Polystyrene Cell Culture Dishes Research Articles

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

BackgroundOsteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes.ResultsHuman monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates.ConclusionOur study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

Raman spectroscopy detects biochemical changes due to different cell culture environments in live cells in vitro.

The in vitro cell culture environment can impact on cell biochemistry and cell cycle. The manifestation of such substrate-induced changes in cell cycle in the Raman microspectroscopic profiles of cell cultures is investigated at the level of nucleolus, nucleus and cytoplasm. HeLa immortalised human cervical cells and HaCaT dermal cells were cultured on three different substrates, conventional polystyrene cell culture dishes, CaF2 slides as a commonly used Raman substrate, and glass slides coated with collagen rat tail, as a mimic of the extra-cellular matrix (ECM) environment. A cell cycle study, based on percentage DNA content, as determined using propidium iodide staining and monitored by flow cytometry, was performed on cells of both types, grown on the different substrates, confirming that the in vitro cell culture environment impacts significantly on the cell cycle. Live cell in vitro Raman spectroscopic analysis of cells on the 2D CaF2 and 3D collagen substrates was performed and data was analysed using principal component analysis (PCA). The spectroscopic analysis revealed differences in profiles which reflect the differences in cell cycle for both in vitro culture environments. In particular, the Raman spectra of cells grown on CaF2 show indicators of cell stress, which are also associated with cell cycle arrest at the G0/G1 phase. This work contributes to the field of Raman spectroscopic analysis by providing a fresh look at the significance of the effect of in vitro culture environment to cell cycle and the sensitivity of Raman spectroscopy to such differences in cell metabolism.

Cell density-dependent differential proliferation of neural stem cells on omnidirectional nanopore-arrayed surface

Recently, the importance of surface nanotopography in the determination of stem cell fate and behavior has been revealed. In the current study, we generated polystyrene cell-culture dishes with an omnidirectional nanopore arrayed surface (ONAS) (diameter: 200 nm, depth: 500 nm, center-to-center distance: 500 nm) and investigated the effects of nanotopography on rat neural stem cells (NSCs). NSCs cultured on ONAS proliferated better than those on the flat surface when cell density was low and showed less spontaneous differentiation during proliferation in the presence of mitogens. Interestingly, NSCs cultured on ONAS at clonal density demonstrated a propensity to generate neurospheres, whereas those on the flat surface migrated out, proliferated as individuals, and spread out to attach to the surface. However, the differential patterns of proliferation were cell density-dependent since the distinct phenomena were lost when cell density was increased. ONAS modulated cytoskeletal reorganization and inhibited formation of focal adhesion, which is generally observed in NSCs grown on flat surfaces. ONAS appeared to reinforce NSC-NSC interaction, restricted individual cell migration and prohibited NSC attachment to the nanopore surface. These data demonstrate that ONAS maintains NSCs as undifferentiated while retaining multipotency and is a better topography for culturing low density NSCs.

Microwave Cavity with Controllable Temperature for In Vitro Hyperthermia Investigations

Hyperthermia is a form of cancer treatment in which affected human tissue is exposed to <TEX>$</TEX><TEX>></TEX><TEX>40^{\circ}C$</TEX> temperature. In this paper, our goal is to assess the efficacy of fullerene agents to reduce heating time for cancer treatment. Such agents can accelerate heating of cancer cells and improve hyperthermia treatment efficacy. Typically, in vitro testing involves cancer cell culturing, heating cell cultures in accordance to specifications, and recording cancer cell viability after hyperthermia. To heat cell cultures, we design and evaluate a 2.4-GHz microwave cavity with controllable temperature. The cavity is comprised of a polystyrene cell culture dish (diameter = 54 mm, height = 13.5 mm) and a printed monopole antenna placed within the cavity for microwave heating. The culture temperature can be controlled through the intensity and duration of the antenna's microwave radiation. Heating experiments were carried out to validate the cavity's performance for F-12K culture medium (Kaighn's F-12K medium, ATCC). Importantly, fullerene agents were shown to reduce heating time and improve hyperthermia treatment efficacy. The culture medium temperature increased, on average, from <TEX>$24.0^{\circ}C$</TEX> to <TEX>$50.9^{\circ}C$</TEX> (without fullerene) and from <TEX>$24.0^{\circ}C$</TEX> to <TEX>$56.8^{\circ}C$</TEX> (with 3 mg/mL fullerene) within 15 minutes.

Collagen‐lactoferrin fibrillar coatings enhance osteoblast proliferation and differentiation

Lactoferrin is a milk-derived glycoprotein with anabolic effects on the bone tissue. In this study, artificial extracellular matrices (aECM) consisting of collagen type I fibrils formed in the presence of lactoferrin at two different concentrations (0.5 and 1 mg mL(-1) ) were prepared on the surface of poly(lactic-co-glycolic acid) (PLGA) foils. The aim of the study was to investigate the effects of aECM on the adhesion, growth and osteogenic differentiation of human osteoblast-like Saos-2 cells. On days 1 and 3 after seeding, higher numbers of cells were found on samples with collagen and collagen-lactoferrin coatings (particularly on those formed at the higher concentration of lacroferrin) than on control microscopic glass coverslips. Cells on coatings formed in the presence of lactoferrin had more numerous and better developed vinculin-containing focal adhesion plaques. On day 7, cells on coatings with and without lactoferrin produced significantly higher levels of osteocalcin than cells on control polystyrene cell culture dishes, the highest average values being found on samples with the lower concentration of lactoferrin. Expression of collagen I and alkaline phosphatase was on a similar level in cells on all tested samples and control polystyrene. Thus, lactoferrin promotes adhesion, growth and osteogenic differentiation of Saos-2 cells and is promising as a bone implant coating component.

Three-dimensional reconstruction of surface nanoarchitecture from two-dimensional datasets

The design of biomaterial surfaces relies heavily on the ability to accurately measure and visualize the three-dimensional surface nanoarchitecture of substrata. Here, we present a technique for producing three-dimensional surface models using displacement maps that are based on the data obtained from two-dimensional analyses. This technique is particularly useful when applied to scanning electron micrographs that have been calibrated using atomic force microscopy (AFM) roughness data. The evaluation of four different surface types, including thin titanium films, silicon wafers, polystyrene cell culture dishes and dragonfly wings confirmed that this technique is particularly effective for the visualization of conductive surfaces such as metallic titanium. The technique is particularly useful for visualizing surfaces that cannot be easily analyzed using AFM. The speed and ease with which electron micrographs can be recorded, combined with a relatively simple process for generating displacement maps, make this technique useful for the assessment of the surface topography of biomaterials.

It is Time for a Change: Petri Dishes Weaken Cells

We wish to draw the attention to a potential deficiency in the biocompatibility of polystyrene cell culture dishes which is caused by a softening of the material under relevant culture conditions. The finding confirms the central hypothesis of our previous model study. In it we assumed a local increase in pH at the interface between the hydrophilic polymer and liquid. The finding is of considerable biological interest. Polystyrene tissue culture dishes are now in use for 50 years. To the best of our knowledge their biocompatibility has never been challenged. Here we report the first experimental proof that exposure to water softens the surface of polystyrene Petri dishes. We expect that our results will stimulate the development of a new generation of cell culture devices, including Petri dishes and culture flasks, and the establishment of improved biomimetic settings for tissue engineering and stem cell research. New non-swelling biomaterials or nanocoatings designed to reduce the swelling of polymer culture dishes could improve cell performance. The need for further study is clear.

Cellular interactions of monodisperse poly(GDMA) latex particles-containing DMAEM brushes

Monodisperse latex particles, 791 nm in diameter, were synthesized by single-stage precipitation polymerization of glycerol dimethacrylate (GDMA). Then N,N-(dimethylamino) ethyl methacrylate (DMAEM) brushes were created on the surface of poly(GDMA) particles by copper-mediated atom transfer radical polymerization (ATRP). The poly(GDMA–DMAEM) particles exhibit thermosensitive properties since DMAEM brushes give cationic character to the poly(GDMA) latex particles. In L929 mouse fibroblast cell cultures no toxicity against cell proliferation was observed when the particle concentration was 25 ng per cell. Poly (GDMA) and poly(GDMA–DMAEM) latex particles attached well onto the polystyrene cell culture dishes. Cellular interactions between fibroblasts and latex particles were investigated on these particle-coated culture plates by means of microscopic observations (optic microscope and scanning electron microscope, SEM) and MTT assay. The results indicated that the poly(GDMA–DMAEM) particles can facilitate cell attachment and proliferation due to the presence of positively charged amino groups on their surfaces. These surface coated latex particles may find useful applications as adhesion modifiers for cells in biomedical devices.

Cell engineering: nanometric grafting of poly-N-isopropylacrylamide onto polystyrene film by different doses of gamma radiation

Poly-N-isopropylacrylamide was successfully grafted onto a polystyrene cell culture dish and γ-preirradiated in air. In this study, the effect of a γ-pre-irradiation dose of radiation (radiation absorbed dosages of 10, 20, 30, 40 KGy) under appropriate temperature and grafting conditions was investigated. The Fourier transform infrared spectroscopy analysis showed the existence of the graft poly-N-isopropylacrylamide (PNIPAAm) on the substrate. The optimal value of the dose for grafting was 40 KGy at 50°C. The scanning electron microscopy and atomic force microscopy (AFM) images clearly showed that increasing the absorbed dose of radiation would increase the amount of grafting. Surface topography and graft thickness in AFM images of the radiated samples showed that the PNIPAAm at the absorbed dose of radiation was properly grafted. The thickness of these grafts was about 50-100 nm. The drop water contact angles of the best grafted sample at 37°C and 10°C were 55.3 ± 1.2° and 61.2 ± 0.9° respectively, which showed the hydrophilicity and hydrophobicity of the grafted surfaces. Differential scanning calorimetry analysis also revealed the low critical solution temperature of the grafted sample to be 32°C. Thermoresponsive polymers were grafted to dishes covalently which allowed fibroblast cells to attach and proliferate at 37°C; the cells also detached spontaneously without using enzymes when the temperature dropped below 32°C. This characteristic proves that this type of grafted material has potential as a biomaterial for cell sheet engineering.

NANOMETRIC GRAFTING OF POLY (N-ISOPROPYLACRYLAMIDE) ONTO POLYSTYRENE FILM BY DIFFERENT DOSES OF GAMMA RADIATION

Poly N-isopropylacrylamide was successfully grafted onto a polystyrene cell culture dish by gamma ray. In this study, the effect of a radiation dose (radiation absorbed dosages of 10, 20, 30, 40 KGy) under appropriate temperature and grafting conditions was investigated. The FTIR analysis showed the existence of the graft PNIPAAm on the substrate. The optimal value of the dose for grafting was 40 KGy at 50 °C . The SEM and AFM images clearly showed that increasing the absorbed dose of radiation would increase the amount of grafting. Surface topography and graft thickness in AFM images of the radiated samples showed that the PNIPAAm at the absorbed dose of radiation was properly grafted. The thickness of these grafts was about 50–100 nm. The drop water contact angles of the best grafted sample at 37 °C and 10 °C were 55.3 ± 1.2 o

Polyelectrolyte Multilayer-Treated Electrodes for Real-Time Electronic Sensing of Cell Proliferation.

We report on the use of polyelectrolyte multilayer (PEM) coatings as a non-biological surface preparation to facilitate uniform cell attachment and growth on patterned thin-film gold (Au) electrodes on glass for impedance-based measurements. Extracellular matrix (ECM) proteins are commonly utilized as cell adhesion promoters for electrodes; however, they exhibit degradation over time, thereby imposing limitations on the duration of conductance-based biosensor experiments. The motivation for the use of PEM coatings arises from their long-term surface stability as promoters for cell attachment, patterning, and culture. In this work, a cell proliferation monitoring device was fabricated. It consisted of thin-film Au electrodes deposited with a titanium-tungsten (TiW) adhesion layer that were patterned on a glass substrate and passivated to create active electrode areas. The electrode surfaces were then treated with a poly(ethyleneimine) (PEI) anchoring layer and subsequent bilayers of sodium poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). NIH-3T3 mouse embryonic fibroblast cells were cultured on the device, observed by optical microscopy, and showed uniform growth characteristics similar to those observed on a traditional polystyrene cell culture dish. The optical observations were correlated to electrical measurements on the PEM-treated electrodes, which exhibited a rise in impedance with cell proliferation and stabilized to an approximate 15 % increase as the culture approached confluency. In conclusion, cells proliferate uniformly over gold and glass PEM-treated surfaces, making them useful for continuous impedance-based, real-time monitoring of cell proliferation and for the determination of cell growth rate in cellular assays.

Culture of mouse embryonic stem cells on photoimmobilized polymers

Mouse embryonic stem (ES) cells were cultured on four types of polymer with different surface properties. The polymers were poly(acrylic acid), polyallylamine, gelatin, and poly(2-methacryloyloxyethyl phosphorylcholine-co-methacrylic acid) (PMAc50), and were coupled with azidophenyl groups and photoimmobilized on conventional polystyrene cell-culture dishes. Mouse ES cells were cultured on the immobilized polymer surfaces, and cell morphology, cell growth, staining for alkaline phosphatase, activation of the transcription factor stat3, and expression of the octamer-binding protein 3/4 (Oct3/4) transcription factor and the zinc finger-containing transcription factor (GATA4) were observed. Morphology and growth rate were significantly affected by the polymer surface properties. The ES cells attached to gelatin or polyallylamine surfaces; however, colonies formed on the former but not the latter. In addition, significant enhancement of growth was observed on the gelatin surface. In contrast, ES cells aggregated to form an embryoid body on the photoimmobilized poly(acrylic acid) surface and the PMAc50 surface, although cell growth was reduced. Significant enhancement of aggregation of ES cells on the PMAc50 surface was observed in morphology and gene expression analyses.

Lamins A and C but Not Lamin B1 Regulate Nuclear Mechanics

Mutations in the nuclear envelope proteins lamins A and C cause a broad variety of human diseases, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. Cells lacking lamins A and C have reduced nuclear stiffness and increased nuclear fragility, leading to increased cell death under mechanical strain and suggesting a potential mechanism for disease. Here, we investigated the contribution of major lamin subtypes (lamins A, C, and B1) to nuclear mechanics by analyzing nuclear shape, nuclear dynamics over time, nuclear deformations under strain, and cell viability under prolonged mechanical stimulation in cells lacking both lamins A and C, cells lacking only lamin A (i.e. "lamin C-only" cells), cells lacking wild-type lamin B1, and wild-type cells. Lamin A/C-deficient cells exhibited increased numbers of misshapen nuclei and had severely reduced nuclear stiffness and decreased cell viability under strain. Lamin C-only cells had slightly abnormal nuclear shape and mildly reduced nuclear stiffness but no decrease in cell viability under strain. Interestingly, lamin B1-deficient cells exhibited normal nuclear mechanics despite having a significantly increased frequency of nuclear blebs. Our study indicates that lamins A and C are important contributors to the mechanical stiffness of nuclei, whereas lamin B1 contributes to nuclear integrity but not stiffness.

In Vitro Investigation of Cell Compatibility of Pure β-TCP Granules

In this study, osteoblastic cells were isolated from rat bone marrow and characterized. The cells were cultured on β-TCP granules and the osteoblast/ β-TCP constructs. For this purpose, bone marrow was harvested under sterile conditions. Cell aggregates were broken up by pipetting and a cell suspension was cultured in DMEM/F12. After three days, the cells that adhered to the surface of the flask were cultured in osteoblast medium. When the cells became confluent, they were passaged and cultured in 24-well polystyrene cell culture dishes. Characterization of the osteoblasts, cell proliferation and alkaline phosphatase activity were measured on days 1, 7, 14, 21 and 30. To investigate the cell compatibility of the β-TCP granules, osteoblastic cells were cultured on β-TCP granules and a polystyrene cell culture dish (control group). Cell proliferation and alkaline phosphatase (ALP) activity were measured on days 1, 7, 14, 21 and 30 in both groups. Cell growth significantly increased at each time point, but on day 30 a decrease was observed. The ALP activity also increased at each time point and also decreased on day 30. This study may be regarded as the first step leading to a therapy for various bone defects.

Cellular Attachment to Ultraviolet Ozone Modified Polystyrene Surfaces

The surfaces of standard untreated polystyrene cell culture dishes have been oxidatively modified for up to 8 min exposure time using an ultraviolet ozone treater in order to promote cell adhesion. Surface oxygen chemisorption and topographical modification has been characterized using monochromatic X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), respectively. The oxidation process is shown to proceed at low exposure times ( 60 s is in the form of loosely bound low molecular weight oxidized material (LMWOM) which is produced by oxidative scission of the PS backbone. Water washing leads to a reduction in surface oxygen content mainly by the removal of ROCO and R2CO functiona...

Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces

Poly( N-isopropylacrylamide) (PIPAAm), exhibiting a lower critical solution temperature (LCST) at 25 °C in physiological phosphate buffered saline solution (pH 7.4) and at 32 ° C in pure water, was grafted onto the surfaces of commercial polystyrene cell culture dishes. This PIPAAm-grafted surface exhibited hydrophobic surface properties at temperatures over the LCST and hydrophilic surface properties below the LCST. Endothelial cells and hepatocytes attached and proliferated on PIPAAmgrafted surfaces at 37 ° C, above the LCST. The cultured cells were readily detached from these surfaces by lowering the incubation temperature without the usual damage associated with trypsinization. In this case, the optimum temperature for cell detachment was 10 ° C for hepatocytes and 20 ° C for endothelial cells. Cell detachment was partially inhibited by sodium azide treatment, suggesting that cell metabolism directly affects cell detachment. Morphological changes of the adherent cells during cell detachment experiments indicated further involvement of active cellular metabolic processes. Cells detached from hydrophobic-hydrophilic PIPAAm surfaces not only via reduced cell surface interactions caused by the spontaneous hydration of grafted PIPAAm chains, but also by active cell morphological changes which were a function of cell metabolism.