Whole blood samples containing PGCs were collected from a BPR embryo at Hamburger Hamilton (HH) stage 13–15 (Hamburger and Hamilton, 1951 ). The blood was dispersed in 500 µL of PGC culture medium. For each culture experiment, PGCs derived from a single embryo were used. The PGC culture medium used was same as that described in a previous study (Ezaki et al., 2020 ) with some modifications. Briefly, KnockOut DMEM (Thermo Fisher Scientific, Waltham, MA, USA) was supplemented with 1X B-27 Supplement Minus Vitamin A (Thermo Fisher Scientific), 1% Chicken Serum (Thermo Fisher Scientific), 1X EmbryoMAX nucleosides (Merck, Darmstadt, Germany), 1X MEM non-essential amino acids (Thermo Fisher Scientific), 0.5 mM monothioglycerol (Wako Pure Chemical Industries, Osaka, Japan), 1X Antibiotic-Antimycotic Mixed Stock Solution (Nacalai Tesque, Kyoto, Japan), 10 ng/mL human FGF2 (PeproTech, Rocky Hill, NJ, USA), 1 unit/mL heparin (Merck), 0.2 µM blebbistatin, and 0.2 µM H-1152 (Wako Pure Chemical Industries). Whole blood samples containing PGCs were cultured in 24-well plates without feeder cells at 38°C, 5% CO2, and 3% O2, and subcultured every 2–4 days. The PGCs were passaged based on their growth. The PGCs were frozen in STEM-CELLBANKER (Nippon Zenyaku Kogyo, Fukushima, Japan) and stored at −80°C.
Stem cellbanker
Manufactured by Nippon Zenyaku Kogyo
Sourced in Japan
STEM-CELLBANKER is a cryopreservation medium designed for the long-term storage of stem cells and other cell types. It is a ready-to-use solution that helps maintain cell viability and functionality during the freezing and thawing process.
Automatically generated - may contain errors
Lab products found in correlation
Chicken serum, by Thermo Fisher Scientific (1 mentions)
Blebbistatin, by Fujifilm (1 mentions)
Monothioglycerol, by Fujifilm (1 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
B27 supplement minus vitamin a, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Embryomax nucleosides, by Merck Group (1 mentions)
Human fgf2, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic mixed stock solution, by Nacalai Tesque (1 mentions)
Heparin, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Accumax, by Innovative Cell Technologies (1 mentions)
Aria 2, by BD (1 mentions)
Luna automated cell counter, by Logos Biosystems (1 mentions)
100 μm cell strainer, by BD (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Egm 2mv, by Lonza (1 mentions)
Poly l lysine pll, by ScienCell (1 mentions)
7 protocols using stem cellbanker
1
Avian Primordial Germ Cell Culture
2
Intestinal Mesenchymal Stromal Cell Production
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Intestinal organoids were cut and spread on a 35-mm dish coated with iMatrix-511 silk. The dishes were coated with 1.7 μg/ml iMatrix-511 silk at 37 °C for 1 h. Then the attached fragments were cultured at 37 °C in 5% CO2 with ESTEM-HE medium (GlycoTechnica). The medium was changed every 2 days. After 21 days of culture, the cells were trypsinized with 0.25% trypsin/1 mM EDTA for 3 min. The passage was onto a non-coated plate. The cells were cultured in XF32 medium at 0–3 days and then in ESTEM-HE medium at day 4 until just before passage. This passaging was performed at least three times, and the cells were purified before use for feeder cells. Thereafter, the medium was changed to DMEM supplemented with 10% FBS at 37 °C in 5% CO2. The medium was changed every 3 days. The cells were passaged every 5–7 days and seeded at 3.6 × 104 cells/cm2. The cells were cryopreserved with STEM-CELLBANKER (ZR646, Nippon Zenyaku Kogyo Co., Ltd.) until use. The protocol for intestinal mesenchymal stromal cell production is shown in a flow chart (Additional file 1 : Figure S1A).
3
Cardiomyocyte Differentiation from Human iPSCs
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Human iPSCs were differentiated by forming embryoid bodies (EBs), as previously described [24 (link), 25 (link)] (see Supplementary Materials for further details). On day 29, EBs were dissociated and dispersed onto a fibronectin (Sigma-Aldrich, St Louis, MO, USA)-coated 6 cm dish. On the following day, seeded cells were collected by Accumax (Innovative Cell Technologies, San Diego, CA, USA) for 10 minutes and subjected to fluorescence-activated cell sorting (FACS) (Aria II, BD Biosciences, San Jose, CA, USA). To purify cardiomyocytes, SIRPa-positive and lineage (CD31, CD49a, CD140b, CD90, or TRA-1-60)-negative cells were sorted [26 (link)] and cryopreserved with STEM-CELLBANKER (Nippon Zenyaku Kogyo, Koriyama, Japan) at −80°C. A few weeks later, the cryotubes were transferred to a liquid nitrogen storage tank.
4
Adipose-Derived Regenerative Cell Isolation
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Human subcutaneous adipose tissues were obtained from healthy female donors undergoing elective liposuction with informed consent. Adipose tissues were minced and digested with Celase (1 U/ml; Cytori Therapeutics, San Diego, CA, USA) for 20 min at 37 °C. After filtration through a 100-μm cell strainer (BD Biosciences, San Jose, CA, USA), ADRCs were isolated by centrifugation (300g for 5 min) removing adipocytes. Total cell number and cell viability were measured with a LUNA automated cell counter (Logos Biosystems, Inc., USA). The freshly isolated ADRCs were maintained in Lactated Ringer's Solution for in vitro experiments, including assessments of gene expression, and were suspended in STEM-CELLBANKER (Nippon Zenyaku Kogyo Co., Fukushima, Japan) for cryopreservation at −80 °C. The frozen ADRCs were thawed, followed by washing with PBS, and incubated in Lactated Ringer's Solution for 6-h recovery period at 37 °C and used in the assessment of gene expression. The freshly isolated cells (fresh) ADRCs from fat tissue and the cryopreserved (frozen) ADRCs were used for all experiments without passage.
5
Nasal Mucosal Tissue Explant Culture
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Nasal mucosal tissues underwent explant culture using a modification of our previously described method30 (link). In short, sterilized mucosal tissues were cut into 1.5-mm cubes, placed in cell culture dishes (Primaria Dish, Corning, Corning, NY, USA) and incubated in 750 μL of medium at 37 °C with 5% CO2 (Fig. 1 A). A further 1 mL of medium was added to each dish 1 h later, and another 2 mL of medium was added the following day. The medium was changed on days 6 and 10. Following 13 days of cultivation, cells were collected by trypsin–EDTA treatment and preserved with freezing medium (STEM-CELLBANKER, Nippon Zenyaku Kogyo, Asakamachi, Fukushima, Japan). In order to track migration behavior, cell outgrowth was monitored for 8 h on day 7 of the explant culture process using differential interference contrast microscopy (CytoWatcher, Atto, Motoasakusa, Tokyo, Japan).
6
Cryopreservation of Stem Cells
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The first step of the freezing process was identical to the passaging of that for the PSCs. After centrifugation, pelleted cells from one well of a six-well plate were resuspended in STEM-CELLBANKER (Nippon Zenyaku Kogyo, Cat nr. 181218), which is a specific medium developed to optimize freezing and thawing conditions for stem cells. The cells were then placed at - 80°C in a freezing container, allowing for a decrease of 1°C/min. The following day the cells were stored in the liquid nitrogen for long-term storage.
7
Cryopreservation of Retinal Cell Types
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Original vials of primary Human Retinal Microvascular Endothelial Cells (HRMVECs, Cell Systems, ACBRI 181) were obtained at passage 3 (P3), Human Retinal Pericytes (HRPs, Cell Systems, ACBRI 183) at P3 and Human Retinal Astrocytes (HRAs, ScienCell, 1870) at P1. The list of all cells used in this study is provided in Supplementary Table I . HRMVECs were cultured in T175 flasks coated for 1 h at 37 °C with 1× Attachment Factor Protein (Gibco, S1006100), in EGM-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKit (EGM-2 MV, Lonza, CC-3202). HRPs and HRAs were cultured in T175 flasks coated for 1 h at room temperature (RT) with 15 μg ml−1 Poly-L-Lysine (PLL, ScienCell, 0403), in Pericyte Medium (PM, ScienCell, 1201) or Astrocyte Medium (AM, ScienCell, 1801), respectively. Cells were harvested with Accutase (Innovative Cell Technologies, AT104). We prepared working stocks by expanding HRMVECs and HRPs to P5 and HRAs to P3, resuspending 1 × 106 cells ml-1 in STEM-CELLBANKER (Nippon Zenyaku Kogyo, 11897) freezing medium and freezing 5 × 105 cells per vial. New frozen vials were used for each experiment.
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