ANGIOstream™

Abstract

ANGIOstream™ is a unique angiogenesis assay system that uses a proprietary human extracellular matrix (ECM) and primary endothelial cells for the in vitro capture and monitoring of the complex multi-stage, multi-step process of stable micro and large macrovessel formation.

The complete process of vessel formation is easily monitored in the dish, without system disruption, and can be interrogated using chosen substances.

Introduction

Angiogenesis is defined as the multi-step process resulting in new Blood Vessel Development from preexisting vasculature.

This process is integral to the development of many normal and pathological tissues. Endothelial cells are the keycell type involved in this process. During angiogenesis, these cells disrupt the surrounding extracellular matrix structure and migrate toward an angiogenic stimulus, where they proliferate and re-organize to initially form and stabilize a lumen [1,2], the necessary three-dimensional vessel structure. As the process continues, more mature blood vessel development occurs, involving more endothelial cells and a complex process of tissue reorganization [3].

Current in vitro assays provide only a glimpse into the early initial stages of vessel formation. In fact, the most widely used extracellular matrix structure (ECM)—obtained from Engelbreth-Holm-Swarm mouse sarcoma—inhibits endothelial cell growth [4,5] and reportedly induces other types of cells to undergo the process observed for endothelial cells, which causes the cells to undergo apoptosis soon after that process has been initiated [4].

Although following the process for a short time provides some rapid data acquisition, using a deficient system limits the opportunity to observe many important stages of this complex multistep process [4,7]. Delineating these missing stages could provide essential new insight into the process of blood vessel formation in vivo. Furthermore, the formation of stable long-lived structures is essential to monitoring the chronic exposure effects of various substances (e.g. drugs) on these biostructures relevant to vessels, either pre- or post-structure formation (e.g. in tube regression analysis) [8]. In fact, observing the effects pertinent to lower concentrations of tested substances might be only detectable using this stable assay system.

These opportunities render access to controlling this ubiquitously important process in tissue development and homeostasis. Understanding and successfully controlling this multistep process could be crucial in developing the cure to many persistent diseases such as cancer, obesity, diabetes cardiovascular disease, and ocular disease [9], while the establishment of stable blood vessels is quintessential for the prospects of engineering thick and viable artificially engineered tissues and organs for future human transplantations [10].

ANGIOstream™ is a unique assay system (US patent: US 10041037 B2) that provides the opportunity to capture and interrogate many more steps involving blood vessel formation in vitro. The process starts with the firststage, in which the proliferation and migration of the endothelial cells occurs, thus mimicking the in vivo case. The secondstage involves endothelial cells differentiating, coalescing, and forming a dense mesh of microvessels. These microvessels remain stable for several weeks. The thirdstage involves some endothelial cells collectively migrating to the liquid phase in a manner reminiscent to angiogenesis “sprouting” and exhibits further complex 3D tissue dynamics that subsequently produce suspended cords of large vessels. These vessels are thick (>15 µm diameter) and long (centimeters) and often cross the cell culture well from one side to the other. The process continues for weeks with the formation of additional large macrovessels and the reorganization of tissue, thus producing even thicker vessels, some of which are visible to the naked eye.

The complex 3D vessel formation process is easy to follow microscopically, as it proceeds in the liquid transparent space of the cell culture well. The entire process involves cells that have easy and immediate access to nutrients and interrogating substances.

One key aspect ofour assay is the use of the unique human extracellular matrixmanufactured by SMSbiotechInc. using a process covered by Patent: US 10041037 B2.

The assay system does not require cells other than normal primary human umbilical vascular endothelial cells (HUVECs). Notably, it is preferable to use pooled HUVEC when averaging out individual donor variations is required.

The assay involves the convenient use of ECM pre-coated flat-bottomed wells (6- or 24- well plate) that can be stored for months in the freezer at -20°C.

Primary HUVECs are then simply added to the wells. Given that the cells actively proliferate in the matrix, variable amounts of cells can be used. Cells sink into the ECM, become flat, spread, and start to migrate and proliferate. Gross changes in the structure of individual endothelial cells occur, and cells reorganize to collectively form a microvessel mesh within days. Tissue remodeling can be observed after one week. Suspended large vessels are formed in 2-3 weeks. Vessel presence and formation continue for months.

KeyAssayFeatures

• Uses human ECM pre-coated in a cell culture plate for immediate use.
• Produces highly stable (for up to months) micro- and macrovessels.
• Exhibits a unique and complex multi-stage, multi-step vessel formation process.

KeyAssayAdvantages

✓ Human bio-matrix biocompatible with endothelial cell migration and proliferation.
✓ Captures the two stages of microvessel and that of macrovessel formation.
✓ Ability to interrogate a multi-step complex vessel-forming process.
✓ Ability to examine acute and chronic effects of substances on the formation and regression of vessel formation.
✓ Simple to use matrix pre-coated plates, practically ready for use.
✓ Does not require the use of any cells other than endothelial cells.
✓ Does not require the use of large amounts of cells.
✓ The complete process, including tissue remodeling, is accessible for microscopic observation and does notrequire tissue disruption.

Figure 1: Human endothelial cells (HUVEC) immersed and spread across the ECM matrix, actively moving and proliferating within hours after application. Cell confluence is achieved in a few days. Magnification 100x.

Figure 2: Human endothelial cells (HUVEC) undergo gross shape changes, forming large vacuoles, coalescing, connecting, and forming a web of microvessels with typical tubular structures within the ECM matrix layer within days. Magnification 100x.

figure 2

Figure 3: Human endothelial cells (HUVEC) exhibiting obvious gross shape changes conducive to vessel formation, such as flattening and forming different sizes of vacuoles (including large vacuoles) within days. Arrows point to formed vacuoles. Magnification 400x.

figure 3

Figure 4: Human endothelial cell (HUVEC) microvessel mesh was isolated, fixed, sectioned, and stained using the Mason trichrome staining procedure. A microtubular network emerged. Magnification 100x.

figure 4

Figure 5: Endothelial cells (HUVEC) “sprouting” upward from the endothelial tissue/ECM matrix surface to the edges of the well, which is the initial event usually preceding tissue detachment and remodeling. Arrow points to one sprout among four sprouts present in the image. After one week. Magnification 200x.

figure 5

Figure 6: Endothelial cells (HUVEC) forming a tissue structure and exhibiting a specific sequence of targeted 3D dynamic remodeling after one week. Right arrows (2) point to suspended, partially detached tissue. Left arrows (3) point to the attachment sites of the suspended tissue. Magnification 100x.

Figure 7: Tissue dynamics forming a suspended macrovessel distant to the ECM matrix and microvessel layer. Suspended macrovessel is attached to the bottom (see arrow) and side of the well. After 2-3 weeks. Magnification 100x.

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Figure 8: A suspended macrovessel (diameter: ~10 μm), distant from the matrix and the microvessel layer, gradually becoming thicker. Vessel bifurcations are common. Arrow points to the attachment site at the well wall. Magnification 100x.

figure 8

Figure 9: A suspended macrovessel, distant from the matrix and the small vessel layer, became thicker (diameter: 25-45 μm) and its surface became quite smooth. Magnification 40x. Suspended vessels are stable for months.

figure 9

References

1- Davis GE, Bayless KJ, Mavila A. (2002) Molecular basis of endothelial cell morphogenesis in three-dimensional extracellular matrices. Anat Rec. Nov 1;268(3):252-75.

2-Davis GE, Stratman AN, Sacharidou A, Koh W. (2011) Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting. Int Rev Cell Mol Biol.; 288: 101–165.

3- Davis GE, Senger DR. (2005) Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ Res. Nov 25;97(11):1093-107.

4- Ngo TX, Nagamori E, Shimizu T, Okano T, Taya M, Kino-Oka M. (2014) In Vitro Models for Angiogenesis Research: A Review, International Journal of Tissue Regeneration, Vol. 5, No. 2, pp 37-45.

5- Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. (2003) Angiogenesis assays: a critical overview. Clin Chem. Jan;49(1):32-40.

6- Staton CA, Reed MWR, Brown NJ. (2009) A critical analysis of current in vitro and in vivo angiogenesis assays. Int J Exp Pathol. Jun; 90(3): 195–221.

7- Staton CA, Stribbling SM, Tazzyman S, Hughes R, Brown NJ, Lewis CE. (2004) Current methods for assaying angiogenesis in vitro and in vivo. Int J Exp Pathol. Oct; 85(5): 233–248.

 

8-Vailhé B, Vittet D, Feige JJ. (2001) In Vitro Models of Vasculogenesis and Angiogenesis. Laboratory Investigation 81(4):439-52, DOI: 10.1038/labinvest.3780252.

9- Tahergorabi Z1, Khazaei M. (2012) A review on angiogenesis and its assays. Iran J Basic Med Sci. Nov;15(6):1110-26.

10- Rivron NC, Liu J J, Rouwkema J, de Boer J, van Blitterswijk CA. (2008) Engineering vascularised tissues in vitro. Eur Cell Mater. Feb 21; 15:27-40.

SMSbiotechInc. 1825 Diamond str. STE101, San Marcos, CA, 92078, USA. Tel: (760) 290 3406



ANGIOstream™ is a product of SMSbiotech inc.