Confluent Cultures Research Articles

Propofol Protects Lung Endothelial Barrier Function by Suppression of High-Mobility Group Box 1 (HMGB1) Release and Mitochondrial Oxidative Damage Catalyzed by HMGB1.

BackgroundThe processes of mechanical ventilation-induced lung injury (VILI) triggers the release of high-mobility group box 1 (HMGB1), a prominent damage-associated molecular pattern (DAMP) family member, which can cause damage to pulmonary vascular endothelial cells. We aimed to determine whether propofol protected against endothelial cell injury induced by HMGB1 in vitro and in vivo.Material/MethodsICR mice (male) were mechanically ventilated for 4 h after anesthetization at both low tidal volume (LVT, 6 ml/kg) and high tidal volume (HVT, 30 ml/kg). A propofol bolus (10 mg/kg) was administered to the animals prior to the onset of ventilation, followed by infusion at 5 mg/(kg·h). We obtained confluent cultures of mouse lung vascular endothelial cells (MLVECs) and then performed cyclic stretching at 20% stretch for 4 h with or without propofol.ResultsHMGB1 reduced the expression of tight junctions between endothelial cells, including VE-cadherin and ZO-1, and increased endothelial permeability, and both were blocked by propofol. We found that MLVECs exhibited mitochondrial oxidative damage by HMGB1, which was successfully suppressed through administration of MnTBAP as well as propofol. Propofol ameliorated HVT-associated lung vascular hyperpermeability and HMGB1 production in vivo. Propofol also inhibited HMBG1 release caused by cyclic stretching in MLVECs in vitro.ConclusionsOur results prove that the cyto-protective function of propofol protects against lung ventilation-induced dysfunction of the lung endothelial barrier. This function of propofol is mediated through inhibition of HMGB1 release caused by mechanical stretching and mitochondrial oxidative damage triggered by HMGB1.

Increasing yield of msc-evs in scalable xeno-free manufacturing

Background & Aim Extracellular vesicles (EVs) are increasingly being investigated as both drug delivery vehicles and as a cell-free therapy. EVs from mesenchymal stem/stromal cells (MSC-EVs), for example, have been shown to recapitulate many of the therapeutic effects of the cells themselves. A critical barrier in the development of MSC-EVs as a commercial therapy, however, is generating the large amount of EVs that will be required per dose. In a recent survey, the majority of those working with EVs were working with less than 100mL of sample (ISEV 2016). In this study, two strategies were investigated to maximize EV yield. First, timing parameters for culturing human bone-marrow derived MSCs and collecting MSC-EVs were evaluated to increase EV yield. Second, conditioning the cells was investigated by adding an EV activator during the EV collection period. Methods, Results & Conclusion MSCs were first cultured in an expansion media (RoosterNourishTM, RoosterBio) in a fed-batch system. The cultures were then switched to a collection media (RoosterCollectTM-EV, RoosterBio) with or without an EV activator (EV BoostTM, RoosterBio). After two days, the conditioned media was harvested. The EVs in the collected conditioned media were analyzed for particle concentration and particle size distributions by nanoparticle tracking analysis, protein expression, RNA expression, and wound healing capability. Particle concentration (particles per L of media) was used as a measure of EV yield. Particle concentration increased with increased culture time in expansion media, with confluent cultures generating the most EVs. The EV activator at a low concentration increased EV yield about 2-fold compared to without the activator after 24hrs. The EV yield from the low EV activator culture at 24hrs was still about 45% greater than the yield of the culture without the activator after 48hrs. After 6hrs of collection, the high EV activator concentration increased EV yield to about the same level as without the activator after 48hrs of collection. Optimizing protocols for EV yield will become increasingly important as MSC-EVs move towards the clinic and lot sizes of clinically-relevant doses are required. Pre-conditioning of cells or conditioning cells during EV collection offers one possibility to further increase EV yield and decrease process time.

Machine Learning with Optical Phase Signatures for Phenotypic Profiling of Cell Lines.

Robust and reproducible profiling of cell lines is essential for phenotypic screening assays. The goals of this study were to determine robust and reproducible optical phase signatures of cell lines for classification with machine learning and to correlate optical phase parameters to motile behavior. Digital holographic microscopy (DHM) reconstructed phase maps of cells from two pairs of cancer and non-cancer cell lines. Seventeen image parameters were extracted from each cell's phase map, used for linear support vector machine learning, and correlated to scratch wound closure and Boyden chamber chemotaxis. The classification accuracy was between 90% and 100% for the six pairwise cell line comparisons. Several phase parameters correlated with wound closure rate and chemotaxis across the four cell lines. The level of cell confluence in culture affected phase parameters in all cell lines tested. Results indicate that optical phase features of cell lines are a robust set of quantitative data of potential utility for phenotypic screening and prediction of motile behavior. © 2019 International Society for Advancement of Cytometry.

Sub-confluent culture of human mesenchymal stromal cells on biodegradable polycaprolactone microcarriers enhances bone healing of rat calvarial defect

In the current emerging trend of using human mesenchymal stromal cell (MSCs) for cell therapy, large quantities of cells are needed for clinical testing. Current methods of culturing cells, using tissue culture flasks or cell multilayer vessels, are proving to be ineffective in terms of cost, space and manpower. Therefore, alternatives such as large-scale industrialized production of MSCs in stirred tank bioreactors using microcarriers (MCs) are needed. Moreover, the development of biodegradable MCs for MSC expansion can streamline the bioprocess by eliminating the need for enzymatic cell harvesting and scaffold seeding for bone-healing therapies. Our previous studies described a process of making regulated density (1.06 g/cm3) porous polycaprolactone biodegradable MCs Light Polycarprolactone (LPCL) (MCs), which were used for expanding MSCs from various sources in stirred suspension culture. Here, we use human early MSCs (heMSCs) expanded on LPCL MCs for evaluation of their osteogenic differentiation potential in vitro as well as their use in vivo calvarial defect treatment in a rat model. In summary, (i) in vitro data show that LPCL MCs can be used to efficiently expand heMSCs in stirred cultures while maintaining surface marker expression; (ii) LPCL MCs can be used as scaffolds for cell transfer for transplantation in vivo; (iii) 50% sub-confluency, mid-logarithmic phase, on LPCL MCs (50% confluent) exhibited higher secretion levels of six cytokines (interleukin [IL]-6, IL-8, Vascular endothelial growth factor (VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), growth-regulated oncogene-α (GRO-α) and stromal cell-derived factor-1α (SDF-1α)) as compared with 100% confluent, stationary phase cultures (100% confluent); (iv) these 50% confluent cultures demonstrated better in vitro osteogenic differentiation capacity as compared with 100% confluent cultures (higher levels of calcium deposition and at earlier stage); the improved bone differentiation capacity of these 50% confluent cultures was also demonstrated at the molecular level by higher expression of early osteoblast genes Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), collagen type I, osterix and osteocalcin); and (v) in vivo implantation of biodegradable LPCL MCs covered with 50% heMSCs into rats with calvarial defect demonstrated significantly better bone formation as compared with heMSCs obtained from monolayer cultures (5.1 ± 1.6 mm3 versus 1.3 ± 0.7 mm3). Moreover, the LPCL MCs covered with 50% heMSCs supported better in vivo bone formation compared with 100% confluent culture (2.1 ± 1.3 mm3). Taken together, our study highlights the potential of implanting 50% confluent MSCs propagated on LPCL MCs as optimal for bone regeneration. This methodology allows for the production of large numbers of MSCs in a three-dimensional (3D) stirred reactor, while supporting improved bone healing and eliminating the need for a 3D matrix support scaffold, as traditionally used in bone-healing treatments.

Heparin prevents oxidative stress-induced apoptosis in human decidualized endometrial stromal cells.

Clinical trials have shown that administering heparin during the luteal phase has beneficial effects on implantation and live birth rates. Heparin exerts direct effects on decidual human endometrial stromal cells (HESCs), which are independent of its anticoagulant effect. However, the accurate effects of heparin on the decidualization process remain unidentified. Here, we demonstrate that HESCs become dramatically resistant to oxidative stress upon decidualization, and we hypothesize a possible direct action of heparin on the decidualization of HESCs, which would lead to improved implantation. To test this hypothesis, we established primary HESC cultures and propagated them, and then we decidualized confluent cultures with 8-bromo-cAMP, with medroxyprogesterone acetate, and with or without heparin. We treated the cells with hydrogen peroxide (H2O2) as a source of reactive oxygen species (ROS). Adding heparin to decidualized HESCs induced prolactin secretion. Decidualized HESCs treated with heparin were prevented from undergoing apoptosis induced by oxidative stress. Heparin induced nuclear accumulation of the forkhead transcription factor FOXO1 and expression of its downstream target, the ROS scavenger superoxide dismutase 2. These results demonstrate that heparin-treated decidualized HESCs acquired further resistance to oxidative stress, suggesting that heparin may improve the implantation environment.

Uptake and distribution of carboxylated quantum dots in human mesenchymal stem cells: cell growing density matters

BackgroundHuman mesenchymal stem cells (MSCs) have drawn much attention in the field of regenerative medicine for their immunomodulatory and anti-inflammatory effects. MSCs possess specific tumor-oriented migration and incorporation highlighting the potential for MSCs to be used as an ideal carrier for anticancer agents. Bone marrow is the main source of MSCs for clinical applications. MSCs tracking in vivo is a critical component of the safety and efficacy evaluation of therapeutic cell products; therefore, cells must be labeled with contrast agents to enable visualization of the MSCs migration in vivo. Due to their unique properties, quantum dots (QDs) are emerging as optimal tools in long-term MSC optical imaging applications. The aim of this study was to investigate the uptake dynamics, cytotoxity, subcellular and extracellular distribution of non-targeted carboxylated quantum dots in human bone marrow MSCs at different cell growing densities.ResultsQDs had no negative impact on MSC viability throughout the experiment and accumulated in all observed cells efficiently; however, in some MSCs QDs induced formation of lipid droplets. At low cell growing densities QDs distribute within MSCs cytoplasm already after 1 h of incubation reaching saturation after 6 h. After 24 h QDs localize mainly in the perinuclear region of the cells in endosomes. Interestingly, in more confluent culture QDs localize mostly outside MSCs. QDs abundantly mark MSC long filopodia-like structures attaching neighboring cells. At high cell density cultivation, we for the first time demonstrated that carboxylated QDs localize in human bone marrow MSC extracellular matrix. Moreover, we observed that average photoluminescence lifetime of QDs distributed in extracellular matrix are longer than lifetimes of QDs entrapped in endocytic vesicles; thus, for the first time showing the possibility to identify and distinguish localization of QDs in various extracellular and intracellular structures using fluorescence-lifetime imaging microscopy without additional staining assays.ConclusionCarboxylated QDs can be used as nonspecific and effective dye for staining of human bone marrow MSCs and their specific extracellular structures. These results are promising in fundamental stem cell biology as well as in cellular therapy, anticancer drug delivery and tissue engineering.

Специфичная токсичность кальций-фосфатных бионов для культур венозных и артериальных эндотелиальных клеток человека

Цель исследования - оценка токсичности кальций-фосфатных бионов (КФБ) и магний-фосфатных бионов (МФБ) для культур эндотелиальных клеток. Методика. Эндотелиотоксичность бионов изучена при помощи добавления равных концентраций МФБ или КФБ к: 1) разреженным или конфлюэнтным культурам иммортализованных венозных эндотелиальных клеток человека линии EA.hy 926 с последующим культивированием в течение 24 ч или 4 ч соответственно; 2) конфлюэнтным культурам коммерческих первичных эндотелиальных клеток коронарной и внутренней грудной артерии человека с последующим культивированием в течение 24 ч. Эндотелиотоксические эффекты бионов оценивали при помощи сочетанного окрашивания клеток флюоресцентными красителями Hoechst 33342 и бромистым этидием, а также посредством колориметрического теста. Кроме того, методом проточной цитометрии оценивали пути и стадии гибели клеток вышеуказанных культур. Результаты. В отличие от МФБ, КФБ индуцировали гибель эндотелиальных клеток всех 3 линий путем апоптоза. Устойчивость культур к токсическому действию КФБ определялась степенью их конфлюэнтности (конфлюэнтные культуры более устойчивы чем разреженные) и типом клеточной линии (эндотелиальные клетки внутренней грудной артерии продемонстрировали большую устойчивость в сравнении с эндотелиальными клетками коронарной артерии). Заключение. Токсичность КФБ для культур эндотелиальных клеток специфична, то есть определяется их специфическим минеральным составом, а не общей для всех типов бионов корпускулярной природой. Добавление КФБ к конфлюэнтным культурам первичных артериальных эндотелиальных клеток и к иммортализованным венозным эндотелиальным клеткам вызывало их гибель, при этом экспозиция МФБ не оказывает значимого токсического действия. Эндотелиальные клетки внутренней грудной артерии менее чувствительны к воздействию КФБ в сравнении с эндотелиальными клетками коронарной артерии человека. Aim. To compare toxicity of calcium phosphate bions (CPB) and magnesium phosphate bions (MPB) for endothelial cells. Me-thods. To assess endothelial toxicity of the bions, we first added equal concentrations of either MPB or CPB to: 1) non-confluent or confluent cultures of immortalized human venous endothelial cells EA.hy 926 with the exposure time of 24 h or 4 h, respectively; 2) confluent cultures of commercially available primary human coronary artery and internal thoracic artery endothelial cells, with the exposure time of 24 h. Endothelial toxicity was then evaluated by combined Hoechst 33342 and ethidium bromide staining following fluorescence microscopy and by colorimetric cytotoxicity assay. In addition, we attempted to determine the pathway of bion-induced cell death utilizing flow cytometry. Results. In contrast to the MPB, CPB induced apoptosis of all studied endothelial cell lines. Resistance of endothelial cells to the CPB was defined by their confluence (confluent cultures demonstrated higher resistance), and cell type (internal thoracic artery endothelial cells were more resistant to the CPB as compared to the coronary artery endothelial cells). Conclusions. Endothelial toxicity of the CPB is defined by their specific mineral composition but not by their corpuscular nature, which is common for all nanoparticles. Addition of the CPB to the confluent cultures of primary human arterial cells and to immortalized human venous endothelial cells evoked their death. On the contrary, exposure to the MPB did not cause any toxic effects. Human internal thoracic artery endothelial cells are more resistant to the CPB in comparison with coronary artery endothelial cells.

Improved Anticancer Effect of Recombinant Protein izTRAIL Combined with Sorafenib and Peptide iRGD.

One of the main problems in oncology is the development of drugs that cause the death of cancer cells without damaging normal cells. Another key problem to be solved is to suppress the drug resistance of cancer cells. The third important issue is to provide effective penetration of drug molecules to cancer cells. TRAIL (TNFα-related apoptosis inducing ligand)/Apo2L is a highly selective anticancer agent. However, the recombinant TRAIL protein having high efficiency against cancer cells in vitro was not effective in clinical trials. Recently we have discovered an acquisition of TRAIL resistance by cancer cells in confluent cultures, which is apparently a manifestation of the general phenomenon of multicellular resistance. The aim of this study was to evaluate whether the anticancer effect of the recombinant protein TRAIL in vivo can be improved by the suppression of multicellular TRAIL-resistance using sorafenib and a tumor-penetrating peptide iRGD, c(CRGDKGPDC). The results testified a great increase in the resistance of human fibrosarcoma HT-1080 cells to izTRAIL both in confluent cultures and in spheroids. Sorafenib administered at nontoxic concentration effectively suppressed confluent- or spheroid-mediated TRAIL-resistance of HT-1080 cells in vitro. Sorafenib combined with iRGD significantly improved the anticancer effect of the recombinant protein izTRAIL in HT-1080 human fibrosarcoma grafts in BALB/c nude mice. Consistent with this finding, multicellular TRAIL-resistance may be a reason of inefficacy of izTRAIL alone in vivo. The anticancer effect of the recombinant protein izTRAIL in vivo may be improved in combination with sorafenib, an inhibitor of multicellular TRAIL resistance and iRGD, the tumor-penetrating peptide.

The Late Osteoblast/Preosteocyte Cell Line MLO-A5 Displays Mesenchymal Lineage PlasticityIn VitroandIn Vivo

The process of osteoblast switching to alternative mesenchymal phenotypes is incompletely understood. In this study, we tested the ability of the osteoblast/preosteocyte osteogenic cell line, MLO-A5, to also differentiate into either adipocytes or chondrocytes. MLO-A5 cells expressed a subset of skeletal stem cell markers, including Sca-1, CD44, CD73, CD146, and CD166. Confluent cultures of cells underwent differentiation within 3 days upon the addition of osteogenic medium. The same cultures were capable of undergoing adipogenic and chondrogenic differentiation under lineage-appropriate culture conditions, evidenced by lineage-specific gene expression analysis by real-time reverse-transcription-PCR, and by Oil Red O and alcian blue (pH 2.5) staining, respectively. Subcutaneous implantation of MLO-A5 cells in a gel foam into NOD SCID mice resulted in a woven bone-like structure containing embedded osteocytes and regions of cartilage-like tissue, which stained positive with both alcian blue (pH 2.5) and safranin O. Together, our findings show that MLO-A5 cells, despite being a strongly osteogenic cell line, exhibit characteristics of skeletal stem cells and display mesenchymal lineage plasticity in vitro and in vivo. These unique characteristics suggest that this cell line is a useful model with which to study aging and disease-related changes to the mesenchymal lineage composition of bone.

S-Allylmercapro-N-Acetylcysteine Attenuates the Oxidation-Induced Lens Opacification and Retinal Pigment Epithelial Cell Death In Vitro.

The capacity of S-Allylmercapto-N-acetylcysteine (ASSNAC) to protect human retinal pigment epithelial (RPE) cells (line ARPE-19) and porcine lenses from oxidative stress was studied. Confluent ARPE-19 cultures were incubated with ASSNAC or N-acetyl-cysteine (NAC) followed by exposure to oxidants and glutathione level and cell survival were determined. Porcine lenses were incubated with ASSNAC and then exposed to H2O2 followed by lens opacity measurement and determination of glutathione (reduced (GSH) and oxidized (GSSG)) in isolated lens adhering epithelial cells (lens capsule) and fiber cells consisting the lens cortex and nucleus (lens core). In ARPE-19 cultures, ASSNAC (0.2 mM; 24 h) increased glutathione level by 2–2.5-fold with significantly higher increase in GSH compared to NAC treated cultures. Similarly, ex-vivo exposure of lenses to ASSNAC (1 mM) significantly reduced the GSSG level and prevented H2O2 (0.5 mM)-induced lens opacification. These results demonstrate that ASSNAC up-regulates glutathione level in RPE cells and protects them from oxidative stress-induced cell death as well as protects lenses from oxidative stress-induced opacity. Further validation of these results in animal models may suggest a potential use for ASSNAC as a protective therapy in retinal degenerative diseases as well as in attenuation of oxidative stress-induced lens opacity.

No Hothouse Flower.

No Hothouse Flower Marguerite Barnett To be a woman is a rich and dangerous job. I experienced this in my mother's womb, she who created me out of hope and love, which transcended betrayal and racism. I sensed this as I watched my adoptive mother subsume her identity to a soul-sucking concept formed at the confluence of culture and religion. But I didn't really learn it until I began the path that led to my current identity as a surgeon. I should not have survived any of this, and I say that with a sense of wonder and gratitude. I was born to a Japanese woman in postwar Japan, who met my father, a US military doctor, at a USO dance. He did not wear a wedding band, but he did not try to hide her. He took her to "nice" restaurants where Japanese women were not usually taken, so, at nineteen, she thought they were having a grand love affair. She did not realize he already had a wife and child back in the States. She was so naïve she did not know she was pregnant. She had morning sickness, and he smuggled her into the base hospital for testing. She found out when he sat her in the office chair and said, "I have a Japanese doctor friend who can fix this." The way that he said it made her realize "this" meant she was pregnant, "fix" meant an abortion. Something was wrong—he should be happy they were having a child! I do not know why she didn't have an abortion. Abortion was not frowned upon in Japan, but having a mixed blood, unwanted child certainly was. Most unusually, she kept me without any support. The combination of youth, poverty, and lack of support left her vulnerable, and she lost custody of me when I was trafficked by a babysitter at age one. Ultimately, I was adopted by an Army chaplain and his wife, who provided a stable upbringing despite the constant movement of military life. This conservative and fundamental religious upbringing primed me for marriage and a life of being a "helpmate" to a husband. There were some warning signs that I might not take the expected path. I knew I wanted to be a doctor even though, at that time, I knew nothing about my natural father other than his nationality. I did not want to be a nurse. I thought it was because I was a failure to thrive, which, in retrospect, I think was because of my refusal to eat when separated from my mother. But the fact that I had a heart murmur and got ill very easily led the doctors to conclude that I had a bad heart and would not make it past my fifth birthday. It was so bad my adoptive mother said she could not hang white sheets on the clothesline lest I thought it was the doctor coming to give me a shot! The warning signs became red flags, and I ultimately hit a brick wall when I chose to attend an Ivy League Medical School to be with my then fiancé. I did not realize how a liberal "heathen" institution such as the one I attended in the 1970s could destroy me. This was the time of widespread student protests against the war, Timothy Leary was urging everyone to "drop out and drop acid," and Patty Hearst had been kidnapped and brainwashed into a gun-toting radical. For my crimes, I was disowned by my parents, and the shock of losing their support was compounded when my minister molested me. I do not know how I survived those dark days. My fledgling marriage fell apart under the pressure, and I was abandoned in the cold north in a program not known for its supportive nature. Desperate, I joined the military scholarship program, but it only paid for books and tuition. The financial aid package provided by the school did not cover living expenses. It should not have been a surprise that the final injury—caused by my lack of financial security, expressed in such seemingly minor things as unsafe housing and...

Transculturalism and its effects on the internationalisation of markets: A conceptual framework

There is considerable literature in the field of cultural profiles, intercultural exchange and in how disparate cultural environments influence business exchange. Existing relevant literature addresses select marketing issues such as how communications are developed, their effectiveness within individualistic and collectivistic cultures, and the like. This study examines how intercultural overlapping may produce a transculturalistic environment that can help enhance market development. It addresses the challenges associated with intercultural symbiosis as individualistic and collectivistic cultures interact to achieve their goals. It argues that exchange between low-context and high-context cultures produces intercultural confluence, creating a confluent culture, which is catalytic to expediting market development. The study uses propositions through which it argues that individualistic and collectivistic cultures converge within a social context. The paper generates insights and offers an incipient framework with which to rationalise the confluence of individualistic and collectivistic cultures.

The Potential of Basal Medium Astemporary Prolong Culture of Umbilical Cord Derived Mesenchymalstem Cells

To compare the viability and characteristics of umbilical cord mesenchymal stromal cells (UC-MSCs) when they were kept in culture in three types of αMEM after confluence for various time points. Viability and characteristic assessments were done after keeping UC-MSC confluent cultures for 0, 24, 48, 72, and 96hours in αMEM with glutamax (Himedia), and without glutamax (Himedia or Gibco).There were thirty cultures, which each ten of them were kept in the three types of basal media, and for each time point and type of basal medium, two flasks were harvested. All viability and characteristic assessments were done after harvest. Viability assessments were done in duplicate, while characteristics assessment by flowcytometry for CD73, CD90 and CD105 were done only once.Further, differences between the various time points in terms of viability and CD73, CD90 and CD105 percentage after various time points were compared and tested by appropriate statistical analysis. Viability was >70% upto 72 hours and 48 hours when the cells were kept in Himediaglutamax containing αMEM and αMEM without glutamax respectively. Flow cytometry showed that CD73, CD90 and CD negative percentage did not differ to initial percentage, but after 24 hours CD105 was slightly decreased to > 60% in Himediaglutamax containing αMEM, while in the two αMEM media without glutamaxthe percentages were below 40%. For our UC-MSC culture, glutamax containing αMEM is better compared to αMEM without glutamax as temporary storage solution, and storage should be restricted to 24 hours.

Identification of topographical architectures supporting the phenotype of rat tenocytes

Tenocytes, the main cell type of the tendon, require mechanical stimuli for their proper function. When the tenocyte environment changes due to tissue damage or by transferring tenocytes from their native environment into cell culture, the signals from the tenocyte niche are lost, leading towards a decline of phenotypic markers. It is known that micro-topographies can influence cell fate by the physical cues they provide. To identify the optimal topography-induced biomechanical niche in vitro, we seeded tenocytes on the TopoChip, a micro-topographical screening platform, and measured expression of the tendon transcription factor Scleraxis. Through machine learning algorithms, we associated elevated Scleraxis levels with topological design parameters. Fabricating micro-topographies with optimal surface characteristics on larger surfaces allowed finding an improved expression of multiple tenogenic markers. However, long-term confluent culture conditions coincided with osteogenic marker expression and the loss of morphological characteristics. In contrast, passaging tenocytes which migrated from the tendon directly on the topography resulted in prolonged elongated morphology and elevated Scleraxis levels. This research provides new insights into how micro-topographies influence tenocyte cell fate, and supports the notion that micro-topographical design can be implemented in a new generation of tissue culture platforms for supporting the phenotype of tenocytes. Statement of SignificanceThe challenge in controlling in vitro cell behavior lies in controlling the complex culture environment. Here, we present for the first time the use of micro-topographies as a biomechanical niche to support the phenotype of tenocytes. For this, we applied the TopoChip platform, a screening tool with 2176 unique micro-topographies for identifying feature characteristics associated with elevated Scleraxis expression, a tendon related marker. Large area fabrication of micro-topographies with favorable characteristics allowed us to find a beneficial influence on other tenogenic markers as well. Furthermore, passaging cells is more beneficial for Scleraxis marker expression and tenocyte morphology compared to confluent conditions. This study presents important insights for the understanding of tenocyte behavior in vitro, a necessary step towards tendon engineering.

Iran and India in the early historic period: A preview of their politico-cultural interface

The Achaemenid Empire’s expansion towards the Indus basin initiated a new confluence of Iranic and Indian cultures. Alexander’s conquests added a Greek component to this confluence, marked by Aśoka’s Aramaic and Greek edicts. The Seleucids and their successor Greek states in Bactria and other parts of Afghanistan, while continuing their homage to Greek divinities on coins, also incorporated concepts, customs and art inherited from the Achaemenids. Their Saka and Parthian successors continued the same policy as indicated by their cons. It was the Kushans beginning with Kanishka (with Huvishka continuing the practice) who shifted to Iranian gods and goddesses.

PRIMARY FIBROBLAST CELL CYCLE SYNCHRONIZATION AND EFFECTS ON HANDMADE CLONED (HMC) BOVINE EMBRYOS

Abstract Spatial and temporal synchrony and compatibility between the receptor oocyte and the donor cell nucleus are necessary for the process of embryo cloning to allow nuclear reprogramming and early embryonic development. The objective of the present study was to evaluate three cell cycle synchronization methods on a primary bovine fibroblast culture for 24, 48, or 72 h. These fibroblasts were used as nuclear donors to evaluate their in vitro developmental potential and the quality of the embryos produced through handmade cloning (HMC). No differences were found between the methods used for fibroblast synchronization in G0/G1 (p > 0.05). Production of clones from fibroblasts in four groups- no treatment at 0 h and using serum restriction SR, high culture confluence HCC, and SR+HCC at 24 h- resulted in high cleavage rates that were not different. Embryo production rates were 37.9%, 29.5%, and 30.9% in the 0h, SR24h, and SR+HHC24h groups, respectively, and 19.3% in the HCC group, which was significantly different from the other three (p < 0.05). There were no differences in the quality parameter among the clones produced with fibroblasts subjected to the different synchronization. Finally, when overall clone production was compared versus parthenotes and IVF embryos, the only difference was between clones and parthenogenetic embryos with zona pellucida (30.2% vs 38.6%). The number of blastomeres from the blastocytes produced through IVF was significantly greater than those from embryos activated parthenogenetically and from clones (117, 80, 75.9, and 67.1, respectively). The evaluation of three synchronization methods at different time points did not demonstrate an increase in the percentage of fibroblasts in the G0/G1 phases of the cell cycle; however, good quality and high cloning rates were obtained, suggesting that it is not always necessary to subject the cells to any synchronization treatments, as they would yield equally good cloning results.

Effects of Nd:YAG low-level laser irradiation on cultured human osteoblasts migration and ATP production: in vitro study.

Low-level laser therapy has become one of the fastest growing fields of medicine in recent years. Many in vivo and in vitro studies have shown that laser irradiation activates a range of cellular processes in a variety of cell types and can promote tissue repair. However, few in vitro experiments have evaluated the effects of laser irradiation on cells in real time. The purpose of this study was to examine the effects of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on the migration of cultured human osteoblasts. A dedicated 96-well plate was used, and confluent cultures of the human osteoblast-like cell line, Saos-2, were injured with a wound maker. The wounded cells were then exposed to the Nd:YAG laser (wavelength of 1064nm) for 60s at 0.3W (10pps, 30mJ). The total energy density was about 10.34J/cm2. Images of the wounds were automatically acquired inside the CO2 incubator by the IncuCyte ZOOM™ software. In addition, after laser irradiation, the production of adenosine triphosphate (ATP) was measured using the CellTiter-Glo™ Luminescent Cell Viability Assay. Migration of cells from the border of the original scratch zone was accelerated by laser irradiation. In addition, compared with the control group, significant enhancement of ATP production was observed in the irradiated group. The present study showed that Nd:YAG laser irradiation (wavelength of 1064nm, 0.3W, 10 pps, 30mJ, 10.34J/cm2, irradiation time 60s) may contribute to the regeneration of bone tissues owing to enhanced osteoblast cell migration.

Abstract 3115: Physiologic comparisons of five bladder cancer cell lines

Abstract The use of tumor-derived cultured cells continues to be a major resource for understanding the biological activities of tumors and the effects of environmental factors and pharmacological agents on tumors. With comparison of data from the International Cancer Genome Consortium and the Cancer Genome Atlas comes the realization that some frequently used cells lines may not closely share genomic similarity with patient tumors, whereas less frequently used cell lines may share high similarity. For example, the bladder cancer cell line T24 (HTB-4) is more frequently used than other cell lines. We selected five commercially available bladder cancer cell lines, all derived from patient primary tumors, for a comparison of selected proteins, with the intent of determining how similar or dissimilar the cultured cells are. In selection of cultured cell lines, we considered reported grade of tumor from which the cell line was derived and the sex and age of the patient. We evaluated protein expression in newly confluent cultures and in sub-confluent cultures. The following cell lines were selected: RT4 (transitional cell papilloma), 5637 (grade II carcinoma), T24 (grade III carcinoma), HT 1376 (grade III carcinoma) and TCCSUP (grade IV carcinoma). We hypothesized that variations exist between bladder cancer cell lines isolated from low and high-grade carcinomas. Further, we identified physiological characteristics of the different bladder cancer cell lines at both newly confluent and sub confluent culture states as additional parameters for a bladder cancer model. Selection of an appropriate tissue culture cell line(s) and state of confluency are critical considerations in cellular research designed to understand the biological activities of tumors. Growth rates of the five cell lines were determined. Next, the basal-luminal status of the cell lines was determined using molecular subtyping markers (transcription factors GATA3 and p63, Cytokeratin 6, and Estrogen Receptor alpha). The presence of Cathepsin L, a cysteine protease, was evaluated. Further, we evaluated the expression of key proteins in the MAPK pathway (p38 MAPK, SAPK/JNK, and ERK1/2) as indicators of cellular stress. Last, the receptor status of Alpha 7 receptor, the putative receptor for nicotine, was determined. Our results indicate there is a difference in growth rates between the cell lines, with line 5637 having a delayed growth rate. Three cell lines were determined to be basal, and two cell lines were determined as mixed baso/luminal. The RT4 cell line had greater expression of cathepsin L. We observed no significant changes in the protein expression levels of the MAPK pathway nor of the alpha-7 receptor. Our results suggest that there are differences between the five bladder cancer cell lines suggesting that more than one cell line should be used and evaluated prior to beginning experiments to evaluate the potential effects of environmental factors and pharmacological agents. Citation Format: Bonnie L. Richmond, Kyle Damen, Ryan Moore, Alex White, Julia H. Carter. Physiologic comparisons of five bladder cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3115.

Abstract 3055: A model of CSC converted from iPSC in the conditioned medium of HCC paving the way to establish HCC CSC

Abstract Hepatocellular carcinoma (HCC) represents the major histological subtype, accounting for 70%–85% of cases of primary liver cancer. Liver CSCs sustain their self-renewal remains largely unknown. The self-renewal and pluripotency of liver CSC are maintained by several signaling cascades. However, the mechanism of regulating these signaling is unknown. In this study, we tried to convert mouse iPSCs into CSCs with the conditioned medium (CM) from human HCC cell line Huh7 cells without any genetic manipulation aiming at establishing liver CSCs. First of all, CM was collected from confluent culture of Huh7 cells. Then, mouse iPSCs cells without MEF feeder cells were cultured in the presence of 50% CM for 4 weeks. The medium was changed every day with fresh medium containing 50% of CM. Mouse iPSCs cultured in the complete medium with LIF were used as a control.The survived cells (5x105 cells) were suspended in HBSS and injected into the liver of BALB/c nude mice. After 25 days malignant tumor was formed in the liver while benign teratoma was formed by the injection of iPSCs. Tumors were then excised and partly fixed in 10% neutral formalin buffer solution for HE staining and immunohistochemical analysis. The rest of tumors were subjected to rt-qPCR anaylsis and primary culture. Immunohistochemical analysis with GFP antibody showed that malignant tumor sustained GFP expression while teratoma from miPSCs did not. Immunocytochemistry of the primary cells from malignant tumor showed high expression of both glypican-3 (GPC3) and cytokeratin19 (CK19) when compared to that in miPSCs. Expression of stem cell markers (Nanog, Oct3/4, Sox2, Klf4) and CSC markers (CD44, EpCAM) were also confirmed by rt-qPCR in both the CSCs converted from iPSCs (miPS-Huh7cm cells) and the primary culture cells. These results indicate that the primary cells from the malignant tumor are rich in CSCs with high expression of GPC3 and CK19, which paves the way to establish a model of HCC CSC. This model should be very important and useful to assess the significant molecular mechanisms necessary to maintain HCC CSC, which will help develop effective therapy of liver cancer. Citation Format: Said M Afify, Anna Sanchez Calle, Kazuki Kumon, Hend M Nawara, Apriliana C Khairani, Hafizah Mahmud, Aung Ko Ko Oo, Du Juan, Maram H Zahara, Akimasa Seno, Tomonari Kasai, Yoshiaki Iwasaki, Masaharu Seno. A model of CSC converted from iPSC in the conditioned medium of HCC paving the way to establish HCC CSC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3055.

Labeling of endothelial cells with magnetic microbeads by angiophagy.

Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism 'angiophagy'. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization. In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5min, as compared to gravitational settling (p < 0.0001). We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications.