Parallelization of Yeast Diode and Implementation of a Concentration Gradient BENG 129A: Design Development in Cell System...
Microfluidics Applications <ul><li>Advantages: </li></ul><ul><ul><li>reduces time </li></ul></ul><ul><ul><li>small require...
Microfluidic Theory Design principles: Laminar Flow = Linear Design Ohm’s law Poiseuille (Laminar) flow Circular Channel R...
<ul><li>Current Microfluidic designs lack multiple cell growth chambers. Concentration gradients have been explored and de...
2) Draw Up A General Design <ul><li>Expansion of the “Tesla microChemostat” design </li></ul><ul><li>Monolayer growth in c...
<ul><li>Microscope Viewing Area:  </li></ul><ul><li>40mm X 20mm </li></ul><ul><li>Microscope Scope is already </li></ul><u...
<ul><li>Design Restraints </li></ul><ul><ul><li>Flow Rates must match at each node to ensure a 50%-50% mixture. </li></ul>...
Standards and Constraints (Contd.) <ul><li>Linear Media Concentration Gradient </li></ul>Media Mixing Efficiency Tree Desi...
Parallel:  The parallel chip design is modeled after a parallel electric circuit. Originally used for a cell culture array...
Parallel Slant Resistors Fluid Rectifiers Serial Tree  Dilution Square Resistors Slant Resistors Fluid Rectifiers Square R...
Features 0 = inadequate; 1 = weak; 2 = sufficient; 3 = good; 4 = excellent 231 164 174 217   Total 2 2 2 2 2 Industrial Ap...
3) Flow Analysis for General Design   MATLAB scripts facilitate chip design process by calculating pressures/flows through...
Matlab: MOCA Microfluidic Open Circuit Analyzer Our system is modeled to the right, with hypothetical inputs (power source...
Flow Analysis (correct)
Final Design <ul><li>Linear Media Concentration Gradient </li></ul>Media Mixing Efficiency Tree Design Serial Design w/ 9 ...
Final Design: Cell Loading Final Design Final Design: Media Running
Process Timeline: Next Quarter <ul><li>Fabrication (3 Step Process) </li></ul>Identify a  Need Design Flow Analysis Finali...
Microfluidic Overview Microfluidic device fabrication can be broken down into 3 steps:
<ul><li>Testing (Dye => Media => Cells) </li></ul>Process Timeline: Next Quarter Identify a  Need Design Flow Analysis Fin...
Acknowledgements <ul><li>PI </li></ul><ul><li>Jeff M. Hasty  PH.D. </li></ul><ul><li>Graduate Students   </li></ul><ul><li...
 
Alternative Designs Parallel:  The parallel chip design is modeled after a parallel electric circuit. Originally used for ...
Alternative Designs
Matlab: MOCA Microfluidic Open Circuit Analyzer Since we know that microfluidic channels can be modeled like an electric c...
Groisman, A., and Quake, S. R., 2004, &quot;A Microfluidic Rectifier: Anisotropic Flow Resistance at Low Reynolds Numbers,...
Features
Gradient Curves Tree Design Serial Design w/ 9 channels Serial Design w/ 5 channels
 
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Parallelization of Yeast Diode and Implementation of a Concentration Gradient

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Current Microfluidic designs lack multiple cell growth chambers. Concentration gradients have been explored and developed but there has not been a fusion of multiple cell chambers combined with a concentration gradient. Our objective:
To create a microfluidic device with 10 Tesla Diodes, cell growth ports, with an easily controllable concentration gradient

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  • Stated previously microfluidic device features on order of 1um In general, for typical flow conditions and simple channel geometries – fluid flows have low reynolds numbers and are considered laminar Laminar flows follow fluid mechanical analog of Ohm’s law Because of this, devices generally easy to design and operate because of similarity with linear circuits
  • Device fabrication is a relatively simple three step process: A photopolymerizable epoxy resin, photoresist, is coated on a silicon substrate to a desired depth and selectively patterned using a high resolution photomask and UV light. Unpolymerized photoresist is removed and the whole process can be repeated as necessary. Once done, the silicon/photoresist structure will contain a positive master mold of the desired channel network. The master mold is then cast in poly(dimethylsiloxane) (PDMS), a transparent elastomer, creating a negative relief of the master mold. The PDMS replica is then released from the master mold and holes are drilled to allow for fluidic access to the channel network. Finally, the PDMS mold is treated to an oxygen plasma atmosphere to both clean and activate the surface for sealing to a similarly cleaned coverslip. Devices made this way cost very little ($2-$5) individually and entire batches, upwards of 30 devices can be made within 1-3days.
  • Aldksf ja;lksdjf a;lkdsjf a;lkdfj
  • Parallelization of Yeast Diode and Implementation of a Concentration Gradient

    1. 2. Parallelization of Yeast Diode and Implementation of a Concentration Gradient BENG 129A: Design Development in Cell Systems Bioengineering Jeff M. Hasty  PH.D. Group #6: Douglas Cohen, Hirak Desai, Lawrence Hui, Robert Langsner, Rushang Patel
    2. 3. Microfluidics Applications <ul><li>Advantages: </li></ul><ul><ul><li>reduces time </li></ul></ul><ul><ul><li>small requirements of solvents, reagents, and cells </li></ul></ul><ul><ul><li>cost effective </li></ul></ul><ul><ul><li>versatility in design </li></ul></ul><ul><ul><li>parallel operations  </li></ul></ul><ul><li>Exerts more control over the cellular microenvironment </li></ul><ul><ul><li>Chemically </li></ul></ul><ul><ul><li>Thermally </li></ul></ul><ul><ul><li>Geometrically  </li></ul></ul><ul><li>Uses: </li></ul><ul><ul><li>DNA sequencing and seperation </li></ul></ul><ul><ul><li>Enzymatic and Immunoassays </li></ul></ul><ul><ul><li>Cell counting and cell sorting </li></ul></ul><ul><ul><li>Exploring single cell and its activity </li></ul></ul><ul><ul><li>Imaging (cells confined to monolayer) </li></ul></ul><ul><ul><li>Montioring gene expression </li></ul></ul>
    3. 4. Microfluidic Theory Design principles: Laminar Flow = Linear Design Ohm’s law Poiseuille (Laminar) flow Circular Channel Rectangular Channel For water flow at 1mm/s through a channel 100µm wide, N Re = 0.01
    4. 5. <ul><li>Current Microfluidic designs lack multiple cell growth chambers. Concentration gradients have been explored and developed but there has not been a fusion of multiple cell chambers combined with a concentration gradient. </li></ul><ul><li>Our objective: </li></ul><ul><ul><li>To create a microfluidic device with 10 Tesla Diodes, cell growth ports, with an easily controllable concentration gradient </li></ul></ul>Problem Statement Identify a Need Design Flow Analysis Finalize Design Fabrication Testing
    5. 6. 2) Draw Up A General Design <ul><li>Expansion of the “Tesla microChemostat” design </li></ul><ul><li>Monolayer growth in cell chamber </li></ul><ul><li>Creeping media flow in chamber throughout expt </li></ul><ul><li>Long runtimes </li></ul>Identify a Need Design Flow Analysis Finalize Design Fabrication Testing 12 4 2 Load Run
    6. 7. <ul><li>Microscope Viewing Area: </li></ul><ul><li>40mm X 20mm </li></ul><ul><li>Microscope Scope is already </li></ul><ul><li>built, thus viewing is limited </li></ul><ul><li>to the restraints of the microscope. </li></ul>Standards and Constraints
    7. 8. <ul><li>Design Restraints </li></ul><ul><ul><li>Flow Rates must match at each node to ensure a 50%-50% mixture. </li></ul></ul><ul><ul><li>Lengths must be sufficiently long to allow complete mixture of media by diffusion. (Dependant on flow rates) </li></ul></ul><ul><ul><li>Inputs pressurized by gravity is limited in range (5 – 40 inh20) </li></ul></ul><ul><ul><li>Inputs pressurized by Air Pumps have much higher ranges but limited to two distinct levels (ie. 100 and 150 inh20) </li></ul></ul><ul><ul><li>Chip can only sustain 15 PSI (normal pressures are 0.6 – 1 PSI) </li></ul></ul>Standards and Constraints
    8. 9. Standards and Constraints (Contd.) <ul><li>Linear Media Concentration Gradient </li></ul>Media Mixing Efficiency Tree Design Serial Design w/ 9 channels Serial Design w/ 5 channels
    9. 10. Parallel: The parallel chip design is modeled after a parallel electric circuit. Originally used for a cell culture array for mammalian cells, we tried to expand on this design to include a chemical gradient. Each cell chamber is independent from the rest, therefore it increases the ease of fabrication and analysis of flow rates. Serial Dilution: This design capitalizes on constant media/buffer dilution to create discrete concentration levels. The media is diluted after each successive chamber. This design decreases the amount of work to analyze and model the circuit significantly. However, with this design the gradient is no longer linear, rather it becomes logarithmic as the M (media) mixes with B (buffer) at each successive port. Tree Dilution: This design allows us to create linear gradient with 3 media inputs and 5 ports overall. The was a novel design drawn by Group 6. Alternative Designs
    10. 11. Parallel Slant Resistors Fluid Rectifiers Serial Tree Dilution Square Resistors Slant Resistors Fluid Rectifiers Square Resistors Serial Dilution Basic Concept Design Thought Process
    11. 12. Features 0 = inadequate; 1 = weak; 2 = sufficient; 3 = good; 4 = excellent 231 164 174 217   Total 2 2 2 2 2 Industrial Application 3 3 3 3 3 Ease of Design 4 4 4 4 4 Cost 3 2 2 4 4 Modification Aptitude 4 4 4 4 5 Media gradient efficiency 4 2 2 3 6 Spatial Flexibility 4 4 4 4 6 Ease of Fabrication 4 3 3 4 7 # of inputs 3 0 0 4 8 Flow Analysis 4 2 3 4 10 Media mixing efficiency 3 3 3 1 10 Cell Loading Complications Serial Dilution w/ square resistors Tree Dilution w/ fluid rectifiers Tree Design w/ slant resistors Parallel Design Weight Parameters
    12. 13. 3) Flow Analysis for General Design MATLAB scripts facilitate chip design process by calculating pressures/flows throughout device Identify a Need Design Flow Analysis Finalize Design Fabrication Testing
    13. 14. Matlab: MOCA Microfluidic Open Circuit Analyzer Our system is modeled to the right, with hypothetical inputs (power sources) and arbitrary segment lengths (resistances). With 4 unknown inputs and 35 segment lengths, it becomes nearly impossible to solve this system by hand. On the right is an example of a circuit in which the currents at each joining section do not match and the power inputs are off so that a back-flow occurs on the far right.
    14. 15. Flow Analysis (correct)
    15. 16. Final Design <ul><li>Linear Media Concentration Gradient </li></ul>Media Mixing Efficiency Tree Design Serial Design w/ 9 channels Serial Design w/ 5 channels
    16. 17. Final Design: Cell Loading Final Design Final Design: Media Running
    17. 18. Process Timeline: Next Quarter <ul><li>Fabrication (3 Step Process) </li></ul>Identify a Need Design Flow Analysis Finalize Design Fabrication Testing 1. Print Photomasks 2. Fabricate Mold 3. Produce Chips from Mold Mask glued to borosilicate glass Mask file to be sent for printing Patterned Silicon Wafer Finished Devices!
    18. 19. Microfluidic Overview Microfluidic device fabrication can be broken down into 3 steps:
    19. 20. <ul><li>Testing (Dye => Media => Cells) </li></ul>Process Timeline: Next Quarter Identify a Need Design Flow Analysis Finalize Design Fabrication Testing S. Cerevisiae strain K699 E. coli wt strain JM2.300 Automated Microscope
    20. 21. Acknowledgements <ul><li>PI </li></ul><ul><li>Jeff M. Hasty  PH.D. </li></ul><ul><li>Graduate Students </li></ul><ul><li>Lee Pang </li></ul><ul><li>Scott Cookson </li></ul>
    21. 23. Alternative Designs Parallel: The parallel chip design is modeled after a parallel electric circuit. Originally used for a cell culture array for mammalian cells, we tried to expand on this design to include a chemical gradient. Each cell chamber is on an independent channel from the rest, therefore it increases the ease of fabrication and analysis of flow rates. Design 4: The newest design is a simplified version of Design 2 with the use of only 4 inputs as opposed to the original design using 5 inputs. This design decreases the amount of work to analyze and model the circuit significantly. However, with this design the gradient is no longer linear, rather it becomes logarithmic as the M (media) mixes with B (buffer) at each successive port. Design 1 (Tree Design w/ slant resistors): The resistors in design were taken off a known resistor design commonly used to increase resistances and length of channels to provide adequate length for diffusion 1 . This serial design was designed with a linear gradient in mind. Design 2 (Tree Design w/ triangular mixers): This design uses the ‘Tree design’ that we previously designed. In a paper by Alex Groisman, a use of a microfluidic rectifier was used to induce mixing using triangular mixers 2 . Further mixers were added on the Cell line on the bottom to induce mixing once the chemicals had reached the chamber line.
    22. 24. Alternative Designs
    23. 25. Matlab: MOCA Microfluidic Open Circuit Analyzer Since we know that microfluidic channels can be modeled like an electric circuit, we can utilize the similarity in concept to model our system in matlab to understand flow and resistance analysis.
    24. 26. Groisman, A., and Quake, S. R., 2004, &quot;A Microfluidic Rectifier: Anisotropic Flow Resistance at Low Reynolds Numbers,&quot; Phys. Rev. Lett., 92(9), pp. 094501 Alternative Designs
    25. 27. Features
    26. 28. Gradient Curves Tree Design Serial Design w/ 9 channels Serial Design w/ 5 channels
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