Device to MonitorControl Differentiation of Stem Cells to islet Cells - PowerPoint PPT Presentation

Loading...

PPT – Device to MonitorControl Differentiation of Stem Cells to islet Cells PowerPoint presentation | free to download - id: 146a28-NGI5Y



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Device to MonitorControl Differentiation of Stem Cells to islet Cells

Description:

Stem cells show promise to differentiate into insulin secreting cells ... of a membrane-based gradient generator for use in cell-signaling studies. Lab chip. ... – PowerPoint PPT presentation

Number of Views:56
Avg rating:3.0/5.0
Slides: 17
Provided by: CAE1
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Device to MonitorControl Differentiation of Stem Cells to islet Cells


1
Device to Monitor/Control Differentiation of Stem
Cells to ß-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 ß-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
  • Results cannot be compared to Mashima et al paper
  • Expensive imaging for 3D set up
  • Unconventional cell culture protocol
  • Redesign in 2D

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

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
Agarose Results
  • 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

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
Apply growth factors and allow for cell growth
and differentiation replace source/sink every 24
hours
Replace media with fixative and then conduct
live/dead assay
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?art269153issue185
  • 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.
About PowerShow.com