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Single Cell Analysis

 

VyCAP has designed a novel technology by combining very efficient single cell seeding followed by the ability to select and isolate specific individual cells of interest for further interrogation.

 

 

Single cell seeding

 

 

 

 

Microwells, created in silicon, are used to seed single cells in individual wells. The microwells have a diameter between 5 and 10 times the diameter of a cell. The bottom of each microwell is closed by a silicon nitrite membrane that contains a single pore in the center of the well. The silicon chip is mounted in a slide that is compatible with the disposable filtration unit.

 

To seed the cells in the microwells, the slide is inserted in the disposable filtration holder which is connected to the same pump unit as is used for the filtration of whole blood for the detection of CTC.

 

 

 

The sample fluid is transferred to the sample compartment of the filtration unit and a negative pressure is applied to the waste side. The fluid enters the wells and exits through the pore at the bottom. The single cells are dragged along towards the pore. As soon as a single cell, with a diameter larger than the pore diameter, has landed onto the pore the flow stops and no other cell will enter that same well.

 

 

 

 

Single Cell Isolation

 

After cell seeding, the slide with the silicon microwells chip is transferred to an fully automated fluorescence microscope with dedicated software to acquire multicolor fluorescence images of the single cells in the individual wells. A gallery of images is next presented to the user to make a selection of the cells that need to be isolated. To isolate the cells, the slide with the microwells is moved by the microscope stage to the location of the punch needle which is mounted onto the same fluorescence microscope. The punch needle is lowered into the well with the cell that needs to be isolated and punches out the bottom of the well together with the cell. The tip of punch needle is shaped such that it only touches the bottom next to wall of the microwell. The cell is always in the center of the well and the needle will not touch the cell during punching. The punched cell, together with the fragments of the bottom of the punched microwell, are collected in the selected well of a PCR plate or any other kind of reaction tube. A video of the operation of the system can be viewed here. Next, any commercially available WGA kit can be used for DNA amplification and next followed by sequencing.

 

 

 

The Puncher system at Glance

 

The Puncher system is a fully automated instrument which is built on top of a Nikon TiE fluorescence microscope, equipped with a Hamamatsu Orca FLASH II camera, multiple fluorescence filter cubes and a Lumencor Sola SE II light source for fluorescence excitation.

 

The system has dedicated software for image acquisition and isolation of the single cells.

 

Different adaptors for your reaction tube of choice are available.