Aluminum alloys are widely utilized in diverse industries, such as automobiles, aerospace, and architecture, owing to their high specific strength and resistance to oxidation. However, to meet the increasing demands of the industry, it is necessary to design new aluminum alloys with excellent properties. Thus, a new method is required to efficiently test additively manufactured aluminum alloys with various compositions within a short period during the alloy design process. In this study, a combinatory approach using a direct energy deposition system for metal 3D printing process with a dual feeder was employed. Two types of aluminum alloy powders, namely Al6061 and Al-12Cu, were utilized for the combinatory test conducted through 3D printing. Twelve types of Al-Si-Cu-Mg alloys were manufactured during this combinatory test, and the relationship between their microstructures and properties was investigated.

Canada’s Pickering Unit 3 was performed a three-stage decontamination from June to August 1989 in preparation for pressure tube replacement. The first step was a reducing CAN-DECON treatment to dissolve the magnetic film inside the reactor, which was applied following partial defueling of the reactor core. The second step was an oxidative dilute alkaline permanganate treatment to remove the chromium-rich oxides of the stainless steel parts. And the final CAN-DECON step was applied continuously after completely removing fuel from the reactor core. In situ pipe gamma-ray spectroscopy techniques were applied to measure radioactivity within feeder piping during various stages of Pickering Unit 3 decontamination. Measurements were performed at a maximum dose rate of 5 mSv/h, and both the detector and the scanned feeder pipe were properly shielded from other neighboring pipes. 60Co was the dominant radionuclide in feeder piping prior to decontamination. And radionuclides 103Ru, 95Zr, 95Nb, 59Fe, 140La and 124Sb were detected. The Co-60 radioactivity was 2.09×105 Bq/cm2 before decontamination and 3.11×103 Bq/cm2 after decontamination in the inlet feeder pipe T18. And in the outlet feeder pipe P21, it is 2.56×104 Bq/cm2 before decontamination and 2.04×103 Bq/cm2 after decontamination.

In this study, 3D design, vibration analysis and experiment, and feed rate performance test were performed to develop a vibratory bowl feeder for supplying ultra-thin plate parts of less than 50 microns. The natural frequency and resonant frequency of the vibratory bowl feeder were obtained through the vibration analysis and experiment, and it was confirmed that the results of the analysis and experiment agree well. Through the feed rate experiment, it was confirmed that up to 610 ultra-thin parts per minute were fed at 250Hz and a supply voltage of 220V, where the excitation frequency and the resonant frequency match. And through analysis and experimental research, it was confirmed that the development of a vibratory bowl feeder for supplying ultra-thin parts was successful.

Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. Regulation of cytotoxicity in NK cells is mediated by inhibitory receptors that bind major histocompatibility complex class I (MHC-I) molecules on target cells. Human myelogenous leukemia K562 cells are readily attacked by NK cells, because K562 cells expressed very low levels of MHC-I molecules for inhibitory NK cell receptors. In this study, we compared the ability of γ-irradiated- or mitomycin C (MMC)-treated K562 feeder cells to support expansion and activation of canine NK cells. We isolated CD5 negative cells from canine peripheral blood mononuclear cells by immunomagnetic separation and co-cultured with γ-irradiated (100 Gy)- or MMC (20 μg/mL)-treated K562 cells in the presence of interleukin (IL)-2, IL-15 and IL-21 for 21 days. As a result, number of CD5 negative cells, co-cultured with γ-irradiated- or MMC-treated K562 cells (56.72 ± 13.77 fold or 32.99 ± 10.83 fold), was increased than those of CD5 negetive cells (2.99 ± 1.42 fold). Also, we found that gene expression markers of activated NK cells such as NKp30, NKp44, NKp46, Ly49, NKG2D, CD244, perforin, and granzyme B and production of interferon gamma were similarly upregulated in NK cells co-cultured with γ-irradiated- or MMC-treated K562 cells, respectively. Next, we observed that cytotoxicity of NK cells co-cultured with γ-irradiated K562 cells was more sensitively reacted to canine mammary carcinoma cells than those of MMC-treated K562 cells. These results revealed that γ-irradiated K562 cells are more efficient feeder cells than MMC-treated K562 cells for enhancing NK cells expansion and activation.

The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

RNA Sendai virus (SeV) vector system has no risk of being integrated into the host genome. Sendai virus (SeV) vectors expressing pluripotent factors have been used to produce integration-free induced pluripotent stem cells (iPSCs) with high efficiency from various cell types in human and mouse. In this study, we generated iPSCs from pig ear fibroblast cells using the SeV vector expressing 4 human factors (POU5F1, SOX2, C-MYC, and KLF4). Colonies were emerged at Day 14 of transduction and expressed the classical pluripotency markers (POU5F1, NANOG, and SOX2) and surface marker (SSEA1). Furthermore, they showed a domed shape and could passage over 40 times under 2i (CHIR99021 and PD0325901)-LIF and MEF feeder culture condition having in vitro differentiation ability into 3 germ layers. Next, we examined the ability of six feeder free culture conditions to maintain piPSCs in a pluripotent state. piPSCs were plated on Matrigel coated dishes in different media: 1. CM: control media (LIF culture media); 2. CM-F: CM+100 ng Fetuin-A; 3. CM-N: CM+100 ng Nanog-TAT; 4. CM-2i: CM+3 uM CHIR99021+1 uM PD0325901; 5. CM-2iN: CM-2i+100 ng Nanog-TAT; 6. CM-2iN+100 ng Fetuin-A. However, piPSC could not maintain the typical self-renewal morphology on feeder free conditions regardless of culture media tested here. Further, expression of pluripotency-related genes (Oct4, Nanog and Klf4) of piPSCs cultured on feeder free conditions could not be compared with that of iPSCs cultured on MEF feeder plate. Our results suggest that integration free pluripotent stem cell from pigs could be generated by SeV vector system and maintained their pluripotency under 2i-LIF and MEF feeder culture condition, but further optimization of culture conditions may be required.

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.

Pluripotency of human embryonic stem cell (hESC) is one of the most valuable ability of hESCs for applying cell therapy field, but also showing side effect, for example teratoma formation. When transplant multipotent stem cell, such as mesnchymal stem cell (MSC) which retains similar differentiation ability, they do not form teratoma in vivo, but there exist limitation of cellular source supply. Accordingly, differentiation of hESC into MSC will be promising cellular source with strong points of both hESC and MSC line. In this study, we described the derivation of MSC like cell population from feeder free cultured hESC (hESC- MSC) using direct differentiation system. Cells population, hESC-MSC and bone marrow derived MSC (BM-MSC) retained similar characteristics in vitro, such as morphology, MSC specific marker expression and differentiation capacity. At the point of differentiation of both cell populations, differentiation rate was slower in hESC-MSC than BM-MSC. As these reason, to verify differentially expressed molecular condition of both cell population which bring out different differentiation rate, we compare the molecular condition of hESC-MSC and BM-MSC using 2-D proteomic analysis tool. In the proteomic analysis, we identified 49 differentially expressed proteins in hESC-MSC and BM-MSC, and they involved in different biological process such as positive regulation of molecular function, biological process, cellular metabolic process, nitrogen compound metabolic process, macromolecule metabolic process, metabolic process, molecular function, and positive regulation of molecular function and regulation of ubiquitin protein ligase activity during mitotic cell cycle, cellular response to stress, and RNA localization. As the related function of differentially expressed proteins, we sought to these proteins were key regulators which contribute to their differentiation rate, developmental process and cell proliferation. Our results suggest that the expressions of these proteins between the hESC-MSC and BM-MSC, could give to us further evidence for hESC differentiation into the mesenchymal stem cell is associated with a differentiation factor. As the initial step to understand fundamental difference of hESC-MSC and BM-MSC, we sought to investigate different protein expression profile. And the grafting of hESC differentiation into MSC and their comparative proteomic analysis will be positively contribute to cell therapy without cellular source limitation, also with exact background of their molecular condition.

Mesenchymal stem cells (MSCs) have the multipotent capacity and this potential can be applied for obtaining valuable cell types which can use for cell therapy on various regenerative diseases. However, insufficient availability of cellular source is the major problem in cell therapy field using adult stem cell sources. Recently, human embryonic stem cells (hESCs) have been highlighted to overcome a limitation of adult cellular sources because they retain unlimited proliferation capacity and pluripotency. To use of hESCs in cell therapy, above all, animal pathogen free culture system and purification of a specific target cell population to avoid teratoma formation are required. In this study, we describe the differentiation of a mesenchymal stem cell-like cells population from feeder-free cultured hESCs(hESC-MSCs) using direct induction system. hESC-MSCs revealed characteristics similar to MSCs derived from bone marrow, and undifferentiated cell markers were extremely low in hESC-MSCs in RT- PCR, immunostaining and FACS analyses. Thus, this study proffer a basis of effective generation of specialized human mesenchymal stem cell types which can use for further clinical applications, from xenofree cultured hESCs using direct induction system.

  In this paper, we consider a planning problem arising from printed circuit board manufacturing industries. Given a set of several types of PCBs, component feeders and surface mounting machines in series in a PCB assembly line, the problem is to define t

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies.

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/–) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/– (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/– (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture. Key words : Mouse embryonic stem cell, Feeder cell, Pluripotency marker, MEF feeder cell