Product Name 3D Human HUVECs Angiogenesis
Product Format 6, 12, and 24 well
Our 3D Human HUVECs Angiogenesis model is construct using GFP- Tagged human umbilical
vein endothelial cells (HUVECs) are co-cultured with RFP- Tagged supporting cells. GFP
positive capillary like tubule formation can be monitored in real time under fluorescence
microscope throughout the whole process of the experiment.
1) Cells used in the 3D model are all human cells; results obtained are more relevant to
human situations rather than those data from animal models, i.e. CAM et al.
2) The whole angiogenesis process can be monitored (from cell inoculation to the end of
experiment), therefore, more crucial information can be acquired at multiple time points
from a single experiment.
3) No need to perform post-experimental staining for endothelial markers, this is
particularly important, if those markers are changed in experimental conditions involved
in the studies.
The 3D Human HUVECs Angiogenesis contains all of the materials necessary to perform
multiple angiogenesis assays in 6, 12, or 24 well formats. The 3D model is designed that the
testing materials, i.e. compounds, conditioned media, or tissue explants, can be added into the
system at any time, ranging from the onset of vasculogenesis to advanced angiogenesis. The
resulting effect on tubule formation (tubular length, number of branches et al) can be monitored
throughout the whole process under inverted fluorescence microscope.
Angiogenesis is a multistep process whereby new blood vessels develop from preexisting
vasculature. Angiogenesis plays a key role in numerous physiological and pathological processes
and understanding the mechanism of angiogenesis will therefore provide new approaches to the
treatment of a wide range of pathologies. Angiogenesis is a complex process in which the
following events are believed to play a critical role:
• Proteolytic degradation of the extracellular matrix
• Directed migration of endothelial cells
• Proliferation of endothelial cells
• Deposition of new extracellular matrix
• Formation of tubules and anastomosis of the newly formed vessels
The 3D Human HUVECs Angiogenesis Kit series products are the proprietary systems of
Alphabioregen in which GFP-tagged human endothelial cells from variable vascular beds are cocultured with RFP-tagged human supporting cells in a specially designed medium. The
endothelial cells initially form small islands within the culture matrix. They subsequently begin
to proliferate and then enter a migratory phase during which they move through the matrix to
form threadlike tubule structures with lumens. They gradually join up (by 1 – 2 weeks) to form a
network of anastomosing tubules, which closely resembles the capillary bed found in vivo.
Reagents and Materials Provided:
• 1 x vial of mixture of GFP-tagged HUVECs and RFP-tagged human mesenchymal
supporting cells (-80°C or liquid N2).
• 1 x 24-well AlphaBio coated plate (Room temperature) (3) 1 x 500ml of Endo-Growth
Protocol Day 1:
• Pre warm Endo-Growth Medium to 37ºC in a water bath
• Accurately pipette 24ml Endo-Growth Medium into a 50ml Falcon tube
• Rapidly thaw the vial of cryopreserved cells in a 37ºC water bath
• Transfer all cells gently into 24ml pre warmed Endo-growth medium
• Mix well the cells gently using a serological pipette
• Add 1.0ml of cell suspension to each well of the pre coated 24-well plate
• Make sure the cells are evenly dispersed in the wells
• Place the plate in an incubator (37ºC, 5% CO2 and humidified)
• Take the plate from the incubator and examine cells under inverted fluorescence
microscopy (GFP positive HUVECs should sparsely and evenly distributed among
RFP positive human supporting cells)
• Wash the cells one with 2 ml of PBS
• Add 2.0ml of fresh Endo-Growth medium (control) or Experimental media (EndoGrowth medium, plus pro- or anti-angiogenic regents according to customer’s needs)
• Place the plate back into the incubator. Day 4, 6, 8, 10, 12, and 14…… 13. Replace
the medium every 2 days until the end of the experiments.
Recommended Coating Volume
Well Well Area (cm²) Coating Volume (ml)
6 4.67 0.6
12 1.2 0.25
24 0.33 0.05