WO2007054159A1

WO2007054159A1 - Apparatus for obtaining cell suspensions with subsequent admixture of additives and corresponding methods

Info

Publication number: WO2007054159A1
Application number: PCT/EP2006/009386
Authority: WO
Inventors: Thomas Montag-Lessing; Ingo Spreitzer; Bettina Löschner; Bettina Ziegele
Original assignee: Paul-Ehrlich-Institut Bundesamt Für Sera Und Impfstoffe
Priority date: 2005-11-09
Filing date: 2006-09-27
Publication date: 2007-05-18
Also published as: DE202005017529U1

Abstract

The present invention relates to an apparatus for obtaining cell suspensions with subsequent admixture of additives, especially protectants for cryopreservation. The apparatus of the invention eliminates in particular the need for centrifugation and other elaborate and possibly contaminating processing steps. The present invention further relates to a method for obtaining cell samples using the apparatus.

Description

Apparatus for obtaining cell suspensions with subsequent admixture of additives and corresponding processes

The present invention relates to a device for obtaining cell suspensions with subsequent admixture of additives, in particular protective agents for cryopreservation. In particular, the device of the invention eliminates the need for cell isolations and other expensive and potentially contaminating treatment steps. The present invention further relates to a method of obtaining cell samples using the device.

For the clinical testing of vaccines against pathogens infested by cell-mediated immunity (or the cellular immune status of vaccinated individuals), or more generally for determining the immune status of patients, mononuclear cells (PBMCs) are often isolated from the vaccine blood samples , For this purpose, highly specialized laboratories with appropriately qualified personnel must be set up on site. Furthermore, all vessels and reagents must be pyrogen-free. The cells are usually frozen in liquid nitrogen and transported as well.

Often o.g. clinical trials in developing countries (e.g., in Africa). The establishment of specialized laboratories on site, the recruitment of suitable personnel and the sample transport in liquid nitrogen require extremely high financial and technical effort. Furthermore, the isolation of the mononuclear cells is prone to failure under pyrogen-free conditions; it may lead to activations of the monocytes e.g. by pyrogens, rendering the sample worthless.

WO 2005-089837 describes a two-component prefilled syringe.

US 5,522,804 describes a syringe for aspiration, mixing and injection of substances. The syringe contains a chamber integrated into its piston for substances that can be injected after injection of a first substance. It is an object of the present invention to provide a device which facilitates facilitated, safe and thus improved sample preparation. It should thereby be developed a device with which in particular individual blood samples can be obtained and mixed with additives, so that the samples without further processing directly in the device with the least possible effort and at the lowest possible cost cryopreserved and transported (eg for the clinical Testing vaccines against pathogens defended by cell-mediated immunity to determine the individual immune status of patients).

This object of the present invention is achieved in a first aspect by a device for preparing a blood sample which has a sample chamber (12) with an inlet (1), a movable plunger (3) with a stamp plate with pores (2) arranged inside the sample chamber. , and a second chamber (5), within the sample chamber (12), for sample conditioning means connected to the sample chamber (12) via a closable inlet (8).

According to the invention, a device has been developed in which, in particular, individual blood samples can be obtained and admixed with additives, so that the samples can be frozen directly in the device according to the invention without further processing. The monocytes and lymphocytes of the samples retain their function and are not significantly activated. The cryopreservation and the transport are carried out according to the invention without liquid nitrogen. This makes it possible to obtain individualized blood samples (for clinical vaccine testing, for example) with very little financial and technical effort. The principle is also applicable to other cell suspensions.

Preferred is a device according to the invention, which is characterized in that the second chamber is formed by a within the sample chamber (12) lying movable hollow punch (7), in particular by a sealing washer (6). In a preferred embodiment of the device, the sealing disk (6) is entrained by suitable surface design (eg roughness) of the overflow channels (8). Alternatively, the gasket can be connected via a groove with the hollow punch (7). For step 5 ("mixing supernatant with the protective agent", see below), then in another embodiment, an existing connection between Gasket and hollow punch (eg by turning the punch) are released. In use, no protective agent initially enters the sample because the volume of the reservoir remains the same and / or the inlet (8) is closed.

According to the invention it is preferred that the device is provided with a multi-step fixation principle for the hollow die in the lower position, as will be explained below.

Preferably, a device according to the invention is characterized in that the closable inlet (8) of the second chamber (5) can be opened by a rotary and / or piercing mechanism by means of the hollow punch (7). As a result, the protective means (5) passes through the transfer channels (8) into the supernatant (for example plasma). By the design of the surface (e.g., roughness, no firm connection to the hollow die) of the overflow channels (8), the gasket (6) is still moveable when a little more pulling is applied. Alternatively, in one embodiment, a connection can be made between the hollow die and sealing washer (e.g., via a groove) which can be dissolved (e.g., by spinning) prior to mixing the supernatant with the treating agent (e.g. By rotating the device several times overhead, uniform mixing of the supernatant with the treating agent (e.g., preservative) is achieved. In a further preferred embodiment of the device of the present invention, this is thus characterized in that the sealing disc (6) via a groove with the hollow punch (7) is detachably connected.

In a further preferred embodiment of the device of the present invention, this is characterized in that the closable inlet (8) comprises overflow channels (8) with a guide for the sealing disc (6).

In a further preferred embodiment of the device of the present invention, this is characterized in that the punch (3) is movably mounted within the hollow punch (7). It is preferred that the punch (3) is guided by a sealing ring (4).

In a further preferred embodiment of the device of the present invention, this is characterized in that the punch (3) and the hollow punch (7) by a releasable connection clip (9) are interconnected. The connection clip (9) provides for the defined simultaneous retraction of both punches (3 and 7), but if necessary removed from the punch (3) and from the hollow punch (7). In a further embodiment, instead of the connecting clip (9), a rotatable punch may be provided with a groove connection or other locking, which is then released accordingly.

In a further preferred embodiment of the device of the present invention, this is characterized in that a stop and / or a corresponding marking is provided for the hollow punch (7) within the first sample space (12). Thus, a stop is preferably provided for the hollow punch (7), which guarantees the stop of both punches in such a way that no protective means (5) is pressed into the sample space. In one embodiment, the hollow die can be fixed in this position by turning (for example by 90 °). The device contains a mark for stopping the stamper (e.g., for blood at about 50% of the total sample volume) so as not to subject the cells to any mechanical pressure. Turbulence can lead to a first breakthrough of protective agent through the overflow channels as well as to mixing with supernatant (for example plasma), which is not disadvantageous, since in the next step a thorough mixing of protective agent and supernatant takes place. A device is preferred in which, by further turning (for example 90 °) of the hollow punch (7), it is released from the first holder and can be pulled out of the device until it stops against the sealing disk (6). As a result, the protective means (5) passes through the transfer channels (8) into the supernatant (for example plasma). By the design of the surface (e.g., roughness, no firm connection to the hollow die) of the overflow channels (8), the gasket (6) is still moveable when a little more pulling is applied. Alternatively, a connection can be made between the hollow die and sealing washer (e.g., via a groove) which can be loosened (e.g., by rotation) prior to mixing the supernatant with the preservative. By repeatedly turning the device upside down, a uniform mixing of the supernatant with the protective agent is achieved.

In yet another preferred embodiment of the device of the present invention, this is characterized in that the hollow punch (7) within the first sample space (12), in particular by a rotation, is fixable to the stop.

In a still further preferred embodiment of the apparatus of the present invention this is characterized in that the inlet (1) has a screw cap with a thread. This facilitates both the filling of the sample, as well as the closing and further storage.

In yet another preferred embodiment of the device of the present invention, it is characterized in that the porous stamp plate (2) has a pore size of between 500 nm to 10 μm, preferably from 1 μm to 7 μm, most preferably from 2 μm to 5 has μm. By pushing in the punch (3), cells and supernatant (e.g., blood and plasma) are separated because the liquid can pass through the porous stamp plate (3) but not the cells. For this purpose, the pore size is chosen (e.g., 2-5 μm) so that smooth passage of the liquid takes place without clogging of the stamp plate, but prevents passage of cells. In order to facilitate the separation of cells and liquids, one can leave the filled syringe overhead for some time or centrifuge to achieve a sedimentation of the cells.

By retracting the punch (3) protective agent-containing supernatant and cells are mixed. In this case, the supernatant as a liquid penetrate the pores of the stamp plate and there is automatically optimal mixing of protective agent-containing supernatant and cell suspension. The now protective agent-containing cell suspension can be frozen directly in the device. After storage and thawing, the cell suspension can be recovered from the device by pressure on the punches.

In yet another preferred embodiment of the device of the present invention, this is characterized in that the punches (3, 7) are provided with a predetermined breaking point and / or a corresponding marking or the grips of the punches (3, 7) are detachably provided. The device can thus be provided with a predetermined breaking point for the stamp, which can be canceled for storage. In this case, it is also advantageous if the device has on the connection side a screw cap, which is removed to recover the cell suspension.

In yet another preferred embodiment of the apparatus of the present invention, it is characterized in that the sample conditioning agent is selected from a cryopreservative, especially DMSO, hydroxyethyl starch (HES), such as final 10% or 20% DMSO in PBS or other aqueous buffer. By repeatedly turning the device upside down, a uniform mixing of the supernatant with the protective agent is achieved. The amount of protective agent is chosen so that the cells are optimally protected during cryopreservation.

In a further aspect of the device according to the invention, this is completely pyrogen-free so as not to interfere with the measurements.

A further aspect of the present invention then relates to a method for preparing and freezing a blood sample, comprising the steps of a) providing a device according to the invention as above, b) providing a blood sample to be frozen, c) drawing the blood sample into the device from a) up to d) closing the inlet of the device, e) removing the connecting clip (9), f) separating cells and supernatant by pushing in the stamp (3), g) mixing protective agent and supernatant, h) mixing protective agent-containing supernatant and cells, and i) freezing the protective agent-containing cell suspension.

Preferred is a method of the invention wherein the blood sample is provided with an anticoagulant as described herein and well known in the literature.

Further preferred is a method according to the invention, wherein the fixing takes place by turning the hollow punch (7), if this comprises corresponding fixing means.

Further preferred is a method according to the invention, wherein the supernatant is plasma. Even more preferred is a method according to the invention, wherein a uniform mixing of the supernatant with the protective agent is carried out by multiple overhead rotation of the device.

Particularly preferred is a method according to the invention, wherein the prepared preservative-containing cell suspension is frozen directly in the device. Example of this are also described here. A final aspect of the present invention then relates to the use of a device according to the invention for preparing and freezing a blood sample, as stated above.

The advantages of the device according to the invention include that the e.g. Syringe (as a preferred embodiment) contains all the necessary means to prepare the spot for a freezing process. In this case, no further expensive devices except a -80 ° refrigerator or other suitable freezer at temperatures of -100 ° C or higher are required. Blood samples can be obtained directly in the device according to the invention with minimal effort, the additives can be mixed gently and the samples are frozen. Further, cryopreservation can be accomplished without the use of liquid nitrogen (e.g., in the -80 ° C freezer or on dry ice). The principle can also be used for other cell suspensions.

It is not necessary to set up highly specialized laboratories with suitably qualified personnel on site. Furthermore, eliminates all steps and the equipment and chemicals to isolate the cells. As a result, costs and effort are reduced enormously (probably by 90% or more). Furthermore, the risk of changes in cell status through manipulation is greatly reduced.

The features disclosed in the foregoing description, the figures and the claims may be of importance both individually and in any combination for the realization of the invention in the various embodiments. For the purposes of the present application, all cited references are hereby incorporated by reference in their entirety.

Particular embodiments of the present invention will now be further clarified with reference to the figures, the list of reference numerals and the examples, without being limited thereby.

Figure 1A to C show schematically a preferred apparatus of the present invention and its use in sampling and sealing of the apparatus. Figures 2 A to C show schematically the preferred apparatus of the present invention of Figure 1 and its use in staple removal, filtration and mixing of the cell-free supernatant with sample conditioning agent (preservative).

Figure 3A shows schematically a preferred apparatus of the present invention of Figures 1 and 2 and their use in mixing the whole sample with the sample conditioning agent (preservative). Not shown are further steps of freezing at between -7O ⁰ C and -100 ° C without liquid nitrogen in a refrigerator or on dry ice.

FIG. 4 shows a further preferred device of the present invention, in which the second chamber (5) is opened by means of a piercing device (13). The second chamber (5) is separated from the sample space (12) in this case by a membrane (14) (A). First, fill the syringe with blood or cell suspension, first securing the piercing device so that the preservative does not enter the sample chamber too early. As the punch (3) moves upward, the membrane (14) or other partition is pierced or pushed up by the piercing device (13) while filtration is taking place (B). In this case, the protective agent is released into the sample chamber (12) and mixed with retraction of the punch (3) with the plasma.

example

Step 1: Sampling (Figure IA and B)

The sampling (eg blood with anticoagulants) is carried out as with a conventional syringe. Sealing ring (4) and movable sealing washer (6) guarantee the necessary negative pressure for mounting the sample. If these parts were not present, there would be no negative pressure in the sample chamber (12) when retracting the stamp, since air would pass through the porous stamp plate (2) into the sample chamber (12). The sealing disc (6) is pulled along by suitable surface design (eg roughness) of the overflow channels (8). Alternatively, the gasket can be connected via a groove with the hollow punch (7). For step 5 ("mixing the supernatant with the protective agent", see below), the connection between the sealing washer and the hollow punch would then be released (eg by turning the punch). ensures the defined simultaneous retraction of both punches (3 and 7). There is no protective agent in the sample, because the volume of the reservoir remains the same. For the hollow punch (7) there is a stop which guarantees the stop of both punches in such a way that no protective means (5) is pressed into the sample space (12). The hollow punch can be fixed in this position by turning it (eg by 90 °) (eg by a groove in the stop, need for fixation, see step 4).

Step 2: Remove the cannula, close the device (Figure IC)

After removing the Luer-Lock cannula (10), the device is closed with a commercially available Luer-Lock cover (1). The closure is a prerequisite for step 4 (see there), but also serves to protect the sample.

Step 3: Remove clamp (Figure 2A)

The connection clip (9) is removed from the punch (3) and the hollow punch (7).

Step 4: Separate cells from the supernatant (Figure 2B)

By pushing in the punch (3), cells and supernatants (e.g., blood and plasma) are separated because the liquid can pass through the porous stamp plate (2) but not the cells. For this purpose, the pore size is chosen (e.g., 2-5 μm) so that smooth passage of the liquid takes place without clogging of the stamp plate, but prevents passage of cells. In order to facilitate the separation of cells and liquids, one can leave the filled syringe overhead for some time or centrifuge to achieve a sedimentation of the cells. The device contains a mark for stopping the stamper (e.g., for blood at about 50% of the total sample volume) so as not to subject the cells to any mechanical pressure. The hollow punch remains fixed in its intermediate position (see step 1). Turbulence can lead to a first breakthrough of protective agent through the overflow channels as well as to mixing with supernatant (for example plasma), which is not disadvantageous, since in the next step a thorough mixing of protective agent and supernatant takes place.

Step 5: Mix supernatant with preservative (Figure 2C)

By further rotation (eg 90 °) of the hollow punch (7) this is released from the first holder and can be pulled out until it stops against the sealing washer (6) from the device. As a result, the protective means (5) passes through the overflow channels (8) in the supernatant (eg plasma). Due to the design of the surface (eg roughness, no firm connection to the hollow punch) of the overflow channels (8), the sealing disc (6) is still movable, if a little stronger train is exercised. Alternatively, a connection can be made between the hollow punch and the sealing washer (eg via a groove), which can be loosened before mixing the supernatant with the protective agent (eg by turning). By repeatedly turning the device upside down, a uniform mixing of the supernatant with the protective agent is achieved. The amount of protective agent is chosen so that the cells are optimally protected during cryopreservation.

Step 6: Blend Protecting Agent-containing Supernatant with Cells (Figure 3A)

By retracting the punch (3) protective agent-containing supernatant and cells are mixed. As in step 4, the supernatant as a liquid can penetrate the pores of the stamp plate. Optimal mixing of protective agent-containing supernatant and cell suspension takes place automatically. The now protective agent-containing cell suspension can be frozen directly in the device. After storage and thawing, the cell suspension can be recovered from the device by pressure on the punches. The device may be provided with a predetermined breaking point for the stamp, which can be canceled for storage. In this case, it is advantageous if the device has a screw cap on the connection side, which is removed for obtaining the cell suspension.

LIST OF REFERENCE NUMBERS

1. Luer lock connection for cannula and lid

2. Stamp plate with pores

3rd stamp

4. Sealing ring

5. second chamber with sample preparation agent (protective agent)

6. movable sealing washer

7. Hollow stamp

8. Inlet, transfer channels with guide for movable sealing disc

9. Connection bracket

10. Luer lock cannula

11. lockable inlet

12. first sample chamber

13. piercing device

14. membrane

Claims

claims

A device for preparing and freezing a blood sample, comprising a sample chamber (12) having an inlet (1), a movable plunger (3) with a within the sample chamber (12) arranged stamp plate with pores (2), and one within the Sample chamber (12) lying second chamber (5), for a sample conditioning means which is connected via a closable inlet (8) with the sample chamber (12).

2. Apparatus according to claim 1, characterized in that the second chamber (5) by a within the sample chamber (12) lying movable hollow punch (7) is formed, preferably additionally by means of a sealing washer (6).

3. Apparatus according to claim 1 or 2, characterized in that the sealing disc (6) connected via a groove with the hollow punch (7) is detachably connected.

Device according to any one of Claims 1 to 3, characterized in that the closable inlet (8) of the second chamber (5) is closed by a rotary and / or piercing mechanism, e.g. can be opened by means of the hollow punch (7).

5. Device according to one of claims 1 to 4, characterized in that the closable inlet (8) overflow channels with a guide for the sealing disc (6).

6. Device according to one of claims 1 to 5, characterized in that the punch (3) is movably mounted within the hollow punch (7).

7. Device according to one of claims 1 to 6, characterized in that the punch (3) is guided by a sealing ring (4).

8. Device according to one of claims 1 to 7, characterized in that the punch (3) and the hollow punch (7) by a releasable connection bracket (9) with each other are connected.

9. Device according to one of claims 1 to 8, characterized in that for the hollow punch (7) within the first sample space (12) a stop and / or a corresponding mark is provided.

10. The device according to claim 9, characterized in that the hollow punch (7) within the first sample space (12), in particular by a rotation, is fixable to the stop.

11. Device according to one of claims 1 to 10, characterized in that the inlet (1) has a screw cap with a thread.

12. Device according to one of claims 1 to 11, characterized in that the porous stamp plate (2) has a pore size of between 500 nm to 10 .mu.m, preferably from 1 .mu.m to 7 .mu.m, most preferably from 2 microns to 5 microns ,

13. Device according to one of claims 1 to 12, characterized in that the stamp (3, 7) are provided with a predetermined breaking point and / or a corresponding marking or the handles of the stamp (3, 7) are provided detachably.

14. Device according to one of claims 1 to 13, characterized in that the sample preparation means is selected from a Kryokonservierungsmittel, in particular DMSO.

15. Device according to one of claims 1 to 14, characterized in that it is completely pyrogen-free.

16. A method for preparing and freezing a blood sample or cell suspension, comprising the steps of a) providing a device according to any one of claims 1 to 15, b) providing a frozen blood sample or cell suspension, c) drawing the blood sample into the device from a) to to a stop and fixing the hollow punch (7), d) closing the inlet (11) of the device, e) removing the connecting clip (9), f) separating cells and supernatant by pushing in the stamp (3), g) mixing protective agent and supernatant, h) mixing protective agent-containing Supernatant and cells, and i) freezing the preservative-containing cell suspension.

17. The method of claim 16, wherein the blood sample is provided with an anticoagulant.

18. The method according to any one of claims 16 or 17, wherein the fixing takes place by rotating the hollow punch.

19. The method according to any one of claims 16 to 18, wherein the supernatant is plasma.

20. The method according to any one of claims 16 to 19, wherein a uniform mixing of the supernatant with the protective agent is carried out by multiple overhead rotation of the device.

21. The method according to any one of claims 16 to 20, wherein the protective agent-containing cell suspension is frozen directly in the device.

22. The method according to any one of claims 16 to 21, wherein the mixing of the supernatant with the protective agent by a piercing device (13).

23. Use of a device according to one of claims 1 to 15 for preparing and freezing a blood sample.