Identification, characterization, and production of amniotic membrane-derived stem cells for application in ocular surface diseases

• Grant number: 23/07094-2
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: July 01, 2023
• End date: June 30, 2025
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Mônica de Cássia Alves
• Grantee: Caroline Nascimento Barquilha
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated research grant: 21/04045-5 - Evaluation of new techniques of cellular therapy to ocular surface diseases, AP.JP2

Abstract

Ocular surface diseases (OSD) in their most severe forms, such as dry eye, chemical burns, limbal cell deficiency and neurotrophic disease, still have major treatment challenges. In the quest for precision and regenerative medicine, several cell types and sources have been discovered, tracked, and used in experimental, preclinical and clinical studies, such as adult stem cells (SC). This cell population has shown promise due to its ability to undifferentiate, clonogenicity, self-renewal, and differentiation potential, in addition to immunomodulatory and pro-angiogenic characteristics. An interesting cellular source, in which the tissue itself is already widely used in ophthalmic dressings, is the amniotic membrane (AM). SC-AMs are considered a safe, effective, easily collected, basic and amplified source for use in ophthalmic cell therapy, but these processes have not yet been standardized. Thus, the present project is dedicated to the standardization of the collection, isolation, cultivation, characterization, and cryopreservation processes of SC-AM under GMP (Good Manufacturing Practices) conditions, so they can be used in future pre-clinical and clinical studies. For this, AM samples will be collected from 10 human placentas. Different processing protocols (enzymatic digestion, mechanical dissociation, cell explantation) will be tested and validated, in addition to the investigation of cell duplication rates, cell cycle, cell senescence, and genetic stability. Besides, morphological analyses will be performed through microscopy and immunocytochemistry, flow cytometry will be used to immunophenotypic analyses, the Tri-lineage Differentiation Potential (osteogenic, adipogenic and chondrogenic) will be checked, and RNA will be extracted for RT-PCR analysis. The respective statistical analyzes will be carried out considering each variable (parametric, non-parametric or multifactorial data). Also, aliquots will be stored at each passage to compose a biobank of these cells. At each stage, rigorous control will be carried out to guarantee the quality of the processes, from checking all microbiological and serological tests of each patient who will donate AM, as well as microbiological control for cell culture. Our hypothesis is that SC-AM can act regenerating the ocular surface, and may be used in OSD cell therapy. We hope that, with the application of this experimental design, it will be possible to obtain a bank of human SC-AM in conditions for clinical applications.