A simple method for the isolation, purification and cultivation of retinal microvascular pericytes from rat

Abstract

Purpose: to establish a simple method for isolation, purification and cultivation of primary retinal microvascular pericytes(RMPs)from a readily available tissue source, rat, and to facilitate the study of their properties in vitro.

Methods: Retinas were isolated from weanling rats by ophthalmic microscopic surgical instruments followed by mechanical morcel and collagenase digestion. After homogeneous retinal microvascular fragments of 50-100μm were obtained from the digested retinas by filtered through nylon sieve, they were incubated in Dulbecco's modified Eagle's medium with 20% fetal b

Results: Migrating cells emerged from microvascular fragments after 24-48 hours of plating and formed loose colonies by 1 week. And they formed a pro-confluent multilayer on day 14-16. The subcultures grew faster and reached confluence on day 12-14. The cultured cells and their subpassages showed typical pericyte morphology with irregular triangular cell bodies and multiple long processes. They uniformly expressed the cellular markersα-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-β(PDGFR-β), nerve/glial antigen 2 (NG2) and desmin, but lack of markers stained positive for von Willebrand’s factor(vWF), glutamine synthetase (GS) and glial fibrillary acidic protein(GFAP).The purity of cultured cells was up to 99% demonstrated by positive double staining for α-SMA and PDGFR-β. The cells can be cryopreserved, recultured and passaged repeatedly for 7 passages without a significant loss in expression of pericyte markers and for 9 passages without loss of typical features.

Conclusions: These results indicate that the isolated cells were RMPs showing both morphologic and functional characteristics of pericytes. Rat RMPs can be obtained easily by our method. Here we establish a simple and convenient method for the primary culture of rat RMPs, which would be more helpful to identify their roles the pathogenesis of various retinal angiogenic diseases such as diabetic retinopathy.

Keywords: 694 retinal culture • 691 retina: proximal (bipolar, amacrine, and ganglion cells) • 698 retinal development