Growth Of 3T3 Cells Research Articles

Facile preparation of zwitterionic core-shell nanogels for dual-functional coatings: Combining antifouling and antibacterial features in silicon implant model surface modification

To develop surface-coating technology for fabricating functionalized biomedical implant and overcome implant-associated bacterial infections, this study presented an innovative antifouling and antibacterial dual-functional nanogel coating methodology for engineering silicon-based biomedical implants/biochips. Firstly, zwitterionic core-shell PAA@(GMA/SBMA) nanogels were designed and prepared via reflux-precipitation polymerization and were characterized through NMR, FTIR, DLS, AFM and TEM. The nanogels exhibited uniform and spherical morphology, salt-ion responsiveness, and high lysozyme loading capability. Subsequently, the PAA@(GMA/SBMA) nanogels were covalently grafted onto the surface of silicon wafers, via an epoxy-amine ring-opening reaction, to prepare nanogel-coated silicon wafers (Si@nanogels). A comprehensive array of characterization techniques, including SEM-EDS, contact angle measurements, and XPS, validated the effective nanogel-coating. These coatings demonstrated notable resistance to protein adsorption, highlighting their robust antifouling properties. Upon loading with the antibacterial agent lysozyme, the nanogel coating displayed remarkable antibacterial efficacy against both Gram-negative (E. coli) and Grampositive (S. aureus) bacteria and effectively inhibited biofilm formation. Furthermore, the nanogel coatings exhibited good biocompatibility and dynamic stability, thus facilitating the surface-mediated adhesion and growth of fibroblast 3T3 cells. Significantly, this work provided a facile, economic and efficient surface modification approach to fabricate nanogel-coated antifouling/antibacterial dual-functional biomedical implant model towards potential clinical applications.

Antioxidant-Engineered Milk-Derived Extracellular Vesicles for Accelerating Wound Healing via Regulation of the PI3K-AKT Signaling Pathway.

Inspired by the experience of relieving inflammation in infants with milk, antioxidant-engineered milk-derived extracellular vesicles (MEVs) are developed to evaluate their potential for accelerating wound healing. In this work, MEVs with polydopamines (PDA) are engineered using the co-extrusion method. Subsequently, the authors incorporated them into a Schiff-based crosslink hydrogel, forming a skin dosage form that could facilitate the wound healing process. The antioxidant properties of PDA assist in the anti-inflammatory function of engineered MEVs, while the gel provides better skin residency. The PDA@MEVs+GEL formulation exhibits excellent biocompatibility, pro-angiogenic capacity, and antioxidant ability in vitro. Furthermore, in vivo experiments demonstrate its efficacy in wound repair and inflammation inhibition. Mechanistically, PDA@MEVs+GEL simultaneously promotes the growth, migration, and anti-inflammation of 3T3 cells by activating PI3K-AKT pathway. Moreover, PDA@MEVs+GEL exhibits enhanced functionality in promoting wound healing in vivo, attributed to its ability to inhibit inflammation, stimulate angiogenesis, and promote collagen synthesis. In conclusion, this study delves into the mechanism of MEVs and underscores the improved efficacy of engineered extracellular vesicles. Additionally, the feasibility and prospect of engineered MEVs in treating skin wounds are verified, suggesting that antioxidant-engineered MEVs could be a promising therapeutic agent for wound healing applications.

Development and characterization of solvent-based 3D printed polylactic acid/45S5 bioactive glass composites for soft and hard tissue engineering.

With the benefit of offering hydrolysis breakdown and bio-resorption of its products, polylactic acid (PLA) is among the most frequently utilized polymers for many biomedical applications. Composites made of polylactic acid (PLA) and bioactive substances such as bioactive glass (BG) are developing as novel biomaterials because they comprise the mechanical properties and bioactivity of bioactive glass (BG) with the conformability and bio absorption of PLA. In this work, composites of PLA/BG were produced by employing the solvent-based three-dimensional printing process. To accomplish this, 0-2 wt% of BG particles (size ≤ 2 µm) were added to PLA. The resulting composite mix was then fed into a solvent-based 3D printer for the layer-by-layer construction of composites. According to the SEM/EDX investigation, BG particles were evenly dispersed throughout the polymer matrix which resulted in the interfacial bonding between them. FTIR and XRD analysis showed that PLA and BG did not interact chemically. All the composites were evaluated for cytocompatibility by in vitro cellular tests, which also proved their suitability as a substrate for NIH 3T3 cell adherence and growth. The composites were also found to be good in terms of hemocompatibility and platelet adhesion. In conclusion, additional studies on these materials were encouraged by the successful outcomes, which suggested that 3D-printed composite scaffolds consisting of PLA and BG particles might be useful in soft and hard tissue engineering.

Growth and Drug Interaction Monitoring of NIH 3T3 Cells by Image Analysis and Capacitive Biosensor.

Capacitive biosensors are manufactured on glass slides using the semiconductor process to monitor cell growth and cell–drug interactions in real time. Capacitance signals are continuously monitored for each 10 min interval during a 48 h period, with the variations of frequency from 1 kHz to 1 MHz. The capacitance values showed a gradual increase with the increase in NIH 3T3 cell numbers. After 48 h of growth, 6.67 μg/mL puromycin is injected for the monitoring of the cell–drug interaction. The capacitance values rapidly increased during a period of about 10 h, reflecting the rapid increase in the cell numbers. In this study, we monitored the state of cells and the cell–drug interactions using the developed capacitive biosensor. Additionally, we monitored the state of cell behavior using a JuLiTM Br&FL microscope. The monitoring of cell state by means of a capacitive biosensor is more sensitive than confluence measuring using a JuLiTM Br&FL microscope image. The developed capacitive biosensor could be applied in a wide range of bio-medical areas; for example, non-destructive real-time cell growth and cell–drug interaction monitoring.

Titanium implants modified by laser microtexturing enhance the bioactivity of gastric epithelial cells and fibroblast cells.

The clinical application of anastomotic instruments improves the efficiency of the digestive tract surgery. However, the stapler with titanium nails implanted is still controversial in terms of anastomotic complications, and further improvement and optimization are needed. The purpose of this study was to explore the optimal microtextured parameters that could enhance the bioactivity of titanium implants in vitro. Laser microtexturing technology was used to construct the groove-type microstructural surfaces with different parameters, and human gastric mucosal epithelial cells (GES-1 cells) and mouse fibroblasts (3T3 cells) were cultured on the surface of the titanium plates in vitro. The data of cell adhesion, cell proliferation and cell activity were obtained and statistically analyzed. The textured titanium plates meet the expected design. GES-1 and 3T3 cell adhesion were better in the surface of titanium plates in microstructural group than that in the polished group. GES-1 and 3T3 cells also showed higher proliferative activity in the microstructural group compared with the polished group. The laser textured titanium plates have good groove-type microstructure, which increase the surface roughness, change the surface wettability, promote the adhesion, proliferating and orderly growth of GES-1 and 3T3 cells, and show good biological properties.

Copper nanoparticles stabilized with cashew gum: Antimicrobial activity and cytotoxicity against 4T1 mouse mammary tumor cell line.

Copper nanoparticles stabilized with cashew (CG-CuNPs) were synthesized by reduction reaction using ascorbic acid and sodium borohydride, using the cashew gum (CG) as a natural polymer stabilizer. Dynamic light scattering, atomic force microscopy, Fourier-transform infrared spectroscopy, UV-Vis spectrophotometry, and x-ray diffraction were used to characterize the nanoparticles (CG-CuNPs), and copper was quantified by electrochemical measurement. The UV-vis spectra of the CG-CuNPs confirmed the formation of nanoparticles by appearance of a surface plasmon band at 580 nm after 24 h of reaction. The Fourier-transform infrared spectrum of CG-CuNPs showed the peak at 1704 cm−1 from cashew gum, confirming the presence of the gum in the nanoparticles. The average size of CG-CuNPs by dynamic light scattering and atomic force microscopy was around 10 nm, indicating small, approximately spherical particles. Antimicrobial assays showed that CG-CuNPs had activity against Staphylococcus aureus ATCC 29213 with a minimal inhibitory concentration of 0.64 mM. The cytotoxicity assay on BALB/c murine macrophages showed lower cytotoxic effects for CG-CuNPs than CuSO4·5H2O. Viability cell assays for CG-CuNPs at (0.250 mM) inhibited by 70% the growth of 4T1 LUC (4T1 mouse mammary tumor cell line) and NIH 3T3 cells (murine fibroblast cells) over a 24-h period. Therefore, CG-CuNPs can be used as an antimicrobial agent with lower cytotoxic effects than the CuSO4·5H2O precursor.

The oxidoreductase p66Shc acts as tumor suppressor in BRAFV600E-transformed cells.

Metabolic reprogramming, as exemplified by the shift from oxidative phosphorylation to glycolysis, is a common feature of transformed cells. In many tumors, altered metabolism is also reflected in increased reactive oxygen species (ROS) levels, which contribute to proliferation and survival signaling. However, despite high ROS levels, cancer cells can be efficiently killed by further increasing ROS production. We have shown previously that both wild‐type and oncogenic CRAF and BRAF prevent excessive mitochondrial ROS production. Subsequently, it has been demonstrated that raising ROS levels in BRAFV600E‐transformed melanoma cells by inhibiting BRAF or MEK rendered them susceptible to cell death induction. To understand how oncogenic BRAF affects mitochondrial ROS production in melanoma, we studied the mitochondrial ROS‐producing oxidoreductase p66Shc, which is frequently overexpressed in tumors. Using NIH 3T3 BRAFV600E fibroblasts and the melanoma cell lines A375 and M238 carrying the same BRAF mutation, we show that under treatment with the ROS‐inducing agent phenethyl isothiocyanate (PEITC), oncogenic BRAF renders cells refractory to p66ShcS36 phosphorylation, which is essential for p66Shc activation and mitochondrial ROS production. Consistent with this, the activation of JNK1/2, which phosphorylate S36, was blunted, while other mitogen‐activated protein kinases were not affected. Inhibition of JNK1/2 efficiently prevented ROS production, while BRAF and MEK inhibitors increased ROS levels. Vemurafenib‐resistant M238R melanoma cells were impaired in S36 phosphorylation and ROS production following PEITC treatment. Moreover, they failed to increase ROS levels after MEK/BRAF inhibition. Finally, shRNA‐mediated knockdown of p66Shc led to increased growth of BRAFV600E‐transformed NIH 3T3 cells in soft agar assay. Taken together, these data suggest that phosphorylation‐activated p66Shc functions as a tumor suppressor in melanoma cells.

Facile green reduction of graphene oxide using Ocimum sanctum hydroalcoholic extract and evaluation of its cellular toxicity

Currently, biological application of graphene family materials is widely explored from drug delivery to biosensing. In this study, we reduced graphene oxide (GO) using hydroalcoholic extract of Ocimum sanctum (holy basil) by refluxing with 5% extract solution. Total phenols and flavonoids present in extract were assayed using appropriate methods as these phytocompounds are known to influence reducing capacity. 2, 2-diphenyl 1- picrylhydrazyl (DPPH) assay and IC 50 value determination was carried out to know the potency of plant extract as antioxidant. Total phenolic content in hydroalcoholic extract of O sanctum was found to be 20% and total flavonoid content was found to be 7.5%. DPPH assay showed ∼90% inhibition of free radicals by extract at 10 μg/mL concentration and ∼42% inhibition at 0.75 μg/mL and IC 50 value of 1.3 μg/mL showing that the extract is a potential antioxidant. Characterization of GO and O sanctum reduced GO (ORGO) showed that PXRD of ORGO had a broad peak centred at 2θ = 25° as against 2θ = 10.69° of GO indicating efficient removal of oxygen containing group. SEM images of GO showed typical flake like morphology whereas ORGO appeared like overlapped folded curtains, typical of their respective morphologies. To assay the concentration dependent toxicology profile of GO and ORGO, efflux of haemoglobin from suspended red blood corpuscles was estimated spectroscopically at 541 nm. Viability of mouse fibroblast cells (Balb 3T3 cells) under the influence of different concentrations of GO/ORGO was examined with water soluble tetrazolium salt using cell counting kit assay (CCK-8). GO here showed higher haemolytic activity of 6.9% and higher inhibition of growth of 3T3 cells at all tested concentrations than ORGO, which is probably due to removal of oxygen functionalities by plant extract in the latter. From this study, we conclude that O sanctum hydroalcoholic extract has been efficiently employed as a reductant in removal of functionalities of GO and thus synthesized ORGO showed lesser hematologic and cellular toxicity and the material can be a good entrant for biological applications after further in vivo studies.

Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells

CREBZF, also known as Zhangfei or SMILE, is a member of the CREB/ATF protein family. CREBZF has mainly been considered as a basic region-leucine zipper transcription factor that functions in coordination with other transcription factors and plays a role in latent HSV-1 infection, apoptosis and the mammalian endoplasmic reticulum stress and unfolded protein response. In this study, we constructed recombinant lentiviral vectors for CREBZF short hairpin RNA (shRNA) expression and over-expression to improve understanding of the mechanisms regulating CREBZF. The CREBZF ORF sequence was cloned into the lentiviral shuttle plasmid pCD513B-1, and various shRNA oligonucleotides and one negative control (shN) were cloned into the pCD513B-U6 expression vector. The recombinant lentivirus was packaged and transduced into NIH 3T3 cells. CREBZF mRNA and protein expression were examined using real-time reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting, respectively. The over-expression vector and the most effective shRNA vector significantly affected the expression of CREBZF mRNA and protein. Both of the CREBZF recombinant lentiviral vectors were successfully constructed. The over-expression vector significantly increased the expression of exogenous CREBZF and inhibited the growth of NIH 3T3 cells compared to controls. The most effective shRNA lentiviral vector, pCD513B-U6-CREBZF-shRNA-3, was transformed, leading to significant knockdown of the CREBZF gene. We conclude that CREBZF the recombinant lentiviral vectors are promising tools for regulating the expression of CREBZF in NIH 3T3 cells.

Threonine appears to be essential for proliferation of human as well as mouse embryonic stem cells.

Mouse embryonic stem (mES) cell proliferation depends exclusively on the nutritionally essential amino acid, L-threonine, in the medium. Other essential and non-essential amino acids need not be added to the medium for mES cell proliferation. Furthermore, the threonine analog, 3-hydroxynorvaline (3-HNV), selectively inhibits mES cell proliferation (Wang et al., 2009). HeLa, MEF and 3T3 cell growth all are not affected by 3-HNV. Selective inhibition of ES cell proliferation by 3-HNV is expected to have teratogenic and embryotoxic effects on development, and these effects have been observed in chicken and mouse embryos (Louw et al., 2005). It has been proposed that rapid catabolism via threonine dehydrogenase (TDH) accounts for how threonine supplementation is needed to support mES cell proliferation and that TDH is the site of 3-HNV inhibition of mES cell proliferation (Wang et al., 2009; Han et al., 2013; Chen and Wang, 2014). In support of this possibility, TDH induction enhances reprogramming of mouse somatic cells into induced pluripotent stem (iPS) cells, and 3-HNV inhibits this induction (Han et al., 2013). The concentrations of threonine or 3-HNV needed for mES cell proliferation or its inhibition are, however, over an order of magnitude lower than the apparent Km or Ki values for interaction of threonine or 3-HNV with TDH (Wang et al., 2009). Hence, another mechanism may account for the influences of threonine and 3-HNV on mES cell proliferation. To test this hypothesis, we determined whether 3-HNV inhibits human embryonic stem (hES) cell proliferation. Humans produce truncated and apparently inactive TDH proteins that cannot make appropriate contact with 3-HNV, threonine or NAD+, the cofactor needed for TDH activity (Edgar, 2002).

The Validated Embryionic Stem Cell Test to Predict Embryotoxicityinvitro

Backgrounds: A straight-forward way to identify whether a drug or environmental chemical can be harmful to unborn baby is to evaluate its effect on laboratory animals. All invivo methods need large number of animal and are therefore time consuming and expensive. However, the thousands of chemicals in need of testing, to reduce the spending of live animals, an assortment of in vitro assays has been proposed. In recent years, embryonic stem cell test (ESC) has been used to investigate the mutagenic, cytotoxic and embryotoxic effects of compounds in vitro. Methods: The EST uses two cell lines and three endpoints to predict embryotoxic chemicals: mouse embryonic stem cells (D3 cells) and murine fibroblasts (3T3 cells). The validated prediction model was developed based on the inhibition of differentiation of D3 cells into cardiomyocytes, and the inhibition of D3 cells and 3T3 cell viability. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. Results: The three experimental endpoints: ID50 ( 50% inhibition of differentiation of ES cells into cardiac myoblasts and IC50 D3 and IC50 3T3 ( 50% inhibition of cell growth in ES and 3T3 cells in the MTT assay, respectively). According to these results chemicals are classified into three classes of the in vivo ( notembryotoxic, weak and strong embryotoxic). Conclusion: The in vitro EST described is rapid, simple, and sensitive and can be usefully applied as a component of the risk/hazard assessment process. Keywords: Cytotoxic, Embryionic stem cell, Mutagenic.

Formic acid and acetic acid induce a programmed cell death in pathogenic Candida species.

Cutaneous fungal infections are common and widespread. Antifungal agents used for the treatment of these infections often have undesirable side effects. Furthermore, increased resistance of the microorganisms to the antifungal drugs becomes the growing problem. Accordingly, the search for natural antifungal compounds continues to receive attention. Apoptosis is highly regulated programmed cell death. During yeast cell apoptosis, amino acids and peptides are released and can stimulate regeneration of human epithelium cells. Thus, detection of chemical compounds inducing apoptosis in yeast and nontoxic for humans is of great medical relevance. The aim of this study was to detect chemical compound inducing apoptosis in pathogenic Candida species with the lowest toxicity to the mammalian cells. Five chemical compounds--acetic acid, sodium bicarbonate, potassium carbonate, lithium acetate, and formic acid--were tested for evaluation of antifungal activity on C. albicans, C. guilliermondii, and C. lusitaniae. The results showed that acetic acid and formic acid at the lowest concentrations induced yeast cells death. Apoptosis analysis revealed that cells death was accompanied by activation of caspase. Minimal inhibitory concentrations of potassium carbonate and sodium bicarbonate induced Candida cells necrosis. Toxicity test with mammalian cell cultures showed that formic acid has the lowest effect on the growth of Jurkat and NIH 3T3 cells. In conclusion, our results show that a low concentration of formic acid induces apoptosis-like programmed cell death in the Candida yeast and has a minimal effect on the survivability of mammalian cells, suggesting potential applications in the treatment of these infections.

Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factorMYBL1

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

Silica Aerogel Improves the Biocompatibility in a Poly-ε-Caprolactone Composite Used as a Tissue Engineering Scaffold

Poly-ε-caprolactone (PCL) is a biodegradable polyester that has received great attentions in clinical and biomedical applications as sutures, drug delivery tool, and implantable scaffold material. Silica aerogel is a material composed of SiO2that has excellent physical properties for use in drug release formulations and biomaterials for tissue engineering. The current study addresses a composite of silica aerogel with PCL as a potential bone scaffold material for bone tissue engineering. The biocompatibility evaluation of this composite indicates that the presence of silica aerogel effectively prevented any cytotoxic effects of the PCL membrane during extended tissue culture periods and improved the survival, attachment, and growth of 3T3 cells and primary mouse osteoblastic cells. The beneficial effect of silica aerogel may be due to neutralization of the acidic condition that develops during PCL degradation. Specifically, it appears that silica aerogel to PCL wt/wt ratio at 0.5 : 1 maintains a constant pH environment for up to 4 weeks and provides a better environment for cell growth.

Ultraviolet C Irradiation Induces Different Expression of Cyclooxygenase 2 in NIH 3T3 Cells and A431 Cells: The Roles of COX-2 Are Different in Various Cell Lines

Ultraviolet C (UVC) is a DNA damage inducer, and 20 J/m2 of UVC irradiation caused cell growth inhibition and induced cell death after exposure for 24–36 h. The growth of NIH 3T3 cells was significantly suppressed at 24 h after UVC irradiation whereas the proliferation of A431 cells was inhibited until 36 h after UVC irradiation. UVC irradiation increased COX-2 expression and such up-regulation reached a maximum during 3–6 h in NIH 3T3 cells. In contrast, UVC-induced COX-2 reached a maximum after 24–36 h in A431 cells. Measuring prostaglandin E2 (PGE2) level showed a biphasic profile that PGE2 release was rapidly elevated in 1–12 h after UVC irradiation and increased again at 24 h in both cell lines. Treatment with the selective COX-2 inhibitor, SC-791, during maximum expression of COX-2 induction, attenuated the UVC induced-growth inhibition in NIH 3T3 cells. In contrast, SC-791 treatment after UVC irradiation enhanced death of A431 cells. These data showed that the patterns of UVC-induced PGE2 secretion from NIH 3T3 cells and A431 cells were similar despite the differential profile in UVC-induced COX-2 up-regulation. Besides, COX-2 might play different roles in cellular response to UVC irradiation in various cell lines.

Mitogenic Activity of Sulfated Chitosan and Cellulose Derivatives is Related to Protection of FGF‐2 from Proteolytic Cleavage

Novel chitosan (CHS) and cellulose sulfates (CSs) are studied regarding their mitogenic activity and their protective effect against proteolytic digestion of FGF-2. An intermediate degree of sulfation (DS(S) ) and lower concentration of CHS have superior effect on 3T3 cell growth while the mitogenic activity of CS increases with DS(S) and concentration. Experiments with trypsin as model proteinase show that protection of FGF-2 from proteolytic digestion depends on DS(S) and the concentration of derivatives in the same manner as cell growth. Studies on stability of FGF-2 added to cultures of 3T3 cells show that the FGF-2 concentration remains higher in the presence of derivatives. Results indicate that the mitogenic activity of CHS and CS is due to protection of FGF-2 from proteolytic cleavage.

Neutron reflectometry characterization of PEI–PSS polyelectrolyte multilayers for cell culture

Using Neutron Reflectometry (NR), polyelectrolyte multilayer (PEM) films made by layer-by-layer (LbL) deposition of a strong polycation (polyethylene imine [PEI]) and a polyanion (polystyrene sulfonate [PSS]) have been characterized. PEI terminated samples with a total of 5, 7, and 9 layers were deposited on a quartz substrate and studied under three different environmental conditions (i.e., dry air, 100% relative humidity, and bulk water). We were able to model all the measurements at three different contrast conditions using one simple, physically reasonable and consistent model, which led to a firm understanding of the structure of the systems. The PEM thickness was found to vary linearly with the number of layers deposited. Thin film structures formed using the LbL method were constituted of two distinctive regions, i.e., the bottom and top strata. When measured in dry air and D2O vapors, the ∼30 to 50 A thick bottom stratum was found to consist of loosely packed polymers (i.e. 30% polymer by volume). This region could have resulted from an island type of deposition during the initial stages of LbL assembly. In contrast, the thickness of the top strata, which consisted of densely packed polymers (i.e. 100% polymer by volume when measured in dry air), was found to vary linearly with the number of layers deposited. Upon exposure to D2O saturated vapors, it was observed that the top and bottom strata absorbed significant quantities of heavy water, accompanied with PEM swelling. We estimated that in this case, the top strata comprise ca. 57% (v/v) polymer and 43% (v/v) D2O for 7- and 9-layered samples. No further swelling of the PEM samples was observed when they were exposed to bulk D2O. Nevertheless, the entire polymeric system underwent a rearrangement leading towards the homogenization of the multilayered structure, suggested by the decreased scattering contrast between the top and bottom strata. We also performed studies to assess the cytocompatibility of 7-layered PEM structures. Two different cell types, fibroblasts (3T3) and human embryo kidney cells (HEK-293), were seeded on the polyelectrolyte multilayer, and the cell coverage was monitored by optical microscopy at varying times. Our observations confirmed that cells adhered and spread on PEM substrates, which showed no sign of immediate toxicity. Therefore, such multilayers proved to be a suitable support for 3T3 and HEK-293 cell growth.

Directed Intermixing in Multicomponent Self-Assembling Biomaterials

The noncovalent coassembly of multiple different peptides can be a useful route for producing multifunctional biomaterials. However, to date, such materials have almost exclusively been investigated as homogeneous self-assemblies, having functional components uniformly distributed throughout their supramolecular structures. Here we illustrate control over the intermixing of multiple different self-assembling peptides, in turn providing a simple but powerful means for modulating these materials' mechanical and biological properties. In β-sheet fibrillizing hydrogels, significant increases in stiffening could be achieved using heterobifunctional cross-linkers by sequestering peptides bearing different reactive groups into distinct populations of fibrils, thus favoring interfibril cross-linking. Further, by specifying the intermixing of RGD-bearing peptides in 2-D and 3-D self-assemblies, the growth of HUVECs and NIH 3T3 cells could be significantly modulated. This approach may be immediately applicable toward a wide variety of self-assembling systems that form stable supramolecular structures.

High‐throughput live‐cell imaging reveals differential inhibition of tumor cell proliferation by human fibroblasts

Increasing evidence indicates that cancer development requires changes both in the precancerous cells and in their microenvironment. To study one aspect of the microenvironmental control, we departed from Michael Stoker's observation (Stroker et al, J Cell Sci 1966;1:297-310) that normal fibroblasts can inhibit the growth of admixed cancer cells (neighbour suppression). We have developed a high-throughput microscopy and image analysis system permitting the examination of live mixed cell cultures growing on 384-well plates, at the single cell level and over time. We have tested the effect of 107 samples of low passage number (<5) primary human fibroblasts from pediatric and adult donors, on the growth of six human tumor cell lines. Three of the lines were derived from prostate carcinomas, two from lung carcinomas and one was an EBV transformed lymphoblastoid line. Labeled tumor cells were grown in the presence of unlabeled fibroblasts. The majority of the tested fibroblasts inhibited the proliferation of the tumor cells, compared to the control cultures where labeled tumor cells were co-cultured with unlabeled tumor cells. The proliferation inhibiting effect of the fibroblasts differed depending on their site of origin and the age of the donor. Inhibition required direct cell contact. Mouse 3T3 fibroblasts inhibited the growth of SV40-transformed 3T3 cells and human tumor cells, showing that the inhibitory effect could prevail across the species barrier. Our high-throughput system allows the quantitative analysis of the inhibitory effect of fibroblasts on the population level and the exploration of differences depending on the source of the normal cells.

Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation

Rho GTPases impact a number of activities important for oncogenesis. We describe a small molecule inhibitor that blocks oncogenic transformation induced by various Rho GTPases in fibroblasts, and the growth of human breast cancer and B lymphoma cells, without affecting normal cells. We identify the target of this inhibitor to be the metabolic enzyme glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. We show that transformed fibroblasts and breast cancer cells exhibit elevated glutaminase activity that is dependent on Rho GTPases and NF-κB activity, and is blocked by the small molecule inhibitor. These findings highlight a previously unappreciated connection between Rho GTPase activation and cellular metabolism and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.