Re: mammospheres attachment (commercial response)

[Roy Edward-2]

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Dear Mario,

You may wish to refer to a 2010 paper in JBS by Fredika Robertson's
laboratory (MD Anderson Cancer Center) which describes the use of a
hydrogel mountant we have developed (CyGEL-TM).
Using this, they immobilised IBC tumour-cell reconstituted spheroids for
high resolution multi-colour imaging.
Ref: Robertson et al. Imaging and Analysis of 3D Tumor Spheroids Enriched
for a Cancer Stem Cell Phenotype. J. Biomol. Screen. 2010 15 (7) 820-829

Others have utilised this patented mountant for model organism
immobilisation (Danio embryos, C elegans, Drosophila larvae) and
parasites, tissue pieces and non-adherent cells.

In brief, CyGEL is a thermo-reversible hydrogel that is a liquid when cold
and gels above 22 degC. It is optically inert, clear and compatible with
living tissues, cells and can equally be used with fixed samples.  It can
support the addition of supra-vital DNA dyes, viability dyes and
functional dyes (Price et al., Molec. Biochem. Parasitol. 2010 169 66-69)

It could be combined with Tobias's idea of plasma treating the surface of
a slide / coverslip.  Thus, one could try anchoring the mammospheres onto
the surface with a small volume of liquid and then gentlyoverlaying with
liquid CyGEL.
Alternatively, it should be possible to do a gentle spin  of the CyGEL
mounted-mammospheres down through chilled (liquid) CyGEL in a refrigerated
centrifuge and then warming to allow it to set. This has been done with
danio embryos and may negate the need for the plasma-treated anchoring
surface. I've typically used a piece of steel from the fridge/blue pack
for melting and the warmth of the lamp on a stereoscope for setting.  The
reversible process takes place over 1 degC and is complete in a couple of
minutes. It is critical not to over-dilute the CyGEL by more than 20% v/v
aqueous addition.  For live cells which are sensitive to their supporting
media (e.g. epithelial cells requirement for DMEM F12 + insulin & EGF)
then we have a culture medium-ready version where one adds 10X medium to
CyGEL Sustain to prepare it for use.  This might be the case where cells
are to be observed for several hours.
 
A broad review on CyGEL can be found in American Biotechnology Laboratory:
www.nxtbook.com/nxtbooks/isc/abl_20100708/#/14

Further details are available on our website. Otherwise, please come back
to me off-line for detailed discussion of your experimental design.

Best regards,
Roy

Roy Edward
Biostatus Ltd.
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>------------------------------
>
>Date:    Mon, 12 Mar 2012 13:41:55 +0100
>From:    Mario Faretta <[hidden email]>
>Subject: mammospheres attachment
>
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>
>Dear all,
>we are trying to image fixed cell spheroids (>100um diameter) on a leica
>sp5 inverted confocal microscope. The high resolution objectives we use
>have short but sufficient working distance to analyse the spheres if
>they attached to the  coverslip.  However standard coatings as
>polilysine are not efficient in immobilizing them (even if they can
>initially adhere we loose them extensively during staining). We tried
>also particular gels as Q gel but we were not able to find a
>reproducible protocol to have them clearly attached to the substrate
>instead of being simply embedded in the gel.
>I know that new high NA water-immersion long working-distance objectives
>could provide the simplest solution but unfortunately their cost is
>quite limiting.
>thanks for all the answers
>Mario
>
>--
>---PLEASE Note the change in telephone number---
>
>--
>Mario Faretta
>Department of Experimental Oncology
>European Institute of Oncology
>c/o IFOM-IEO Campus for Oncogenomics
>via Adamello 16 20139 Milan Italy
>Phone: ++39-0294375027
>email: [hidden email]
>http://www.ifom-ieo-campus.it
>
>------------------------------