Reagent Development: Print Parameters
The Allevi bioprinter is a versatile machine capable of printing with a wide array of materials and cells types. In addition to pre-optimized materials, many users also choose to use their own materials with the Allevi. Below offers users a step-by-step guide to easily develop print parameters with your Allevi. In addition to print parameters, viability testing should also be completed with new materials to test toxicity and create 3D printed controls for experiments.
The first step in calibrating a material is determining pressure needed for extrusion. Fill a syringe with your material and load into the Allevi. Increase the pressure incrementally by about 5 psi until the material extrudes evenly. Take note of at least three to four pressures the material extrudes at, varying from a slow to fast extrusion.
Troubleshooting tip 1: Each time you increase the pressure, be sure to turn off the pressure then turn it back on again. Otherwise, the pressure settings on the dial may not accurately portray actual pressure output.
Troubleshooting tip 2: There are a variety of factors that can affect pressure needed for extrusion. Take note of what needle gauge and shape you are using. To lower pressure needed for extrusion, try using a lower gauge or a tapered needle. For more information on needles, check out our Guide to Picking Your Needle.
Troubleshooting tip 3: Material volume loaded into the syringe can also have an effect on pressure. Generally, material volume fluxes of ~ 2 ml can affect pressure needed for extrusion.
Troubleshooting tip 4: Be aware of what temperature you are completing the extrusion test at. Many materials change viscosity when heated or cooled. If you needed to heat or cool the material before loading into the syringe, take note of the amount of time allowed to reach printing temperature for future tests. Allevi 1, 3 and 6 offer wide range of printing temperatures: from 4 °C to 160 °C. Allevi 2 can heat up extruder up to 160 °C.
Troubleshooting tip 5: If the desired material takes a long time to cool or heat to desired print temperature, try placing loaded syringe in ice bath or heated water bath to speed process.
Results: This calibration step determines a series of extrusion pressures that will be used in later calibration tests.
*Want to learn how to write these custom extrusion files? Check out this post on understanding gcode.
Once the extrusion test has been completed, a crosslinking test should be used to determine necessary crosslinking time and, if necessary, photoinitiator concentration. Extrude equal amounts of material into a well plate and crosslink at varying times. Next, dry the samples in a vacuum oven overnight, then record the dry weight of each sample (w1). Immerse the samples in a solution in which they would dissolve if not crosslinked. For example, gelatin methacrylate should be immersed in PBS or deionized water and heated to 37 °C. After 24 h, remove the samples from solution and dry in a vacuum oven overnight. Record the weight of the dry samples (w2). Compare this second weight to the original weight to obtain the sol fraction.
The lower the sol fraction, the more the sample has crosslinked. A sol fraction of 100% correlates to a sample that has not crosslinked at all, while a sol fraction of 0% correlates to a sample that has completely crosslinked. Generally, sol fractions of less than 5% are acceptable. This preliminary test can also be completed more extensively with developed architectures post-calibration to determine and optimize mechanical properties of printed constructs and effects of crosslinking time on these properties.
Results: This calibration step determines necessary crosslinking time and conditions.
Once a range of pressures for extrusion has been determined, feed rate, path height and print speed must be optimized. This can be completed through a line test in which various lines are extruded at different print speeds (Figure 1). A line test using G-code provided by Allevi should be completed for each pressure and path height to be tested. Once completed, the lines can be analyzed via brightfield microscopy for accuracy and precision.
Troubleshooting Tip 1: Start with a wide range of speeds and path heights, then test a narrower range to optimize.
Troubleshooting Tip 2: If you find your line width is larger than desired, a smaller gauge may allow for finer extrusion. However, this might affect extrusion pressure.
Troubleshooting Tip 3: Larger line widths than expected may also be caused from incorrect path heights, which can cause the material to smudge. Try increasing path height.
Results: From this test, speed and path height are determined at the desired pressure and gauge. Line resolution at these desired parameters is also ascertained.
*Our pre-sliced gcode is written to make editing it easy. The gcode is set up in section as shown below.
You can change the layer height for each row (Ex: Row 22) and the speed for each section (Ex: Row 27 and 39).
Note: In gcode, speed is written in unit of ‘mm/min’ rather than ‘mm/s’. This means that ‘mm/s’ speeds are multiplied by 60.
X/Y Resolution Test
Once pressure, gauge, path height, print speed and feed rate are determined, the material spatial resolution in x and y directions can be determined. This can be completed through a resolution test with the G-Code provided from Allevi.
Results: This calibration step determines maximum spatial resolution. This parameter will help when designing more complex structures.
Single Layer Lattice Test
Now that resolution has been determined, your current optimized printing conditions can be tested with the lattice test. Print a single layer lattice of varying pore sizes to check print parameters determined in previous tests.
Troubleshooting Tip 1: Sometimes print speed and pressure need to be adjusted for better resolution of more complex prints. This test will determine if optimized parameters from previous tests work when applied to slightly more complex prints.
Troubleshooting Tip 2: If complex prints cause issues, be sure to check how the software is slicing your design file. Less-than-optimal software settings and G-code will affect printing results.
Results: This calibration step allows for further testing of optimized parameters before moving to more complex 3D layer tests.
To further optimize path height, the calibrated print settings determined in the line test should be tested through a z-stack calibration with the Pediatric Bronchi print. This print will allow for further optimization of path height and will also test the material ability to structurally support multiple layers in the z-direction. Accuracy of z-direction can be determined by measuring final height of construct and comparing to theoretical height from the design.
Troubleshooting tip 1: If you run into issues with z-stack calibration, try altering path height as well as crosslinking time between layers. Try pausing the print and crosslinking the first few layers for increased amounts of time before increasing height.
Troubleshooting tip 2: If you are still having issues after increasing crosslinking time of initial layers, you can also try enlarging the width of layers for increased support.
Troubleshooting tip 3: If the needle starts to run into previously printed layers, your path height is likely too small. Try increasing path height.
Troubleshooting tip 4: If the material extrudes only in droplets as layers increase, either the path height is too large or the print speed is too slow. Try either decreasing path height or increasing print speed.
Results: This calibration step further optimizes path height and can determine z-resolution.
Multi-layer Lattice Test
This final calibration tests all optimized parameters from previous calibrations, as well as complexity in 3D dimensions. Print multi-layer lattices of varying sizes with previously determined print parameters. This calibration test is meant as a final step to confirm all previous optimized parameters. If this test is not optimized, you may need to return to previous steps to recalibrate certain print settings.
Results: This calibration step allows for final testing of optimized parameters.
A volume test allows for estimation of volume extruded from the printer. To complete a volume test, first measure a series of known volumes of desired material. Plot the weights of these measurements against volume and use a line of best fit to estimate weight increase by volume (see figure 7 and equation 2).
Next, load your material into the Allevi at the temperature and pressure you plan to print. Extrude the material for equal amounts of time, then measure to obtain a series of weights per time printed (see figure 7 and equation 3). From these weights and the weight by volume estimate from the previous plot, you can estimate the amount of volume extruded over a period of time (see equation 4).
Equation 2 variables m1 and b1 can be determined from the first graph in figure 6. Equation 3 variables m2 and b2 are determined from the second graph in figure 6. From these two equations, the volume of material in a print can be estimated based on time of extrusion for the print.
Results: This calibration step determines a method for volume estimation based on time of extrusion for the print.
*see example extrusion files for reference.