AgarSqueezer - agar-based cell compression device

The Agarsqueezer device has been successfully used to study compression of adherent and
non-adherent cells, including:

Human cells:
Fibrosarcoma (HT-1080)
Osteosarcoma (U-2 OS)
Colorectal adenocarcinoma
(HT29 & HCT116)
Prostate cancer (PC-3 & DU 145)
Breast cancer (MDA-MB-231)
Leukemia (TF1 & ML2)
Megakaryocytes
Fibroblasts (HS27A)
Breast cells (MCF10A)
Primary T-lymphocytes

Murine cells:
Osteocyte-like cells (MLO-Y4)
Primary dendritic cells
Primary muscle cells

Plant cells:
Arabidopsis Thaliana root cells

3D cell culture:
Mice gastruloids

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The system is fully compatible with live imaging and time-lapse microscopy and all
immunostaining steps can be performed in situ. Alternatively, cells can be collected for
classical biochemical and molecular analysis (qPCR, Western Blot, flow cytometry, etc).

Yes, the AgarSqueezer device can be re-used as long as it is thoroughly sterilized by autoclaving
before each experiment. Simply keep in mind that the agarose solution should be prepared
no more than one day before your experiment. 

The device has been designed for long-term confinement studies and the porous nature of
agarose enables passive medium renewal as well as oxygen diffusion. You can safely grow cells
up to 10 days in the AgarSqueezer device.

Yes, the use of agarose allows free diffusion of small molecules (size <30 nm in 2% agarose).
You can easily add drugs, antibodies or other compounds at any point during cell confinement
studies thanks to an open access to the reservoir. Drug availability and activity within the
system has been validated using in situ addition of the tyrosine kinase inhibitor imatinib, and
diffusion experiments showed that 3 hours are required for the diffusion of small molecules
such as BSA or FITC.

Yes, cell confinement is initiated by screwing the top part of the device. To apply a temporary confinement, you can simply unscrew this part to bring the cells back to unconfined conditions.

The currently available pillar heights are:
- 2.5μm for inducing highly confined conditions
- 5μm for inducing moderately confined conditions
- 30μm to serve as an unconfined control
- 100μm to confine 3D cellular structures or immobilize them for imaging

Those dimensions have been determined based on the size of most classical human cell lines.
The resulting level of compression applied will depend on your sample type, size and the use
of mono- or multi-cell layers.

You can easily modulate the matrix stiffness by tuning the type and concentration of agarose used to mold the pillars. Using a standard agarose at the recommended 2% concentration will result in a storage modulus within the range of 50-200kPa. To accentuate the matrix stiffness and the resulting confinement applied, the agarose concentration can be increased. For example, a 3% agarose was found to be more efficient at confining cell cultures from Arabidopsis roots. If you wish to decrease the matrix stiffness, we recommend using a “low melting point” or “ultra-low gelling” temperature agarose to yield a storage modulus in the range of ~1kPa. For a similar stiffness on top and bottom walls, the coverslip can be coated with an additional soft agarose layer.

To analyze the role of various ECM proteins on cell response to mechanical confinement, you
can coat the coverslip with adhesive proteins (fibronectin, collagen, Matrigel, etc). In addition,
the agarose can be added with collagen, PEGDA with covalently immobilized RGD peptides or
silk.

Our AgarSqueezer kits contain all elements making up the compression device, a wafer to mold your agarose gel and some needles specifically designed to form your entry ports in the agarose. Optionally, a holder (102.5 mm x 143.5 mm)​ designed to image 2 AgarSqueezer devices in parallel can be added to your order. This holder will fit into Okolab imaging chambers or equivalents. 

All you need to have on your side is:
• Agarose powder
• Distilled water
• Round 22 or 30 mm coverslips
• A screwdriver
• For imaging: a standard petri dish holder can be used to place and image the AgarSqueezer on inverted microscopes. Use objectives up to 40X for optimal performance. Although objectives of 60X and above can be used, they will result in a shorter observation area and we cannot guarantee an optimal working distance depending on your microscope.

It is possible to measure cell-generated forces in response to the imposed confinement in the AgarSqueezer by using 3D traction force microscopy. Indeed, you can incorporate fluorescent beads into the agarose pillars. If the gel is soft enough to be deformed by the cells (using ultralow agarose and/or tuning the agarose concentration can help modulating viscoelastic properties), a dynamic quantification of the compression force sensed by the cell could be retrieved. 

Alternatively, other methods such as optical tweezers might be envisaged.