Materials used for fabrication of the culture chamber were obtained from the following commercial sources: glass capillary (diameter, 600 μm; length, 3.2 cm) from Hirschmann Laborgeräte, Germany; poly (methyl methacrylate) plates from Astra products, USA. Human umbilical vein endothelial cells (HUVECs, CC-2517A), human neonatal dermal lymphatic microvascular endothelial cells (HMVEC-dLy-Neo, CC-2812), endothelial basal medium (EBM-2, CC-3156) and SingleQuots growth supplement (CC-4176) from Lonza, Switzerland; type I collagen and 10x Ham’s F12 medium (Cellmatrix Type I-A) from Nitta Gelatin, Japan; vascular endothelial growth factor-A (VEGF-A) and VEGF-C from PeproTech, USA; ZM306416, Cabozantinib and SAR131675 from selleckchem, USA; MMP-2/MMMp-9 inhibitor (Abcam, ab145190); All other chemicals were purchased from Sigma, USA unless otherwise indicated.
Hmvec dly neo
Manufactured by Lonza
Sourced in Switzerland
HMVEC-dLy-neo is a type of cell line derived from human microvascular endothelial cells from the dermis. It is designed for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ebm 2, by Lonza (2 mentions)
Cc 2812, by Lonza (1 mentions)
Cc 2517a, by Lonza (1 mentions)
Singlequots growth supplement cc 4176, by Lonza (1 mentions)
Sar131675, by Selleck Chemicals (1 mentions)
Cc 3156, by Lonza (1 mentions)
Matrigel, by BD (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Collagenase a, by Roche (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Collagen type 1, by Advanced BioMatrix (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Egmtm 2 singlequots, by Lonza (1 mentions)
5 protocols using hmvec dly neo
1
Fabrication of Angiogenesis Culture Chamber
2
Lymphatic Vascular Tubulogenesis Assay
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Basement membrane matrix (Matrigel®, BD Biosciences) was added to 2-well chamber slides and solidified by incubation at 37 °C for 30 minutes. hPSC-derived LYVE-1+PODOPLANIN+cells (2 × 104) were labeled with CM-DiI, plated with hLECs (HMVEC-dLy-neo, 1 × 105, Lonza) onto Matrigel, and cultured under α-MEM medium containing 10% FBS, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol, 4 ng/mL of FGF2, 10 ng/ml VEGF-A, 20 ng/mL of VEGF-C, and 10 ng/ml EGF, and incubated at 37 °C for 12 h. After removing the medium, 4% PFA was added for fixation. The tube structures were evaluated by microscopy.
3
Therapeutic Potential of hPSC-derived Lymphatic Cells
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hPSC-derived LYVE-1+PODOPLANIN+cells were isolated using magnetic columns (MACS®, Milteny Biotec). Full-thickness excisional skin wounds (8 mm) were created on the backs of male athymic nude mice, and PBS (100 μl), LYVE-1+PODOPLANIN+cells (1 × 105) or hLECs (HMVEC-dLy-neo, Lonza) labeled with DiI were injected into the wound bed around the wound. The wound tissues, including a perimeter of 1 to 2 mm of normal skin tissue, were harvested for immunohistochemistry at day 4, 7, or 14 after the injection. Wound closure was monitored at days 0, 2, 4, 6, 8 and 12 and calculation of the area of the wound was performed with the NIH Image J analyzer.
4
Isolation and Characterization of Lymphatic Endothelial Cells
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Human microvascular endothelial cell–dermal lymphatic–neonatal (HMVEC–dLyNeo) cells, and the media optimized for their growth (EBM-2), were purchased from Lonza (Euroclone SpA). These cells have been characterized as previously described [8 (link)] and reported in Supplementary Figure S1. Mouse lymphangioma-derived endothelial cells (LAECs) were isolated and immortalized following the procedure described previously [26 (link)]. Briefly, the mice were injected twice intraperitoneally, with a 15-day interval, with 200 μl of emulsified (1:1 with PBS) incomplete Freund's adjuvant (Sigma). Hyperplastic vessels were isolated from the liver and diaphragm at day 30 and treated with 0.5 mg/ml of collagenase A (Roche Diagnostics), and the resulting single-cell suspension was cultured. After 7–10 days of culture, subconfluent cells (LAECs) were recovered with trypsin/EDTA and immortalized by means of SV40 infection. Immortalized LAECs were characterized for lymphatic endothelial markers as reported previously by us [27 (link)].
5
Culturing Human Neonatal and Juvenile Dermal Lymphatic Endothelial Cells
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Single donor human neonatal dermal lymphatic microvascular endothelial cells (ndLECs) were obtained from Lonza (Visp, Switzerland, catalogue #CC-2812; HMVEC-dLyNeo) and cultured in complete EBM-2 medium consisting of EBM-2 basal Medium (CC-3156, Lonza) supplemented with EGM-2 MV Single QuotsTM (CC-4147, Lonza). Cell culture dishes were pre-coated with 10μg/ml collagen type 1 (Advanced Biomatrix) and 10μg/ml fibronectin (Merck Millipore) in PBS.
Juvenile skin LECs (jdLECs) from the foreskin of a single male donor (Lot Nr. 431Z006.2) were obtained from Promocell (Heidelberg, Germany, # C-12216) and cultured in complete EBM-2 (CC-3156, Lonza) supplemented with EGMTM-2 SingleQuotsTM (CC-4176, Lonza), without VEGF-A (CC-4176, Lonza), additionally supplemented with (3%) FBS for a final concentration of 5% FBS (Gibco), as used for ndLECs. Cell culture dishes were pre-coated with 10μg/ml collagen type 1 (Advanced Biomatrix, San Diego, CA, USA) and 10μg/ml fibronectin (Merck Millipore) in PBS.
Juvenile skin LECs (jdLECs) from the foreskin of a single male donor (Lot Nr. 431Z006.2) were obtained from Promocell (Heidelberg, Germany, # C-12216) and cultured in complete EBM-2 (CC-3156, Lonza) supplemented with EGMTM-2 SingleQuotsTM (CC-4176, Lonza), without VEGF-A (CC-4176, Lonza), additionally supplemented with (3%) FBS for a final concentration of 5% FBS (Gibco), as used for ndLECs. Cell culture dishes were pre-coated with 10μg/ml collagen type 1 (Advanced Biomatrix, San Diego, CA, USA) and 10μg/ml fibronectin (Merck Millipore) in PBS.
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