Tsung-Jen Wang1, Ming-Sheng Chen2, Ming-Li Chou2, Hsiu-Chen Lin3, Jerard Seghatchian4, Thierry Burnouf5. 1. Department of Ophthalmology, Taipei Medical University Hospital, Taipei, Taiwan; Department of Ophthalmology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan. 2. Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan. 3. Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; Department of Clinical Pathology, Taipei Medical University Hospital, Taipei, Taiwan. 4. International Consultancy in Blood Components Quality/Safety Improvement, Audit/Inspection and DDR Strategies, London, UK. Electronic address: jseghatchian@btopenworld.com. 5. Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan; International PhD Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan. Electronic address: thburnouf@gmail.com.
Abstract
BACKGROUND: Human platelet lysates (HPLs) are emerging as the new gold standard supplement of growth media for ex vivo expansion of cells for transplant. However, variations do exist in the way how HPLs are prepared. In particular, uncertainties still exist regarding the type of HPL most suitable for corneal endothelium cells (CEC) expansion, especially as these cells have limited proliferative capacity. MATERIAL AND METHODS: Three distinct HPL preparations were produced, with or without calcium chloride/glass beads activation, and with or without heat treatment at 56°C for 30min. These HPLs were used to supplement basal D-MEM growth medium, each at a protein concentration equivalent to that of 10% fetal bovine serum (FBS; control). Impact on CEC (BCE C/D-1b cells) in vitro morphology, viability and capacity to express Zonula occludens-1 (ZO-1) tight junction marker was assessed by Western blotting. RESULTS: BCE C/D-1b cells grown in all HPL supplements exhibited four of essential characteristic properties: adhesion capacity, microscopic morphology and viability similar to that observed when using 10% FBS. In addition, Western blots analysis revealed an expression of the ZO-1 marker by BCE C/D-1b cells in all conditions of culture. CONCLUSION: CECs can expand ex vivo in a basal medium supplemented with the three HPLs without noticeable difference compared to FBS supplement. These data support further studies to evaluate the potential to use HPLs as a clinical-grade xeno-free supplement of CEC for corneal transplant.
BACKGROUND:Human platelet lysates (HPLs) are emerging as the new gold standard supplement of growth media for ex vivo expansion of cells for transplant. However, variations do exist in the way how HPLs are prepared. In particular, uncertainties still exist regarding the type of HPL most suitable for corneal endothelium cells (CEC) expansion, especially as these cells have limited proliferative capacity. MATERIAL AND METHODS: Three distinct HPL preparations were produced, with or without calcium chloride/glass beads activation, and with or without heat treatment at 56°C for 30min. These HPLs were used to supplement basal D-MEM growth medium, each at a protein concentration equivalent to that of 10% fetal bovine serum (FBS; control). Impact on CEC (BCE C/D-1b cells) in vitro morphology, viability and capacity to express Zonula occludens-1 (ZO-1) tight junction marker was assessed by Western blotting. RESULTS: BCE C/D-1b cells grown in all HPL supplements exhibited four of essential characteristic properties: adhesion capacity, microscopic morphology and viability similar to that observed when using 10% FBS. In addition, Western blots analysis revealed an expression of the ZO-1 marker by BCE C/D-1b cells in all conditions of culture. CONCLUSION: CECs can expand ex vivo in a basal medium supplemented with the three HPLs without noticeable difference compared to FBS supplement. These data support further studies to evaluate the potential to use HPLs as a clinical-grade xeno-free supplement of CEC for corneal transplant.
Authors: Jessica M Gilbertie; Julie M Long; Alicia G Schubert; Alix K Berglund; Thomas P Schaer; Lauren V Schnabel Journal: Front Vet Sci Date: 2018-07-04