Since different aspects and applications of BoneMA were being tested, samples were prepared using different cell lineages for various experiments. To that end, human dental pulp stem cells (HDPSC) and human mesenchymal stem cells (HMSC) were used to assess cytocompatibility and bioprintability, while green fluorescent protein (GFP)-expressing human umbilical vein endothelial cells (HUVECs) (cAP0001GFP, Angio-proteomie, USA) were employed to investigate the vasculogenic potential of the synthesized biomaterial. HDPSCs (Lonza, USA) and HMSCs were each cultured in α-MEM medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin. HUVECs were cultured in Endothelial Cell culture medium (Vasculife-VEGF, Lifeline Cell Technologies) on 0.1% gelatin-coated substrates. Cells were maintained in an incubator at 37°C, 5% CO2 incubator, and the medium was replaced every 2 days.
Cap0001gfp
Manufactured by Angio-Proteomie
Sourced in United States
The CAP0001GFP is a laboratory instrument designed for the detection and analysis of green fluorescent protein (GFP) in biological samples. It utilizes advanced optics and detection mechanisms to accurately measure and quantify GFP expression levels.
Automatically generated - may contain errors
4 protocols using cap0001gfp
1
Bioprinting Vascularized Bone Constructs
2
Vascular Cell Culture Protocol
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We used 3 types of cells normally comprising blood vessels: Fibroblasts (NHDFc, C-12302, and HAoAF, C-12380 PromoCell, Heidelberg, Germany), Smooth muscle cells (hASMCs, cAP-0026RFP, Angio-Proteomie, Boston, USA), and Endothelial cells (HUVECs, cAP-0001GFP, Angio-Proteomie, Boston, USA). Frozen stock of each types of the cells was thawed and scaled up in appropriate media: D-MEM (High Glucose with Phenol Red and Sodium Pyruvate, StemSure, Wako) supplemented with 10% of fetal calf serum for smooth muscle cells and fibroblasts and Endothelial media (Endothelial Cell Growth Medium, PromoCell) for endothelial cells in 75 cm2 flasks (Primaria, Corning, NC, USA). Before co-culture cells were adapted for 1:1 mix of the above media for 72 h. For layer formation cells were detached from the surface with Trypsin/EDTA solution (Wako, Japan), and washed with its cultured media filtered through 1.2 µm membrane filter.
3
HUVEC Nanoparticle Uptake Study
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Human umbilical vein endothelial (HUVEC) cells (cAP-0001GFP, Angio-Proteomie) were cultured in Endothelial Cell Medium (ECM, P60104, Innoprot). For the uptake experiments, the cells were seeded into a 6-well plate at a density of 0.15 × 106 cells per well. The next day, the medium was exchanged with ECM containing 10, 50, 100, 200, and 400 μg ml−1 silane-coated nanoparticles. The medium for control cells was exchanged with ECM without additives.
4
Culturing Endothelial and Stem Cells
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All experiments used green fluorescent protein (GFP)-expressing human umbilical vein endothelial cells (HUVECs—cAP0001GFP, Angioproteomie) (P5-10), which were cultured in Endothelial Growth Medium (EGM) (cAP-02, Angioproteomie) on gelatin (0.1% (w/v)) coated substrates. SCAP (P4-8) (primary cells donated by Dr. Diogenes, University of Texas), and DPSC (P4-8) (primary cells, catalog # PT-5025, Lonza, Basel, Switzerland) were cultured in alpha-MEM supplemented with L-glutamine, 10% (v/v) fetal bovine serum and 1% (v/v) penicillin–streptomycin. Primary cultures of bone marrow human mesenchymal stem cells (BMSC) (donated by Dr. Brian Johnstone, OHSU Orthopedics) on passages 3 and 4 were cultured in alpha-MEM supplemented with L-glutamine, 10% (v/v) fetal bovine serum and 1% (v/v) penicillin–streptomycin. All cells were maintained in an incubator at 37 °C, 5% CO2, with media replacement every 2 days.
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