Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro.
BACKGROUND
Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro.


RESULTS
We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age) which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames' medium (>26 mL) per retina, a higher speed (constant 55 rpm) of agitation by rotary shaker, and a greater concentration (10%) of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age). The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP) into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons.


CONCLUSIONS
This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing molecular biological bioassays that used to be conducted in isolated cultured cells.
Moritoh
Satoru
S
Division of Correlative Physiology, National Institute for Physiological Sciences, Okazaki, Japan.
Tanaka
Kenji F
KF
Jouhou
Hiroshi
H
Ikenaka
Kazuhiro
K
Koizumi
Amane
A
D000818:Animals; D002470:Cell Survival; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D036103:Nanotechnology; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000254:growth & development* / Q000502:physiology; D012165:Retinal Ganglion Cells / Q000378:metabolism; D014162:Transfection / Q000295:instrumentation
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins