Device to Monitor/Control Differentiation of Stem Cells to β -islet Cells

1. Device to Monitor/Control Differentiation of Stem Cells to β-islet Cells Dhaval Desai – Team Leader Jon Baran – BWIG Tim Pearce – BSAC Tess Rollmann – Communications Client : Victoria Browning, Ph.D. Advisor: Naomi Chesler, Ph.D.

2. Motivation • Type I diabetes patients cannot produce insulin • Current treatment methods take insulin from a donor • Stem cells show promise to differentiate into insulin secreting cells eliminate the need for a donor

3. Problem Statement • Differentiate foregut committed progenitor cells into insulin-producing pancreatic β-islet cells • Replace or supplement transplanted donor beta cells • Test different concentrations of growth factors (GFs) for their ability to affect conversion of progenitor cells into mature insulin-secreting cells • Continuous linear growth factor gradient

4. Design Contraints • Capable of holding ≥ 100 cells (1000-5000 ideal) • Compatible with imaging • Capable of withstanding immunofluorescense • Able to withstand 7-28 day incubation period at 37 degrees Celsius • Minimal amount of GF required • Total cost of under $500

5. Previous Work • Created linear gradient in a microfluidic channel • Channel filled with matrigel to create a high resistance barrier • Characterized the gradient formation using modeling software

6. Design Change Problems in 3D Redesign in 2D • Results cannot be compared to Mashima et al paper • Expensive imaging for 3D set up • Unconventional cell culture protocol

7. Goals for Redesign • Create a GF gradient within a microfluidic channel which allows for 2D cell culture • Integrate and test viability of cells in the channel • Validate our microenvironment culture conditions by comparing with standard culture conditions as seen in the Mashima et al paper

8. Method 1 • High resistance membrane • 0.8 micron pore polyester membranes • Placed between 2 PDMS layers • Cells suspended in media introduced from the sink

9. Membrane Results Successes Failures Inconsistent results Leakage Fluid connection hard to determine Difficult to work with Time consuming set up • Gradient formation • Plasma bonding sealed PDMS layers to prevent leakage • Using non-fluorescent dye to ensure fluid connection • Dextran labeled with Texas red dye

10. Method 2 Agarose barrier • Create plug at source • Agarose introduced on source port in liquid form • Once agarose cools, it solidifies to create barrier • Different concentrations of agarose to find optimal viscosity • SeaPrep and SeaKem agarose tested • 1.5% SeaKem agarose – saw gradient formation

11. Successes 1.5% agarose was optimal for gradient formation Linear gradient formed using Dextran labeled with Texas Red Failures Inconsistent results Difficult to ensure agarose has created a seal without entering the channel Agarose Results

12. Method 3 • New T-shaped channel design • Larger agarose channel will fill without migration into the smaller channel • Gradient will be formed within the smaller channel • Difficult to control agarose movement • Difficult to introduce fluid into the cell channel Agarose channel Cell channel

13. Cell Integration • Cells will adhere to the bottom of a glass slide coated with gelatin • Test cell viability for 1-5 days • Evaporation was one of the main obstacles • Cells were able to survive in the channel for 5 days without media exchange • Cells attached, lived, and divided • Ready for integration into the microchannel

14. Recommendations • The 3D system created last semester was the most consistent method Introduce cells + Matrigel in the channel and add media to source and sink Replace media with fixative and then conduct live/dead assay Apply growth factors and allow for cell growth and differentiation; replace source/sink every 24 hours Use inverted microscope to image cells. Use confocal microscope if necessary. Replace media with fixative and perform immunofluorescence for specific markers

15. Thanks • Our advisor, Professor Chesler • Our Client, Dr. Victoria Browning • Professor Justin Williams • Graduate students, Vinay Abhyankar and Erwin Berthier

16. References • Mashima H, Ohnishi H, Wakabayashi K, Mine T, Miyagawa J, Hanafusa T, Seno M, Yamada H, and Kojima I. Betacellulin and Activin A Convert Amylase-secreting Pancreatic AR42J Cells into Insulin-secreting Cells. J. Clin. Invest. 97: 1647-1654. • Qiu J. Automating Cell Counting to Produce Fast Reliable Results. Next Generation Pharmaceutical. Retrieved on February 20, 2008 from http://www.ngpharma.com/currentissue/article.asp?art=269153&issue=185 • Abhyankar VV, Lokuta MA, Huttenlocher A, and Beebe DJ. Characterization of a membrane-based gradient generator for use in cell-signaling studies. Lab chip. 6: 389-393.