Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-11-17
2003-10-14
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S033000, C435S395000, C435S297500
Reexamination Certificate
active
06632656
ABSTRACT:
The present invention relates to cell based assays. In particular the invention relates to a microfabricated apparatus for performing cell growth and cell based assays and to methods for performing such assays.
The current focus in high throughput screening applications towards the screening of increasing numbers of compounds is being driven by the twin technologies of combinatorial chemistry and genomics, in order to produce new potential drug targets and novel candidate drugs as potential therapeutic compounds. The primary screening process has been addressed by the development of high throughput screening assay processes and assay miniaturisation utilising the microtitre well plate format with 384, 864, 1536 or greater miniaturised wells. Miniaturised assays are capable of allowing throughput levels of over 100,000 tests/day in primary screening. At this level of throughput, a primary screen might be expected to yield 100-1000‘hits’ per day. Each of these putative drugs is required to undergo further refined screening and testing in a variety of assays in order to investigate the biological compatibility of the compound. Such assays include bioavailability, metabolism and toxicology, and they are carried out predominantly using cultured cell lines. In comparison with assays used in the primary screening process, secondary screening assays have a much higher level of complexity and more stringent requirements, both in the mechanics of the assay and in the information generated. There is a requirement in the art for secondary screening assay methodologies which are capable of handling both the increasing rate of putative drug lead generation and the generation of biological data concerning the drug candidate. In addition, development of assays yielding higher information content have the potential to increase the level of characterisation of a lead drug during the screening phase of drug development.
Microfabricated devices have been described previously which are suitable for use in miniaturized biological analyses. For example, WO 96/15450 discloses a device which comprises an etched glass structure with a collection of chambers connected by a micro-channel and enclosed by a glass cover plate. Devices have been described by Wilding et al., for example in WO 93/22058, which discloses a mesoscale device for amplifying DNA by PCR, the device consisting of a number of chambers connected by a channel. WO 93/22055 and WO 93/22053 relate to devices for analysing a fluid cell-containing sample comprising a mesoscale flow system with entry port for capture and/or lysis of cells in biological fluids, or containing a binding moiety for specifically binding an analyte, where the analyte may be an intracellular component in a cell in a sample, or a cell population in a sample. The above devices are concerned with the measurement or detection of cells or cellular analytes in cells introduced into the device immediately prior to analysis. They are not described for use in cell culture or growth, nor for use in the study of cellular responses to agents under test. Moreover, they do not describe or permit the use of adherent cultured cells.
There is a requirement for a device capable of providing an environment which supports the long term survival of cultured cells, coupled with means to utilise cells cultured within the device for secondary drug screening or other studies.
In one aspect, the present invention provides apparatus microfabricated for performing cell growth and cell based assays in a liquid medium, said apparatus comprising:
a) a base plate supporting a plurality of micro-channel elements, each comprising a cell growth chamber, an inlet channel for supplying liquid sample thereto and an outlet channel for removal of liquid sample therefrom;
b) a cover plate positioned over said base plate said cover plate extending over said elements so as to define said chambers and connecting channels; said cover plate being supplied with holes to provide access to said channels; and
c) means, incorporated in said cell growth chambers, for cell attachment and cell growth.
In a second aspect of the invention there is provided a method for studying the effect of a test substance on a cellular activity or physical parameter by the use of the apparatus as defined, which method comprises:
a) providing a suspension of cells in a fluid medium;
b) introducing said cells into said apparatus and causing said cells to be transported to one or more cell growth chambers in said apparatus;
c) providing one or more samples of test substances whose effect upon the cells is to be measured under conditions so as to cause said cells to be exposed to said substances;
d) determining the effect of the test substances on said cells by means of optical detection.
Preferably the method for studying the effect of a test substance includes the step of culturing cells adhering to a surface within the apparatus prior to the introduction of the test substances. Preferably there are provided following step c) one or more assay reagents and dispersing the reagents to one or more reaction chambers in the apparatus.
In a further aspect of the present invention there is provided a method for measuring a cellular analyte by the use of the apparatus as defined, which method comprises:
a) providing a suspension of cells containing an analyte to be measured in a fluid medium;
b) introducing cells into the apparatus and causing the cells to be transported to one or more cell growth chambers in the apparatus;
c) allowing cells to grow;
d) providing one or more assay reagents and dispersing the reagents to one or more chambers in the apparatus;
e) measuring the cellular analyte by optical means.
In order that the invention may be better understood, several embodiments will now be described by way of example only and with reference to the accompanying drawings in which:
FIG. 1
a
is a diagrammatic representation in plan of an individual micro-channel element of the microfabricated apparatus for performing cell growth and cell based assays;
FIG. 1
b
is a sectional view of a microfabricated disc including a plurality of assay elements for performing cell growth and cell based assays according to the present invention;
FIG. 2
represents an alternative configuration of an individual assay element of the microfabricated apparatus for use with cells growing on the surface of microcarrier beads; and
FIG. 3
represents a further configuration of an individual assay element of the microfabricated apparatus in which means are provided for preventing or impeding the passage of cells in the cell growth chamber.
Referring to
FIG. 1
b
, the apparatus of the present invention comprises a rotatable disc (
18
) microfabricated to provide a sample introduction port (not shown) located towards the centre of the disc and connected to an annular sample reservoir (
9
) which in turn is connected to a plurality of radially dispersed micro-channel assay elements (
6
) each of said micro-channel elements comprising a cell growth chamber, a sample inlet channel and an outlet channel for removal of liquid therefrom and a cover plate positioned onto said disc so as to define closed chambers and connecting channels. Each micro-channel element is connected at one end to the central sample reservoir (
9
) and at the opposing end to a common waste channel (
10
).
Each of the radially-dispersed micro-channel elements (
6
) of the microfabricated apparatus (shown in
FIG. 1
a
) comprises a sample inlet channel (
1
) connected at its left hand-end end to the reservoir (
9
), a cell growth chamber (
2
) for performing cell growth and connected through a channel (
4
) to an assay chamber (
3
) and an outlet channel (
5
) connected at its right-hand end to the waste channel (
10
).
In an alternative format of the present invention as shown in
FIG. 2
, the micro-channel elements (
6
) are modified to permit the use of the apparatus with cells growing on the surface of microcarrier beads (
17
). Thus, each micro-channel element (
6
) comprises a sample inlet channel
Andersson Per
Thomas Nicholas
Beisner William H.
Fulbright & Jaworski LLP.
Gyros AB
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