Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.
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ABSTRACT:
SUBMITTER: Keller KE
PROVIDER: S-EPMC6042513 | biostudies-literature | 2018 Jun
REPOSITORIES: biostudies-literature
Publications
Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.
Keller Kate E KE Bhattacharya Sanjoy K SK Borrás Theresa T Brunner Thomas M TM Chansangpetch Sunee S Clark Abbott F AF Dismuke W Michael WM Du Yiqin Y Elliott Michael H MH Ethier C Ross CR Faralli Jennifer A JA Freddo Thomas F TF Fuchshofer Rudolf R Giovingo Michael M Gong Haiyan H Gonzalez Pedro P Huang Alex A Johnstone Murray A MA Kaufman Paul L PL Kelley Mary J MJ Knepper Paul A PA Kopczynski Casey C CC Kuchtey John G JG Kuchtey Rachel W RW Kuehn Markus H MH Lieberman Raquel L RL Lin Shan C SC Liton Paloma P Liu Yutao Y Lütjen-Drecoll Elke E Mao Weiming W Masis-Solano Marisse M McDonnell Fiona F McDowell Colleen M CM Overby Darryl R DR Pattabiraman Padmanabhan P PP Raghunathan Vijay K VK Rao P Vasanth PV Rhee Douglas J DJ Chowdhury Uttio Roy UR Russell Paul P Samples John R JR Schwartz Donald D Stubbs Evan B EB Tamm Ernst R ER Tan James C JC Toris Carol B CB Torrejon Karen Y KY Vranka Janice A JA Wirtz Mary K MK Yorio Thomas T Zhang Jie J Zode Gulab S GS Fautsch Michael P MP Peters Donna M DM Acott Ted S TS Stamer W Daniel WD
Experimental eye research 20180309
Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the sc ...[more]
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Project description:It has been observed in several tissues that direct isolation of cells in serum-free media and on nonadhesive substrates results in the formation of spherical clusters of cells known as free-floating spheres. Such free-floating spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Our goal was to isolate and characterize such free-floating spheres from HTM cell primary cultures. For this purpose, HTM cells were incubated in serum-free media and on a nonadhesive substrate. Individual free-floating spheres generated in these conditions were isolated in 96-well plates, and their proliferative capacity was evaluated by monitoring their size increase over time. The expression of the TM markers, MGP and CHI3L1, was examined using recombinant adenoviruses containing the respective promoters. Morphology of the free-floating spheres was analysed in semithin sections, and the gene expression profile was obtained using Human Genome U133 Plus 2.0 Affymetrix microarrays. HTM cells incubated in serum-free media and on nonadhesive substrate generated free-floating spheres that could be grown for more than 3 months. Addition of serum to the culture media promoted the attachment of the spheres to the substrate, migration of cells from the spheres, and differentiation into cells phenotypically similar to normal TM cells. Gene profiling analysis demonstrated strong similarities between the gene expression profiles of the spheres and HTM cell monolayers. Both infection with the recombinant adenoviruses and gene array analysis demonstrated the expression of CHI3L1 and MGP, indicating that free-floating spheres likely originate from HTM cells. Gene array analysis also showed expression of the marker for neural precursor cells nestin, as well as leukemia inhibitory factor, a gene involved in the maintenance of the undifferentiated state of progenitor cells. Analysis of semithin sections indicated that these TM free-floating spheres were highly dynamic structures demonstrating a distinct radial gradient of cell proliferation, survival, and apoptosis. Extensive up- and down-regulation of gene expression was associated with the processes of sphere attachment and cell migration after the addition of serum. These results suggest that HTM primary cultures might contain relatively undifferentiated or progenitor cells. The availability of TM progenitor cell cultures could constitute a useful tool to investigate cell therapy approaches targeting the TM in glaucoma.
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