Technology

Single cell analysis

VyCAP’s single cell isolation and analysis platform delivers an unparalleled efficiency and simplicity that revolutionizes the way you select and isolate single cells.

The platform is specially designed to enable the isolation of single cells that cannot be isolated by other technologies. For example, isolation of single cells from a sample that only contains a limited number of cells (1 – 10.000) in a sample volume of 100µl – 40 ml and isolation of single cells with a very low frequency of appearance, for example CTC and fetal trophoblasts.

The Puncher platform solution comprises of two essential parts:

  1. A single cell disposable, that contains a microwell chip that distributes single cells in individual wells
  2. A Puncher system, to acquire fluorescence images of the single cells in the microwells and to transfer the selected cells from the microwells to the reaction tube or plate of choice, for example 0.2 ml Eppendorf PCR tubes or PCR well plates

The innovative single cell disposable distributes single cells in individual microwells. After completion of the cell distribution the Puncher system acquires high quality multi-colored fluorescence images of the single cells in the microwells. Based on these images the user identifies the cells of interest and transfers the selected cells to the reaction tube or plate of choice in a fast, automatic and controlled manner with minimal cell losses.

Unique Selling Points

  • Short workflow (<1hr) to obtain single cells, for DNA and RNA analysis and clonal expansion
  • Single cell isolation and transfer efficiency > 95%
  • Validated protocols
  • Single cell selection based on high quality immuno-fluorescence images

 

Typical workflow for single cell DNA and RNA analysis

Single cell disposable with isolation chip

The isolation chip comprises 6400 microwells in an effective area of 8×8 mm. Each well has a diameter of 70µm and a depth of 360µm. The bottom of the well is a thin, optical transparent, silicon nitride (SiNi) membrane with a thickness of 1 µm, having a single pore with a diameter of 5 µm. To facilitate the microfluidic handling and cell distribution, the chip is mounted in a plastic microscopy slide that is part of the disposable.  A cell suspension is transferred to sample side of the the disposable on top of the microwells. A small negative pressure is applied across the microwells. The fluid enters the wells from the supply side and leaves the wells through the pore at the bottom of the well. Hydrodynamic forces drag individual cells into the wells towards the pore in the bottom of the well. After a cell has landed onto the pore the sample flow through that particular well stops and no other cell will enter the same well. The next cell is then diverted to a neighbouring well. In this manner single cells are sorted in individual wells across the entire microwell chip in a few minutes. The sample volume is between 1 –  40 ml.

Isolating single cells

After distributing and staining cells, the slide is transferred to the Puncher system to acquire fluorescence images of the single cells in the individual wells. Based on their fluorescence signature the software makes a preselection of the cells to be isolated and presents these in an image gallery to the user who makes the final selection.

The selected cells are isolated using our innovative Puncher technology. The Puncher system moves the slide with the microwell chip from the Puncher image acquisition location towards the location of the Punch needle. 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 of the microwell but not the cell. The punched cell, together with the bottom of the punched microwell, are collected in an Eppendorf tube, PCR plate or any other kind of reaction tube.

 

Single cell downstream analysis

VyCAP single cell isolation is compatible with commercially available Whole Genome Amplification kits. VyCAP has established WGA protocols for both live and fixed cells using the Repli-G kit of Qiagen and the AMPLI-1 kit of Silicon Biosystems. The quality of the WGA product using these kits is determined by using VyCAP’s WGA QC kit. The kit determines the presence of 10 DNA fragments present on 10 different chromosomes. For the Repli-g kit all 10 bands should be visible in the most optimal case. For the Ampli-1 kit, 7 bands are visible for maximum quality. One band is lost because the DNA fragment is too long to become amplified and 2 fragments are lost due to the presence of restriction sites within these fragments, for the Mse-I enzyme as is used in the Ampli-1 WGA kit.

 

Sequencing

The WGA product is ready for sequencing. The example here displays the Sanger sequencing result for two genes PTEN and ROBO2, for three single cells of cell lines SKBR-3, PC-3 and LnCAP, that have been isolated using the Puncher platform technology, followed by AMPLI-1 WGA.

 

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