Product Name 3D Human RetinalMicrovascular AngiogenesisCatalog Number EP008Product Format 6, 12, and 24 wellStorage 37°CThis product is commercially produced for Research Use Only, and under no circumstances that the product can be used for anyhuman applications.GENERAL INFORMATIONRetinal microvascular morphogenesis is a complex and highly coordinated process, whichoccurs during embryonic development, post-natally and in association with severalvisually-impairing diseases, including retinopathy of prematurity (ROP), age-relatedmacular degeneration and diabetic retinopathy.Retinal vascular formation is a complex process that requires a precise temporospatialregulation of various elements, including the growth factors. Insulin-like growth factor-1(IGF-1) and vascular endothelial growth factor (VEGF) are ligands of specific receptortyrosine kinases (RTKs), which, if activated, will initiate downstream pathways thatultimately promote cell survival, cell proliferation, vascular permeability and cellmigration; all of which permit blood vessel development. Formation of blood vessels frompre-existing vessels may be normal (angiogenesis) or pathological (neovascularization)processes.Our 3D Human Retinal Microvascular Angiogenesis model is constructed using GFPTagged human Retina Microvascular Endothelial cells. They are co-cultured with RFPTagged human supporting cells. GFP positive human retinal capillary like tubule formationcan be monitored in real time under fluorescence microscope throughout the wholeprocess 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 endof experiment) therefore, more crucial information can be acquired at multiple timepoints from a single experiment.3) No need to perform post-experimental staining for endothelial markers, this isparticularly important, if those markers are changed in experimental conditionsinvolved in the studies.The 3D Human Retina Microvascular Angiogenesis model contains all of the materialsnecessary to perform multiple angiogenesis assays in 6, 12, or 24 well formats. The 3Dmodel is designed that the testing materials, i.e. compounds, conditioned media, or tissueexplants, can be added into the system at any time, ranging from the onset ofvasculogenesis to advanced angiogenesis. The resulting effect on tubule formation (tubularlength, number of branches et al) can be monitored throughout the whole process underinverted fluorescence microscope.Introduction:Angiogenesis is a multistep process whereby new blood vessels develop from preexistingvasculature. Angiogenesis plays a key role in numerous physiological and pathologicalprocesses and understanding the mechanism of angiogenesis will therefore provide newapproaches to the treatment of a wide range of pathologies. Angiogenesis is a complexprocess 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 Retinal Microvascular Angiogenesis Kit series products fromAlphabioregen is a proprietary system in which GFP-tagged human endothelial cells fromvariable vascular beds are co-cultured with RFP-tagged human supporting cells in aspecially designed medium. The endothelial cells initially form small islands within theculture matrix. They subsequently begin to proliferate and then enter a migratory phaseduring which they move through the matrix to form threadlike tubule structures withlumens. They gradually join up (by 1 – 2 weeks) to form a network of anastomosing tubules
which closely resembles the capillary bed found in vivo. 3D Human Retinal MicrovascularAngiogenesis Kit contains all of the materials necessary to perform multiple angiogenesisassays in a 24-well format. The kit is designed so that the testing materials, i.e. compounds,conditioned media, or tissue explants, can be added into the system at any time, rangingfrom the onset of vasculogenesis to advanced angiogenesis. The resulting effect on tubuleformation (tubular length, number of branches et al) can be monitored throughout thewhole process under inverted fluorescence microscope.Reagents and Materials Provided:(1) 1 x vial of mixture of GFP-tagged Human Retina Microvascular ECs and RFP-taggedhuman supporting cells (-80°C or liquid N2)(2) 1 x 24-well AlphaBio coated plate (Room temperature, for 2 months) (3) 1 x 500ml ofEndo-Growth Medium (4°C)Protocols: Day 11. Pre warm Endo-Growth Medium to 37ºC in a water bath2. Accurately pipette 24ml Endo-Growth Medium into a 50ml Falcon tube3. Rapidly thaw the vial of cryopreserved cells in a 37ºC water bath4. Transfer all cells gently into 24ml pre warmed Endo-growth medium5. Mix well the cells gently using a serological pipette6. Add 1.0ml of cell suspension to each well of the pre coated 24-well plate.7. Make sure the cells are evenly dispersed in the wells.8. Place the plate in an incubator (37ºC, 5% CO2 and humidifiedDay 29. Take the plate from the incubator and examine cells under inverted fluorescencemicroscopy (GFP positive Human Retina Microvascular ECs should sparsely and evenlydistributed among RFP positive human mesenchymal supporting cells).10. Wash the cells once with 2 ml of PBS11. 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).12. 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.