Development of a Temperature- Controlled Printing System for Solid Freeform Fabrication of Cell-Laden Matrigel Constru

1. Development of a Temperature- Controlled Printing System for Solid Freeform Fabrication of Cell-Laden Matrigel Constructs Mechanical Engineering Department Drexel University MEM-22 Dr. Wei Sun, Dr. Alan Lau, Dr. Alisa Morss Clyne, Robert Chang Team Members: Veronica Castro --- Mechanical Engineering Olga Filippova --- Biomedical Engineering Jason Hollestein --- Mechanical Engineering Munir Nahri --- Biomedical Engineering Akash Patel --- Biomedical Engineering 11/25/2008

2. Presentation Outline Problem Background Motivation for Design Need for Matrigel Constructs Unmet Engineering Challenges Problem Statement Objective Criteria for Success Method of Solution Constraints and Challenges Major Engineering Design Tasks Construct Characterization Alternative Solutions Feasibility Specific Deliverable Economic Analysis Societal, Ethical and Environmental Impacts Project Management Summary and Conclusions Acknowledgements

3. Motivation for Design • Biomaterials used for constructs directly impact effectiveness • Matrigel has great potential since it is naturally derived • Progress in creating Matrigel constructs is limited by the inability to print them using solid freeform fabrication • Tissue Engineering efforts are focused on creating better constructs • Constructs must better mimic natural environments to maintain cell viability, proliferation, and function • Construct efficacy is controlled by structure and biomaterial used • Solid Freeform Fabrication is the sequential delivery of cell-laden biomaterials to specified points in space to produce a scaffold • Allows for the micro-scale recreation of natural tissue structure

4. Need for Matrigel Constructs • Better mimics in vivo extracellular matrix composition • Thermal cross-linking of Matrigel better for cell viability • Compared to chemically cross-linked polymers • Matrigel improves cell viability, differentiation, and function as a construct material • Basement membrane extract from EHS mouse sarcoma (cancer of connective tissue) • Natural mixture of biopolymers • Composed of laminin, collagen IV, heparin sulfate proteoglycans, and entactin

5. Unmet Engineering Challenges in Matrigel Printing • Unique thermo-physical properties • Acts as a reverse fluid • Matrigel is a liquid at cold temps. and a cross-linked gel at room temps. • Transition temp at 4oC • Current Matrigel printing systems in ambient room temp fail due to gel occlusion • System needed to accommodate to the unique thermo-physical properties • Current printing systems are large and cannot be used inside of the sterilized environment of a hood

6. Problem Statement Objective: Design and develop a working prototype of a temperature-controlled printing system for solid freeform fabrication of cell-laden Matrigel constructs Criteria for successful design: • A 4°C environment must be maintained around the material delivery device to keep Matrigel in its liquid state • For sterilization, overall printing system must be small enough to fit into laboratory hood • Printing system must create Matrigel constructs with acceptable cell viability

7. Constraints and Challenges Printing must take place in a sterile environment System must fit into laboratory hood NuAir NU425-600 System must be kept at 4oC to prevent occlusion of material delivery system Printing processes should maintain acceptable cell viability levels Substrate should be viewable during printing Developed system must be economically feasible

8. Major Engineering Design Tasks Develop a temperature control thermodynamic cycle and enclosure to keep Matrigel delivery system at 4oC Develop a substrate heating system to decrease gel time after printing Develop a Matrigel delivery system that maintains cell viability Develop an automated motion system able to produce Matrigel constructs Assemble and integrate subsystems into a fully functional overall printing system Fabricate necessary joining components Perform mechanical and biological analysis to characterize printed construct

9. Overall System Schematic Thermal Enclosure 3 2 Thermodynamic Cycle 1 Support Arm Dispensing Valve Reservoir Temperature Control System Material Delivery System Motion System

10. System/Subsystem Outline

11. Temperature Control System 4 5 2 7 • Air Pressure • Valve Control • Temp Sensor 8. TCE 9. Pump 10. Spill Release 5. Filter 6. VCC 7. Pump • Pump • Dryer • Duct • Valve 6 1 3 A B 10 B A 8 9 C

12. Temperature Control System Create an environment that will maintain Matrigel at 4°C Enclose the valve, Matrigel reservior, and nozzle tip Prevent air leakage from inlet and outlet sites Use air as working fluid flows through enclosure Cool air using a thermodynamic cycle Perform heat transfer analysis Select the proper temperature sensor for enclosure Consultation of ASHRAE 1999 Fundamentals throughout design process

13. Matrigel Delivery System 2 4 1 3 5 Matrigel Loaded Air Pressure Applied Enters Valve Valve Opened Matrigel Extruded through nozzle tip

14. Matrigel Delivery System • Printed Matrigel must be printed with a uniform diameter • Determine the flow rate needed • Determine the necessary air pressure needed • Determine if printing will harm the cells embedded in Matrigel • Compare cell viability of printed construct with standard pipetting methods • Determine time required for Matrigel to change phase • If more than time required to print one layer, gelling time must be reduced • If above is true, determine the power input to the substrate to achieve faster gelling time • Perform heat transfer analysis to determine if heat added to substrate is enough to change phase of subsequent Matrigel layers

15. Motion System X motion Y motion Z motion Support Arm 2 3 4 1

16. Motion System • Determine the appropriate printing speed necessary to achieve cell uniformity • Ability to combine both printer, nozzle and cooling system seamlessly • Design apparatus to hold dispensing valve and thermal enclosure while printing • Perform FEA to analyze structural integrity

17. Construct Characterization • Optical • Visualization of construct shape • Biological • Cell viability – Live/Dead Cell Assay • Cell Proliferation – Alamar Blue Assay • Mechanical • Evaluate construct integrity

18. Heat Transfer Modeling Assume steady-state system Droplet of diameter D and T1 surroundings temperature on semi-finite medium of thermal conductivity K Temperature T2 is driven from bottom to top of surface Conduction: Heat flows to Matrigel Free Convection: Newton’s Cooling Law Heat loss per unit width

19. Alternative Solutions • Temperature Control • Determine thermodynamic most efficient and feasible vapor cycle • Determine best and safest working fluid • Determine is Matrigel should be pre-cooled • Determine best material for the enclosure • Determine best way to heat substrate (if necessary)

20. Alternative Solutions • Matrigel Delivery • Pneumatic or hydraulic pump • Determine if other valves, Matrigel reservoirs and nozzle tips are necessary • Motion System • Cartesian or Polar Coordinate driven • Free moving arm or XYZ axis • Purchase more accurate motion system after temperature control device is perfected

21. Feasibility • There are numerous thermodynamic cycles that can bring a working fluid to our desired temperature • Material delivery systems have been proven successful and is readily available • Prototype is economically feasible • Once Matrigel is printed successfully, existing motion systems with proper size and micron-scale printing accuracy could be adapted

22. Specific Deliverable A working prototype of a miniature, temperature-controlled Matrigel printing system

23. Economic Analysis

24. Societal and Ethical Impact • Societal Impact • Printing Matrigel allows for the creation of improved tissue engineering constructs • Small yet crucial step in developing better in vitro micro-organ systems • Ethical Impact • Intellectual property • Production of Matrigel involves the breeding and sacrifice of mice

25. Environmental Inpact • Hazardous materials must be kept and disposed of safely • Biological Waste • Matrigel • Cells • Chemical Waste • Refrigirant • Sterilizers • Some disposable components • Energy Use

26. Project Management

27. Summary and Conclusions • Need: Ability to print cell-laden Matrigel constructs which provide better microenvironments • Current limitations: No printing system accommodates Matrigel’s unique thermo-physical properties • Objective: Create a Matrigel printing system with an integrated temperature control, material delivery, and motion systems to maintain Matrigel at 4oC and print constructs with acceptable cell viability levels • Specific deliverable: Working prototype of miniature temperature-controlled Matrigel printing system

28. Aknowlegements • Advisors • Dr. Wei Sun, Dr. Alan Lau, Dr. Alisa Morss Clyne • Robert Chang • Sponsor • NASA Grant

29. Questions? Mechanical Engineering Department Drexel University MEM-22 Dr. Wei Sun, Dr. Alan Lau, Dr. Alisa Morss, Robert Chang Team Members: Veronica Castro --- Mechanical Engineering Olga Filippova --- Biomedical Engineering Jason Hollestein --- Mechanical Engineering Munir Nahri --- Biomedical Engineering Akash Patel --- Biomedical Engineering 11/25/2008

30. Appendix • Free Convection: • Parameters: • Droplet diameter: D = 250*10-6m • Droplet temperature: T1 = 4°C • Surface temperature: T2 = 37°C • Shape actor: S = 2D = 500*10-6m • Air velocity: U∞ = 2m/s • Dynamic Viscosity ν • Reynolds Number Re L • Length: l = 250*10-6m • Nusselt number Nū • Convection coefficient ħ