This application is a continuation-in-part of Application Serial No. 712,413, entitled "Multi Cell Tray which was filed on March 15, 1985. BACKGROUND OF THE INVENTION

The present invention relates generally to tiss culture holders such as dishes, flasks, and multi well tra which have one or more chambers or wells for holding tiss cultures or other reagents for testing.

Tissue culture holders of this type generally re directly on metal grill shelves in incubators, so that whe the incubator door is opened and cool air rushes in a through the shelf openings, uneven heating and cooli effects will result in the culture or reagents under tes It has been shown that this results in uneven heatin effects which are aligned with the grill pattern of th shelf on which the dish or tray stands. It is difficult t overcome these problems since any insulation would interfer with the clear viewing of the culture or cultures in th wells, which normally have transparent walls to allow th cultures under test to be viewed through the bottom of th culture holder using an inverted microscope. SUMMARY OF TRE INVENTION It is an object of the present invention to provide culture holder in which the effects of uneven heating ar reduced or, minimized.

According to the present invention a culture holder i provided which has at least one culture chamber fo containing a tissue culture or other reagent and a insulating chamber below the culture chamber for insulatin the base of the culture chamber.

The culture holder may be a flask or dish fo containing a single tissue culture or other reagent with double walled base defining the insulating chamber, or i may be of the multi-well tray type. The multi-well tra consists of a base having a plurality of wells or chamber for containing cultures and a cover placed over the base t cover the . base and wells. The base has a downwardl depending skirt which extends below the base of the wells and a bottom wall defining the insulating chamber below th wells. The insulating chamber is preferably a sealed chambe and may contain air or any suitable clear fluid which wil not interfere with clear viewing of the culture or cultures This provides a thermal sink which surrounds the cultur chamber or wells in the case of a multi-well tray. The flui may be cold or warm, depending on the temperatur environment desired for the particular test reagents.

In an alternative arrangement, the insulating chambe in the base of a multi well tray may be provided wit passageways to permit gas to flow through the insulatin chamber and into the space between the base and cover abov the wells. The chamber in this case may contain a liqui both to humidify the gas and to act as a thermal sin surrounding and insulating the wells.

The height of the insulating chamber below the cultu chamber or wells is critical. It must be a relatively sma gap so that it will not interfere with clear viewing of t culture through the bottom of the culture holder, while the same time providing sufficient insulation. The thickne or depth of the insulating chamber below the bottom culture chamber or well should preferably be no greater th 5/16 inch.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be better understood fr the following detailed description of some preferr embodiments of the invention, taken in conjunction with t accompanying drawings, in which like reference numera refer to like parts and in which: Figure 1 is a cross-sectional view through a multi wel tray according to a first embodiment of the presen invention having an insulating chamber below the wells;

Figure 2 is a cross-sectional view similar to Figure showing a culture flask according to a second embodiment the invention;

Figure 3 is a cross-sectional view similar to Figure showing a multi-well tray according to a third embodiment the present invention; and

Figure 4 is a perspective view showing the base of th tray in Figure 3.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Figure 1 of the drawings shows a culture holde comprising a multi well tray 10 having a base 12 containin a number of individual wells 14 into which tissue culture or other reagents may be placed for testing, experimenta

and other purposes. A removable cover or lid 16 is plac over the base to cover the open upper ends of the wells and is spaced above the level of the wells by suitable ri or spacers 18, or by a raised rim having passageways for g distribution, as described in my copending applicati Serial No. 712,413 referred to above. This allows gas, f example air, to be distributed into the space 20 above t wells. The tray 10 may be of any suitable shape, such square, circular or rectangular. The tray is made of suitable transparent plastics material or glass to allow t contents of the wells to be viewed at all times.

In the past such culture trays have been plac directly on the metal grill shelves of incubators whi under test. When the incubator door is opened to remo other culture holders or insert new ones, cool air wi circulate around the incubator and through the she openings beneath the wells, resulting in uneven evaporati effects in the cultures under test. In the present case t culture tray 10 is provided with an insulating chamber beneath the wells to reduce or avoid this problem.

As shown in Figure 1, the base 12 has a peripher skirt 24 which extends below the level of the bottom wal 26 of the wells, and the open lower end of the skirt sealed by a bottom wall or panel 28 which is preferab sonically welded around its periphery to the lower edge the skirt. This forms the lower insulating chamber 22, whi may be filled with air or any other suitable fluid. T chamber 22 in this embodiment is sealed, and is preferab manufactured in a low or zero humidity environment, or in vacuum, to avoid or reduce condensation in the chamber whi

could restrict viewing of the wells through the bottom o the tray.

In Figure 1 the chamber 22 is filled with air or othe gas. However, it may alternatively be filled with suitable clear liquid. In this case the liquid could be cooled or heated to provide the desired temperatur environment for a particular culture or reagent under ^* test. The base in this case will be formed with a hole in it upper wall for filling the chamber with liquid. The hole will then be sealed to retain the liquid.

The dimensions of the insulating chamber are critical. If the chamber is too large, it will impede the viewing of the cultures during testing through the bottom wall via a inverted microscope. Thus the gap between the bottom walls of the wells and the bottom wall of the chamber must be sufficiently small so that it will not interfere with the clear viewing of the cultures in the wells through the microscope, i.e. so that the wells will still be within the focal plane of the microscope used. In the preferred embodiment, the depth of the insulating chamber below the bottom walls of the wells is no greater than 5/16 inch. This ensures clear viewing of the contents of the wells via a standard inverted microscope as used in tissue culture applications. Although the insulating chamber is shown in the base of a multi-well tray in the embodiment of Figure 1, in practice such a chamber can be provided in the bottom of any culture holder, such as a culture plate or a culture flask. Figure 2 shows a culture flask 30 of the type generally used to hold a single tissue culture or reagent in chamber 32. A double

wall 34, 36 is provided on the side of the flask which normally rests on an incubator shelf, forming an insulating chamber 38 below the specimen-holding chamber 32. Again the chamber 38 is hermetically sealed and the flask is suitably manufactured in a low or zero humidity environment when chamber 38 is to be an air or gas chamber, to avoid or minimize condensation within chamber 38. The height of insulating chamber is also chosen according to the focal plane of the viewing microscope, and is preferably no greater than 5/16 inch. Chamber 38 may alternatively be filled with a suitable clear liquid which can be cooled or heated to provide temperature control.

Figures 3 and 4 of the drawings show a third embodimen of the invention. In this embodiment a multi-well tray 4 has a base 42 including a plurality of wells 44, and chamber 46 is defined below the wells by bottom wall 48 in similar manner to the embodiment of Figure 1. However, i this embodiment the chamber 46 is not sealed but ha passageways for dispersing gas through the chamber 46 an into the space 50 above the wells between the base 42 an cover 52 of the tray.

An upstanding ridge or rim 54 extending around the wel area spaces the cover above the base and forms an enclose chamber above the wells. Alternatively, a downwardl depending ridge could be provided in the cover, o cooperating rims could be provided on the base and cover. series of openings 55 in the upper wall 56 of the bas outside the rim 54 allow air to flow in the direction o the arrow into the chamber 46. A second series o passageways or openings 58 are provided in the upper wall 5

within the well area, suitably in the gaps between wells illustrated in Figure 4. Gas entering the chamber 46 vi openings 55 will be dispersed via openings 58 up through th base of the tray and into the space above the wells, wit the openings arranged so that gas is dispersed substantiall evenly to all the wells, to reduce or avoid uneve evaporation effects. At the same time, the wells will b insulated from cooling air flowing upwardly through th incubator by the chamber below the wells. The contac between the base and the cover forms a seal against ingres of substantial quantities of gas at their contactin surfaces when the cover is placed over the base, such tha the major gas flow is via the chamber 46 through opening 58. Hydrophobic filters may be provided in the openings 5 to filter the incoming gas or air to remove particles whic may contaminate the cultures. The openings 55 may b replaced with slots or channels to receive a continuou filter strip or strips. The filter pore size will be n smaller than 0.45 microns for effective passage of gas.

If desired a suitable liquid such as water may b provided in the chamber 46 so that air or other gas flowin through the chamber into the space above the wells will b humidified. The water will act as a thermal sink surroundin the wells 44 when the chamber is full, thus helping t insulate the wells and produce even culture results in al the wells.

As mentioned above, the culture tray or holder in all the embodiments is preferably of a transparent material to allow the user to have a clear and continuous view of the well or holder contents during experimentation. The depth of the chamber below the bottom wall of the well or culture chamber will be relatively small to ensure that the chamber will not interfere with clear viewing of the specimen or specimens, and in all cases this height is suitably no greater than 5/16 inch. According to another aspect of the present invention, a method of making a multi-well culture tray with an insulating chamber below the wells is provided. The lid of the tray is suitably molded from transparent plastics material in he standard manner. The base is formed by injection molding plastics material between male and female die parts to form the desired number of wells. This is again a standard technique in manufacturing multi-well trays.

In order to prevent vacuum puckering in the lower wall of each well as the two die parts are separated, a pop- release valve is provided to blow air into the gap between the parts at they are separated, preventing formation of a vacuum. This results in smoother well walls restricting uneven cell aggregation effects as a result of uneven well walls.

Once the upper wall of the base is formed, the bottom wall, which may be a flat plate or a plate with an upturned rim, is suitably attached to the lower edge of the base skirt 24. ^" This is preferably done by sonic welding or alternative bonding techniques around the joint in a vacuum

or in a low or zero humidity environment, to avoid or redu condensation in the chamber below the wells.

Although some preferred embodiments of the inventi have been described above by way of example only, it will understood by those skilled in the field that modification may be made to the disclosed embodiments without departi from the scope of the invention, which is defined by t appended claims.