Christopher Burns & Chase Jackson with Dr. Alonzo Cook, Chemical Engineering

Introduction

Diseases of the retina, such as age-related macular degeneration and diabetic retinopathy, affect millions of people.1 In humans, photoreceptors lost as a result of these diseases are unlikely to be replaced naturally. Thus, these diseases can lead to vision loss. Our team aims to assist in the development of a treatment to not only halt retinal degeneration but to promote regeneration and restore vision. Müller glia cells (MGs) are retinal cells that “have been identified as a source of retinal regeneration in fish, chicks, and rodents.”2 We set out to study to what extent extracellular matrix proteins and other proteins promote retinal regeneration via MGs. This information will enable our team to build a kinetic model for retinal regeneration via MGs.

A 2013 review article described a five-step process of retina regeneration via MGs and summarized research findings related to that process.3 We believe that exposing MGs to the ideal concentrations of molecular factors for specific time periods can optimize each step in the process of retinal regeneration via MGs. In order to test our hypothesis, it is necessary to establish primary MG cultures. Our work this year focused on establishing those cultures.

Methodology

To establish primary MG cultures, we collaborated with Dr. Ed Levine of the Moran Eye Center at the University of Utah. Dr. Levine provided us with a retina dissociation protocol that uses papain—an enzyme found in papaya—as a key ingredient. Dr. Levine invited us to his lab multiple times, where he provided mice from which we harvested retinal cells. The mice were engineered to express either or both a red or green fluorescent marker in certain retinal cells, including MGs.

Beginning in July 2014, we harvested retinal cells multiple times from post-mortem rats we received from labs at BYU. Using animals from BYU was a convenient way to practice our retina dissection and dissociation techniques. Each time we harvested cells (either from mice from Dr. Levine or rats from BYU), we first dissected the retinas, dissociated them per the papain dissociation protocol, and suspended the cells in culture medium. The medium consisted of Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F12), fetal bovine serum (FBS), and penicillin-streptomycin (Pen-Strep). We placed the cultures in plastic well plates and incubated them at 37° C in 5% CO2 and 20% O2. On two occasions, we treated well plates with fibronectin prior to harvesting and culturing cells from Dr. Levine’s mice. Beginning in August, we used an increased FBS concentration in our culture medium. During Fall Semester 2014 we replaced the Pen-Strep in our culture media with an antifungal-antibiotic mixture in an attempt to curb contamination. In October, we began using a new retina dissection method a member of our team identified.

Results

Our team used microscopy and a Moxi Z cell counter (ORFLO) to study our primary retina cell cultures. Though we viewed objects in our mice retina cell cultures via light microscopy, we are uncertain what exactly we were viewing. It was not until summer 2014 that we understood our microscope images better. Cell counts from our mice cultures ranged from 103 to 106 cells/mL of culture medium. We were unsuccessful in our attempts to view these cultures using fluorescent microscopy, even after adding 4-OH tamoxifen to induce expression of the red marker in one of the mouse cultures. Additionally, we cannot confidently say that treating our well plates with fibronectin improved cell attachment to the plates or growth.

Our attempts to culture rat retina cells yielded cultures of cells on the order of 106 cells/mL. After receiving instruction regarding our new microscope, we were confident that we saw cells in our cultures. Most cells were circular just after culturing. Soon after culturing, we saw cells with a web-like morphology. The cultures we harvested on August 11 were our most successful cultures so far. We were able to wash these cultures either five or seven days after harvesting to remove debris and non-attached cells. After washing, the cultures had grown enough to allow us to passage the cultures two weeks after harvesting. At least one of the sub-cultures appeared to grow well. On September 4, contamination was found in two of the August 11 cultures.

Also, a culture we harvested on November 18 did not show signs of robust growth until early December. This suggests that we may need to wait longer than a few days to see robust culture growth.

Discussion

In developing primary retina cell cultures, knowing exactly what we are looking at under the microscope is key. The addition of a new microscope to our lab, along with instruction from a graduate student, helped us know which objects in our cultures were cells. In the near future, we plan to perform immunocytochemistry on cultures to determine whether MGs are present and if so, at what level of approximate purity. Furthermore, it may be useful to learn more about our lab’s cell counter in order to understand what it is counting.

Conclusion

Our work this year consisted primarily of attempts to harvest primary retina cells from mice and rats. On multiple occasions we successfully harvested retina cells and, on one occasion, we successfully passaged primary cultures. Controlling contamination will be crucial as we continue working to develop cultures that we can use to test our hypothesis regarding optimal molecular factor concentrations for retinal regeneration via MGs. Additionally, re-evaluating our culture medium ingredients, determining the optimal animal age for harvesting robustly-growing retina cell cultures, identifying and acquiring growth factors and inhibitors of tumor-suppressing genes that promote retina cell proliferation may also help us more successfully develop primary MG cultures.

References

1. Centers for Disease Control and Prevention, Vision Health Initiative. Common eye disorders. http://www.cdc.gov/visionhealth/basic_information/eye_disorders.htm (Last Updated April 23, 2013).
2. Gallina, D., et al., A comparative analysis of Müller glia-mediated regeneration in the vertebrate retina, Experimental Eye Research (2013), http://dx.doi.org/j.exer.2013.06.019
3. Ibid.