Cell culture of fully developed and functional adult photoreceptor cells can serve as an in vitro model for the study of retinal diseases and the search of prospective therapies. However, obtaining cells that resemble identical characteristics as in vivo cells is still a major challenge. In long-term cultures, photoreceptors have been observed to undergo morphological changes by suffering of outer segment loss and becoming rounded as shown in this study as well as previous studies [10
]. In this study, we have evaluated the current available techniques for adult photoreceptor dissociation to determine which one yields the highest number of intact photoreceptors. We also examined the timeframe of the deformation of intact cells after seeded and the possible contribution of light to this deformation.
Our results showed that the optimal technique to obtain a high number of intact elongated photoreceptors was an enzymatic dissociation followed by a gentle mechanical trituration (Protocol A). Not using enzymes, as in Protocol C, prevents from breaking the cell bodies apart from the tight interphotoreceptor matrix and isolating individual intact photoreceptors. A gentle enzymatic technique consists in exposing the retinal tissues to low doses of papain (0.06–0.1 mg/ml) for short 20 min incubation [4
]. In these experiments, we refrained from activating papain with L-cysteine as it has been shown to be cytotoxic [31
]. We also minimized handling of the tissues by using only wide bore pipette tips and avoiding any agitation of tubes containing the tissue. Our data also suggested that centrifugation after the dissociation to obtain a cell pellet should be avoided as the mechanical stress seemed to contribute to cell deformation. Based on the data, it should be noted that the centrifugation step should be minimized or eliminated from the protocol.
Immunohistochemistry results showed that 2 days after seeding, cells had deformed into a circular shape, even though cells were intact and elongated when initially seeded. The outer segment was still observed attached to the cell body/nucleus but deformed from elongated to round in shape () and this observation is consistent with Townes-Anderson et al. [22
]. Cells that had lost their outer segment during the dissociation process expressed rhodopsin, but within their plasma membrane (), as previously observed [4
]. The time course of the deformation demonstrated that it took approximately 3 h after seeding for all intact cells and outer segment debris to become rounded (). Based on our data, a gentle handling and dissociation are key factors to maintain high numbers of cells. However, the cell deformation cannot be eliminated based on the handling and dissociation factors alone.
To preserve as much elongated photoreceptors in culture, we investigated whether the light exposure during dissociation and culture process play a role in the deformation of outer segments. There are no direct studies to date that have investigated the effect of light on outer segment deformation. However, it has been shown that deformations have been observed in outer segments as a result of changes in osmotic pressure [36
]. Under hypertonic saline solutions, isolated outer segments shrank in size and shape in response to light. It was hypothesized that the change was due to the closing of cGMP-gated channels in response to light [36
]. Similarly, hypotonic solutions caused outer segments to deform and become rounded after cultivation for 10 min under either light or dark conditions [38
]. Hypotonic solutions are known to hyperpolarize cell membranes [39
], a phenomenon that also occurs in photoreceptors in response to light. In a comparison of our study with microscopic images from Cohen [38
], we noted a similar cell deformation; this may suggest that light exposure may play a role in altering the cell shape in culture. Therefore, it is possible that deformations caused by light conditions as well as hypotonic solutions are related, although more research should be performed to correlate these two phenomena.
Our preliminary results showed that photoreceptors isolated in dark conditions better maintained their elongated shape even 12 h after seeding, and remained in an elongated shape for 7 days. In contrast, photoreceptors isolated under room light conditions changed the shape within 3 h, with its deformation starting about 45 min after seeding. Our data suggested that there may be a relationship between the photoreceptor’s ability to maintain structure and light exposure during the dissociation process. Although our results seemed to favor our hypothesis, further research should be conducted to explore the possible mechanism of light involvement and duration in which elongated cells can be maintained. A viability assay after 4 and 7 days in culture indicated that performing experiments in dark promoted higher number of cells to remain viable with time. Based on our data, well preserved elongated cells can be viable for a longer period of time if light exposure is minimized or eliminated during the dissociation and culture process.
The current study demonstrated two important characteristics of the isolation and culture of photoreceptor cells. Our results showed the possibility of using adult photoreceptors maintained in vitro for the experiments mentioned in the Introduction and in transplantation techniques.
The results of this study open many questions regarding the mechanisms that minimize cell deformation in dark conditions. The use of channel blockers to test for possible mechanisms involved is a valuable avenue being considered for immediate future studies. If cell deformation is observed in the dark by blocking cGMP channels, we may be able to suggest that cGMP channels are one of the main factors involved. If no deformation occurs by blocking cGMP channels, we cannot rule out completely the involvement of cGMP channels without testing other channels (e.g., K+ channels or hyperpolarizing-activated channels). Therefore, an in-depth pharmacological studies considering involvement of cGMP channels and additional outer and inner segment channels is essential and will be highly informative.