Bioprinter for the Bioprinter for the Micropatterning of Micropatterning of MacromoleculesMacromolecules

Group 3Group 3Sailaja AkellaSailaja Akella

Caroline LaMannaCaroline LaMannaTeresa MakTeresa Mak

Rupinder SinghRupinder Singh

AdvisorsAdvisorsEmilia Entcheva, Ph.DEmilia Entcheva, Ph.D

Helmut Strey Ph.DHelmut Strey Ph.D

OverviewOverview

BackgroundBackground Printer ModificationsPrinter Modifications Ink Printing TrialsInk Printing Trials Collagen TrialsCollagen Trials Ethanol Wash TrialsEthanol Wash Trials Future ExperimentsFuture Experiments GoalsGoals

Design ProjectDesign Project Design a system that facilitates cell Design a system that facilitates cell

deposition and micropatterning to deposition and micropatterning to be used in the creation of cellular be used in the creation of cellular and polymer based circuitsand polymer based circuits

Customer CriteriaCustomer Criteria Suitable for printing proteins for cell Suitable for printing proteins for cell

adhesionadhesion• FibronectinFibronectin• Collagen IVCollagen IV• Collagen I –fluorescence labeled Collagen I –fluorescence labeled • Laminin Laminin

Biocompatible/Sterile Biocompatible/Sterile Prints accurate & precise patternsPrints accurate & precise patterns

• High resolutionHigh resolution Cost-effective (<$500)Cost-effective (<$500) Neural cell patterning1

Piezoelectric Inkjet PrinterPiezoelectric Inkjet Printer

Epson® Stylus R200Epson® Stylus R200 Print head (DDD)Print head (DDD) Built-in Caddy System (SPD)Built-in Caddy System (SPD)

• Necessary to print on our Necessary to print on our substratesubstrate

Resolution of 5760x1440 dpi Resolution of 5760x1440 dpi • Maximum Diameter of Droplet Maximum Diameter of Droplet

(4.5um)(4.5um)

• Diameter of droplet based on Diameter of droplet based on experimentsexperiments

SoftwareSoftware• Epson CD print softwareEpson CD print software Epson Stylus R220

Alterable Components of Alterable Components of Ink-Jet Printer Ink-Jet Printer

DDD (Droplet Depositing Device): DDD (Droplet Depositing Device): It is necessary to minimize the printed droplet diameter for high It is necessary to minimize the printed droplet diameter for high

resolution (635 dpi) resolution (635 dpi) • Surface tension and viscosity of solutionSurface tension and viscosity of solution

Clogging should be preventedClogging should be prevented• Prevent ink spray phenomenonPrevent ink spray phenomenon

SPD (Substrate Positioning Device):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 onSystem should allow for uniquely dimensioned substrates to printed on

SSD (Solution Supplying Device):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 preventedClogging should be prevented

Software handling for all three should be feasibleSoftware handling for all three should be feasible

Experimental ProtocolExperimental Protocol

Goal: Our design needs to print proteins with a Goal: Our design needs to print proteins with a high resolution of ~635 dpihigh resolution of ~635 dpi By increasing the concentration of proteins in our By increasing the concentration of proteins in our

solution, the viscosity of our ink will increase; hence, solution, the viscosity of our ink will increase; hence, we will achieve higher resolutionwe will achieve higher resolution

By increasing the surface tension, we will prevent By increasing the surface tension, we will prevent wetting of our substratewetting of our substrate

• A high surface tension means low attraction and a low A high surface tension means low attraction and a low surface tension means a high degree of attraction surface tension means a high degree of attraction

Image printed solution of fluorescent collagen Image printed solution of fluorescent collagen (type I)(type I) Fluorescence microscopy Fluorescence microscopy

Printer ModificationsPrinter Modifications Solution Supplying DeviceSolution Supplying Device

Empty & clean ink cartridges (6)Empty & clean ink cartridges (6) Removable ink chipRemovable ink chip Drilled holes & inserted Drilled holes & inserted platinum-cured silicone tube platinum-cured silicone tube

Printer ModificationsPrinter Modifications

Solution Supplying Solution Supplying DeviceDevice 2-6 mm silicone tube2-6 mm silicone tube StandStand Deposition of “ink” Deposition of “ink”

solution into tube & solution into tube & nozzlenozzle

Printer ModificationsPrinter Modifications

Substrate Positioning Substrate Positioning DeviceDevice Built-in CD caddyBuilt-in CD caddy Plastic CD templatePlastic CD template

• Cut to hold microscope Cut to hold microscope slidesslides

Ink Printing TrialsInk Printing Trials Printing the letter “i” suggested that we Printing the letter “i” suggested that we

would have a problem with surface tension would have a problem with surface tension

Ink Printing TrialsInk Printing Trials

Printing ink was relatively easyPrinting ink was relatively easy A small amount was inserted into a tube and A small amount was inserted into a tube and

then allowed to flow freely under the influence then allowed to flow freely under the influence of atmospheric pressureof atmospheric pressure

We did not need to apply a positive gauge We did not need to apply a positive gauge pressure to print our patternspressure to print our patterns

Ink Printing TrialsInk Printing Trials Using a 0.01mm Using a 0.01mm

micrometer, we realized micrometer, we realized that we could easily that we could easily print droplets with print droplets with diameters of 150um diameters of 150um separated by 200um.separated by 200um.

We need to reduce each We need to reduce each by a factor of about 10.by a factor of about 10.

The limits of our The limits of our resolution are still not resolution are still not clear clear

Collagen TrialsCollagen Trials 80% ethanol was the means to sterilize our 80% ethanol was the means to sterilize our

bioprinter before we printed with collagenbioprinter before we printed with collagen This was successfully done by forcing the This was successfully done by forcing the

ethanol through a tube with very little pressureethanol through a tube with very little pressure After a few washes (3 or more to completely After a few washes (3 or more to completely

remove the ink from our system), we tried to remove the ink from our system), we tried to print a PBS solution of fluorescently stained print a PBS solution of fluorescently stained collagen. collagen.

We failed to print anything since our collagen We failed to print anything since our collagen solution was too viscoussolution was too viscous

We ended up clogging the black ink nozzlesWe ended up clogging the black ink nozzles

Ethanol Wash TrialsEthanol Wash Trials

We tried to use a different nozzle to print, We tried to use a different nozzle to print, but this was also unsuccessfulbut this was also unsuccessful

What was the problem if these other What was the problem if these other nozzles were not clogged by collagen?nozzles were not clogged by collagen?

Hypothesized that since each nozzle was Hypothesized that since each nozzle was treated with ethanol, we had caused a treated with ethanol, we had caused a reduction in capillary pressurereduction in capillary pressure

Ethanol Wash TrialsEthanol Wash Trials

Pressure due to Pressure due to reservoir must be reservoir must be high (14.6kPa) high (14.6kPa) since we no since we no longer have longer have capillary pressurecapillary pressure

Ethanol Wash TrialsEthanol Wash Trials

To test our hypothesis, we conducted three To test our hypothesis, we conducted three different trials in which we tried to print ethanoldifferent trials in which we tried to print ethanol

Our experiment attempted to discover the affect Our experiment attempted to discover the affect of external pressure on printingof external pressure on printing With the influence of negative or 0 atm pressureWith the influence of negative or 0 atm pressure

• Nothing printedNothing printed With the influence of atmospheric pressureWith the influence of atmospheric pressure

• Nothing printedNothing printed With the influence of positive pressureWith the influence of positive pressure

• We were able to print somethingWe were able to print something

Future ExperimentsFuture Experiments

Order Ink Chip ResetterOrder Ink Chip Resetter Create software template for printingCreate software template for printing Test runs using collagen solutionTest runs using collagen solution

Determine how well collagen printsDetermine how well collagen prints Test runs with different viscosities & Test runs with different viscosities &

surface tensions of our “ink solution” of surface tensions of our “ink solution” of proteinprotein Determine necessary viscosity & dilutionDetermine necessary viscosity & dilution

Future ExperimentsFuture Experiments

Test runs using collagen solution, printed Test runs using collagen solution, printed onto PEG coated microscope slidesonto PEG coated microscope slides

Create an attachment for our tubing Create an attachment for our tubing system so that it will be mobilesystem so that it will be mobile

Determine patterning resolutionDetermine patterning resolution GoalGoal 635dpi 635dpi

Seed cells onto protein matrixSeed cells onto protein matrix

GoalsGoals Successfully print fibronectin & collagen at Successfully print fibronectin & collagen at

635dpi (20um diam. X 20um seper.) onto 635dpi (20um diam. X 20um seper.) onto slides that facilitate cell growthslides that facilitate cell growth

Spend <$500 during developmentSpend <$500 during development Create URECA poster exhibitCreate URECA poster exhibit

ReferencesReferences

1.1. Sanjana NE, et.al. A fast flexible ink-jet method for patterning Sanjana NE, et.al. A fast flexible ink-jet method for patterning dissociated neurons in culture. J Neurosci Meth (2004); 136: dissociated neurons in culture. J Neurosci Meth (2004); 136: 151-163.151-163.