Technology

Single cell analysis

VyCAP’s single cell isolation solution is simple to use, high recoveries and results in high quality single cell DNA and RNA data. The system is desiged to isolate rare single cells. The system is compatible with all single cell down stream analysis technologies.

The singe cell isolation solution comprises of two essential parts:

  1. A single cell disposable, to distribute single cells in the wells of the isolation chip
  2. A Puncher system, to automatically select and isolate cells

VyCAP offers a complete validated and automated solution to isolate single cells. The solution comprises hardware, software and protocols. The work flows for single cells isolation a compatible with downstream single cell RNA or DNA analysis. Protocols to isolate single CTC and fetal cells   are present on our website.

Unique Selling Points to isolate single cells

  • Short workflow (<1hr) to obtain single cells,
  • Large and small sample volumes 0.1-40ml
  • Automatically select and isolate cells
  • Cell selection based on high quality immuno-fluorescence images
  • Validated protocols for single cell DNA and RNA analysis

Isolate single cells with the Puncher system

Isolation of single cells from whole blood for DNA and RNA analysis

Workflow to isolate single cells for downstream DNA or RNA analsysis

The workflow to isolate cells from whole blood is validated and simple. It takes less than 1 hr to isolate circulating tumor cells or fetal cells from whole blood. The single cell disposable contains a microwell chip with 6400 wells. Our single cell isolation system can be demonstrated in our demolab. Contact us to learn if we can help.

Isolate of single cells from organoids, fetal cells, zebrafish embryos, tissue

VyCAP offers a complete, simple, validated and automated solution to isolate single cells. Use our solution to isolate single cells from organoids, zebra fish embryos and tissue. Validated protocols are available to isolate cells for single cell DNA or RNA analysis.

Single cell disposable contains isolation chip with 6400 wells

The isolation chip has 6400 wells with a single pore in each well. 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 to a PCR plate or PCR tube. Punching is fully automated and controlled by dedicated software.

Process sample volumes of 0.1 – 40ml

The isolation chip is mounted in a plastic slide that is mounted in the single cell disposable. The single cell disposable clicks onto the pump unit. A cell suspension is transferred to the sample side of the disposable and a small negative pressure is applied by the pump unit. Depending on the cell suspension the pressure is adjusted, see our protocols. It takes typically 1-5 minutes to process a cell suspension and fill the microwells with single cells.

One cell in one well

Single cells are distributed in individual microwells. 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 sample flow through that well stops and no other cell will enter. The next cell is diverted to a neighboring open well. In this manner single cells sort in individual wells across the entire chip.

Sorting single cells in a microwell chip with the pump unit

Puncher system is fully automated

After filling of the microwells, the slide is transferred to the Puncher system to acquire fluorescent images. It contains high end scanning stages to quickly position the chip, well plate / Eppendorf tube and punch needle. The system is controlled by user friendly software.

Slide, PCR plate and punch needles can be loaded easily

The slide with the microwell chip and well plates or Eppendorf tubes can be easily loaded onto the system. To location of the punch needle and the well  pate position are  automatically calibrated after loading.

Puncher system has high end optical components

The Puncher system contains high end scanning stages, a Lumencor LED excitation light source and a Hamamatsu CMOS scientific camera. The stages are automatically controlled by the image acquisition software. Furthermore the system has auto focus and an automatic filter cube changer  that can hold a maximum 6 different filter cubes.

Software to automatically select and isolate single cells

Based on the fluorescence cell signature the software can make a pre-selection of the cells of interest. The preselected cells are presented in gallery. From this gallery cells that need to be isolated by punching are selected.

Isolate single cells with the software of the Puncher system

Isolate single cells Punch by Punch

The selected cells are isolated using the innovative Puncher technology. The Puncher system automatically moves the slide towards the location of the punch needle. The punch needle punches out the bottom of the well together with the cell. The alignement of the punch neelde and the deisgn of the tip of the punch needle is such that it does not touch the cell. It takes around 1 second to isolate a single cell after the selection has been made. The isolated cells are collected in an Eppendorf tube or PCR plate. After punching an image of the punched microwell is acquired to ensure that the microwell bottom and cell are removed.Isolate single cells from the microwell chip with a Punch needle

Validated WGA protocols for single cell DNA and RNA analysis

VyCAP has WGA protocols for viable 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 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 are  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.

Isolate single cells and amplify its DNA with commercial kits of Silicon Biosystems (Ampli1)

 

Single cell DNA analysis example

After single cell isolation and WGA the product is ready for DNA analysis. The example here displays the Sanger sequencing result for two genes PTEN and ROBO2.  Three single cells of cell lines SKBR-3, PC-3 and LnCAP, are isolated using the Puncher platform technology, followed by AMPLI-1 WGA.

DNA analysis of isolated single cells

 

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