Colonies of iPSCs (between passage 15 and 20) were passaged using EDTA in order to obtain ~10 colonies per well of a six‐well plate. Four days after plating colonies reached ~500–800 μm in diameter (day 0), the medium was switched from E8 (pluripotent maintenance) to MesenPRO RS™ (mesenchymal stem cell maintenance medium; Thermo Fisher Scientific), MesenPRO RS™ was refreshed every 2 days until day 7. On day 7 cells were passaged to 0.1% gelatine coated T75 using TrypLE, (equivalent to a 1:7.5 split ratio or ~0.5 × 106 to 1 × 106 cells per T75), termed P0. MesenPRO RS™ was then refreshed every 3 days until day 14. On day 14 cells were passaged 1:8 to T75 (tissue culture plastic), termed P1. Mesenchymal cells (iPSC‐MCs) were then maintained in MesenPRO RS™ on tissue culture plastic and passaged 1:6 when ~80% confluent using TrypLE. TGF‐β stimulation was performed using 10 ng/ml TGFβ3 on passage 3 iPSC‐MCs in MesenPRO RS™ for 7 days.
Mesenpro rs
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MesenPRO RS™ is a cell culture media designed for the expansion and differentiation of mesenchymal stem cells (MSCs). It provides a defined, animal-component-free, and serum-free environment for the growth and maintenance of MSCs.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (2 mentions)
β glycerol phosphate, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Glutamine, by Merck Group (1 mentions)
Ccl 163, by American Type Culture Collection (1 mentions)
Xe 5000, by Sysmex (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Cellnest, by Fujifilm (1 mentions)
Hmscs, by Lonza (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Direct zol rna kit, by Zymo Research (1 mentions)
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
20 protocols using mesenpro rs
1
Mesenchymal Differentiation of iPSCs
2
Adipose-Derived Stem Cell Culture and Osteogenic Differentiation
3
Adipose-derived Stem Cells in Collagen Scaffolds
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Adipose-derived mesenchymal stem cells (ThermoFisher, USA) were cultured in MesenPRO RS™ basal media supplemented with MesenPRO RS™ growth supplements (ThermoFisher, USA), 200 mM Glutamine (Sigma-Aldrich, USA) and 1% Penicillin-Streptomycin (ThermoFisher, USA). ASCs-encapsulated collagen scaffolds were prepared by embedding passage 4 ASCs at 106 cells/ml seeding density in 3 mg/ml collagen Type-I solution. The cell-encapsulated collagen solutions were then deposited around the silicone posts, polymerized at 37oC for 1 hour and incubated in the culture media.
4
Cytocompatibility of PCL and PRP-PCL Scaffolds
5
Characterization of Mesenchymal Stem Cells
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BMA and BMC samples were hematologically analyzed (Sysmex XE 5000) for concentrations of white blood cells, total nucleated cells (TNCs), red blood cells (RBCs), neutrophils (NE), and hematopoietic stem cells (HPCs). One BMC sample was cultured in vitro at 37°C with 5% CO2 in humidified air with MesenPro RS (ThermoFisher Scientific) culture medium. Media was changed every 3 days, and pictures were taken at day 1, day 13, and day 20. At day 20, MSC analysis was performed via flow cytometry with the BD Stemflow MSC Analysis Kit (BD Biosciences) and the CytoFLEX Flow Cytometer (Beckman Coulter). Flow cytometry was used to detect cell surface receptors positive for CD73, CD90, and CD105 and negative for CD34, CD45, CD11b or CD79α, and HLA-DR.
6
Comparative Stem Cell Culture on Collagen RCP
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Three MSC lines were used in this study: human bone marrow stem cells (HMSCs) from Lonza (PT-2501); human epiphyseal chondrocyte (HEC)7 (link) cell lines established from cartilage of hyperdactylia at the National Center for Child Health and Development (NCCHD); and an upper limb bone marrow cell (UBMC) line established at NCCHD. For the latter two cell types, parental written informed consent was obtained and their use in this study was approved by the Institutional Review Board of the NCCHD.
HMSCs, HECs, and UBMCs were cultured in low-serum concentration (2%) medium (Mesen PRO RS®; Thermo Fischer Scientific, MA) and in serum-free medium (PRIME-XV® MSC Expansion XSFM; FUJIFILM Irvine Scientific, CA) at 37°C in air. The cells were cultured on CellnestTM (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan); Cellnest is a solution containing 0.1% type I COL-based RCP. RCP has a repetitive RGD sequence designed to enhance cellular adhesion.8 As a control, RCP-coated culture plates were prepared: 0.1% RCP solution (2 mL; Cellnest) was dispensed onto the culture dishes at 37°C. After 2 h, the RCP solution was removed from each well and Dulbecco's phosphate-buffered saline (DPBS) was added to each well. The plates were kept at room temperature for 30 min, and the DPBS was then removed.
HMSCs, HECs, and UBMCs were cultured in low-serum concentration (2%) medium (Mesen PRO RS®; Thermo Fischer Scientific, MA) and in serum-free medium (PRIME-XV® MSC Expansion XSFM; FUJIFILM Irvine Scientific, CA) at 37°C in air. The cells were cultured on CellnestTM (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan); Cellnest is a solution containing 0.1% type I COL-based RCP. RCP has a repetitive RGD sequence designed to enhance cellular adhesion.8 As a control, RCP-coated culture plates were prepared: 0.1% RCP solution (2 mL; Cellnest) was dispensed onto the culture dishes at 37°C. After 2 h, the RCP solution was removed from each well and Dulbecco's phosphate-buffered saline (DPBS) was added to each well. The plates were kept at room temperature for 30 min, and the DPBS was then removed.
7
Fabrication of Cellular Bioinks for Tissue Engineering
8
Osteogenic Differentiation of Human Stem Cells
9
Osteogenic Differentiation of Mesenchymal Stem Cells
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RNA-seq of the MSC differentiation samples was performed according to the following protocol. 50,000 cells were seeded in 6-well plates in maintenance medium: MesenPRO-RS™ (Thermo Fisher Scientific, Massachusetts, USA). After 3 days, medium was replaced with osteogenic differentiation medium: DMEM high glucose supplemented with 10% FBS, 1× non-essential amino acids (NEAA), 2 mM L-glutamine, 0.28 mM ascorbic acid, 10 mM β glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich, USA). At each time-point: 0, 0.5, 1, 2, 4, 6, 8, 12, 16, 24, 48, 72, 125, 168, 336, and 504 h during osteogenesis, total RNA was isolated using TRIzol (Invitrogen™ Life Technologies, Carlsbad, USA) with the Direct-zol RNA kit (ZymoResearch, USA) according to the manufacturer’s instruction. RNA-seq library preparation was carried out using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, USA) according to the manufacturer’s instruction. The quantity and quality of the cDNA library were assessed using the Agilent 2200 tapestation (Agilent Technologies, Santa Clara, USA). Paired-end sequencing of the pooled library was carried out using the Illumina NextSeq 500 v2 kit (Illumina, San Diego, USA) according to the manufacturer’s instruction.
10
Engineered Adipose Tissue Scaffold
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Human adipose-derived stem cells recovered from the lipoaspirate of one patient at the Adipose Stem Cell Research Laboratory (University of Pittsburgh, Pittsburgh, Pa.) were expanded in MesenPRO RS (Thermo Fisher) growth medium (37°C, 5% carbon dioxide) until 85 percent confluence was obtained. Allograft adipose matrix, reconstituted in basal medium and Dulbecco’s Modified Eagle Medium (Sigma-Aldrich) at 25 percent weight/volume, was placed into 24-well transwell culture inserts in a 3-mm layer. Human adipose-derived stem cells were seeded atop the matrix, 0.2 M cells per 7-mm diameter. Confocal microscopy was performed using a Leica TCS SPII microscope (Leica, Allendale, N.J.). Fixed human adipose-derived stem cell–seeded allograft adipose matrix was stained with Phalloidin 633 and 4′,6-diamidino-2-phenylindole (all from Thermo Fisher), to visualize attachment and stretching at day 3. Lipid content was visualized by boron-dipyrromethene (Thermo Fisher), at days 7, 14, and 21 after cell seeding. Scanning electron microscopy was performed using FEI XL30 FEG-SEM (FEI, Hillsboro, Ore.) at 5-kV accelerating voltage on days 0, 7, and 14. Immunohistochemistry against collagen VI, collagen IV, and laminin and the adipogenic markers adiponectin, leptin, and FABP4 was performed at days 0, 7, and 14. Imaging was performed using an Olympus IX71 microscope (Olympus, Center Valley, Pa.).
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