Product Name 3D Human HUVECs AngiogenesisCatalog EP009Product Format 6, 12, and 24 wellStorage 37°C
GENERAL INFORMATIONOur 3D Human HUVECs Angiogenesis model is construct using GFP- Tagged human umbilicalvein endothelial cells (HUVECs) are co-cultured with RFP- Tagged supporting cells. GFPpositive capillary like tubule formation can be monitored in real time under fluorescencemicroscope throughout the whole process of the experiment.Advantages:1) Cells used in the 3D model are all human cells; results obtained are more relevant tohuman 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 ofexperiment), therefore, more crucial information can be acquired at multiple time pointsfrom a single experiment.3) No need to perform post-experimental staining for endothelial markers, this isparticularly important, if those markers are changed in experimental conditions involvedin the studies.The 3D Human HUVECs Angiogenesis contains all of the materials necessary to performmultiple angiogenesis assays in 6, 12, or 24 well formats. The 3D model is designed that thetesting materials, i.e. compounds, conditioned media, or tissue explants, can be added into thesystem at any time, ranging from the onset of vasculogenesis to advanced angiogenesis. Theresulting effect on tubule formation (tubular length, number of branches et al) can be monitoredthroughout the whole process under inverted fluorescence microscope.
Introduction:Angiogenesis is a multistep process whereby new blood vessels develop from preexistingvasculature. Angiogenesis plays a key role in numerous physiological and pathological processesand understanding the mechanism of angiogenesis will therefore provide new approaches to thetreatment of a wide range of pathologies. Angiogenesis is a complex process in which thefollowing 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 vesselsThe 3D Human HUVECs Angiogenesis Kit series products are the proprietary systems ofAlphabioregen in which GFP-tagged human endothelial cells from variable vascular beds are cocultured with RFP-tagged human supporting cells in a specially designed medium. Theendothelial cells initially form small islands within the culture matrix. They subsequently beginto proliferate and then enter a migratory phase during which they move through the matrix toform threadlike tubule structures with lumens. They gradually join up (by 1 – 2 weeks) to form anetwork 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 mesenchymalsupporting cells (-80°C or liquid N2).• 1 x 24-well AlphaBio coated plate (Room temperature) (3) 1 x 500ml of Endo-GrowthMedium (4°C).
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)
Day 2:• Take the plate from the incubator and examine cells under inverted fluorescencemicroscopy (GFP positive HUVECs should sparsely and evenly distributed amongRFP 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. Replacethe medium every 2 days until the end of the experiments.Recommended Coating VolumeWell Well Area (cm²) Coating Volume (ml)6 4.67 0.612 1.2 0.2524 0.33 0.05