3 Design Project & Customer Criteria Design a system that facilitates cell deposition and micropatterning to be used in the creation of neuron and polymer based circuits.Customer Criteriasuitable for printing biological macromolecules, sterileprogrammable for desired patterning, accurate & precisecost-efficient (<$500)
4 Cell Patterning Tissue Engineering Applications Individual cells Functional customized organsTissue rejection or lack of available donor organsBioengineering ApplicationsGenomics high-density DNA microarrays colony arrays for genomic librariesBiosensors Enzyme arrays for bioanalysis
5 PhotolithographyA photosensitive surface (a photoresist) is selectively exposed to light using a template and exposed areas are etched (carved by chemical means)Production of silicon chips that make up modern-day computers, circuitry
7 Soft LithographyThe manipulation of surfaces to create channels and substrate patterns to facilitate protein deposition and cell adhesionMicrocontact PrintingMicrofluidic PatterningMicrocontact printing with different inking solutions.2
8 Soft Lithography Microcontact Printing uses a PDMS microstamp, coated with protein, cellular factors, to leave pattern for later adhesiontransfer a specific cell type onto a substrate in specified patterndeposit cellular factors that will allow or hinder the deposition of a specific cell typeMicrocontact printing process schematic.3
9 Soft Lithography Microcontact Printing Current Technology Advantages Used to print self-assembled monolayers of alkanethiols on goldUsed to pattern ECM proteins on polystyrene to grow rat embryonic cortical neurons in patternsAdvantagesApplied to circuitry grid formationsSuitable for cell growthDisadvantagesExpensive & complexLimits to cell precision and confinement
10 Soft Lithography Microfluidic Patterning Uses channels on PDMS microfluid networks to direct cells, protein, etc. onto substrate in specified patternsMicrochannels for pattern formation4
11 Soft Lithography Microfluidic Patterning Advantages Disadvantages Rapid prototypingEffectively patterns on non-planar substratesDisadvantagesEfficiency of pattern transfer, not repeatableHighly complex and specialized, not automated
12 Contact Printing Quill Solid Pin-and-ring (Matrix Dot) Disadvantages Due to the impact at contact, pin structure deformation, and clogging from contaminants collected at contact, pin-based arraying is prone to suffer from slide-to-slide inconsistencyI guess you could use this slide……..
13 Ink-Jet Printer Printing Technique Biological reconstruction of digital dataNon-contact reprographic techniqueThermal/Bubble & PiezoelectricAdvantagesHigh throughputLow costAutomationComplex patterning flexibilityNo Surface contactPrinting and maintaining viable biological solutionsDisadvantagesLess precise cell patterningProtein cell attachmentResolution limited to nozzle
14 Thermal/Bubble Commercial ink-jet printers Printing Mechanism Hewlett-Packard DeskJet 550CCanon Bubble Jet 2100Printing MechanismHigh temperature heating system5Current pulse is applied for microseconds, which raises temperature ~300oCHeated plate in the nozzle causes vapor bubble to form and eject a droplet of ink
15 Bubble/Thermal Advantages Disadvantages Deposit large amounts Deposit multiple layersSuitable for printing:polymer encapsulated cells6cell solution6DisadvantagesLimited ResolutionumLow precision single cell patterningHigh TemperatureMotor neurons printed in ring. Neurons forming connections7 viable mam
16 Bubble/Thermal Okamoto et al.8 Fabrication of DNA Microarrays Presynthesized oligonucleotides are ejected onto glass surfaceConventional DNA hybridization measuredUsed to detect SNPsAdvantageLow cost compared with photolithographyLess uncertaintyDisadvantageExposure of DNA to high temperature ~200oCShear stress (10m s-1)Microarray using inkjet technology
17 Piezoelectric Commercial Printer Printing Mechanism Epson (patented use of piezo crystals)Printing MechanismA crystal located at each nozzle receives an electric charge causing vibrationCrystal vibrates forces tiny amount of ink5
18 Piezoelectric Disadvantages Advantages Clogging Small drop sizes BiomaterialsEnvironmental effectEffect of Printing TechniqueHigh frequenciesCompressionFriction9AdvantagesSmall drop sizesPicoliterHigh-ejection rateSeveral thousandComplex patternsLow cost
19 Piezoelectric Neville et al.10 Micropattern for neural cell culture Utilize substrate-bound patterns as a simulation environment and test-bedTechniqueDeposited collagen/poly-D-lysine (+) & polyglycol (-)Plated neurons and glial cellsAdvantageLow cost compared with lithographic techniquesExperimentation with different patterns (testing environment)DisadvantageNo direct printing of neuronsDrying of neuronsChanging osmolarity
20 Technical Considerations Mechanical LimitsFluid & Cell MechanicsMechanical Forces created in PrintingPrinting SystemsBubble JetPiezoelectricSoftware & Printer IntegrationCell EncapsulationBiopaper
21 Boundary Conditions Nozzle diameters: 30-100 um Potential range of viscosity: poisePotential surface tension: mN m-
24 Surface Energy/Tension behavior and many properties of liquids can be attributed to intermolecular forces.quantifies the disruption of chemical bonds that occurs when a surface is createdsurfaces are intrinsically less energetically favourable than the bulk of a material; otherwise there would be a driving force for surfaces to be created, and surface is all there would besphere has a smaller ratio of surface area to volume than any other three-dimensional figure, free-falling liquids tend to form spherical drops.
26 Ejection of DropletWeber number is a dimensionless quantity that is often useful in analyzing fluid flows where there is an interface between two different fluids.Dimensionless quantity associated with the smoothness of flow of a fluid.
28 Viscosity Measure of a liquid’s resistance to flow (poise) Has little effect on the ability of a penetrant material to enter a defect but it does have an effect on speed at which the penetrant fills a defectFill time is directly proportional to penetrant viscosity
30 Viscosity η = K·(db-dl)·t  where η is the viscosity in cP, K is the viscometer constant (we used a value of 0.3), db is the density of the ball (2.53), dl is the density of the liquid (g/ml) and t is the time of ball descent
32 Thermal PrintingAn electrical resistor heats ink at more than 1 million degree CA film of ink about 0.1 micrometer thick is heated to about 340 CCardiac Cells: 10x60x100 umCollage Protein: Type I is 300nm long, 1.5nm in diameter and consists of 3 coiled subunits
33 Thermal PrintingNormal evaporation will occur whenever the vapor pressure in the ambient gas is less than the saturation pressure of the liquid at the liquid temperatureSince the saturation pressure of a liquid increases with increasing temperature, the rate of evaporation will also increase with temperatureEvaporation from the liquid surface causes a decrease in liquid surface temperature
34 Theories for Droplet Ejection Note that while several theories exist concerning the mechanisms of liquid droplet ejection, no model exists for prediction of the ejected droplet rate, size or velocityHeterogeneous nucleationHomogeneous nucleation
35 Heterogeneous Nucleation Heterogeneous boiling occurs when vapor bubbles are formed below the surface at a nucleation siteWhen the nucleation site temperature exceeds the saturation temperature of the liquid, vapor bubble formation and growth may occur
36 Superheated LiquidIf the heat rate is fast enough, the liquid may become superheated, that is the liquid temperature can exceed the boiling temperatureA superheated liquid is in a metastable stateTemperature continues to increase, the spinodal is reached and the liquid becomes unstable and catastrophically relaxes to a liquid-vapor mixture
38 Homogeneous Nucleation Once in the metastable region a liquid need not reach the spinodal in order to change to a liquid-vapor mixtureHomogeneous nucleationthe spontaneous creation of vapor nuclei within the liquid, without the aid of preexisting nucleation sites
39 Bubble-Jet PrintingAs the vapor bubbles grow and coalesce, a large bubble may be formed below the surfaceWhen the bubble reaches a critical size it will burst, propelling liquid droplets into the plume
40 Piezoelectric Transducer Mechanical VibrationsThis alignment of molecules will cause the material to change dimensions- electrostriction
43 Printer SoftwareThe software drivers of the HP 550C are rewritten to allow for protein solutions of different viscosities and electrical charges to be printedThe source code for the HP550C printer was provided by the manufacturer at no costThe new driver software constantly adjusts the voltages applied to the nozzle gate to account for different electrical resistance values in the solutionsThis allows the appropriate amount to be dispensed, regardless of concentration, viscosity, or pH
44 Potential Software Code original PLC for particular printer Use DDK (Desktop Driver Kit)Change manner of printingDpiQuality of Paper / Printing
47 Cell EncapsulationA technology used for the microencampsulation of live cells and tissues within protective membraneDrug delivery systemsThe polymeric semipermeable membrane provides a physical barrier, preventing any direct contact between the entrapped cells and its surrounding environment
48 Encapsulation Polymer Poly(Ethylene Glycol) Diacrylate:BiocompatibleNontoxicNon-immunogenicHydrophilicCan be chemically cross-linked into hydrogels
50 Encapsulation Polymer Poly(lactic-co-glycolic acid) (PLGA)Copolymer of PLA and PGAMicrophase separationCrystallinityWater-solubilityBiodegradability
51 BiopaperThe printing substrates that the encapsulated cells would adhere ontoMade of biocompatible ECM-containing hydrogels
52 Biopaper Soy Agar Gel Made from Trypticase soy agar solution Initiate cell growth mediumObserve colony morphologyDevelop pure cultureCulture StorageGood diffusion characteristicsAbsence of toxic bacterial inhibitors and relative absence of metabolically useful minerals and compounds.
53 Biopaper Collagen Gel Produced from rat-tail Type I collagen Promotes attachment and growth of cells
55 Brainstorm Considerations Are lithographic techniques feasible or worthwhile?How does the temperature in thermal printing processes affect cell viability?How accurate can a modified ink-jet printing system become?Should encapsulation in the “ink” solution before deposition or on the surface after cell deposition?How can we guarantee that one cell is deposited at each position?
56 References“EE-527: Microfabrication- Photolithography.” <http://www.ee.washington.edu/research/microtech/cam/PROCESSES/photolithographypdf.html>St. John PM, et al. Preferential glial cell attachment to microcontact printed surfaces. J Neuro Meth 1997; 75:Vogt AK, et al. Micropatterned substrates for the growth of functional neuronal networks of defined geometry. Biotechnol Prog 2003; 19:Hibara A, et al. Surface modification method of microchannels for gas-liquid two-phase flow in microchips. Anal Chem 2005; 77(3):“Ink-Jet Printers.” <www.How Stuff Works.com>Roth EA. Xu T, DasM. Gregory C. Hickman. JJ. Boland T. Inkjet printing for high-throughput cell patterning.Biomaterials Aug;25(17):Xu T. Jin J. Gregory C. Hickman JJ. Boland T. Inkjet printing of viable mammalian cells. Biomaterials Jan;26(1):93-9.Hsieh et al. Ultra-High-Throughput Microarray Generation and Liquid Dispensing Using Multiple Disposable Piezoelectric Ejectors. J of Biomolecular Screening 9(20;2004Okamoto et al. Microarray Fabrication with covalent attachment of DNA using Bubble Jet Technology Nature Biotech 18;200.Sanjana NE, Fuller SB. A fast flexible ink-jet printing method for patterning dissociated neurons in culture.J Neurosci Methods Jul 30;136(2):Poly(methyl acrylate-co-hydroxyethyl acrylate) hydrogel implant material of strength and softness. J Biomed Mater Res 1981;15(4): Xu T, et al. Inkjet printing of viable mammalian cells. Biomaterials 2005; 26: 93-9.