NOVEL HIGH-DENSITY MICROCARRIER RETENTION DEVICE FOR PERFUSION CULTURE AND METHOD OF USE THEREOF

Abstract

The invention relates to the field of microcarrier perfusion culture of adherent cells. Specifically, the present invention relates to a high-density microcarrier retention device for perfusion culture of adherent cells, a microcarrier perfusion culture system for adherent cells containing the device, and methods of use thereof. The retention device of the present invention includes a sedimentation chamber, a pipeline connected to a bioreactor, a microcarrier retention filter membrane, a liquid backflushing device, an air backflushing device, a peristaltic pump and a pipeline connected to a receiver. The device has high efficiency in promoting the separation of microcarriers from cell culture medium and is helpful for perfusion culture of adherent cells and microcarriers. The retention device makes the culture volume in the bioreactor more flexible, can perform perfusion culture of 20%-100% of the maximum culture volume of the bioreactor, and the retention device can be linearly amplified according to the amplification of the bioreactor volume.

Claims

1. A high-density microcarrier retention device for perfusion culture, the device comprising: a sedimentation chamber; a first pipeline connected to a bioreactor and the sedimentation chamber; a microcarrier retention filter membrane located within the sedimentation chamber; a pump and a second pipeline connected to a receiver and the sedimentation chamber; and a liquid backflushing device and an air backflushing device, each connected to the sedimentation chamber.

2. The high-density microcarrier retention device for perfusion culture according to claim 1, wherein the high-density microcarrier retention device for perfusion culture is a high-density microcarrier retention device for adherent cell perfusion culture.

3. The high-density microcarrier retention device for perfusion culture according to claim 1 wherein the sedimentation chamber is connected to the bioreactor through one or more inclined or vertical pipelines, and the angle a between the pipelines and the horizontal plane is between 60-90 degrees.

4. The high-density microcarrier retention device for perfusion culture according to wherein the microcarrier retention filter membrane has a three-dimensional structure with one or more continuous or discontinuous vertical, inclined or curved retention walls.

5. The high-density microcarrier retention device for perfusion culture according to claim 4, wherein the three-dimensional structure has an upper cross section and a lower cross section with the same or different shapes.

6. The high-density microcarrier retention device for perfusion culture according to claim 5, wherein an area of the upper cross section is greater than or equal to that of the lower cross section.

7. The high-density microcarrier retention device for perfusion culture according to claim 5 wherein the lower cross section converges to a point.

8. The high-density microcarrier retention device for perfusion culture according to wherein a horizontal wall of the three-dimensional structure is configured to have a retention effect.

9. The high-density microcarrier retention device for perfusion culture according to claim 1, wherein the device is partially or wholly configured as a disposable device.

10. The high-density microcarrier retention device for perfusion culture according to claim 1, wherein the device is a reusable device.

11. The high-density microcarrier retention device for perfusion culture according to claim 1, wherein the microcarrier retention filter membrane is replaceable.

12. A method for retaining high-density microcarriers by using the high-density microcarrier retention device for perfusion culture according to claim 1 comprising the following steps: i) pumping out culture medium and microcarriers from the bioreactor through the first pipeline connected to the bioreactor to the retention device; ii) harvesting the culture medium into the receiver through the second pipeline connected to the receiver above the retention device and settling the microcarriers by gravity in the retention device; iii) retaining a small amount of microcarriers still kept in the culture medium by the microcarrier retention filter member; iv) backflushing the microcarrier retention filter membrane through the liquid backflushing device; and v) pushing remaining culture medium and microcarriers in the retention device back to the bioreactor by means of air through the air backflushing device.

13. The method according to claim 12, wherein the method is performed by an automated control program.

14. The method according to claim 12, wherein steps i) - v) are repeated one or more times.

15. The method according to claim 12, wherein a linear fluid rate of the culture medium in the sedimentation chamber of the retention device is less than the sedimentation rate of the microcarriers.

Description

DESCRIPTION OF THE DRAWINGS

[0052] According to the accompanying drawings, in combination with the specific embodiments of the present invention, the purpose, features and advantages of the present invention will become apparent. Those skilled in the art will understand that the dimensions of the components in the drawings are not drawn to scale, but are only for the purpose of explaining the present invention, and not limiting the scope of the present invention.

[0053] FIG. 1 is a schematic diagram of a configuration of the high-density microcarrier retention device (100) for perfusion culture of the present invention.

[0054] FIG. 2 is a schematic diagram of a cell microcarrier perfusion culture system including the high-density microcarrier retention device (100) for perfusion culture.

[0055] FIG. 3 shows an embodiment of the cell microcarrier perfusion culture system of the present invention.

[0056] FIG. 4 shows another embodiment of the cell microcarrier perfusion culture system of the present invention.

[0057] FIG. 5 shows the directions of movement of the microcarriers and cell culture medium in the pipeline connected to the bioreactor in the retention device of the present invention.

[0058] FIG. 6 shows various structures of the microcarrier retention filter membrane in the retention device of the present invention.

[0059] FIG. 7 shows the result of the bead to bead scale-up culture of Vero cells in 50 L and 200 L bioreactors.

[0060] FIG. 8 shows the result of perfusion culture of Vero cells in a 50 L bioreactor using the retention device of the present invention.

DETAILED DESCRIPTION

[0061] The improved device and method of the present invention can be used in combination with any perfusion bioreactor or continuous cell culture system. Such a system design can maintain the entire culture process under optimal growth conditions to achieve high-density cell growth. These systems are particularly suitable for perfusion culture of adherent cells combined with microcarriers in a stirred bioreactor.

[0062] The terms “high density microcarrier retention device”, “cell microcarrier retention device”, “microcarrier retention device”, “retention device” or “device” are used interchangeably herein.

[0063] The terms “microcarrier-bound cell”, “cell microcarrier”, “microcarrier cell” or simply “cell” are used interchangeably herein and include any of cells, such as plant cells, insect cells and mammalian cells, which may be attached to the microcarrier and grow in a stirred suspension medium, and can settle by gravity in an unstirred medium with a reasonable sedimentation rate. More specifically, the cells to which the microcarrier is bound are adherent cells, usually mammalian cells, which are bound to the microcarrier particles. The microcarrier particles are, for example, glass, polystyrene, gelatin, dextran or cellulose beads, such as commercially available Cytodex-1 microcarriers, Cytodex-3 microcarriers or Cytopore microcarriers.

[0064] A high-density microcarrier retention device for perfusion culture of the present invention can be described according to FIG. 1. The device is an external independent sedimentation device for microcarrier culture, and other related devices are devices located in the bioreactor or devices externally and physically connected to the bioreactor. As an external device, the device (100) of the present invention is connected to the bioreactor through one or more inclined or vertical pipelines (1). The device (100) also includes a body in the form of a sedimentation chamber (2), which may be a cylinder or any shape with a smooth inner wall, and is made of various materials that meet the requirements of cell culture. A retention filter membrane (3) is installed on the interior top of the sedimentation chamber (2) to prevent the microcarriers from pumping out of the sedimentation chamber. On the top of the sedimentation chamber (2) and above the retention filter membrane (3), there are a plurality of pipelines respectively connected to a plurality of pumps. Under the action of a peristaltic pump (4), the cell microcarriers and the culture medium in the bioreactor enter the sedimentation chamber (2), and the culture medium leaves the sedimentation chamber through the retention filter membrane (3) and enters a receiver. A backflushing pump (5) has a liquid backflushing function, which performs liquid backflushing above the retention filter membrane (3) to prevent the retention filter membrane from clogging. A gas mass flow meter (6) and the pipeline connected thereto have an air backflushing function, which pushes all the culture medium and microcarriers in the sedimentation chamber back to the bioreactor by means of air through the pipeline (1).

[0065] The dvice shown in FIG. 1 can be used in conjunction with a processor to carry out an automated control program. The automated control program is a precise control program for cyclic periodic operation, including but not limited to the following subprograms: [0066] 1. Pump outing the culture medium and microcarriers from the bioreactor to the sedimentation device; [0067] 2. Harvesting the medium; [0068] 3. Settling the microcarriers in the device; [0069] 4. Retaining a small amount of microcarriers remaining in the culture medium by the microcarrier retention filter membrane; [0070] 5. Liquid backflushing the retention filter membrane; and [0071] 6. Pushing all the medium and microcarriers back into the bioreactor by means of air.

[0072] Optionally, the device of the invention includes a balance/loadcell for accurate and flexible culture volume automatic control.

[0073] FIG. 2 shows an example of the cell microcarrier perfusion culture system of the present invention. The device (100) is fluidly connected to the bioreactor (8) through a pipeline (1). The bioreactor (8) can be a large-scale bioreactor. Alternatively, the bioreactor (8) is a disposable bioreactor. These bioreactors are well known to those skilled in the art and include many commercially available products such as Cytiva XDR disposable bioreactors.

[0074] Under the action of the peristaltic pump (4), the medium in the bioreactor enters the receiver (7) through the sedimentation chamber (2). After that, the medium in the receiver (7) can undergo the isolation and purification operations known in the art, such as centrifugation, filtration, chromatography etc., to obtain the target product.

[0075] In this example, the backflushing pump (5) operates regularly to perform liquid backflushing above the retention filter membrane (3) to prevent the retention filter membrane from clogging. In addition, the gas mass flow meter (6) operates regularly, and all the culture medium and microcarriers in the sedimentation chamber are pushed back to the bioreactor (8) through the pipeline (1) by means of sterile air.

[0076] The retention device of the present invention is connected to the bioreactor and the receiver through pipelines, and such separation/connection is replaceable. That is to say, the retention device of the present invention can exist independently of the bioreactor and the receiver. In one embodiment, the cell microcarrier perfusion culture system may include one or more retention devices, for example, 2, 3 or more retention devices. FIGS. 3 and 4 show different ways of connecting the retention device to the bioreactor. In FIG. 3, a plurality of retention devices (201, 202, ...) are connected to the bioreactor (801) through separate pipelines (101, 102, ...). In FIG. 4, a plurality of retention devices (201, 202, ...) are connected to the bioreactor (801) through an “all-in-one” pipeline (101). Different connection ways provide flexibility for the configuration of the retention device of the present invention. Those skilled in the art can reasonably select the corresponding configuration according to the type of the bioreactor used, production efficiency, culture conditions, etc.

[0077] FIG. 5 shows the directions of movement of the microcarrier and cell culture medium in the pipeline connected to the bioreactor. The left panel shows that the sedimentation chamber is connected to the bioreactor through a vertical pipeline, and the angle α between the pipeline and the horizontal plane is 90 degrees. The right panel shows that the sedimentation chamber is connected to the bioreactor via an inclined pipeline (α<90 degrees). Specifically, as shown in FIG. 5, the cell microcarriers in the pipeline settle back to the bioreactor along the A direction, and the culture medium leaves the retention device along the B direction to be harvested. In the case of α<90 degrees, the cell microcarriers gather near the tube wall D along the direction of arrow C due to gravity, so that the cell microcarriers settle down along the tube wall D. In this case, it is easier for the cell microcarriers to settle in the pipeline connected to the bioreactor. Therefore, in a preferred embodiment, α<90 degrees. In either case, the retention of the filter membrane on the top of the sedimentation chamber can prevent the loss of microcarriers. This provides more options for the production process.

[0078] In the process of perfusion culture, the retention filter membrane has the risk of clogging. The retention device of the present invention adopts connecting pipelines with different angles in combination with an adjustable liquid flow rate, so that most of the microcarriers flow back to the bioreactor after a period of sedimentation time, which can significantly reduce the concentration of microcarriers in the sedimentation chamber and reduce the clogging of the retention filter membrane by microcarriers.

[0079] The retention filter membrane of the microcarrier retention device of the present invention adopts a unique three-dimensional structure to increase the retention area, improving the retention efficiency and preventing the clogging. The upper cross section (A), lower cross section (B) and side view (C) of the various three-dimensional structures of the retention filter membrane are shown in FIG. 6. The shapes A, B, and C can be combined in various suitable forms to form the three-dimensional structure of the retention filter membrane of the present invention. The retention filter membrane can have an inverted cone structure, that is, a three-dimensional structure with an upper cross section larger than a lower cross section. The cone structure may be a circular cone, an elliptic cone, a triangular pyramid, a quadrangular pyramid, a pentagonal pyramid, and more pyramids. In one embodiment, the cone structure is an inverted pyramid three-dimensional structure. In a further embodiment, the microcarrier retention filter membrane may have an inverted cone parallel elongated three-dimensional structure, or a cylinder, cuboid or cube structure. For example, the microcarrier retention filter membrane has an inverted pyramid parallel extended three-dimensional structure. In a further embodiment, the microcarrier retention filter membrane has a spherical or hemispherical three-dimensional structure. These designs increase the retention area, and compared to the plane retention way, the vertical, inclined or curved retention wall can significantly reduce the attachment of microcarriers to the filter membrane.

[0080] In addition, the liquid backflushing procedure can wash away a small amount of microcarriers adhering to the filter membrane to avoid the clogging of the filter membrane. The air backflushing procedure can push the microcarriers back into the bioreactor in a short time, avoiding the cell viability decline caused by the cells staying outside the bioreactor for a long time, and further avoiding the clogging of filter membrane. Through the use of device products and cell culture perfusion experiments, it is confirmed that the design of this microcarrier retention device has no clogging of filter membrane and has no effect on cell viability.

[0081] The device of the invention solves the problems of high-density microcarrier perfusion culture, and is especially suitable for large-scale bioreactors and disposable bioreactors. The device has been tested in the Cytiva Fast Trak laboratory and cooperative laboratories, using Vero cells and microcarriers to successfully carry out high-density microcarrier perfusion culture, and successfully scaled up the Vero cell microcarrier perfusion culture process to 50 L disposable bioreactors for producing rabies vaccine.

[0082] In a specific experiment, Vero cells and Cytodex-1 microcarriers were used to compare culture results of the batch culture mode of cell microcarriers in 50 L and 200 L XDR bioreactors with the perfusion culture mode of cell microcarriers in 50 L XDR bioreactors.

[0083] In the batch culture of cell microcarriers, lower microcarrier concentrations are usually used. For Vero cells, a Cytodex-1 microcarrier concentration of 2-3 g/L is usually used. Higher microcarrier concentrations require special control of environmental conditions or frequent medium changes. The inventors used 3 g/L Cytodex-1 microcarriers and the XDR50 bioreactor to culture Vero cells. By optimizing the culture conditions, the cell density can only reach 3 × 10.sup.6 cells/ml (FIG. 7, day 4). Through the microcarrier bead to bead scale-up culture, the Vero cell culture is scaled up to the XDR200 bioreactor, and the cell density can still only reach 3 × 10.sup.6 cells/ml (FIG. 7, days 8-9). The lower concentration of microcarriers and limited nutrient replenishment ways make the whole culture unable to obtain higher cell density, which in turn affects the virus titer in the harvested liquid after inoculation.

[0084] Through the novel microcarrier retention device for perfusion culture of the present invention combined with an XDR50 bioreactor, Cytodex-1 microcarriers are used for Vero cell perfusion culture. The microcarrier concentration can be increased to 12-18 g/L, making the cell density more than 8 × 10.sup.6 cells/ml (FIG. 8, day 6). The cell density is increased by almost 3 times through perfusion culture, which results in the efficient production of vaccines in disposable bioreactors.

[0085] Specifically, the cell microcarrier perfusion culture system of the present invention supported the perfusion culture of Vero cells with 12 g/L Cytodex-1 microcarriers in a 50L bioreactor for 22 days, so that the cell density exceeded 8×10.sup.6 cells/ml. The first 7 days was the growth period of cell perfusion culture, and the last 15 days was the collection period of rabies vaccine of perfusion culture. In the entire run, the retention rate of cell microcarriers was 100%.