| Applications |
Neuroretina (NR) is an evagination of the central nervous system (CNS) which is composed of photoreceptors; glial (Muller) cells; horizontal, bipolar, amacrine; and ganglion neuronal cells. The cells were able to respond to electrical stimulation by producing action potentials which were inhibited by 10(-7) M tetrodotoxin. The specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of the neurotransmitter gamma-aminobutyric acid, was high. |
| Comments |
Neuroretinas were dissected from normal quail embryos, dissociated and immortalized by infection with Rous sarcoma virus (RSV) mutant ts NY-68 to establish NR cell lines. Cells were subsequently cloned after 18 months in culture to establish the cloned cell line QNR/K2. Neuroretina (NR) is an evagination of the central nervous system (CNS) which is composed of photoreceptors; glial (Muller) cells; horizontal, bipolar, amacrine; and ganglion neuronal cells. The QNR/K2 cell line displays properties of Muller (astroglia) cells. The cells were able to respond to electrical stimulation by producing action potentials which were inhibited by 10(-7) M tetrodotoxin. The specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of the neurotransmitter gamma-aminobutyric acid, was high. QNR/D cells (ATCC CRL-2532) and QNR/K2 cell (ATCC CRL-2533) were transplanted into chicken embryo eyes. Implanted QNR/D cells migrate only to the retinal ganglion and amacrine cell layers and project neurites in the plane of retina. In contrast, QNR/K2 cells migrate through the ganglion and amacrine layers, locate in the inner nuclear layer, and project processes across the retina. |
| Subculturing |
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
- Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels.
- Incubate cultures at 39°C.
Subcultivation Ratio: 1:3
Medium Renewal: Every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994. |
| References |
Pessac B, et al. A neuronal clone derived from a Rous sarcoma virus-transformed quail embryo neuroretina established culture. Nature 302: 616-618, 1983. PubMed: 6300691
Pessac B, et al. Cells with neuronal properties in permanent cultures of quail embryo neuroretinas infected with Rous sarcoma virus. Brain Res. 275: 53-59, 1983. PubMed: 6313126
Trisler D, et al. Retinal engineering: engrafted neural cell lines locate in appropriate layers. Proc. Natl. Acad. Sci. USA 93: 6269-6274, 1996. PubMed: 8692804
Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.
Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.
Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online.
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