Lee Lankford1, Y Julia Chen1, Zoe Saenz1, Priyadarsini Kumar1, Connor Long1, Diana Farmer1, Aijun Wang2. 1. Surgical Bioengineering Laboratory, Department of Surgery, University of California, Davis School of Medicine, Sacramento, California, USA. 2. Surgical Bioengineering Laboratory, Department of Surgery, University of California, Davis School of Medicine, Sacramento, California, USA. Electronic address: aawang@ucdavis.edu.
Abstract
BACKGROUND: In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). METHODS: PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). RESULTS: PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3-5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. DISCUSSION: PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.
BACKGROUND: In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). METHODS: PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). RESULTS: PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3-5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. DISCUSSION: PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.
Authors: Priyadarsini Kumar; James C Becker; Kewa Gao; Randy P Carney; Lee Lankford; Benjamin A Keller; Kyle Herout; Kit S Lam; Diana L Farmer; Aijun Wang Journal: FASEB J Date: 2019-02-12 Impact factor: 5.191
Authors: Sarah C Stokes; Christina M Theodorou; Jordan E Jackson; Christopher Pivetti; Priyadarsini Kumar; Kaeli J Yamashiro; Zachary J Paxton; Lizette Reynaga; Alicia Hyllen; Aijun Wang; Diana L Farmer Journal: J Pediatr Surg Date: 2021-09-20 Impact factor: 2.549
Authors: Laura A Galganski; Priyadarsini Kumar; Melissa A Vanover; Christopher D Pivetti; Jamie E Anderson; Lee Lankford; Zachary J Paxton; Karen Chung; Chelsey Lee; Mennatalla S Hegazi; Kaeli J Yamashiro; Aijun Wang; Diana L Farmer Journal: J Pediatr Surg Date: 2019-10-21 Impact factor: 2.545
Authors: Jordan E Jackson; Christopher Pivetti; Sarah C Stokes; Christina M Theodorou; Priyadarsini Kumar; Zachary J Paxton; Alicia Hyllen; Lizette Reynaga; Aijun Wang; Diana L Farmer Journal: J Surg Res Date: 2021-07-15 Impact factor: 2.417