WO2020008067A1

WO2020008067A1 - Sampling device and method

Info

Publication number: WO2020008067A1
Application number: PCT/EP2019/068174
Authority: WO
Inventors: Ali YASSER
Original assignee: Ge Healthcare Bio-Sciences Corp.; Ge Healthcare Limited
Priority date: 2018-07-06
Filing date: 2019-07-05
Publication date: 2020-01-09

Abstract

Disclosed is a product and method for sampling cells from a bioreactor, for the purposes of determining the cell count or to remove a sample and retain sterility of the sample for quality control (QC) assessment during a cell expansion in a bioreactor using vacuum tubes in combination with a sampling manifold (1) comprising:a primary fluid conduit (2);a connection port (3) positioned at one end of said fluid conduit (2) comprising means (4) for selective fluid connection of the interior of said fluid conduit (2) to the interior of a sampling reservoir (5); and a plurality of sampling ports (6a-e) disposed along said fluid conduit (2); wherein each of said plurality of sampling ports(6a-e) comprises means (7a-e) for selective fluid connection with an outlet (8a-e) adapted to connect to means (9) to permit exclusively unidirectional fluid flow from said primary fluid conduit.

Description

SAMPLING DEVICE AND METHOD

Field of the Invention

The present invention relates to a method for sampling cells from a cell culture in a bioreactor for the purposes of, for example, determining the cell count, providing a sample for off-line analyses or removing a sample and retain sterility of the sample for quality control (QC) assessment. More particularly, the invention relates to a method for aseptic sampling of cells at one or more instances in time during a cell expansion in a bioreactor using vacuum tubes.

Description of Related Art

At present advanced cell therapy products are grown in a number of bioreactors that differ in their design and structure. In particular, immunotherapy applications require the rapid expansion of T lymphocytes, either na^'fve cells or engineered cells that express a receptor to tumor cell antigen. Over the course of the cell expansion the operator may wish to understand what is happening with the cell culture environment, supply of nutrients, accumulation of waste metabolites or to remove a cell sample for further analysis.

Flexible cell culture bags are currently used to culture and expand primary peripheral blood mononuclear cells (particularly T cells) for transplantation into patients. Cells are grown within a contained cell bag and high cell densities are achieved by using media perfusion, where fresh media is added to the culture and spent media is removed. The rate of media perfusion is dependent upon the concentration of cells within the cell bag, the perfusion rate increasing with increased cell concentration. Monitoring of the growth rate and concentration of the cultured cells requires sampling from the cell bag, and generally at more than one timepoint.

The preferred prior art method for sampling is to connect a syringe to a needless port of the bioreactor and remove a sample of the cell suspension from the bioreactor. Use of a syringe to sample from the disposable bioreactor requires careful and detailed procedures to ensure sterility is maintained; a syringe is a two-way device and consequently has the potential to allow the operator to push air into the bioreactor and risk contaminating the culture. Disconnection of the syringe from the bioreactor exposes the contents of the syringe to the local environment and increases the risk of microbial contamination.

In a second preferred prior art method the user may fit a length of sterile tube connected to a 3-way valve. A syringe is fitted to the valve and used to draw sample into the tubing. The tubing is then clamped and sealed to provide a sterile cell sample. However, in this method the operator has multiple steps to complete to obtain the final sample.

When opened, the sampling port exposes the culture to the external environment which carries the risk of contamination of the culture, and each sampling instance requires drawing a portion of the sample from the cell bag. Different tubes are attached to ports on the cell bag or are passed through the ports at different instances in time for different sampling instances. Any leakage or contamination in the tubing or in the connection between the culture vessel and the tubing may introduce contamination in the cell bag. Every sampling instance is accompanied by a user attaching some sort of tubing either directly or indirectly to the cell bag, thereby increasing the risk of contamination of the cell culture. In addition, there is a likelihood of a portion of the sample being left in the tubing after the sampling instance. This residual sample may then be inadvertently carried over to the next sampling instance, thereby jeopardizing the purity of the sample obtained in the next sampling instance. Also, each sampling instance increases the likelihood of contamination of the cell culture. Hence, it is desirable to ensure that sampling is carried out in a manner which avoids introduction of contaminants into the pre-established sterile system. Furthermore, using current methods to carry out sampling from a bioreactor such as the WAVE or Xuri™ (GE Healthcare) bioreactors is often cumbersome and can leave the isolated sample exposed to the atmosphere. Sampling the bioreactor using known approaches therefore also runs the risk of providing a non-sterile sample that may fail the sterility QC check.

Consequently, in addition to the complex nature and risk of contamination associated with known sampling techniques, there also may exist an inherent limitation on the number or frequency of samplings which may be accommodated, either by reason of a limited number of sterilizable sequences to which a particular connector can be subjected to before severe degradation occurs or simply by reason of the long time needed to perform a sample withdrawal. These limitations may pose significant problems in situations where rapid and frequent sampling is required in order to monitor a potentially fast- changing situation. Still further, of course, elaborate and/or time-consuming sampling techniques can add significantly to the overall cost of the culture process.

The present inventors have recognized potential problems with some currently known systems for sampling from a bioreactor. In some configurations clamps are used to control flow into and out of the sampling manifold. In this case, if the operator forgets to open/close a clamp this creates the possibility of the flow going in the wrong direction, e.g. part of sample finding its way back into the bioreactor and being a contamination risk.

It would therefore be desirable to have a simple and robust cell sampling method which provides a sterile sample and which exposes the cell culture to a minimal contamination risk.

Summary of the Invention

In one aspect the present invention provides a sampling manifold comprising: a primary fluid conduit (2); a connection port (3) positioned at one end of said fluid conduit (2) comprising means (4) for selective fluid connection of the interior of said fluid conduit (2) to the interior of a sampling reservoir (5); a plurality of sampling ports (6a-e) disposed along said fluid conduit (2);and wherein each of said plurality of sampling ports (6a-e) comprises means (7a-e) for selective fluid connection with an outlet (8a-e) adapted to connect to means (9) to permit exclusively unidirectional fluid flow from said primary fluid conduit.

In another aspect the present invention provides a sampling method comprising the following steps:

(i) providing a sampling manifold (1 ) as defined herein;

(ii) providing a sterile, empty sampling reservoir (5), wherein said sampling reservoir (5) comprises a fitting compatible with the connection port (3) of said sampling manifold (1 ) to permit selective fluid connection between the interior of said sampling manifold (1 ) and the interior of said sampling reservoir (5);

(iii) aseptically coupling said fitting and said connection port (3);

(iv) aseptically transferring a liquid into the interior of said sampling reservoir (5);

(v) positioning said sampling reservoir (5) such that said liquid is proximal to said fitting;

(vi) setting the means for selective fluid connection (4 and 7a-e) to permit fluid flow from said bioreactor (5) into a container to be positioned at a selected outlet (8a-e) of said sampling manifold (1 ); and,

(vii) connecting a container in which a vacuum has been created to said selected outlet (8a-e) so that a sample is transferred from said sampling reservoir (5) to the interior of said container. The present invention provides a cost-effective system and method for rapid cell sampling which will maintain a sterile sample, reduce handling time and effort compared to prior art. The method of the invention involves use of a simple disposable system that in its single sampling embodiment removes the need to flush and residual cell suspension from the sampling line prior to taking a sample, which is often considered a concern when a multi-sampling device has been assembled. The method of the invention in another embodiment permits multi- sampling where the sampling device of the invention is used, which provides advantages over prior art multi-sampling methods and systems. The method of the invention is a simple, low risk and effective procedure to collect one or more samples from any bioreactor fitted with a needless port. The sampling method of the invention may be used for any type of cultivated cells even very sensitive cells such as human peripheral blood mononuclear cells (PBMCs) and lymphocytes.

Aspects of the present invention permit more controlled, unidirectional flow of sample from a bioreactor thereby making the sampling process simpler for users with reduced scope for error.

The sampling manifold of the invention is a functionally closed accessory that reduces the risks associated with repeated sampling. It allows sampling consistency and safety of use. The system is aseptic and allows to use fresh port for each sample.

Furthermore, the present invention allows line purging post sampling.

The invention also provides the opportunity to obtain an aseptic fresh sample with no risk or contamination due to repeated sampling using the same port with minimal user errors.

Brief Description of the Figures

Figure 1 shows an embodiment of the sampling manifold of the present invention.

Figure 2 shows an embodiment of an arrangement at an outlet of the sampling manifold.

Figure 3A and 3B illustrate the cell count data obtained when a sampling manifold of the invention was tested vs. a known sampling means.

Figure 4A and 4B illustrate the cell viability data obtained when a sampling manifold of the invention was tested vs. a known sampling means.

Detailed Description of the Preferred Embodiments

To more clearly and concisely describe and point out the subject matter of the claimed invention, definitions are provided hereinbelow for specific terms used throughout the present specification and claims. Any exemplification of specific terms herein should be considered as a non-limiting example.

The term“sampling manifold” is taken to mean a device suitable for taking one or more samples from a sampling reservoir in an aseptic manner.

The term“sampling reservoir” refers to a storage space for a fluid and in the context of the present invention to a storage space that can provide the fluid with specific controlled conditions. Non-limiting examples of such fluids include cell cultures or other solutions that require routine sampling to monitor cell growth or other solution criteria.

The term“fluid conduit” as used herein refers to tubing suitable for the creation of a closed fluid pathway that permits the passage of material from a sampling reservoir to a sampling container. Suitable conduits include tubing made from flexible liquid-tight tubing.

The term“port” as used herein refers to an opening allowing the passage of a fluid therethrough. The terms“connection port” and“sampling port” refer to a port that includes a means for liquid-tight connection or linkage to another feature or device. In the context of the present invention that connection is suitably selective and aseptic. The connection port of the present invention permits selective fluid connection between the sampling manifold and the sampling reservoir where the sampling reservoir includes a fitting compatible with the connection port. The fitting of the sampling reservoir is associated with an outlet port so that liquid may pass out of the sampling reservoir. In the method of the present invention is it desirable to connect the sampling manifold to the sampling reservoir prior to use to eliminate the chance of introducing contamination. Each sampling port of the present invention permits selective fluid connection between the sampling manifold and an outlet configured for extracting a sample.

The phrase“means for selective fluid connection” used in connection with the present invention refers to a valve that may be set either to permit or stop the flow of a liquid along a closed fluid pathway. Each means for selective fluid connection can be set to permit sampling from a chosen outlet or alternatively to prevent fluid flow from the sampling reservoir as desired. At the connection port the means for selective fluid connection may in one embodiment be termed a clave stopcock. At each sampling port the means for selective fluid connection may in one embodiment be termed a manifold stopcock. The term“asepticallv coupling” refers to the process of connecting two devices together while maintaining sterility within said devices.

The term“outlet” as used herein refers to an opening in the sampling manifold from which a sample is collected. In the exemplary embodiment illustrated in Figure 1 each sampling port (6a-e) is connected via a length of tubing (17a-e) to each outlet (8a-e). The tubing may be formed from a thermoplastic material that can be heat sealed following sampling from a particular sampling port, thereby preventing re-use. In particular, the outlet of the present invention is adapted to connect to means to permit exclusively unidirectional fluid flow from the sampling manifold into a container. For example, the outlet may comprise a needle (such as a hypodermic needle) that can puncture a container in which a vacuum has been created (such as a Vacutainer™). Figure 2 illustrates a non-limiting example of an outlet arrangement comprising a needle (9a) positioned at an outlet (8) and within a container holder (18). Not illustrated but optional is a cap that can be positioned at the container holder opening for safety purposes, ensuring a user does not accidently touch the needle inside.

The term“sterile” as used herein means free from bacteria or other living microorganisms. Sterility can be achieved using a number of well-known methods in the art. The method should be compatible with the materials being sterilized, e.g. chemical or radiation sterilization more suitable for plastic materials.

The term“empty” used herein in connection with the sampling reservoir is taken to mean containing no solid or liquid matter.

The phrase“liquid is proximal to said fitting” used in connection with the method of the invention refers to positioning said sampling reservoir such that the fitting and the associated outlet port of the sampling reservoir are submerged under the volume of liquid in the sampling reservoir. In a non-limiting example a 2L or 10L Xuri™ cell bag is used as the reservoir and can be tilted on its tray to achieve the liquid proximal to the fitting, and tilted back following sampling.

A“container” in the context of the present invention is meant a sealed glass or plastic container of any suitable shape that is evacuated to create a vacuum inside the container facilitating the draw of a predetermined volume of liquid therein. Suitably the container is sterile. In one embodiment the container is made from glass. In another embodiment the container is made from plastic, for example polyethylene terephthalate (PET). In one embodiment the volume of the container is suitable for collection of a minimum sample size of 2ml_, for example 2-10 ml_. Exemplary containers have volumes of 2, 3 and 6ml_. The 2ml_ container can be used for sampling from a small culture volume (i.e. 150 to 300ml_) at the beginning of culture initiation process. The 3 and 6 ml_ vials can be used later during the cell expansion process for larger culture volume (i.e. 500 to l OOOmL).

In one embodiment the primary fluid conduit (2) of said sampling manifold (1 ) comprises a linear passageway. Such an arrangement is illustrated in Figure 1 where an elongate straight linear conduit runs from the connection port and has a series of evenly-spaced sampling ports (6a-e) disposed therealong.

It is not essential for the fluid conduit to be linear nor is it essential that the sampling ports are evenly-spaced. The person skilled in the art will appreciate that other arrangements will function equally well.

In one embodiment the primary fluid conduit (2) of said sampling manifold (1 ) comprises a non-linear passageway.

In one embodiment the primary fluid conduit (2) of said sampling manifold (1 ) comprises a circular passageway. In one embodiment, said circular passageway is comprised within a valve.

In one embodiment said sampling manifold further comprises an additional sampling port (10) proximal to said connection port (3). This feature permits optional additional samples to be taken after all the sampling ports of the sampling manifold have been used and an exemplary embodiment is illustrated in Figure 1 , positioned adjacent to the connection port (3). To use this feature for sampling, the stopcock should first be set to the“on” position, i.e. to permit flow from the sampling reservoir and out of the additional sampling port. The additional sampling port in Figure 1 has a Luer cap that can be removed and then swabbed with an alcohol wipe prior to attaching a Luer lock syringe. Then the liquid in the sampling reservoir is moved to submerge the fitting and outlet of the sampling reservoir such that when the syringe plunger is pulled back a sample is taken up into the syringe. The line from the sampling reservoir to the additional sampling port can be purged by moving the liquid in the sampling reservoir away from the fitting and associated outlet, and using the syringe to pull the remaining liquid out of the system.

In one embodiment said means (4, 7a-e) for selective fluid connection comprises a flow control valve.

In one embodiment said flow control valve is a manually- or automatically- controlled hermetic seal with linear or circular configuration to direct the flow in the desired sampling path.

In one embodiment said flow control valve is a stopcock. This embodiment is illustrated in Figure 1 where manually-controlled 3-way stopcocks are used at the sampling ports and a manually-controlled on-off stopcock is used at the connection port. The person skilled in the art will appreciate that a number of known stopcock configurations that permit selective fluid flow are equally suitable. Prior to taking a sample using the sampling manifold illustrated in Figure 1 , the on-off connection port (can also be regarded as the“clave” port) should be set to the“off’ position. The 3-way stopcock at the selected sampling port should be set to the“sampling” position, i.e. to permit flow from the sampling reservoir out of that sampling port. The remaining 3-way stopcocks should be set to the “through” position, i.e. to permit flow of liquid past these sampling ports from the sampling manifold. Then the sampling reservoir should be positioned so that the fitting of the sampling reservoir and associated outlet are under the liquid contained therein (e.g. for a Xuri™ cell bag the tray is tilted). The elastomeric stopper of an evacuated container can then be pushed onto the needle (9a) so that the needle punctures through the stopper and into the container. Liquid from the sampling reservoir will begin to flow through the sampling manifold and into the vial. The vial can be held in place until the flow stops. Following sample collection, the sampling reservoir can be moved back to its original position (e.g. for a Xuri™ cell bag horizontal or if necessary for the liquid not to cover the fitting). Then the length of thermoplastic tubing (17a-e) between the sampling port (6a- e) and its respective outlet (8a-e) can be sealed, e.g. using a tubular sealer, to separate the parts distal from the sampling manifold for disposal. The 3-way stopcock at the just-used sampling port can then be set to the“through” position.

In one embodiment said connection port (3) further comprises means for selective aseptic fluid communication between said sampling reservoir (5) and the interior of one or more further sampling manifolds as defined herein.

In one embodiment said sampling reservoir (5) is a bioreactor. The term “bioreactor” refers to any manufactured or engineered device or system that supports a biologically active environment. A bioreactor in the context of the present invention is a device or system in which cells or tissues are grown in the context of cell culture. The bioreactor may be a rigid reusable vessel made e.g. from stainless steel or a flexible cell bag designed for a single use before being discarded.

In one embodiment said means (9) to permit exclusively unidirectional fluid flow comprises a needle (9a) positioned at an outlet (8a-e) and a container in which a vacuum has been created wherein said container is configured to be punctured by said needle (9a) without loss of said vacuum to the surroundings. Figure 1 illustrates an exemplary sampling manifold configured to have a needle at each outlet (8a-e) that can pierce the seal of a vacuum tube positioned in one of a plurality of container holders (18a-e).

In one embodiment said sampling manifold (1 ) is sterile. In this embodiment the sampling manifold may be provided to the user within protective packaging. The sampling reservoir can also be provided sterile and in protective packaging. Both the sterile packaged sampling manifold and sampling reservoir can be removed from their packaging and placed in sterile environment such as a biosafety cabinet prior to connection using aseptic technique.

In one embodiment said container comprises a self-sealing stopper capable of being punctured by a needle (9a) positioned at said outlet (8a-e). such self-sealing stoppers are well known to those of skill in the art and are made from an elastomeric material. Entry can be made into the container by means of a hypodermic needle so that fluids can be inserted in and removed from the container without breaking the sterility of the container. After the needle is removed from the stopper it immediately reseals.

In one embodiment said sampling method further comprises after step (vii):

(viii) removing said container;

(ix) positioning said sampling reservoir such that said liquid is distal to said fitting;

(x) connecting a further container to said selected outlet to purge residual liquid from between said sampling reservoir (5) and said outlet; and,

(xi) sealing the selected outlet (8a-e).

In the sampling manifold of the present invention the purging step is advantageous due to the fact that flow from the sampling manifold is only in the direction of the outlet, thereby helping to ensure that no liquid that has passed out of the sampling reservoir during the sampling process gets back into the sampling reservoir. This is an improvement over known methods.

In one embodiment said sealing is as close as possible to the sampling port (6a-e) corresponding to said selected outlet (8a-e).

In one embodiment of the sampling method the steps are carried out sequentially.

In one embodiment of the invention the container is a vacuum tube and the outlet connects to the interior of the vacuum tube via a needle-provided access device wherein a fluid path is created through the needle of the access device. A fluid path passage is thereby created through the needle of the access device so that fluid can flow from the sampling reservoir into the vacuum tube. A well-known use of such vacuum tubes is for drawing blood samples directly from a vein. A non-limiting example of such a vacuum tube is a Vacutainer™ tube. Containers under vacuum, such as the Vacutainer™ are well-established products used at hospitals and care centers for rapid blood sampling. The containers comprise a vacuum tube that aseptically draws blood through a sleeved covered needle. The vacuum tube is available in various volumes and often pre-coated with various compounds to prevent for example blood clotting. The invention exploits the features of the vacuumised containers and associated sleeved needle for use as a cell sampling device that connects to a bioreactor for cell culture through a one-way needless port. The access device is preferably a sterile, single use product and offers a simplified process for the operator to remove a sample for analysis. The design of the access device with vacuum tube and sleeved needle is such that once drawn the sample will retain its sterility for further analysis.

In one embodiment of the invention the vacuum tube may be empty, in another embodiment it may be filled with selected reagents, such as cell viability stains, for example DRAQ5, Hoechst, or propidium iodide. In a further embodiment the vacuum tube contains single antibodies, or multiple antibodies, such as anti-CD3, anti-CD4 or anti-CD8.

In one embodiment of the method of the invention the cells are instantly mixed with the reagent and then the tube is removed from the bioreactor and the cells are analyzed, for example in respect of viable cell number or the presence of antigen.

The exemplary 5-port sampling manifold illustrated in Figure 1 has been designed to be compatible with Xuri™ W25 Cell Expansion System instrument. The tilted position of the tray is used during the sampling process. The operating (Unicorn) software for Xuri™ W25 instrument allows user to program the try to pause in the required tilted position during sampling, and return back to the horizontal orientation to purge the sampling line. Also, this sampling manifold is compatible to the current Luer connection used for the connection (or clave) port which allows the sampling manifold to be assembled to any size of Xuri™ cellbags (2L, 10L, 20L and 50L) and to the same range of Wave™ cellbags in bioprocess applications.

Persons skilled in the art will readily be able to appreciate how the device and method of the invention can be applied to other known cell expansion systems, and indeed to other comparable systems comprising a reservoir from which samples need to be taken. The following non-limiting example describes an embodiment of the present invention.

Example 1 : Cell Test of an Exemplary Sampling Manifold of the Invention

The embodiment of the invention illustrated in Figure 1 was tested by connection to Xuri™ cellbags that were filled with live T-Cells, which were expanded following a typical expansion procedure.

The sampling manifold and a clave port were both attached to each cellbag.

Samples were taken daily from both the sampling manifold and the clave port simultaneously.

Cell count and viability (cell markers such as CD3, CD8 and CD25 to evaluate shear force) was tested and compared between sampling methods. The sampling manifold was also was tested with nutrient broth over time to validate the sterility assurance of the sampling device during the worst case of use.

A two sample t-test (paired groups) was used for comparing the data from the two sampling methods.

The testing results showed no risk to cell shear and culture sterility when using the sampling device. The cell count data is illustrated in Figures 3A and 3B. The cell viability data is illustrated in Figures 4A and 4B.

Claims

What Is Claimed Is:

1 A sampling manifold (1 ) comprising: a primary fluid conduit (2); a connection port (3) positioned at one end of said fluid conduit (2) comprising means (4) for selective fluid connection of the interior of said fluid conduit (2) to the interior of a sampling reservoir (5); and a plurality of sampling ports (6a-e) disposed along said fluid conduit (2); wherein each of said plurality of sampling ports (6a-e) comprises means (7a-e) for selective fluid connection with an outlet (8a-e) adapted to connect to means (9) to permit exclusively unidirectional fluid flow from said primary fluid conduit.

2 The sampling manifold (1 ) as defined in Claim 1 wherein said primary fluid conduit (2) comprises a linear passageway.

3 The sampling manifold (1 ) as defined in Claim 1 wherein said primary fluid conduit (2) comprises a non-linear passageway.

4 The sampling manifold (1 ) as defined in Claim 3 wherein said primary fluid conduit (2) comprises a circular passageway.

5 The sampling manifold (1 ) as defined in Claim 3 or Claim 4 wherein said circular passageway is comprised within a valve.

6 The sampling manifold (1 ) as defined in any one of Claims 1 -5 wherein said sampling manifold further comprises an additional sampling port (10) proximal to said connection port (3)

7 The sampling manifold (1 ) as defined in any one of Claims 1 -6 wherein said means (4, 7a-e) for selective fluid connection comprises a flow control valve.

8 The sampling manifold (1 ) as defined in Claim 7 wherein said flow control valve is a manually- or automatically-controlled hermetic seal with linear or circular configuration to direct the flow in the desired sampling path.

9 The sampling manifold (1 ) as defined in Claim 7 or Claim 8 wherein said flow control valve is a stopcock.

10. The sampling manifold (1 ) as defined in any one of Claims 1 -9 wherein said connection port (3) further comprises means for selective aseptic fluid communication between said sampling reservoir (5) and the interior of one or more further sampling manifolds, each of which is as defined in any one of Claims 1 -9.

11. The sampling manifold (1 ) as defined in any one of Claims 1 -10 wherein said sampling reservoir (5) is a bioreactor.

12. The sampling manifold (1 ) as defined in any one of Claims 1 -11 wherein said means (9) to permit exclusively unidirectional fluid flow comprises a needle (9a) positioned at an outlet (8a-e) and a container in which a vacuum has been created wherein said container is configured to be punctured by said needle (9a) without loss of said vacuum to the surroundings.

13. The sampling manifold (1 ) as defined in any one of Claims 1 -12 which is sterile.

14. A sampling method comprising the following steps:

(i) providing a sampling manifold (1 ) as defined in Claim 13;

(iii) aseptically coupling said fitting and said connection port (3);

(vii) connecting a container in which a vacuum or partial vacuum has been created to said selected outlet (8a-e) so that a sample is transferred from said sampling reservoir (5) to the interior of said container.

15. The sampling method as defined in Claim 14 wherein said container comprises a self-sealing stopper or septum capable of being punctured by a needle (9a) positioned at said outlet (8a-e).

16. The sampling method as defined in Claim 14 or Claim 15 wherein said sampling reservoir (5) is a bioreactor.

17. The sampling method as defined in any one of Claims 14-16 wherein said liquid is a liquid cell population.

18. The sampling method as defined in Claim 17 wherein said container contains a cell viability stain.

19. The sampling method as defined in Claim 18 wherein the cell viability stain is selected from DRAQ5, Hoechst, or propidium iodide.

20. The sampling method as defined in Claim 17 wherein said container contains single antibodies.

21. The sampling method as defined Claim 17 wherein said container contains multiple antibodies.

22. The sampling method as defined in either Claim 20 or Claim 21 wherein said container contains anti-CD3, anti-CD4 or anti-CD8.

23. The sampling method as defined in any one of Claims 14-22 which further comprises after step (vii):

(viii) removing said container;

(xi) sealing the selected outlet (8a-e).

24. The sampling method as defined in Claim 23 wherein said selected outlet (8a-e) is sealed as close as possible to the sampling port (6a-e) corresponding to said selected outlet (8a-e).

25. The sampling method as defined in any one of Claims 14-24 wherein said steps are carried out sequentially.