regulation, but both degranulation and Mac-1 surface up-regulation appear to depend on the process of adhesion. Correspondingly, incubation of neutrophils with antibodies 60.1 and 60.3 inhibited not only adhesion of cells stimulated with FMLP at 37 degrees C but degranulation as well. These results indicate that Mac-1 influences degranulation as well as it controls adhesion not by its mere quantity on the cell surface, but rather by an yet undefined molecular modulation.     

Monitoring of temperature effects on animal cell metabolism in a packed bed process

Animal cell (Chinese Hamster Ovary) concentration was determined on-line in a packed bed process using dielectric spectroscopy. This enabled the evaluation of the effect of temperature on specific metabolic rates during 3 months of continuous culture. The effect of low cultivation temperature on cell growth and metabolism was monitored, and the data were used for process development. At 37 degrees C cells grew exponentially with a specific growth rate of 0.038 d-1 and specific glucose uptake and lactate production rates increased continually. Reduction of the temperature to 33.5 degrees C resulted in a lowering of these metabolic rates while having no effect on cell proliferation. Subsequent reduction of the temperature to 32 degrees C resulted in stabilization of the cell concentration at a high density (3.6 x 10(7) cell per mL of packed bed). In addition, the specific production rate of the protein of interest increased by a factor of 6 compared to the value at 37 degrees C. During the stationary phase at 32 degrees C, all other specific metabolic rates could be controlled to low and constant levels.     

The immobilization of enzymes and cells of Bacillus stearothermophilus onto poly(maleic anhydride/styrene)-Co-polyethylene and poly(maleic anhydride/vinyl acetate)-Co-polyethylene

The graft copolymer, poly(maleic anhydride/styrene)-co-polyethylene was prepared. The copolymer immobilized bovine serum albumin (BSA), but the amount coupled appeared to be effected by the amount of styrene in the graft copolymer, temperature, and pH of the coupling medium. Competition existed between hydrolysis of the grafted anhydride groups and the protein. A graft copolymer with 66% add-on immobilized 4.5 mg/glucose oxidase/g copolymer, 4.6 mg alkaline phosphates/g copolymer and 0.2 mg cell of Bacillus stearothermophilus/g copolymer. A number of copolymers containing poly(maleic anhydride/vinyl acetate)-co-polyethylene were prepared to cover a range of grafting levels. These immobilized larger quantities of BSA, alkaline phosphatase, and cells of B. stearothermophilus than did the styrene graft copolymer. The copolymer was also hydrolyzed to release the hydroxyl group from the poly(vinyl acetate) component of the grafted chains. Using p-benzoquinone as the "activating agent," the copolymer coupled to BSA and to acid phosphatase. Using p-toluene-sulfonyl chloride, the copolymer was very effective in immobilizing trypsin.     

Micropatterned thermoresponsive polymer brush surfaces for fabricating cell sheets with well-controlled orientational structures

Newly developed fabrication technique of thermoresponsive surface using RAFT-mediated block copolymerization and photolithography achieved stripe-like micropatterning of poly(N-isopropylacrylamide) (PIPAAm) brush domains and poly(N-isopropylacrylamide)-b-poly(N-acryloylmorpholine) domains. Normal human dermal fibroblasts were aligned on the physicochemically patterned surfaces simply by one-pot cell seeding. Fluorescence images showed the well-controlled orientation of actin fibers and fibronectin in the confluent cell layers with associated extracellular matrix (ECM) on the surfaces. Furthermore, the aligned cells were harvested as a tissue-like cellular monolayer, called "cell sheet" only by reducing temperature below PIPAAm's lower critical solution temperature (LCST) to 20 °C. The cell sheet harvested from the micropatterned surface possessed a different shrinking rate between vertical and parallel sides of the cell alignment (approximately 3:1 of aspect ratio). This indicates that the cell sheet maintains the alignment of cells and related ECM proteins, promising to show the mechanical and biological aspects of cell sheets harvested from the functionalized thermoresponsive surfaces.     

Gangliosides support neural retina cell adhesion

Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition during neuronal development. To model such interactions, we developed a technique to test the ability of cell surface lipids (particularly glycosphingolipids) to mediate specific cell recognition and adhesion (Blackburn, C.C., and Schnaar, R.L. (1983) J. Biol. Chem. 258, 1180-1188). When cells were incubated on plastic microwells adsorbed with various glycolipids, carbohydrate-specific cell adhesion was readily detected. We report here the use of this method to study adhesion of embryonic chick neural retina cells to purified cell surface lipids. Rapid and specific cell adhesion was observed when the neural retina cells were incubated on surfaces adsorbed with gangliosides (an important class of neuronal cell surface glycoconjugates) but not on surfaces adsorbed with various neutral glycosphingolipids, phospholipids, or sulfatide. This suggests that the observed cell adhesion was specific for the carbohydrate moiety of the adsorbed ganglioside and was not due to nonspecific ionic or hydrophobic interactions. Although the surface density of adsorbed lipid required to support cell adhesion was the same for all gangliosides examined, the extent of adhesion varied when different purified gangliosides were used. Ganglioside-specific adhesion was not dependent on the presence of calcium (at 37 degrees C) and was attenuated by pretreatment of the cells with trypsin. The extent of ganglioside-directed neural retinal cell adhesion varied with embryonic age. These results imply that gangliosides may play a role in cell-cell recognition in the developing nervous system.     

The effect of the plasma needle on tumoral cell lines apoptosis

In this paper, we present the effect of the plasma needle on tumor cell surface. The plasma is generated at the tip of a metal needle by using a radio-frequency generator of 13.56 MHz, 100's V amplitude. In our study we investigated the interaction of non-thermal plasma (plasma needle) with living monolayer tumour cells in culture medium. We applied short needle to sample distance (1 mm) at temperature of 25 degrees C, 30 degrees C and 37 degrees C, respectively. Our data sugest that the plasma needle reduces the viability and induces apoptosis of tumour cells. These activities may be very useful in dermatology, where a part of the tissue must be removed with high-precision, without damage to the adjacent cells and without inflammatory reaction.     

Stabilization of enzyme immobilized in temperature-sensitive hydrogels

-Galactosidase was immobilized in a crosslinked poly(N-isopropylacrylamide-co-acrylamide) hydrogel which exhibits an LCST(lower critical solution temperature) behavior. The hydrogel collapses above the LCST, and expands below the LCST. The temperature-dependent phase transition was around 37 °C. The stability of immobilized enzyme was investigated at different temperatures which allow different degrees of collapse in the hydrogel matrix. It was hypothesized that the immobilzed enzyme is more stable in the collapsed matrix due to the physical restraint imposed on the enzyme entrapped.     

Bioactive polymer fibers to direct endothelial cell growth in a three-dimensional environment

This study reports the fabrication of bioactive polymer fibers onto which signaling molecules can control and direct cell responses. To encourage and control directional biological responses, GRGDS peptides were immobilized onto the surface of 100 microm diameter poly(ethylene terephtalate) (PET) fibers (monofilaments). PET fiber surfaces were first coated with a thin polymeric interfacial bonding layer bearing amine groups by plasma polymerization. Carboxy-methyl-dextran (CMD) was covalently grafted onto the surface amine groups using water-soluble carbodiimide chemistry. GRGDS were covalently immobilized onto CMD-coated fiber surfaces. X-ray photoelectron spectroscopy (XPS) analyses enabled characterization of the multilayer fabrication steps. Human umbilical vein endothelial cells were seeded and grown on fibers to investigate cell patterning behavior (i.e., adhesion, spreading, cytoskeleton organization, and cell orientation). Cell adhesion was reduced on CMD-coated fibers, whereas amine- and GRGDS-coated fibers promoted cell adhesion and spreading. Cell adhesion was enhanced as the GRGDS concentration increased. Epifluorescence microscopic visualization of cells on RGD-coated substrates showed well-defined stress fibers and sharp spots of vinculin, typical of focal adhesions. In comparison to plasticware commonly used in cell cultures, fiber curvature promoted cell orientation along the fiber axis.     

Thermo-responsive PNiPAAm-g-PEG films for controlled cell detachment

A series of graft copolymers consisting of either poly(N-isopropylacrylamide) (PNiPAAm) or poly(N,N-diethylacrylamide) (PDEAAm) as a thermo-responsive component in the polymer backbone and poly(ethyleneglycol) (PEG) were immobilized as thin films and cross-linked on a fluoropolymer substrate using low-pressure argon plasma treatment. The surface-immobilized hydrogels exhibit a transition from partially collapsed to completely swollen, which is in the range of 32-35 degrees C and corresponds to the lower critical solution temperature of the soluble polymers. The hydrogels were used as cell carriers in culture experiments with L929 mouse fibroblast cells to probe for cell adhesion, proliferation, and temperature-dependent detachment of cell layers. The fibroblast cells adhere, spread, and proliferate on the hydrogel layers at 37 degrees C and become completely detached after reducing the temperature by 3 K. The cell release characteristics were further correlated to the swelling and collapsing behavior of the hydrogel films and the polymer solutions as measured in PBS solution and RPMI cell cultivation medium. It could be shown that, long before the swelling has completed upon temperature reduction, the cells detach. This can be attributed to the large content of PEG present in the hydrogel, which weaken the cell adhesion strength to the hydrogel layers.