Isolation and Staining of Rare Single Cells
With the SIEVEWELL chips a new generation of nanowell arrays is now available. SIEVEWELL is a novel design of a chamber slide that has an additional nanowell structure at the base of the liquid chamber. Each of these individual nanowells has 2 micropores at the bottom of the well that is connected to a small liquid gap underneath. This captures the cells inside the nanowells and generates a one-directional flow from the top of the liquid chamber, through the pores into the liquid gap underneath. Thanks to this design it is now possible not only to separate single cells and capture them inside the nanowells but also to process them directly inside these wells, e.g., antibody labeling, staining, washing, etc., without losing any cells. This completely cell loss-free on-chip processing makes SIEVEWELL technology extremely efficient for rare single cell applications, such as isolation of circulating tumor cells, fetal cells and others.
The chip design in combination with the CellCelector technology allows for complete automation of the identification of single cells as well as 100% pure isolation of the desired target cells.
Size of the nanowells: 20 µm width and 25 µm depth. Perfectly suited for efficient single cell capture.
370,000 nanowells per chip (17 x 17 mm)
Structure of the SIEVEWELL membrane. Top view of the nanowells.
Structure of the SIEVEWELL membrane. Side view of the nanowells.
Brightfield image of the SIEVEWELL membrane. The pores at the bottom of the nanowells are well visible.
This makes the SIEVEWELL technology very suitable for microscopic and optical measurements and allows the acquisition of high-quality microscopic imaging data.
In each of the 370,000 nanowells of the SIEVEWELL chip two microscopic pores with a diameter of 2 µm each are positioned on the bottom of the nanowell. They connect the fluid volume above and below the chip. The fluid volume below the nanowells is connected to two side ports through a micro-gap situated below the chip membrane.
After loading the cell suspension onto the chip, a one-directional fluid flow from the inner liquid chamber to the side ports can be generated and controlled by aspirating liquid with a standard pipette. The cells will follow the liquid flow and are trapped in the nanowells. When a cell entered a nanowell it will block the micropores hence reducing the liquid flow through that nanowell. Other cells are therefore automatically redirected towards other, empty nanowells leading to a self-sorting nanowell array. Following the cell loading on-chip staining can be performed in the same way without any cell loss during fixation, permeabilization, blocking, incubation and washing.
Load enriched or processed single cell suspension into the chip. The SIEVEWELL technology is compatible with living and fixed cells.
The single cell suspension will be added into the chamber of the SIEVEWELL chip.
The buffer will be aspirated from the side ports of the SIEVEWELL chip.
By aspirating with the pipette, a directional flow is generated from the inner chamber, through the two micropores at the bottom of each single nanowell towards the pipette at the side port.
The cells move down with the liquid flow and are trapped inside the nanowells due to the micropores being small enough to avoid cells passing through.
The size of the nanowells allows only one cell to be captured per well. Upon entering the nanowell the cell blocks its pores and therefore reduces the liquid flow through this well. Because of this,the subsequent cells are automatically redirected into surrounding, empty nanowells resulting in a self-sorting single cell capture of very high efficiency.
A549 cells loaded into SIEVEWELL
Add reagents, incubate and wash to stain the cells without loss. In-device fixation is also possible before staining.
Reagents or wash solution will be added into the chamber of the SIEVEWELL chip.
Scan with CellCelector
Blue: DAPI; Green: A549 cells; Red: Namalwa cells
CTC captured in a nanowell
Excess reagents or washing buffer will be aspirated from the side ports of the SIEVEWELL chip. As cells are trapped inside the nanowells they can't get lost during the process.
Fluorescence image after washing.
The chip will be scanned with the CellCelector to detect the target cells. The cells are immobilized in the nanowells and keep their original positions during scanning.
Due to the high flatness of the SIEVEWELL chip it is easy to scan the entire area of the chip without losing the focus.
Scan with CellCelector™
Target cells will be transferred to PCR tubes or cell culture plates. The SIEVEWELL chip has been specifically optimized for use with the automated single cell picking system, the CellCelector.
Single cell recovery using CellCelector
Isolated A549 cell in destination well
The SIEVEWELL technology is compatible with single cell DNA next-generation sequencing, RNA sequencing and other molecular-biological analysis methods. Living cells can be also cloned.
Single cell RT-PCR was performed using gene specific primers for GAPDH.
PC: positive control, c-DNA from pooled A549 cells
NC: negative control, lysis buffer
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