Soft Tissue Kit Protocol

Wikis > Soft Tissue Kit Protocol

Overview

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Figure 1:  Live/dead image of human neonatal dermal fibroblasts encapsulated in bioprinted Gelatin/Fibrin

Processed Gelatin/Fibrin Soft Tissue Kit contains all the materials needed to print with this gelatin-based hydrogel, which solidifies through enzymatic crosslinking with a thrombin, calcium chloride, and transglutaminase solution. The recommended preparation provided in the user instruction below yields a streamlined matrix bioink that supports 3D printed cell-laden constructs. However, preparation can be modified by users to suit their needs.

Storage and Handling

Processed Gelatin should be stored at  4°C. Fibrinogen powder should be stored at -20°C. Transglutaminase, calcium chloride and thrombin powder  should be stored at -20°C. ɛ-Aminocaproic Acid powder should be stored at -20°C

Kit Includes

  • Printing needles
  • Sterile filters
  • Processed Gelatin
  • Fibrinogen Powder
  • Transglutaminase, calcium chloride and thrombin crosslinking powder
  • ɛ-Aminocaproic Acid optional media additive

You will Need

  • Petri dish or well plate for printing
  • 10 mL BD Syringes
  • DMEM and any other desired media supplements
  • PBS (phosphate-buffered saline) without magnesium or calcium

Instructions for Use

Just looking to practice printing or test a file design? Try preparing the 10-15% gelatin mixture without fibrinogen and printing it on its own! While temporary, this fast preparation will allow you to quickly test new designs.

  • Prepare crosslink solution by dissolving crosslink powders in suggested volume of PBS and sterile filtering the solution with a 0.2 µm syringe filter*

Suggested concentrations: For every 100 ml of solution, add 0.1 g of Transglutaminase, 0.022 g Calcium Chloride, and 2 KU of thrombin.

  •  Add sterile PBS without magnesium or calcium at 37°C to fibrinogen powder to create a 35 mg/ml solution.  The solution should take about 30 minutes to 1 hour to dissolve.

Critical step: If available, use a large dish and slowly sprinkle fibrinogen over warm PBS, being careful not to agitate. If the powder takes more than 1 hour to dissolve, the fibrinogen has likely clotted. Redo this step with fresh fibrinogen.

  • Sterilize fibrinogen solution by exposing to UV light for at least 30 minutes.
  • Heat gelatin solution to 37°C and filter with a 0.45 µm syringe filter under sterile conditions.*

Note: This step is a bit difficult and sometimes frustrating to users. The filtration process requires some arm muscle, and you will likely lose some of the of the material during the filtration process. We are working on finding an alternative sterilization method for this step. To help, try heating up the syringe filter and syringe prior to filtration and make sure to quickly filter the gelatin solution after removing from heat. If the gelatin begins to solidify again, reheat it before continuing filtration.

  • Mix sterile gelatin and fibrinogen solution for a final concentration of 10-15% gelatin and 10 mg/ml fibrinogen.

Note: 10% gelatin will provide better resolution and decreased clogging and is suggested at room temperatures of 21 – 24 °C. At room temperatures of 24-28°C, a 15% solution is suggested to provide greater shape fidelity.

  •  Mix in desired cell concentration in a sterile container. Continue to reheat solution as necessary to maintain  37°C temperature*
  •  Load cell-laden bioink into a 10 ml sterile luer lok syringe at 37°C. Cool bioink on ice for about 8 min. Load syringe into the printer and allow for another 5-10 minutes for it to reach room temperature.

Critical step: The bioink can take a while to cool to room temperature. It is important to cool the material on ice prior to loading to speed up this process.

  •  Load syringe into BioBots printer.
  •  Prepare your design file through Repetier Host, then upload the sliced gcode file to biobots software.
  •  As soon as a structure is finished printing, immediately add crosslink solution. Allow the structure to crosslink for 30 minutes before removing the solution and rinsing with PBS.
  • Optional: Prepare cell culture medium and add ɛ-Aminocaproic Acid to decrease degradation rate of the material.

* For prints without cells, these steps may be skipped.

Print Settings

Remember: When setting up your pressure, calibrate each time by beginning at 0 psi, turning the pressure on, then slowly increasing the pressure until there is consistent extrusion of material from the syringe. This calibration method will help prevent loss of material from a pressure setting that is too high.

Slic3r Settings
Speed (mm/s) Layer height (mm) Nozzle Diam (mm)   Gauge

8.33

0.2 0.3 27 0.25"

*Critical step: Your needle can have significant effects on print settings. Be sure to use the suggested needle type or re-calibrate print parameters.

Print Pressure and Temperature Settings
Pressure (psi) Print Temp (°C)

10-15

23

*Note: Your pressure will vary based on gelatin concentration, cell concentration, and needle type. Ideal print pressure for lower gelatin concentrations is 20 -23°C. For higher print temperatures, such as 24 -28°C, a higher concentration of gelatin is suggested. 

Notes

  1. Gelation time and gel stiffness can be adjusted by varying the concentration of processed gelatin. For help adjusting print parameters please contact info@biobots.io.
  2.  A fill volume change of more than 2 ml may affect pressure settings.
  3.  A lower gauge size or tapered gauge will require lower pressure, while a higher gauge will require higher pressure for extrusion. Lowering the gauge size will also generally lower resolution

for further questions or information about these products, please contact info@biobots.io