Plating Efficiency Research Articles

Efficient long-term cryopreservation of pluripotent stem cells at −80 °C

Current long term cryopreservation of cell stocks routinely requires the use of liquid nitrogen (LN2), because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at higher storage temperatures, e.g. −80 °C. This instability leads to ice recrystallization, causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2 for cell storage significantly increases operational expense and raises issues related to impaired working efficiency and safety. Here we demonstrate that addition of Ficoll 70 to cryoprotectant solutions significantly improves system thermal stability at the working temperature (~−80 °C) of laboratory deep freezers. Moreover, a medium comprised of Ficoll 70 and dimethyl sulfoxide (DMSO) in presence or absence of fetal bovine serum (FBS) can provide reliable cryopreservation of various kinds of human and porcine pluripotent stem cells at −80 °C for periods that extend up to at least one year, with the post-thaw viability, plating efficiency, and full retention of pluripotent phenotype comparable to that achieved with LN2 storage. These results illustrate the practicability of a promising long-term cryopreservation method that completely eliminates the need for LN2.

Deposition of silver films on copper nanopowders by three-times electroless plating

In order to enhance the oxidation resistance of a copper substrate, silver films were deposited by electroless plating onto pure copper nanopowder. However, the efficiency of electroless plating for nanopowders is very low. Silver-coated copper nanopowders can be successfully achieved by three-times electroless silver plating on copper nanopowder. The microstructure and the chemical composition of the silver-coated copper nanopowders were systematically analyzed. The silver films have shown excellent oxidation resistance, according to the results of accelerated temperature cycling tests. The silver-coated copper nanopowders were characterized using scanning electron microscopy and X-ray diffraction. Following conclusions were obtained under the optimum condition that the reaction temperature was 50 °C, the dosage of EDTA was 4 g/L, hypophosphite (NaH2PO2) was 20.6 g/L. When the silver content reached 25 %, silver was homogeneously distributed around copper nanopowders.

Identification of optimal rate-enhanced silver ELP processes for silver–ceramic composite membrane fabrication

ABSTRACTThis work targets the identification of optimal rate-enhancement technique for the electroless fabrication of silver–ceramic porous membranes. The membranes were fabricated with porous ceramic supports of 4.5 µm and porosity of 19.3% and with alternate electroless plating (ELP) processes including conventional (CEP), sonication (SOEP), and surfactant (SIEP) plating baths. The alternate processes were evaluated based on the process (conversion, plating efficiency (η), average plating rate) and membrane characteristics (percent pore densification (PPD), metal loading index (MLI)), and their combinations (η/(MLI.PPD)). Among alternate processes, SOEP has been evaluated to be the best to yield silver–ceramic membranes.

Honokiol suppresses pancreatic tumor growth, metastasis and desmoplasia by interfering with tumor–stromal cross-talk

The poor clinical outcome of pancreatic cancer (PC) is largely attributed to its aggressive nature and refractoriness to currently available therapeutic modalities. We previously reported antitumor efficacy of honokiol (HNK), a phytochemical isolated from various parts of Magnolia plant, against PC cells in short-term in vitro growth assays. Here, we report that HNK reduces plating efficiency and anchorage-independent growth of PC cells and suppresses their migration and invasiveness. Furthermore, significant inhibition of pancreatic tumor growth by HNK is observed in orthotopic mouse model along with complete-blockage of distant metastases. Histological examination suggests reduced desmoplasia in tumors from HNK-treated mice, later confirmed by immunohistochemical analyses of myofibroblast and extracellular matrix marker proteins (α-SMA and collagen I, respectively). At the molecular level, HNK treatment leads to decreased expression of sonic hedgehog (SHH) and CXCR4, two established mediators of bidirectional tumor-stromal cross-talk, both in vitro and in vivo . We also show that the conditioned media (CM) from HNK-treated PC cells have little growth-inducing effect on pancreatic stellate cells (PSCs) that could be regained by the addition of exogenous recombinant SHH. Moreover, pretreatment of CM of vehicle-treated PC cells with SHH-neutralizing antibody abolishes their growth-inducing potential on PSCs. Likewise, HNK-treated PC cells respond poorly to CM from PSCs due to decreased CXCR4 expression. Lastly, we show that the transfection of PC cells with constitutively active IKKβ mutant reverses the suppressive effect of HNK on nuclear factor-kappaB activation and partially restores CXCR4 and SHH expression. Taken together, these findings suggest that HNK interferes with tumor-stromal cross-talk via downregulation of CXCR4 and SHH and decreases pancreatic tumor growth and metastasis.

Understanding Speciation in Ionic-Liquid Electrolytes for Non-Aqueous Redox Flow Batteries

Conventional aqueous redox flow batteries (RFB) are limited by low energy and power densities; thus, recent focus has been on development of non-aqueous electrolytes. Although organic solvents allow for increased voltage windows, they possess a several drawbacks, including sensitivity to moisture, low solubility of active species, and high flammabilities and toxicities. Ionic liquids (IL) offer a “greener” alternative to volatile organic electrolytes as they are non-toxic and nonflammable [1, 2]. Iron chloride based IL electrolytes containing up to 6.3 M iron have been synthesized. It was found that electrolyte conductivity and viscosity, as well as the nature of the electroplated iron, are greatly influenced by electrolyte composition. An electrolyte containing iron chloride, choline chloride, and ethylene glycol in a 1:1:4 molar ratio was attractive due to its high plating efficiencies, fast kinetics, and acceptable conductivity [3]. Electrochemical reactivity of the solute ions as well as the physical properties of the electrolyte are controlled by speciation of the metals in solution. To understand what ions are responsible for the observed properties in these iron IL electrolytes, chemical speciation was investigated using x-ray absorption spectroscopy (XAS) as well as x-ray photoelectron spectroscopy (XPS). The two techniques indicate the presence of organometallic compounds which may hinder the electrokinetics as well as the fluid properties. For example, the conductivity is a function of the mole fraction of iron as well as the iron to chloride ratio (Fig 1 below). Complexes such as [FeCl4]-have been shown to exhibit enhanced fluid properties [4], however, when the iron:chloride ratio is less than 1:4, di-iron species have been known to form [5]. The properties of these iron IL electrolytes as a function of composition coupled with results from XAS and XPS to explain observed trends will be presented. [1] M. J. Earle and R. K. Seddon, Pure Appl. Chem., vol. 72, p. 1391, 2000. [2] K. N. Marsh, A. Deev, A. C.-T. Wu, T. E., and A. Klamt, Korean J Chem. Eng. , vol. 19, 2002. [3] J. S. W. M. A. Miller, and R. F. Savinell, "Iron Ionic Liquid Electrolytes for Redox Flow Battery Applications," Journal of The Electrochemical Society, vol. 163, pp. A578-A579, 2016. [4] I. M. H. C. A. Angell, P. A. Cheeseman, "Physico-chemical and computer simulation studies of the role of cation coordination numbers on melt physical properties," Electrochemical Society Proceedings, p. 138, 1976. [5] M. S. Sitze, E. R. Schreiter, E. V. Patterson, and R. G. Freeman, "Ionic Liquids Based on FeCl3 and FeCl2. Raman Scattering and ab Initio Calculations," vol. - 40, pp. - 2304, 2001. Figure 1

Effect of Degraded Additives on Under-Fill TSV

In the past few years the 3D IC integration (3DI) stacking has emerged as a potentially novel approach for extending computing performance without the high cost of CMOS scaling. The 3DI stacking technology is thought to be the essential technology of the next generation high-end semiconductors which will provides improved performance, and reduced form factor for applications such as logic-memory integration, image sensors, MEMS, and LED. The through silicon via (TSV) is one of the main components for 3DI stacking. Copper plating has been used for TSV metallization due to its low resistivity, low cost, excellent plating efficiency, easy to integrate, excellent reliability and a unique bottom up fill capability in presence of additives in the plating bath. One of the major challenges in the TSV metallization by plating is embedded voids and underfill TSV. Among the many parameters in TSV plating; bath composition especially suppressor to accelerator ratio (S/A) significantly affects TSV under-fill. It has been seen that the additives also degrades over the shelf life aging depends on their storage condition. Since the degradation of both suppressor and accelerator is not happening in the same rate, the S/A ratio vary and thus found to be caused TSV under-fill. Fig. 1(a) shows a cross-section SEM image of a TSV plated with BKM bath additives for 1600s. In contrast, Fig. 1(b) and 1(c) shows cross-section SEM images those were plated with same amount of BKM accelerator but with 10% and 25% less suppressor than the BKM respectively. It can be seen that the growth of copper inside the TSV increased~ 33% (with 25% lower suppressor) than the POR without increase of Cu film thickness on side wall, Chamfers and field area. Similarly, for a given suppressor, the copper fill rate can also varied by varying the accelerator concentration. In this paper, we will describe how additives affect the TSV under-fill and correlate the TSV under-fill with degradation of additives during their shelf life aging. Fig. 1(a) Cross-section SEM image of a TSV deposited with BKM plating additives of accelerator and suppressor and plating process for 1600s. Fig.1 (b) and (c) shows cross –section SEM images of a TSV those were plated with same amount of BKM accelerator but 10 and 25% less suppressor respectively than the BKM condition but exactly same plating process and time as Fig 1a Figure 1

Hepatocyte Isolation After Laparoscopic Liver Resection

Liver tissue obtained from partial hepatectomy is a common source for isolation of primary human hepatocytes. Until now, liver resections were most commonly performed by conventional open surgery. Although the laparoscopic approach is currently emerging in liver surgery, data on the outcome of hepatocyte isolation from laparoscopically resected liver tissue are not available. A total of 22 hepatocyte isolations were performed using the two-step collagenase perfusion technique from October 2015 to March 2016. Liver tissue was obtained from n = 15 open liver resections (OLRs) and n = 7 laparoscopic liver resections (LLRs). Isolation parameters (cell yield, viability, and Percoll survival) were assessed and hepatocyte function (plating efficiency, urea, albumin, and aspartate aminotransferase) was measured over a culture period of 6 days (OLR: n = 13; LLR: n = 3). Total cell yield (OLR: 36.81 ± 6.77 × 10(6) cells/g vs. LLR 16.84 ± 10.66 × 10(6) cells/g, p = 0.0318) as well as viable yield (OLR 31.70 ± 6.05 × 10(6) cells/g vs. LLR 14.70 ± 9.89 × 10(6) cells/g, p = 0.0260) was significantly higher in the OLR group. Subgroup analysis revealed that the worse outcome of isolation of laparoscopically resected liver tissue was associated with right-lateral LLRs, whereas hepatocyte isolation from left-lateral LLRs was as effective as from open surgery. Hepatocyte function did not differ between hepatocytes from openly resected versus left-lateral laparoscopically resected liver tissue. We here present the first data on hepatocyte isolation from laparoscopic liver surgery. Although the overall outcome is worse compared with open surgery, our data suggest that liver tissue from laparoscopic resection of the left lobe is an excellent source for primary human hepatocytes.

The Baseplate of Lactobacillus delbrueckii Bacteriophage Ld17 Harbors a Glycerophosphodiesterase

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca(2+) ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage.

Morphology and acoustic artefacts of copper deposits electroplated using megasonic assisted agitation

Purpose This study aims to understand the influence of megasonic (MS)-assisted agitation on printed circuit boards (PCBs) electroplated using copper (Cu) electrolyte solutions to improve plating efficiencies through enhanced ion transportation. Design/methodology/approach The impact of MS-assisted agitation on topographical properties of the electroplated surfaces was studied through a design of experiments by measuring surface roughness, which is characterised by values of the parameter Ra as measured by white light phase shifting interferometry and high-resolution scanning electron microscopy. Findings An increase in Ra from 400 to 760 nm after plating was recorded for an increase in acoustic power from 45 to 450 W. Roughening increased because of micro-bubble cavitation energy and was supported through direct imaging of the cavitation. Current thieving effect by the MS transducer induced low currents, leading to large Cu grain frosting and reduction in the board quality. Current thieving was negated in plating trials through specific placement of transducer. Wavy electroplated surfaces, due to surface acoustic waves, were also observed to reduce the uniformity of the deposit. Research limitations/implications The formation of unstable transient cavitation and variation of the topology of the Cu surface are unwanted phenomena. Further plating studies using MS agitation are needed, along with fundamental simulations, to determine how the effects can be reduced or prevented. Practical implications This study can help identify manufacturing settings required for high-quality MS-assisted plating and promote areas for further investigation, leading to the development of an MS plating manufacturing technique. Originality/value This study quantifies the topographical changes to a PCB surface in response to MS agitation and evidence for deposited Cu artefacts due to acoustic effects.

Effect of plating bath hydrodynamic on the morphology of palladium layer electrolessly deposited on porous ceramic support

The present work was focused on the preliminary stage of palladium composite membrane preparation through electroless plating and the effects of bath hydrodynamic properties on the deposited layer morphology. Eight samples of palladium composite membrane were prepared which were different in terms of plating time, solution stirring rate and bath agitating mode. Scanning electron microscopy and energy-dispersive X-ray spectroscopy were used for characterization of deposited layer. In comparison with the support rotating mode, the support fixing and the magnet rotating mode, which are caused running the solution toward the support, were led to a deposited layer with better quality, larger grains of deposited layer, more compacted plating and higher plating efficiency.

Biological responses of human solid tumor cells to X-ray irradiation within a 1.5-Tesla magnetic field generated by a magnetic resonance imaging-linear accelerator.

Devices that combine magnetic resonance imaging with linear accelerators (MRL) represent a novel tool for MR-guided radiotherapy. However, whether magnetic fields (MFs) generated by these devices affect the radiosensitivity of tumors is unknown. We investigated the influence of a 1.5-T MF on cell viability and radioresponse of human solid tumors. Human head/neck cancer and lung cancer cells were exposed to single or fractionated 6-MV X-ray radiation; effects of the MF on cell viability were determined by cell plating efficiency and on radioresponsiveness by clonogenic cell survival. Doses needed to reduce the fraction of surviving cells to 37% of the initial value (D0s) were calculated for multiple exposures to MF and radiation. Results were analyzed using Student's t-tests. Cell viability was no different after single or multiple exposures to MRL than after exposure to a conventional linear accelerator (Linac, without MR-generated MF) in 12 of 15 experiments (all P > 0.05). Single or multiple exposures to MF had no influence on cell radioresponse (all P > 0.05). Cells treated up to four times with an MRL or a Linac further showed no changes in D0s with MF versus without MF (all P > 0.05). In conclusion, MF within the MRL does not seem to affect in vitro tumor radioresponsiveness as compared with a conventional Linac. Bioelectromagnetics. 37:471-480, 2016. © 2016 Wiley Periodicals, Inc.

Characterization of novel Staphylococcus aureus lytic phage and defining their combinatorial virulence using the OmniLog® system.

ABSTRACTSkin and soft tissue infections (SSTI) caused by methicillin resistant Staphylococcus aureus (MRSA) are difficult to treat. Bacteriophage (phage) represent a potential alternate treatment for antibiotic resistant bacterial infections. In this study, 7 novel phage with broad lytic activity for S. aureus were isolated and identified. Screening of a diverse collection of 170 clinical isolates by efficiency of plating (EOP) assays shows that the novel phage are virulent and effectively prevent growth of 70–91% of MRSA and methicillin sensitive S. aureus (MSSA) isolates. Phage K, which was previously identified as having lytic activity on S. aureus was tested on the S. aureus collection and shown to prevent growth of 82% of the isolates. These novel phage group were examined by electron microscopy, the results of which indicate that the phage belong to the Myoviridae family of viruses. The novel phage group requires β-N-acetyl glucosamine (GlcNac) moieties on cell wall teichoic acids for infection. The phage were distinct from, but closely related to, phage K as characterized by restriction endonuclease analysis. Furthermore, growth rate analysis via OmniLog® microplate assay indicates that a combination of phage K, with phage SA0420ᶲ1, SA0456ᶲ1 or SA0482ᶲ1 have a synergistic phage-mediated lytic effect on MRSA and suppress formation of phage resistance. These results indicate that a broad spectrum lytic phage mixture can suppress the emergence of resistant bacterial populations and hence have great potential for combating S. aureus wound infections.

Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems.

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. Here, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding and modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.

SLPW: A Virulent Bacteriophage Targeting Methicillin-Resistant Staphylococcus aureus In vitro and In vivo.

Staphylococcus aureus (S. aureus) is a Gram-positive pathogen causing a variety of infections in humans and animals. Extensive use of antibiotics has led to the emergence of methicillin-resistant S. aureus (MRSA). As an alternative antibacterial agent against drug-resistant S. aureus, a lytic phage, designated SLPW, was isolated from fecal sewage in a pig farm. The SLPW was morphologically classified under Podoviridae and contains a double-stranded DNA genome. The genome of SLPW was 17,861 bp (29.35% G+C) containing 20 open reading frames and lacked regions encoding lysogeny-related integrase gene and cI repressor gene. Phage SLPW showed a broad host range and high efficiency of plating against various types of S. aureus. One-step growth curve showed a short latency period (10 min) and a long lytic period (120 min). Phage SLPW remained stable under a wide range of temperatures or pH and was almost unaffected in chloroform or ultraviolet light. Further, it efficiently lysed MRSA strains in vitro and in vivo. Intraperitoneal phage administration at 1 h post-infection cured the mice and reduced the bacterial expression of inflammatory cytokines in mice. Specifically, the phage SLPW displayed a wide antibacterial spectrum. It was therapeutically effective against intra-abdominal infection in mice harboring different multilocus sequence typing (MLST) types of S. aureus strains. Therefore, phage SLPW is a potential therapeutic agent against MRSA infections.

Intrinsic Radiosensitivity and Cellular Characterization of 27 Canine Cancer Cell Lines.

Canine cancer cell lines have progressively been developed, but are still underused resources for radiation biology research. Measurement of the cellular intrinsic radiosensitivity is important because understanding the difference may provide a framework for further elucidating profiles for prediction of radiation therapy response. Our studies have focused on characterizing diverse canine cancer cell lines in vitro and understanding parameters that might contribute to intrinsic radiosensitivity. First, intrinsic radiosensitivity of 27 canine cancer cell lines derived from ten tumor types was determined using a clonogenic assay. The 27 cell lines had varying radiosensitivities regardless tumor type (survival fraction at 2 Gy, SF2 = 0.19–0.93). In order to understand parameters that might contribute to intrinsic radiosensitivity, we evaluated the relationships of cellular radiosensitivity with basic cellular characteristics of the cell lines. There was no significant correlation of SF2 with S-phase fraction, doubling time, chromosome number, ploidy, or number of metacentric chromosomes, while there was a statistically significant correlation between SF2 and plating efficiency. Next, we selected the five most radiosensitive cell lines as the radiosensitive group and the five most radioresistant cell lines as the radioresistant group. Then, we evaluated known parameters for cell killing by ionizing radiation, including radiation-induced DNA double strand break (DSB) repair and apoptosis, in the radiosensitive group as compared to the radioresistant group. High levels of residual γ-H2AX foci at the sites of DSBs were present in the four out of the five radiosensitive canine cancer cell lines. Our studies suggested that substantial differences in intrinsic radiosensitivity exist in canine cancer cell lines, and radiation-induced DSB repair was related to radiosensitivity, which is consistent with previous human studies. These data may assist further investigations focusing on the detection of DSB for predicting individual response to radiation therapy for dogs, regardless of tumor type.

679. Heat Stress Elevate Cell Population That Express Cancer Stem Cell Markers in Different Types of Cancer Cell Line

Discovery of cancer stem cells (CSC) as driving force behind cancer cells tumorigenesis and heterogeneity in solid tumors led to a paradigm shift. The recognition of non-CSC to CSC transient introduces the cancer cell plasticity concept. This behavior appeared to be brought into existence majorly by the hostility of tumor microenvironment and stress imposed by hypoxia, pH, and nutrient depletion in the tumor niche. This recognition draw attention to the correlation between cancer hallmarks such as metastasis, chemotherapy, radiotherapy resistant and the transition of non-CSC to CSC by determination of CSC markers in cells subpopulations. Different types of cancer cell lines were shown to have a subset of cells expressing CSC markers such as CD44, ALDH, C133, Oct 3/4 with tumorigenicity in vivo. Recently, metabolic stress in a long-term nutrient deprivation showed to induces conversion of non-CSC to CSC like state. Additionally the hypoxic microenvironment was shown to upgraded stem-like properties of gastric cancer cells. Since it was recognized that metabolic alterations in tumors often combined by hyperthermia, we hypothesized that hyperthermia could be one of the stress components imposed by microenvironment that may manipulate non-CSC to CSC status and we asked if this elevation in temperature would affect phenotypic characteristics or up-regulate the genes responsible for stress resistant and the genes of cancer stem cell markers. To answer this, three different types of cancer cell lines were investigated by incubating in elevated temperature without CO2 supplement. They were rhebdomyosarcoma cell line (RD), cervical carcinoma cell line (HeLa), and mice mammary adenocarcenoma (AMN3). In all experiments cell lines were seeded in 12 well tissue culture plates and incubated at 37°C with 5% CO2 until they reach absolute confluent monolayer, after that subsets of plates incubated either at 40°C or 37°C without CO2 for 24 or 48 hr. Cultivation of cell lines in RPMI1640 at 37°C for 24hr without CO2 served as control untreated experiments. We followed changes in cells viability, phenotypic alterations, cologencity, and genetic markers of heat resistant (HSP90-beta and HIF), genetic markers of cancer stem cells (CD44, ALDH, Oct4, and CD133) by RT- qPCR. Results showed that upon incubation at 40°C for 24 hr or 48 hr without CO2 supplement cells were detached floated and acquired spherical shapes and aggregated. Noticeably the formed aggregation resembled to some extent the cell spheres that induced in cancer stem cells at sphere forming assay (fig.1,A,Bfig.1,A,B). The count of viable floated cells for RD, HeLa, and AMN3 cells were 3.93E–6, 2.1E–6, and 5.7E–6 cell/ml at 40°C for 48 hr respectively, and generally it was temperature and time dependant (Fig 1,CFig 1,C). All viable floated cells from the cell lines investigated were capable to form colonies in colony forming assay, the plating efficiency for RD, HeLa, and AMN3 cells were 48%, 64%, and 57% after incubation at 37°C for 10 days with CO2 supplement respectively (Fig.1,DFig.1,D). Up regulation of heat stress gene HSP90-beta and HIF was detected in floated cells derived from all experiment and time of incubation. This was combined by up regulation of CSC markers CD44, ALDH, Oct4 and CD133 in floated cells derived from all experiment. These results may indicate a shift of non-CSC to CSC during heat stress and the increment of cancer stem cell population in the cell line under investigation which need more careful investigation.View Large Image | Download PowerPoint Slide

Increased reactive oxygen species and exhaustion of quiescent CD34-positive bone marrow cells may contribute to poor graft function after allotransplants.

Poor graft function (PGF) is a fatal complication following allogeneic haematopoietic stem cell transplantation. However, the underlying mechanism is unclear. Effective cross-talk between haematopoietic stem cells (HSCs) and bone marrow microenvironment is important for normal haematopoiesis. Normal HSCs reside in a hypoxic bone marrow microenvironment that protects them from oxidative stress that would otherwise inhibit their self-renewal and results in bone marrow failure. Whether an increased level of reactive oxygen species (ROS) causes PGF following allotransplant is unclear. Using a prospective case-pair study, we identified increased levels of ROS in CD34+ bone marrow cells in subjects with PGF. Elevated ROS levels was associated with an increased frequency of DNA strand breaks, apoptosis, exhaustion of quiescent CD34+ cells and defective colony-forming unit plating efficiency, particularly in the CD34+CD38− fraction. Up-regulated intracellular p53, p21, caspase-3 and caspase-9 levels (but not p38) were detected in CD34+ cells, particularly in the CD34+CD38− fraction. To further study the potential role of ROS levels in post-transplant haematopoiesis, CD34+ bone marrow cells from subjects with good graft function were treated with H2O2. This increased ROS levels resulting in defective CD34+ cells, an effect partially reversed by N-acetyl-L-cysteine. Moreover, CD34+ bone marrow cells from the donors to subjects with poor or good graft function exhibited comparable haematopoietic reconstitution capacities in the xeno-transplanted NOD-PrkdcscidIL2rgnull mice. Thus, even if the transplanted donors' bone marrow CD34+ cells are functionally normal pre-transplant, ROS-induced apoptosis may contribute to the exhaustion of CD34+ bone marrow cells in subjects with PGF following allotransplant.

Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells.

The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS)-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate), two polymers (PVA and ficoll), two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide), a disaccharide (trehalose), and a calcium chelator (EGTA) to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum) were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the plating efficiency to the level of unfrozen controls. Moreover, ASCs cryopreserved in this defined medium retained their multipotency and chromosomal normality. These results are of significance for tissue engineering and clinical applications of stem cells.

BVES Regulates Intestinal Stem Cell Programs and Intestinal Crypt Viability after Radiation.

Blood vessel epicardial substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves(-/-) mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild-type (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves(-/-) mice. To examine stem cell function after BVES deletion, we used ex vivo 3D-enteroid cultures. Bves(-/-) enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar "CBC" and "+4" stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves(-/-) enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves(-/-) mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves(-/-) mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626-1636.

Adipose mesenchymal stromal cells response to ionizing radiation

Background aimsThis study evaluates the biological response of adipose tissue–derived mesenchymal stromal cells (aMSCs) to ionizing radiation (IR). MethodsIrradiated BALB/c mice aMSCs were characterized for functionality and phenotype. The clonogenic capacity of irradiated aMSCs was assessed and compared with those of metastatic breast cancer cell line (4T1) and normal mouse fibroblasts (NIH3T3-wt). We investigated the IR-induced DNA damage response, apoptosis, changes in cell cycle (CC) dynamics and protein and gene expression. ResultsIrradiated and non-irradiated aMSCs were able to differentiate into adipocytes, chondrocytes and osteocytes with no significant difference. Irradiated aMSCs maintained the expression of mesenchymal stromal cells (MSCs) surface antigens and, as expected, were negative for hematopoietic stem cells (HSCs) surface antigens when tested up to 7 days after IR for all irradiation doses with no significant difference. Clonogenically, irradiated aMSCs had higher relative survival fraction and plating efficiency than 4T1 and NIH3T3-wt. Irradiated aMSCs expressed higher □H2AX and significantly showed faster and more time-efficient IR-induced DNA damage response evident by up-regulated DNA-PKcs and RAD51. Two hours after IR, most of aMSCs DNA damage/repair-related genes showed up-regulation that disappeared within 6 h after IR. Irradiated aMSCs showed a significant rise and an earlier peak of p-ATM-dependent and -independent (p84/5E10-mediated) G2/M CC arrest compared with 4T1 and NIH3T3-wt. ConclusionsAfter IR exposure, aMSCs showed a robust and time-efficient radiation-induced DNA damage repair response, stable phenotypical characteristics and multi-lineage differentiation potential, suggesting they may be reliable candidates for cell therapy in radiation oncology regenerative medicine.