Clonal expansion

VyCAP’s single cell selection and clonal expansion platform delivers an unparalleled efficiency and simplicity that revolutionizes the way you select and isolate single cells for clonal expansion. The system is specially designed for selection and 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. Examples of these are CTC, trophoblasts, anitbody producing cells selected for highest production and cells that express features that appear in very low frequency in an abundance of other cells or are present in large volume.

The Puncher platform solution comprises of two essential parts:

1. A single cell disposable, containing a silicon chip with microwells that distributes single cells in individual wells
2. A Puncher system, to image and transfer the selected cells towards a culture plate

The innovative single cell isolation microwell chip, 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 culture plate of choice in a fast, automatic and controlled manner with minimal cell stress and high efficiency.

Unique Selling Points

  • Short workflow (<1hr) to obtain pure viable cells for clonal expansion
  • Microwells filled with single cells >99%
  • Low cell loss during isolation, recovery percentage >95%
  • Single cell selection based on high quality immuno-fluorescence images
  • Puncher system placed in flow cabinet to maintain sterile conditions

Typical workflow

Singel cell seeding

The microwell chip comprises 6400 wells in an effective area of 8×8 mm. Each well has a diameter of 70 µm and a depth of 360 µm. 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 in the centre. An impression of the chip is shown in figure 1 panel A. To facilitate the microfluidic handling and cell distribution, the chip is mounted in a plastic microscopy slide (panel B) that fits in our single cell disposable. The principle of the self-distributing microwell chip is depicted in panel C of figure 1. A cell suspension is transferred to the microwells, and 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 center of 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 neighboring well. In this manner single cells are sorted in individual wells across the entire microwell chip until all single cells occupy individual wells. Over 99% of the filled microwells contain a single cell after completion of the cells seeding process. After the microwells are filled with single cells, these cells are imaged through the optical transparent silicon nitride bottom using an inverted fluorescence microscope in the Puncher system in an automated fashion.

Images of live cells in a well

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 has the choice to either isolate the single cells immediate after filling and imaging to a culture plate or put the microwell chip in an incubator and culture the cells while these are in the microwells (panel B).

Image and obtain pure cells for clonal expansion

After cell seeding and staining, the slide is transferred to the Puncher system to acquire multicolor fluorescence images of the single cells in the individual wells. A gallery of images is next presented to the user who makes a selection of the cells that need to be isolated. To isolate the cells, the slide is moved  from the imaging part of the Puncher system towards the punch needle to isolate the cells of interest towards a culture plate. The sterile 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 microwell bottom and not the cell. The punched cell, together with the bottom of the punched microwell, are collected in the selected well of a plate with medium. To limit the stress on the cell during punching, culture media can be applied in between the bottom of the microwells and the culture plate.

 

Cell culturing in a culture plate after punching

Viable cells can be sorted and stained in the chip and punched into culture plate for proliferation. In images below an MCF-7 cells are punched into culture medium and cultured. VyCAP has validated protocols available for clonal expansion in combination with single cell isolation using our Puncher platform.  The Puncher platform has been used to isolate and culture organoid, CTCs and hybridoma cells.

Cell proliferation inside the microwells

Cell proliferation from single MCF-7 cells, LnCAP, MDA and organoid cells has been successfully tested. After seeding  of the cells in the microwells, the slide with the microwell chip is placed in an incubator to culture the cells inside the microwells. The image below presents the proliferating of a single MCF-7 cell into a clump of cells 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 .

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