CME MS Thesis - Migration of B Cells in a Microfluidic Device-Induced CXCL12 Gradient

Monday, March 11, 2019 The event started -448 days ago

11:00 AM12:00 PM

Engineering Building

Room 118

MIGRATION OF B CELLS IN A MICROFLUIDIC DEVICE-INDUCED CXCL12 GRADIENT

by: Rachel Lauren Stokes

Recent studies have continued to shed light on the intricacies of the germinal center (GC) where resting B cells activate and subsequently differentiate into plasma cells and memory B cells. Once we can recapitulate the dynamics of GC in an in-vitro setup, developing personalized cellular immunotherapy would be possible. While several methods have been developed to activate B cells and induce GC-like reaction, most in-vitro culture systems lack the zonality of physiological GCs. Migration between the dark and light zones (DZ and LZ) has been proven to be essential for effective somatic hypermutation and B cell survival during GC reactions. In order to add this crucial element to an in-vitro GC platform, this thesis attempts to incorporates a gradient of a critical chemokine, CXCL12, in a microfluidic chamber as a synthetic GC niche. The chemokine gradient was induced via feed and sink channels containing CXCL12- supplemented medium and blank medium, respectively. 10 kDa FITC-dextran was employed to model the diffusion of the CXCL12 and to characterize the concentration gradient using fluorescence microscopy. Naïve B cells isolated from the spleens of C57BL/6J mice were activated with one of three conditions: soluble HA-tagged CD40L, soluble HA-tagged CD40L and anti-HA antibody, and anti-HA antibody coated magnetic beads loaded with HA-tagged CD40L. Migration competency of the cultured B cells was determined by flow cytometry analysis of dark and light zone surface markers. The main hypothesis is that CXCR4- upregulating cells (DZ-like B cells) will migrate toward increasing concentrations of the CXCL12. Time-lapse imaging was utilized to monitor the behavior of B cells in this gradient. Interestingly, cells did not display overt chemotaxis in response to thus far tested gradients. Cells seeded at a density of 8 million cells/mL reformed germinal center clusters rather than migrating, while cells seeded at a density of 4 million cells/mL showed weak movement toward the source channel.

ADVISOR: Dr. Kyung-Ho Roh


Details

Category
Conference/Lecture
department
College of Engineering, Chemical and Materials Engineering
Audience
Public, Students, Faculty and Staff, Alumni

Contact

Kyung-Ho Roh 256-824-5292 This email address is being protected from spambots. You need JavaScript enabled to view it.

Venue

Engineering Building

John Wright Dr. NWHuntsville, AL 35809

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