E0771/mCherry-luc2 cells (2 × 104 cells / well) were seeded in 96-well plates optimized for tumor spheroid formation (IWAKI EZSPHERE, AGC Techno Glass Co. Ltd., Shizuoka, Japan) with 10% FBS-RPMI with murine galectin-3 (Biolegend, San Diego, CA, USA), and incubated for 72 h. The mCherry fluorescence was detected to visualize tumor spheroids (Keyence, Osaka, Japan). The size of tumor spheroids was analyzed in three independent images for each concentration of galectin-3 with Hybrid Cell Count software (Keyence).
Ezsphere
Manufactured by AGC Techno Glass
12 citations
Sourced in Japan
About the product
The EZSPHERE is a lab equipment product designed for spherical glass processing. It is capable of producing spherical glass components with high precision and consistency.
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Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
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Y 27632, by Fujifilm (2 mentions)
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Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
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Penicillin streptomycin mixture, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
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12 protocols using «ezsphere»
1
Galectin-3 Modulates Tumor Spheroid Formation
2
Enteral Transplantation of CD73+ Spheroids for Murine Colitis
Exactly 14 days after sorting, CD73+ cells were cultured for 2 days in EZSPHERE® (AGC Techno Glass, Shizuoka, Japan), in order to generate spheroids. Spheroids were collected and suspended in PBS. Cells (1.0 × 106 cells in 200 μL of PBS) were transplanted via enteral administration 0 and 5 days after DSS treatment. Trans-anal transplantation was performed as previously described, with slight modifications [22 (link)]. The cell suspension was infused into the murine colonic lumen by using a thin, flexible catheter (diameter: 2.1 mm) or a stainless steel needle (diameter: 1.9 mm). After transplantation, the mice were housed as usual, and colon tissues were collected and analyzed by flow cytometry. Control mice received an equal volume of PBS, or a solution of 0.01 mg/mL 5-aminosalicylic acid (5-ASA; Zeria Pharmaceutical Co., Ltd., Tokyo, Japan). The concentration of 5-ASA (100 mg/kg) was based on a previous paper, and 5-ASA was administered enterally in the same manner as the spheroids [23 (link)].
Corresponding organizations : Juntendo University
3
Differentiation of hiPSCs into CD31+ Endothelial Cells
CD31+ cells were prepared from differentiated hiPSCs (Ff-I14). To generate embryoid bodies (Ebs), hiPSCs were seeded onto EZSPHERE (AGC TECHNO GLASS Co., Ltd., Shizuoka, Japan). Approximately 3 × 105 hiPSC cells/mL were cultured in the StemFit AK03N medium containing 2 ng/mL BMP4 and 10 µM Y27632 in the absence of component C (Ajinomoto, Co., Ltd., Tokyo, Japan) (day 0). On day 1, 9 ng/mL BMP4, 10 ng/mL bFGF, and 6 ng/mL Activin A (R&D Systems, Inc., Minneapolis, MN, USA) were added to the medium. From days 2 to 7, Ebs were cultured in a single-use bioreactor and magnetic stirrer (ABLE Corporation & Biott Corporation, Tokyo, Japan). On day 2, the medium was supplemented with 9 ng/mL BMP4, 10 ng/mL bFGF, and 6 ng/mL Activin A (R&D Systems, Inc., Minneapolis, MN, USA), and removed on day 4. On day 4, the medium was supplemented with 25 ng/mL vascular endothelial growth factor (VEGF) (R&D Systems, Inc., Minneapolis, MN, USA) and 8 ng/mL bFGF, and removed on day 7. On day 7, Ebs were enzymatically dissociated and subjected to MACS (Miltenyi Biotec Inc., Tokyo, Japan) to separate CD31+ cells. CD31+ cells were maintained in EGM2 (Lonza, Inc., Basel, Switzerland) on collagen-type IV-coated tissue culture dishes. They were passaged every two days until being harvested for cryopreservation on day 13.
Corresponding organizations : Keio University, Osaka University, Tokyo Women's Medical University, National Institute of Advanced Industrial Science and Technology, National Institute of Biomedical Innovation, Health and Nutrition
4
Hypoxic Culture of hiPSC-Derived Cardiomyocytes
hiPSC-CMs at day 23 were seeded onto EZ Sphere (AGC Techno Glass, Shizuoka, Japan) at 5 × 104 cells/cm2 to form a spheroid. The following day (day 24), spontaneous beating was confirmed, and the spheroids were moved to a normoxic (20% O2) or hypoxic (1% O2) incubator for 4 or 12 days to be cultured in DMEM supplemented with 10% FBS and penicillin–streptomycin (Sigma–Aldrich). The culture medium was changed every 4 days. To investigate intracellular Ca2+ transients, spheroids were transferred to 6-well plates and loaded with 5 μM Fluo-8 (AAT Bioquest, Sunnyvale, CA, USA) in DMEM supplemented with 10% FBS for 1 h in a humidified incubator with 5% CO2 at 37 °C. Ten minutes before observation, each well was immersed in phenol red-free DMEM (Nacalai Tesque, Kyoto, Japan) containing 10% FBS and penicillin–streptomycin. Changes in fluorescence levels were monitored at a rate of 100 ms with an ORCA-R2 CCD camera (Hamamatsu Photonics K.K., Shizuoka, Japan) and processed with Aquacosmos image processing software (Hamamatsu Photonics K.K.) for baseline correction and colored visualization. The Ca2+ transient was recorded during electrical stimulation at 1 Hz (C-Pace EP, IonOptix, Westwood, MA, USA).
Corresponding organizations : Tokyo Women's Medical University
5
Generation of IL4-Modulated Dendritic Cells
IL4−DCs were generated using previously reported low−adherence cell culture maturation protocols [25 (link)]. PBMCs from patients were suspended in AIM−V medium (serum−free medium, Thermo Fisher Scientific, Inc., Waltham, MA, USA), placed into adherent dishes (Primaria, BD Biosciences, San Jose, CA, USA), and incubated for 18–24 h. After removing non−adherent cells, 100 ng/mL of GM−CSF and 50 ng/mL of IL-4 (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) were added the following day. Cells were cultured for 5 days to generate immature DCs. Immature DCs were differentiated by matured by stimulation with OK−432 (10 μg/mL, streptococcal preparation; Chugai Pharmaceutical Co., Ltd., Tokyo, Japan), PGE2 (10 ng/mL; Kyowa Pharma Chemical Co., Ltd., Toyama, Japan), 20 μg/mL of the WT1 peptides reconstituted with dimethyl sulfoxide (DMSO) (for WT1−235 killer peptide: CYTWNQMNL, residues 235–243: for WT1−34 helper peptide: WAPVLDFAPPGASAYGSL, residues 34–51; Peptide Institute, Inc., Osaka, Japan) for 24 h in either Prime surface (Sumitomo Bakelite, Tokyo, Japan) for the low−adherent dish or EZSPHERE (AGC TECHNO GLASS Co., Ltd., Shizuoka, Japan) for the cluster−controlled dish.
Corresponding organizations : Kanazawa Medical University, Kanazawa Medical University Hospital
Top 5 most cited protocols using «ezsphere»
1
Isolation and Characterization of hAFSCs
The study was approved by the Institutional Review Board of Keio University School of Medicine (no. 20140285), and informed consent was obtained from all the volunteer donors. Five-milliliter amniotic fluid samples were obtained from two pregnant women who underwent amniocentesis at 15 and 16 weeks of gestation. CD117-positive (CD117+) cells were isolated as hAFSCs, as described previously [17 (link)–22 (link)]. Briefly, within 2 h, the samples were centrifuged at 200×g for 5 min. After removing the supernatant, the cell pellet was cultivated in growth medium comprising alpha modified Eagle minimum essential medium (α-MEM; Invitrogen, Carlsbad, CA), 15% fetal bovine serum (FBS) (Invitrogen), 1% L-glutamine (Invitrogen), 1% penicillin/streptomycin (Invitrogen), and 40% AmnioMax-II (Life Technologies, Carlsbad, CA). After the cell population became sub-confluent, the cells were counted, and the CD117+ cells were isolated as hAFSCs using a magnetic cell sorting kit (Miltenyi Biotec, Auburn, CA).
CD117+ cells were characterized by flow cytometry for surface markers, as described in our previous studies [17 (link), 18 (link), 21 (link)]. The antibodies used for flow cytometry are listed in Table S1. CD117+ cells were cultured in “adipogenic differentiation medium” and “osteogenic differentiation medium” (PromoCell, Heidelberg, Germany) according to the manufacturer’s protocol. To induce chondrogenic differentiation, a total of 1.0 × 106 cells were seeded in EZSPHERE (AGC Techno Glass, Tokyo, Japan), then cultured for 12 days in “chondrogenic differentiation medium” (PromoCell). CD117+ cells were also characterized by real-time polymerase chain reaction (RT-qPCR) for the expression of molecular differentiation markers into adipogenic, osteogenic, or chondrogenic lineages. RT-qPCR was performed in duplicate in a volume of 25 μL per reaction using a 96-well Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Richmond, CA). Reaction mixtures contained 5 ng genomic DNA as the template, 0.4 mM of each primer (FASMAC, Atsugi, Kanagawa, Japan), SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Bio), and sterile H2O. The primer sets are listed in Table S2. We analyzed the relative gene expression in each sample by the 2−ΔΔCT method. Gene expression values were normalized to β-actin levels as an internal control.
CD117+ cells were characterized by flow cytometry for surface markers, as described in our previous studies [17 (link), 18 (link), 21 (link)]. The antibodies used for flow cytometry are listed in Table S1. CD117+ cells were cultured in “adipogenic differentiation medium” and “osteogenic differentiation medium” (PromoCell, Heidelberg, Germany) according to the manufacturer’s protocol. To induce chondrogenic differentiation, a total of 1.0 × 106 cells were seeded in EZSPHERE (AGC Techno Glass, Tokyo, Japan), then cultured for 12 days in “chondrogenic differentiation medium” (PromoCell). CD117+ cells were also characterized by real-time polymerase chain reaction (RT-qPCR) for the expression of molecular differentiation markers into adipogenic, osteogenic, or chondrogenic lineages. RT-qPCR was performed in duplicate in a volume of 25 μL per reaction using a 96-well Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Richmond, CA). Reaction mixtures contained 5 ng genomic DNA as the template, 0.4 mM of each primer (FASMAC, Atsugi, Kanagawa, Japan), SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Bio), and sterile H2O. The primer sets are listed in Table S2. We analyzed the relative gene expression in each sample by the 2−ΔΔCT method. Gene expression values were normalized to β-actin levels as an internal control.
Corresponding organizations : Keio University Hospital, Keio University
2
Feeder-Free Derivation of iPSC-Derived Neural Crest Cells
Human iPSC lines 253G1 and 201B7, which are generated from adult skin fibroblasts [27 (link),28 (link)], were obtained from the Riken BioResource Center (Ibaraki, Japan). The iPSCs were cultured under feeder-free and serum-free conditions with AK03N medium (Ajinomoto, Tokyo, Japan) in six-well culture plates coated with iMatrix-511 (Nippi, Tokyo, Japan) as previously described [29 (link)]. The iPSCs were subcultured once a week, with the medium changed daily. The iPSCs were differentiated into NCCs according to a previously described method [25 (link),26 (link)] with some modifications. Briefly, after the undifferentiated iPSCs reached confluence in culture, the iPSCs were dissociated with Accutase (Millipore, Temecula, CA) and seeded first on a 3-D culture plate (EZSPHERE®; AGC Techno Glass, Shizuoka, Japan) [30 (link)] at a density of 2 × 105 cells/mL in an embryoid body (EB) induction medium, which was composed of DMEM/F12 (Sigma-Aldrich, St. Louis, MO) supplemented with 1% N-2 (Gibco, Grand Island, NY), 2% MACS® NeuroBrew-21 (Miltenyi Biotec, Auburn, CA), 2 mM l -glutamine (Sigma-Aldrich), 20 ng/mL recombinant human epidermal growth factor (EGF: PeproTech, Rocky Hill, NJ), 1% penicillin/streptomycin (Gibco), and 10 μM Y-27632 (Wako, Osaka, Japan). The spent medium was changed once on EB culture day 4, with uniformly sized EBs forming in 7 days. Subsequently, the EBs were replated on a noncoated 10 cm dish in NCC induction medium, DMEM/F12 with 1% N-2, 2 mM l -glutamine, 1% penicillin/streptomycin, 20 ng/mL EGF, 20 ng/mL recombinant human fibroblast growth factor-basic (bFGF: Wako), and 5 μg/mL heparin (Sigma-Aldrich). The culture dish was left untouched for the first 3 days to allow the EBs to attach to the culture dish and the induced NCCs to migrate from the attached EBs, after which the spent medium was changed on days 4, 8, 11, 13, and 14 in conjunction with the replating of the EBs. NCC induction was completed by day 14. The iPSC-NCCs were used for subsequent experiments.
This study was performed in accordance with ethics protocols approved by the Keio University Ethics Committee and the Institutional Review Board at RIKEN Center for Developmental Biology. Informed consent was obtained from all healthy volunteers.
This study was performed in accordance with ethics protocols approved by the Keio University Ethics Committee and the Institutional Review Board at RIKEN Center for Developmental Biology. Informed consent was obtained from all healthy volunteers.
Corresponding organizations : Keio University, RIKEN Center for Biosystems Dynamics Research, Fujita Health University, Japan Society for the Promotion of Science
3
Directed Differentiation of Human iPSCs
Human iPSC (Ff-I14) cultured on iMatrix511 were dissociated with TrypLE Select and a single cell suspension was cultured on EZSPHERE (10 cm diameter) (AGC Techno Glass, Shizuoka, Japan) for 4 days in StemFit AK03N medium not containing component C with Y27632 (10 μM) at 37 °C in humid air with 5% CO2 and 1% O2. At day 4, cell aggregates were collected from EZSPHERE dishes and resuspended in StemFit AK03N medium not containing component C and cultured in a 100 mL stirred bioreactor system (ABLE) until day 13 or 14. The following growth factors and small molecules were used at the corresponding days: days 0–1, 2 ng/mL BMP4 (R&D systems); days 1–4, 10 ng/mL BMP4, 6 ng/mL Activin A (R&D systems), 10 ng/mL bFGF (ReproCell); days 4–8, 1 μM IWP-3 (Stemgent, Lexington, MA, USA), 0.6 μM Dorsomorphine (Sigma–Aldrich, St. Louis, MO, USA), 5.4 μM SB432542 (Sigma Aldrich); after day 8, 5 ng/mL VEGF (R&D Systems) and 10 ng/mL bFGF. At days 6, 8, 10, and 12, the culture medium was exchanged.
Corresponding organizations : Tokyo Women's Medical University, Waseda University
4
Cardiac Differentiation and Co-Culture of iPSCs
Following cardiac differentiation of human iPS cells, cells were cultured for 2 days in 24-well culture plates (Corning) at a density of 2.1 × 105 cells/cm2 in DMEM supplemented with 10% FBS at 37 °C in humid air with 5% CO2. One day before starting the co-culture experiment, iPS cells cultured on iMatrix511 were dissociated with TrypLE Select and a single cell suspension was cultured on EZ Sphere (AGC Techno Glass, Shizuoka, Japan) for 1 day in StemFit AK03 medium with Y27632 (10 μM) (Wako) to form cell aggregates. The following day, 50 cell aggregates of iPS cells were co-cultured with iPS-derived cardiac cells in 24-well culture plates in StemFit AK03 for 1 day. These preparations were then cultured for 2 days according to the following conditions: StemFit AK03 at 37 °C or 42 °C, 10% FBS DMEM at 37 °C or 42 °C.
Corresponding organizations : Tokyo Women's Medical University
5
Culturing Insulin-Secreting Cell Aggregates
The cell line 832/13, derived from INS-1 rat insulinoma cells, was obtained from Dr. Christopher B. Newgard (Duke University Medical Center, Durham, NC) [21] . Cells were grown in RPMI medium 1640 containing l -glutamine (Invitrogen Ltd.), 1 mM sodium pyruvate (Invitrogen Ltd.), 10 mM HEPES (Invitrogen Ltd.), 10 vol% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA), 55 μM 2-mercaptoethanol (Invitrogen Ltd.), 100 IU/ml penicillin (Gibco, Grand Island, NY), and 100 μg/ml streptomycin (Gibco). Cells were cultured in a humidified atmosphere containing 5% CO2/95% air at 37 °C.
Gelatin hydrogel microspheres and INS-1 cells were separately suspended in the culture medium. The initial seeding density of cells was 1 × 103 or 1 × 104 cells/well at the cells/microspheres number ratio of 100/1. The mixed suspensions of microspheres and INS cells (14 ml) were added to EZSPHERE (4000-905, AGC Techno Glass Co. Ltd., Shizuoka, Japan). Pictures of INS-1 cell aggregates with or without the gelatin hydrogel microspheres incorporation were taken with the microscope as described above.
Cell aggregates with or without gelatin hydrogel microspheres were transferred to each well of a 12 mm Transwell (#3402, Coring Inc. Corning, NY) and washed once by phosphate-buffered saline solution (PBS, Gibco). Then, Krebs-Ringer-bicarbonate HEPES (KRB) buffer solution containing 10 mM glucose [21] was added to each well, and the cell aggregates were incubated for 1 h at 37 °C. The concentration of insulin secreted in the supernatants was measured by ELISA kit (Rat Insulin ELISA KIT, Shibayagi Co. Ltd., Gunma, Japan). Experiments were performed on three wells for each sample.
Gelatin hydrogel microspheres and INS-1 cells were separately suspended in the culture medium. The initial seeding density of cells was 1 × 103 or 1 × 104 cells/well at the cells/microspheres number ratio of 100/1. The mixed suspensions of microspheres and INS cells (14 ml) were added to EZSPHERE (4000-905, AGC Techno Glass Co. Ltd., Shizuoka, Japan). Pictures of INS-1 cell aggregates with or without the gelatin hydrogel microspheres incorporation were taken with the microscope as described above.
Cell aggregates with or without gelatin hydrogel microspheres were transferred to each well of a 12 mm Transwell (#3402, Coring Inc. Corning, NY) and washed once by phosphate-buffered saline solution (PBS, Gibco). Then, Krebs-Ringer-bicarbonate HEPES (KRB) buffer solution containing 10 mM glucose [21] was added to each well, and the cell aggregates were incubated for 1 h at 37 °C. The concentration of insulin secreted in the supernatants was measured by ELISA kit (Rat Insulin ELISA KIT, Shibayagi Co. Ltd., Gunma, Japan). Experiments were performed on three wells for each sample.
Corresponding organizations : Kyoto University, Takeda (United States)
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