HK1069847B - Disposable vessel - Google Patents

Description

Disposable container

Technical Field

The invention relates to a laboratory vessel. More particularly, the present invention relates to an improved container for cell culture and methods of use thereof.

Background

There are two main types of cells that grow in vitro: suspension cells (anchorage-independent cells) and adhesion cells (anchorage-dependent cells). Suspension or anchorage-independent cells can be proliferated in vitro without being attached to a surface. In contrast, adherent or anchorage-dependent cells need to be attached to a surface in order to grow in vitro.

Suspension or anchorage-independent cells are typically grown in glass tubes, metal or rigid plastic containers. However, there are some drawbacks in using these cell culture vessels. Glass and metal cell culture vessels are expensive and require maintenance because they are not disposable or sterile. In order to maintain a sterile or aseptic environment for cell culture, it is often necessary to sterilize the container using an autoclave. Therefore, the cell culture vessel must be cleaned and sterilized before and/or after use. In addition, since the glass and metal cell culture vessel is not disposable, a sufficient space is required to store the glass and metal vessel. Thus, there is a need for a cell culture container that is inexpensive, disposable, foldable, and pre-sterilizable, since glass, metal, and rigid plastic containers are very expensive, not disposable, and require a great deal of maintenance.

Furthermore, for anchorage-independent growth of biological cells, these cells need to be in continuous suspension. In order for the cells to remain suspended, the cell culture vessel must have a means for maintaining the cells in suspension. Many cell culture vessels have an impeller with vanes that rotate to keep the cells in suspension. If the impeller is rotated or moved too strongly or the fins are too stiff or too long, the cells may be sheared by the force of the impeller or fins. Also, if the impeller rotates or moves too weakly or the fins are too short, the cells cannot remain suspended. Accordingly, there is a need for an improved cell culture vessel that provides gentle agitation to prevent shearing and maintain the cells in suspension.

Disclosure of Invention

The present invention provides a vessel for cell culture, comprising: a top plate having a circumferential edge; a collapsible bag having an inner surface, an outer surface and a top periphery, the top periphery of the bag being sealed to the edge of the top panel; and an impeller comprising a hollow flexible shaft having a top region and a bottom region, said top region being connected to said top plate, the impeller having a flexible flap, said top region of said shaft containing means for limiting the movement of said shaft to periodic oscillatory rotation.

According to another aspect of the present invention, there is provided a vessel for cell culture, comprising: a top plate; a pre-sterilized collapsible bag sealed to the top panel; an impeller including a hollow flexible shaft connected to said top plate, said hollow flexible shaft having a top region; two flexible flaps connected to the impeller; and a retractor o-ring disposed on a top region of the hollow flexible shaft.

According to another aspect of the present invention, there is provided a container comprising: a top plate having a circumferential edge; a collapsible bag having an interior surface, an exterior surface and a top periphery, said top periphery of said bag being sealed to the edge of said top panel; and a flexible impeller located in said collapsible bag and comprising a hollow flexible shaft and a flexible flap, said hollow flexible shaft having a top region, said impeller being mounted to said top plate at said top region of said hollow flexible shaft, said top region of said shaft including means for limiting movement of said shaft to periodic oscillating rotation, such that said impeller turns in periodic oscillating rotation.

The present invention also provides a container comprising: a top plate; a pre-sterilized collapsible bag sealed to the top panel; an impeller including a hollow flexible shaft connected to said top plate, said hollow flexible shaft having a top region; two flexible flaps connected to the impeller; an o-ring disposed on a top region of the flexible shaft; and a hole disposed in the top plate for access to the hollow flexible shaft of the impeller.

The present invention also provides an impeller comprising a shaft and one or more flexible tabs projecting from said shaft, said shaft having a top end region and a bottom end, said top end region of said shaft including means for restricting the movement of said shaft to periodic oscillatory rotation, such that said impeller rotates in periodic oscillatory rotation.

Drawings

The features of the present invention are believed to be novel. The figures are for illustration purposes only and are not drawn to scale. The invention itself, however, both as to organization and method of operation, may best be understood by reference to the detailed description which follows taken in conjunction with the accompanying drawings in which:

FIG. 1 illustrates one embodiment of the present invention;

FIG. 2 illustrates the elements of the container of the present invention;

FIG. 3 shows a vessel of the present invention filled with fluid and particles on an adjustable magnetic stir plate;

FIG. 4A is a partial side view of a container showing a first rotational movement of the impeller, flexible flaps and particles when a magnetic force is applied to the container, according to the present invention;

FIG. 4B is a partial side view of the container showing continued rotational movement of the impeller, flexible flaps and particles when a magnetic force is applied to the container, in accordance with the present invention;

FIG. 4C is a partial side view of a container according to the present invention showing continued rotational movement of the impeller, flexible flaps and particles as the magnetic force moves the impeller in a direction opposite to the rotational direction shown in FIG. 4B;

FIG. 4D is a partial side view of the container showing continued rotational movement of the impeller, flexible flaps and particles shown in FIG. 4C when a magnetic force is applied to the container, in accordance with the present invention;

FIG. 5A is a top view of a container showing the movement of the impeller, flexible flaps and particles by a first rotation when a magnetic force is applied to the container, according to the present invention;

fig. 5B is a top view of a container according to the present invention showing the movement of the impeller, flexible wings and particles by counter-rotation when a magnetic force is applied to the container shown in fig. 5A.

Detailed Description

The present invention provides a container for cell culture comprising a collapsible bag (collapsible bag) having an inner surface, an outer surface, a top periphery, and a top panel. The bag has a circumferential edge wherein the top periphery of the bag is sealed to the edge of the top panel. The present invention also provides an impeller comprising a hollow flexible shaft having a top region and a bottom region, wherein the bottom region comprises a flexible flap. The present invention further provides a method of mixing fluids comprising the steps of: providing a container having a collapsible bag, said collapsible bag containing an impeller comprising a hollow flexible shaft; inserting a magnet into the hollow shaft of the impeller; an external, adjustable magnetic source is introduced to interact with the magnet and move the magnet and hollow shaft. The method may further comprise the step of removing the magnet from the hollow shaft of the impeller before the container is disposed of (discarded).

A container according to an exemplary embodiment of the present invention has an impeller with thin flexible fins and a hollow shaft in which a reusable magnet may be placed. The presence of the magnet in the impeller shaft and the presence of a restraining mechanism (e.g., an o-ring) disposed on the impeller shaft allows the impeller to rotate smoothly and the flexible fins to subsequently undulate when an adjustable magnetic force (e.g., a magnetic stir plate) is applied to the vessel. This creates gentle agitation of the cells to keep them in suspension and prevent them from being sheared.

FIG. 1 shows a container 100 for cell culture comprising a collapsible bag 105 having an inner surface 110, an outer surface 115, a top periphery 120, and a top panel 125. The top plate 125 has a circumferential edge 130 to which the top periphery 120 of the bag 105 is sealed. One method of sealing includes simply melt bonding the edge 130 to the top plate 125. However, other methods may be employed including gluing, hot melt bonding or other sealing methods known to those skilled in the art. The collapsible bag 105 further includes a base 127 with an outer edge 128 that allows the bag 105 to stand freely. In the exemplary embodiment, outer edge 128 is formed when bag 105 is manufactured. Bag 105 is formed of a foldable plastic and sealed along seam 129. The lower portions of these seams form a stable surface or platform for the bag 105. Other methods of ensuring vertical stability of the bag 105, including the use of a base similar to the top plate 125, are contemplated as being compatible with the present invention.

However, it is important to avoid low flow areas or vortex pockets (eddy pockets) to ensure good mixing within the vessel. For example, fig. 1 shows that the bag 105 does not extend into the outer rim 128. Further, the bottom 121 of the inner surface 110 is made in a circular (arc) shape to achieve good stirring.

In one embodiment of the present invention, the container 100 is made of polyethylene. In addition, the container 100 may be sterilized in advance. When most cell culture processes are carried out under sterile conditions using the so-called sterilization technique, the pre-sterilization of the container 100 maintains the culture chamber and the fluid pathways in a sterile, closed environment. The foldability and disposability of the container 100 is ideal for pre-sterilization, as the optimal goal is to perform the culturing process in a system where the culture chamber and fluid pathway are functionally closed with respect to the external environment, and maintain sterile integrity from the time the device is manufactured until it has been disposed of. One method of pre-sterilization involves gamma radiation. Other methods known to those skilled in the art may also be employed.

In another embodiment, the top plate 125 includes at least one aperture 135. According to the present invention, the aperture 135 may be used to fill the container 100. The aperture 137 may be a hole for gas supply. In this exemplary embodiment, the hole 138 may also be used for inserting a magnet 139. These are merely examples of the many different apertures that may be provided in the top plate 125. Those skilled in the art will be well aware of the requirements for a particular cell culture and can readily provide the wells required for a particular application.

In the exemplary embodiment, container 100 includes an impeller 140 having one flexible flap 145. The flexible flap 145 may be made of polyethylene. The flexible flap 145 may be a single flap, a pair of flaps, or a plurality of flaps. The impeller 140 includes a hollow flexible shaft 150 having a top region 155 and a bottom region 160, the top region 155 being connected to the top plate 125 and the flexible tabs 145 being connected to the bottom region 160 of the shaft 150. In an exemplary embodiment of the invention, flexible flap 145 is adjacent to shaft 150. The shaft 150 of the impeller 140 may contain a magnet 139. In an exemplary embodiment of the invention, the top region 155 of the shaft 150 includes means for limiting the motion of the shaft 150 to a generally periodic oscillation (e.g., an elliptical rotation). According to the present invention, the means for limiting the movement of the shaft 150 may be an o-ring 152, a notch, or other means of forming a relatively weak point in the top region 155 of the shaft 150 of the impeller 140 to prevent the impeller from twisting.

Fig. 2 shows the elements of the container 100. As described above, the elements of the container 100 are the collapsible bag 105, the top panel 125, and the impeller 140. The bag 105 includes an inner surface 110, an outer surface 115, and a top periphery 120. As described above, the top 120 of the bag 105 may be heat sealed or attached to the edge 130 of the top plate 125. The bottom 127 of the bag 105 may have an outer edge 128 to support the bag 105. The outer edge 128 forms a triangle-like shape at the corners of the outer surface 115 of the bag 105 that is spaced apart from the inner surface 110 of the bag 105. The impeller 140 includes a hollow flexible shaft 150 having a top region 155 and a bottom region 160, wherein the top region 155 is attached, such as by heat sealing, to the bottom 200 of the hole 138 in the top plate 125 and the bottom region 160 of the shaft 150 is attached to the flexible flap 145.

Fig. 3 shows a container 100 filled with a fluid 300 and particles 305 on an adjustable magnetic stir plate 310. In this example, the fluid 300 is a cell culture medium and the particles 305 are biological cells.

Fig. 4A-4D illustrate the sequential movement of the flexible flaps 145 of the shaft 150 of the impeller 140 and the particles 305 in the vessel 100 when a magnetic force is applied to the vessel 100 with an adjustable magnetic stirring plate 310. The adjustable magnetic stirring plate 310 comprises a magnetic rod mounted on a shaft, which is driven by a motor. Such stir plates are well known to those skilled in the art. The speed of the motor is generally controlled by a rheostat. According to the present invention, as shown in fig. 4A, when a magnetic force, for example, an adjustable magnetic stirring plate 310 is applied to the vessel 100, the rotation of the magnetic rod (not shown) within the magnetic stirring plate 310 causes an elliptical rotational motion of the magnet 139 within the vessel 100. The movement of the magnet 139 corresponding to the rotation of the magnetic stirring plate 310 causes the impeller 140 to start moving in accordance with the elliptical swing type rotation. When the impeller 140 moves in the direction of arrow 400, the fluid resistance against the flexible flaps 145 forces the flexible flaps 145 to move in the opposite direction of arrow 400, thereby subjecting the particles 305 to agitation. Fig. 4B shows that when the impeller 140 begins to move in the direction of arrow 405, the flexible flaps 145 and particles 305 are forced to move in the opposite direction of arrow 405. Fig. 4C shows that the impeller 140 continues to perform the elliptical oscillating rotation. As the impeller 140 continues to move in the direction of arrow 410, the flexible flaps 145 and particles 305 are forced to move in the opposite direction of arrow 410. Fig. 4D shows that the impeller 140 continues to make an elliptical oscillating rotation and starts to move in the direction of arrow 415. Also, the flexible flap 145 and the particle 305 are forced to move in the opposite direction of arrow 415. The continuous motion of the impeller 140 and flexible fins 145 by the elliptical oscillating rotation results in a continuous suspension of the particles 305.

As shown in fig. 4A-4D, the presence of the constriction 152 creates a type of "fulcrum" or "weak point" along the shaft. This device affects the movement of the impeller 140 when the magnet 139 interacts with an adjustable external magnetic force, such as a stir plate 310. As a result, an elliptic oscillating rotation of the impeller 140 is formed. The constriction device 152 may take many forms including a ring or a segment of material. An exemplary constriction device may be a typical o-ring placed around the shaft. Alternatively, a notch in the shaft itself forms a "fulcrum" that allows the desired elliptical oscillating rotation. The result of the elliptical oscillating rotation of the impeller 140 is a gentle movement of the flexible flaps 145 and a gentle agitation of the fluid 300 and particles 305. When the fluid 300 is a cell culture medium and the particles 305 are biological cells, a smooth movement of the flexible flaps 145 and a smooth agitation of the fluid and particles 305 is necessary.

Fig. 5A and 5B show top views of the container 100 illustrating the effect of the elliptical oscillating rotation of the impeller 140 and the rotation of the magnets in the stir plate on the flexible flaps 145 and particles 305 when a magnetic force is applied to the container 100.

The present invention is also directed to an impeller 140 comprising a hollow flexible shaft 150 having a top region 155 and a bottom region 160, wherein the bottom region 160 comprises a flexible flap 145. The impeller 140 may be made of polyethylene. In an exemplary embodiment, the bottom region 160 of the impeller 140 includes two flexible flaps 145. The hollow flexible shaft 150 of the impeller 140 may also contain a magnet 139 and the magnet 139 may be removable. The ability to remove the magnet 139 from the impeller 140 allows the impeller 140 to be disposed of or discarded and the magnet 139 to be reused with another impeller or container. The ability to reuse the magnet 139 is also quite advantageous since the discarding of the magnet 139 adds additional cost if it must be replaced for each use.

The invention also relates to a method of mixing or agitating a fluid. First, a container is provided that includes a collapsible bag containing an impeller comprising a hollow flexible shaft. A magnet is then inserted into the hollow shaft of the impeller. An external adjustable magnetic source, such as a magnetic stir plate, is introduced to interact with the magnet and move the hollow shaft. The magnet is then removed from the hollow shaft of the impeller. The method may further include a container having a top plate and a hollow shaft with an impeller having a top region and a bottom region, wherein the top region is attached to the top plate.

The invention also relates to a preferred method of using the container of the invention, which is a cell culture method. First, a pre-sterilized container is provided that includes a collapsible bag having a top panel and an impeller including a hollow flexible shaft having a top region and a bottom region, wherein the top region is attached to the top panel and the bottom region includes a flexible flap. A magnet is then inserted into the hollow shaft of the impeller and a cell line and medium are introduced into the vessel through a fill hole. The cell line was then allowed to proliferate. The cell line and medium were removed from the vessel. Finally, the magnet is removed from the hollow shaft of the impeller through the impeller magnet hole and the container is disposed of.

While the present invention has been described in conjunction with a specific preferred embodiment, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the scope of the present invention is intended to embrace any alternatives, modifications and variations that fall within the true scope and spirit of the present invention.

Claims (21)

1. A vessel for cell culture, comprising:

a top plate having a circumferential edge;

a collapsible bag having an inner surface, an outer surface and a top periphery, the top periphery of the bag being sealed to the edge of the top panel; and

an impeller comprising a hollow flexible shaft having a top region and a bottom region, said top region being connected to said top plate, the impeller having a flexible flap, said top region of said shaft containing means for limiting the movement of said shaft to periodic oscillatory rotation.

2. The container of claim 1, wherein the bag is made of polyethylene.

3. The container of claim 1, wherein the container is pre-sterilized.

4. The container of claim 1, wherein the top panel comprises at least one aperture.

5. The vessel of claim 1, wherein the impeller is made of polyethylene.

6. The container of claim 1, wherein the flexible flap is attached to a bottom region of the shaft.

7. The container of claim 6, wherein the flexible flap is adjacent to the shaft.

8. The container of claim 1, wherein the shaft comprises a magnet.

9. The container of claim 1, wherein said means comprises an o-ring.

10. A vessel for cell culture, comprising:

a top plate;

a pre-sterilized collapsible bag sealed to the top panel;

an impeller including a hollow flexible shaft connected to said top plate, said hollow flexible shaft having a top region;

two flexible flaps connected to the impeller; and

a retractor o-ring disposed on a top region of the hollow flexible shaft.

11. A container, comprising:

a top plate having a circumferential edge;

a collapsible bag having an interior surface, an exterior surface and a top periphery, said top periphery of said bag being sealed to the edge of said top panel; and

a flexible impeller located in said collapsible bag and comprising a hollow flexible shaft and a flexible flap, said hollow flexible shaft having a top region, said impeller being mounted to said top plate at said top region of said hollow flexible shaft, said top region of said shaft including means for limiting movement of said shaft to periodic oscillating rotation, such that said impeller turns in periodic oscillating rotation.

12. The container of claim 11, wherein the bag is made of polyethylene.

13. The container of claim 11, wherein the container is pre-sterilized.

14. The container of claim 11, wherein the top panel includes at least one aperture.

15. The vessel of claim 11, wherein the impeller is made of polyethylene.

16. The container of claim 11, wherein the flexible flap is attached to a bottom region of the shaft.

17. The container of claim 16, wherein the flexible flap is adjacent to the shaft.

18. The container of claim 11, wherein the shaft comprises a magnet.

19. The container of claim 18, wherein said means comprises an o-ring.

20. A container, comprising:

a top plate;

a pre-sterilized collapsible bag sealed to the top panel;

an impeller including a hollow flexible shaft connected to said top plate, said hollow flexible shaft having a top region;

two flexible flaps connected to the impeller;

an o-ring disposed on a top region of the hollow flexible shaft; and

a hole disposed in the top plate for access to the hollow flexible shaft of the impeller.

21. An impeller comprising a shaft and one or more flexible tabs projecting from said shaft, said shaft having a top region and a bottom end, said top region of said shaft including means for restricting movement of said shaft to periodic oscillatory rotation such that said impeller rotates in periodic oscillatory rotation.