IDENTIFICATION OF CD90 POSITIVE AND NEGATIVE CELLS AND ELIMINATION OF CONTAMINATING FIBROBLASTS FROM HUMAN PRIMARY CELL CULTURES

Abstract

Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. This study was designed to identify expression of CD90 on a panel of human primary cells by flow cytometry. Human Corneal Epithelial Cells (CEC), Human Retinal Pigmented Epithelial (RPE) cells, Normal Human Epidermal Keratinocytes (NHEK), Normal Human Bronchial Epithelial (NHBE) cells, Human Umbilical Vein Endothelial Cells (HUVEC), Uterine Smooth Muscle Cells (UtSMC), and Pulmonary Artery Smooth Muscle Cells (PASMC) were analyzed for CD90 expression. CD90 was positively expressed on UtSMC. Non-activated HUVEC, NHEK, NHBE and PASMC were negative for CD90 expression. RPE had varying levels of CD90 expression and were neither positive nor negative. CEC were mixed for CD90 positivity, due to fibroblast contaminants in the culture. Three methods were tested to find an efficient and reliable means of eliminating unwanted or contaminating fibroblasts from human primary cell cultures including: Trypsin-EDTA selective detachment, growth medium with and without fetal bovine serum, and cell separation using anti-CD90 monoclonal antibody (MAb) bound to magnetic beads. The magnetic bead cell separation method provided the most reliable results, yielding clean, fibroblast- free cultures. The goal of this thesis was to identify CD90 as a human primary cell biomarker and to find an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.