Culture of murine aortic explants in 3-dimensional extracellular matrix: a novel, miniaturized assay of angiogenesis in vitro

Abstract

Assays of angiogenesis in vitro are critical to the study of vascular morphogenesis and to the evaluation of therapeutic compounds that promote or inhibit vascular growth. Culture of explanted aortic segments from rats or mice in a 3-dimensional extracellular matrix (ECM) is one of the most effective ways to generate capillary-like endothelial sprouts in vitro. We have modified the classic aortic explant model by placing the aortic segments from mice within small (5.6 mm diameter, 30 microl volume) lenticular hydrogels of type I collagen supported at the edge by nylon mesh rings. This method of culture, referred to as the "miniature ring-supported gel" (MRSG) assay, optimizes handling, cytological staining, and conventional imaging of the specimen and permits use of minimal volumes of reagents in a 96-well tissue culture format. We have used the MRSG assay to quantify the impaired angiogenic response of aged mice relative to young mice and to show that aged mice have significantly decreased sprout formation, but have similar levels of invasion of vascular smooth muscle cells into the supportive ECM. The MRSG assay, which combines low volume, physically robust gels in conjunction with mouse aortic segments, may prove to be a highly useful tool in studies of the process and control of vascular growth.

Figure 1

Depiction of the MRSG angiogenesis assay. A) Diagram showing an oblique view of an MRSG assembly comprised of a lenticular collagen gel, supportive Nitex mesh ring, and mouse aortic segment. B) View of an actual MRSG preparation after 7 days of culture. Microvascular sprouts are visible within the collagen gel.

Figure 2

Aortic segments from young mice show significantly more sprouting than segments from aged mice in MRSG assays. A,B) Representative aortic segments (AS) cultured 7 days and viewed by phase contrast. Sprouts (Sp) from the young segment (panel A) are significantly longer and more branched than are sprouts from the aged segment (panel B). In A and B, scale bars = 250μm. This trend is represented quantitatively in panel C (vertical bars = standard deviations; asterisk = significance, p = 0.017, n = 4 for both young and aged aortic segments).

Figure 3

Immunofluorescent imaging of ECs and supportive cells in sprouts from an aged mouse aortic segment. A sprout (Sp) from the segment bifurcates into 2 branches. ECs are labeled for CD31 (red) and supporting cells are labeled for SM actin (green). Cell nuclei are stained with DAPI (blue). Inset shows a portion of a branch (rectangle) that is enlarged. Green SM actin-positive cells (arrowheads) form an incomplete layer on the outside of the red CD31-positive ECs. Scale bar = 200μm.

Figure 4

Outgrowth of SM actin-positive cells from young and aged aortic segments is similar. A) Sprout (Sp) from an aged aortic segment exhibits substantial numbers of SM actin-positive cells (green). Additionally, many SM actin-positive cells have migrated as single cells into the collagen gel (arrowheads). Cell nuclei are labeled with DAPI. The average number of SM actin-positive cells per 10X field that had invaded the collagen and were not in sprouts was similar in aortic segments from aged mice as that of segments from young mice, as represented quantitatively in panel B (vertical bars = standard error; p=>0.05, n = 4 for young and n = 5 for aged aortic segments). In A, scale bar = 100μm.