Biomedical research is facing a reproducibility crisis in that new research findings are rarely translated into therapeutic applications. This protocol reduces the human factor and introduces automation and standardization into manufacturing. This method focuses specifically on photo cross-linkable hydrogels for 3D cell culture applications as hydrogels have become the most used platform in cancer and other disease tissue models within the last decade.
We have successfully addressed the current hardware and software limitations with the development of an open source technology platform. This platform has been specifically designed for hydrogels and enables automated manufacturing workflows for tissue engineering research. To install the API, open the command line interface.
To install the work API, enter pip install openworkstation and press Enter. To operate the pipetting bio fabrication module, enter the command to install the opentrons API. Then use the command line to open a Python script and verify if both APIs have been installed successfully.
To generate the protocol code, open the protocol design application to generate a customized protocol script that will be executed by the platform. The interface runs on every commonly used internet browser. Enter the protocol name on the setup page and click Continue.
In the input tray setup, select three by four heating block to define the input tray. To define the materials and stock concentrations, select Gel 1 from the define inputs menu and enter GelMA as the name. Set the stock concentration to 20%and the number of samples to three and click Add to save the entries and to fill the first column.
Select Gel 2 from the define inputs menu and enter Alginate as the name. Set the stock concentration to 4%and the number of samples to three and click Add to save the entries and to fill the second column. After setting the photoinitiator and diluent parameters as demonstrated, select photo crosslinking, set the time to 30 seconds, and the intensity to two and click OK.Next, set the well plate type to 96 well plate and click Group 1 to allow the parameters for creating double network hydrogels to be specified.
Then check the apply advanced mixing protocol box if necessary, set the number of samples to 96, and click Continue. To set the deck layout, select the appropriate tray type for each slot. When all of the tray types have been selected, click the pipette left box and select 10 to 100 microliter positive displacement from the dropdown menu.
Set the aspirating speed to 600 and the dispensing speed to 800. Then set the pipette right parameters in the same manner. Then click Generate Protocol to generate and save the protocol script.
Before executing the protocol, spray the consumables with 70%ethanol and position them according to the setup defined in the user setup. Place the reaction tubes with the materials in the aluminum block on the temperature docks according to the selected setup. Next, spray your gloves with 70%ethanol and open the reaction tubes carefully without touching opened tubes.
When the substances have reached the appropriate experimental temperature, run the generated protocol using the user interface. The workstation will begin with the homing process, followed by getting an empty well plate from the storage module. After removing the lid from the well plate, the plate is transported to the next module.
The protocol specifies the volume being pipetted from each stock solution and automatically changes the tips after each material to prevent cross-contamination. To mix viscous solutions in a reproducible manner, the workstation executes a specific mixing protocol which has been optimized for viscous hydrogels. The protocol design app takes the filling level of the reservoir into account and automatically adapts the mixing height to prevent unnecessary dipping into the viscous materials.
After automatic generation of 3D models and well plates, the workstation closes the well plate with the lid again and stores the well plate in the programmed position in the storage module. Once the protocol is finished, remove the plate from the storage module. For validation and verification of the conducted protocol, load the plate onto a spectrophotometer and read the absorbance two times at 450 nanometers.
After saving the absorbance values, open the analysis spreadsheet file which is provided as supplementary material in the publication and copy the absorbance readings into the table in the raw data sheet. Then click on the analysis sheet to view the mean values, the standard deviation, and the coefficient of variance values, which are automatically calculated and displayed for a uniform sample distribution for specific rows and for specific columns of a 96 well plate. To find the setup that ensures a high reproducibility for glycerol solutions, protocols were generated without temperature control and without tip touch, with temperature control and without tip touch, or with temperature control and with tip touch.
The calculated coefficient of variation values for the three setups revealed a significant influence of the temperature dock and the tip touch function, highlighting the ability of the protocol to generate highly reproducible results when used both features. Using the tip touch function with the temperature dock, the standard deviation was significantly reduced in setup three. Plotting of the sample absorbance values for setup three yielded no increasing or decreasing values throughout the experiment and therefore indicated no influence of the sample position on the absorbance values.
Next, a GelMA dilution series were prepared by diluting a 20%GelMA stock solution and assessing the differences between different GelMA dilutions. The absorbance value measured at each concentration step were significantly different and linear regression demonstrated a high fit confirming the ability of distinct concentration steps to be generated. In addition, the influence of the tip touch was evaluated for double network hydrogels with 5%GelMA, 2%alginate, and 0.15%LAP, which were automatically generated with the setup.
The integration of the tip touch results in a significant decrease of the standard deviation supporting the generation of a reproducible dataset. Visualization of the absorbance values and heat maps confirmed the reduced deviation when using tip touch to remove excess material from the tip. Our technology enables the automation of hydrogel fabrication for 3D cell culture and tissue engineering.
It's a low-cost solution to increase the throughput and reproducibility of technically challenging workflows. By providing a customizable open source approach, this technology paves the way for the widespread adaptation of process automation in tissue engineering research.
