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Device to monitor and control the differentiation of stem cells into β -islet cells of the pancreas

Device to monitor and control the differentiation of stem cells into β -islet cells of the pancreas. Clients: Victoria Browning, Ph.D. Brenda Kahan, Ph.D. Advisor: Naomi Chesler, Ph.D. Jon Baran Dhaval Desai Tim Pearce Tess Rollmann. Outline. Background/Problem statement

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Device to monitor and control the differentiation of stem cells into β -islet cells of the pancreas

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  1. Device to monitor and control the differentiation of stem cells into β-islet cells of the pancreas Clients: Victoria Browning, Ph.D. Brenda Kahan, Ph.D. Advisor: Naomi Chesler, Ph.D. Jon Baran Dhaval Desai Tim Pearce Tess Rollmann

  2. Outline • Background/Problem statement • Accomplishments from last semester • Changes in design specifications • Evaluation of our device • Paper by Mashimaet al. • Cell seeding • Cell viability and maintenance • Gradient formation • Immunofluorescence based results • Future work • References • Questions

  3. Background and motivation • Current method for long term treatment of Type 1 diabetes • Human Islet Transplantation • Problems: Shortage of donors, immune reaction • Possible solution: Stem cells • Client would like to differentiate foregut committed progenitor cells into insulin-producing pancreatic β-islet cells • Test a number of growth factors (GF) for their ability to differentiate the progenitor cells to β-islet cells that secrete insulin • Generate a linear growth factor gradient • Use immunofluorescence to determine ideal GF concentration for differentiation

  4. Last semester… (c) a. Microfluidic Device b. Gradient formation in device using Texas-Red labeled Dextran c. Gradient Image Quantification using MATLAB (a) (b)

  5. Last semester… • Time-lapse experiment • Source filled with a fluorescently-labeled Dextran. • Various time-points imaged. • Gradient formation is clearly seen over time.

  6. Changes in design specification • 3D (Matrigel)  2D (bottom of channel) • Cells suspended in Matrigel  Cells in media • High resistance needed to prevent flow: Matrigel  Porous membranes • Testing on: Progenitor cells  testing on AR42J cells (to be discussed shortly)

  7. Device evaluation: Paper by Mashimaet al. [1] • Showed the differentiation of a cell-line into an insulin-secreting cell-line • AR42J cells: rat pancreatic cells (amylase secreting cells) • Differentiated into insulin secreting cells when treated with 1 nM betacellulin and 2 nM activin A • Confirmed by visual change in cell morphology, immunocytochemistry, and RT-PCR (reverse transcription - polymerase chain reaction

  8. Device evaluation: Paper by Mashimaet al. • Positive control • Test our device with AR42J cell line and a generate a gradient of betacellulin and activin • Validate gradient formation and differentiation • System can be applied to the progenitor cells [1]

  9. Device Testing Protocol Cell Viability Cell Seeding Gradient Formation Immunofluorescence

  10. Cell Seeding • Use tissue culture treated plastic or gelatin coated glass slide for seeding • Determine the easiest way to adhere the cells to the bottom of the channel • Methods employed so far: • Flow cells into the channel and then add additional media • Fill channel with media and then flow cells in • Test cell adhesion by putting additional media in the source and the sink

  11. Cell Seeding Before: Cells introduced in the microchannel After: Media put in the source and the sink. Cells flowed out of the channel.

  12. Cell Viability • Test cell viability within different channel lengths and widths • Lengths (1mm and 2mm) • Widths (150µm, 300µm, 450µm) • Test viability in channels for 1-4 days without media change • Live-dead assay: Trypan Blue • Determine optimal time to replenish media Live Cells Dead Cells [2]

  13. Gradient Formation • Use porous membranes (0.4-8 micron pores) to separate the channel from the source and sink • Allows for the growth factor and media change without disturbing the channel gradient • Test gradient formation of membranes using Dextran coated with Texas-Red dye [3]

  14. Immunofluorescence • Create a growth factor gradient using betacellulin and activin to determine the effect on the AR42J cell line • Allow differentiation of cells within the gradient • Image results using immunofluorescence • Compare results from the growth factor gradient with the positive control results from Mashima’s paper

  15. Future Work • Continue testing seeding and viability using AR42J cells • Gradient formation • Determine why the gradient has not formed with 0.4 micron membranes • Use an agarose gel to create a physical barrier between the channel and source instead of the membranes if necessary • Test gradient with AR42J cells and growth factors betacellulin and activin • Compare results to Mashima et al.[1] paper

  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.

  17. Questions?

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