Biological analysis device having improved contamination prevention

A device for conducting biological analysis, such as cell culture, is disclosed having improved contamination prevention features. In a preferred embodiment, a microwell membrane plate for cell analysis is adapted to be inserted into a shallow rectangular tray with elevated side walls which form a single large reservoir for holding an appropriate liquid used in cell growth and analysis. The side surfaces of the tray include a raised ridge extending about the entire periphery of the tray. A series of uniform-height steps formed on the ridge serve as support for the microwell membrane plate when inserted into the tray and also create a narrow capillary gap between the plate and the tray. A ledge extending laterally from the base of the ridge forms a second, wider gap when the microwell membrane plate is inserted into the tray. The height of the first capillary gap is less than the height of the second gap formed within the zone of space extending to the exterior of the device, thus when the tray and plate are tilted, as when jostled during handling, liquid from the reservoir will preferentially remain in the space formed by the first capillary gap. Liquid which does spill into the second gap will run back into the reservoir when the device is leveled.

BACKGROUND OF THE INVENTION 
This invention relates generally to devices for carrying out biological 
analyses, particularly in vitro processes involving living cells such as 
cell culture and subsequent assays evaluating cell function. More 
particularly, this invention relates to multi-welled cell culture plates 
with permeable membranes sealed to the bottom of each of the wells. 
The technology explosion associated with the biotech industry has seen a 
concomitant growth in the "biotechware" industry, where the need for a 
variety of dishes, flasks, tubes and plates to support biological studies 
is paramount. In many experiments involving biomolecules there is a 
stringent requirement to maintain sterile conditions as much as possible. 
This is particularly the case in carrying out in vitro processes involving 
living cells. 
Devices used in conjunction with the in vitro growth and subsequent 
analysis of living cells are available from a number of manufacturers such 
as A/S Nunc and Corning. Such devices are available in various forms 
ranging from large plastic flasks, to which media and/or reagents are 
added to culture or assay the living cells, to plates having a plurality 
of self-contained impermeable plastic wells (usually available in 6-, 12- 
or 24-well versions), in which cell culture media or biological reagents 
are added to each individual well. 
It has been shown that benefits ensue when cells are grown and studied with 
the aid of a permeable microporous membrane suspended in the cell growth 
medium. The microporous membrane usually is sealed to one end of a plastic 
cylinder which then is placed into a well of a culture plate with culture 
medium. Cells are placed in the chamber above and sometimes below the 
membrane. The microporous membrane allows free diffusion of ions and 
molecules so that cells more closely resemble their in vivo state than 
when grown on solid, impermeable plastic surfaces. The membrane allows 
liquid access to both sides of the cell, thereby improving cell 
differentiation and facilitating studies of cell transport and 
permeability as well as cell-cell interactions. The Millicell.RTM. culture 
plate insert sold by Millipore Corporation is an example of such a 
membrane device. 
More recently, the concept of providing membrane wells for cell studies has 
been expanded to provide multiple-well plates having an array of wells 
with open bottoms and a microporous membrane sealed to the bottom of each 
well (hereinafter referred to as a "microwell membrane plate"). The 
microwell membrane plate is adapted to be inserted into a tray having a 
mating pattern of closed-end wells. Tissue culture media and/or reagents 
can be provided above and below the membranes, and cells can be added as 
desired to perform multiple studies simultaneously. The filter plate 
element of a MultiScreen.RTM. plate sold by Millipore Corporation is an 
example of such a microwell membrane plate. 
Often it is desired to grow cells on the surface of the microporous 
membranes for several days before undertaking cell biology studies in 
order to allow attachment dependent cells to form a confluent layer on the 
membrane and to express their fully differentiated anatomical and 
physiological functions. A multi-welled tray of the type described above 
having mating closed-end wells can be used with a microwell membrane plate 
during the cell growth period; however, the culture media must be changed 
often to replenish nutrients used by the growing cells. It is advantageous 
during this initial growth period to use a tray with a few large 
reservoirs or even a single large reservoir which accepts all the filter 
wells of the microwell membrane plate. The large tray reservoirs provide a 
greater volume of culture medium per well than can a reservoir which 
accepts only one filter well, thus the medium does not have to be changed 
as often. Also, it is easier to change the culture medium in a single 
large reservoir rather than to change it in as many as 96 small reservoirs 
per tray. 
Regardless of the type of device employed, experimentation involving living 
cells requires attention to sterility. Microbiological contamination is 
always an important concern when handling living cells. However, in this 
regard, it is not possible to hermetically seal the plates housing the 
growing cells from the ambient environment because an exchange of gas from 
the cells to the external environment and vice-versa is necessary. Thus 
covers which are used with such plates must provide a space between the 
cover and the container housing the cells. During handling of these cell 
analysis devices, the liquid solutions employed may spill and remain in 
the space between the cover and the container. This provides a pathway for 
contamination to enter the container. 
The foregoing problem associated with spillage becomes magnified when 
dealing with a microwell membrane plate, such as the aforementioned 
MultiScreen.RTM. device, when used with a tray which has a single 
reservoir for media to interface with all cell-containing wells through 
the membrane. In this instance, the tray reservoir is relatively large and 
is filled almost to its rim with liquid. Since the tray cannot be sealed 
to the plate (or a cover for the plate), spillage is likely when the tray 
and plate are tilted or jostled during handling. Thus liquid will readily 
seep into the space between the tray and plate and remain there, thereby 
creating a contamination pathway which could adversely effect the cells or 
any subsequent analysis performed thereon. 
SUMMARY OF THE INVENTION 
The present invention overcomes the disadvantages and limitations of prior 
art devices by providing an improved biological analysis device which 
utilizes capillary forces to eliminate a contamination liquid pathway by 
which microbiological contaminants may enter the interior of the device 
from the ambient environment. In a preferred embodiment, a microwell 
membrane plate for cell analysis is adapted to be inserted into a shallow 
rectangular tray with elevated side walls which form a single large 
reservoir for holding an appropriate liquid used in cell growth and 
analysis. Each well of the plate fits within the reservoir to expose its 
corresponding membrane to the cell reagent liquid. The side surfaces of 
the tray include a raised ridge extending about the entire periphery of 
the tray. A series of uniform-height steps formed on the ridge serve as 
support for the microwell membrane plate when inserted into the tray and 
also create a narrow capillary gap between the plate and the tray. 
In the foregoing embodiment of the invention, the raised ridge protrudes 
along the innermost edge of the tray (i.e. closest to the reservoir) and a 
ledge extending laterally from the base of the ridge forms a second, wider 
gap when the microwell membrane plate is inserted into the tray. This 
second gap extends uniformly to the outermost edge of the plate and tray 
combination and in conjunction with the first gap forms a pathway for the 
exchange of gases between the cells in the reservoir and the external 
environment. In accordance with an important aspect of the invention, the 
height of the first gap is less than the height of the second gap formed 
within the zone of space extending to the exterior of the device. In this 
manner, when the tray and plate are tilted, as when jostled during 
handling, liquid from the reservoir will preferentially remain in the 
space formed by the first capillary gap. Liquid which does spill into the 
second gap will run back into the reservoir when the device is leveled.

DETAILED DESCRIPTION OF THE INVENTION 
FIG. 1 shows a preferred embodiment of a device 10 used for cell culture. 
Although this embodiment is described in the context of cell culture, the 
device may be used in any number of ways for studying in vitro living 
cells, such as used to perform assays for analyzing cell function. 
The device 10 includes a microwell plate 11 having ninety-six (96) 
individual plastic wells 12 with open ends 12A and a permeable membrane 13 
sealed to the bottom of each of the wells. The plate is formed from a 
suitable material such as polystyrene and the membrane is a microporous 
membrane made from cellulose esters, polyvinylidene fluoride (PVDF) or any 
other suitable material. The pore size of the membrane for the device 10 
can range from 0.1 to 20 microns. Devices used for other applications 
requiting ultrafiltration membranes will have pore sizes less than 0.1 
microns. A rectangular tray 14 made of a suitable material such as glycol 
modified polyethylene terephthalate (PETG) includes elevated side walls 15 
defining a shallow reservoir 16 which occupies essentially the entire area 
of the tray. The dimensions of the reservoir are such that all of the 96 
wells may be inserted within the reservoir when the plate and tray are 
combined during cell culture. 
The tray 14 includes a raised ridge 17 located along the innermost edge of 
the tray adjacent to the reservoir 16 about the entire periphery of the 
tray. A ledge 18 is disposed substantially at right angles to the side 
walls 15 and extending laterally from the base of the raised ridge. As 
best shown in FIGS. 2 and 3, the outer edge 19 of the plate 11 extends to 
approximately the same width as that of the ledge when the plate is 
inserted into the tray. 
Four steps 20 of uniform height formed on the raised ridge 17 at opposite 
ends of the tray 14 serve to support the plate 11 and also create a 
capillary gap 21 between the tray and plate near the reservoir 16. As 
shown, a second, much wider gap 22 is also formed in the vicinity of the 
outer edge 19 of the plate when the plate is inserted in the tray. The 
space between the tray and plate at this location defines the external 
interface between the ambient environment and the interior of the device 
10. In accordance with an important aspect of the invention, the height of 
the capillary gap 21 is less than the height of the gap 22; additionally, 
there is no space in the zone between the capillary gap and the exterior 
of the device 10 whose height is as low as or lower than the gap 21. A 
cover (not shown) is placed over the device during cell culture and the 
combination of the cover, the plate, the tray and both gaps 21 and 22 form 
a narrow, tortuous path which minimizes the chances for contamination 
during cell culture while still allowing the necessary exchange of gases. 
As mentioned, sterility is an important consideration for cell culture 
applications; however, it is not desirable to hermetically seal the device 
10 because this would obviate the ability to exchange CO.sub.2 and O.sub.2 
gas between the cells and the ambient environment. Thus spillage of growth 
media during handling is a potential source of microbiological 
contamination. If the medium stagnates and remains in contact with the 
external environment, a pathway for microbiological contaminants to enter 
the device will result due to the presence of a continuous liquid path 
directly to the reservoir 16. To overcome these difficulties, the height 
of the capillary gap 21 is kept as small as possible consistent with the 
need to allow gas exchange. Heights of between 0.01 mm and 1.0 mm have 
been found to be particularly advantageous. On the other hand, the height 
of the second gap 22 is made significantly larger than that of the gap 21, 
for example, between 0.1 mm and 5.0 mm. Heights of the gap 22 can be 
several times the height of the gap 21. That is to say, if the capillary 
gap is 0.2 mm the second gap will be 1.0 mm or thereabouts. Thus when the 
device 10 is tilted and liquid spills from the reservoir 16 it will 
preferentially remain in the capillary gap 21 where it is isolated from 
the contaminating external environment because of the absence of liquid in 
the second, larger gap 22. 
Other considerations which influence the creation of the above described 
liquid barrier involve the selection of materials having appropriate 
surface wetting properties relative to the cell culture liquid. It would 
be preferred to have both the plate 11 and the tray 14 made of materials 
that were not wetted by the cell culture liquid. That is, when the contact 
angle of the liquid on both surfaces is greater than 90.degree., the 
liquid will not enter the gap 21 except under the influence of some 
pressure. However, materials having such wetting properties often are not 
acceptable for other reasons. The device 10 also will prevent 
contamination of the cell culture liquid if the plate 11 and the tray 14 
are made of materials that the cell culture liquid wets but on which it 
will not spread. The cell culture liquid will not spread if it makes a 
contact angle of less than 90.degree. but greater than 0.degree. with the 
solid surfaces. Under these conditions, the cell culture liquid may spill 
beyond the capillary gap 21 into the second gap 22; however, any culture 
liquid which remains in the gap 22 will return to the reservoir 16 when 
the device is once again leveled. For certain applications the inclusion 
of a sharp edge 30 at the interface between the capillary gap 21 and the 
second gap 22 will enhance the liquid barrier performance. 
FIG. 4 shows an alternate embodiment for the device 10 wherein the ridge is 
of different construction. In this embodiment, a ridge 40 is formed 
between two slopes defined by tapered ledge 41 and perpendicular side wall 
42. The ledge 41 tapers from a narrow height with respect to the plate 11 
at the innermost edge of the tray 14 to a wider height at the outermost 
edge of the tray. In this instance, the wide end would not need to be much 
wider than the narrow end. It is also possible to taper the ledge in the 
opposite direction as long as the taper does not create a space whose 
height is less than the capillary gap 21. 
The invention is not intended to be limited by the foregoing examples as 
still other modifications may be possible or will become apparent to those 
of skill in the art without departing from the scope of the present 
invention.