The present invention relates to apparatus for transport or permeation testing, such as culturing cells onto a membrane and then using the membrane as a substrate to simulate an epithelial cell layer to carry out transport or permeation testing thereon. In particular, the present invention relates to a multiwell filter device which allows for adding or removing liquid from the feeding tray of the apparatus without disturbing a material such as cells contained within the wells.
It is often desirable to evaluate how various chemicals that are orally ingested into a human will be absorbed into the blood stream through the intestinal wall. Such evaluation can be useful in, for example, drug testing to determine how various drugs will be absorbed into the blood stream. Transport of various substances through other types of epithelial cell layers can also be useful in therapeutically treating patients.
The MultiScreen® Caco-2 96-well device commercially available from Millipore Corporation is designed to support attachment, growth and differentiation of Caco-2 and other adherent cell lines. After the formation of a differentiated Caco-2 cell monolayer, the device can then be used to measure the rate of known and unknown drug transport across the Caco-2 cell barrier. All procedures are designed to be carried out in a single device and can be performed using automation for cell seeding, cell feeding, washing and drug transport experiments. Caco-2 cells possess many of the properties of the small intestine. As such, these cells represent a useful and well-accepted tool for studying the absorption and/or secretion of drugs/chemicals across the intestinal mucosa.
The MultiScreen® Caco-2 device is designed to support adherent cell growth and differentiation, particularly the ATCC® cell line Caco-2 designated as HTB-37. These cells are ideally suited for the performance of drug absorption experiments to determine the rate at which a known or unknown drug will penetrate an intestinal cell barrier. The MultiScreen Caco-2 device consists of a filter plate with 96 basolateral access holes, receiver plate, lid and additional receiver plate for analysis. It is ANSI-SBS-compliant; the Caco-2 device is compatible with automated liquid handling systems and span pipettes. An example of such a device is shown in WO 02/102962 assigned to Millipore Corporation, the disclosure of which is hereby incorporated by reference.
Extraction of most or all of the media from the feeding/receiver plate of a multi-well device is an important factor in the overall functionality and efficiency of the device. In addition, a reduction in the volume under the filter plate will allow the use of less media to feed the cells, which can be a substantial cost advantage.
It is therefore an object of the present invention to provide a multi-well device that includes a feeding/receiver plate having a U-shaped configuration under the active filter area.
It is a further object of the present invention to provide a multi-well filtration device wherein the relative positioning of the filter plate wells and the feeding/receiver plate wells is shifted in order to reduce or eliminate capillary hold-up.
The problems of the prior art have been overcome by the present invention, which provides a multiwell filter device comprising a filter plate having a plurality of wells, each well including a support such as a membrane for retaining sample. Preferably each well has a corresponding adjacent access port, which provides direct access to the bottom of the corresponding feeding/receiver plate well, such as via a pipette tip inserted into the access port. The filter plate is configured to be positioned over a feeding/receiver plate having a plurality of wells, such that each respective well of the plurality of wells of the feeding/receiving plate aligns with and is communication with a corresponding well of the filter plate. Moreover, the alignment is such that each of the access ports in the filter plate provides access to a respective well in the feeding/receiver plate. However, unlike the prior art, when the filter plate is positioned over the feeding/receiver plate, the wells in the filter plate are not centrally located with respect to the wells in the feeding/receiver plate. Instead, the wells in the filter plate are shifted in the x and y directions relative to the feeding/receiver wells an amount effective for reducing or eliminating capillary holdup of liquid in the gap between the filter wells and the feeding/receiver wells.
In a further embodiment of the present invention, the wells in the feeding/receiver plate are designed to create a U-shaped bottom below each access port to provide a more uniform volume of feeding media and allow for improved medial removal through the access port, such as via syringe or pipette.
6B is a cross-sectional view of the filter plate positioned over the feeding/receiver plate along line C-C of
a is a top view of a feeding/receiver plate well of the prior art; and
b is a top view of the feeding/receiver plate well in accordance with the present invention.
Turning first to
The filter plate 20 is configured to be positioned over and aligned with a feeding/receiver plate 30 (hereinafter referred to as a receiver plate, although those skilled in the art will appreciate that the plate can function as a feeding plate as well). Suitable posts, cones, pyramids, pins, dimples or similar alignment mechanisms 28 (
When the filter plate 20 is properly positioned over the receiver plate 30, each well 22 of the filter plate 20 extends into a corresponding well 32 of the receiver plate 30, ensuring that each membrane 25 extends into only one receiver well 32. Accordingly, the inside diameter of each receiver plate well 32 is larger than the largest outside diameter of each filter plate well 22 in order to enable each filter plate well 22 to enter into and reside a respective receiver plate well as seen in
In accordance with a preferred embodiment of the present invention, when the filter plate 20 is properly positioned on the receiver plate 30, the relative positioning of the filter plate wells 22 and receiver plates wells 32 is shifted from the conventional centered positioning in order to reduce or eliminate capillary hold-up between the interior wall of each receiver plate well and the exterior wall of each respective filter plate well (an example of such capillary hold-up is illustrated in
Since the shape (as seen from a cross-sectional top view in
Suitable shapes for the receiver wells 32 include tear drops, ovals, diamonds, squares, rectangles, circles, regular polygons, irregular polygons, etc.
In a further embodiment of the present invention, the wells 32 of the receiver plate 30 have a U-shaped bottom below the active filter area, as best seen in
The volume under the filter area can be smaller (e.g., less than 10 microliters of sample remains after removal by pipette) with a U-shaped bottom draining to a U-shaped aspirating point at 41, and less media can be used to feed the cells. Moreover, a more uniform volume of feeding media to growing cells results, which allows for more uniform cell growth, compared to a slanted bottom which results in a varying volume and cell growth. The U-shape at the liquid removal point offers an improved amount of removal of the media.
Despite the relative positioning of the filter plate and feeding plate wells, the device remains automation compliant (e.g., complaint with ANSI-SBS standards).