An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

NOTE: The protocol starts with an introduction to (1) the software and (2) the hardware setup to familiarize the user with required installations and the workstation. Following a section on (3) material preparation and (4) the usage of the protocol designer application, (5) the calibration of the pipetting module and (6) the execution of the automated protocol is highlighted in detail. Finally, (7) validation and verification procedures are described, including absorbance reading and data analysis. A general protocol workflow with individual tasks is displayed in Figure 1.

1. Software setup

NOTE: This section includes a detailed instruction to install the application programming interface (API) as well as the required protocol designer application and the calibration terminal. The following instructions are written for a Raspberry Pi (RPi) single-board computer; however also Windows 8, 10 and macOS 10.13+ have been successfully used with the API and the applications.

1. Set up the computer environment.
   NOTE: Be familiar with the basics of Python³⁹, how to set up and use a Raspberry Pi^40,41, and how to connect to the internet⁴². The following tutorial steps focus on protocol-specific steps and additional information on the usage of a Raspberry Pi is available online⁴⁰.
   1. Open a terminal window from the taskbar or application menu.
   2. Update the system’s package list:
      sudo apt-get update
   3. Upgrade all the installed packages:
      sudo apt-get dist-upgrade
   4. Restart the Raspberry Pi:
      sudo reboot
   5. Check installed Python version:
      python3 –version
      Make sure that at least Python 3.5 is installed; if not, install the latest version⁴³.
   6. Install python pip, which publishes Python packages with the Python Package Index⁴⁴:
      sudo apt-get install python3-pip
   7. Install dependencies:
      pip install numpy
      pip install python-resize-image
      NOTE: If you are using Windows, you need to install the windows-curses package via: python -m pip install windows-curses
2. Install the application programming interface (API).
   NOTE: The API provides a simple Python framework designed to write experimental protocols script and operate the workstation. The following two APIs are required to successfully execute the generated protocol code.
   1. Install workstation API:
      pip install openworkstation
   2. Install Opentrons API to operate the pipetting module:
      pip install opentrons==2.5.2
   3. Verify, if API is installed successfully:
      python3
      >>> import openworkstation
      >>> import opentrons
      NOTE: The size of the API and the protocol design application is 2.2 MB and 1.2 MB, respectively. No issues were experienced during the installation when used with limited disk space (200 MB). However, the disk space requirements depend on the operating system.
3. Select a directory for file download (calibration terminal, protocol design application, etc.).
   NOTE: Files can be copied and pasted elsewhere afterwards.
4. Clone files from GitHub repository:
   git clone https://github.com/SebastianEggert/OpenWorkstation
   NOTE: The ‘git clone’ command clones and subsequently saves all files into the directory, which is open in the terminal at this time. Since the repository also includes the hardware files for the assembly, not the entire repository is required to execute the presented protocols. All required files to replicate the experiments are available as Supplemental File and in the GitHub repository under “/examples/publication-JoVE”.
5. Open the downloaded folder. If the entire repository was downloaded, navigate to the ‘publication-JoVE’ folder via
   cd openworkstation/examples/publication-JoVE
   NOTE: This folder includes files required for the operation of the workstation and the usage of the protocol designer application and the calibration terminal.

2. Hardware setup

1. Place the workstation in a biological safety cabinet to avoid sample contamination.
2. Install pipettes on the workstation.
   1. Select the pipette size based on the experimental setup. In general, take a pipette size which volume to be aspirated is on the higher end of the range. If mixing tasks with volumes greater than 1 mL are required for a specific setup (e.g., aspirating/dispensing of 2 mL), choose the M1000E with a maximal aspirating/dispensing volume of 1,000 µL to minimize pipetting steps and save time.
      NOTE: A detailed instruction for air-displacement pipettes is available online⁴⁵. The developed pipetting module is able to integrate the following off-the-shelf positive displacement pipettes: M10E (1–10 µL), M25E (3–25 µL), M50E (20–50 µL), M100E (10–100 µL), M250E (50–250 µL), M1000E (100–1,000 µL).
   2. Use an M4 Allen key to loosen and tighten screws.
   3. Attach the two pipette fixation plates (white acrylic plates) to the aluminum rail and tighten M5 screws loosely.
   4. Insert the pipette into the two pipette fixation plates and ensure that the ergonomic tail of the pipette is resting on the opposite side of the acrylic mounting plate.
   5. Tighten the four screws of the two pipette fixation plates firmly.
   6. Slide the two square fastening nuts, which are attached to the acrylic mounting plate, into the extrusion slot of the z-axis and tighten screws.
      NOTE: Fasten pipette tightly to avoid any movement during operation.

3. Material preparation

NOTE: The viscous materials (glycerol, GelMA, alginate) are used for the experiments presented in this study, and, therefore, prepared volumes and handling tasks (e.g., add 5 mL of stock solution in 5 mL reaction tubes) are specifically for this experimental setup.

1. Gelatin methacryloyl (GelMA)
   NOTE: GelMA functionalization, dialysis, and lyophilization are not the scope of this paper, and a step-by-step protocol is available in Loessner et al.³³. The protocol starts using lyophilized GelMA, which can be prepared in-house or purchased commercially.
   1. Calculate required mass of GelMA (m_GelMA) based on the desired final stock concentration (c_GelMA) and volume (V_GelMA) using the equation:
      m_GelMA = c_GelMA x V_GelMA
      NOTE: VGelMA depends on the experimental setup and it is recommended to prepare 20−30% excess material. The presented protocols start with 5 mL of 20% (w/v) GelMA as a stock solution.
   2. Weigh the required amount of lyophilized GelMA, add it into a 50 mL reaction tube and add the required amount of phosphate buffered saline (PBS).
   3. Mix GelMA either by soaking into the solvent at 4 °C overnight or by heating to 60 °C for 6 h in a water bath.
      NOTE: Sterile GelMA solutions can be stored protected from light at 4 °C for at least six months.
   4. Fill 5 mL of GelMA into 5 mL reaction tubes.
2. Photoinitiator: Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)
   NOTE: Avoid additional exposure to room light, since LAP is light sensitive.
   1. Calculate required mass of LAP (m_LAP) based on the desired final stock concentration (c_LAP) and required volume (V_LAP) using the equation:
      m_LAP = c_LAP x V_LAP
      NOTE: It is recommended to prepare a 3% (w/v) stock solution.
   2. Weigh the required amount of LAP, add it into a 15 mL reaction tube and add PBS.
   3. Wrap the tube in aluminum foil to prevent photo-induced decomposition.
   4. Dissolve LAP by placing the reaction tube in a water bath at 37 °C for 2 h or until fully dissolved.
   5. Fill 1 mL of LAP stock solution in 5 mL tubes.
3. Alginate
   1. Calculate required amount of alginate (m_alginate) based on the desired final stock concentration (c_alginate) and volume (V_alginate) using the equation:
      m_alginate = c_alginate x V_alginate
      NOTE: Valginate depends on the experimental setup and it is recommended to prepare 20−30% excess material. The presented protocols start with 5 mL of 4% (w/v) alginate as a stock solution.
   2. Weigh the required mass of alginate, add it into a 50 mL reaction tubes, and add PBS.
   3. Place the alginate mix into a water bath at 37 °C for 4 h.
      NOTE: The usage of a vortex mixer will accelerate the dissolution process, but also generate air bubbles. Dissolved alginate can be stored at 4 °C for at least six months.
   4. Fill 5 mL of alginate into 5 mL reaction tubes.
4. Fill 5 mL of glycerol in 5 mL reaction tubes.
5. Orange G solution
   1. Prepare a 10 mg/mL stock solution of Orange G in a 50 mL reaction tube.
      NOTE: The volume depends on the number of experiments. Depending on the diluent type, prepare stock solution either in ultrapure water, PBS or a suitable diluent reagent. In the presented experiments, ultrapure water was used for diluting glycerol and PBS for diluting GelMA and alginate. PBS was used as a diluent for GelMA and alginate, and can be either prepared using tablets or purchased off-the-shelf.
   2. Mix for 10 s by vortexing.
   3. Wrap the tube in aluminum foil to prevent photo-induced decomposition.
      NOTE: Stock solution can be used after 24 h to ensure proper dissolution of Orange G.
   4. Dilute stock solution to a 1 mg/mL working solution in a 50 mL reaction tube.
   5. Transfer working solution to appropriate flasks/tubes for the experimental setup.
      NOTE: For the presented experiments, the working solution was filled into 5 mL tubes. Orange G stock and working solution can be stored at 4 °C and used within three months upon preparation.
   6. Fill 5 mL of the 1 mg/mL Orange G working solution in 5 mL reaction tubes.

4. Generate protocol code with the protocol designer application

NOTE: The specified parameters in steps 4.2−4.7 are the same for all conducted experiments, except for the material’s stock concentration and the final output concentration. These parameters are summarized in Table 1 and, in the following, parameters are used to prepare double network hydrogels with 5% (w/v) GelMA, 2% (w/v) alginate, 0.15% (w/v) LAP, and PBS as a diluent.

1. Open the protocol designer application by running ‘ProtocolDesignApp.html’.
   NOTE: The app “ProtocolDesignApp.html” guides the user through the parameter selection process and automatically generates the ready-to-use protocol to operate the workstation. The user interface runs on every commonly used internet browser (i.e. Chrome, Firefox, Safari, edge, Internet Explorer).
2. Enter protocol name (e.g., double-network-hydrogels) on setup page.
3. Click ‘続行’ to confirm protocol name and proceed to the next step.
4. Define input tray by selecting ‘3×4 Heating Block’ from drop-down menu and the following input parameters:
   1. Select ‘Gel 1’ from drop-down menu, enter name ‘GelMA’, enter stock-concentration ‘20%’, set ‘Number of Samples’ to ‘3’ to fill one column. Click ‘+add’ to save entries.
   2. Select ‘Gel 2’ from drop-down menu, enter name: ‘Alginate’, enter stock-concentration ‘4%’, set ‘Number of Samples’ to ‘3’ to fill one column. Click ‘+add’ to save entries.
   3. Select ‘Photoinitiator’ from drop-down menu, enter name: ‘LAP’, enter stock-concentration ‘3%’, set ‘Number of Samples’ to ‘3’ to fill one column. Click ‘+add’ to save entries.
   4. Select ‘Diluent 1’ from drop-down menu, enter name: ‘PBS’, set ‘Number of Samples’ to ‘3’ to fill one column. Click ‘+add’ to save entries.
      NOTE: The visualization of the input tray automatically updates, once ‘+add’ is clicked. If more inputs are added than the tray’s capacity, the warning ‘Too many samples for this tray’ will be shown to the user.
5. Define crosslinking parameters by checking ‘Photo crosslinking’ and typing the time in seconds, ‘30’, and the intensity W/m² with ‘2’.
6. Finish the input setup by clicking ‘CONTINUE’.
7. Define output tray setup by selecting ’96 well plate’ in the drop-down menu for well plate type.
8. Click on ‘Group1’ to define outputs by creating a group of samples.
   1. Specify output composition by entering desired concentrations and sample volume into the fields for each input: GelMA = ‘5’, Alginate = ‘2’, LAP = ‘0.15’, Total Volume = ‘60’.
   2. Check box to apply advanced mixing protocol.
9. Specify number of samples by entering the number of samples into the field ‘Number of Samples’: ‘96’.
   NOTE: The visualization of the sample tray automatically updates, once ‘+add group’ is clicked. If more samples are added than the tray’s capacity, the warning ‘Too many samples for this tray’ will be shown to the user.
10. Finish the output setup by clicking ‘CONTINUE’.
    1. Select tray position on the deck layout and prepare the platform accordingly:
    2. Check checkmark in field ‘SLOT A1’ and select ‘Empty_Cell’ from drop-down menu.
    3. Check checkmark in field ‘SLOT A2’ and select ‘Trash_Cell’ from drop-down menu.
    4. Check checkmark in field ‘SLOT B1’ and select ‘Tips_Cell_100µL’ from drop-down menu.
    5. Check checkmark in field ‘SLOT B2’ and select ‘Tips_Cell_1000µL’ from drop-down menu.
    6. Check checkmark in field ‘SLOT C1’ and select ‘Input_Cell’ from drop-down menu.
    7. Check checkmark in field ‘SLOT C2’ and select ‘Empty_Cell’ from drop-down menu.
    8. Check checkmark in field ‘SLOT D1’ and select ‘Mixing_Cell’ from drop-down menu.
    9. Check checkmark in field ‘SLOT D2’ and select ‘Output_Cell’ from drop-down menu.
    10. Define type and characteristics of first pipette (M100E) by checking ’Pipette Left’, selecting ‘10-100µL positive displacement’ from drop-down menu and setting aspirating speed = ‘600’, dispensing speed = ‘800’.
    11. Define type and characteristics of second pipette (M1000E) by checking ’Pipette Right’, selecting ‘100-1000µL positive displacement’ from drop-down menu and setting aspirating speed = ‘800’, dispensing speed = ‘1200’.
11. Click on ‘GENERATE PROTOCOL’ to confirm the setup and generate the protocol script.
    NOTE: The developed protocol designer app generates automatically a new folder whenever a new protocol is generated. All files which are required for this experiment and to operate the workstation are saved in this folder which is named after the protocol name. The folder can be copied into different directories without causing issues.
12. Do not close the interface, as it will be used to execute the protocol (see step 6.6.).

5. Calibration of the pipetting module

NOTE: Containers (e.g., well plates, tip rack, trash) and pipettes (e.g., M1000E) must be calibrated initially. If a container and/or a pipette position are modified/changed, the new position must be calibrated.

1. Navigate to the protocol folder and open the calibration terminal by executing the file ‘calibrate.py’ in a terminal windox (see step 1.1.1):
   phython.calibrate.py
   NOTE: The 'calibrate.py' interface guides the user through the calibration of the deck setup and pipettes. Make sure that the file is in the same folder as the protocol file and the module files. It is automatically generated in step 4.10.
2. Select movement increments for plungerx,y,z movement with the numeric keypad (1−8): ‘1’ for 0.1 mm, ‘2’ for 0.5 mm, ‘3’ for 1 mm, ‘4’ for 5 mm, ‘5’ for 10, ‘6’ for 20 mm, ‘7’ for 40 mm, and ‘8’ for 80 mm.
3. Calibrate the pipette.
   1. Press keyboard shortcut P to select pipette size.
   2. Press keyboard shortcut V to enter the plunger calibration mode.
      NOTE: It is recommended to start with small increments (2, 5, and 10 mm) to get familiar with the increment size and movement action of the pipette head.
   3. Calibrate the following plunger positions for a positive displacement pipette: T–Top = rest position; B–Bottom = plunger is pushed until resistance is met; P–Pick-up = plunger is pushed to a position where a piston tip can be attached; E–Eject = plunger is pushed until an attached tip is ejected. Vary plunger positions using the upwards and downwards arrows on the keyboard, and save the final position using S on the keyboard.
   4. Leave the pipette plunger calibration mode by pressing keyboard shortcut V.
4. Calibrate container position relative to the pipette tip.
   1. Press keyboard shortcut P to select pipette type. Make sure that a tip is connected to the selected pipetted.
   2. Press keyboard shortcut C to select container type.
   3. Select an appropriate movement increment and move pipette tip to the following positions. For well plates, calibrate to the ‘A1’ well position at the bottom; For tip rack, calibrate to the ‘A1’ position; For trash, choose a position (defined as a point) where the tip can be ejected into the trash.
   4. Press keyboard shortcut S to save position.
   5. Repeat steps 5.3.1−5.3.3 for all containers listed under ‘C’ for the selected pipette type.
   6. Repeat 5.3.1−5.3.5 for the second pipette type.
   7. Close the calibration script.

6. Protocol execution with the workstation

NOTE: Protocol files are accessible via the repository and are also available as Supplemental File.

1. Position trash container, tip racks, input tray, mixing tray, and output on the deck (defined in step 4.3).
2. Calibrate pipettes and instruments as defined in section 5.
3. If required, switch the temperature dock ON and select the temperature for input and mixing tray.
   NOTE: The experiments in this tutorial were conducted without temperature control and at 40 °C for glycerol, and 37 °C for GelMA and alginate pipetting.
4. Position tubes with input reagents in the aluminum blocks on the temperature docks according to the selected setup.
5. Wait until input reagents have reached the desired temperature.
   NOTE: To ensure proper temperature distribution, an incubation time of 30 min is recommended for GelMA and alginate.
6. Execute the protocol file by clicking on ’RUN PYTHON SCRIPT’
   NOTE: The selected protocol is now executed by the workstation. The accompanying video highlights automated mixing of GelMA and the distribution of 60 µL into a 96 well plate.
7. Run is completed, when ‘Finished’ is displayed.

7. Validation and verification process

1. Remove the well plate from workstation and transport the well plate with the samples to a spectrophotometer.
2. Read absorbance with a spectrophotometer at 450 nm. Read each plate 2x to compare results and ensure consistent results.
3. Export and save absorbance readings.
4. Data analysis.
   NOTE: Experimental data can be processed individually or copied and pasted into the provided template to evaluate the mean, standard deviation, and coefficient of variance (CV) value using spreadsheet software.
   1. Open the Supplemental File ‘supplementary_template-analysis.xlsx”, which is also vailable within the GitHub repository under ‘openworkstation/examples/publication-JoVE.
   2. Copy absorbance readings into the ‘raw data’ sheet, ensure that all cell references are correctly defined in all tables, and click on the ‘analysis’ sheet for information on the average, standard deviation, and coefficient of variance (CV) values.
      NOTE: Depending on the sample distribution on a well plate, the following preset evaluation types are available with the template: the ‘Uniform’ type is used when all samples have the same composition, the ‘By rows’ type is used when samples in different rows have different compositions, the ‘By columns’ type is used when samples in different columns have different compositions, and the ‘Customized’ type is used when the sample positions are user specific.