Isolation method of mesenchymal stromal cells from human amniotic membrane with xeno-free reagents to pre-clinical and clinical ophthalmological application

Abstract

Purpose : Ocular surface diseases (OSD) in their most severe forms, such as dry eye, limbal cell deficiency, and neurotrophic disease, still have major treatment challenges. In the regenerative medicine field, mesenchymal stromal cells (MSC) have been strongly investigated due to their potential for undifferentiating, self-renewal, and immunomodulatory characteristics. MSC from the human amniotic membrane (hAM) are considered a promising source for ophthalmological cell therapy, but there is no consensus about their acquisition. Thus, we aimed to standardize an isolation process of hAM-MSC for future pre-clinical and clinical applications.

Methods : AM samples were collected from five human placenta, which were obtained from patients undergoing elective cesarean section at Women's Integrated Healthcare Center (CAISM-UNICAMP), Campinas, SP, Brazil. Three different enzymatic digestion protocols to isolate hAM-MSC were tested, with the combinations of 1) Dispase + Collagenase I; 2) Trypsin + Collagenase I; and 3) TrypLE + Collagenase I. Each protocol was run three times. For validation, it was performed cell line authentication by Short Tandem Repeats (STR) analyses, cell counting, and morphological evaluation.

Results : The more efficient protocol, regarding cell viability and adhesion capacity, was protocol 3. Briefly, after tissue dissection, washing, and decontamination, AM fragments were incubated three times with TrypLE, a xeno-free solution, for 30 minutes at 37°C in agitation for removing epithelial cells. Then, AM fragments were incubated with Collagenase type I in DMEM for 30 minutes at 37°C in agitation. The homogenate was filtered and centrifuged. The pellet of hAM-MSC was resuspended, and cells were counted, cultured, or frozen. The cellular identity was confirmed by STR analysis. Cell counting demonstrated a viability of cell population over 97% since passage 0. Most of the isolated hAM-MSC presented fibroblastoid morphology, typical of MSC.

Conclusions : As hAM-MSC have the potential to be used in cell therapy of OSD, it is important to standardize a protocol for pre-clinical and clinical studies. By focusing on xeno-free processes, the first step to follow Good Manufacturing Practices, our work proposes a method of hAM-MSC isolation seeding light to the promising field of regenerative medicine in ophthalmology.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.