1. Device to monitor/control differentiation of stem cells to β-islet cells
Jon Baran – BWIG
Dhaval Desai – Communications
Kyle Herzog – Team leader
Tim Pearce – BSAC
Client: Victoria Browning, Ph.D.
Advisor: Naomi Chesler, Ph.D.

3. Outline Background Problem statement Client requirements
(1) Human Islet Transplantation (HIT), (2) stem cells, (3) microfluidics
Problem statement
Client requirements
Current gradient generation methods
(1) Flow, (2) non-flow, (3) dropdown
Our decision
Future work
References
Questions

4. Human Islet Transplantation
Current method used to treat patients with type 1 diabetes [2]

5. Stem Cells
[1]

6. Stem Cells Client has derived foregut-committed cell lines
Region from which pancreas develops
Certain GFs induce differentiation into insulin producing cells
Low purity, complete differentiation requirements largely unknown
GF gradients play a large role in wild-type embryonic development

7. Microfluidics
Behavior of fluids on the micro-level is very different than on the macro-level
Laminar flow dominates
Fluids will not mix readily via turbulence, and diffusion alone is responsible for mixing
This property allows microchannels to establish concentration gradients [3,4]

8. Microfluidics Devices are relatively inexpensive and easy to make
PDMS used for channels can be imaged through and can withstand immunofluorescense
Dr. Beebe is an expert in the field and is willing to guide us through the design process

9. Problem Statement
Client would like to differentiate foregut committed progenitor cells into insulin-producing pancreatic β-islet cells
These cells could replace or supplement transplanted donor beta cells
Would like to test a large number of growth factors (GFs) for their ability to affect conversion of these progenitor cells into mature insulin-secreting cells
Continuous linear growth factor gradient would be ideal

10. Client Requirements
Capable of holding >=100 cells ( would be 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

11. Flow System Multiple fluid streams converge to form gradient
Allows rapid fluid exchange
Laminar flow ensures only diffusion
Syringe pumps induce flow

12. Flow System Pros Cons More widely researched
Gradient is set up very quickly
Flow would make staining for imaging easy
Cons
No autocrine/paracrine signaling
High reagent volume required
Shear forces could have adverse effects
Syringe pump required to maintain flow

13. Non-flow System
Linear gradient forms via diffusion between source and sink
Long term testing a possibility
Used in migration studies
System pioneered by student in Dr. Beebe’s lab

14. Non-flow System Pros Cons Autocrine/paracrine signaling uninhibited
Low volume of reagent required
No syringe needed to maintain flow
Cons
Gradient takes time to develop
Staining for imaging may be a problem
Not as widely researched as flow systems

15. Dropdown System
Fluid is flown across the top of the area which contains the cells
The fluid flows across a variable permeable membrane
The variation in the permeability causes a concentration gradient to form across the cells
Two gradients will be formed
One leftright and one topbottom

16. Dropdown System Pros Cons
Gradient could be easily established and maintained
Two different gradients formed
Could be patentable
Cons
May be difficult using different growth factors as they may be different sizes
Construction of a variable permeable membrane may be difficult
Large volume of GF required

17. Our Decision Non-flow system 5000 cells plated Channel dimensions
10 micron diameter
Channel dimensions
2mm x 0.3mm x 0.3mm
0.01mm Matrigel coating
Channel volume
0.157 nL
Sink
0.01ng GF in 1microL (10ng/mL)
Source concentration
0.15ng GF in 1microL (150ng/mL)

18. Future Work Final design constraints will be determined
Construction of prototype
Verification of functionality
Cell seeding efficiency
Concentration gradients
Cell viability
Testing
The system will be given to the client to test if the system identically meets their needs
Due to the complexity of the project, multiple systems will be constructed with input from the client

19. References
[1] Stephanie Watson. How Stem Cells Work.  11 Nov 2004  16 Oct 2007
[2] Islet cell transplantation. Wikipedia, The Free Encyclopedia. 16 Sep 2007 < 16 Oct 2007
[3] Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES, Jeon NL Human nueral stem cell growth and differentiation in a gradient generating microfluidic device. Lab Chip. 5(4):
[4] Whitesides GM The origins and the future of microfluidics. Nature. 442:

20. Questions?