Apparatus and method for volume adjustable cell culture

Abstract

A cell culture apparatus is disclosed comprising a cell culture vessel comprising a flexible tube; a support stand; and a pair of opposed retainers for retaining opposite portions of the tube in a fluid-tight manner such that fluid is not passable through the respective portions inside the tube, the spacing between the pair being adjustable to provide an adjustable sealed volume in the tube between the pair of retainers.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201780041896.X, filed on June 22, 2017, entitled "Apparatus and Method for Volume-Adjustable Cell Culture". Technical Field

[0002] The present invention relates to apparatus and methods for in vitro cell culture with variable volume, and specifically for optimizing said culture. Background Technology

[0003] Conventional cell culture involves expanding cells into increasingly larger containers to make room for the increasing number of cells. This stepwise approach typically takes the form of expanding a small sample of cells of interest from a vial (typically only a few milliliters) to a large volume (e.g., 500–2000 L) in a cell bag or flask. During the cell expansion phase, cells are initially infused into flasks or rollers to allow the cell line to regenerate. Subsequently, the cells are transferred and expanded in cell bags on a shaker or in a stirred flask. During expansion, cells are stepwise transferred to the next larger flask / roller / cell bag / flask and expansion continues. Challenges exist in using large containers for small numbers of cells, namely, the required amount of expensive cell culture media is disproportionate to the resulting cell return; and, inefficient space may exist where there are no cells or very few cells.

[0004] Variable-volume cell culture containers address some of the challenges mentioned above and have been proposed in WO2015 / 009153 and similarly disclosed in US20130157353. These documents show cell culture bags placed on a stage. Rollers press against the stage to compress the entire width of the bag. Movement of the rollers relative to the bag allows the bag to gain a larger volume. However, the inventors have recognized that these known variable-volume cell culture bags present further challenges in maintaining and sustaining a large container size during the initial small-volume culture period; preventing any unused areas of the container from contacting the cell culture during the initial small-volume culture period; increasing the volume without interfering with the container's inlet and outlet ports; and controlling the increased volume throughout the cell expansion phase, thus optimizing the volume of the cell container. Summary of the Invention

[0005] The objective of this invention is to provide a cell culture apparatus comprising embodiments that address the challenges posed above with respect to the known cell culture bags identified above.

[0006] According to a first aspect of the invention, a cell culture apparatus is provided, comprising: a cell culture container including a flexible tube; a support platform; and a pair of opposing retainers for holding opposite portions of the tube in a fluid-impermeable manner, such that fluid cannot pass through the corresponding portions inside the tube, the spacing between the pairs being adjustable to provide an adjustable, sealed volume between the retainer pairs and within the tube. In this first aspect, the retainer pairs provide positive stability at each end of the tube while simultaneously allowing for adjustable volume.

[0007] Preferably, the culture vessel includes multiple fluid ports positioned between a pair of retainers when the vessel is mounted on a stage. Therefore, when the retainers are removed (when a larger volume is required), this movement does not disrupt the ports and their corresponding fluid communication.

[0008] Preferably, the flexible tube is folded or rolled up at the retainer at least once. Therefore, the required compressive force is reduced to provide a fluid-impermeable seal at the retainer, because the tube material is folded at the retainer, rather than simply compressed.

[0009] Preferably, a feedback mechanism is used to control the device to provide accurate position adjustment and thus better control over the volume between the retainers.

[0010] According to a second aspect, the present invention includes a cell culture container formed of a tube made of a polymeric material, the tube initially laid flat and then folded or rolled up at at least one end of the tube, having inlet and outlet ports in the folded or unrolled portion of the tube. Preferably, both ends of the tube are folded or rolled up on each side of the side inlet and outlet ports. Thus, when the tube is filled with liquid, the tube can be unfolded or spread out to increase the usable cell culture volume within the tube.

[0011] According to another aspect of the present invention, a method for providing variable volume in cell culture is provided, the method comprising the steps of: Provides cell culture containers that are initially in the form of flattened tubes; The central portion of the tube is sealed at either side by mechanically compressing the tube and optionally by folding the tube. The liquid for cell culture is introduced into the central portion, so that the opposite end of the tube is substantially free of the liquid due to the seal. The compressed / folded position is moved in the opposite direction to increase the size of the central portion and thus increase the cell culture volume.

[0012] Other aspects of the invention are contemplated. Therefore, the description of a feature in a claim is not intended to limit that feature to combinations with all other features in that claim, but rather, where context permits, such a feature may be combined with other features obtained in other claims or in the description to provide other aspects of the invention. Thus, further advantages and benefits of those other aspects of the invention will become apparent to those skilled in the art, specifically in consideration of the detailed description that follows. Attached Figure Description

[0013] The invention will now be described in more detail with reference to the accompanying drawings, in which: Figure 1 A diagram showing an embodiment of a cell culture apparatus; Figure 2 The first embodiment is shown, but in which... Figure 1 In comparison, the device has a different structure; Figure 3 A cross-sectional view showing the features of the first embodiment is displayed; Figure 4 , Figure 5 and Figure 6 Each shows a partial view of a feature of the first embodiment; Figure 7 A schematic diagram of the control circuit for the first embodiment is shown; and Figure 8-14 A second embodiment of the cell culture apparatus is shown. Detailed Implementation

[0014] Figure 1 A variable-volume cell culture apparatus 5 is shown, comprising a cell culture bag or container in this case in the form of a cell culture tube 10, which is laid flat on a support platform 22, which in turn is supported on a rocking support plate 20. The tube 10 is formed from a polymer as an elongated seamless tube, wherein opposite sides are brought together to form a double-layered, closed, openable cell culture tube. The tube 10 further includes inlet and outlet ports 12, which allow fluid and gas to enter and exit the interior of the tube, thus providing a cell culture container around the ports.

[0015] The corresponding opposite ends of tube 10 are held and folded inside opposing tube holders 30 and 30', which can be moved by means of holder moving mechanism 40. Therefore, since the spacing between the holders is adjustable, it can be seen that the volume of the cell culture container is also adjustable. Figure 1 The figure shows the minimum volume required to begin cell culture.

[0016] In addition, refer to Figure 2Holders 30 and 30' are shown to be moved apart to provide a larger volume for cell culture in the now larger tube portion 10 present between holders 30 and 30'. To facilitate obtaining a larger volume, holders 30 and 30' are moved synchronously such that equal amounts of tube are extended from both sides, as described below. This movement can be performed as a slow, continuous movement, or, as in this embodiment, as a segmented movement using a stepper motor 42. The holders can be moved apart until the desired cell culture volume is obtained based on the distance between the holders. The holders are held in place by guides (e.g., tubular rails 44) that allow linear translation in the direction of arrow X and, generally, translation in the opposite direction after the cell culture process is complete, to return to the position where the initial small volume is required, i.e., as... Figure 1 As shown in the diagram. In this embodiment, translation is achieved by a stepper motor 42, which is fixed in a central position and causes a fixed-axis pinion to rotate. The fixed-axis pinion, in turn, causes a pair of toothed racks to translate, with the racks meshing with the top and bottom teeth of the pinion, respectively. The rotation of the pinion causes the two racks to move in opposite directions.

[0017] Figure 3 A cross-sectional view of retainer 30 is shown, with retainer 30' being a mirror image of the displayed retainer 30. Retainer 30 includes a fixed lower jaw plate 32, to which a loosely engaged hinge pin 34 connects the lower jaw plate 32 to a movable upper jaw plate 36. In use, jaw plates 32 and 36 are pushed together, for example by a tension spring, and can be forced apart by an electric actuator 37 mounted to the lower jaw plate 32 for loading the tube 10. The actuator 37 includes a release pin 33 that pushes the upper jaw plate 36 to increase the gap between the two jaw plates. Jaw plates 32 and 36 further include complementary teeth 31 that closely abut but do not contact each other, forming a meandering path 35 between them, thus effectively folding the tube at an angle greater than 90 degrees to increase the sealing effect of the teeth. The end portion 14 of the tube 10 fits between jaw plates 32 / 36, such that the end stop 16 and the folded and pleated portion 18 of the end 14 are held in place within the cavity 38 formed between the jaw plates, and a further clamping portion 11 of the end 14 is held between teeth 31 in the path 35. In use, the teeth 31 apply sufficient clamping force to keep the tube 10 impermeable at the clamping portion 11, but the tube can still be pulled through the circuitous path 35 when the retainers 30 / 30' move in the direction of arrow X. An elastomer is used to cover the molded jaw plates 31 to provide a sealing clamping of the tube, but allows the tube 10 to slide relative to the jaw plates when needed.

[0018] Figure 4The retainer drive mechanism 40 is shown in more detail. Specifically, the aforementioned stepper motor 42 and its drive pinion 41 are visible in this view, mounted to the disk 20 in a fixed relationship via mounting plate 26. The teeth of the pinion cooperate with the teeth of the corresponding racks 46, each rack 46 being fixed to one of the lower jaw plates 32 of the corresponding retainer 30 / 30'. As described above, rotation of the pinion causes the racks, and thus the retainers, to move apart, or move together synchronously by an equal amount.

[0019] Figure 5 The back of the disk 20 is shown, which includes a guide rail 44 fixed to the disk 20 and a guide bushing 45 surrounding the guide rail 44. The guide bushing 45 is attached to the lower jaw plate 32 of the respective retainer 30 / 30', and the guide bushing 45 moves along the guide rail 44 when the retainer 30 / 30' is driven by the drive mechanism 40.

[0020] Figure 6 The arrangement of the central ports is shown in more detail, including a liquid supply port 12a; a harvest port 12b; a sample collection port 12c; and a gas supply port 12d.

[0021] Figure 7 A schematic diagram of the control circuitry 60 for the hardware described above is shown, and includes a position sensor 48 for sensing the positions of the two holders, such as a linear encoder having a component mounted to the track 44. Figure 7 It also includes a control unit 50, which acquires information from the position sensor 48 to determine the dimensions between the retainers 30 / 30' and sends a command signal to the drive mechanism 40 to adjust the position of the retainers according to a predetermined cell culture protocol, which can be modified to suit a cell density determined based on a sample obtained from the cell sample port 12c. The control unit can detect feedback from the sensor to stop the drive mechanism at the desired position.

[0022] The above embodiments have the following advantages: during cell culture, the retainers 30 / 30' keep the tube in place and store unused areas of the tube so that the area remains stable and unobstructed until needed; throughout the cell expansion process, the two retainers move synchronously away from each other to control the bag volume, thus providing optimal cell culture volume in addition to securely holding both ends of the tube; the tube is symmetrically released at either side while the tube ports remain in relatively unchanged positions; and the device can be manufactured to be fully automated. It will be understood that the unused portion 14 of the tube 10 can be stored in a different manner than folding (e.g., rolling).

[0023] Figure 8-14The diagram shows a view of a second embodiment 105 of the present invention, which is simpler than the first embodiment but requires manual adjustment to provide cell culture volume regulation. Figure 8 The initially flattened cell culture tube 100 is shown, comprising a central region 100 and side regions 114. In use, the central region 100 contains the cell culture fluid, while the side regions 114 are sealed to an impermeable fluid relative to the central region by a sealing compression force applied by a pair of rollers 130 and 130', which overcome tension in the tube portion 110. The rollers effectively fold the tube at an angle greater than 90 degrees to further enhance their sealing effect. The end 116 of the tube 100 is held at a stop end 118. The stop end 118 and the opposing rollers 130 / 130' are connected in pairs by a scissor-action frame with an adjustable pivot point 150. Adjusting the pivot point 150 moves the rollers 130 / 130' apart and brings the associated stop end 118 closer to the rollers 130 / 130', resulting in a larger dimension between the two rollers 130 / 130' and thus a larger cell culture volume at the central portion 110.

[0024] Figure 9 and Figure 10 Showing Figure 8 More details of the device shown in the image. Figure 9 In the diagram, ports 12a, 12b, 12c, and 12d, located at the center, are shown as the ports corresponding to those described above. Figure 10 More details of the adjustment mechanism are shown, in this case, the adjustment mechanism includes a simple slot 152 in the back plate 160, and bolts and wing nuts 154, which can be tightened at any position in the slot to keep the frame 140 in the desired position.

[0025] Figure 11 and Figure 12 The device 105 is shown in its initial position, with a minimum distance between rollers 130 / 130'. Figure 13 and Figure 14 The device 105 is shown in its final position, where the mechanism allows for maximum distance between the rollers and thus allows for additional volume in the tube for cell culture. Manual adjustment can be made according to a predetermined schedule or in response to an increase in cell density measured via the sample port 12c.

[0026] This invention should not be construed as limited to the embodiments described above, but can be varied within the scope of the appended claims, as will be apparent to those skilled in the art. For example, other tube retainers (e.g., opposing pressure rollers) may be employed, or resiliently actuated features other than the jaw plates described above may be used. The stepper motor 42 may be replaced by a motor of a different type, or a non-electric actuator (e.g., a pneumatic actuator). Moreover, the first embodiment can be manufactured to be manually powered, and the second embodiment can be manufactured to be automatic. A notable feature of the illustrated embodiment is that a fluid-impermeable seal is formed by folding the tube material (i.e., bending the tube material at an angle of 90 degrees or greater, which promotes a sealing effect).

Claims

1. A cell culture apparatus, comprising: Cell culture containers, including flexible tubes; Support platform; as well as A pair of opposing retainers for opposite portions of the flexible tube and for retaining the central portion of the flexible tube in a fluid-impermeable manner, such that fluid is prevented from flowing from the central portion to the opposite portions of the flexible tube within the pair of opposing retainers, the spacing between the pair of opposing retainers being adjustable to provide an adjustable sealing volume in the central portion of the flexible tube between the pair of opposing retainers; Wherein, each of the opposite portions of the flexible tube is a coiled tube portion including at least one coiled portion or a folded tube portion including at least one folded portion within a corresponding retainer of a pair of opposing retainers; and In this configuration, each of the paired opposing retainers is hinged.

2. The cell culture apparatus according to claim 1, wherein, The culture vessel includes multiple fluid ports located between the pairs of opposing retainers when the vessel is mounted on the support platform.

3. The cell culture apparatus according to claim 1, wherein, The opposite portions of the flexible tube include the folded tube portions.

4. The cell culture apparatus according to claim 1, wherein, The cell culture apparatus is controlled using a feedback mechanism.

5. The cell culture apparatus according to claim 1, wherein, Each of the paired opposing retainers includes two jaw plates defining a meandering path between the two jaw plates, and wherein the tube is folded within the folded tube portion, within the meandering path.

6. The cell culture apparatus according to claim 5, wherein, The detour path defines a fold greater than 90 degrees.

7. The cell culture apparatus according to claim 1, wherein, The folded or unrolled portion of the flexible tube between the paired opposing retainers includes inlet and outlet ports.

8. The cell culture apparatus according to claim 1, wherein, Each of the opposite portions of the flexible tube is wound into at least one wound portion within the corresponding retainer of the pair of opposite retainers.

9. The cell culture apparatus according to claim 1, wherein, Each of the paired opposing retainers comprises two jaw plates connected by a hinge pin.

10. The cell culture apparatus according to claim 1, wherein, Each of the paired opposing retainers includes a retainer that secures a corresponding end of the flexible tube.

11. The cell culture apparatus according to claim 1, wherein, Each of the paired opposing retainers includes two jaw plates forming a cavity, wherein the folded and pleated respective ends of the respective tubes are held within the respective cavity of the respective retainer.

12. The cell culture apparatus according to claim 1, wherein, Each of the opposite portions is a folded tube portion, which includes a folded and pleated end of the tube held within a respective retainer in the opposite retainer.

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