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Response of Müller cells to growth factors alters with time in culture
R K Small1, P Patel, B A Watkins
1Department of Clinical Neurology, Institute of Neurology, London, England.
Abstract:
We have developed an explant culture technique, using the retinae of newborn guinea pigs, that reliably yields cultures of Müller cells showing uniform morphology and phenotype. Since the guinea pig retina is avascular and lacks astrocytes, Müller cells are the only glial cell-type and the only vimentin-positive population present. Virtually all passaged cells (greater than 98%) contain vimentin-positive intermediate filaments and no glial fibrillary acidic protein (GFAP) has been detected using a range of GFAP antibodies known to label astrocytes in the guinea pig optic nerve. Most vimentin-positive cells were also labeled with an antibody to carbonic anhydrase II, an enzyme which in the retina is specific for Müller cells. Proliferating Müller cells were identified within the inner nuclear layer of retinal fragments as early as 2 days in culture using bromodeoxyuridine (BrdU) and vimentin double labeling. Cultured Müller cells change their growth characteristics with successive passaging. The length of the cell cycle increases from 25.4 h for cells at first passage, to 66.7 h for cells at fourth passage. Altered responses to mitogens were also observed with passaging. First-passage cultures responded to basic fibroblast growth factor (bFGF) but not to several other factors tested including interleukin-2 (IL-2). In contrast, older cultures were highly responsive to IL-2 but showed a minimal response to bFGF. The altered responsiveness to mitogens observed in vitro may be relevant to changes in growth control of Müller cells in the developing and mature retina. The guinea pig retina provides an ideal mammalian tissue for generating Müller cell cultures that are free of astrocytes, endothelial cells, and pericytes, the most frequent contaminants of retinal glial cultures. The monolayers obtained show a high degree of homogeneity and are well suited for studies of Müller cell function.
Insights
Researchers developed a novel guinea pig retinal explant culture for pure Müller cell isolation. This method provides a reliable source of Müller cells for studying retinal glial cell function and growth regulation.
Area of Science:
- Neuroscience
- Cell Biology
- Ophthalmology
Background:
- Müller cells are the primary glial cells in the mammalian retina.
- Previous retinal glial cell cultures are often contaminated with other cell types.
- Guinea pig retinas offer a unique advantage due to their avascularity and lack of astrocytes.
Purpose of the Study:
- To establish a reliable explant culture technique for isolating pure Müller cells from guinea pig retina.
- To characterize the morphology, phenotype, and growth properties of cultured Müller cells.
- To investigate the response of Müller cells to mitogens during passaging.
Main Methods:
- Utilized newborn guinea pig retinae for explant cultures.
- Employed immunocytochemistry with antibodies for vimentin, glial fibrillary acidic protein (GFAP), and carbonic anhydrase II.
- Assessed cell proliferation using bromodeoxyuridine (BrdU) labeling.
- Determined cell cycle length and responsiveness to mitogens (bFGF, IL-2) across passages.
Main Results:
- Successfully generated Müller cell cultures with uniform morphology and phenotype (>98% vimentin-positive).
- Confirmed Müller cell identity via carbonic anhydrase II labeling and absence of GFAP.
- Identified proliferating Müller cells in retinal fragments within 2 days of culture.
- Observed changes in cell cycle length and differential mitogen responsiveness with passaging.
Conclusions:
- The guinea pig explant culture technique yields highly pure Müller cell cultures, free from common contaminants.
- Cultured Müller cells exhibit altered growth characteristics and mitogen responses with passaging.
- This model is well-suited for studying Müller cell-specific functions and growth control mechanisms in the retina.