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Device to monitor and control the differentiation of stem cells into

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... differentiation of stem cells into -islet cells of the pancreas ... Stem cells ... based gradient generator for use in cell-signaling studies. Lab chip. ... – PowerPoint PPT presentation

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Title: Device to monitor and control the differentiation of stem cells into


1
Device to monitor and control the differentiation
of stem cells into ß-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
(No Transcript)
3
Outline
  • Background/Problem statement
  • Accomplishments from last semester
  • Changes in design specifications
  • Evaluation of our device
  • Paper by Mashima et al.
  • Cell seeding
  • Cell viability and maintenance
  • Gradient formation
  • Immunofluorescence based results
  • Future work
  • References
  • Questions

4
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 ß-islet cells
  • Test a number of growth factors (GF) for their
    ability to differentiate the progenitor cells to
    ß-islet cells that secrete insulin
  • Generate a linear growth factor gradient
  • Use immunofluorescence to determine ideal GF
    concentration for differentiation

5
Last semester
(c)
a. Microfluidic Device b. Gradient formation in
device using Texas-Red labeled Dextran c.
Gradient Image Quantification using MATLAB
(a)
(b)
6
Last semester
  • Time-lapse experiment
  • Source filled with a fluorescently-labeled
    Dextran.
  • Various time-points imaged.
  • Gradient formation is clearly seen over time.

7
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)

8
Device evaluation Paper by Mashima et 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

9
Device evaluation Paper by Mashima et 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
10
Device Testing Protocol
Cell Viability
Cell Seeding
Gradient Formation
Immunofluorescence
11
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

12
Cell Seeding
Before Cells introduced in the microchannel
After Media put in the source and the sink.
Cells flowed out of the channel.
13
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
14
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
15
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 Mashimas
    paper

16
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

17
References
  1. 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.
  2. 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?art269153issue185
  3. 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.

18
Questions?
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