Meeting Minutes 09/11/2013 12PM
-Discussed with Dr. Hall potential of moving printer for access -Moved to AR- 125
-Mouse embryo fibroblast cells for printing -Compatible with PCL scaffold
-Discussion of which growth factor would be best -Challenge = how to deposit/which is feasible to use -Fibroblast growth factor II
-Discussion of ultimate goals—what do we want? -What tissue should we aim to make?
Tasks:
-Should we continue with cartilage? What about bone (osteocytes)? -Realistic constraints
-What can be an ultimate goal?
-What is too much? What is too little? -Determined PLC would be realistically be used for project
-Do more research -Come up with potential ideas for tissue
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Minutes 09/13/2013
Meeting with Dr. Wagner
-Presented idea of developing bone rather than cartilage (easier) -Many uses for PCL, must determine what -Need to determine ultimate goals
-Refinement and enhancement of printer (Fall goal) -TE device (Spring)
-Cells -Structure
-What polymer(s)
-Design -Goals dictate design which dictates specifics
-Need to start early on cell culturing, takes a lot of time -need assay for proliferation
-Goals become requirements (IMPORTANT for tables for presentation) -Printer
-Device -Mathematical modeling needed for shear stress on cells -Prove each requirement made
-Justifications are important -Need marker to show they are growing (cells)
-testing phase -BUDGET- do a lot of research now to create budget; will likely need to appeal for more money -Must demonstrate that it WORKS -Goals → requirements → device and cells → testing with assays/function of cells -Gannt chart
-Complete after determining goals -Determine responsibilities for each member -Remember that lab is BSL 1 → no human cells -Find available cells that can be used with PCL (many are compatible) -Perhaps look at Invitrogen life
Tasks:
-ATTC -Stem cell technology
-Articulate goals -Once articulated, write up requirements -More research-
-Break up tasks -Make Gannt chart -Determine budgeting (by due date)Meeting at 7:00PM (9/13/13) Steps for Cell Culturing:
-Ensure midlog growth phase confluence - 60-80% -Add 5-10mL DPBS (Dulbecco’s Phosphate-Buffered Saline)
-Provide buffer system to maintain cell culture media in the physiological pH range or to
irrigate, transport, or dilute fluid while maintaining cell tonicity and viability -Add TrypLE express prewarmed (cell compatibility)
-Cell dissociation from one another -Coat culture surface and incubate 5-8 minutes, 36-38 degrees Celsius -or until cell healthily detached
-Pipet detached cell into single cell solution, verify on inverted microscope -Verification of cell health
-Round/plump- refracting light around membrane -Discard cells if:
-Detaching in large numbers (attached lines) and/or look shrivelled and grainy/ dark in color -In quiescence
-After extrusion of scaffold:
-Coat scaffold with collagen- Collagen surface coatings improve biocompatbility, facilitates cell adhesion (“Lee CH, Biomedical Applications of Collagen”)
-Remove collagen- ‘draw back up’ through syringe -Let sit for 3 hours- ensure complete emersion -Extrude suspension of cells + standard growth media, same syringe, to create composite cell scaffold
-Perhaps sterilize through well with alcohol, or input extra syringe -Cultivate for 2 hours under high humidity
-Cite Protocol for justification (DUTY) -Dry cells out (humidity levels should be higher, above 95%)
- Specifications -Temperature 36-38 degrees celsius -Add warmed chondrogenesis media
-Protocol -Incubate 37 degrees celsius, 5% CO2
-Controls pH and osmolality of culture -Life technologies -http://www.lifetechnologies.com/us/en/home/references/gibco-cell-culture- basics/cell-culture-environment/ph-co2-levels.html
-Refeed every 2-3 days with more media (consumption/nutrition)
-Greater than 14 days - Gene expression analysis, protein detection, or immunohistochemistry
-Verification of results -Alcian blue stain analysis
-Blue staining indicates synthesis of proteoglycans by chondrocytes
-Began researching temperature/humidity control devices for environmental chamber -Omega RHCN-1F Relative Humidity/Temperature Controller
Tasks: (Due 9/20) -Begin to think of research and tasks needed to be completed within the next few weeks
-Chris: work on Gaant chart
-Scott: Begin website design which needs to be complete by Oct 2.
-Scott & Kim: begin design of Environmental Chamber, determine parts and what is needed
-Take into account protection of microcontrollers
-Research viable options for humidity/temperature control
-George: Budgeting
Next week: Due (Friday 9/20) -Propose biology to wagner
Issues:
-Validate printer→ address issues -Confirm what materials we have -Start creating list of materials for biology
-Endstops -Slic3r-Pronterface -Nozzle? -Others
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Minutes 9/16
-Attempting to get the printer working to where previous team had
-Problems with starting the printer
-Side with syringe side will come out, preventing from pulling up
-Nothing should be moving when turned on, but random parts move at different times
-Nothing should be moving when turned on, but axes moving randomly
-Syringe pump goes up/down
-All axes moving, one after another, but randomly not in uniform
-Does not zero itself will not stay still, continuously moves up/down or side to side
-Research humidity effects on solutions
-Rotating chamber may be affected my water
Need to consider specifications for environmental control
-Humidity- will it affect other parts? The solutions in the wells?
-How vital is partial pressure- CO2 for cells?
-Talked to Hayin
-Should only plug in after uploaded
-Able to turn printer on and turn on heater
-Experimented with moving the axes of the printer using Pronterface
Arduino Setup
-Download Arduino, Slic3r and Pronterface
-When I downloaded Pronterface, it already came up with Slic3r, so maybe download that first
-Look for the .ino file in the Marlin folder given from the previous senior project group
-Create a shortcut for arduino onto desktop (for convenience purposes mostly)
-Drag marlin.ino file into the shortcut
-If no other files are uploaded with it, manually input each .h and .cpp file by going to sketch, add file…
-When all files uploaded, click on Verify (check mark on arduino)
-When printer connected to laptop via usb, click upload
-If it does not upload because of a driver error, download driver for FT232 USB
-Open Pronterface
-Set Com4 @250000
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Updates/Minutes 09-18-2013 -Suggestion of uv sterilization than ethanol
-more benefits -temperature/humidity control may affect ethanol -No waste deposit
-Assigning roles for each person -Scott: overall design of chamber (mechanical)
codes
Wells
-Size, shape, material -Tubing for wastes(?) leading out of chamberà vacuum pump?
-Wondering if it’s a possibility -Kim: Design/incorporate environmental control unit into the chamber
-Chris: Implement method for UV sterilization -Sterilization of scaffold, material,e tc
-George: Smoother transition, upload 1 code for phase 1 and 2 instead of 2 separate -Fix printer so that Z axes are not 2 different parts controlled
-Collagen -cell suspension -standard growth media -Chondrogenesis media -Waste into other wells
à Easier to take out rotating chamber -Devise different method for getting materials into dish
-Using stepper stick for other axis instead of having to attach another on -IDEA
-Attach translational wells to the x axis (next to the dish) -Extending from side to allow for easier coding -Would not need to create separate controls or think about the mechanical aspects/modifications that would need to be made -Would only have to increase dimensions of the case
Talked to Joe for machine shop access -Set screw not touching shaft
-Took off casing and tightened -Should write subprogram for ‘home’ positions
-Will direct printer to go back to 0 -Problems with controlling y axis
Cell Culture
Goals-Implement uv sterilization -Better with humidity control without affecting solvents
-Design environmental chamber -Implement environmental control for chamber
Printer- Enhancements/Refinements: -Better method for extracting waste
-Altering chamber -Chamber for materials (media) – alter
-Smoother transition between phase 1 and 2 -Alter mechanical problems
-Missing screws, fix end stops, etc.
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Updates/Minutes 09-18-2013
-Suggestion of uv sterilization than ethanol
-more benefits
-temperature/humidity control may affect ethanol
-No waste deposit
-Assigning roles for each person
-Scott: overall design of chamber (mechanical)
-Size, shape, material
-Tubing for wastes(?) leading out of chamber vacuum pump?
-Wondering if it’s a possibility
-Kim: Design/incorporate environmental control unit into the chamber
-Chris: Implement method for UV sterilization
-Sterilization of scaffold, material,e tc
-George: Smoother transition, upload 1 code for phase 1 and 2 instead of 2 separate codes
-Fix printer so that Z axes are not 2 different parts controlled
Wells
-Collagen
-cell suspension
-standard growth media
-Chondrogenesis media
-Waste into other wells
Easier to take out rotating chamber
-Devise different method for getting materials into dish
-Using stepper stick for other axis instead of having to attach another on
-IDEA
-Attach translational wells to the x axis (next to the dish)
-Extending from side to allow for easier coding
-Would not need to create separate controls or think about the mechanical aspects/modifications that would need to be made
-Would only have to increase dimensions of the case
Talked to Joe for machine shop access
-Set screw not touching shaft
-Took off casing and tightened
-Should write subprogram for ‘home’ positions
-Will direct printer to go back to 0
-Problems with controlling y axis
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Minutes 9/23/2013- With Dr. Wagner
-Design: List out requirements/improvements needed -Assign each task to each individual
-Should not break into, in process à aseptic environment -Requirement: fix à print cells for aseptic processing
-Limit human contact -Define Goals clearly
-In wells
-Find a way to mix suspension/cells -Cells would settle -Account for shearing
-Function of volume and evaporation rate -Do not necessarily need to account for humidity for printer
-Cell Culturing
-Should have 37 degrees C -Slows metabolic processes
-May stress cells
-Be careful with differentiation kit à works with non humans MSCs -Call manufacturer à ask about kit -Are you aware of whether it would work with rat or mouse -Invitrogen = Life Technologies
-Look for references/publications done with kit -May ask technical support
-Trypsin inhibited by a lot of media -PBS rinse so that trypsin works well
-Differences between 2 kits -Think about how to prove goal
- build scaffold that supports chondrocytes
-How to test if it works/differentiated/proliferate - Need positive/negative controls, do assays work as intended
-Test chondrocytes/differentiation without scaffold to test for differentiation -Does scaffold affect assay?
Fix:
-On scaffold without -Assay on scaffold-Z axis syringe à move upwards -Perhaps control through Pronterface -Change parameters for y axis (steps away from home position to
account for sterilization -End stops
-UV light -Chamberà temperature control -Rods from syringe (metal) à too short -Fix extrusion of PCL à how it goes into nozzle
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
-Discussed with Dr. Hall potential of moving printer for access -Moved to AR- 125
-Mouse embryo fibroblast cells for printing -Compatible with PCL scaffold
-Discussion of which growth factor would be best -Challenge = how to deposit/which is feasible to use -Fibroblast growth factor II
-Discussion of ultimate goals—what do we want? -What tissue should we aim to make?
Tasks:
-Should we continue with cartilage? What about bone (osteocytes)? -Realistic constraints
-What can be an ultimate goal?
-What is too much? What is too little? -Determined PLC would be realistically be used for project
-Do more research -Come up with potential ideas for tissue
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Minutes 09/13/2013
Meeting with Dr. Wagner
-Presented idea of developing bone rather than cartilage (easier) -Many uses for PCL, must determine what -Need to determine ultimate goals
-Refinement and enhancement of printer (Fall goal) -TE device (Spring)
-Cells -Structure
-What polymer(s)
-Design -Goals dictate design which dictates specifics
-Need to start early on cell culturing, takes a lot of time -need assay for proliferation
-Goals become requirements (IMPORTANT for tables for presentation) -Printer
-Device -Mathematical modeling needed for shear stress on cells -Prove each requirement made
-Justifications are important -Need marker to show they are growing (cells)
-testing phase -BUDGET- do a lot of research now to create budget; will likely need to appeal for more money -Must demonstrate that it WORKS -Goals → requirements → device and cells → testing with assays/function of cells -Gannt chart
-Complete after determining goals -Determine responsibilities for each member -Remember that lab is BSL 1 → no human cells -Find available cells that can be used with PCL (many are compatible) -Perhaps look at Invitrogen life
Tasks:
-ATTC -Stem cell technology
-Articulate goals -Once articulated, write up requirements -More research-
-Break up tasks -Make Gannt chart -Determine budgeting (by due date)Meeting at 7:00PM (9/13/13) Steps for Cell Culturing:
-Ensure midlog growth phase confluence - 60-80% -Add 5-10mL DPBS (Dulbecco’s Phosphate-Buffered Saline)
-Provide buffer system to maintain cell culture media in the physiological pH range or to
irrigate, transport, or dilute fluid while maintaining cell tonicity and viability -Add TrypLE express prewarmed (cell compatibility)
-Cell dissociation from one another -Coat culture surface and incubate 5-8 minutes, 36-38 degrees Celsius -or until cell healthily detached
-Pipet detached cell into single cell solution, verify on inverted microscope -Verification of cell health
-Round/plump- refracting light around membrane -Discard cells if:
-Detaching in large numbers (attached lines) and/or look shrivelled and grainy/ dark in color -In quiescence
-After extrusion of scaffold:
-Coat scaffold with collagen- Collagen surface coatings improve biocompatbility, facilitates cell adhesion (“Lee CH, Biomedical Applications of Collagen”)
-Remove collagen- ‘draw back up’ through syringe -Let sit for 3 hours- ensure complete emersion -Extrude suspension of cells + standard growth media, same syringe, to create composite cell scaffold
-Perhaps sterilize through well with alcohol, or input extra syringe -Cultivate for 2 hours under high humidity
-Cite Protocol for justification (DUTY) -Dry cells out (humidity levels should be higher, above 95%)
- Specifications -Temperature 36-38 degrees celsius -Add warmed chondrogenesis media
-Protocol -Incubate 37 degrees celsius, 5% CO2
-Controls pH and osmolality of culture -Life technologies -http://www.lifetechnologies.com/us/en/home/references/gibco-cell-culture- basics/cell-culture-environment/ph-co2-levels.html
-Refeed every 2-3 days with more media (consumption/nutrition)
-Greater than 14 days - Gene expression analysis, protein detection, or immunohistochemistry
-Verification of results -Alcian blue stain analysis
-Blue staining indicates synthesis of proteoglycans by chondrocytes
-Began researching temperature/humidity control devices for environmental chamber -Omega RHCN-1F Relative Humidity/Temperature Controller
Tasks: (Due 9/20) -Begin to think of research and tasks needed to be completed within the next few weeks
-Chris: work on Gaant chart
-Scott: Begin website design which needs to be complete by Oct 2.
-Scott & Kim: begin design of Environmental Chamber, determine parts and what is needed
-Take into account protection of microcontrollers
-Research viable options for humidity/temperature control
-George: Budgeting
Next week: Due (Friday 9/20) -Propose biology to wagner
Issues:
-Validate printer→ address issues -Confirm what materials we have -Start creating list of materials for biology
-Endstops -Slic3r-Pronterface -Nozzle? -Others
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Minutes 9/16
-Attempting to get the printer working to where previous team had
-Problems with starting the printer
-Side with syringe side will come out, preventing from pulling up
-Nothing should be moving when turned on, but random parts move at different times
-Nothing should be moving when turned on, but axes moving randomly
-Syringe pump goes up/down
-All axes moving, one after another, but randomly not in uniform
-Does not zero itself will not stay still, continuously moves up/down or side to side
-Research humidity effects on solutions
-Rotating chamber may be affected my water
Need to consider specifications for environmental control
-Humidity- will it affect other parts? The solutions in the wells?
-How vital is partial pressure- CO2 for cells?
-Talked to Hayin
-Should only plug in after uploaded
-Able to turn printer on and turn on heater
-Experimented with moving the axes of the printer using Pronterface
Arduino Setup
-Download Arduino, Slic3r and Pronterface
-When I downloaded Pronterface, it already came up with Slic3r, so maybe download that first
-Look for the .ino file in the Marlin folder given from the previous senior project group
-Create a shortcut for arduino onto desktop (for convenience purposes mostly)
-Drag marlin.ino file into the shortcut
-If no other files are uploaded with it, manually input each .h and .cpp file by going to sketch, add file…
-When all files uploaded, click on Verify (check mark on arduino)
-When printer connected to laptop via usb, click upload
-If it does not upload because of a driver error, download driver for FT232 USB
-Open Pronterface
-Set Com4 @250000
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Updates/Minutes 09-18-2013 -Suggestion of uv sterilization than ethanol
-more benefits -temperature/humidity control may affect ethanol -No waste deposit
-Assigning roles for each person -Scott: overall design of chamber (mechanical)
codes
Wells
-Size, shape, material -Tubing for wastes(?) leading out of chamberà vacuum pump?
-Wondering if it’s a possibility -Kim: Design/incorporate environmental control unit into the chamber
-Chris: Implement method for UV sterilization -Sterilization of scaffold, material,e tc
-George: Smoother transition, upload 1 code for phase 1 and 2 instead of 2 separate -Fix printer so that Z axes are not 2 different parts controlled
-Collagen -cell suspension -standard growth media -Chondrogenesis media -Waste into other wells
à Easier to take out rotating chamber -Devise different method for getting materials into dish
-Using stepper stick for other axis instead of having to attach another on -IDEA
-Attach translational wells to the x axis (next to the dish) -Extending from side to allow for easier coding -Would not need to create separate controls or think about the mechanical aspects/modifications that would need to be made -Would only have to increase dimensions of the case
Talked to Joe for machine shop access -Set screw not touching shaft
-Took off casing and tightened -Should write subprogram for ‘home’ positions
-Will direct printer to go back to 0 -Problems with controlling y axis
Cell Culture
Goals-Implement uv sterilization -Better with humidity control without affecting solvents
-Design environmental chamber -Implement environmental control for chamber
Printer- Enhancements/Refinements: -Better method for extracting waste
-Altering chamber -Chamber for materials (media) – alter
-Smoother transition between phase 1 and 2 -Alter mechanical problems
-Missing screws, fix end stops, etc.
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Updates/Minutes 09-18-2013
-Suggestion of uv sterilization than ethanol
-more benefits
-temperature/humidity control may affect ethanol
-No waste deposit
-Assigning roles for each person
-Scott: overall design of chamber (mechanical)
-Size, shape, material
-Tubing for wastes(?) leading out of chamber vacuum pump?
-Wondering if it’s a possibility
-Kim: Design/incorporate environmental control unit into the chamber
-Chris: Implement method for UV sterilization
-Sterilization of scaffold, material,e tc
-George: Smoother transition, upload 1 code for phase 1 and 2 instead of 2 separate codes
-Fix printer so that Z axes are not 2 different parts controlled
Wells
-Collagen
-cell suspension
-standard growth media
-Chondrogenesis media
-Waste into other wells
Easier to take out rotating chamber
-Devise different method for getting materials into dish
-Using stepper stick for other axis instead of having to attach another on
-IDEA
-Attach translational wells to the x axis (next to the dish)
-Extending from side to allow for easier coding
-Would not need to create separate controls or think about the mechanical aspects/modifications that would need to be made
-Would only have to increase dimensions of the case
Talked to Joe for machine shop access
-Set screw not touching shaft
-Took off casing and tightened
-Should write subprogram for ‘home’ positions
-Will direct printer to go back to 0
-Problems with controlling y axis
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting Minutes 9/23/2013- With Dr. Wagner
-Design: List out requirements/improvements needed -Assign each task to each individual
-Should not break into, in process à aseptic environment -Requirement: fix à print cells for aseptic processing
-Limit human contact -Define Goals clearly
-In wells
-Find a way to mix suspension/cells -Cells would settle -Account for shearing
-Function of volume and evaporation rate -Do not necessarily need to account for humidity for printer
-Cell Culturing
-Should have 37 degrees C -Slows metabolic processes
-May stress cells
-Be careful with differentiation kit à works with non humans MSCs -Call manufacturer à ask about kit -Are you aware of whether it would work with rat or mouse -Invitrogen = Life Technologies
-Look for references/publications done with kit -May ask technical support
-Trypsin inhibited by a lot of media -PBS rinse so that trypsin works well
-Differences between 2 kits -Think about how to prove goal
- build scaffold that supports chondrocytes
-How to test if it works/differentiated/proliferate - Need positive/negative controls, do assays work as intended
-Test chondrocytes/differentiation without scaffold to test for differentiation -Does scaffold affect assay?
Fix:
-On scaffold without -Assay on scaffold-Z axis syringe à move upwards -Perhaps control through Pronterface -Change parameters for y axis (steps away from home position to
account for sterilization -End stops
-UV light -Chamberà temperature control -Rods from syringe (metal) à too short -Fix extrusion of PCL à how it goes into nozzle
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------