Product Name 3D Human Retinal Microvascular Angiogenesis
Catalog Number EP008
Product Format 6, 12, and 24 well
This product is commercially produced for Research Use Only, and under no circumstances that the product can be used for any
Retinal microvascular morphogenesis is a complex and highly coordinated process, which
occurs during embryonic development, post-natally and in association with several
visually-impairing diseases, including retinopathy of prematurity (ROP), age-related
macular degeneration and diabetic retinopathy.
Retinal vascular formation is a complex process that requires a precise temporospatial
which closely resembles the capillary bed found in vivo. 3D Human Retinal Microvascular
Angiogenesis Kit contains all of the materials necessary to perform multiple angiogenesis
assays 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, 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.
Reagents and Materials Provided:
(1) 1 x vial of mixture of GFP-tagged Human Retina Microvascular ECs and RFP-tagged
human supporting cells (-80°C or liquid N2)
(2) 1 x 24-well AlphaBio coated plate (Room temperature, for 2 months) (3) 1 x 500ml of
Endo-Growth Medium (4°C)
Protocols: Day 1
1. Pre warm Endo-Growth Medium to 37ºC in a water bath
2. Accurately pipette 24ml Endo-Growth Medium into a 50ml Falcon tube
3. Rapidly thaw the vial of cryopreserved cells in a 37ºC water bath
4. Transfer all cells gently into 24ml pre warmed Endo-growth medium
5. Mix well the cells gently using a serological pipette
6. 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 humidified
9. Take the plate from the incubator and examine cells under inverted fluorescence
microscopy (GFP positive Human Retina Microvascular ECs should sparsely and evenly
distributed among RFP positive human mesenchymal supporting cells).
10. Wash the cells once with 2 ml of PBS
11. 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.
regulation of various elements, including the growth factors. Insulin-like growth factor-1
(IGF-1) and vascular endothelial growth factor (VEGF) are ligands of specific receptor
tyrosine kinases (RTKs), which, if activated, will initiate downstream pathways that
ultimately promote cell survival, cell proliferation, vascular permeability and cell
migration; all of which permit blood vessel development. Formation of blood vessels from
pre-existing vessels may be normal (angiogenesis) or pathological (neovascularization)
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 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 Retina Microvascular Angiogenesis model 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 Retinal Microvascular Angiogenesis Kit series products from
Alphabioregen is a proprietary system in which GFP-tagged human endothelial cells from
variable vascular beds are co-cultured 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