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Augmentation of Clinical Grade Neonatal Porcine Islets with a Novel Maturation Media

  • Author / Creator
    Hassouna, Tarek
  • Current clinical islet cell transplantation relies solely on human cadavers as a source of graft tissue. Much research has been focused on neonatal porcine islets as an alternative due to their ability to be mass-produced, showing strikingly similar physiology and biological activity to human insulin, and extensive research in various type I diabetic animal models. Our lab has demonstrated the feasibility of transplanting neonatal porcine islets in diabetic induced mice with successful reversal of diabetes. However, islet composition and time-to-normalization have remained suboptimal, which has been thought to be attributed to a low number of β-cells within reach islet transplanted. We formulated a maturation protocol for islets in long-term culture in hopes of maintaining a purely β-cell phenotype that will exclusively produce insulin and reverse diabetes sooner than islets grown in standard media. Pancreata from 1 to 3-day old neonatal pigs were procured and cultured in standard HAMS-F10 media for 4 days. Subsequently, each pancreas was split and further cultured in DMEM-F12 maturation media containing a variety of growth added in a stepwise fashion, or cultured in control HAMS-F10 media. After 20 days, a portion of islets were transplanted into immunodeficient diabetic mice under the kidney capsule. Remaining islets underwent static incubation to assess insulin secretory capacity, DNA quantification to assess β-cell composition, RT-PCR, total cellular insulin content per pancreas, and immunohistology. Insulin content from static incubation and β-cell composition was expressed amount produced per pancreas. Higher levels of insulin expression, insulin positive cells, and β-cells were observed in matured islets compared to control islets. Increased insulin, β-cell progenitors, and PCNA positive cells were observed in mature islets compared to control islets. Less TUNEL staining and amylase were observed among mature islets compared to control. Mice transplanted with matured islets had significantly lower blood glucose values at weeks 18 and 20 compared to control islets. It is clear that long-term culture of islets in a maturation media have proven to be successful in vitro. The increase insulin production did translate into faster achievement of normalization after transplantation. However, more transplantation experiments are needed in other animal models to suggest that a purely β-cell phenotype is beneficial for a type I diabetic recipient.

  • Subjects / Keywords
  • Graduation date
    2016-06
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R3V980290
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Master's
  • Department
    • Department of Surgery
  • Specialization
    • Experimental Surgery
  • Supervisor / co-supervisor and their department(s)
    • Dr. Gregory Korbutt
  • Examining committee members and their departments
    • Dr. Jean Buteau, Surgery
    • Dr. Adetola Adesida, Surgery
    • Dr. Tom Churchill, Surgery