1. Bioprinter for the Micropatterning of Macromolecules Group 3 Sailaja Akella Caroline LaManna Teresa Mak Rupinder Singh Advisors Emilia Entcheva, Ph.D Helmut Strey Ph.D

2. Overview  Background  Printer Modifications  Ink Printing Trials  Collagen Trials  Ethanol Wash Trials  Future Experiments  Goals

3. Design Project  Design a system that facilitates cell deposition and micropatterning to be used in the creation of cellular and polymer based circuits  Customer Criteria Suitable for printing proteins for cell adhesion Suitable for printing proteins for cell adhesion FibronectinFibronectin Collagen IVCollagen IV Collagen I –fluorescence labeledCollagen I –fluorescence labeled LamininLaminin Biocompatible/Sterile Biocompatible/Sterile Prints accurate & precise patterns Prints accurate & precise patterns High resolutionHigh resolution Cost-effective (<$500) Cost-effective (<$500) Neural cell patterning 1

4. Piezoelectric Inkjet Printer  Epson® Stylus R200 Print head (DDD) Print head (DDD) Built-in Caddy System (SPD) Built-in Caddy System (SPD) Necessary to print on our substrateNecessary to print on our substrate Resolution of 5760x1440 dpi Resolution of 5760x1440 dpi Maximum Diameter of Droplet (4.5um)Maximum Diameter of Droplet (4.5um) Diameter of droplet based on experimentsDiameter of droplet based on experiments Software Software Epson CD print softwareEpson CD print software Epson Stylus R220

5. Alterable Components of Ink-Jet Printer  DDD (Droplet Depositing Device): It is necessary to minimize the printed droplet diameter for high resolution (635 dpi) It is necessary to minimize the printed droplet diameter for high resolution (635 dpi) Surface tension and viscosity of solutionSurface tension and viscosity of solution Clogging should be prevented Clogging should be prevented Prevent ink spray phenomenonPrevent ink spray phenomenon  SPD (Substrate Positioning Device): Movement of substrate should be minimized for high resolution Movement of substrate should be minimized for high resolution System should allow for uniquely dimensioned substrates to printed on System should allow for uniquely dimensioned substrates to printed on  SSD (Solution Supplying Device): Could make system robust for different viscosities and surface tensions Could make system robust for different viscosities and surface tensions Different static pressuresDifferent static pressures Clogging should be prevented Clogging should be prevented  Software handling for all three should be feasible

6. Experimental Protocol  Goal: Our design needs to print proteins with a high resolution of ~635 dpi By increasing the concentration of proteins in our solution, the viscosity of our ink will increase; hence, we will achieve higher resolution By increasing the concentration of proteins in our solution, the viscosity of our ink will increase; hence, we will achieve higher resolution By increasing the surface tension, we will prevent wetting of our substrate By increasing the surface tension, we will prevent wetting of our substrate A high surface tension means low attraction and a low surface tension means a high degree of attractionA high surface tension means low attraction and a low surface tension means a high degree of attraction  Image printed solution of fluorescent collagen (type I) Fluorescence microscopy Fluorescence microscopy

7. Printer Modifications  Solution Supplying Device Empty & clean ink cartridges (6) Empty & clean ink cartridges (6) Removable ink chip Removable ink chip Drilled holes & inserted platinum-cured silicone tube Drilled holes & inserted platinum-cured silicone tube

8. Printer Modifications  Solution Supplying Device 2-6 mm silicone tube 2-6 mm silicone tube Stand Stand Deposition of “ink” solution into tube & nozzle Deposition of “ink” solution into tube & nozzle

9. Printer Modifications  Substrate Positioning Device Built-in CD caddy Built-in CD caddy Plastic CD template Plastic CD template Cut to hold microscope slidesCut to hold microscope slides

10. Ink Printing Trials  Printing the letter “i” suggested that we would have a problem with surface tension

11. Ink Printing Trials  Printing ink was relatively easy A small amount was inserted into a tube and then allowed to flow freely under the influence of atmospheric pressure A small amount was inserted into a tube and then allowed to flow freely under the influence of atmospheric pressure We did not need to apply a positive gauge pressure to print our patterns We did not need to apply a positive gauge pressure to print our patterns

12. Ink Printing Trials  Using a 0.01mm micrometer, we realized that we could easily print droplets with diameters of 150um separated by 200um.  We need to reduce each by a factor of about 10.  The limits of our resolution are still not clear

13. Collagen Trials  80% ethanol was the means to sterilize our bioprinter before we printed with collagen  This was successfully done by forcing the ethanol through a tube with very little pressure  After a few washes (3 or more to completely remove the ink from our system), we tried to print a PBS solution of fluorescently stained collagen.  We failed to print anything since our collagen solution was too viscous  We ended up clogging the black ink nozzles

14. Ethanol Wash Trials  We tried to use a different nozzle to print, but this was also unsuccessful  What was the problem if these other nozzles were not clogged by collagen?  Hypothesized that since each nozzle was treated with ethanol, we had caused a reduction in capillary pressure

15. Ethanol Wash Trials  Pressure due to reservoir must be high (14.6kPa) since we no longer have capillary pressure

16. Ethanol Wash Trials  To test our hypothesis, we conducted three different trials in which we tried to print ethanol  Our experiment attempted to discover the affect of external pressure on printing With the influence of negative or 0 atm pressure With the influence of negative or 0 atm pressure Nothing printedNothing printed With the influence of atmospheric pressure With the influence of atmospheric pressure Nothing printedNothing printed With the influence of positive pressure With the influence of positive pressure We were able to print somethingWe were able to print something

17. Future Experiments  Order Ink Chip Resetter  Create software template for printing  Test runs using collagen solution Determine how well collagen prints Determine how well collagen prints  Test runs with different viscosities & surface tensions of our “ink solution” of protein Determine necessary viscosity & dilution Determine necessary viscosity & dilution

18. Future Experiments  Test runs using collagen solution, printed onto PEG coated microscope slides  Create an attachment for our tubing system so that it will be mobile  Determine patterning resolution Goal  635dpi Goal  635dpi  Seed cells onto protein matrix

19. Goals  Successfully print fibronectin & collagen at 635dpi (20um diam. X 20um seper.) onto slides that facilitate cell growth  Spend <$500 during development  Create URECA poster exhibit

20. References 1. Sanjana NE, et.al. A fast flexible ink-jet method for patterning dissociated neurons in culture. J Neurosci Meth (2004); 136: 151-163.