Successful Transportation and in vitro Expansion of Human Retinal Pigment Epithelium and its Characterization; A step towards Cell-based Therapy for Age related Macular Degeneration

Order of Publishing in Issue: 
5
Volume :6
Issue :1
January, 2012 - March, 2012
Page No: 
44-54
Authors: 
Rajappa Senthilkumar[2,3], Sadananda Rao Manjunath [2,3], Subramani Baskar [2,3], Vidyasagar Devaprasad Dedeepiya [2],3, Sundaram Natarajan [6], Sudhakar John[2], Periyasamy Parikumar [7], Insaan Aditya [8] Yuichi Mori [4], Hiroshi Yoshioka [4] David W Green [5], Madasamy Balamurugan [9], Shigeo Tsukahara [1] and Samuel JK Abraham[1,2*]
Address: 
[1] Yamanashi University, Faculty of Medicine, Chuo, Japan
Address: 
[2] Nichi-In Centre for Regenerative Medicine (NCRM), Chennai, India
Address: 
[3] Dept. of Biotechnology, Acharya Nagarjuna University, Guntur, India
Address: 
[4] Waseda University, Tokyo, Japan
Address: 
[5] Faculty of Science, University of Technology, Sydney, Australia
Address: 
[6] Aditya Jyot Eye Hospital, Mumbai, India
Address: 
[7] The Light Eye Hospital, Dharmapuri, India
Address: 
[8] Shah Satnamji Hospital, Sirsa, India
Address: 
[9] Sri Lakshmi Narayana Institute of Medical Sciences, Pondicherry, India
Email-ID: 
drabrahamsj@ybb.ne.jp

Abstract

Age related Macular Degeneration (AMD) is a disease of the retina that leads to deterioration in vision and eventually permanent blindness. As yet there are no definitive ways of repairing the damage caused by AMD. Recently evidence is mounting that cell-based therapy using Retinal Pigment Epithelium (RPE) could be a feasible option for treating this disease. For example, autologous RPE transplantation has been successful at providing a functioning replacement for the diseased retina in animal models and humans. However, degeneration can re-occur requiring more RPE cells from the patient. Therefore, considering the option of onetime harvested RPE tissue from the periphery of the patient’s eye, safe transportation between clinics/hospitals, efficient in vitro RPE expansion at the destination and long-term cryopreservation for future applications, we have developed a biodegradable RPE carrying medium in 3D, made from a growth factor-free Thermoreversible gelation polymer (TGP - Mebiol gel). RPE cell layers harvested from cadaver eyes were embedded in the TGP hydrogel and divided into three groups: Group 1, were processed immediately, Group 2 after 18-24 hours and Group 3 after 40-48 hrs of harvesting. Each group had one control sub-group grown in conventional media and one TGP sub-group grown embedded in TGP scaffold. No growth factors were used in the culture, when grown for three weeks. RPE cell counts were done at regular intervals during the expansion phase, and were then characterized by RT-PCR to confirm their RPE phenotype. The cells in all the three TGP preserved groups and the controls were equally viable after different periods of preservation, with a maximum duration of 48 Hrs. In cultivation, TGP preserved RPE cells formed a monolayer with a typical honeycomb/cobblestone appearance characteristic of native RPE. The degree of pigmentation is increased in the TGP group compared to the control group indicating that the RPE possesses a native RPE phenotype. The proliferative capacity of RPE was also increased when embedded in TGP. Cells from both the groups expressed Cellular Retinaldehyde-Binding Protein (CRALBP) and RPE65, which are abundantly expressed in the RPE cells and Mueller cells of the retina. We have established a simple and efficient transportation module for RPE at varying climatic conditions without the need for cool preservation using a polymer hydrogel cocktail and a culture method without using any growth factors. These cells can be a potential source for transplantation in treating retinal disorders upon further confirmation of their functional characteristics.

Keywords: 
Retinal pigment epithelium, macular degeneration, hydrogel scaffold.
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