Intact Cell Monolayers Research Articles

LECT2 Ameliorates Blood–Retinal Barrier Impairment Secondary to Diabetes Via Activation of the Tie2/Akt/mTOR Signaling Pathway

PurposeCurrent treatments for diabetic retinopathy (DR) have considerable limitations, emphasizing the need for new therapeutic options. The effect of leukocyte cell-derived chemotaxin 2 (LECT2) on diabetes-induced blood–retinal barrier impairment and the possible underlying mechanism were investigated both in vivo and in vitro.MethodsTwenty diabetic and 22 nondiabetic eyes were included in this study. Additionally, we established a streptozotocin-induced diabetic mouse model and observed vascular leakage in mice treated with or without recombinant LECT2 (rLECT2) intravitreal injection (40 µg/mL, 1 µL). The levels of LECT2 and interendothelial junction proteins (ZO1, VE-cadherin, and occludin) were analyzed by western blot and/or immunofluorescence. Endothelial junctions in mouse retinas were observed by transmission electron microscopy (TEM). Moreover, confluent human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs) were treated (0–72 hours) with glucose (0 or 30 mM) in the presence or absence of rLECT2 (40–360 ng/mL). After treatment, intact cell monolayers were monitored for permeability to 40-kD FITC-dextran. Interendothelial junction targets and Tie2/Akt/mTOR signaling pathway components were investigated by western blot.ResultsIn diabetic human and mouse retinas and high-glucose (30 mM)–treated HRMECs and HUVECs, the levels of LECT2 and interendothelial junction proteins were decreased. rLECT2 treatment (80 ng/mL) significantly attenuated the hyperglycemia-induced reduction in endothelial cell barrier function and inhibited the migration and tube formation of HRMECs and HUVECs. In addition, rLECT2 increased the levels of interendothelial junction proteins via activation of the Tie2/Akt/mTOR signaling pathway. Furthermore, intravitreal rLECT2 injections increased the levels of interendothelial junction proteins and reversed diabetes-induced junction disruption.ConclusionsrLECT2 can increase the levels of interendothelial tight junction proteins through activation of the Tie2/Akt/mTOR signaling pathway and can ameliorate inner blood–retinal barrier impairment secondary to diabetes. LECT2 might be a potential target to prevent the progression of DR.

Conditioned Medium of Bone Marrow Mesenchymal Stem Cells Involved in Acute Lung Injury by Regulating Epithelial Sodium Channels via miR-34c

BackgroundOne of the characteristics of acute lung injury (ALI) is severe pulmonary edema, which is closely related to alveolar fluid clearance (AFC). Mesenchymal stem cells (MSCs) secrete a wide range of cytokines, growth factors, and microRNA (miRNAs) through paracrine action to participate in the mechanism of pulmonary inflammatory response, which increase the clearance of edema fluid and promote the repair process of ALI. The epithelial sodium channel (ENaC) is the rate-limiting step in the sodium–water transport and edema clearance in the alveolar cavity; the role of bone marrow-derived MSC-conditioned medium (BMSC-CM) in edema clearance and how miRNAs affect ENaC are still seldom known.MethodsCCK-8 cell proliferation assay was used to detect the effect of BMSC-CM on the survival of alveolar type 2 epithelial (AT2) cells. Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect the expression of ENaC in AT2 cells. The effects of miR-34c on lung fluid absorption were observed in LPS-treated mice in vivo, and the transepithelial short-circuit currents in the monolayer of H441 cells were examined by the Ussing chamber setup. Dual luciferase reporter gene assay was used to detect the target gene of miR-34c.ResultsBMSC-CM could increase the viability of mouse AT2 cells. RT-PCR and western blot results showed that BMSC-CM significantly increased the expression of the γ-ENaC subunit in mouse AT2 cells. MiR-34c could restore the AFC and lung wet/dry weight ratio in the ALI animal model, and Ussing chamber assay revealed that miR-34c enhanced the amiloride-sensitive currents associated with ENaC activity in intact H441 cell monolayers. In addition, we observed a higher expression of miR-34c in mouse AT2 cells administrated with BMSC-CM, and the overexpression or inhibition of miR-34c could regulate the expression of ENaC protein and alter the function of ENaC. Finally, we detected that myristoylated alanine-rich C kinase substrate (MARCKS) may be one of the target genes of miR-34c.ConclusionOur results indicate that BMSC-CM may alleviate LPS-induced ALI through miR-34c targeting MARCKS and regulate ENaC indirectly, which further explores the benefit of paracrine effects of bone marrow-derived MSCs on edematous ALI.

Electrospun Scaffold for Biomimic Culture of Caco-2 Cell Monolayer as an In Vitro Intestinal Model.

The Caco-2 cell monolayer has been extensively used for the high-throughput assessing of nutrient absorption, screening of drug permeability, and studying the intestinal physiological process in vitro. The most used Caco-2 cell model is the Transwell model with polycarbonate microporous membranes. However, Caco-2 cells in the classical Transwell model need 21 days to gain an intact and mature monolayer. Electrospun nanofiber scaffolds mimicking the natural extracellular matrix could improve cell adhesion, proliferation, and expression, whereas there are no reports that intestinal cells were cultured on the electrospun nanofiber scaffolds. Here, electrospun polylactic acid (PLA) nanofiber scaffolds were chosen as the ideal scaffolds for Caco-2 cell monolayers to construct a modified Transwell. Cell morphology and polarity were studied. Monolayer barrier properties were assessed by measuring transepithelial electrical resistance (TEER) and the leakage of phenol red. As found, intact Caco-2 cell monolayers were formed on the PLA nanofiber scaffolds after 4 days of culture. After 4 days, the TEER increased to 422 Ω·cm2 and the apparent permeability coefficients of phenol red decreased to 1.0 ± 0.1 × 10-6 cm/s, suggesting that Caco-2 cell monolayers developed a formidable barrier to small molecules on the surface of PLA nanofiber scaffolds. Microvilli and tight junctions were clearly visible after day 3. Besides, Caco-2 cell monolayers on the surface of PLA nanofiber scaffolds presented higher differentiation properties than on the surface of the polycarbonate microporous membrane in traditional Transwell including higher alkaline phosphatase activity and higher P-gp activity. Results of quercetin absorption and probiotics adhesion demonstrated that Caco-2 cell monolayers formed on the surface of PLA nanofiber scaffolds also had better physiological function and prediction function in vitro. Overall, the present study indicated that the Transwell with the structurally and functionally biomimetic electrospun PLA nanofiber scaffold could be potentially developed as a promising in vitro intestinal model.

Microfluidic Prototyping by Xurography to Engineer Fully‐lumenized Microvessels In Vitro

Vascular permeability (VP) is a measure of the barrier function (BF) of an intact endothelial cell (EC) monolayer. This parameter is especially important in physiology, as deregulation of permeability leads to leaky blood vessels with a transvascular imbalance of chemical gradients, fluidic pressure, and in some instances excessive trafficking of blood‐borne cells into the surrounding tissue. Regulation of VP is a highly coordinated process. However, monitoring VP in vivo can be challenging as it typically requires invasive and labor intensive intravital microscopy. Here we present an in vitro alternative by developing a microfluidic model of a blood vessel analog that permits characterization of endothelial BF under the influence of different extracellular matrix (ECM) and perivascular cellular constituents. Our model relies on the manufacturing process known as xurography, an inexpensive and rapid print‐and‐peel prototyping method for assembling multiple layers of polymers. This method has been recently applied to bypass the need for high‐end fabrication facilities that are not readily available at all research institutions. However, to date most experimental platforms manufactured with xurography have focused on biochemical studies with few cell culture‐based applications. Briefly, after using a cutting plotter to form a channel (.8 mm x 8 mm) on a PDMS layer (~ 400 um thick), the device is constructed through a series of bindings to other PDMS layers (3 x ~ 250 um) and biopsy punching that form two regions. The inlet and outlet ports for cell seeding (2mm and 4mm) are located at the extremes of the model. An area for ECM hydrogels is allocated in the middle, with a PDMS rod placed in the center (Fig 1). After the construction, collagen type I at 6mg/ml is inserted into the central region. Once polymerized the PDMS rod is pulled to leave a lumen, permitting the addition of ECs into the formed ductal structure. In order to assess BF, the vessels were stained and imaged with confocal microscopy for the intercellular adherent junction molecule, VE‐cadherin as well as cytoskeletal proteins. To quantify VP, a fluorescent tracer molecule was infused in the vessel, and this parameter was determined by measuring changes of intensity as the dye permeated from the vessel to the surrounding hydrogel. Importantly, our results indicated the vessel analog demonstrated VP on the same order of magnitude as similar studies with ECs growing in static conditions (Fig 2). Notably, the microdevice allows the addition of cells that can support EC function. These findings will be fundamental as we explore the crosstalk between cells that serve in regulating blood vessel BF. Moreover, the inexpensive fabrication process can be adapted seamlessly by research institutions across the nation to probe further into vascular biology.Support or Funding InformationFunding was provided by the National Heart, Lung, and Blood Institute (R01HL141941) and The Mark Foundation for Cancer Research ASPIRE Award. MC‐M acknowledges funding from a NIH Diversity Supplement (R01HL141941).Microfluidic model design and Confocal Imaging ‐ A) Assembly of microfluidic vessel analogue through binding of layers after fabrication. B) Staining for cytoskeletal protein, cell nuclei, and VE‐cadherin.Figure 1Apparent Permeability: Quantification of Vascular Permeability. Images of fluorescent dye permeating through the endothelial cell wall (time point ‐ 0 to 5 min.)Figure 2

COMP-Ang1 Stabilizes Hyperglycemic Disruption of Blood-Retinal Barrier Phenotype in Human Retinal Microvascular Endothelial Cells.

PurposeCurrent treatments for diabetic retinopathy (DR) have considerable limitations, underpinning the need for new therapeutic options. In this article, the ability of an engineered angiopoietin-1 variant (COMP-Ang1) to ameliorate the injurious effects of hyperglycemia on barrier integrity in a human retinal microvascular endothelial cell (HRMvEC) model is comprehensively investigated.MethodsConfluent HRMvECs were treated (0–72 hours) with d-glucose (5 or 30 mM) in the absence and presence of COMP-Ang1 (10–200 ng/mL). l-glucose (30 mM) was used as osmotic control. Posttreatment, intact cell monolayers were monitored for permeability to FITC-dextran 40 kDa. Cells were also harvested for analysis of interendothelial junction targets by RT-qPCR and Western blotting. The impact of receptor tyrosine kinase Tie2 gene silencing on COMP-Ang1 efficacy was also evaluated.ResultsTreatment with 30 mM d-glucose (but not l-glucose) demonstrated a time-dependent elevation in the mean rate of FITC-dextran diffusion across intact HRMvEC monolayers, in parallel with significant reductions in mRNA/protein levels of occludin, claudin-5, ZO-1, and VE-Cadherin. These effects were all attenuated by COMP-Ang1 in a concentration-dependent fashion, with 200 ng/mL recovering barrier function by ∼88%, and recovering reduced interendothelial junction protein levels by more than 50%. Finally, Tie2 knockdown by small interfering RNA silencing blocked the ability of COMP-Ang1 to mitigate against hyperglycemia-induced permeabilization of HRMvECs and depletion of junctional expression levels.ConclusionsIn summary, this article presents a reproducible in vitro cell study that quantifies the concentration-dependent efficacy of COMP-Ang1 to mitigate the injurious effects of hyperglycemic challenge on HRMvEC barrier properties via Tie2-mediated signaling.

MRP2-mediated transport of etoposide in MDCKII MRP2 cells is unaffected by commonly used non-ionic surfactants.

The aim of the present study was to investigate the ability of non-ionic surfactants to inhibit MRP2-mediated transport in vitro in MDCKII MRP2 cells. Transport studies across MDCKII MRP2 cell monolayers were performed using 3H-etoposide and 3H-digoxin. 19 different non-ionic surfactants, including several polysorbates (PS), cremophor EL, vitamin E-TPGS, and n-nonyl β-D-glucopyranoside (NG), were investigated. Barrier function of the cells was investigated measuring TEER and transport of 14C-glycine. The amount of isotope was quantified using liquid scintillation counting. In MDCKII MRP2 cells a polarized transport of etoposide and digoxin in the secretory (basolateral to apical) direction with efflux ratios of 5.5 ± 0.7 and 18.5 ± 4.2, respectively, was measured. P-gp inhibitors such as valspodar and zosuquidar did not affect etoposide transport, and furthermore PS20 decreased secretory transport of digoxin, but not of etoposide. Transport of etoposide was therefore mainly MRP2-mediated and used as a probe to investigate pharmaceutical excipients. Non-ionic surfactants did not modulate etoposide transport across intact cell monolayers of MRP2 overexpressing MDCKII cells, although preliminary studies suggest that most were able to alter MRP2-mediated efflux of the fluorescent 5-chloromethylfluorescein (CMF). In conclusion, etoposide transport across MDCKII MRP2 cells was modulated by cyclosporin A, an inhibitor of MRP2 and P-gp, but not by specific P-gp inhibitors (valspodar and zosuquidar), which suggests that etoposide transport is primarily influenced by MRP2. In addition, commonly used non-ionic surfactants did not decrease MRP2-mediated etoposide transport in MDCKII MRP2 cells. These results suggest that etoposide transport in MDCKII MRP2 cells is a model system to investigate MRP2 interactions, and that surfactants may not have a large potential for increasing oral bioavailability of drugs through inhibition of MRP2 transport activity.

Toxoplasma gondii plaque assays revisited: Improvements for ultrastructural and quantitative evaluation of lytic parasite growth

Lytic growth of intracellular Toxoplasma gondii tachyzoite stages over a period of days results in plaques within mononolayers of host cells. Plaque assays are in frequent use to isolate single clones and to investigate invasion, replication and egress over a longer time frame. To allow correlating plaque morphology and/or size with ultrastructural examination of individual parasites we introduce a simple protocol for correlative light and electron microscopy (CLEM) of entire plaques. We also illustrate the advantages of visualizing only the boundaries of plaques by staining for infected cells (‘positive staining’) rather than the traditional staining of the intact cell monolayer, thus outlining the area of lysed cells (‘negative staining’). Tachyzoites expressing β-galactosidase of Escherichia coli are an easy to visualize histochemical marker for this purpose. Quantitative measurements of plaque area with our compiled user-friendly ImageJ macros are compared to commercial software for ease and shown to be more accurate for some applications. Finally, a chemically defined medium is shown to be superior to the fetal bovine serum-containing medium for plaque assays, resulting in larger plaques. The reported additions and changes of the plaque assay procedure offer improved ways to analyze subtle differences in invasion, pathogen growth and egress. Our chemically defined medium will improve standardization of e.g. drug screening assays.

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

Hydration state inside HeLa cell monolayer investigated with terahertz spectroscopy

The hydration state in living cells is believed to be associated with various cellular activities. Nevertheless, in vivo characterization of intracellular hydration state under physiological condition has not been well documented to date. In this study, the hydration state of an intact HeLa cell monolayer was investigated by terahertz time-domain attenuated total reflection spectroscopy. Combined with the extended theory of Onsager, we found 23.8 ± 7.4% of HeLa intracellular water was hydrated to biomolecules (corresponding to 1.25 g H2O/g solute); exhibiting slower relaxation dynamics than bulk water.

Application of speckle image correlation for real-time assessment of metabolic activity in herpes virus-infected cells

Earlier we reported developing a speckle interferometry technique and a device designed to assess the metabolic activity of a cell monolayer cultivated on a glass substrate. This paper aimed at upgrading the technique and studying its potential for real-time assessment of herpes virus development process.Speckle dynamics was recorded in the image plane of intact and virus-infected cell monolayer. HLE-3, L-41 and Vero cells were chosen as research targets. Herpes simplex virus-1-(HSV-1)- infected cell cultures were studied.For 24 h we recorded the digital value of optical signal I in one pixel and parameter η characterizing change in the distribution of the optical signal on 10 × 10-pixel areas. The coefficient of multiple determination calculated by η time dependences for three intact cell cultures equals 0.94. It was demonstrated that the activity parameters are significantly different for intact and virus-infected cells. The difference of η value for intact and HSV-1-infected cells is detectable 10 minutes from the experiment start.

Ellagitannins as synergists of ACV on the replication of ACV-resistant strains of HSV 1 and 2

The plant-derived polyphenolic compounds castalagin, vescalagin and grandinin (C-glucosidic ellagitannins containing nonahydroxyterphenoyl) manifested a strong inhibitory effect on the replication of acyclovir-resistant strains of herpes simplex viruses (HSV) type 1 and 2 in MDBK cells in focus forming units (i.e., microscopically registered microplaques) reduction assay and in two variants of cytopathic effect inhibition test. The effect on the acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) strain was markedly higher compared to that on the ACV-resistant herpes simplex virus type 2 (HSV-2). The three compounds showed comparable levels of antiviral activity against ACV-resistant HSV strains, in contrast with previous results where castalagin exerted the highest degree of activity against wild type HSV strains (Vilhelmova et al., 2011). Combinations of ellagitannins and ACV were tested on the ACV-resistant strains of both HSV-1 and 2 and produced synergistic effects that were revealed by applying the three-dimensional approach of Prichard and Shipman (1990). The ellagitannin(s)-ACV combination applied against ACV-resistant HSV-1 produced a much stronger synergistic effect compared to the effect observed against ACV-resistant HSV-2. The study of the effects of the combination ellagitannin(s)-ACF on intact cell monolayers did not show any toxicity resulting from interaction between the two substances. Altogether, the results obtained in this study demonstrate the highly promising potential of these plant polyphenols as antiherpetic agents.

Photocrosslinked ultrathin anionic polysaccharide supports for accelerated growth of human mesenchymal stem cells.

Properties of cell culture supports obtained from ultrathin multilayer films containing anionic natural polysaccharides (PSacs) and a synthetic polycation were studied. Supports were prepared via a layer-by-layer (LbL) self-assembly deposition method. Polymers used were: heparin (Hep), chondroitin sulphate (CS), hyaluronic acid (HA), and ι-carrageenan (Car) as polyanions, and diazoresin (DR) as a polycation. PSac layers were crosslinked with DR layers by irradiation with UV light absorbed by DR resin. DR/PSac films are very efficient cell culture growth supports as found from experiments with human mesenchymal stem cells (hMSCs). Irradiation of the films resulted in changing zeta potential of outermost layers of both DR and PSac to more negative values, and in increased film hydrophobicity, as found from the contact angle measurements. Photocrosslinking of the supports led to their increased stability. The supports allow for obtaining intact cell monolayers faster than when typical polystyrene tissue culture plates are used. Moreover, these monolayers spontaneously detach permitting formation of new cell layers on these surfaces relatively early during culture, compared to cells cultured on commonly used tissue culture plastic.

Magnetic field enhanced convective diffusion of iron oxide nanoparticles in an osmotically disrupted cell culture model of the blood-brain barrier.

PurposeThe present study examines the use of an external magnetic field in combination with the disruption of tight junctions to enhance the permeability of iron oxide nanoparticles (IONPs) across an in vitro model of the blood–brain barrier (BBB). The feasibility of such an approach, termed magnetic field enhanced convective diffusion (MFECD), along with the effect of IONP surface charge on permeability, was examined.MethodsThe effect of magnetic field on the permeability of positively (aminosilane-coated [AmS]-IONPs) and negatively (N-(trimethoxysilylpropyl)ethylenediaminetriacetate [EDT]-IONPs) charged IONPs was evaluated in confluent monolayers of mouse brain endothelial cells under normal and osmotically disrupted conditions.ResultsNeither IONP formulation was permeable across an intact cell monolayer. However, when tight junctions were disrupted using D-mannitol, flux of EDT-IONPs across the bEnd.3 monolayers was 28%, increasing to 44% when a magnetic field was present. In contrast, the permeability of AmS-IONPs after osmotic disruption was less than 5%. The cellular uptake profile of both IONPs was not altered by the presence of mannitol.ConclusionsMFECD improved the permeability of EDT-IONPs through the paracellular route. The MFECD approach favors negatively charged IONPs that have low affinity for the brain endothelial cells and high colloidal stability. This suggests that MFECD may improve IONP-based drug delivery to the brain.

Measurement of Intracellular Ca2+ Release in Intact Cells Using 45Ca2+

This protocol describes a technique using (45)Ca(2+) to measure the release of Ca(2+) from the intracellular stores in monolayers of intact cells cultured in 12-well 4-cm(2) clusters. The (45)Ca(2+)-flux technique described here can only be applied to cell types that adhere to plastic. We describe the loading of the stores with (45)Ca(2+), and the subsequent (45)Ca(2+) efflux.

Influence of prior pandemic A(H1N1)2009 virus infection on invasion of MDCK cells by community-associated methicillin-resistant Staphylococcus aureus

Secondary bacterial pneumonia due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a highly publicized cause of death associated with influenza. In this study, we performed the gentamicin-killing assay using Madin-Darby canine kidney (MDCK) cells and MRSA strains to investigate whether prior infection from pandemic A(H1N1)2009 virus (A[H1N1]pdm09) lead to increased invasion of MDCK cells by MRSA. We found that the invasion rate of two MRSA strains (ATCC BAA-1680 [USA 300] and ATCC BAA-1699 [USA 100]) into intact MDCK cell monolayers was 0.29±0.15% and 0.007±0.002%, respectively (p<0.01, n≥3). In addition, the relative invasion rate of both ATCC BAA-1680 and ATCC BAA-1699 was significantly increased by prior A(H1N1)pdm09 infection of MDCK monolayers from 1±0.28 to 1.38±0.02 and from 1±0.24 to 1.73±0.29, respectively (p<0.01). These results indicate that ATCC BAA-1680 displays much stronger invasiveness of MDCK cells than ATCC BAA-1699, although invasion of both strains was increased by prior A(H1N1)pdm09 infection. In conclusion, this study provided the first evidence that prior A(H1N1)pdm09 infection facilitates the invasion of MDCK cells by MRSA, presumably due to cellular injury caused by the virus.

Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

Thermo‐Responsive Hydrogel Layers Imprinted with RGDS Peptide: A System for Harvesting Cell Sheets

Cell sheet technology is a novel approach to preparing and harvesting monolayer cell sheets by using poly(N-isopropylacrylamide)(PNIPAAm)-modified surfaces as thermoresponsive cell culture substrates. At lower temperatures the cultured cells detach spontaneously as the surface of the substrate changes from hydrophobic to hydrophilic. In this way, an intact cell monolayer can be harvested non-invasively together with its underlying extracellular matrix (ECM). As a frequently used way to achieve scaffold-free tissue engineering, cell sheet technology holds great promise in cell-based regenerative medicine. Considering the limited availability of autologous cells and the timeliness requirement of clinical treatments, current attempts at improving the efficiency of thermo-responsive cell sheet harvest systems face a dilemma. On the one hand, the cell culture substrate must be very cell-adhesive to markedly promote rapid adhesion and proliferation of the limited autologous cells for a timely therapy. On the other hand, the same substrate should become very cell-repulsive after the formation of a confluent cell monolayer such that the mature cell sheet can be rapidly released without hurting the cells and their underlying ECM. Specifically, simple regulation of the chemical composition or the topography of the surface can only either promote cell adhesion or accelerate cell detachment and these surfaces commonly have very low bioactivity. Introducing cell-adhesive biomolecules by means of covalent binding or physical adsorption can improve the surface bioactivity but result in the inevitable deceleration of cell detachment and serious leakage of the biomolecules, respectively. Thus far, no single method without additional auxiliary means can effectively enhance cell adhesion during culture as well as facilitate the rapid harvest of cell sheets. To conquer this long-standing problem, we conceive of introducing cell-adhesive biomolecules to a thermo-responsive cell culture substrate in a reversible way and modulating them through temperature-dependent interactions. In this case, biomolecules can be stably immobilized on the substrate at cell culture temperature (37 8C), while they can be released as the temperature drops (e.g., 20 8C), thus facilitating both the initial cell adhesion and the final detachment of the cell sheet. With this strategy in mind, we find that the reversible interaction known as “specific binding” in noncovalent molecular imprinting is very appealing. As is well known, polymeric receptors with tailor-made recognition sites and “specific binding” properties comparable with those of natural receptors can be easily prepared by molecular imprinting. More importantly, molecularly imprinted polymers (MIPs) containing thermo-responsive recognition sites (i.e., the sites with temperature-dependent interactions between MIPs and targeted molecules) can be readily obtained using PNIPAAm-based materials. We herein report a novel system for harvesting cell sheets which relies on a PNIPAAm-based MIP hydrogel layer with thermo-responsive affinity toward specific biomolecules (Scheme 1). The commonly used cell-adhesive peptide ArgGly-Asp-Ser (RGDS) was chosen as the target biomolecule to demonstrate the proof-of-principle of our strategy. In our design, the thermo-responsive recognition sites in the MIPs were the tactic used to achieve temperature-dependent interactions between RGDS molecules and cell culture substrate. Specifically, besides the temperature-induced change of the surface wettability, the thermo-responsive recognition sites on the MIP hydrogel layer also resulted in the stable recognition and binding of RGDS at 37 8C and the triggered release of RGDS as the temperature was decreased. In contrast to the introduction of biomolecules by means of covalent binding or physical absorption, the thermo-responsive affinity of the MIPs toward RGDS not only significantly promotes cell adhesion during cell culture (37 8C) but also facilitates the detachment of cell sheets at low temperature (20 8C). To the best of our knowledge, although MIPs have exhibited some expanded bioapplications with the emergence of various biomolecule (e.g., proteins or peptides) imprinted polymers, this study is the first demonstration of molecular imprinting as a methodology to biofunctionalize thermoresponsive cell culture substrates to harvest cell sheets for potential biomedical applications. To achieve the best affinity between RGDS and the MIP matrix during cell culture, the imprinting process was performed by redox-initiated polymerization at 37 8C in [*] Dr. G. Pan, Q. Guo, Prof. Dr. H. Yang, Prof. Dr. B. Li Department of Orthopaedics The First Affiliated Hospital of Soochow University 188 Shizi Street, Suzhou, Jiangsu 215006 (China) and Orthopaedic Institute, Soochow University 708 Renmin Road, Suzhou, Jiangsu 215007 (China) E-mail: yueer@suda.edu.cn binli@suda.edu.cn

AMP-18 facilitates assembly and stabilization of tight junctions to protect the colonic mucosal barrier

Inflammatory bowel disease (IBD) is characterized by an injured epithelium. Development of agents that could enhance mucosal healing is a major goal in IBD therapeutics. The 18-kDa antrum mucosal protein (AMP-18) and a 21-mer peptide derived from AMP-18 stimulate accumulation of tight junction (TJ) proteins in cultured epithelial cells and mouse colonic mucosa to protect the mucosal barrier, suggesting it might be a useful agent to treat IBD. We searched for molecular mechanisms by which AMP peptide or recombinant AMP-18 act on TJs in intact cell monolayers, or those disrupted by low-calcium medium. Roles of the p38 mitogen-activated protein kinase (MAPK) / heat shock protein (hsp)25 pathway and PKCζ were investigated by immunoblotting and confocal microscopy. AMP peptide activated p38 MAPK, which subsequently phosphorylated hsp25. Accumulated phospho-hsp25 was associated with perijunctional actin. AMP-18 also induced rapid phosphorylation of PKCζ and its colocalization with perijunctional actin in Caco2/bbe cells, which was accompanied by translocation and formation of complexes of "polarity proteins" in the TJ-containing detergent-insoluble fraction. Treatment with AMP-18 also stimulated accumulation of ZO-1, ZO-2, and JAM-A in nascent TJs known to associate with the multimeric p-PKCζ/Par6/ Cdc42/ECT2·GTP/Par3 polarity protein complex. AMP-18 facilitates translocation and assembly of multiple proteins into TJs and their association with and subsequent stabilization of the actin filament network. We speculate that improved barrier function induced by AMP-18 is mediated by enhanced TJ assembly. Thus, AMP-18 may provide a promising lead to develop agents effective in healing injured colonic epithelium in IBD.

In vitro angiogenesis assay for the study of cell-encapsulation therapy

Cell encapsulation within alginate beads has potential as a sustained release system for delivering therapeutic agents in vivo while protecting encapsulated cells from the immune system. There is, however, no in vitro model for cell-encapsulation therapy that provides a suitable platform for quantitative assessment of physiological responses to secreted factors. Here we introduce a new microfluidic system specifically designed to evaluate and quantify the pro-angiogenic potential of factors secreted from human fetal lung fibroblasts encapsulated in beads on an intact endothelial cell monolayer. We confirmed that cell-encapsulating beads induced an angiogenic response in vitro, demonstrated by a strong correlation between the encapsulated cell density in the beads and the length of the vascular lumen formed in vitro. Conditions established by in vitro tests were then further shown to exert a pro-angiogenic response in vivo using a subcutaneous mouse model, forming an extensive network of functional luminal structures perfused with red blood cells.

Biochemical Characterization of the Cell-Biomaterial Interface by Quantitative Proteomics

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. The biochemical basis of the transduction of the physical and mechanical signals to cellular responses is not well understood. The lack of a systematic characterization of cell-biomaterial interaction is the major bottleneck. This study demonstrated the use of a novel subcellular fractionation method combined with quantitative MS-based proteomics to enable the robust and high-throughput analysis of proteins at the adherence interface of Madin-Darby canine kidney cells. This method revealed the enrichment of extracellular matrix proteins and membrane and stress fibers proteins at the adherence surface, whereas it shows depletion of extracellular matrix belonging to the cytoplasmic, nucleus, and lateral and apical membranes. The asymmetric distribution of proteins between apical and adherence sides was also profiled. Apart from classical proteins with clear involvement in cell-material interactions, proteins previously not known to be involved in cell attachment were also discovered.