Simvastatin Coadministration Modulates the Electrostatically Driven Incorporation of Doxorubicin into Model Lipid and Cell Membranes.

Understanding the interactions between drugs and lipid membranes is a prerequisite for finding the optimal way to deliver drugs into cells. Coadministration of statins and anticancer agents has been reported to have a positive effect on anticancer therapy. In this study, we elucidate the mechanism by which simvastatin (SIM) improves the efficiency of biological membrane penetration by the chemotherapeutic agent doxorubicin (DOX) in neutral and slightly acidic solutions. The incorporation of DOX, SIM, or a combination of them (DOX:SIM) into selected single-component lipid membranes, zwitterionic unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), neutral cholesterol, and negatively charged 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) was assessed using the Langmuir method. The penetration of neutral lipid monolayers by the codelivery of SIM and DOX was clearly facilitated at pH 5.5, which resembles the pH conditions of the environment of cancer cells. This effect was ascribed to partial neutralization of the DOX positive charge as the result of intermolecular interactions between DOX and SIM. On the other hand, the penetration of the negatively charged DMPS monolayer was most efficient in the case of the positively charged DOX. The efficiency of the drug delivery to the cell membranes was evaluated under in vitro conditions using a panel of cancer-derived cell lines (A172, T98G, and HeLa). MTS and trypan blue exclusion assays were performed, followed by confocal microscopy and spheroid culture tests. Cells were exposed to either free drugs or drugs encapsulated in lipid carriers termed cubosomes. We demonstrated that the viability of cancer cells exposed to DOX was significantly impaired in the presence of SIM, and this phenomenon was greatly magnified when DOX and SIM were coencapsulated in cubosomes. Overall, our results confirmed the utility of the DOX:SIM combination delivery, which enhances the interactions between neutral components of cell membranes and positively charged chemotherapeutic agents.

Trypan Blue - Adapting a Dye Used for Labelling Dead Cells to Visualize Pinocytosis in Viable Cells.

BACKGROUND/AIMS: Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. METHODS: We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. RESULTS: We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. CONCLUSION: In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.

High-throughput quantification of the effect of DMSO on the viability of lung and breast cancer cells using an easy-to-use spectrophotometric trypan blue-based assay.

One of the main aspects investigated in potential therapeutic compounds is their effect on cells viability and proliferative ability. Although various methods have been developed to investigate these aspects, these methods present with shortcomings in terms of either cost, availability, accuracy, precision, or throughput. This study describes a simple, economic, reproducible, and high-throughput assay to quantify cell death and proliferation. In this assay, adherent cells are fixed, stained with trypan blue, and measured for trypan blue internalization using a spectrophotometric absorbance plate reader. Corresponding cell counts to the absorbance measurements are extrapolated from a standard curve. This assay was used to measure the effect of dimethyl sulfoxide (DMSO) on the viability of breast and lung cancer cells. Decrease in cell count associated with the increase in DMSO percentage and exposure time. The assay's results closely correlated with the conventional trypan blue exclusion assay (Pearson correlation coefficient (r) > 0.99; p < 0.0001), but with higher precision. The assay developed in this study can be used for various applications such as optimization, cell treatment investigations, proliferation, and cytotoxicity studies.

Establishment of a porcine corneal endothelial organ culture model for research purposes.

Human corneas usually are not available for research, as they are used for transplantation only. At the same time, scientific studies on cultured human endothelial cells can produce misleading results due to inevitable dedifferentiation. Therefore, an organ-culture model of porcine corneas-displaying endothelial cell death rates comparable to those of cultured human corneas-would be very desirable. Fresh pig eyes were prepared under sterile conditions to obtain corneoscleral buttons, corneal buttons and so called "split corneal buttons" (new preparation method) and cultivated for 15 days. Morphology of the endothelial cell layer was observed by light microscopy on day 1, 8 and 15. On day 15 staining with trypan blue and alizarin red S was performed. Photographs were evaluated in a randomized, blinded manner. Here, the morphology of the corneal endothelium and the number of endothelial cells per mm2 were analyzed. After 15 days of cultivation the endothelial cell layer was maintained only in corneal buttons and split corneal buttons. Alizarin red S stained areas and the existence of polymorphisms like rosette figures and reformation figures were significantly less frequent in split corneal buttons than in corneal buttons. Loss of endothelial cells was significantly greater in corneal buttons [575 ± 25/250 cells/mm2 (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm2 (median ± 25%/75%-quantile); 10.2%]. The new preparation method of split corneal buttons allows the cultivation of porcine corneas for 2 weeks with cell death rates comparable to those of the corresponding human tissue in cornea banks without the need to add de-swelling additives to the media. This is therefore a simple and highly reliable method model to be applied in intervention studies on corneal endothelial cells in their natural compound.

Selective imaging of internalized proteopathic α-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies.

Direct cell-to-cell transmission of proteopathic α-synuclein (α-syn) aggregates is thought to underlie the progression of neurodegenerative synucleinopathies. However, the specific intracellular processes governing this transmission remain unclear because currently available model systems are limited. For example, in cell culture models of α-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied α-syn seed species. Herein, we employed fluorescently labeled α-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized α-syn seeds in cultured primary neurons and to quantitatively characterize the concentration dependence, time course, and inhibition of pff uptake. To study the long-term fates of exogenous α-syn pffs in neurons, we developed a pff species labeled at amino acid residue 114 with the environmentally insensitive fluorophore BODIPY or the pH-sensitive dye pHrodo red. We found that pffs are rapidly trafficked along the endolysosomal pathway, where most of the material remains for days. We also found that brief pharmacological perturbation of lysosomes shortly after the pff treatment causes aberrations in intracellular processing of pff seeds concomitant with an increased rate of inclusion formation via recruitment of endogenous α-syn to a relatively small number of exogenous seeds. Our results validate a quantitative assay for pff uptake in primary neurons, implicate lysosomal processing as the major fate of internalized proteopathic seeds, and suggest lysosomal integrity as a significant rate-determining step in the transmission of α-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.

Autoinducer-2 of quorum sensing is involved in cell damage caused by avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) infections are responsible for great losses in the poultry industry. Quorum sensing (QS) acts as a global regulatory system that controls genes involved in bacterial pathogenesis, metabolism and protein biosynthesis. However, whether QS of APEC is related to cell damage has not been elucidated. In the present study, we explored the correlation between the damage of chicken type II pneumocytes induced by APEC and the autoinducer-2 (AI-2) activity of APEC. The results showed that when chicken type II pneumocytes were co-cultured with 108 CFU/ml of APEC-O78 for 6 h, the release of LDH reached the highest level (192.5 ± 13.4 U/L) (P < 0.01), and the percentages of dead cells followed the same trend in trypan blue exclusion assay. In addition, the AI-2 activity of cell-free culture fluid (CF) reached the maximum value after 6 h co-culture with 108 CFU/ml of APEC-O78. At the same time, the mRNA expressions of eight virulence genes (papC, fimA, fimC, hlyE, ompA, luxS, pfs, and qseA) of 108 CFU/ml APEC-O78 were significantly increased compared with those of 107 CFU/ml, and the mRNA expressions of four virulence genes (hlyE, tsh, iss, and luxS) of 108 CFU/ml APEC-O78 were higher than those of 109 CFU/ml (p < 0.05) after incubation for 6 h. These results suggested that AI-2-mediated QS is involved in the cell damage induced by APEC-O78, indicating AI-2 may be one new potential target for preventing chicken colibacillosis.

Novel adult feeding disruption test (FDT) to detect insecticide resistance of lepidopteran pests in cotton.

BACKGROUND: Resistance monitoring is an important aspect of insect resistance management and the preservation of insecticide efficacy. The adult vial test (AVT) is most often used for resistance monitoring for a variety of insects. A potential alternative method is feeding disruption where resistant insects are distinguished from susceptible insects on the basis of their ability to feed on insecticide in nectar containing a colorimetric marker to measure feeding. The advantages of a feeding disruption test (FDT) for lepidopteran adults might include a more rapid assay than AVT, an assay format easier to prepare, a bioassay applicable to both oral and contact insecticides and the provision of food and water during the course of the test. The objective of the present work was to determine the feasibility of an adult FDT. RESULTS: Heliothis virescens moths fed permethrin and spinosad in dyed nectar yielded dose-dependent ingestion, fecal production and mortality data. A permethrin diagnostic dose distinguished pyrethroid-resistant from pyrethroid-susceptible moths, based on fecal production. CONCLUSION: Proof of concept was demonstrated for an adult FDT in which resistant moths were distinguished from susceptible moths on the basis of the ability of the insect to feed on insecticide in dyed nectar and produce dyed feces.

Photodynamic properties of vital dyes for vitreoretinal surgery.

The purpose of this study was to evaluate photodynamic properties of indocyanine green (ICG), brilliant blue G (BBG) and trypan blue (TB) as currently used vital dyes for chromovitrectomy. Under consideration of intraoperative illumination intensities and dye concentrations, a simulative in vitro investigation was set up. Therefore, standardized dilutions of original ICG, BBG and TB vials were irradiated at a wavelength of 366 nm with an intensity of 14 µW/cm2 between 0 and 48 h. After this, all samples were measured spectroscopically in a 220- to 750-nm bandwidth. Analyzing the vital dyes over the time course, an exponential photolysis was observed for ICG, whereas BBG and TB presented photostable properties. Regarding ICG, 5% of the concentration was degraded to toxic metabolites every 20 min. For this reason, our study provides evidence that intraocular dye concentrations and modern endoillumination systems alone cannot fully prevent ICG photodegradation.

siRNA-mediated down-regulation of ceramide synthase 1 leads to apoptotic resistance in human head and neck squamous carcinoma cells after photodynamic therapy.

BACKGROUND: The effectiveness of photodynamic therapy (PDT) for cancer treatment correlates with apoptosis. We previously observed that the knockdown of ceramide synthase 6, an enzyme from the de novo sphingolipid biosynthesis pathway, is associated with marked reduction in C18-dihydroceramide and makes cells resistant to apoptosis post-PDT. Down-regulation of ceramide synthase 1 (CERS1) can also render cells resistant to anticancer drugs. AIM: To explore the impact of CERS1 knockdown on apoptosis and the sphingolipid profile, post-PDT, with the silicone phthalocyanine Pc 4, in a human head and neck squamous carcinoma cell line. MATERIALS AND METHODS: Besides siRNA transfection and PDT treatment, the following methods were used: immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectroflurometry and flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation. RESULTS: CERS1 knockdown led to inhibition of PDT-induced caspase 3-like (DEVDase) activation, of apoptosis and cell death. CERS1 knockdown was associated with global and selective decreases in ceramides and dihydroceramides, in particular C18-, C18:1- and C20-ceramide post-PDT. CONCLUSION: Our novel findings are consistent with the notion that CERS1 regulates apoptotic resistance to PDT, partly via C18- and C20-ceramide, and that CERS1 is a molecular target for controlling resistance to PDT.

The use of agroinfiltration for transient expression of plant resistance and fungal effector proteins in Nicotiana benthamiana leaves.

Agroinfiltration is a versatile, rapid and simple technique that is widely used for transient gene expression in plants. In this chapter we focus on its use in molecular plant pathology, and especially for the expression of plant resistance (R) and fungal avirulence (Avr) (effector) genes in leaves of Nicotiana benthamiana. Co-expression of an R gene with the corresponding Avr gene triggers host-defence responses that often culminate in a hypersensitive response (HR). This HR is visible as a necrotic sector in the infiltrated leaf area. Staining of the infiltrated leaves with trypan blue allows visual scoring of the HR. Furthermore, fusion of a fluorescent tag to the recombinant protein facilitates determination of its sub-cellular localization by confocal microscopy. The matching gene pair I-2 and Avr2, respectively from tomato and the fungal root-pathogen Fusarium oxysporum f. sp. lycopersici, is presented as a typical example.

RGDS-fuctionalized alginates improve the survival rate of encapsulated embryonic stem cells during cryopreservation.

Cryopreservation of stem cells, especially embryonic stem cells, is problematic because of low post-thaw cell survival rates and spontaneous differentiation following recovery. In this investigation, mouse embryonic stem cells (mESCs) were encapsulated in arginine-glycine-aspartic acid-serine (RGDS)-coupled calcium alginates (1.2 percent, w/v), allowed to attach to the substratum and then cryopreserved in 10 percent (v/v) dimethyl sulfoxide (DMSO) solution at a slow cooling rate of 1 C per min. RGDS coupling to alginate was confirmed by Transmission Fourier Transform Infra-Red spectroscopy (T-FTIR) and quantified by using ninhydrin-Ultraviolet/Visible light (ninhydrin-UV/VIS) assay. Flow cytometry data showed that mESCs cryopreserved in RGDS-alginate beads had a higher expression of stem cell markers compared with cells cryopreserved in suspension or cells cryopreserved in unmodified alginates. Cell viability after thawing was assessed using trypan blue exclusion assay and monitored using Alamar blue assay for 6 hours. It was shown that post-thaw cell survival rate was significantly higher for cells encapsulated in RGDS-modified alginate (93 ± 2 percent, mean and standard error) than those in suspension (52 ± 2 percent) or in unmodified alginates (62 ± 3 percent). These results showed that cells encapsulated and attached to a substratum have better survival rate and stem cell marker expression 24 hours after cryopreservation than those in suspension. Encapsulation in RGDS-alginate was optimized for peptide concentration, cryoprotective agent loading time and cooling rate. The best result was obtained when using 12.5 mg peptide per g alginate, 30 minutes loading time and 1 C per min cooling rate.

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume.

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted.

Analysis of endothelial barrier function in vitro.

Increased microvascular solute permeability underlies many forms of pathophysiological conditions, including inflammation. Endothelial monolayer cultures provide an excellent model system which allows systemic and mechanistic study of endothelial barrier function and paracellular permeability in vitro. The endothelial-specific complexus adherens junction protein VE-cadherin and their intracellular complex form pericellular structures along the cell borders which are critical to regulate endothelial barrier function by controlling pericellular permeability of vasculature. Here, we describe methods for both visualizing and quantifying junctional permeability and barrier changes in endothelial monolayers in vitro.

Rhinacanthus nasutus protects cultured neuronal cells against hypoxia induced cell death.

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington's disease, Parkinson's disease and Alzeheimer's disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL⁻¹ root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL⁻¹ had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL⁻¹ reduced HT-22 cell proliferation. We also used H2DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

[Research of biocompatibility of nanoparticles with fluorescent domen Er/Yb in the neutrophilic granulocytes system].

Fluorescent tags are extensively used for the diagnostic, labeling and marking in vivo and in vitro systems. In this study, fluorescent nanoparticles with Er/Yb lightning center were tested on neutrophilic granulocytes. The main purpose was to idenfity possible toxic effect. The negative impact of fluorophores on the metabolism of neutrophilic and their enzyme systems, on the receptor-mediated cell responses and on the rigidity of cell membranes was shown. The viability of neutrophils (estimated with the use of propidium iodide) after 2 hours of incubation with the fluorescent nanoparticles in concentrations of 10(-4) and 10(-3) mM was 27.0 +/- 6.6 and 19.07 +/- 3.34 %, respectively.

New 3',8''-linked biflavonoids from Selaginella uncinata displaying protective effect against anoxia.

Seven 3',8''-linked bioflavonoids, including one new compound, (2''S)-2'', 3''-dihydroamentoflavone-4'-methyl ether and six known compounds: (2S)-2,3- dihydroamentoflavone-4'-methyl ether, (2S,2''S)-2,3,2'',3''-tetrahydroamento- flavone-4'-methyl ether, (2S,2''S)-tetrahydroamentoflavone, (2S)-2,3-dihydro- amentoflavone and (2''S)-2'',3''-dihydroamentoflavone (6) and amentoflavone, were isolated from the 60% ethanolic extract of Selaginella uncinata (Desv.) Spring. The structures of these compounds were elucidated mainly by analysis of their 1D and 2D NMR spectroscopic data, and their absolute configurations were determined by circular-dichroism (CD) spectroscopy. All the seven compounds showed protective effect against anoxia in the anoxic PC12 cells assay, in which compound 6 displayed particularly potent activity.

Studies on cryoprotectant toxicity to zebrafish (Danio rerio) ovarian tissue fragments.

Cryopreservation of ovarian tissue is a viable alternative to cryopreservation of oocytes and embryos in many species but it has not been studied in fish. Selection of cryoprotectant is an important step in designing cryopreservation protocols. In order to identify the optimum cryoprotectant (CPA) in a suitable concentration for zebrafish ovarian tissue cryopreservation, studies on toxicities of five commonly used cryoprotectants methanol, ethanol, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were carried out. Experiments were conducted on ovarian tissue fragments consisting of stage I and stage II ovarian follicles. Ovarian tissue fragments were incubated in 90% L-15 medium (pH 9) containing 1-4M cryoprotectants for 30min at 22°C. Three different tests were used to assess ovarian tissue fragment viability: trypan blue (TB) staining, fluorescein diacetate (FDA) combined with propidium iodide (PI) staining and adenosine 5´- triphosphate (ATP) assay. Results from these tests showed that ATP assay was more sensitive than FDA+PI or TB staining for assessing cryoprotectant toxicity to follicles in tissue fragments. Methanol and ethanol were the least toxic cryoprotectants tested. Cryoprotectant toxicity increased in the order of methanol/ethanol, DMSO, PG and EG. Ethanol was used for zebrafish ovarian tissue for the first time and the results showed that the effect of methanol and ethanol on ovarian tissue fragments were comparable. As methanol has been shown to be the most effective cryoprotectant for zebrafish ovarian follicles in our laboratory, the use of ethanol will also be considered in assisting future freezing protocol design. The present study also showed that stage II ovarian follicles are more sensitive to cryoprotectant treatment than stage I follicles in tissue fragments. The results obtained in this study provided useful information for ovarian tissue fragment cryopreservation protocol design in the future.

Photodynamic effects of pterin on HeLa cells.

Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. In the present study, we ascertained that 2-aminopteridin-4(3H)-one, abbreviated as Ptr, is readily incorporated into and/or onto cervical cancer cells (HeLa) and that these cells die upon UV-A irradiation of Ptr. Cell death was assessed using two tests: (1) the Rhodamine 123 fluorescence assay for mitochondrial viability and (2) the Trypan Blue assay for membrane integrity. The data suggest that, for Ptr-dependent photoinitiated cell death, events related to mitochondrial failure precede those associated with the failure of the cell membrane.

Comparison of the automated fluorescence microscopic viability test with the conventional and flow cytometry methods.

The cell viability test is an essential tool in any laboratory, performing cell-based studies and clinical laboratory tests. The trypan blue exclusion method is the most popular assay for its simple concept among various diagnostic tools. However, several disadvantages include time-consuming and labor-intensive steps with low precision. In this study, we evaluated a new technique for the automatic cell viability measurement with microscopic cell counter and microchip. Upon blood draw from 11 healthy volunteers, Mononuclear cells were separated immediately from the heparinized whole blood, and the viable cells were diluted from 100 to 1%. The cell viability tests were performed simultaneously with following three methods: the conventional manual trypan blue exclusion method; the flow cytometry measurement with propidium iodide stain; and the newly developed microscopic cell counter with microchip. Linearities, precisions, and correlations from three methods were analyzed and compared. The correlations data from the microscopic cell counter were in good agreement with both the conventional trypan blue method (r=0.99, P<0.05) and the flow cytometry (r=0.99, P<0.05), respectively. The precision (2.0-6.2%) and linearity from the microscopic cell counter method with microchip were superior in comparison with the conventional method. The microscopic cell counter with microchip performed well with high precision, linearity, and efficient running time than both the manual trypan blue and the flow cytometry methods.

Fabricating gradient hydrogel scaffolds for 3D cell culture.

Optimizing cell-material interactions is critical for maximizing regeneration in tissue engineering. Combinatorial and high-throughput (CHT) methods can be used to systematically screen tissue scaffolds to identify optimal biomaterial properties. Previous CHT platforms in tissue engineering have involved a two-dimensional (2D) cell culture format where cells were cultured on material surfaces. However, these platforms are inadequate to predict cellular response in a three-dimensional (3D) tissue scaffold. We have developed a simple CHT platform to screen cell-material interactions in 3D culture format that can be applied to screen hydrogel scaffolds. Herein we provide detailed instructions on a method to prepare gradients in elastic modulus of photopolymerizable hydrogels.