Spheroid-based angiogenesis assays for the analysis of pro- and anti-angiogenic factors

Spheroids are utilized as size - and cell number-defined focal delivery devices of quiescent ECs in various in vitro assays. For instance, embedding of the spheroids in 3D hydrogels and stimulation with growth factors results in angiogenic sprouting and network formation. Likewise, spheroids generated from VSMCs also support cellular quiescence and differentiation and are utilized for similar assays.

The figures below show examples of the formation of lumenized sprouts originating from collagen-embedded EC spheroids. 

_{Outgrowing sprouts of neighbouring spheroids grow directionally towards each other to establish networks of anastomosing capillary-like structures (A. phase contrast microscopy). Cross-sections of collagen gels embedded in paraffin show capillary-like, lumenized structures originating from EC spheroids (B-E). Depending on the plain of section, sprouting EC originating from lumenized capillary sprouts can be identified (D. arrow). HUVEC spheroids embedded in collagen gels and stimulated with 50 ng/ml FGF-2 (E) or VEGF (F) from branching capillary-like sprouts originating from the spheroid body and invade the collagen matrix within 24-48 hours. The “tip” cells extend filopodia into the collagen gel (E, F, scale bar: 100 µm). Paraffin serial sections of sprouts, originating from collagen-embedded spheroids, revealed that capillary-like sprouts form continuous lumen (G-L, scale bar: 20 µm) dividing at the branching points (H, I). Time-lapse videos (20 hours) showing the EC sprouting in detail are available for control-, VEGF (25 ng/ml)- and VEGF (50 ng/ml)-stimulated spheroids.} 

 

The number and length of the sprouts originating from one EC spheroid (determined as the cumulative sprout length [CSL]) indicate the level of a pro-angiogenic stimulation and are utilized as a basic parameter to quantify angiogenic activation of ECs. Consequently, spheroid-based EC sprouting can be exploited to screen compounds for their anti- or pro-angiogenic efficacy.

_{Analysis of capillary sprouting of endothelial cells from different origin. Collagen gel-embedded spheroids of different endothelial cell types were stimulated with 25 ng/ml VEGF for 24 hours (A, *p<0.05 vs. corresponding controls; CSL – cumulative sprout length). Representative spheroids of HMVEC are shown (B, C, scale bar: 200 µm). Collagen-embedded spheroids of HUVEC were stimulated with increasing doses of VEGF for 24 hours (D, *p<0.05 vs. control). Mean CSL was calculated for 10 randomly selected spheroids per experimental group.}

 

_{Analyzing the effects of the anti-angiogenic compounds SU5402 and SU5614. The kinase inhibitors SU5402 (10 µM) and SU5614 (10 µM) were analyzed for their capacity to inhibit VEGF-induced (25 ng/ml, 24 h) HUVEC sprouting (A, *p<0.05 vs. control, #p<0.05 vs. VEGF). Representative spheroids are shown (F-H, scale bar: 100 µm). Mean CSL was calculated for 10 randomly selected spheroids per experimental group.}