Microfluidic chips for cell and droplet manipulation

1
Microfluidic chips for cell and droplet
manipulation

• Inventor Robert Westervelt
• School of Engineering and Applied Science
• Harvard University
• HBS Project Team Paul Conrad, Katheryn Nathe,
  Michael Pistiner, Ben Zeskind

2
The Invention

• Use of a solid-state coil array to move cells and
  particles via magnetic or electric fields
• Manipulation of individual liquid droplets
  sandwiched between non-miscible liquid layers
• Possible areas of utility research, medicine,
  diagnostics, fine chemicals
• Killer Application yet to be identified

3
Path to Market (near term)

• Transfection experiments for biomedical research
  on non-adherent cells
• The Westervelt device could transfect cells by
  electroporation with simultaneous imaging in real
  time.
• Electroporation occurs at 40 kV/m (Krassowska,
  Biophys J, 2007)
• Device capable of 450 kV/m (5V across 11 mm2
  pixels)
• Path to Market
• Work with inventor to demonstrate utility in
  transfection studies.
• Sell initially as a research product.
• Develop branded community of scientists and
  engineers to provide future customer and
  application insight.
• Competition Transfection by Microinjection
• Does not work on non-adherent cells.
• Requires large numbers of cells which must then
  be transferred for imaging (e.g., Fujitsu
  Cellinjector).
• Need for continued development
• Develop cost-effective imaging or other data
  output technology.

Images source http//www.computers.us.fujitsu.com
/downloads/biosciences/BR_cellinjector.pdf
(Data from interview with M. Barch, MIT
transfection user).
4
Path to Market (mid term)Building a Branded
Community

• What is a branded community?A Branded Community
  is the process of activating your user base with
  services that allow users to promote your brand
  to each other while generating additional and
  incremental value for your brand
• Open Source Software
• Web-based community of equipment purchasers
• Provides new downloads of current cell
  manipulation protocols
• Allows publication of new, creative
  applications
• Gives company customer and new market
  intelligence
• Engenders loyalty and enthusiasm for the
  technology

5
Path to Market (long term)

• In Vitro fertilization
• Large market for IVF technologies gt1 Billion
• Modest success rates especially for ICSI
• Significant cost (30,000) and preparation time
  (3 months) make failures very expensive
• Current ICSI technologies carries risk of causing
  birth defects
• Development needed before Commercializing
• Evidence suggests that mild electric fields do
  not affect development but further research is
  critical.
• Several studies on possible effects of 60 Hz
  electric fields on reproduction and development
  in rats, using field strengths from 10 to 150
  kV/m did not report any consistent adverse
  effects.

Image Source www.jonesinstitute.org/icsi.html
quote souce Juutilainen, 2005 ICSI
Intracytoplasmic Sperm Injection
6
Business Model

• The Product
• A cell transfection and manipulation system
  consisting of 1) hardware (computer, cell
  manipulation device, microscope, camera, solvent
  delivery) 2) software and 3) consumable reagents.
  
• Proposed Product Pricing
• Reagents 10 per transfection
• Hardware 20-25,000 per system (comparable to
  Amaxa Nucleofector)
• Software Open source with community of users
  engaged to think of new applications
• Cost of Goods Sold
• We estimate that the system can be supplied for
  11,750 (computer 500, device 250,
  scope/camera 10,000, solvent delivery 1000,
  reagents 1-2)

The cell transfection/manipulation array
represents a versatile, high margin platform for
novel cell treatments. Building a community of
talented scientists, we will draw out a variety
of new applications.
7
Intellectual Property

• Patentability
• MANIPULATION AND/OR DETECTION OF BIOLOGICAL
  SAMPLES OR OTHER OBJECTS
• Utility filing in U.S. and Internationally April
  2005
• MICROFLUIDIC MANIPULATION DEVICE
• Provisional US Application filed June 2007

• Freedom to Operate
• Electrowetting technology Not a threat.
  Electrodes change wettability of a surface
  thereby pushing droplets across surface
• Traditional Microfluidics Not a threat. This
  technology does not rely on traditional
  techniques for fabrication of channels, pumps and
  valves
• Flow cytometry Not a threat for applications
  involving advanced cell manipulations/treatments.

Strategy considerations A variety of follow-on
licensing opportunities outside the
medical/therapeutic space (e.g., chemical process
technology) will likely emerge as users become
more familiar with this technologys capabilities
8
Backup and Data Slides
9
Proposed Path to Market

• Summary of the Invention
• Device to allow software controlled manipulation
  and movement of cells and fluid droplets in an
  imageable chamber using electric fields.
• Can this invention be monetized at this time?
• May be an immediate opportunity in transfection
  studies for biomedical research, specifically on
  non-adherent cells being prepared for imaging
  studies. Microinjection currently does not work
  on non-adherent cell types such as B-cells, which
  has created a market for products like the
  Fujitsu Cellinjector. However, this product is
  ineffective because a large number of cells must
  be transfected and then transferred to a culture
  dish prior to imaging. The Westevelt device
  could transfect using electroporation and then
  immediately image the same cells in situ. (Data
  from interview with M. Barch, MIT transfection
  user).
• Large In vitro fertilization market (to replace
  intracytoplasmic sperm injection), but 50 kV/m
  field strengths generate safety concerns even
  though Juutilainen, 2005 reports that several
  studies have addressed possible effects of 60 Hz
  electric fields on reproduction and development
  in rats, using field strengths from 10 to 150
  kV/m In general, the studies did not report any
  consistent adverse effects. Malformations were
  increased and fertility was decreased in one
  experiment These effects were not confirmed in
  the second experiment of the same study.
• Path to Market
• Work with Westervelt lab to demonstrate utility
  in transfection studies.
• Develop imaging or other data output technology.
• Sell initially as a research product.
• Use proceeds to test ICSI and other applications.

10
Market

• Unmet Need/Potential Applications
• 1)Gene transfection research tool
• 2)Reproductive technology research
• Size of Market
• To be determined
• Competition/Availability of Substitutes
• Digital Microfluidics
• Richard Fair (Duke)
• Aaron Wheeler (University of Toronto)
• CJ Kim (UCLA)
• Advanced Liquid Logic (Research Triangle Park,
  NC)
• Traditional Microfluidics
• Long term markets
• 1) Measurement of cell-cell communication (e.g.
  Alzheimer's disease, depression)
• 2) Assembly of 2 dimensional biologic tissue
  (e.g. skin grafts)
• 3)Artificial cellular interactions in controlled
  environments (e.g. cytokine release by T-cells)

11
Business Model

• We are selling a versatile cell manipulation
  system which will consist of a computer
  component, a solvent delivery unit, and the
  electrical cell manipulation array. Software for
  conducting particular procedures or tests will be
  a complementary product.
• Proposed Pricing (based on Amaxa Nucleofector
  analogy)
• 10 per transfection
• Hardware 26,000 per system
• This system will provide more versatile
  transfection approaches in a multiplexed fashion.
  Use in research will also generate interest and
  uses outside this setting.
• How much does it cost to make?
• The system comprises a computer, a solvent
  delivery unit, and the electrical array. The
  computer can be built to order for 100, the
  solvent delivery unit would likely cost 100, and
  the electrical array in mass production will cost
  lt100.
• Who pays?
• End users will pay through research funding
• These funding will likely originate with the U.S.
  government

12
Technology Protection

• Status of IP
• "MANIPULATION AND/OR DETECTION OF BIOLOGICAL
  SAMPLES OR OTHER OBJECTS" moving objects with
  magnetic and electric fields
• Utility filing in U.S. and Internationally April
  2005
• MICROFLUIDIC MANIPULATION DEVICE moving
  droplets trapped between liquid layers
• Provisional US Application filed June 2007
• Competing IP
• Electrowetting technology (e.g., Fair, Kim)
• Uses electrodes to change wettability of a dry
  surface thereby pushing droplets across surface
• Fundamentally different approach
• Traditional Microfluidics
• Westervelt technology does not rely on
  traditional techniques for fabrication of
  microfluidic channels, pumps and valves
• Flow cytometry (in some applications)
• A threat only for simple cell separation and
  counting. Not a threat for applications
  involving more advanced cell manipulations/treatme
  nts
• Strategy considerations
• Harvard has freedom to operate using this
  technique as far as can be determined from patent
  databases at present.
• May be many follow-on licensing opportunities
  outside the medical/therapeutic space (e.g.,
  chemical process technology)

13
Additional Device Data

• 1) Cell behavior in chambers
• coat the walls with BSA (bovine serum albumin)
• If the cell is suspended in the liquid, there is
  no threshold field to make it move. 
• 2)  Achieveable velocities
• The velocity depends linearly on the force, as
  describe in below.
• depends on the gradient of the electric field,
• the voltage required to hold a 5 micron radius
  cell against thermal motion is about 90 V/m,
  which is quite small. 
• The field required to maintain a motion of a cell
  through water at the speed 10 microns/sec is 5
  kV/m. 
• an estimate of the maximum flow velocity at 40
  kV/m is 0.7 mm/sec.
• 3)  Design time and costs
• it took a student about 4 mo. - a professional
  electrical engineer could do this more quickly. 
• The ICs where made at the TSMC foundry for about
  10k for 40 chips. 
• The microfluidic chamber is added at Harvard
  using facilities of our Center for Nanoscale
  Systems.  I think that there are foundries where
  that could be done for a fee. 
• The complete system includes a microcomtroller
  (standard commercial chip) and a circuit board
  (cheap to make commercially) to hold and control
  the CMOS/microfluidic chip, an optical microscope
  with a video camera (standard make) to see what
  happens, and software for a computer to record
  and control the motion (any standard computer,
  our software). 

14
Interviews

• Robert Tepper, former CSO Millenium
  Pharmaceuticals, partner Third Rock Ventures
• Craig Muir, founder of Codon Devices and
  high-throughput screening expert at Millenium
  Pharmaceuticals
• Mariya Barch, researcher at MIT experienced in
  gene transfection technologies
• JC, a successful ICSI patient

15
Dead Ends Examined

• Flow cytometry replacement (feedback loop)
• Cell-cell communication studies
• Analyzing cerebrospinal fluid
• Cellular surgery
• Nerve repair
• Building tissue in 3D
• Blood filtration/analysis/diagnostics
• Artificial lymph node

16
References

• Hunt, T.P., Issadore, D., and Westervelt, R.M.
  (2007) Integrated circuit / microfluidic chip to
  programmably trap and move cells and droplets
  with dielectrophoresis In Press
• Whitesides, G.M. (2006) The origins and the
  future of microfluidics Nature, 442 369-372.
• Krassowska, W., Filev, P.D. (2007) Modeling
  electroporation in a single cell Biophysical
  Journal, 92(2)404-417.
• Juutilainen, J. (2005) Developmental Effects of
  Electromagnetic Fields Bioelectromagnetics,
  Supplement 7 S107-S115.