Cell/tissue culturing device and method

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CELL/TISSUE CULTURING DEVICE AND METHOD

FIELD OF INVENTION

The present invention relates to devices for culturing and harvesting cells and/or tissues, bioreactors and fermentors. In particular this invention relates to such devices which are disposable but which nevertheless may be used a plurality of times prior to disposal of same.

INTRODUCTION

Cell and tissue culture techniques have been available for many years and are well known in the art. The prospect of using such culturing techniques economically is for the extraction of secondary metabolites, such as pharmaceutically active compounds, various substances to be used in cosmetics, food additives and natural pesticides, from a harvest of the cultured cells or tissues. While potentially lucrative, this prospect has nevertheless not crystallised. Prior art technology for the production of cell and/or tissue culture in commercial quantities, to be used for the extraction of secondary metabolites, is based on glass bioreactors and stainless steel bioreactors. These bioreactors represent expensive capital items, and are in general each provided with substantially the peak volume capacity that is required at the time. The alternative, namely to provide a number of smaller bioreactors whose total volume capacity matches requirements, while offering a degree of flexibility for increasing or reducing overall capacity, is nevertheless much more expensive than the provision of a single larger bioreactor. Furthermore, running costs associated with glass and stainless steel bioreactors are also high, due to low yields coupled to the need for sterilising the bioreactors after every culturing cycle. Consequently, the products extracted from cells or tissues grown in such bioreactors are expensive, and cannot at present compete commercially with comparable products produced with 1 alternative techniques. In fact, only one Japanese company is known to use the aforementioned cell/tissue culture technique commercially, using stainless steel bioreactors. This company produces Shikonin, a compound which is used exclusively in Japan.

Disposable bioreactors may also be used for culturing cells and/or tissues and may thus provide an alternative solution to glass and stainless steel bioreactors. One such known disposable bioreactor is produced by Osmotec, Israel, and comprises a conical bag having an inlet through which culture medium, air, inoculant and other optional additives may be introduced. However, these bags are used mainly in laboratory research and their volume is determined by the overall volume capacity requirements, as for the stainless steel bioreactors. Mixing of the medium is performed by very small air bubbles which in many cases results in damage to cell walls, particularly in the case of plant cell cultures. In particular, these bags are for single-use only, and are therefore not directed towards an economical solution to the question of providing commercial quantities of secondary metabolites to be extracted from the culture, as discussed above.

An aim of the present invention is to provide a device, and associated method, for culturing and harvesting cells and/or tissue which does not have the aforegoing disadvantages, and which furthermore is economical and simple to produce and to use.

Another aim of the present invention is to provide such a device which is disposable, but nevertheless may be used a plurality of times for cyclically culturing and harvesting desired cells and/or tissues. 2

Another aim of the present invention is to provide such a device wherein inoculant is only required to be provided for the first culturing cycle, while inoculant for subsequent cycles is provided by a portion of the culture broth which remains in the device after harvesting same in a preceding cycle.

Another aim of the present invention is to provide a battery of such devices for industrial scale production of cells and/or tissues.

SUMMARY OF THE INVENTION A multiple-use disposable device, and corresponding method, for culturing and harvesting cells and/or tissue in cycles comprising a sterilisable transparent and/or translucent reservoir having a top end and a bottom end, which reservoir may be filled with a suitable sterile biological cell and/or tissue culture medium and/or sterile inoculant and/or sterile air and/or other sterile required additives, said reservoir comprising: air inlet means for introducing sterile air in the form of bubbles into said culture medium through a first inlet opening connectable to a suitable air supply; gas outlet means for removing excess air and/or waste gases from said reservoir connectable to a suitable exhaust means, said exhaust means comprising means for substantially preventing introduction of contaminants into said reservoir via said gas outlet means; additive inlet means for introducing said inoculant and/or said culture medium and/or said additives into said reservoir, said additive inlet means comprising means for substantially preventing introduction of contaminants into said reservoir via said additive inlet means; and harvesting means for harvesting a desired first portion of the said medium containing cells and/or tissue, wherein a 3 119310/3 remaining second portion of said medium containing cells and/or tissue serve as inoculant for a next culture and harvest cycle wherein said culture medium and/or said required additives provided, said harvest means comprising means for substantially preventing introduction of contaminants into said reservoir via said harvesting means; wherein said device may be utilised a plurality of times for culturing and harvesting said cells and/or tissue in cycles, wherein inoculant is externally provided for a first cycle thereof, and wherein for each subsequent cycle, said second portion of medium with cell/tissue remaining after a preceding harvest serves as inoculant for the next cycle, and wherein medium and air and any other required additives are provided in suitable quantities during each cycle to enable culture of said cells and/or tissue from said inoculant, and wherein said device may be disposed of when contaminated.

DESCRIPTION OF FIGURES

Figure 1 illustrates the main components of a preferred embodiment of the present invention in cross-sectional side view.

Figure 2 illustrates the main components of a second embodiment of the present invention in cross-sectional side view and in front elevation.

Figure 3 illustrates the main components of a third embodiment of the present invention in cross-sectional side view and in front elevation.

Figure 4 illustrates the main components of an embodiment of the battery of the present invention.

Figure 5 illustrates the main components of another embodiment of the battery of the present invention. 4

DESCRIPTION

The present invention relates to a multiple-use disposable device for culturing and harvesting cells and/or tissue in cycles comprising a sterilisable transparent and/or translucent reservoir having a top end and a bottom end, which reservoir may be filled with a suitable sterile biological cell and/or tissue culture medium and/or sterile inoculant and/or sterile air and/or other sterile required additives, said reservoir comprising:- (i) air inlet means for introducing sterile air in the form of bubbles into said culture medium through a first inlet opening connectable to a suitable air supply; (ii) gas outlet means for removing excess air and/or waste gases from said reservoir connectable to a suitable exhaust means, said exhaust means comprising means for substantially preventing introduction of contaminants into said reservoir via said gas outlet means; (iii) additive inlet means for introducing said inoculant and/or said culture medium and/or said additives into said reservoir, said additive inlet means comprising means for substantially preventing introduction of contaminants into said reservoir via said additive inlet means; and (iv) harvesting means for harvesting a desired first portion of the said medium containing cells and/or tissue, wherein a remaining second portion of said medium containing cells and/or tissue serve as inoculant for a next culture and harvest cycle wherein said culture medium and/or said required additives provided, said harvest means comprising means for substantially preventing introduction of contaminants into said reservoir via said harvesting means; wherein said device may be utilised a plurality of times for culturing and harvesting said cells and/or tissue in cycles, wherein inoculant is externally provided for a first 5 119310/2 cycle thereof, and wherein for each subsequent cycle, said second portion of medium with cell/tissue remaining after a preceding harvest serves as inoculant for the next cycle, and wherein medium and air and any other required additives are provided in suitable quantities during each cycle to enable culture of said cells and/or tissue from said inoculant, and wherein said device may be disposed of when contaminated.

Thus, with reference to Figures 1, 2, and 3, corresponding respectively to a first, second and third embodiments of the device, the device, generally designated (10), comprises a transparent and/or translucent reservoir (20), having a top end (26) and a bottom end (28). The said reservoir (20) comprises a side wall (22) which is preferably substantially cylindrical, though other shapes such as rectangular or polyhedral, for example, may also be suitable. The said bottom end is suitably shaped to minimise sedimentation thereat. For example, in the first embodiment, the said bottom end (28) is substantially convex. In the second embodiment, the bottom end (28) is substantially semi-cylindrical. In the third embodiment, the bottom end (28) may be substantially frustro-conical or alternatively has at least one upwardly and outwardly sloping wall (29). The aforementioned configurations of the bottom end (28), in conjunction with the location of the outlet (76) (hereinafter described) near the bottom end (28), enables air supplied via said outlet (76) to induce a mixing motion to the reservoir contents at the bottom end (28) which effectively minimises sedimentation thereat. The reservoir (20) comprises an internal volume (30) which is typically between 5 and 50 litres, though may be greater than 50 litres or less than 5 litres. Said internal volume (30) may be filled with a suitable sterile biological cell and/or tissue culture medium (65) and/or sterile inoculant (60) and/or sterile air and/or other sterile required additives, as hereinafter described. In the aforementioned embodiments, the reservoir (20) is substantially non-rigid, being made from materials such as polyethylene or polycarbonate, for example. The said reservoir (20) may optionally comprise two concentric outer walls (24) (Figure 1) to 6 119310/2 enhance mechanical strength and to minimise risk of contamination of the contents via the reservoir walls. In other embodiments, the reservoir may be rigid and made from a suitable plastic material. In all embodiments, the reservoir (20) is made from a material or materials that enable the reservoir to be sterilised prior to first use.

The reservoir (20) comprises air inlet means for introducing sterile air in the form of bubbles (70) into said culture medium (65) through a first inlet opening (72) connectable to a suitable air supply (not shown). In the aforementioned embodiments, said air inlet means comprises a pipe (74) extending from said inlet opening (72) to a location inside said reservoir (20) at or near said bottom end (28). The pipe (74) thus comprises an air outlet (76) of suitable diameter to produce air bubbles (70) of a required mean diameter. These bubbles not only aerate the medium (65), but also serve to mix the contents of the reservoir, thereby minimising sedimentation at the bottom end (28) as well, as hereinbefore described. In some cases, particularly relating to plant cells, small bubbles may actually damage the cell walls, and a mean bubble diameter of not less than 4mm substantially overcomes this potential problem. Optionally, said outlet (76) may be restrained in position at said bottom end (28) by means of a tether (not shown) or other means known in the art.

The said reservoir (20) further comprises gas outlet means for removing excess air and/or waste gases from said reservoir (20). These gases collect at the said top end (26) of the said reservoir (20). The said gas outlet means may comprise a pipe (90) having an inlet (96) at or near the said top end (26), said pipe (90) being connectable by known means to a suitable exhaust means (not shown). The said exhaust means preferably comprises suitable suction means such as a suction pump, for example. The said exhaust means further comprises means, such as a suitable one-way valve, for substantially preventing introduction of contaminants into said reservoir via said gas outlet means. At least a portion of the top end (26) may be suitably configured to 7 119310/2 facilitate the collection of waste gases prior to being removed via said inlet (96).

Thus, in the second and third embodiments, the upper portion of the top end (26) progressively narrows to a minimum cross sectional area near the location of the inlet (96). Alternatively, at least the upper portion of the top end (26) may be correspondingly substantially frustro-conical or convex.

The said reservoir (20) further comprises additive inlet means for introducing said inoculant and/or said culture medium and/or said additives into said reservoir. In the aforementioned embodiments, said additive inlet means comprises a suitable pipe (80) having an outlet (86) preferably at or near the said top end (26). Said pipe (80) is connectable by known means to a suitable sterilised supply of said inoculant and/or said culture medium and/or said additives. Said additive inlet means further comprises means for substantially preventing introduction of contaminants into said reservoir via said additive inlet means, and comprises, in these embodiments, a suitable one-way valve (84).

The said reservoir (20) further comprises harvesting means for harvesting a desired first portion of the said medium containing cells and/or tissue, wherein a remaining second portion of said medium containing cells and/or tissue serve as inoculant for a next culture and harvest cycle wherein said culture medium and/or said required additives provided. In the aforementioned embodiments, said harvesting means comprises a pipe (50) having an inlet (52) in communication with said internal volume (30), and an outlet (56) outside said reservoir (20). Preferably, said inlet (52) is located near the bottom end (28) of the said reservoir (20), so that only the reservoir contents above said inlet (52) are harvested. Thus, at the end of each harvesting cycle, said second portion of medium containing cells and/or tissue automatically remains at the said bottom end (28) of the said reservoir (20), up to a level below the level (51) of the said inlet (52). Alternatively, said inlet (52) may be 8 119310/2 located at the lowest point in the said reservoir (20), wherein the operator would manually ensure that a suitable portion of medium containing cells and/or tissue would remain in the reservoir (20) after harvesting a desired portion of medium and cells and/or tissue. Said harvest means further comprises means for substantially preventing introduction of contaminants into said reservoir via said harvesting means, and comprises, in the aforementioned embodiments, a suitable one-way valve (54). Said pipe (50) is of a relatively large diameter, typically 2cm, since the harvested cell and/or tissue flow therethrough is turbid.

In the aforementioned embodiments, said remaining second portion of said medium containing cells and/or tissue typically comprises between 10% and 20% of the original volume of said culture medium and said inoculant, though said second portion may be greater than 20% or less than 10% of the said original volume, if required.

Said device (10) optionally further comprises attachment means for attaching same to an overhanging support structure. In the aforementioned embodiments, said support structure may comprise a bar (100) (Figures 2, 3) or rings (not shown). In the first embodiment, said attachment means may comprise a hook (25) preferably integrally attached to the said top end (26) of the said reservoir (20). Alternatively, and as shown for the second and third embodiments in Figures 2 and 3 respectively, said attachment means may comprise a preferably flexible and substantially cylindrical loop (27) of suitable material, typically the same material as is used for the reservoir, either integral with or suitably attached to (via heat welding, for example) the top end (26) of the device. 9

I 119310/1

The present invention also relates to a battery of multiple-use disposable devices for culturing and harvesting cells and/or tissue in cycles, wherein each of a plurality ofthese devices is structurally and operationally similar to said device (10), hereinbefore defined.

Referring to Figure 4, a battery (500) comprises a plurality of said devices (10) are held on a frame (not shown) by means of said attachment means. A suitable air compressor (100) provides air, sterilised by suitable means at (110) such as suitable filters, to each said device (10) via common piping (174) suitably attached to each corresponding air inlet means. Sterilised air is typically provided continuously during the culturing cycle.

In this embodiment, suction means (200) removes excess air and/or waste gases from each of the said devices (10) via common piping (290) suitably connected to each corresponding gas outlet means (90). Alternatively, the common piping may vent to the atmosphere without said suction means (200). Said suction means (200), or where the same is not installed - the common piping (290), is provided with suitable means (210), such as filters, for preventing contaminants from flowing into each said device (10). Alternatively, the gas outlet means (90) of each device (10) may be individually allowed to vent to the atmosphere, preferably via filters which substantially prevent contaminants from flowing into the device (10). 9a 119310/2

In one embodiment, concentrated media in tank (320) is mixed with distilled water from a tank or other suitable supply (330) in a mixing tank (300) and delivered to each said device (10) via a filter (310) and common piping (380) suitably connected to each corresponding additive inlet means (Figure 4). The tank (300) is filled with sufficient concentrated medium to supply all the said devices (10) in the said battery (500) for one cycle. Alternatively, media is contained in a holding tank (340) and pumping means (345) deliver the media at a preferably predetermined and controllable flow rate to a static mixer (350), through which water - either distilled or suitably filtered and purified - flows from a suitable supply (360), preferably with the aid of a suitable pumping means (365) (Figure 5). By adjusting the flow rates of pumping means (345) and (365), for example, the concentration of media available to be delivered into said device(s) (10) may be controlled. Media mixed with water may then be delivered to each said device (10) via a filter (310) and common piping (380) as hereinbefore described. In said mixing tank (300) or said static mixer (350), sterile required additives may be added as well as inoculant prior to delivery to each said device (10)

Alternatively, a single pipe or a set of pipes (not shown) connect said mixing tank (300), or static mixer (350), to a said device (10) or to a corresponding set of devices (10), respectively, at a time, wherein a conveyor system transports the device (10) or set of devices (10) to the said single pipe or set of pipes, respectively, or vice versa. After filling the said device (10) or set of devices (10), the conveyor enables a further device (10) or set of devices (10) to be connected to the mixing tank (300) or static mixer (350) by means of the said single pipe or set of pipes, respectively. io 119310/1

Alternatively, a trolley (380) may be fitted with the end (390) of a suitable flexible pipe (370) connected to said mixing tank (300) or static mixing tank (350). The said trolley (380) may then be moved from one said device (10) to the next, each time the said end (390) being connected to the inlet end of the corresponding pipe (80) to enable media to be provided to each device in turn. Suitable sterilised connecting means (375) and (376) at said end (390) and the inlet of the pipe (80) of the corresponding device (10), respectively, enable each device (10) to be easily connected and subsequently disconnected to the end (390) and thus to the media supply, and many examples of suitable connecting means for connecting two pipes together are well known in the art. Suitable filters (86) and (84) are provided at the end (390) and at the pipe (80), respectively, to prevent or at least minimise potential contamination of the reservoir contents. The trolley (380) may thus be automatically or manually moved from device (10) to device (10), and at each device in turn, an operator may connect the device (10) to the media supply via trolley (380), fill the device with a suitable quantity of media and/or additives, and subsequently disconnect the trolley (380) from the device, to then move on to the next device. The trolley (380) thus provides a simple relatively low-cost alternative for the efficient supply of media to the devices (10). Of course, the end (380) may be adapted to comprise a plurality of connecting means (375) rather than just a single sterilised connecting means (375), so that rather than one, a similar plurality of devices (10) having corresponding connecting means (376) may be connected at a time to the media supply via the trolley (380). Each time, prior to connecting said end (390) to each device or set of devices, the corresponding connecting means (375) and (376) are sterilised.

Each device (10) may be harvested by removing said first portion of medium containing cells and/or tissue into corresponding receiving tanks (400) via suitable 10a 119310/1 stertilised connection means. Alternatively, common piping (not shown) may be utilised to connect each harvesting means of each device (10), via suitable sterilised connecting means, to a common receiving tank (not shown). Alternatively, a single pipe or a set of pipes (not shown) may connect said common receiving tank to a said device (10) or a corresponding set of devices (10), respectively, at a time, wherein a conveyor system transports the device (10) or set of devices (10) to the said single pipe or set of pipes, respectively, or vice versa. After harvesting the said device (10) or set of devices (10), the conveyor enables a further device (10) or set of devices (10) to be connected to the said common receiving tank by means of the said single pipe or set of pipes, respectively. Alternatively, a trolley system (450), similar to said trolley (380) hereinbefore described, may be employed to connect, in turn, a single device (10) or set of devices (10) to a receiving tank (590) via a flexible connecting pipe (550). The trolley end of said pipe (550) and the outlet pipe (50) of each device (10) are fitted with suitable mutually connectable sterilised connecting means (565) and (566), respectively, similar to said connecting means (375) and (376) as hereinbefore described. Suitable filters (56) and (54) are provided at the pipe (550)) and at the pipe (50), respectively, to prevent or at least minimise potential contamination of the reservoir contents in the device (10) or of the contents of tank (590) which may be fitted with yet another filter (not shown) to further minimise risk of contamination therein. The tank (590) may be static or transportable. The trolley system (450) may thus be moved from one device (10) (or set of devices (10), if the trolley (450) is fitted with a plurality of connecting means (565) at the end of said pipe (550)) to the next device (10) (or set of devices (10)) by an operator to harvest in turn said device (10) (or set of devices (10)), and thus provides an efficient and low cost alternative for the harvesting phase. Each time, prior to connecting said pipe (550) to each device or set of devices, the corresponding connecting means (565) and (566) are sterilised. 10b

The present invention also relates to a method for culturing and harvesting cells and/or tissue in a multiple-use disposable device comprising the steps of:- a) providing said device (10), hereinbefore defined ; b) providing sterile air continuously to said reservoir via said air inlet means during each cycle; c) providing sterile inoculant and/or sterile said culture medium and/or sterile said additives via said additive inlet means; d) optionally illuminating said reservoir with external light means; e) allowing said cells and/or tissue to grow in said medium to a desired yield; f) continuously allowing excess air and/or waste gases to leave said reservoir via said gas outlet means; g) checking for contaminants in said reservoir: if contaminants are found in levels in excess of a threshold, the device and its contents are disposed of; if contaminants are not found, or if the levels thereof are below said threshold, step h) is executed; h) harvesting said desired first portion of the said medium containing cells and/or tissue, while leaving said second portion of medium containing cells and/or tissue in said reservoir, wherein said second portion of medium serves as inoculant for a next culture/harvest cycle; 11 i) providing sterile said culture medium and/or sterile said additives for the next culture/harvest cycle via said additive inlet means; j) repeating steps b), d), e), f), g), h) and i) a plurality of times until in g) the said contaminants are found in levels in excess of a threshold, whereupon the device and its contents are disposed of.

The present invention also relates to a method for culturing and harvesting cells and/or tissue in a battery of multiple-use disposable device comprising the steps of:- A) providing a battery of said devices (10), hereinbefore defined ; B) for each said device, providing steps b) to j) as hereinbefore defined.

Typically, a water distiller supplies distilled water to a tank comprising concentrated media, and diluted media is then pumped to the said device (10) via said additive inlet means. Filters, typically 0.2μπι, are installed in the feed pipes and also just upstream of the said additive inlet means to minimise risk of contamination of the reservoir contents in each device (10). Alternatively or additionally, a one-way valve may be also be used to minimise this risk.

For the first culturing cycle of each device (10), inoculant, typically a sample of the type of cell that it is required to harvest in the said device (10), is pre-mixed with media in a steam sterilised container prior to introducing the media into said device (10), though for subsequent cycles, only media is introduced, as hereinbefore described.

Typically, an air compressor provides substantially sterilised air to each said device (10), via a filter, typically 0.2pm, wherein particles, humidity and contaminants are 12 removed. Preferably, another filter just upstream of the said air inlet means further minimises the risk of contamination of the reservoir contents.

For each said device (10), all connections to the reservoir, i.e., to said air inlet means, to said additive inlet means, and preferably also to the gas outlet means and to the harvesting means are sterilised prior to and during connection to peripheral equipment, including, for example, said air supply and said exhaust means. This may be achieved by means of an ultraviolet generation chamber which is provided to enclose each of the connectors and the corresponding peripheral equipment, and irradiating the mating parts of the connectors for a period, typically in the order of minutes, prior to and during connection.

Temperature control for each device (10) is preferably provided by suitable air conditioning means, and optional illumination of the device may be provide by suitable fluorescent light means suitably arranged around the said device (10).

During each culturing cycle of each device (10), the contents of each corresponding reservoir (20) are typically aerated and mixed for 7 to 14 days under controlled temperature and lighting conditions.

At the end of the culturing cycle for each device (10), , the corresponding said harvesting means is typically connected to a presterilised environment by means of suitable connectors which are sterilised prior and during connection, as hereinbefore described. Harvesting is then effected, leaving behind typically 10%-20% of cells and/or tissue to serve as inoculant for the next cycle.

The harvested cells may then be dried, as required. 13

Although only a few embodiments have been described in detail in the foregoing description, the present invention is not limited thereto ands is only defined by the scope of the claims. 14