A Simple and Nonenzymatic Method to Isolate Human Corpus Cavernosum Endothelial Cells and Pericytes for the Study of Erectile Dysfunction

Abstract

PurposeTo establish a simple and nonenzymatic technique to isolate endothelial cells (ECs) and pericytes from human corpus cavernosum tissue and to evaluate the angiogenic ability of the human cavernous EC or pericytes for the study of high glucose-induced angiopathy.Materials and MethodsFor primary human cavernous EC culture, cavernous tissues were implanted into Matrigel in dishes. For primary human cavernous pericyte culture, cavernous tissues were settled by gravity into dishes. We performed immunocytochemistry and Western blot to determine phenotype and morphologic changes from passage 1 to 5. The primary cultured cells were exposed to a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition, and then tube formation assay was done.ResultsWe successfully isolated high-purity EC and pericytes from human corpus cavernosum tissue. Primary cultured EC showed highly positive staining for von Willebrand factor, and pericyte revealed positive staining for NG2 and platelet-derived growth factor receptor-β. Primary cultured EC and pericytes maintained their cellular characteristics up to passage 2 or 3. However, we observed significant changes in their typical phenotype from the passage 4 and morphological characteristics from the passage 3. Human cavernous EC or pericytes formed well-organized capillary-like structures in normal-glucose condition, whereas severely impaired tube formation was detected in high-glucose condition.ConclusionsThis study provides a simple and nonenzymatic method for primary culture of human cavernous EC and pericytes. Our study will aid us to understand the pathophysiology of diabetic erectile dysfunction, and also be a valuable tool for determining the efficacy of candidate therapeutic targets.

A Method to Isolate Pericytes From the Mouse Urinary Bladder for the Study of Diabetic Bladder Dysfunction

“…1B). Similar to our previous findings for erectile tissues [20], the primary cultured cells demonstrated typical pericyte morphologic characteristics with multidirectional projections (Fig. 1B).…”

“…There is no single entirely pericyte-specific marker [ 15 ]. In the present study, therefore, we used PDGFR-β and NG2 as makers for pericytes, because these makers were useful to define pericytes from urinary bladder and erectile tissue [ 14 , 17 , 20 ].…”

“…Mouse urinary bladder pericytes were prepared and maintained as previously described for erectile tissues [ 17 , 20 ]. In brief, urinary bladder tissues were cut into several pieces (2–3 mm), and the tiny tissue fragments were settled by gravity into collagen Icoated 35-mm cell culture dishes (Becton Dickinson, Mountain View, CA, USA) ( Fig.…”

“…To evaluate the effect of glucose concentrations on the angiogenic potency of primary cultured pericytes, the cells were cultured under normal-glucose (5 mmol/L) or high-glucose (30 mmol/L) conditions for 48 hours as shown in previous studies [ 21 , 22 ]. Tube formation assay was performed as previously described [ 17 , 20 ]. The pericytes were seeded onto Growth FactorReduced (GFR) Matrigel (Becton Dickinson) at 5×10 4 cells/well, and the plates were incubated at 37°C for 6 hours in a CO2 incubator.…”

Pericytes in Cerebrovascular Diseases: An Emerging Therapeutic Target

“…Pericytes express a series of protein molecules, including α-SMA (Alarcon-Martinez et al, 2018), nestin (Muñoz-Fernández et al, 2018), vimentin (Bandopadhyay et al, 2001), NG2 (Sato et al, 2016), aminopeptidases A and N (Winkler et al, 2018), intercellular adhesion molecule-1 (ICAM-1) (Fan et al, 2019), vascular cell adhesion molecule-1 (Winkler et al, 2018), platelet-derived growth factor alpha and beta receptors (PDGFR-α and -β) (Wilson et al, 2018), CD34, CD146 (MCAM), CD4, CD11b (Wang et al, 2019), regulator of G-protein signaling 5 (RGS5) (Berger et al, 2005), stem cell antigen-1 (Kunisaki, 2019), and major histocompatibility complex classes I and II (Rink et al, 2017), without the expression of von Willebrand factor (Yin et al, 2019), platelet endothelial cell adhesion molecule (Nikolajsen et al, 2016), and glial fibrillary acidic protein (Winkler et al, 2018). Brain pericytes also express ATP-sensitive potassium channel protein Kir6.1 (Table 1; Bondjers et al, 2006).…”