Application of Microfluidics in Stem Cell Culture

In this chapter, we review the recent developments, including our studies on the microfabricated devices applicable to stem cell culture. We will focus on the application of pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells. In the first section, we provide a background on microfluidic devices, including their fabrication technology, characteristics, and the advantages of their application in stem cell culture. The second section outlines the use of micropatterning technology in stem cell culture. The use of microwell array technology in stem cell culture is explored in the third section. In the fourth section, we discuss the use of the microfluidic perfusion culture system for stem cell culture, and the last section is a summary of the current state of the art and perspectives of microfluidic technologies in stem cell culture

Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves.

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process

Il libro di Daniele e l'apocalittica ebraica antica

1. Un genere letterario e una visione del mondo. 2. La letteratura apocalittica ebraica. 3. Le radici dell'apocalittica ebraica. 4. Apocalittica, enochismo, qumranesimo. 5. Il libro di Daniele. 6. Le principali apocalissi non canoniche

Antibacterial Properties Of Laser Surface-Textured TiO2/ZnO Ceramic Coatings

Bacterial attachment on surfaces cause fouling, which reduces the hygiene status and effectiveness of equipment. Preventing bacterial attachment on surfaces through surface modification is a potential solution to fouling and has thus become a key research area. In this study, the effect of different ZnO contents (wt%) and picosecond laser surface texturing on the antibacterial properties of TiO2/ZnO ceramic coatings were investigated. The attachment and viability of Escherichia coli (E. coli) bacteria on laser surface-textured and non-textured TiO2/ZnO ceramic coatings were explored. Bacterial growth in an immersion suspension was evaluated using the optical density method. The number of colony-forming units on laser surface textured TiO2/ZnO coatings was found to be lower than that on non-textured coatings, which indicates that laser surface-textured coatings demonstrate strong antibacterial properties. Furthermore, the number of viable E. coli bacteria on laser surfacetextured TiO2/ZnO coatings was observed to be lower than that on non-textured coatings. This finding also demonstrates that laser surface texturing enhances the antibacterial properties of TiO2/ZnO coatings. Overall, laser surface texturing increased the surface areas of the coatings and improved the effectiveness of ZnO as an antibacterial agent. These results prove that laser surface texturing is a successful method for fabricating antibacterial surface

Reduction of N-Glycolylneuraminic Acid in Human Induced Pluripotent Stem Cells Generated or Cultured under Feeder- and Serum-Free Defined Conditions

BACKGROUND: The successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine. In particular, hiPSCs are a promising source of cells for regenerative medicine and pharmacology. However, one of the major obstacles to such uses for hiPSCs is the risk of contamination from undefined pathogens in conventional culture conditions that use serum replacement and mouse embryonic fibroblasts as feeder cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a simple method for generating or culturing hiPSCs under feeder- and serum-free defined culture conditions that we developed previously for human embryonic stem cells. The defined culture condition comprises a basal medium with a minimal number of defined components including five highly purified proteins and fibronectin as a substrate. First, hiPSCs, which were generated using Yamanaka's four factors and conventional undefined culture conditions, adapted to the defined culture conditions. These adapted cells retained the property of self renewal as evaluated morphologically, the expression of self-renewal marker proteins, standard growth rates, and pluripotency as evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo (teratoma formation in immunodeficient mice). Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions. Second, we successfully generated hiPSCs using adult dermal fibroblast under the defined culture conditions from the reprogramming step. For a long therm culture, the generated cells also had the property of self renewal and pluripotency, they carried a normal karyotype, and they were Neu5Gc negative. CONCLUSION/SIGNIFICANCE: This study suggested that generation or adaption culturing under defined culture conditions can eliminate the risk posed by undefined pathogens. This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application

BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin

Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast

Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

<p>Abstract</p> <p>Background</p> <p>Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label.</p> <p>Results</p> <p>We used four light scatter parameters: the peak height and area of the forward scatter (<it>FSheight </it>and <it>FSarea</it>) and side scatter (<it>SSheight </it>and <it>SSarea</it>). The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high <it>FSarea </it>and middle <it>SSheight/SSarea </it>fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher <it>FSheight/FSarea </it>and <it>SSheight/SSarea </it>fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their <it>FSarea-SSarea </it>profiles, for more detailed analysis. We found that <it>SSarea </it>was proportional to the cell volume and the <it>FSarea </it>correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases cell viability but decreases the proliferative ability of the PC12 cells.</p> <p>Conclusion</p> <p>We demonstrated a pretreatment method to collect well-characterized, viable, single cells without using fluorescent labels and without significant damage to the cells. This method is quantitative, rapid, single-step, and yields cells of high purity, making it applicable for a variety of single-cell level analyses.</p

Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves.

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process

Wettability characteristics of laser surface textured plasma sprayed TiO2/ZnO coatings

Surface wettability behaviour is generally categorised as hydrophobic and hydrophilic depending on the contact angle value. Surface wettability has been attracting considerable attention in research due to its unique behaviour in the field of self-cleaning, anti-fouling and anti-corrosion. Surfaces with high and low wettabilities can be fabricated using various methods, including chemical etching, anodisation and laser surface texturing. The present study investigated the effects of textured surfaces via laser surface texturing on the wettability properties of TiO2/ZnO coatings. TiO2/ZnO coating was selected due to its high photocatalytic activity, non-toxicity and low cost, which are essential properties for self-cleaning and anti-fouling applications. Picosecond laser ablation was used to produce microdimple textures on the coating surfaces. The wettability of the laser-textured surfaces was greatly reduced, achieving superhydrophilic properties with contact angle of 1.4° ± 2.42° for laser-textured TiO2 coating. On the other hand, coatings with ZnO compositions exhibited increased contact angles for both textured and non-textured surfaces. Moreover, no clear cut correlations between the surface roughness properties of non-textured and laser-textured TiO2/ZnO coatings and the surface wettability properties was observed. This finding provides new approaches in designing textured surface materials that can effectively increase the wettability properties for selfcleaning and anti-fouling application