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Müller cells are the major supportive and protective glial cells across the retina. Unlike in fish, they have lost the capacity to regenerate the retina in mammals. But, mammalian Müller cells still retain certain retinal stem cell properties with various degree of self-renewal and differentiation potentials, and thereby held a merit in cell-based therapies for treating retinal degeneration diseases. In our laboratory, we use an enzymatic procedure to isolate, purify, and culture mouse Müller cells.
Müller glial cell is a major lineage in the retina that functions to maintain retinal homeostasis through synthesis of neurotrophic factors, uptake and recycle of neurotransmitters, spatial buffering of ions, and maintenance of the blood-retinal barrier (Bringmann et al., 2006; De Melo Reis et al., 2008). Müller glia serve as retinal progenitor/stem cells in fish and, to a limited extent, in birds (Vihtelic and Hyde, 2000; Fischer and Reh, 2001). But, mammalian Müller cells have lost such a capacity to regenerate the retina, though still retain certain properties of adult stem cells such as proliferation upon a retinal damage. Researches in restoring of the lost capacity of mammalian Müller cells to repair retinal damage and understanding of the underlying mechanism are undertaken in laboratories with primary cells isolated from model animal retinas. Proteolytic enzymes are widely used in Müller cell dissociation and papain is less damaging and more effective than other proteases. Sarthy and Lam developed a method for dissociation and separation of glial cells with papain digestion followed by gentle mechanical dissociation, they found that among the enzymes used for dissociating turtle retina, papain produced the least trauma (Sarthy and Lam, 1978).
Using the above procedure, we successfully obtained large number of Müller cells isolated from 4 C57BL/6 mice (total 8 eye cups) by continuously passaging the primary cells at 1:1 ratio until passage 6 (P6) when most cells manifested a stress-induce premature senescence (SIPS) phenotype–large and flat with an obvious heterochromatin foci nucleus and positive for β-galactosidase activity. These senescent Müller cells were not proliferative, but could survive in culture for a long period of time if keeping medium timely refreshed, but eventually died in 3–4 months. Cells should be preserved in liquid nitrogen tank for a long-term storage if not used for month, and could be recovered by directly thawing the frozen cell vials in a 37 °C water bath and thereafter seeding cells in culture with little loss of viability. The percentage of the Müller cell purity differs from passage to passage because other retinal neural cells will not survive under such a culture condition. The estimation of the purity after 2 times of passage is more than 95%.
This protocol was initially adopted in the laboratory for pig (Xu et al., 2017), and now modified for mouse. The work was supported by National Institute of General Medical Sciences (P20GM103453 to Y. L.), University of Louisville School of Medicine (E0819 to Y. L.), and Research to Prevent Blindness (to the Department of Ophthalmology and Visual Sciences at Louisville).
1All tissue and cell manipulations must be under sterile condition either in a tissue dissecting or cell culture hood with surface disinfected by 70% ethanol.
2We strongly recommend to collect and dissect two eyeballs together from the same mouse, and to isolate and culture Müller cells separately from other mice to minimize possible cross contamination.
3The primary cells are including Müller cells, photoreceptors and other neurons. Photoreceptor and other neuron cells cannot be adapted for adherent culture and will go apoptosis after passage 2 (P2).
4The dish for culture is specifically coated with 0.1% gelatin to allow Müller cells to better adhere.