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 Advantages
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 formation4

11. Soft Lithography Microfluidic Patterning Advantages Disadvantages
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
I guess you could use this slide……..

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

14. Thermal/Bubble Commercial ink-jet printers Printing Mechanism
Hewlett-Packard DeskJet 550C
Canon Bubble Jet 2100
Printing Mechanism
High temperature heating system5
Current pulse is applied for microseconds, which raises temperature ~300oC
Heated 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 layers
Suitable for printing:
polymer encapsulated cells6
cell solution6
Disadvantages
Limited Resolution um
Low precision single cell patterning
High Temperature
Motor 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 surface
Conventional DNA hybridization measured
Used to detect SNPs
Advantage
Low cost compared with photolithography
Less uncertainty
Disadvantage
Exposure of DNA to high temperature ~200oC
Shear stress (10m s-1)
Microarray using inkjet technology

17. Piezoelectric Commercial Printer Printing Mechanism
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 ink5

18. Piezoelectric Disadvantages Advantages Clogging Small drop sizes
Biomaterials
Environmental effect
Effect of Printing Technique
High frequencies
Compression
Friction9
Advantages
Small drop sizes
Picoliter
High-ejection rate
Several thousand
Complex patterns
Low cost

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: poise
Potential surface tension: 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
Weber 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.

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. Viscosity 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

31. Thermal Printing

32. Thermal Printing
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

33. Thermal Printing
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

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 velocity
Heterogeneous nucleation
Homogeneous nucleation

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. Piezoelectric Transducer

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 Printer Technology Technical Considerations
-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
“EE-527: Microfabrication- Photolithography.” <
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.” < 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;2004
Okamoto 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.