Search results for: primary cell culture

Authors: Yemima Dani Riani, Anggraini Barlian

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Green turtle (Chelonia mydas) is one of well known long-lived turtle. Its age can reach 100 years old. Senescence in green turtle is an interesting process to study because until now no clear explanation has been established about senescence at cellular or molecular level in this species. Since 1999, green turtle announced as an endangered species. Hence, establishment of fibroblast skin cell culture of green turtle may be material for future study of senescence. One common marker used for detecting senescence is telomere shortening. Reduced telomerase activity, the reverse transcriptase enzyme which adds TTAGGG DNA sequence to telomere end, may also cause senescence. The purpose of this research are establish and identify green turtle fibroblast skin cell culture and also compare telomere length and telomerase activity from passage 5 and 14. Primary cell culture made with primary explant method then cultured in Leibovitz-15 (Sigma) supplemented by 10% Fetal Bovine Serum (Sigma) and 100 U/mL Penicillin/Streptomycin (Sigma) at 30 ± 1oC. Cells identified with Rabbit Anti-Vimentin Polyclonal Antibody (Abcam) and Goat Polyclonal Antibody (Abcam) using confocal microscope (Zeiss LSM 170). Telomere length obtained using TeloTAGGG Telomere Length Assay (Roche) while telomerase activity obtained using TeloTAGGG Telomerase PCR ElisaPlus (Roche). Primary cell culture from green turtle skin had fibroblastic morphology and immunocytochemistry test with vimentin antibody proved the culture was fibroblast cell. Measurement of telomere length and telomerase activity showed that telomere length and telomerase activity of passage 14 was greater than passage 5. However, based on morphology, green turtle fibroblast skin cell culture showed senescent morphology. Based on the analysis of telomere length and telomerase activity, suspected fibroblast skin cell culture of green turtles is not undergo aging through telomere shortening.

Keywords: cell culture, chelonia mydas, telomerase, telomere, senescence

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Authors: Daniel Nieto, Ramiro Couceiro

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Microfluidic chips have demonstrated their significant application potentials in microbiological processing and chemical reactions, with the goal of developing monolithic and compact chip-sized multifunctional systems. Heat generation and thermal control are critical in some of the biochemical processes. The paper presents a laser direct-write technique for rapid prototyping and manufacturing of microheater chips and its applicability for perfusion cell culture outside a cell incubator. The aim of the microheater is to take the role of conventional incubators for cell culture for facilitating microscopic observation or other online monitoring activities during cell culture and provides portability of cell culture operation. Microheaters (5 mm × 5 mm) have been successfully fabricated on soda-lime glass substrates covered with aluminum layer of thickness 120 nm. Experimental results show that the microheaters exhibit good performance in temperature rise and decay characteristics, with localized heating at targeted spatial domains. These microheaters were suitable for a maximum long-term operation temperature of 120ºC and validated for long-time operation at 37ºC. for 24 hours. Results demonstrated that the physiology of the cultured SW480 adenocarcinoma of the colon cell line on the developed microheater chip was consistent with that of an incubator.

Keywords: laser microfabrication, microheater, bioengineering, cell culture

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Authors: Misa Nakao, Yuta Kurashina, Chikahiro Imashiro, Kenjiro Takemura

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The Research Goal: With the growing demand for regenerative medicine, an effective mass cell culture process is required. In a repetitive subculture process for proliferating cells, preparing single cell suspension which does not contain any cell aggregates is highly required because cell aggregates often raise various undesirable phenomena, e.g., apoptosis and decrease of cell proliferation. Since cell aggregates often occur in cell suspension during conventional subculture processes, this study proposes a single cell sorter driven by a resonance vibration of a cell culture substrate. The Method and the Result: The single cell sorter is simply composed of a cell culture substrate and a glass pipe vertically placed against the cell culture substrate with a certain gap corresponding to a cell diameter. The cell culture substrate is made of biocompatible stainless steel with a piezoelectric ceramic disk glued to the bottom side. Applying AC voltage to the piezoelectric ceramic disk, an out-of-plane resonance vibration with a single nodal circle of the cell culture substrate can be excited at 5.5 kHz. By doing so, acoustic radiation force is emitted, and then cell suspension containing only single cells is pumped into the pipe and collected. This single cell sorter is effective to collect single cells selectively in spite of its quite simple structure. We collected C2C12 myoblast cell suspension by the single cell sorter with the vibration amplitude of 12 µmp-p and evaluated the ratio of single cells in number against the entire cells in the suspension. Additionally, we cultured the collected cells for 72 hrs and measured the number of cells after the cultivation in order to evaluate their proliferation. As a control sample, we also collected cell suspension by conventional pipetting, and evaluated the ratio of single cells and the number of cells after the 72-hour cultivation. The ratio of single cells in the cell suspension collected by the single cell sorter was 98.2%. This ratio was 9.6% higher than that collected by conventional pipetting (statistically significant). Moreover, the number of cells cultured for 72 hrs after the collection by the single cell sorter yielded statistically more cells than that collected by pipetting, resulting in a 13.6% increase in proliferated cells. These results suggest that the cell suspension collected by the single cell sorter driven by the resonance vibration hardly contains cell aggregates whose diameter is larger than the gap between the cell culture substrate and the pipe. Consequently, the cell suspension collected by the single cell sorter maintains high cell proliferation. Conclusions: In this study, we developed a single cell sorter capable of sorting and pumping single cells by a resonance vibration of a cell culture substrate. The experimental results show the single cell sorter collects single cell suspension which hardly contains cell aggregates. Furthermore, the collected cells show higher proliferation than that of cells collected by conventional pipetting. This means the resonance vibration of the cell culture substrate can benefit us with the increase in efficiency of mass cell culture process for clinical applications.

Keywords: acoustic radiation force, cell proliferation, regenerative medicine, resonance vibration, single cell sorter

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Authors: Hirowati Ali, Aisyah Ellyanti, Dewi Rusnita, Septelia Inawati Wanandi

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Stem cell has been known for its potency to be differentiated in many cells. Recently stem cell has been used for many treatment of degenerative medicine. It is still controversy whether stem cell can be used for therapy or these cells can activate cancer stem cell. SCUBE2 is a novel secreted and membrane-anchored protein which has been reported to its role in better prognosis and inhibition of cancer cell proliferation. Our study aims to observe whether stem cell can up-regulate SCUBE2 gene in MCF7 breast cancer cell line. We used in vitro study using MCF-7 cell treated with stem cell derived from placenta Wharton's jelly which has been known for its stemness and widely used. Our results showed that MCF-7 cell line grows up rapidly in 6-well culture dish. Stem cell was cultured in 6-well dish. After 50%-60% MCF-7 confluence, we co-cultured these cells with stem cells for 24 hours and 48 hours. We hypothesize SCUBE2 gene which is previously known for its higher expression in better prognosis of breast cancer, is up-regulated after stem cells addition in MCF7 culture dishes.

Keywords: breast cancer cells, inhibition of cancer cells, mesenchymal stem cells, SCUBE2

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Authors: Shafieeh Mansoori, Fatemeh Yazdian, Ashrafsadat Hatamian, Majid Azizi

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Monascus purpureus, which has a special metabolite with many therapeutic and medicinal properties including antioxidant, antibiotic, anti-hypercholesterolemia, and immunosuppressive properties, is a traditional Chinese fermentation fungus and is used as a natural dietary supplement. Production of desired metabolites actually determined by optimized growth which is supported by some factors such as substrates and Monascus strains type, moisture content of the fermentation mixture, aeration, and control of contamination issues. In this experiment, M. purpureus PTCC5305 was cultured in both the liquid and solid culture medium. The former medium contain YMP (yeast extract, maltose and peptone), PGC (peptone, glucose complex), and GYP (glucose, yeast extract and peptone) medium. After 8 days, the best medium for the cell production was PGC agar medium on solid culture with 0.28 g dry weight of cell mass whereas the best liquid culture was GYP medium with 3.5 g/l dry weight of cell mass. The lowest cell production was on YMP agar with 0.1 g dry weight of cell mass and then YMP medium with 2.5 g/l dry cell weight.

Keywords: Monascus purpureus, solid state fermentation, submerged culture, Chinese fermentation fungus

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Authors: M. M. Rahman, A. Liu, A. Frostegard, J. Frostegard

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The human immune system plays an essential role in cardiovascular disease (CVD) and atherosclerosis. Our earlier studies showed that major immunocompetent cells including T cells are activated by phosphorylcholine epitope. Further, we have determined for the first time in a clinical cohort that antibodies against phosphorylcholine (anti-PC) are negatively and independently associated with the development of atherosclerosis and thus a low risk of cardiovascular diseases. It is still unknown whether activated T cells play a role in anti-PC production. Here we aim to clarify the role of T cells in anti-PC production. B cell alone, or with CD3 T, CD4 T or with CD8 T cells were cultured in polystyrene plates to examine anti-PC IgM production. In addition to mixed B cell with CD3 T cell culture, B cells with CD3 T cells were also cultured in transwell co-culture plates. Further, B cells alone and mixed B cell with CD3 T cell cultures with or without anti-HLA 2 antibody were cultured for 6 days. Anti-PC IgM was detected by ELISA in independent experiments. More than 8 fold higher levels of anti-PC IgM were detected by ELISA in mixed B cell with CD3 T cell cultures in comparison to B cells alone. After the co-culture of B and CD3 T cells in transwell plates, there were no increased antibody levels indicating that B and T cells need to interact to augment anti-PC IgM production. Furthermore, anti-PC IgM was abolished by anti-HLA 2 blocking antibody in mixed B and CD3 T cells culture. In addition, the lack of increased anti-PC IgM in mixed B with CD8 T cells culture and the increased levels of anti-PC in mixed B with CD4 T cells culture support the role of helper T cell for the anti-PC IgM production. Atherosclerosis is a major cause of cardiovascular diseases, but anti-PC IgM is a protection marker for atherosclerosis development. Understanding the mechanism involved in the anti-PC IgM regulation could play an important role in strategies to raise anti-PC IgM. Studies suggest that anti-PC is T-cell independent antibody, but our study shows the major role of T cell in anti-PC IgM production. Activation of helper T cells by immunization could be a possible mechanism for raising anti-PC levels.

Keywords: anti-PC, atherosclerosis, aardiovascular diseases, phosphorylcholine

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Authors: Azieva A. M., Yastremsky E. V., Kirillova D. A., Patsaev T. D., Sharikov R. V., Kamyshinsky R. A., Lukanina K. I., Sharikova N. A., Grigoriev T. E., Vasiliev A. L.

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Adhesive properties of scaffolds, which predominantly depend on the chemical and structural features of their surface, play the most important role in tissue engineering. The basic requirements for such scaffolds are biocompatibility, biodegradation, high cell adhesion, which promotes cell proliferation and differentiation. In many cases, synthetic polymers scaffolds have proven advantageous because they are easy to shape, they are tough, and they have high tensile properties. The regeneration of nerve tissue still remains a big challenge for medicine, and neural stem cells provide promising therapeutic potential for cell replacement therapy. However, experiments with stem cells have their limitations, such as low level of cell viability and poor control of cell differentiation. Whereas the study of already differentiated neuronal cell culture obtained from newborn mouse brain is limited only to cell adhesion. The growth and implantation of neuronal culture requires proper scaffolds. Moreover, the polymer scaffolds implants with neuronal cells could demand specific morphology. To date, it has been proposed to use numerous synthetic polymers for these purposes, including polystyrene, polylactic acid (PLA), polyglycolic acid, and polylactide-glycolic acid. Tissue regeneration experiments demonstrated good biocompatibility of PLA scaffolds, despite the hydrophobic nature of the compound. Problem with poor wettability of the PLA scaffold surface could be overcome in several ways: the surface can be pre-treated by poly-D-lysine or polyethyleneimine peptides; roughness and hydrophilicity of PLA surface could be increased by plasma treatment, or PLA could be combined with natural fibers, such as collagen or chitosan. This work presents a study of adhesion of both induced pluripotent stem cells (iPSCs) and mouse primary neuronal cell culture on the polylactide scaffolds of various types: oriented and non-oriented fibrous nonwoven materials and sponges – with and without the effect of plasma treatment and composites with collagen and chitosan. To evaluate the effect of different types of PLA scaffolds on the neuronal differentiation of iPSCs, we assess the expression of NeuN in differentiated cells through immunostaining. iPSCs more effectively differentiate into neurons on PLA scaffolds with high adhesive properties for primary neuronal cells.

Keywords: PLA scaffold, neurons, neuronal differentiation, stem cells, polylactid

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Authors: Anna Vildová, H. Hendrychová, J. Kubeš, L. Tůmová

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The abiotic elicitation is one of the methods for increasing the secondary metabolites production in plant tissue cultures and it seems to be more effective than traditional strategies. This study verified the use of silver nitrate as elicitor to enhance flavonolignans and flavonoid taxifolin production in suspension culture of Sylibum marianum (L.) Gaertn. Silver nitrate in various concentrations (5.887.10-3 mol/L, 5.887.10-4 mol/L, 5.887.10-5 mol/L) was used as elicitor. The content of secondary metabolites in cell suspension cultures was determined by high performance liquid chromatography. The samples were taken after 6, 12, 24, 48, 72 and 168 hours of treatment. The highest content of taxifolin production (2.2 mg.g-1) in cell suspension culture of Silybum marianum (L.) Gaertn. was detected after silver nitrate (5.887.10-4 mol/L) treatment and 72 h application. Flavonolignans such as silybinA, silybin B, silydianin, silychristin, isosilybin A, isosilybin B were not produced by cell suspension culture of S. marianum after elicitor treatment. Our results show that the secondarymetabolites could be released from S. marianum cells into the nutrient medium by changed permeability of cell wall.

Keywords: Silybum marianum (L.) Gaertn., elicitation, silver nitrate, taxifolin

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Authors: Mayuva Youngsabanant, Suphaphorn Rabiab, Hatairuk Tungkasen, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The porcine follicular fluid proteins (pFF) of healthy small size ovarian follicles (1-3 mm in diameters) of Large White pig ovaries were collected by sterile technique. They were used for testing the effect on cell viability and characterization of Vero cell lines using MTT assay. Two hundred microliter of round shape Vero cell lines were culture in 96 well plates with DMEM for 24 h. After that, they were attachment to substrate and some changed into fibroblast shape and spread over the surface after culture for 48 h. Then, Vero cell lines were treated with pFF at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins/mL for 24 h. Yields of the best results were analyzed by using one-way ANOVA. MTT assay reviewed an increasing in percentage of viability of Vero cell lines indicated that at concentration of 400-600 µg proteins/mL showed higher percentage of viability (115.64 ± 6.95, 106.91 ± 5.27 and 116.73 ± 20.15) than control group. They were significantly different from the control group (p < 0.05) but lower than the positive control group (DMEM with 10% heat treated fetal bovine serum). Cell lines showed normal character in fibroblast elongate shape after treated with pFF except in high concentration of pFF. This result implies that pFF of small size ovarian follicle at concentration of 400-600 µg proteins/mL could be optimized concentration for using as a supplement in Vero cell line culture medium to promote cell viability instead of growth hormone from fetal bovine serum. This merit could be applied in other cell biotechnology researches. Acknowledgements: This work was funded by a grant from Silpakorn University and Faculty of Science, Silpakorn University, Thailand.

Keywords: cell viability, porcine follicular fluid, MTT assay, Vero cell line

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Authors: Maryam Parsian, Pelin Mutlu, Ufuk Gunduz

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Two-dimensional cell culture affords simplicity and low cost, but it has serious limitations; lacking cell-cell and cell-matrix interactions that are present in tissues. Cancer cells grown in 3D culture systems have distinct phenotypes of adhesion, growth, migration, invasion as well as profiles of gene and protein expression. These interactions cause the 3D-cultured cells to acquire morphological and cellular characteristics relevant to in vivo tumors. Palbociclib is a chemotherapeutic agent for the treatment of ER-positive and HER-negative metastatic breast cancer. Poly-amidoamine (PAMAM) dendrimer is a well-defined, special three-dimensional structure and has a multivalent surface and internal cavities that can play an essential role in drug delivery systems. In this study, palbociclib is loaded onto the magnetic PAMAM dendrimer. Hanging droplet method was used in order to form 3D spheroids. The possible toxic effects of both free drug and drug loaded nanoparticles were evaluated in 2D and 3D MCF-7, MD-MB-231 and SKBR-3 breast cancer cell culture models by performing MTT cell viability and Alamar Blue assays. MTT analysis was performed with six different doses from 1000 µg/ml to 25 µg/ml. Drug unloaded PAMAM dendrimer did not demonstrate significant toxicity on all breast cancer cell lines. The results showed that 3D spheroids are clearly less sensitive than 2D cell cultures to free palbociclib. Also, palbociclib loaded PAMAM dendrimers showed more toxic effect than free palbociclib in all cell lines at 2D and 3D cultures. The results suggest that the traditional cell culture method (2D) is insufficient for mimicking the actual tumor tissue. The response of the cancer cells to anticancer drugs is different in the 2D and 3D culture conditions. This study showed that breast cancer cells are more resistant to free palbociclib in 3D cultures than in 2D cultures. However, nanoparticle loaded drugs can be more cytotoxic when compared to free drug.

Keywords: 2D and 3D cell culture, breast cancer, palbociclibe, PAMAM magnetic nanoparticles

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Authors: Fumio Kasai, Noriko Hirayama, Jorge Pereira, Azusa Ohtani, Masashi Iemura, Malcolm A. Ferguson Smith, Arihiro Kohara

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The VERO cell line was established in 1962 from normal tissue of an African green monkey, Chlorocebus aethiops (2n=60), and has been commonly used worldwide for screening for toxins or as a cell substrate for the production of viral vaccines. The VERO genome was sequenced in 2014; however, its cytogenetic features have not been fully characterized as it contains several chromosome abnormalities and different karyotypes coexist in the cell line. In this study, the VERO cell line (JCRB0111) was compared with one of the sublines. In contrast to 59 chromosomes as the modal chromosome number in the VERO cell line, the subline had two peaks of 56 and 58 chromosomes. M-FISH analysis using human probes revealed that the VERO cell line was characterized by a translocation t(2;25) found in all metaphases, which was absent in the subline. Different abnormalities detected only in the subline show that the cell line is heterogeneous, indicating that the subline has the potential to change its genomic characteristics during cell culture. The various alterations in the two independent lineages suggest that genomic changes in both VERO cells can be accounted for by progressive rearrangements during their evolution in culture. Both t(5;X) and t(8;14) observed in all metaphases of the two cell lines might have a key role in VERO cells and could be used as genetic markers to identify VERO cells. The flow karyotype shows distinct differences from normal. Further analysis of sorted abnormal chromosomes may uncover other characteristics of VERO cells. Because of the absence of STR data, cytogenetic data are important in characterizing animal cell lines and can be an indicator of their quality control.

Keywords: VERO, cell culture passage, chromosome rearrangement, heterogeneous cells

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Authors: Delphine Morales, Florian Lombart, Agathe Truchot, Pauline Maire, Pascale Vigneron, Antoine Galmiche, Catherine Lok, Muriel Vayssade

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Targeted therapy molecules are used as a first-line treatment for metastatic melanoma with B-Raf mutation. Nevertheless, these molecules can cause side effects to patients and are efficient on 50 to 60 % of them. Indeed, melanoma cell sensitivity to targeted therapy molecules is dependent on tumor microenvironment (cell-cell and cell-extracellular matrix interactions). To better unravel factors modulating cell sensitivity to B-Raf inhibitor, we have developed and compared several melanoma models: from metastatic melanoma cells cultured as monolayer (2D) to a co-culture in a 3D dermal equivalent. Cell response was studied in different melanoma cell lines such as SK-MEL-28 (mutant B-Raf (V600E), sensitive to Vemurafenib), SK-MEL-3 (mutant B-Raf (V600E), resistant to Vemurafenib) and a primary culture of dermal human fibroblasts (HDFn). Assays have initially been performed in a monolayer cell culture (2D), then a second time on a 3D dermal equivalent (dermal human fibroblasts embedded in a collagen gel). All cell lines were treated with Vemurafenib (a B-Raf inhibitor) for 48 hours at various concentrations. Cell sensitivity to treatment was assessed under various aspects: Cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK signaling pathway analysis (Western-Blotting), Apoptosis (TUNEL), Cytokine release (IL-6, IL-1α, HGF, TGF-β, TNF-α) upon Vemurafenib treatment (ELISA) and histology for 3D models. In 2D configuration, the inhibitory effect of Vemurafenib on cell proliferation was confirmed on SK-MEL-28 cells (IC50=0.5 µM), and not on the SK-MEL-3 cell line. No apoptotic signal was detected in SK-MEL-28-treated cells, suggesting a cytostatic effect of the Vemurafenib rather than a cytotoxic one. The inhibition of SK-MEL-28 cell proliferation upon treatment was correlated with a strong expression decrease of phosphorylated proteins involved in the MAPK pathway (ERK, MEK, and AKT/PKB). Vemurafenib (from 5 µM to 10 µM) also slowed down HDFn proliferation, whatever cell culture configuration (monolayer or 3D dermal equivalent). SK-MEL-28 cells cultured in the dermal equivalent were still sensitive to high Vemurafenib concentrations. To better characterize all cell population impacts (melanoma cells, dermal fibroblasts) on Vemurafenib efficacy, cytokine release is being studied in 2D and 3D models. We have successfully developed and validated a relevant 3D model, mimicking cutaneous metastatic melanoma and tumor microenvironment. This 3D melanoma model will become more complex by adding a third cell population, keratinocytes, allowing us to characterize the epidermis influence on the melanoma cell sensitivity to Vemurafenib. In the long run, the establishment of more relevant 3D melanoma models with patients’ cells might be useful for personalized therapy development. The authors would like to thank the Picardie region and the European Regional Development Fund (ERDF) 2014/2020 for the funding of this work and Oise committee of "La ligue contre le cancer".

Keywords: 3D human skin model, melanoma, tissue engineering, vemurafenib efficiency

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Authors: Hani M. M. Alothaid, Louise Robson, Richmond Muimo

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This study will investigate cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) dependent calcineurin (Cn), known as protein phosphatase 2 B (PP2B) as well, regulation of chloride ion (Cl⁻) secretion and the release of pro-inflammatory molecules in immune cells such as cytokines. THP-1-derived monocytes, primary human monocytes and the bronchial epithelial cell line (16HBE14o-) were used in this study. The 16HBE14o- cells were chosen as positive control. Hence, to further confirm the expression of cystic fibrosis transmembrane conductance regulator (CFTR), calcium binding protein (S100A10), annexin A2 (AnxA2) and calcineurin A subunit (CnA) in all three cell types, cell lysate was probed against corresponding primary antibodies by immunoblotting. Western blot analyses show the expression of CFTR, AnxA2, CnA and S100A10 in THP-1-derived monocytes and primary human monocytes. In conclusion, CFTR, S100A10, CnA and AnxA2 are expressed in THP-1-derived monocytes and primary human monocytes and regulate Cl⁻ secretion. Also, they may play a role in the pro-inflammatory molecules release. The ongoing work will confirm interaction between these proteins in the cell lines.

Keywords: annexin A2, calcineurin, CFTR, chloride, monocytes, pro-inflammatory molecules, S100A10

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Authors: Bryony Davies

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Many cell and tissue culture practices ignore the effects of gravity on cell biology, and little is known about how cell components may move in response to gravitational forces. Starfish oocytes provide an excellent model for interrogating the movement of cell components due to their unusually large size, ease of handling, and high transparency. Chromosomes from starfish oocytes can be visualised by microinjection of the histone-H2B-mCherry plasmid into the oocytes. The movement of the chromosomes can then be tracked by live-cell fluorescence microscopy. The results from experiments using these methods suggest that there is a replicable downward movement of centrally located chromosomes at a median velocity of 0.39 μm/min. Chromosomes nearer the nuclear boundary showed more restricted movement. Chromosome density and shape could also be altered by microinjection of restriction enzymes, primarily Alu1, before imaging. This was found to alter the speed of chromosome movement, with chromosomes from Alu1-injected nuclei showing a median downward velocity of 0.60 μm/min. Overall, these results suggest that there is a non-negligible movement of chromosomes in response to gravitational forces and that this movement can be altered by enzyme activity. Future directions based on these results could interrogate if this observed downward movement extends to other cell components and to other cell types. Additionally, it may be important to understand whether gravitational orientation and vertical positioning of cell components alter cell behaviour. The findings here may have implications for current cell culture practices, which do not replicate cell orientations or external forces experienced in vivo. It is possible that a failure to account for gravitational forces in 2D cell culture alters experimental results and the accuracy of conclusions drawn from them. Understanding possible behavioural changes in cells due to the effects of gravity would therefore be beneficial.

Keywords: starfish, oocytes, live-cell imaging, microinjection, chromosome dynamics

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Authors: Soroosh Torabi, Brad Berron, Ren Xu, Christine Trinkle

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Microfluidics integrated with 3D cell culture is a powerful technology to mimic cellular environment, and can be used to study cell activities such as proliferation, migration and response to drugs. This technology has gained more attention in cancer studies over the past years, and many organ-on-a-chip systems have been developed to study cancer cell behaviors in an ex-vivo tumor microenvironment. However, there are still some barriers to adoption which include low throughput, complexity in 3D cell culture integration and limitations on non-optical analysis of cells. In this study, a user-friendly microfluidic multi-well plate was developed to mimic the in vivo tumor microenvironment. The microfluidic platform feeds multiple 3D cell culture sites at the same time which enhances the throughput of the system. The platform uses hydrophobic Cassie-Baxter surfaces created by microchannels to enable convenient loading of hydrogel/cell suspensions into the device, while providing barrier free placement of the hydrogel and cells adjacent to the fluidic path. The microchannels support convective flow and diffusion of nutrients to the cells and a removable lid is used to enable further chemical and physiological analysis on the cells. Different breast cancer cell lines were cultured in the device and then monitored to characterize nutrient delivery to the cells as well as cell invasion and proliferation. In addition, the drug response of breast cancer cell lines cultured in the device was compared to the response in xenograft models to the same drugs to analyze relevance of this platform for use in future drug-response studies.

Keywords: microfluidics, multi-well 3d cell culture, tumor microenvironment, tumor-on-a-chip

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Authors: A. Zuchowska, A. Buta, M. Mazurkiewicz-Pawlicka, A. Malolepszy, L. Stobinski, Z. Brzozka

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In recent years, graphene-based materials are finding more and more applications in biological science. As a thin, tough, transparent and chemically resistant materials, they appear to be a very good material for the production of implants and biosensors. Interest in graphene derivatives also resulted at the beginning of research about the possibility of their application in cancer therapy. Currently, the analysis of their potential use in photothermal therapy and as a drug carrier is mostly performed. Moreover, the direct anticancer properties of graphene-based materials are also tested. Nowadays, cytotoxic studies are conducted on in vitro cell culture in standard culture vessels (macroscale). However, in this type of cell culture, the cells grow on the synthetic surface in static conditions. For this reason, cell culture in macroscale does not reflect in vivo environment. The microfluidic systems, called Lab-on-a-chip, are proposed as a solution for improvement of cytotoxicity analysis of new compounds. Here, we present the evaluation of cytotoxic properties of graphene oxide (GO) on breast, liver and colon cancer cell line in a microfluidic system in two spatial models (2D and 3D). Before cell introduction, the microchambers surface was modified by the fibronectin (2D, monolayer) and poly(vinyl alcohol) (3D, spheroids) covering. After spheroid creation (3D) and cell attachment (2D, monolayer) the selected concentration of GO was introduced into microsystems. Then monolayer and spheroids viability/proliferation using alamarBlue® assay and standard microplate reader was checked for three days. Moreover, in every day of the culture, the morphological changes of cells were determined using microscopic analysis. Additionally, on the last day of the culture differential staining using Calcein AM and Propidium iodide were performed. We were able to note that the GO has an influence on all tested cell line viability in both monolayer and spheroid arrangement. We showed that GO caused higher viability/proliferation decrease for spheroids than a monolayer (this was observed for all tested cell lines). Higher cytotoxicity of GO on spheroid culture can be caused by different geometry of the microchambers for 2D and 3D cell cultures. Probably, GO was removed from the flat microchambers for 2D culture. Those results were also confirmed by differential staining. Comparing our results with the studies conducted in the macroscale, we also proved that the cytotoxic properties of GO are changed depending on the cell culture conditions (static/ flow).

Keywords: cytotoxicity, graphene oxide, monolayer, spheroid

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Authors: Kamila Duś-Szachniewicz, Kinga M. Walaszek, Paweł Skiba, Paweł Kołodziej, Piotr Ziółkowski

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Cell adhesion is of fundamental importance in the cell communication, signaling, and motility, and its dysfunction occurs prevalently during cancer progression. The knowledge of the molecular and cellular processes involved in abnormalities in cancer cells adhesion has greatly increased, and it has been focused mainly on cellular adhesion molecules (CAMs) and tumor microenvironment. Unfortunately, most of the data regarding CAMs expression relates to study on cells maintained in standard oxygen condition of 21%, while the emerging evidence suggests that culturing cells in ambient air is far from physiological. In fact, oxygen in human tissues ranges from 1 to 11%. The aim of this study was to compare the effects of physiological lymph node normoxia (5% O2), and hyperoxia (21% O2) on the expression of cellular adhesion molecules of primary diffuse large B-cell lymphoma cells (DLBCL) isolated from 10 lymphoma patients. Quantitative RT-PCR and immunohistochemistry were used to confirm the differential expression of several CAMs, including ICAM, CD83, CD81, CD44, depending on the level of oxygen. Our findings also suggest that DLBCL cells maintained at ambient O2 (21%) exhibit reduced growth rate and migration ability compared to the cells growing in normoxia conditions. Taking into account all the observations, we emphasize the need to identify the optimal human cell culture conditions mimicking the physiological aspects of tumor growth and differentiation.

Keywords: adhesion molecules, diffuse large B-cell lymphoma, physiological normoxia, quantitative RT-PCR

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Authors: Dean Robinson, Miriam Gublebank, Ella Sklan, Tali Tavor Re'em

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Alginate, a natural linear polysaccharide polymer extracted from brown seaweed, is extensively applied due to its biocompatibility, all- aqueous ease of handling, and relatively low costs. Alginate easily forms a hydrogel when crosslinked with a divalent ion, such as calcium. However, Alginate hydrogel holds low mechanical properties and is cell-inert. To overcome these drawbacks and to improve alginate as a bio-ink for bioprinting, we produced a new alginate matrix combined with spider silk, one of the most resilient, elastic, strong materials known to men. Recombinant spider silk biopolymer has a sponge-like structure and is known to be biocompatible and non-immunogenic. Our results indicated that combining synthetic spider-silk into bio-printed cell-seeded alginate hydrogels resulted in improved properties compared to alginate: improved mechanical properties of the matrix, achieving a tunable gel viscosity and high printability, alongside prolonged and higher cell viability in culture, probably due to the improved cell-matrix interactions. The new bio-ink was then used for bilayer bioprinting of epithelial and stromal endometrial cells. Such a co-culture model will be used for the formation of the complex endometrial tissue for studying the embryo implantation process.

Keywords: cell culture, tissue engineering, spider silk, alginate, bioprinting

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Authors: Fan Gao, Lior Pachter

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The primary tool currently used to pre-process 10X Chromium single-cell ATAC-seq data is Cell Ranger, which can take very long to run on standard datasets. To facilitate rapid pre-processing that enables reproducible workflows, we present a suite of tools called scATAK for pre-processing single-cell ATAC-seq data that is 15 to 18 times faster than Cell Ranger on mouse and human samples. Our tool can also calculate chromatin interaction potential matrices, and generate open chromatin signal and interaction traces for cell groups. We use scATAK tool to explore the chromatin regulatory landscape of a healthy adult human brain and unveil cell-type specific features, and show that it provides a convenient and computational efficient approach for pre-processing single-cell ATAC-seq data.

Keywords: single-cell, ATAC-seq, bioinformatics, open chromatin landscape, chromatin interactome

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Authors: Yuta Kurashina, Chikahiro Imashiro, Kiyoshi Ohnuma, Kenjiro Takemura

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Research objectives and goals: To realize clinical applications of regenerative medicine, a mass cell culture is highly required. In a conventional cell culture, trypsinization was employed for cell detachment. However, trypsinization causes proliferation decrease due to injury of cell membrane. In order to detach cells using an enzyme-free method, therefore, this study proposes a novel cell detachment method capable of detaching adherent cells using acoustic radiation pressure exposed to the dish by the assistance of serum-free medium with ITS liquid medium supplement. Methods used In order to generate acoustic radiation pressure, a piezoelectric ceramic plate was glued on a glass plate to configure an ultrasonic transducer. The glass plate and a chamber wall compose a chamber in which a culture dish is placed in glycerol. Glycerol transmits acoustic radiation pressure to adhered cells on the culture dish. To excite a resonance vibration of transducer, AC signal with 29-31 kHz (swept) and 150, 300, and 450 V was input to the transducer for 5 min. As a pretreatment to reduce cell adhesivity, serum-free medium with ITS liquid medium supplement was spread to the culture dish before exposed to acoustic radiation pressure. To evaluate the proposed cell detachment method, C2C12 myoblast cells (8.0 × 104 cells) were cultured on a ø35 culture dish for 48 hr, and then the medium was replaced with the serum-free medium with ITS liquid medium supplement for 24 hr. We replaced the medium with phosphate buffered saline and incubated cells for 10 min. After that, cells were exposed to the acoustic radiation pressure for 5 min. We also collected cells by using trypsinization as control. Cells collected by the proposed method and trypsinization were respectively reseeded in ø60 culture dishes and cultured for 24 hr. Then, the number of proliferated cells was counted. Results achieved: By a phase contrast microscope imaging, shrink of lamellipodia was observed before exposed to acoustic radiation pressure, and no cells remained on the culture dish after the exposed of acoustic radiation pressure. This result suggests that serum-free medium with ITS liquid inhibits adhesivity of cells and acoustic radiation pressure detaches cells from the dish. Moreover, the number of proliferated cells 24 hr after collected by the proposed method with 150 and 300 V is the same or more than that by trypsinization, i.e., cells were proliferated 15% higher with the proposed method using acoustic radiation pressure than with the traditional cell collecting method of trypsinization. These results proved that cells were able to be collected by using the appropriate exposure of acoustic radiation pressure. Conclusions: This study proposed a cell detachment method using acoustic radiation pressure by the assistance of serum-free medium. The proposed method provides an enzyme-free cell detachment method so that it may be used in future clinical applications instead of trypsinization.

Keywords: acoustic radiation pressure, cell detachment, enzyme free, ultrasonic transducer

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Authors: Thanunthon Bowornsakulwong, Charukorn Charukarn, Franck Courtes, Panit Kitsubun, Lalintip Horcharoen

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Vero cell is a continuous cell line that is widely used for the production of viral vaccines. However, due to its adherent characteristic, scaling up strategy in large-scale production remains complicated and thus limited. Consequently, suspension-like Vero cell culture processes based on microcarriers have been introduced and employed while also providing increased surface area per volume unit. However, harvesting Vero cells from microcarriers is a huge challenge due to difficulties in cells detaching, lower recovery yield, time-consuming and dissociation agent carry-over. To overcome these problems, we developed a dissociation-association platform technology for detaching and re-attaching cells during subculturing from microcarriers to microcarriers, which will be conveniently applied to seed trains strategies in large scale bioreactors. Herein, Hillex-2 was used to culture Vero cells in serum-containing media using spinner flasks as a scale-down model. The overall confluency of cells on microcarriers was observed using inverted microscope, and the sample cells were daily detached in order to obtain the kinetics data. The metabolites consumption and by-products formation were determined by Nova Biomedical BioprofileFlex.

Keywords: dissociation-reassociation, microcarrier, scale up, Vero cell

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Authors: Ana Paula Rosifini Alves Claro, André Luiz Reis Rangel, Nathan Trujillo, Ketul C. Popat

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In the present work, in vitro cytotoxicity was evaluated after nanotubes growth on Ti15Mo alloy surface. TiO2 nanotubes were obtained by anodizing technique at room temperature in an electrolyte with 0.25 %NH4F and glycerol at a constant anodic potential of 20 V for 24 hours. The morphology of nanotubes was observed by field emission scanning electron microscopy (FE-SEM; XL 30 FEG, Philips). Crystal structure was analyzed by wide-angle X-ray diffraction. A cell culture model using human fibroblast-like cells was used to study the effect of TiO2 nanotubes growth on the cytotoxicity of the Ti15Mo alloy for 1, 4 and 7 days culture period. The MTT assay was used to evaluate cell viability and cell adhesion was evaluated by scanning electron microscopy. Results show that Ti15Mo alloy with TiO2 nanotubes on surface is nontoxic and exhibit good interaction with surface.

Keywords: titanium alloys, TiO2 nanotubes, cell growth, Ti-15Mo alloy

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Authors: Masoud Sakhinia, Sajjad Ahmad

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Introduction: Though knowledge of the compositional makeup and structure of the limbal niche has progressed exponentially during the past decade, much is yet to be understood. Identifying the precise profile and role of the stromal makeup which spans the ocular surface may inform researchers of the most optimum conditions needed to effectively expand LESCs in vitro, whilst preserving their differentiation status and phenotype. Limbal fibroblasts, as opposed to corneal fibroblasts are thought to form an important component of the microenvironment where LESCs reside. Methods: The corneal stroma was tissue engineered in vitro using both limbal and corneal fibroblasts embedded within a tissue engineered 3D collagen matrix. The effect of these two different fibroblasts on LESCs and hTCEpi corneal epithelial cell line were then subsequently determined using phase contrast microscopy, histolological analysis and PCR for specific stem cell markers. The study aimed to develop an in vitro model which could be used to determine whether limbal, as opposed to corneal fibroblasts, maintained the stem cell phenotype of LESCs and hTCEpi cell line. Results: Tissue culture analysis was inconclusive and required further quantitative analysis for remarks on cell proliferation within the varying stroma. Histological analysis of the tissue-engineered cornea showed a comparable structure to that of the human cornea, though with limited epithelial stratification. PCR results for epithelial cell markers of cells cultured on limbal fibroblasts showed reduced expression of CK3, a negative marker for LESC’s, whilst also exhibiting a relatively low expression level of P63, a marker for undifferentiated LESCs. Conclusion: We have shown the potential for the construction of a tissue engineered human cornea using a 3D collagen matrix and described some preliminary results in the analysis of the effects of varying stroma consisting of limbal and corneal fibroblasts, respectively, on the proliferation of stem cell phenotype of primary LESCs and hTCEpi corneal epithelial cells. Although no definitive marker exists to conclusively illustrate the presence of LESCs, the combination of positive and negative stem cell markers in our study were inconclusive. Though it is less traslational to the human corneal model, the use of conditioned medium from that of limbal and corneal fibroblasts may provide a more simple avenue. Moreover, combinations of extracellular matrices could be used as a surrogate in these culture models.

Keywords: cornea, Limbal Stem Cells, tissue engineering, PCR

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Authors: Mayuva Youngsabanant, Chanikarn Srinark, Supanyika Sengsai, Soratorn Kerdkriangkrai, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The follicular fluid proteins of healthy medium size follicles (4-6 mm in diameters) and large size follicles (7-8 mm in diameter) of large white pig ovaries were collected by using sterile technique. They were used for testing the effect on primary in vitro cell culture growth of porcine oviductal epithelial cells (pOEC). Porcine oviductal epithelial cells of luteal phase was culture in M199 and added with 10% fetal calf serum 2.2 mg/mL, NaHCO₃, 0.25 mM pyruvate, 15 µg/mL and 50 µg/mL, gentamycin sulfate at high humidified atmosphere with 5% CO₂ in 95% air atmosphere at 37°C for 96 h before testing. The optimized concentration of pFF of two follicle sizes (at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins) in culture medium was observed for 24 h using MTT assay. Results were analyzed with a one-way ANOVA in SPSS statistic. Moreover, pOEC was also studied in morphological characteristic on long-term culture. The results of long-term study revealed that pOEC showed 70-80 percentage of healthy morphology on epithelial-like character and contained 30 percentage of an elongated shape (fibroblast-like morphology) at 4 weeks of culture time. MTT assay reviewed an increase in the percentage of viability of pOEC in 2 treated of follicular fluid groups. Two treatment concentration groups were higher than control group (p < 0.05) but not in positive control group. Interestingly, at 200 µg protein of 2 treated follicular fluid groups were reached the highest cell viability which is higher than a positive control and it is significantly different form control group (P < 0.05). These cells are developed and had fibroblast elongate shape which is longer than the cells in control group and positive control group. This report implies that pFF of medium follicle size at 200 µg proteins and large follicle size at 200 and 500 µg proteins could be optimized concentration for using as a supplement in culture medium to promote cell growth and development instead of growth hormone from fetal calf serum. It could be applied in cell biotechnology researches. Acknowledgements: The project was funded by a grant from Silpakorn University Research and Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: in vitro, porcine follicular fluid protein (pFF), porcine oviductal epithelial cells (pOEC), MTT

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Authors: H. Rezzouk, M. Hatti, H. Rahmani, S. Atoui

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This paper discusses the design and analysis of a hybrid PV-Fuel cell energy system destined to power a DC load. The system is composed of a photovoltaic array, a fuel cell, an electrolyzer and a hydrogen tank. HOMER software is used in this study to calculate the optimum capacities of the power system components that their combination allows an efficient use of solar resource to cover the hourly load needs. The optimal system sizing allows establishing the right balance between the daily electrical energy produced by the power system and the daily electrical energy consumed by the DC load using a 28 KW PV array, a 7.5 KW fuel cell, a 40KW electrolyzer and a 270 Kg hydrogen tank. The variation of powers involved into the DC bus of the hybrid PV-fuel cell system has been computed and analyzed for each hour over one year: the output powers of the PV array and the fuel cell, the input power of the elctrolyzer system and the DC primary load. Equally, the annual variation of stored hydrogen produced by the electrolyzer has been assessed. The PV array contributes in the power system with 82% whereas the fuel cell produces 18%. 38% of the total energy consumption belongs to the DC primary load while the rest goes to the electrolyzer.

Keywords: electrolyzer, hydrogen, hydrogen fueled cell, photovoltaic

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Authors: Nelson Barrientos, Matthew J. Davies, Marco C. Marques, Darren N. Nesbeth, Gary J. Lye, Nicolas Szita

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Microbioreactor technology is making important advances towards its application in cell culture and bioprocess development. In particular, the technology promises flexible and controllable devices capable to perform parallelised experimentation at low cost. Currently, state of the art methods (e.g. optical sensors) allow the accurate monitoring of the microbioreactor operation. In addition, the laminar flow regime encountered in these devices allows more predictive fluid dynamics modelling, improving the control over the soluble, physical and mechanical environment of the cells. This work describes the development and characterisation of a novel microbioreactor cassette system (microbioreactor volume is 150 μL. The volumetric oxygen transfer coefficient (KLa) and mixing time have been characterised to be between 25 to 113 h-1 and 0.5 and 0.1 s, respectively. In addition, the Residence time distribution (RTD) analysis confirms that the reactor operates at well mixed conditions. Finally, Staphylococcus carnosus TM300 growth is demonstrated via batch culture experiments. Future work consists in expanding the optics of the microbioreactor design to include the monitoring of variables such as fluorescent protein expression, among others.

Keywords: microbioreactor, cell-culture, fermentation, microfluidics

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Authors: Sergey V. Sazonov, Anna S. Mogilenskikh, Svetlana S. Deryabina, Denis A. Demidov, Sergey M. Demidov

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The majority of breast cancer (BC) cases are estrogen receptor (ER) positive, and the most common among surrogate molecular biological subtypes is Luminal A. Despite ongoing treatment, drug resistance occurs in a significant proportion of patients, leading to disease recurrence. To determine the sensitivity of a particular tumor to therapy, it is necessary to create an in vitro model, which could then serve as a basis for the selection of personalized therapy for breast cancer. The aim is to evaluate the molecular genetic profile of tumor cells from primary cultures of the Luminal A subtype of breast cancer as a potential model for assessing resistance to therapy. Materials and methods: Primary cell cultures were obtained from a surgical sample in patients with Luminal A subtype of breast cancer when the following inclusion criteria were met: absence of previous therapy and availability of voluntary informed consent. The subtype was determined using the IHC method. Growth was maintained in a serum-free Mammocult™ environment (STEMCELL, Canada). The culture was passed after 7-10 days (p1-p5), and flow cytometry was performed on a Beckman Coulter cytometer (USA) at each passage. Antibodies to estrogen (Alexa Fluor® 647, SP1, Abcam, Canada) to HER2 (24D2, Brilliant Violet 421™, Biolegend, USA) were used for evaluation, and at least 5,000 events were analyzed.). NGS testing was performed on the same cells (Prep&Seq™ U-panel BCEv1, 63 genes). Results: Luminal A subtype includes cases with a Ki-67 threshold of less than 10% of tumor cell nuclei, no HER2 expression (level 0 to 1 points), lack of amplification of the HER2 gene, and high estrogen and/or progesterone levels. The median for RE expression was 66.5% (IQR 7.1) from p1 to p5, and this index didn´t change significantly throughout culture (p<0.001). The presence of Her2 oncoprotein was detected in 5.0% across all passages (IQR 5.2). The data obtained are consistent with the characteristics of the Luminal A subtype. NGS testing detected mutations in genes associated with breast cancer: BARD1, BRCA2, BRIP1, CASP8, CCNE1, CDH1, KEAP1, MSH3, NF1, PALB2, RAD51D, RECQL5, TP53 ZNF217, as well as in genes associated with the Luminal A subtype: PIK3CA, MAP3KI, ESR1. At all passages are detected in the MAP3K1 gene, 7 mutations (3 missenses, 3 synonymous, 1 in-frame) except p2 and p5. A mutation in 1 exon (synonymous) is added at p2 and p5, while no changes are detected in the other genes. Conclusion: Primary cell cultures during the five passages can preserve the expression of RE and the composition of mutations in genes and can be used as an in vitro model. The combination of mutation in ESR1 and MAP3K gene is a potential factor of acquired resistance to therapy in patients with Luminal A breast cancer.

Keywords: breast cancer, Luminal A subtype, primary cell culture, HER2 receptors, estrogen receptors, MAP3K1 gene, ESR1 gene

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Authors: Zohra Salim

Abstract:

Objective: To access the relationship of primary tumor thickness with cervical lymphadenopathy in squamous cell carcinoma of buccal mucosa. Methodology: A cross-sectional observational study was carried out on 80 Patients with biopsy-proven oral squamous cell carcinoma of buccal mucosa at Dow University of Health Sciences. All the study participants were treated with wide local excision of the primary tumor with elective neck dissection. Patients with prior head and neck malignancy or those with prior radiotherapy or chemotherapy were excluded from the study. Data was entered and analyzed on SPSS 21. Chi-squared test with 95% C.I and 80% power of the test was used to evaluate the relationship of tumor depth with cervical lymph nodes. Results: 50 participants were male, and 30 patients were female. 30 patients were in the age range of 20-40 years, 36 patients in the range of 40-60 years, while 14 patients were beyond age 60 years. Tumor size ranged from 0.3cm to 5cm with a mean of 2.03cm. Tumor depth ranged from 0.2cm to 5cm. 20% of the participants reported with tumor depth greater than 2.5cm, while 80% of patients reported with tumor depth less than 2.5cm. Out of 80 patients, 27 reported with negative lymph nodes, while 53 patients reported with positive lymph nodes. Conclusion: Our study concludes that relationship exists between the depth of primary tumor and cervical lymphadenopathy in squamous cell carcinoma of buccal mucosa.

Keywords: squamous cell carcinoma, tumor depth, cervical lymphadenopathy, buccal mucosa

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Authors: Kirsten Wilson, Patrick M. Brauer, Sandra Babic, Diana Golubeva, Jessica Van Eyk, Tinya Wang, Avanti Karkhanis, Tim A. Le Fevre, Andy I. Kokaji, Allen C. Eaves, Sharon A. Louis, , Nooshin Tabatabaei-Zavareh

Abstract:

Long-lived plasma cells are rare, non-proliferative B cells generated from antibody-secreting cells (ASCs) following an immune response to protect the host against pathogen re-exposure. Despite their therapeutic potential, the lack of in vitro protocols in the field makes it challenging to use B cells as a cellular therapeutic tool. As a result, there is a need to establish robust and reproducible methods for the generation of B cells. To address this, we have developed a culture system for generating B cells from hematopoietic stem and/or progenitor cells (HSPCs) derived from human umbilical cord blood (CB) or pluripotent stem cells (PSCs). HSPCs isolated from CB were cultured using the StemSpan™ B Cell Generation Kit and produced CD19+ B cells at a frequency of 23.2 ± 1.5% and 59.6 ± 2.3%, with a yield of 91 ± 11 and 196 ± 37 CD19+ cells per input CD34+ cell on culture days 28 and 35, respectively (n = 50 - 59). CD19+IgM+ cells were detected at a frequency of 31.2 ± 2.6% and were produced at a yield of 113 ± 26 cells per input CD34+ cell on culture day 35 (n = 50 - 59). The B cell receptor loci of CB-derived B cells were sequenced to confirm V(D)J gene rearrangement. ELISpot analysis revealed that ASCs were generated at a frequency of 570 ± 57 per 10,000 day 35 cells, with an average IgM+ ASC yield of 16 ± 2 cells per input CD34+ cell (n = 33 - 42). PSC-derived HSPCs were generated using the STEMdiff™ Hematopoietic - EB reagents and differentiated to CD10+CD19+ B cells with a frequency of 4 ± 0.8% after 28 days of culture (n = 37, 1 embryonic and 3 induced pluripotent stem cell lines tested). Subsequent culture of PSC-derived HSPCs increased CD19+ frequency and generated ASCs from 1 - 2 iPSC lines. This method is the first report of a serum- and feeder-free system for the generation of B cells from CB and PSCs, enabling further B lineage-specific research for potential future clinical applications.

Keywords: stem cells, B cells, immunology, hematopoiesis, PSC, differentiation

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Authors: Mostafa Ahmed Shokshok

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Libyan industrial companies face many challenges in today's competitive market. Quality management culture approaches is one of these challenges which may furnish the road to the Libyan industrial companies to effectively empower their employees and improve their ability to respond to the international competition. The primary objective of this paper is to design a practical approach to guide Libyan industrial companies toward successful quality culture implementation.

Keywords: TQM, quality culture, Libyan manufacturing industries, quality framework

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