Exploring The Migration of CAR-Jurkat cells Expressing MicroRNA in an In Vitro Blood-Brain Barrier Model
Restricted (Penn State Only)
- Author:
- Dong, Yicheng
- Graduate Program:
- Biomedical Engineering (MS)
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 16, 2023
- Committee Members:
- Cheng Dong, Thesis Advisor/Co-Advisor
Daniel J Hayes, Committee Member
Daniel Hayes, Program Head/Chair
Justin R Pritchard, Committee Member - Keywords:
- In vitro BBB model
Glioblastoma
CAR-Jurkat cell
Cell Migration - Abstract:
- Glioblastoma is the most prevalent primary malignant tumor of the brain and unfortunately, the survival rate for glioblastoma patients is only 6.8% after 5 years and the average length of survival is estimated to be 8 months. Despite the advancement of our science and technology, survival rates and mortality statistics for GBM have been virtually unchanged for decades and only five drugs and one device have ever been approved by the FDA for the treatment of glioblastoma. For the treatment of Glioblastoma, the biggest problem people need to solve is the Blood-Brain Barrier (BBB). BBB has been a major challenge in creating and delivering drugs for CNS disorders and brain tumors. Its highly selective permeability, which only allows a limited number of drugs to reach the brain, has consistently hindered progress in this field. Overall, the BBB plays a crucial role in protecting the brain from injury and disease and is essential for normal brain function, it also blocks most of the therapeutic agents that are trying to pass through the BBB and get to the glioblastoma. Because of the structure of BBB, the study was done by using the fourth generation of CAR technology and inserting the gene of micro-RNA inside of the T lymphocyte Jurkat cells. T lymphocyte Jurkat cells will work as a delivery carrier and pass through the BBB, once they get to the glioblastoma size, they will start the secretion of the micro-RNA. A new protocol for seeding cells on the membrane of a flow chamber system was developed to make a tight, confluent in vitro BBB model comprised of Bovine Brain Microvascular Endothelial Cells (BBMVEC) to better mimic the in vivo conditions. The thesis focused on studying the migration abilities of Jurkat cells in response to various conditions using a newly designed chamber plate and flow chamber system. The study used a concentration of 2 x 105 cells/mL and a CXCL12 chemoattractant concentration of 500 ng/mL to test the migration abilities of Jurkat, CAR-Jurkat, and CAR-Jurkat-m34a cells in response to tumor culture media and RPMI control. The study also performed flow migration studies of the Jurkat cells under a shear rate of 50 sec-1. To increase the migration of the Jurkat immune cells under flow conditions, the study treated endothelial cells with TNF- α to upregulate the expression of ICAM-1. Overall, the study provides important insights into the migration abilities of Jurkat cells and engineered CAR-Jurkat cells.