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System for Micropatterning and Cell Encapsulation GROUP 3 Sailaja Akella Caroline LaManna Tereissa Mak Rupinder Singh.

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Presentation on theme: "System for Micropatterning and Cell Encapsulation GROUP 3 Sailaja Akella Caroline LaManna Tereissa Mak Rupinder Singh."— Presentation transcript:

1 System for Micropatterning and Cell Encapsulation GROUP 3 Sailaja Akella Caroline LaManna Tereissa Mak Rupinder Singh

2 Overview Design Project and Customer Criteria Current Technology -Photolithograpy -Soft Lithography -Contact Printing -Ink-Jet Printer Technical Considerations -Fluid & Cell Mechanics -Software -Encapsulation -Biopaper Brainstorming

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 Criteria –suitable for printing biological macromolecules, sterile –programmable for desired patterning, accurate & precise –cost-efficient (<$500)

4 Cell Patterning Tissue Engineering Applications –Individual cells  Functional customized organs –Tissue rejection or lack of available donor organs Bioengineering Applications –Genomics  high-density DNA microarrays  colony arrays for genomic libraries –Biosensors  Enzyme arrays for bioanalysis

5 Photolithography A 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

6 Photolithography Procedure: –wafer cleaning –barrier layer formation –photoresist application – soft baking; mask alignment –exposure and development –hard-baking. Photolithography process. 1

7 Soft Lithography The manipulation of surfaces to create channels and substrate patterns to facilitate protein deposition and cell adhesion –Microcontact Printing –Microfluidic Patterning Microcontact 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 adhesion –transfer a specific cell type onto a substrate in specified pattern –deposit cellular factors that will allow or hinder the deposition of a specific cell type Microcontact printing process schematic. 3

9 Soft Lithography Microcontact Printing –Current Technology Used to print self-assembled monolayers of alkanethiols on gold Used to pattern ECM proteins on polystyrene to grow rat embryonic cortical neurons in patterns –Advantages Applied to circuitry grid formations Suitable for cell growth –Disadvantages Expensive & complex Limits 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 patterns Microchannels for pattern formation 4

11 Soft Lithography Microfluidic Patterning –Advantages Rapid prototyping Effectively patterns on non-planar substrates –Disadvantages Efficiency of pattern transfer, not repeatable Highly 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 inconsistency

13 Ink-Jet Printer Disadvantages –Less precise cell patterning Protein  cell attachment –Resolution limited to nozzle Advantages –High throughput –Low cost –Automation –Complex patterning  flexibility –No Surface contact –Printing and maintaining viable biological solutions Printing Technique –Biological reconstruction of digital data –Non-contact reprographic technique –Thermal/Bubble & Piezoelectric

14 Thermal /Bubble Commercial ink-jet printers –Hewlett-Packard DeskJet 550C –Canon Bubble Jet 2100 Printing Mechanism –High temperature heating system 5 Current pulse is applied for microseconds, which raises temperature ~300 o C Heated plate in the nozzle causes vapor bubble to form and eject a droplet of ink

15 Bubble/Thermal Advantages –Deposit large amounts –Deposit multiple layers –Suitable for printing: polymer encapsulated cells 6 cell solution 6 Disadvantages –Limited Resolution 300-400um –Low precision single cell patterning –High Temperature Motor neurons printed in ring. Neurons forming connections 7 viable mam

16 Bubble/Thermal Okamoto et al. 8 –Fabrication of DNA Microarrays Presynthesized oligonucleotides are ejected onto glass surface Conventional DNA hybridization measured Used to detect SNPs –Advantage Low cost compared with photolithography Less uncertainty –Disadvantage Exposure of DNA to high temperature ~200 o C Shear stress (10m s -1 ) Microarray using inkjet technology

17 Piezoelectric Commercial Printer –Epson (patented use of piezo crystals) Printing Mechanism –A crystal located at each nozzle receives an electric charge causing vibration –Crystal vibrates forces tiny amount of ink 5

18 Piezoelectric Advantages –Small drop sizes Picoliter –High-ejection rate Several thousand –Complex patterns –Low cost Disadvantages –Clogging Biomaterials Environmental effect –Effect of Printing Technique High frequencies Compression Friction 9

19 Piezoelectric Neville et al. 10 –Micropattern for neural cell culture Utilize substrate-bound patterns as a simulation environment and test-bed –Technique Deposited collagen/poly-D-lysine (+) & polyglycol (-) Plated neurons and glial cells –Advantage Low cost compared with lithographic techniques Experimentation with different patterns (testing environment) –Disadvantage No direct printing of neurons –Drying of neurons –Changing osmolarity

20 Technical Considerations Mechanical Limits –Fluid & Cell Mechanics –Mechanical Forces created in Printing Printing Systems –Bubble Jet –Piezoelectric Software & Printer Integration Cell Encapsulation Biopaper

21 Boundary Conditions Nozzle diameters: 30-100 um Potential range of viscosity: 0.5-20 poise Potential surface tension: 25-50 mN m-

22 Preventing Continuous Flow

23 Intermolecular Forces

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 created surfaces 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 be sphere has a smaller ratio of surface area to volume than any other three-dimensional figure, free-falling liquids tend to form spherical drops.

25 Surface Energy/Tension

26 Ejection of Droplet

27 Capillary Action Force

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 defect Fill time is directly proportional to penetrant viscosity

29 Viscosity

30 η = K·(db-dl)·t [1] 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 Viscosity

31 Thermal Printing

32 An electrical resistor heats ink at more than 1 million degree C A film of ink about 0.1 micrometer thick is heated to about 340 C –Cardiac Cells: 10x60x100 um –Collage Protein: Type I is 300nm long, 1.5nm in diameter and consists of 3 coiled subunits Thermal Printing

33 Normal evaporation will occur whenever the vapor pressure in the ambient gas is less than the saturation pressure of the liquid at the liquid temperature –Since the saturation pressure of a liquid increases with increasing temperature, the rate of evaporation will also increase with temperature Evaporation from the liquid surface causes a decrease in liquid surface temperature Thermal Printing

34 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 velocity –Heterogeneous nucleation –Homogeneous nucleation Theories for Droplet Ejection

35 Heterogeneous Nucleation Heterogeneous boiling occurs when vapor bubbles are formed below the surface at a nucleation site When the nucleation site temperature exceeds the saturation temperature of the liquid, vapor bubble formation and growth may occur

36 Superheated Liquid If the heat rate is fast enough, the liquid may become superheated, that is the liquid temperature can exceed the boiling temperature A superheated liquid is in a metastable state Temperature continues to increase, the spinodal is reached and the liquid becomes unstable and catastrophically relaxes to a liquid-vapor mixture

37 P-T Characteristic

38 Homogeneous Nucleation Once in the metastable region a liquid need not reach the spinodal in order to change to a liquid-vapor mixture Homogeneous nucleation –the spontaneous creation of vapor nuclei within the liquid, without the aid of preexisting nucleation sites

39 Bubble-Jet Printing As the vapor bubbles grow and coalesce, a large bubble may be formed below the surface When the bubble reaches a critical size it will burst, propelling liquid droplets into the plume

40 Piezoelectric Transducer Mechanical Vibrations This alignment of molecules will cause the material to change dimensions - electrostriction

41 Piezoelectric Transducer

42

43 Printer Software The software drivers of the HP 550C are rewritten to allow for protein solutions of different viscosities and electrical charges to be printed The source code for the HP550C printer was provided by the manufacturer at no cost The new driver software constantly adjusts the voltages applied to the nozzle gate to account for different electrical resistance values in the solutions –This 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 printing –Dpi –Quality of Paper / Printing

45 Printer Circuit Board

46 Communication With Printer Head

47 Cell Encapsulation A technology used for the microencampsulation of live cells and tissues within protective membrane –Drug delivery systems The 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: –Biocompatible –Nontoxic –Non-immunogenic –Hydrophilic –Can be chemically cross-linked into hydrogels

49 Encapsulation Polymer Poly(lactic Acid) (PLA) –Aliphatic polyester Derived from lactic acid –Biodegradable –Thermoplastic

50 Encapsulation Polymer Poly(lactic-co-glycolic acid) (PLGA) –Copolymer of PLA and PGA –Microphase separation –Crystallinity –Water-solubility –Biodegradability

51 Biopaper The printing substrates that the encapsulated cells would adhere onto Made of biocompatible ECM-containing hydrogels

52 Biopaper Soy Agar Gel –Made from Trypticase soy agar solution –Initiate cell growth medium Observe colony morphology Develop pure culture Culture Storage –Good diffusion characteristics –Absence 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

54 Summary Current Technology -Photolithograpy -Soft Lithography -Contact Printing -Bioprinter Printer Technology -Bubble Jet -Piezoelectric Technical Considerations -Fluid & Cell Mechanics -Software -Encapsulation -Biopaper

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 1.“EE-527: Microfabrication- Photolithography.” 2.St. John PM, et al. Preferential glial cell attachment to microcontact printed surfaces. J Neuro Meth 1997; 75: 171-7. 3.Vogt AK, et al. Micropatterned substrates for the growth of functional neuronal networks of defined geometry. Biotechnol Prog 2003; 19: 1562-8. 4.Hibara A, et al. Surface modification method of microchannels for gas-liquid two-phase flow in microchips. Anal Chem 2005; 77(3): 943-7. 5.“Ink-Jet Printers.” 6.Roth EA. Xu T, DasM. Gregory C. Hickman. JJ. Boland T. Inkjet printing for high- throughput cell patterning.Biomaterials. 2004 Aug;25(17):3707-15. 7.Xu T. Jin J. Gregory C. Hickman JJ. Boland T. Inkjet printing of viable mammalian cells. Biomaterials. 2005 Jan;26(1):93-9. 8.Hsieh et al. Ultra-High-Throughput Microarray Generation and Liquid Dispensing Using Multiple Disposable Piezoelectric Ejectors. J of Biomolecular Screening 9(20;2004 9.Okamoto et al. Microarray Fabrication with covalent attachment of DNA using Bubble Jet Technology Nature Biotech 18;200. 10.Sanjana NE, Fuller SB. A fast flexible ink-jet printing method for patterning dissociated neurons in culture.J Neurosci Methods. 2004 Jul 30;136(2):151-63. 11.Poly(methyl acrylate-co-hydroxyethyl acrylate) hydrogel implant material of strength and softness. J Biomed Mater Res 1981;15(4):497-509. 12.Xu T, et al. Inkjet printing of viable mammalian cells. Biomaterials 2005; 26: 93-9.


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