VyCAP’s offers a solution to isolate cells for clonal expansion in a tissue culture plate. Viable cells can be selected based on their fluorescence, morphology and even on what single cells secrete. To detect what individual cells secretes the chip with viable cells is connected with a capturing surface.
The clonal expansion solution comprises of three essential parts:
- A single cell disposable, to distribute single cells in the wells of the isolation chip
- A Clamping unit, to connect the chip with a capaturing surface
- A Puncher system in a flowhood, to automatically select and isolate cells
VyCAP offers a complete, simple and automated solution for clonal expansion. The solution contains of hardware, software and protocols. After single cell isolation into a tissue culture plate the cells proliferate. Tools and protocols are currently available for researchers who are interested in selecting and expanding the best producing cells.
Unique Selling Points for clonal expansion
- Short workflow (<1hr) to select cells for clonal expansion
- Minimal amount of stress on viable cells
- Puncher system and pump-unit placed in flow cabinet
- Methods to investigate the secretome of cells
- Protocols to clonal expand the best antibody producing cells
Typical workflow for clonal expansion
The workflow to select cells for clonal expansion is done in a flowhood. Depending on the cell selection criteria such as cell fluorescence, cell morphology or single cell secretion there are different workflows. If cells are selected based on their fluorescence or morphology the workflow is straightforward and no clamping unit is needed. The clamping unit is used to collected the secreted cell products on a PVDF membrane.
Clonal expand the best antibody producing cells
To detect what individual cells secrete the chip with viable cells is connected with a capturing surface and incubated for 24hrs. At the moment a PVDF membrane is used as a capaturing surface. After incubation the secreted material can be correlated with the cell in the microwell. After the correlation the best cells can be isolated in a tissue culture plate.
Single cell disposable contains isolation chip with 6400 wells
The isolation chip has 6400 wells with a single pore in each well. For clonal expansion the chip is made sterile using ethanol. The bottom of the well is optically transparent and has a thickness of 1 µm. To isolate single cells the well bottom together with a cell is punched in a culture plate. Punching is done automatically with the punching system in a flowhood.
Pump unit to process cells in medium
The isolation chip mounted in a plastic slide fits in the single cell disposable. The single cell disposable clicks onto the pump unit. Cells in medium are transferred to the sample side of the disposable and a small negative pressure is applied by the pump unit. It takes typically 1-5 minutes and a low pressure 10mbar to process cells with medium.
One cell in one well
After processing there is one cell in one well. Hydrodynamic forces drag individual cells into the wells towards the pore in the bottom of the well. After a cell lands on the pore the flow through that well stops and no other cell will enter. The next cell diverts to a neighbouring open well. In this manner single cells sort in individual wells across the entire chip. Over 99% of the filled microwells contain a single cells.
Minimal amount of stress on viable cells
Below an example of MCF-7 cells that are labelled with a live/death staining. Calcein AM staining indicated that over 90% of the cells are viable after filtering (panel A). The user can isolate the single cells or put the microwell chip in an incubator and culture the cells while these are in the microwells (panel B).
Puncher system is located in a flowhood
After processing, the slide is transferred to the Puncher system located in a dedicated Telstar flowhood. The screen of the Puncher to control the software is mounted on the backside of the flowhood. Furthermore the mouse and keyboard can be made sterile. The Puncher has an additional z-stage compared to the normal puncher. This stage is used to move the tissue culture plate up and down. So in total the system has six stages which are all automatically controlled. Furthermore the system has an autofocus, changes filter cubes by itself and a needle calibration unit. The calibration unit aligns the needle and tissue culture plate correctly.
Isolate single cells for clonal expansion
To limit the stress on the cell during punching, culture media is applied between the bottom of the chip and the tissue culture plate. For clonal expansion the Puncher system is placed in the flowhood.
Cell culturing in a tissue culture plate after punching
Viable cells in the chip are punched into a tissue culture plate for proliferation. In the images below and example is depicted. In this example MCF-7 cells are punched into culture medium and cultured. The Puncher platform has been used to isolate and culture organoid, CTCs, hybridoma and Cho cells.
Cell proliferation inside the microwells
After processing, the slide with the microwell chip can be placed in an incubator to culture the cells inside the microwells. The image below presents the proliferating of a single MCF-7 cell during 9 days. The microwell chips can be coated with a thin layer of proteins i.e. matrigel or fibronection to enhance cell adhesion and proliferation properties of the cells. Work has been done in close collaboration with the University of Twente.
Measure cell secretion of all cells in the chip
A new tool is available to connect a capturing surface like a PVDF-membrane with the microwell isolation chip. The current capturing surface is a PVDF-membrane. In a project started in 2017 is shown that antibodies produced by cells in the wells diffuse through the pore in the bottom to the capturing PVDF membrane. The secreted molecules can be fluorescently labelled and correlated with the chip well position for clonal expansion. See workflow below.