Coated membrane for assessing the invasive capacity of a cell

Details Coated membrane for assessing the invasive capacity of a cell Coated membrane for assessing the invasive capacity of a cell

2001-08-29

2004-05-25

Gitomer, Ralph (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S034000, C435S287900

Reexamination Certificate

active

06740501

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to cell culture, and more particularly relates to an improved device for measuring invasion by a cell through an aggregated polymerized extracellular matrix, and method for formation of the device.
BACKGROUND
Invasion is the process of cell movement across the basement membrane barrier and/or through the interstitial tissue. Invasion takes place during malignant tumor cell metastasis and during normal physiological processes such as angiogenesis and wound healing.
In vitro assessment of the invasive property of a cell has conventionally been carried out by quantitatively measuring the ability of the cell to digest away components of a reconstituted basement membrane which mimics the barrier function of natural basement membrane. A reconstituted basement membrane which has been extensively used in invasion determinations has been isolated from the Englebreth-Holm-Swarm (EHS) mouse tumor and disclosed in U.S. Pat. No. 4,829,000 to Kleinmann et al. and in Technical Bulletin 427 entitled “An Improved MATRIGEL® Invasion Chamber” (Becton Dickinson and Co.) wherein MATRIGEL® is a registered trademark of Becton Dickinson and Co. for an EHS preparation.
Assays for invasion using prior art EHS compositions coated onto a support surface, usually a porous membrane, are subject to various deficiencies, most of which are associated with a non-uniform drying of the prior art coating solution onto the porous support surface. A particular problem consequent to uneven drying is discontinuous cell invasion manifested by a patterning effect, such as dots or concentric rings of invading cells. Uneven drying may also lead to deposition of salt crystals at the outer edges of the membrane due to surface tension effects and an unacceptable number of uncoated pores. Because of these and other deficiencies with prior art coatings, discrimination between invasive and non-invasive cells may be compromised.
SUMMARY OF THE INVENTION
A porous membrane for determining invasivity of a cell has a composition thereon which includes a coating of reconstituted extracellular matrix derived from the EHS mouse tumor. The composition is applied with a coating solution which includes a polyol in a pH 7.8-8.2 buffer. The preferred buffer is Tris and the preferred polyol is sucrose.
A second aspect of the invention is an assembly which includes a multiwell tissue culture plate and an insert having a sleeve therethrough. The sleeve mates with a well of the plate, and the coated membrane serves as the bottom wall of the sleeve. The assembly may optionally include a lid for the plate and a feeder tray.
Another aspect of the invention is a method to prepare the coated membrane. The method includes inducing aggregation of components of the EHS and drying and stabilizing the composition on the porous membrane.
There is a need in the art for a composition which forms a uniform coating on a porous support, which is highly digested by an invasive cell substantially resistant to passage of a non-invasive cell and which thereby provides an easy and accurate discrimination between invasive and non-invasive cells. The present invention is directed to fulfilling this need.


REFERENCES:
patent: 4829000 (1989-05-01), Kleinman et al.
patent: 5714465 (1998-02-01), Langley et al.
patent: 6087157 (2000-07-01), Badylak et al.
Bouchara J. P. Recognition of Fibrinogen and Basement Membrane Components as Mediators of the Adherence of A. fumigatus Conidia. Colloids and Surfaces B: Biointerefaces 2(1-3)299-304, 1994.*
Isley, Stephen R., “MATRIGEL® Basement Membrane Cell Invasion Chamber”, Becton Dickinson Technical Bulletin #422 (1996).
Isley, Stephen R. et al., “An Improved MATRIGEL® Invasion Chamber”, Becton Dickinson Technical Bulletin #427 (1998).
Flaherty, et al, “Screening of Anti-Metastatic Compounds of using the BD BioCoat (TM) Fluroblok (TM Invasion System”, The Cell/Line BD Biosciences, vol. 11, No. 2, Jun. 2001, pp. 1-8.

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