Direct Measurement of Blood Flow in Microvessels Grown in Matrigel In Vivo Carlo R. Bartoli, Ph.D.,* , † , ‡ , § ,1 Sujith Dassanayaka, M.S.,† Kenneth Brittian, B.S.,§ Arun C. Nadar,† Mohamed A. Ismahil, Ph.D.,§ Steven C. Koenig, Ph.D.,† , { ,1 and Sumanth D. Prabhu, M.D.† , ‡ , § ,1,2 *M.D./Ph.D. Program, University of Louisville School of Medicine, Louisville, Kentucky; †Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky; ‡Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky; §Department of Medicine, Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky; and {Department of Bioengineering, University of Louisville, Louisville, Kentucky Originally submitted July 16, 2011; accepted for publication September 7, 2011 Background. The Matrigel assay provides a versa- tile platform to examine vessel growth. Similarly, the microsphere method is used extensively in laboratory animals to measure tissue-specific blood flow. How- ever, microsphere models have not been used with Ma- trigel to study angiogenesis in live animals. The goal of this study was to develop a novel technique to directly measure blood flow with microspheres in vessels grown in Matrigel in vivo. Methods. In calves (n [ 10, 110 ± 5 kg), 5 mL of Matrigel was injected subcutaneously. After 10 d, a per- cutaneous cardiac catheterization was performed. Fluorescent-labeled 15 mm microspheres were injected into the left ventricular chamber to distribute throughout the body based on systemic blood flow pat- terns. Afterwards, Matrigel plugs were removed, and animals were recovered. Flow cytometry was used to count microspheres and quantify blood flow within the plug. FITC-conjugated isolectin-B4 staining was performed to quantify Matrigel capillary density. Flow cytometry was performed to quantify circulating plasma CD34 D cells. Linear regressions were used to determine relationships between Matrigel blood flow, Matrigel capillary density, and plasma CD34 D cells. Results. Over 10 d, small-caliber vessels grew into subcutaneous Matrigel plugs. Microspheres lodged throughout the plug and indicated that newly grown vessels in the Matrigel were functional and able to ac- commodate blood flow. Modest associations between Matrigel blood flow, Matrigel capillary density, and circulating plasma CD34 D cells were noted. Conclusion. This method provides a novel and cost-effective technique to measure blood flow within vessels grown in Matrigel in vivo. Ó 2012 Elsevier Inc. All rights reserved. Key Words: angiogenesis; neovascularization; Matri- gel; fluorescent microspheres; isolectin-B4; capillary density; CD34 D . INTRODUCTION Angiogenesis, the growth of new vessels from resi- dent endothelial cells, plays an important role in nu- merous (patho)physiologic processes [1]. Over the past two decades, the role of vascular proliferation in embry- ology, neoplasia, ischemia, tissue growth, wound heal- ing, and exercise physiology has generated intense interest in novel methods to study angiogenesis. To quantify angiogenesis in vivo, the Matrigel plug assay provides a versatile platform to examine new mi- crovessel growth as well as the effects of pro- and anti-angiogenic factors [1–3]. Matrigel, a basement membrane extract of the Engleberth-Holm-Swarm tu- mor, self-assembles from a liquid at 4  to a semi-solid at 37  C and thereby provides a scaffold for the prolifer- ation of vascular structures. Standard (immuno)histo- logic techniques are regarded as the gold standard to quantify microvessel density in the Matrigel plug [4, 5]. However, determination of the function of experi- mentally grown vessels is difficult, and an in vivo tech- nique to directly measure blood flow within vessels grown in Matrigel plugs has not been described. Such 1 Contributed equally to this study. 2 To whom correspondence and reprint requests should be ad- dressed at Division of Cardiovascular Disease, University of Alabama at Birmingham, 311 THT, 1900 University Boulevard, Birmingham, AL 35294-0006. E-mail: sprabhu@uab.edu. 0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved. e55 Journal of Surgical Research 172, e55–e60 (2012) doi:10.1016/j.jss.2011.09.010