This repository contains documentation for manufacturing, assembly and use of the New Harvest in-incubator perfusion system, developed in collaboration between IRNAS Ltd., New Harvest and the Institute of Biomedical Sciences as part of a joint research project. The aim of the described system is providing controlled perfusion capabilities into established cell-biology workflows and equipment. Specifically, it was developed for use inside cell culture incubators commonly used in life sciences and related fields.
Jernej Vajda1, Nejc Klemenčič2, Marko Sagadin2, Mihael Miško2, Luka Mustafa2, Jeremiah Johnston3, Breanna Duffy3, Uroš Maver1,4, Boštjan Vihar1,2
- Institute of Biomedical Sciences, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia
- IRNAS Ltd., Limbuška cesta 76b, 2000 Maribor, Slovenia
- New Harvest foundation, 1401 21st Street, Suite 4556, Sacramento, CA 95811-5226, USA
- Department of Pharmacology, Faculty of Medicine, University of Maribor, Taborska Ulica 8, 2000 Maribor, Slovenia
- Correspondence to: bostjan.vihar@um.si
Detailed instructions on manufacturing, assembly and operation of the individual levels of this system, namely Mechanics, Electronics and Software are available in their respective sub-repositories.
After assembling the mechanics, electronics and installing the software, as described in the complementary repositories, the system can be installed into a cell culture incubator, by placing the controller box outside and the mechanical assembly inside the incubator and connecting them via a 4-pin charger cable. Depending on the incubator model, this could be achieved through a port in the back wall of the incubator as indicated in the figure below.
After the system is assembled and charged, it is ready to use and full functionality can be accessed via GUI on the integrated touch screen display as described below and in the GUI user guide. Additionally, system data (e.g. drive activity, flow rate and direction) are logged automatically if a USB flash drive is inserted into the USB hub on the back of the control box. Furthermore, the GUI can be accessed remotely via wifi, which can be set-up in the config tab of the GUI, as shown in the figure below.
Overview: The calibration function allows the user to adjust the perfusion rates of the pumped liquid to be adjusted in ml/min ranges with variable tubing and (compatible) peristaltic pump heads. As the perfusion rate depends on the rotation per minute (RPM), the inner and outer diameters of the tubing, precise flow control can be difficult to estimate, especially if the precise measurements of the tubing aren't known. The calibration function of the new harverst in-incubator perfusion mitigates this, by allowing RPM adjustment to fit the tubing and pump heads.
Components:
- New Harvest in-incubator perfusion prototype (assembled)
- 1 peristaltic pump cartridge
- 1 target tubing to be used for the experiments (e.g. silicone/BPT/tygon), compatible with the pump head
- 2 beakers (or similar containers)
- Calibration liquid (e.g. water) to fill half a beaker (the precision of the calibration method increases with the volume difference between slow and fast perfusion rates)
Method:
- Install the tubing and cartridge into the pump head
- Insert both open ends into a liquid filled beaker
- Start single flow pumping to completely fill the tubing (GUI - single flow speed tab)
- Weigh/tare the empty beaker
- Place one end of the tubing into the empty beaker
- Go to Calibration tab (GUI) and set low/high RPM settings and perfusion time
- NOTE: appropriate RPM values will strongly depend on the microstepping settings (GUI - Config tab) which should be adjusted to according to the preferred range of the perfusion rate. At 1/256 microstepping, a good calibration range for perfusion rates lies between 200 RPM (low) and 5000 RPM (high)
- Start the calibration procedure by following the on-screen instructions and measuring the accumulated amount of pumped liquid as depicted in the figure below.
- Save the settings file to use for perfusion experiments.
The mechanical part of the in-incubator perfusion system is composed of an aluminium tray, that holds up to 3 P6-well plate-sized sample (e.g. 3D cell culture) containers, a pump drive (stepper motor) and a peristaltic pump head. The pump drive is connected to a driver and power supply via a 4-pin charger cable.
| Parameter | Value |
|---|---|
| Build: | Aluminium |
| Charging: | LRS-150-48, 48V/150W power supply |
| Pump: | Peristaltic pump, with Ismatec(R) pump head |
| Flow: | 0.1 - 100ml/min ± 0.02ml/min |
| Tubing: | with 2-4mm outer diameter suitable for cell culture applications (e.g., platinum cured silicone, tygon, Pharmed BPT(R)) |
| Tubing connections: | Luer-lock (polypropylene) |
| Dead volume: | depending on assembly |
| Monitoring/control: | Graphical user interface (via touch screen or wifi) |
| Control parameters: | flow rate (continuous or dynamic), direction |
The perfusion system control box that is placed outside the incubator provides power supply and control over the pump drive, a GUI that allows direct access via touch-screen and WiFi connection.
| Parameter | Value |
|---|---|
| Computer system: | Raspberry Pi 4 with Raspberry Pi OS and Raspberry Pi touch display |
| Motor driver: | PoLabs PoStep60-256 |
| Power supply: | RS-15-5, 5V/15W power supply |
| Housing: | 1458G5 metal casing, customized PMMA sheets |
| Connectivity: | WiFi, USB (data logging), IEC socket (power supply), 4-pin socket (motor control) |
Additional documentation on manufacturing, assembly and use of the in-incubator perfusion system can be found in connected repositories, which will hopefully provide additional insight and help with reproducibility and use. Links to that documentation are listed below:
- Mechanics manufacturing and assembly
- Electronics assembly
- Software set-up and GUI
- Tools for liquid handling
- Testing and additional insights
The New Harvest in-incubator perfusion system is licensed under open-source licenses: Hardware including documentation is licensed under CERN OHL v.1.2. license. Firmware and software originating from the project are licensed under GNU GENERAL PUBLIC LICENSE v3. Open data and additional documentation are licensed under Creative Commons Attribution-ShareAlike 4.0 Unported License.
Open-source licensing means the hardware, firmware, software and documentation may be used without paying a royalty, and knowing one will be able to use their version forever. One is also free to make changes, but if one shares these changes, they have to do so under the same conditions they are using themselves.
IRNAS template for a GitHub repository. It comes with a basic group of CI workflows for release automation.
This repository contains submodules. To include them when cloning the repository the following command should be used: git clone --recurse-submodules https://github.com/IRNAS/newharvest-incubator-perfusion.git
