WO1996030274A9

WO1996030274A9 - Tube closure

Info

Publication number: WO1996030274A9
Application number: PCT/US1996/004347
Authority: WO
Filing date: 1996-03-29
Publication date: 1996-12-05

Abstract

A closure (10) for a culture tube (13), the closure (10) being gas-permeable and liquid-resistant. The closure (10) incorporates a gas-permeable, hydrophobic membrane (22).

Description

TUBE CLOSURE

Field of the Invention

The present invention relates to a closure for a culture tube. In particular, the present invention is directed to a gas-permeable, liquid-resistant closure.

Background of the Invention

The maintenance of eukaryotic and prokaryotic cells in vi tro has proven to be indispensable to basic and applied biological and biomedical research and operations. Considerable effort has been made to develop systems for culturing cells for numerous purposes including virology, the study of cellular physiology and heterologous gene expression.

In order to culture cells in vi tro, chemically defined media containing the necessary nutrients, vitamins, ions and other essential metabolites, is required to provide the proper environment for the metabolism, growth and function of the cells, tissue or organisms. An additional requirement for in vitro culture of eukaryotic cells, such as animal, plant and insect cells, is maintenance of the proper pH in the media through various buffering systems. Under such conditions, it is also desirable to regulate or maintain the gas composition and pH of the liquid media.

Typically, pH is maintained in vi tro through bicarbonate buffering. In a bicarbonate system, NaHCO_β is present in the media and cells are cultured in a controlled atmosphere of CO2 and air at ambient pressure. Culture vessels such as flasks, tubes, dishes and multiwell plates must be constructed to allow for exchange of the controlled atmosphere with the liquid media. The neck of cell culture flasks and tubes is typically threaded and supplied with a cap. Gas exchange is permitted by leaving the cap loose by partially threading the cap onto the vessel neck or employing culture vessels having two-position caps for venting or sealing as disclosed in U.S. Patent Nos. 4,289,248 and 4,456,137. However, the partial seal of the cap to the neck of the culture vessel is a potential entry point for contaminants into the cell culture. Moreover, the flasks and tubes do not allow for small volume cell culture.

Cell culture flasks are currently marketed which have a vented plug seal screw cap which can be securely screwed onto the flask and allows consistent gas exchange, e.g., the Falcon vented tissue culture flask (Falcon Labware catalog nos. 3108, 3109, 3110, 3111 and 3112) . These devices utilize a 0.2 urn hydrophobic filter membrane incorporated into a modified screw cap. The filter membrane acts as a vent and allows for gas exchange while the cap is tightly screwed onto the flask. The small pore nature of the membrane minimizes the possibility of contamination of the culture by excluding bacteria and spores.

However, the vented plug seal screw-capped flasks are inadequate for small volume cell culture. Moreover, these devices do not allow for cell sedimentation by centrifugation without a transfer step to a centrifuge tube.

Other devices described in the patent literature are as follows.

U.S. Patent No. 4,763,804 discloses an autoclavable tissue culture container and closure which provides for semi-open positioning of the closure. This device does not allow for mixing the contents of the culture container.

U.S. Patent No. 4,057,168 discloses a vented top for a bacteria culture medium tube. A diaphragm member functions as a one-way check valve which permits the escape of gases but prevents the entry of impurities. U.S. Patent No. 4,271,973 discloses a sterility testing vessel having a closure. The vessel is a plain cylindrical container having a flat bottom and is preferably made of borosilicate glass. The closure is in the form of a one-piece molded cap which houses a filter element held in place by a clip. The filter is disclosed to act as a vent to prevent pressure differences from building up during autoclaving and cooling, thereby minimizing the risk of a blowout. The vessel volume is disclosed to be about 250 to about 400 ml.

None of the devices described above permit small- volume suspension cell culture. Currently, gas exchange in these types of cultures can be effected in microcentrifuge tubes having volumes of 250 ul to 1.5 ml by partially unscrewing or loosely fitting the caps. This, of course, may allow entry of contaminants into the culture and does not allow for constant mixing without risk of spillage or leakage of the tube contents. Thus, a need exists for a liquid-resistant, gas-permeable closure for a centrifugable culture tube.

Summary of the Invention

Briefly stated, the present invention relates to a closure for a culture tube. The closure includes an annular endcap having an annular rim for sealing engagement with the culture vessel; an endwall having a top face and a bottom face, the top face and bottom face perforated by one or more openings; and a gas-permeable, hydrophobic membrane located adjacent to the bottom face of the endcap. The closure allows for free gas exchange between the interior and the exterior of the culture tube. Brief Description of the Drawings

The foregoing summary, as well as the detailed description of the preferred embodiment, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawing embodiments which are presently preferred, it being understood however, that the invention is not limited to the specific methods and instrumentalities disclosed. In the drawings:

Fig. 1 is a elevational view of a closure in accordance with the present invention;

Fig. 2 is an enlarged cross-sectional view of the closure of Fig. 1 taken along line 2-2 of Fig. 1; and Fig. 3 is a perspective view of an alternative embodiment of a closure in accordance with the present invention.

Description of the Preferred Embodiments Referring to the drawings, wherein like numerals indicate like elements throughout, there is shown in Figs. 1, 2 and 3, a closure 10 for an annular culture tube 13 (not shown in Fig. 2) . The closure 10 comprises a molded annular endcap 11 having an annular rim 12 for sealing engagement with the culture tube and an endwall 14. The endwall 14 has a top face 16 and a bottom face 18, both of which are perforated by one or more openings 20. Located adjacent to the bottom face 18 of the endwall 14 is a gas-permeable, hydrophobic membrane 22 which is affixed to the entire periphery of the endwall 14 such that there are no gaps between the membrane 22 and endwall 14. Preferably, the membrane is annular in shape. Affixation of the membrane 22 to the endcap 11 can be accomplished by techniques well known to those skilled in the art such as gluing in place using a cell/tissue culture-compatible chemical adhesive, heat or ultrasonic fusion or mechanical means.

The membrane porosity can be varied. Preferably, the pore size of the membrane 22 allows for unimpeded gas exchange between the interior and exterior of a cell culture tube 13. Also preferred is a membrane which can be sterilized. Most preferably, the gas-permeable, hydrophobic membrane has a pore size of 0.2 um or 0.45 u . Particularly preferred is an autoclavable membrane having a pore size of 0.2 um.

Materials for the gas-permeable, hydrophobic membrane can be obtained from, e.g., Millipore Corporation, Bedford, MA and Gelman Sciences, Ann Arbor, MI. Exemplary membranes available from Millipore include Durapore® hydrophobic membrane (0.22 and 0.45 um pore size, autoclavable) and Durapel® hydrophobic membrane (0.2 to 2.0 um pore size). Exemplary membranes available from Gelman Sciences which are autoclavable and hydrophobic include GN-6 Metricel® (0.45 um pore size, made of mixed cellulose esters), TF (PTFE) (0.2, 0.45 and 1.0 um pore size, made of polytetrafluoroethylene on a polypropylene screen) and Metricel® polypropylene (0.1 um pore size, made of pure polypropylene) . The annular endcap is constructed of a cell culture compatible plastic material, which may or may not permit attachment of cells. Construction of the endcap can be achieved by plastic fabrication techniques well known to those skilled in the art. Preferably, the plastic material used for fabrication is polystyrene, polypropylene, polypropylene co-polymer or polycarbonate. Most preferably, the plastic material is polypropylene.

The culture tube can be any annular tube with a volume of up to 50 ml in which biological material such as viruses, bacteria, eukaryotic cells and tissues can be cultured or incubated. Preferably, the culture tube is a plastic, conical-type microcentrigue tube having a volume of 250, 400, 500, 1500 or 2000 ul, such as those available from Sarstedt, USA/Scientific Plastics, Nunc, S/P, Eppendorf and Brinkmann.

The closure of the invention sealably engages the wall of the culture tube. The seal formed is air and liquid tight. In one embodiment of the invention shown in Fig. 1, the annular rim 12 can incorporate friction- fit means 26 to sealably engage the culture tube 13.

Closures employing friction-fit means can be of a press- on or a flip-top type. In an alternative embodiment shown in Fig. 3, the annular rim 12 can incorporate thread means 24 to sealably engage the culture tube 13. In use, the closure 10 is sealably engaged to the culture tube. Appropriate culture/incubation media for culture of viruses, bacteria, eukaryotic cells or tissues in suspension or incubation of biological materials are present in the culture tube. If cell culture is desired, appropriate seed materials are also present in the culture tube. The gas-permeability property of the membrane 22 allows for free gas exchange between the culture media within the tube and a defined atmosphere within a cell or tissue culture incubator such that gases within the incubator equilibrate with the culture media thereby maintaining optimal conditioning of the media.

Secondly, the hydrophobicity of the membrane 22 allows the membrane to resist wetting by liquids inside of or outside of the tube, thereby allowing the contents to be mixed, e.g., continuously on a mechanical mixer, without the possibility of the membrane becoming wet. Continuous mixing causes faster equilibration of the media and provides an advantage in culturing living organisms. See Example, infra . Non-wetting of the membrane prevents possible microbial contamination of the culture as well as the preclusion of gas exchange across the membrane.

Third, the small pore size of the membrane prevents biological and non-biological contaminants from entering the tube. Thus, a sterile environment is maintained within the tube.

The present invention will now be described in more detail with reference to the following specific, non- limiting example.

EXAMPLE Membrane Gas Diffusion and Other Characteristics Gas diffusion across a tube closure was tested. Forty ml of Ca/Mg-free HBSS cell culture media containing phenol red, 0.35 g/1 NaHCOβ, 15 mM HEPES and 0.3% BSA was de-gassed with 100% N2 for approximately 20 minutes. One ml of de-gassed media was added to 1.5 ml conical flip-top polypropylene microcentrifuge tubes. The head space above the media in each tube was gassed with N2 and all tubes capped with a solid cap. Solid caps were left in place on two sets of tubes; solid caps were removed from two other sets of tubes and replaced with closures which had been fitted with a 0.2 um hydrophobic membrane in accordance with the invention (vent cap) ; and one set of tubes had the caps completely removed. The tubes were immediately placed in a cell culture incubator with an atmosphere of 5% Cθ2/95% air, 95% relative humidity at 37°C. Some tubes were mixed by placing them on a gyrating mixer set within the incubator. The mixing action was such that the media rocked up against the membrane. After 10, 30 and 50 min, solid caps were placed on each tube while they were still within the incubator and the tubes removed for assay of pθ2 and PCO2 in the media. Gas analysis was performed on an AVL Automatic Blood Gas System by opening a tube immediately before drawing a 100 ul sample of media into a capillary tube and placing the sample immediately into the analyzer. Once all gas analyses for a sample period were complete, the same tubes were placed back into the cell culture incubator with the original cap position or type and allowed to incubate for an additional period of time.

The data in Tables 1 and 2 show that the rate of gas exchange was indistinguishable between tubes with the closure of the invention in the closed position and tubes which had a closure left open. Thus, the membrane permitted unencumbered gas exchange.

Additionally, it was observed that the hydrophobic nature of the membrane, in conjunction with its small pore size, effectively prevented mass loss of liquid-from the tube when inverted. Further observation indicated that upon continuous mixing of the media by rocking on a mechanical mixing device, gases within the media equilibrated approximately 2-fold faster than in stationary tubes. Further, the closure effectively prevented loss of sterility of the media within the tubes for at least 5 days under cell culture conditions (37° C, 95% relative humidity, 5% Cθ2/95% air) .

The integrity of the membrane was examined microscopically after repeated autoclaving (250° F for 20 in) , centrifugation (tested up to 12,000x g) or wetting (with cell culture media) , alone or in combination. The membranes were determined to be intact and had not separated from the cap. No adverse effect on gas exchange capability was observed.

The effect of autoclaving on membrane diffusion capability was tested. Tubes and closures were subjected to a temperature of 250°C for 20 min in a bench-top steam autoclaving unit. Tubes with Ca/Mg-free HBSS media were prepared as described above with the exception that solid caps were left in place on one set of tubes, one set of tubes was left uncapped and a closure in accordance with the invention put in place on all other sets. Two sets of tubes capped by a closure of the invention (vent cap) (one set autoclaved) were placed on a gyrating mixer and allowed to mix continuously such that the media was in contact with the membrane in the closure approximately one-half the time. All tubes were placed in a cell culture incubator under conditions identical to those described above and, at indicated time points, all tubes were removed from the incubator and capped with a solid cap. The solid cap was removed from each tube immediately before analysis of the media and a 100 ul sample collected into a capillary tube. The solid cap was replaced immediately after the media sample was collected. PCO2 and pθ2 were determined as described previously. After sampling was complete, all tubes were placed back into the incubator with the original cap/closure condition and allowed to incubate for an additional period of time. The data in Tables 3 and 4 show that autoclaving had no adverse effect on gas exchange capability of the closure of the invention.

Table 1 P02/PC02 (mm Hg) Measured in Two Independent Tubes

Duration of N2 solid cap membrane membrane solid cap membrane equilibration gassed closed cap cap closed cap period in media no mixing closed open w/ mixing closed incubator no mixing no mixing w/ mixing

Omin 74.9/ 8.6

10 min 139.8/7.8 134.1/10.2 144.3/ 10.1 118.2/8.0 172.5/ 13.7

98.5/ 8.5 137.8/10.1 152.6/ 10.4 129.4/8.2 166.9/ 20.0 mean 119.2/8.2 136.0/ 10.2 148.5/ 103 123.8/8.1 169.7/ 16.9

168.6/7.7 177.3/ 15.4 169.8/ 17.2 172.1/7.5 174.9/43.8

30 min

168.8/7.9 175.9/ 15.5 172.0/ 15.9 185.2/7.5 173.5/35.1 mean 168.7/7.8 176.6/ 15.5 170.9/ 16.6 178.7/7.5 174.2/39.5

181.4/7.3 168.5/23.5 169.8/27.2 181.2/7.1 166.8/ 35.7

50 min

183.0/7.5 171.7/25.2 172.9/ 26.4 183.3/7.2 167.4/37.6 mean 181.2/7.4 170.1/24.4 171.4/26.8 1823/ 7.2 167.1/36.7

Effect of Autoclaving on Membrane Diffusion Capability

A- solid cap, no mixing C - vent cap, no mix E- vent cap, continuous mix

B - open cap, no mixing D - vent cap, no mix, autoclaved F- vent cap, continuous mix, autoclaved

Vented Tube-Cap 02 Diffusion

A- solid cap, no mixing C - vent cap, no mix E- vent cap, continuous mix

Vented Tube-Cap C02 Diffusion

It is appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concepts thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the scope and spirit of the invention as defined by the appended claims.

Claims

1. A closure for a culture tube comprising: an annular endcap having an annular rim for sealing engagement with the culture tube; an endwall having a top face and a bottom face, the top face and bottom face perforated by one or more openings; and a gas-permeable, hydrophobic membrane located adjacent to the bottom face of the endcap; whereby the closure allows for free gas exchange between the interior and the exterior of the culture tube.

2. The closure of claim 1 wherein the gas- permeable, hydrophobic membrane has a pore size of 0.2 um.

3. The closure of claim 1 wherein the gas- permeable, hydrophobic membrane has a pore size of 0.45 um.

4. The closure of claim 1 wherein the annular endcap is constructed of a cell culture compatible plastic material.

5. The closure of claim 4 wherein the cell culture compatible plastic material is polystyrene.

6. The closure of claim 4 wherein the cell culture compatible plastic material is polypropylene.

7. The closure of claim 1 wherein the annular endcap sealably engages the culture tube by thread means.

8. The closure of claim 1 wherein the annular endcap sealably engages the culture tube by friction-fit means.

9. The closure of claim 1 which sealably engages a culture tube having a volume of up to 50 ml.

10. The closure of claim 9 which sealably engages a conical microcentrifuge culture tube.

11. The closure of claim 10 which sealably engages a conical microcentrifuge culture tube having a volume of 250, 400, 500, 1500 or 2000 ul.

12. A closure for a culture tube having a volume of up to 1500 ul comprising: an annular endcap constructed of a cell-culture compatible plastic material, the annular endcap having an annular rim for sealing engagement with the culture vessel; an endwall having a top face and a bottom face, the top face and bottom face perforated by one or more openings; and a gas-permeable, hydrophobic membrane having a 0.2 um pore size located adjacent to the bottom face of the endcap; whereby the closure allows for free gas exchange between the interior and the exterior of the culture tube.