SELECTIVE PROPAGATION OF RETINAL PERICYTES IN MIXED MICROVASCULAR CELL-CULTURES USING L-LEUCINE-METHYL ESTER

Endothelial cell (EC) propagation has been simplified by developing ce ll-specific selection criteria. Methods commonly used for selectively isolating EC include: (i) differential sieving of disaggregated tissue , (ii) differential plating of cells on extracellular matrices (iii) l ectin affinity isolation of cell populations and (iv) fluorescence-act ivated cell sorting of cells labeled with a carbocyanine dye of acetyl ated low-density lipoprotein (DiI-Ac-LDL). Few criteria for selectivel y propagating pericytes (PC) are currently available. Nonspecific este rases exhibit a high degree of multiplicity when compared with other m ammalian isozymes and may be suitable for the identification and selec tive propagation of cells of the microvasculature. Evaluation of ester ase isotype expression in PC and EC by zymography indicates PC contain alpha-naphthyl acetate and alpha-naphthyl butyrate hydrolyzing estera ses as well as dipeptidylpeptidase I, while EC only contain alpha-naph thyl acetate esterase. The cytotoxic response of PC and EC to various amino acid esters is assessed by monitoring viral dye uptake and by li ght microscopy. Several amino acid esters are cytotoxic to both cell t ypes, whereas 50 mM L-leucine methyl ester (L-Leu OMe) is toxic to EC but not to PC. This amino acid ester is also toxic to mesothelial and retinal pigmented epithelial cells, other common contaminants of PC cu ltures. Analysis of protein composition by two-dimensional gel electro phoresis indicates that L-Leu OMe does not stimulate expression of str ess response proteins in PC. Thus, L-Leu OMe can be utilized to cultiv ate PC selectively from mixed cell populations.