WO1989000189A2

WO1989000189A2 - Culture bags

Info

Publication number: WO1989000189A2
Application number: PCT/US1988/002187
Authority: WO
Inventors: Julio E. Pabon, Jr.; William E. Findley; William E. Gibbons
Original assignee: Baylor College Of Medicine
Priority date: 1987-07-02
Filing date: 1988-06-29
Publication date: 1989-01-12
Also published as: AU2124988A; WO1989000189A3

Abstract

A culture bag for storing cultured biological materials in a controlled atmosphere is provided which includes an enclosure (17) at least a portion of which is formed by a film of transparent flexible plastic material (12) which is largely oxygen impermeable and which strongly attenuates light below a predetermined wavelength for preventing damage to biological materials stored within the enclosure. The enclosure has an open end (19) for receiving the culture, normally in a culture dish. Sealing means (21) seal the enclosure after it has been filled with a desired gas to create an air-tight sealed chamber within the enclosure. Means (23, 25, 27) are provided for humidifying the gas within the enclosure. Tissue culture vessels are also provided, such as culture dishes, flasks and multiwell plates formed of a transparent material and having a distortion free surface for allowing microscope inspection of biological materials stored therein, and which strongly attenuates light below a predetermined wavelength for preventing damage to such biological materials.

Description

CULTURE BAGS Field of Invention

This invention relates generally to containers for maintaining biological materials in vitro and more particularly to gas impermeable culture bags and tissue culture vessels for maintaining biological materials in vitro under controlled conditions and shielded from potentially toxic short wavelength light.

Background

Biological materials such as tissues, cells, eggs or embryos are often cultured in culture media maintained in the presence of specific gas mixtures. Such cultures may be grown either in incubators or in culture bags which are themselves usually stored in incubators. Particularly when it is desired to culture the materials in a reduced oxygen atmosphere incubators are disadvantageous in that they require large amounts of nitrogen to replace the oxygen introduced from the air each time the door to the incubator is opened. The examination of the cultures often requires repeated door openings which effectively prevent the maintenance of a substantially reduced oxygen concentration within the incubator during, and for as long as 30 min. after such door openings.

Bacterial cultures requiring anaerobic conditions are often grown in special plastic bags containing generators which, when activated, consume most or all of the oxygen within the bag. However, currently available bags have two major problems. Most bags must be heat sealed in order to be airtight and therefore must be cut open and replaced each time a culture is examined. Other bags, intended for use with cultures which are to be maintained at reduced oxygen levels, may be filled with gas from an external tank and then closed with a zip-lock closure. However, if excessive evaporation of the culture medium is to be prevented, the gas must first be humidified by being bubbled through water, which, unless careful precautions are taken, may become contaminated with bacteria. Such contaminants may then be introduced into the culture bag.

In vitro fertilization (IVF) and embryo culture techniques require that the gametes (eggs and sperm) and embryos be cultured for varying lengths of time. The use of incubators for such applications can be inconvenient since they are expensive and are ordinarily set up in a laboratory remote from the patient and the desired gas concentrations cannot be maintained during those parts of the procedures that require repeated door openings. A further problem with currently available incubators and culture bags results from the fact that it has been found by the applicants that certain types of biological materials, including culture media and mammalian pre-embryos, are adversely affected by short wavelength light of the type produced by fluorescent lighting widely used in laboratories and by atmospheric concentrations of oxygen. For instance, applicants believe that exposure of biological materials for as little as two hours or less to such short wavelength light and/or atmospheric concentrations of oxygen can produce toxic effects. This may account, at least in part, for the disappointingly low pregnancy rates that have been achieved through IVF techniques, even with the transfer of several pre-embryos. With humans such rates seldom reach 28% and the on-going pregnancy rate is usually well below 22%.

While currently certain available culture bags do make it possible, albeit in an unsatisfactory manner, to maintain biological materials in a selected atmosphere different from air, their use can result in toxic effects on the biological material as a result of exposure to fluorescent room light.

Summary of the Invention In accordance with the invention there is provided a culture bag for storing cultured biological materials in a controlled atmosphere which includes an enclosure at least a portion of which is formed by a film of transparent flexible plastic material which is largely oxygen impermeable and which strongly attenuates light below a pre-determined wavelength for preventing damage to biological materials stored within the enclosure. The enclosure has an open end for receiving the culture, normally in a culture dish. Means are provided for sealing the open end of the enclosure after it has been filled with a desired gas to create an air-tight sealed chamber within the enclosure. In accordance with another aspect of the invention means are provided for humidifying the gas within the enclosure which may include a wick and a breakable vial containing sterile water for moistening the wick when the vial is broken.

In accordance with a further aspect of the invention the enclosure is filled through a tube adjacent to one side of the open end which communicates with the wick for injecting the gas into the enclosure through the moistured wick while the sealing means is partially closed.

In accordance with a still further aspect of the invention there are provided tissue culture vessels such as culture dishes, flasks and multiwell plates formed of a transparent material and having a distortion free surface for allowing microscope inspection of biological materials stored therein, and which strongly attenuates light below a predetermined wavelength for preventing damage to such biological materials.

These and other advantages and features of the invention can be more fully understood from the following detailed description of the preferred embodiment of the invention as illustrated in the accompanying drawings.

Brief Description of the Drawings

FIG. 1 is a top view of a culture bag in accordance with the invention.

FIG. 2 is a side view of the culture bag of FIG. 1.

FIG. 3 is an enlarged crossectional view of the plastic film material of the culture bag FIG. l. FIG. 4 is an enlarged crossectional view of the plastic film material forming the bottom of the culture bag of FIG. 1.

FIG. 5 is a side view of the gas warming box of- FIG. 3. FIG. 6 is a top view of a gas warming box for use in conjunction with the culture bag in accordance with the invention.

FIG. 7 is a side sectional view of a culture dish in accordance with the invention.

Description of Preferred Embodiment Referring now to FIGS. 1 and 2 of the drawings there is illustrated a culture bag 11 in accordance with the preferred embodiment of the invention. In the illustrated embodiment the culture bag 11 is formed of a single sheet of flexible, transparent plastic film material 12 which is folded in two to form a closed end 13 and sealed along the sides 15 by heat, adhesive, or other means to form an enclosure 17. The open end 19 of the enclosure 17 is provided with a reusable sealing arrangement such as the zip- lock closure 21.

A wick 23, which may be formed of sterile cotton or other wicking material is arranged around the closed sides of the enclosure 17. A plastic vial 25 filled with sterile water and having a cap 27 is incorporated within the wick 23 for humidifying the gas within the enclosure 17. The wick 23 is secured to the sides of the culture bag 11 by ties 29 to maintain it in place. The plastic tube 31 extends from one end of the wick 23 toward the open end 19 of the enclosure 17. The end of the tube 31 proximate the open end 19 is flared for receiving a gas supply needle.

Preferably, the top layer 33 of the enclosure 17 is slightly domed to form a tent like structure to inhibit the bottom 34 of the bag 11 from coming up when the bag 11 is filled with the gas and to facilitate the sealing of the zip-lock closure 21 with a culture dish 35 in the bag. To use the culture bag of the invention, the cap

27 is broken off of water vial 25 so that the wick may begin to fill with water. A culture dish 35 is placed within the enclosure 17 and the zip-lock closure 21 is sealed except for a small opening in front of the gas tube 31. To establish the desired atmosphere within the bag 11, means such as a blunt large bore (e.g. 16g) needle, which is connected to a supply of a gas may be used to fill the enclosure 17. The needle is preferably connected to the gas supply through a 0.2 micron filter in order to filter out any contaminants, such as bacteria, in the gas. After turning on the gas supply the needle is inserted into the bag 11 opening beside the tube 31 and the bag 11 is filled by desired gas. When the bag 11 is slightly turgid, it is pressed flat with the hand in order to purge the interior of the air containing atmosphere. This filling and expelling is repeated one or more times and the needle is then inserted through the gas tube 31 into the wick 23. The purge process is then preferably repeated another time with the gas being humidified by passing through the moist wick 23. After the last purge, the bag 11 is filled once more through the gas tube 31 and the needle is withdrawn while the zip-lock closure 21 is tightly closed behind it. The thumb and finger press points 37 are then tested to be sure that they can be easily pressed together. This indicates that there is sufficient space in the enclosure 17 for the gas to expand when it is heated to the desired temperature (usually about 37° C. for IVF applications). If the bag 11 is too full, the zip-lock closure 21 may be slightly separated at one location and the press points 37 again pressed to force out sufficient gas. While still pressing the press points 37 the closure 27 is re-sealed. To insure that there is no slow leakage through the closure 27, a clip 39 is applied to the extending strip 41 of the bag 11. The clip 39 preferably includes two crimp points to insure an air-tiσht seal of the enclosure 17. In accordance with the invention the plastic film material 12 forming the bag 11 is substantially impermeable to gas molecules such as oxygen and strongly attenuates light of wavelengths below a pre- selected level, such as 500 nm. Referring to Fig. 3 of the drawings the plastic film material 12 in the illustrated embodiment is made up of a multi-layer film comprised of a barrier film 43 for blocking the transmission of oxygen sandwiched between inner and outer layers of film material 45 and 47, respectively, that contain light absorbers such as dyes or pigments. The inner and outer layers 45 and 47 serve both to strongly attenuate the light below the pre-selected wavelength and to protect the barrier layer 43 from the effects of high internal and external humidity levels which may tend to undesirably increase the permeability of the barrier layer 43 to oxygen.

The barrier layer 43 is formed of a plastic film material which is substantially impermeable to gas molecules such as oxygen. A preferred material for use as the barrier layer 43 is a film of EVAL EF-XL available from the EVAL Company of America, Lilse, Illinois. EVAL EF-XL is a flexible transparent film made up of ethylene and ethylene resins which has been treated to impart a bi-axial orientation that results in superior barrier properties at elevated temperature and humidity levels of the type to which the bag 11 is exposed in use. Alternative barrier film materials are EVAL EF, a material formed of ethylene and ethylene resins also available from the EVAL Company of America, and Saran High Barrier (HB) sold by The Dow Chemical Company. Saran HB is a copolymer of vinylidene chloride and vinylchloride and has a very low transmission of oxygen and other small molecules. The barrier layer 43 should be thick enough to perform blocking function but not so thick as to make the bag 11 too stiff or difficult to handle. For the EVAL and Saran HB film materials listed above a film thickness of about 0.6 mil and 2.0 mil, respectively, is preferred.

The light produced by fluorescent fixtures of the type normally used in laboratories contains significant amounts of short wavelength components that can adversely affect the culture media and biological materials stored in culture bag 11. To attenuate the transmission of such short wavelength light, the inner and outer film layer 45 and 47 are formed of films of low density polyethylene containing pigments and/or dyes (collectively referred to as absorbers) . In the illustrated embodiment the absorbers are .5% of Tinuvin 326, a UV light absorber available from Ciba-Geigy which is composed of benzotriazole derivatives, and .5% each of the pigments FD&C yellow #5 lake and FD&C yellow #6 lake such as those sold under the trademark Lakolene by H. Kohnstamm, Inc. Tinuvin 326 strongly absorbs light of wavelengths below about 390 nm while FD&C yellow #5 lake and FD&C yellow #6 lake have peak absorbencies at 428 nm and 486 nm respectively.

Alternatively, other absorbers may be substituted for or used in combination with Tinuvin 326. In addition, pigments such as D&C Yellow #10 sold under the designation K7181 by H. Kohnstamm, Inc. could be substituted for or used in combination with the other pigments.

Other die and/or pigment systems could also be used in the plastic film materials of the bag 11 in accordance with the invention provided that they are compatible with the plastic film material, strongly attenuate the light of wavelengths below about 500 nm and leave the film otherwise transparent so as to allow visual inspection of the contents. The absorbers and pigments in the plastic film layers should absorb 90% or more of the incident light having wavelengths of less than about 500 nm. The dyes and pigments used in the film layers 45 and 47 and particularly in the inner layer 45 should be selected so that they do not have potentially adverse effects on the biological materials shown in the bag 11. It is preferable that the dyes and pigments be ones which have been approved by the FDA for use in materials coming into contact with food (FD&C approved) in the concentrations used.

The degree to which the die and pigment system attenuates short wavelength light depends on the concentration of the dyes and pigments in the plastic film layers 45 and 47 and on the thickness of the layers. In the illustrated embodiment the outer film layers 45 and 47 are each 2 mils thick while the barrier film layer 43 is 0.6 mils thick when EVAL EF- XL is used.

Referring now to FIGS. 1 and 4 of the drawings, the plastic film material 12 which forms the floor 34 of the bag 11 preferably includes in the central portion thereof a layer of relatively stiff plastic film material 49, such as a 2 mil thick layer of polystyrene, in order to maintain the floor 34 of the bag 11 essentially flat for ease of handling and of insertion of culture dishes. The polystyrene layer 49 preferably extends to within about one inch of the edges of the bag 11 and of the zip-lock closure 21.

The floor 34 of the bag 11 may also be formed of non-transparent material such as one containing a metal foil. Referring now to FIGS. 5 and 6 of the drawings, there is illustrated a gas warming box which may advantageously be used in pre-warming of the gas used to fill the bag 11. The gas is usually obtained from a pre-mixed commercially available cylinder of compressed gas. As the gas leaves the cylinder it expands and consequently drops in temperature so that the gas being supplied to the bag 11 can be quite cool. Such low temperatures can adversely affect biological materials stored in the bag 11. To obviate this problem the gas from the supply is preferably passed through the gas warming box 51 to pre-warm it before it is fed into the bag 11. The box 51 includes a horizontally disposed electrical heating coil 53. A spirally wound gas conducting tube 55 is disposed below the heating coil 53. The outer extremity of the tube 55 is connected to the externally projecting barbed hose fitting 57 which in turn may be connected to a gas supply cylinder (not shown) . The interior end of the spiral wound tube 55 is connected by an extension of tube 55 through an aperture in the box 51 to a Leur tip 59. A 0.2 micron filter 60 can be attached to the Leur tip 59. A blunt 16g needle 61 is attached to the filter 59 for insertion into the bag 11 proximate the gas filling tube 59. The gas passing through the tube 55 is heated by the heating coil 53 in order to raise its temperature to an appropriate level for the biological materials in the bag 11. Normally a temper-ature of about 37° C. is appropriate. The temperature of the heating coil 53 may be controlled in a well-known fashion by a thermostatic or proportional control (not shown) included in the box 51. An external control 63 may be provided to set the desired temperature or actuation level of the electrical heating coil 53.

Some of the important advantages of the invention can be achieved with the use of a tissue culture vessel formed of a transparent rigid plastic or glass which incorporate dyes and/or pigments for strongly attenuating toxic short wavelength light and have a plane, distortion-free surface for permitting microscope investigation of the contents of the vessel. Referring now to FIG. 7 of the drawings, an example of a tissue culture in accordance with the invention is the culture dish 65 which includes a shallow cylindrical cup shaped lower portion 67 that contains the culture medium and is typically between about 1.5 to 4 inches in diameter. The culture dish 65 also includes a cover 69 which fits loosely over the base portion 67 and allows circulation of the ambient air or gas between the interior and exterior of the culture dish 65. In the illustrated embodiment of the invention the culture dish is formed of clear polystyrene which contains about .5% of ultraviolet light absorber Tinuvin P as well as .5% each of the pigments FD&C yellow #5 lake and FD&C yellow #6 lake. The other absorbers and pigments referred to above for use in the polyethylene layers of the bag 11 may also be used provided that they meet the criteria for compatability with the particular material forming the vessel. The invention also may be embodied in other tissue culture vessels such as tissue culture flasks and multiwell plates formed of a transparent plastic or glass incorporating short wavelength light attenuating dyes and/or pigments and having a plane distortion-free surface for permitting microscope inspection of the contents.

The illustrated embodiments of the invention describes culture bags and tissue culture vessels incorporating absorbers which absorb at least 90% of wavelengths below 500 nm. and having only minor attenuation of light of wavelengths above about 500 nm. While an absorption range limit of about 500 nm is preferred, since it protects the contents of the bag or vessel from potentially toxic short wavelength light while preserving the transparency of the bag or vessel to permit inspection of the contents, the absorption range limit can be set somewhat higher or lower without departing from the scope of the invention. In general the range limit may be set between about 450 and 550 nm while still providing adequate protection to culture media and biological materials in many applications at 450 nm and preserving adequate visible light transmission at the 550 nm to permit inspection of the contents.

Although the illustrated embodiment of the bag 11 of the invention has been described with reference to the use of a cotton wick 23 for humidifying the gas in the enclosure 17, it should be recognized that other humidification means may be used, and in a given application could be preferable from a cost and/or convenience point of view. For instance a dampened gauze pad on the bottom or along one or more closed ends of the bag 11 could be used but may not provide the immediate humidification of the gas accomplished in the illustrated embodiment.

In addition, other plastic film materials can be used to form the bag 11 in place of the laminated structure described so long as they are substantially impermeable to gas molecules such as oxygen and attenuate short wavelength light that is potentially injurious to cultured biological materials. While it is preferred that the bag be transparent to light of wavelengths above about 500 nm to facilitate inspection of the contents_f it is not required that it be so.

It should also be recognized that bag structures different from the disclosed embodiment may also be used in accordance with the invention. For instance. the film forming the bag may be from two separate sheets that are heat sealed together so that, for example, the bags are formed in a roll. In addition the bag may be formed in part of rigid or semirigid materials. It should be recognized, therefore that various modifications may be made to the illustrated embodiment of the culture bag without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

We claim:

1) A culture bag (11) for storing biological materials in a culture medium in a controlled atmosphere, comprising: an enclosure (17) at least a portion of which is formed of a transparent plastic film material (12) that is substantially impermeable to oxygen, said enclosure having an open end (19) for receiving a container of biological materials in a culture medium, said enclosure forming a closed pocket (13) except for said open end, said transparent plastic film material including means (45,47) for strongly attenuating light below a predetermined wavelength for preventing damage to a culture medium and biological materials within said enclosure? and/ means (21) for sealing said open end /of said enclosure for creating a gas-tight sealed chamber within said enclosure.

2) The culture bag of claim 1 further including humidification means (23,25) within said enclosure for raising the relative humidity of the gas within said enclosure.

3) The culture bag of claim 1 in which the portion of such enclosure formed of a transparent plastic film material includes the top side (33) of the bag and in which said plastic film material is flexible and domed for ease of insertion and storage of a culture dish (35) in the bag.

4) The culture bag of claim 1 wherein said portion includes multi-layer flexible plastic film material (12) which includes a substantially oxygen impermeable barrier layer (43) and inner and outer layers (45,47) for shielding the barrier layer from ambient moisture within and outside of the bag, and wherein at least one of said layers includes means for strongly attenuating light below said predetermined wavelength.

5) The culture bag of claim 1 wherein said means for strongly attenuating light below a predetermined wavelength include means for preventing transmission of at least 90% of the light incident on the transparent plastic material having wavelengths below 500 nm.

6) The culture bag of claim 4 wherein said means for strongly attenuating light below a predetermined wavelength include means for preventing transmission of at least 90% of the light incident on the transparent plastic material having wavelengths below 500 nm.

7) The culture bag of claim 6 wherein said means for preventing transmission of incident light includes absorbers of light having wavelengths below 500 nm . incorporated in at least one of said layers.

8) The culture bag of claim 6 wherein said absorbers are incorporated in said inner and outer layers.

9) The culture bag of claim 5 wherein said means for strongly attenuating light includes absorbers of light having wavelengths below about 500 nm incorporated in said transparent plastic film material.

10) The culture bag of claim 4 wherein said portion further includes the floor (34) of said bag and wherein said floor includes a multi-layer plastic film material (49) , said multi-layer film material including a barrier layer which is substantially impermeable to oxygen and a layer of transparent relatively stiff plastic material in the central portion of the floor of the bag for maintaining said central portion relatively flat when said enclosure is filled with gas.

11) The culture bag of claim 2 wherein said humidification means includes a wick (23) and a breakable vial (25) containing sterile water for moistening said wick when said vial is broken.

12) The culture bag of claim 11 further including a tube (31) adjacent one edge of said open end of saiα. enclosure and communicating with said wick for / injecting gas into said enclosure through said wick while said sealing means is partially closed.

13) A tissue culture vessel for storing biological materials in a culture medium under controlled conditions, comprising a transparent container; a cover member for said container; a plane distortion-free surface, for permitting micro- scope examination of biological materials within said container from outside the vessel; and means for strongly attenuating the transmission of the light of wavelengths below a predetermined wavelength from outside said vessel to the interior thereof, said predetermined wavelength being between 450 and 550 nm.

14) The tissue culture vessel of claim 13 wherein said tissue culture vessel is a culture dish (65) having a disk portion (67) and a loose fitting cover (69) both formed of a rigid transparent material, wherein said means for strongly attenuating the transmission of light includes absorbers incorporated in the dish portion and cover for strongly attenuating light having wavelengths below said predetermined wavelength.

15) The tissue culture vessel of claim 14 wherein said predetermined wavelength is 500 nm.

16) A culture bag system, comprising: a culture bag (11) for storing biological materials in a culture medium in a controlled atmosphere including: an enclosure (17) at least a portion of which is formed of a transparent plastic film material (12) that is substantially impermeable to oxygen, said enclosure having an open end (19) for receiving a container of biological materials in a culture medium, said enclosure forming a closed pocket except for said open end, said transparent plastic film material including means for strongly attenuating light below a predetermined wavelength for preventing damage to a culture medium and biological materials within said enclosure; and means (21) for sealing said open end of said enclosure for creating a gas-tight sealed chamber within said enclosure; and means for filling said enclosure with a gas at a controlled temperature including: means for connecting to a source of gas; means (51) for heating the gas received from said source to a controlled temperature; and means (59,60,61) for injecting said heated gas into said culture bag when said sealing means is partially closed.

17) The culture bag system of claims 16 wherein said means for injecting heated gas into said bag includes a Leur tip (59) , a hypodermic needle (61) , and a filter (60) for filtering particles from said gas.