"Improved disposable device for centrifuging and treating a fluid biological material"

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The present invention relates to an improved disposable device for centrifuging and treating a fluid biological material.

More specifically, the present invention relates to an improved sterile disposable device for centrifuging and treating blood.

Recently, various methods for obtaining autologous preparations for use in various therapeutic procedures have become widespread, these methods generally having the drawback of comprising many complex operations in sterile environments and therefore requiring much time, highly skilled operators and very well-equipped laboratories.

An example of the said methods is described in international patent application WO 01/043787. This method relates to the preparation of an autologous platelet gel for accelerating wound healing, and comprises the following steps:

- collecting 40-50 ml of venous blood from the patient using a first sterile syringe;

- transferring the said blood from the said first syringe to a first sterile test tube;

- centrifuging the said first test tube at 180 g for 20 minutes, thus obtaining two phases: a dark bottom phase formed by red and white cells and a light top phase formed by platelet-rich plasma;

- collecting the light top phase using a second sterile syringe; - transferring the said light top phase into a second sterile test tube;

- centrifuging the said second test tube at 580 g for 20 minutes, thus obtaining a platelet sediment and a supernatant liquid formed by platelet-poor plasma;

- collecting the said supernatant liquid, using a third sterile syringe; - suspending the said platelets in a portion of the said supernatant

liquid sufficient to obtain about 6 ml of platelet concentrate;

- collecting the said platelet concentrate, using a fourth sterile syringe, and transferring it into a sterile Petri dish containing an aqueous solution of a calcium salt and batroxobin; - gently shaking the dish for about 30 seconds;

- collecting the platelet gel thus formed.

The aforesaid drawbacks of operation have been overcome by a disposable container described and claimed in International patent application WO 05/039773 in the name of the present applicant. In particular, this application describes and claims a disposable container for centrifuging and treating a fluid biological material, the said container being provided with an open top end and a closed bottom end, characterized in that the said top end is provided with a lid having: a) a first opening through which passes a first cannula which can be connected for operation to the external environment in order to control the exit and entry of air in conjunction with the transfer of a fluid biological material into or out of the said container; b) a second opening through which passes a second cannula which can be accessed by a hollow needle for transferring a fluid biological material into or out of the said container through the said hollow needle, c) a third opening through which passes a third cannula connected for operation to a connector which can receive and accommodate one end of a syringe for transferring a fluid biological material into or out of the said container through the said third cannula.

The disposable container according to the aforesaid International patent application can be used for operation in sterile conditions in any environment where no sterile hoods are present, and can be produced by the simple and economical assembly of commercially available components.

However, the applicant proposes to simplify the structure thereof in order to facilitate its use and reduce its production costs.

This aim is achieved by means of a disposable device for centrifuging and treating a fluid biological material, which comprises: a hollow body extending upwards and provided with an open top end and a closed bottom end, and a stopper which can be joined to the said open top end, in which the said stopper comprises a first and a second opening, a filtering device associated with the said first opening to filter the air entering and departing from the said hollow body in conjunction with a collection or introduction of a fluid from or into the said hollow body, and a tube of which one portion extends through the said second opening and enters the said hollow body to form a passage for transferring a fluid into and out of the said hollow body, characterized in that: the said stopper is provided with a chamber, the said second opening passes through the said chamber, the said tube is substantially rigid and its portion which enters the said hollow body through the said second opening has a length at least equal to the height of the hollow body, the said chamber houses a resilient element for hermetically sealing the said second opening around the said portion of tube while also allowing the said portion of tube to slide with a degree of friction within the said resilient element when a pushing or lifting action is performed on the said tube, and retaining the said portion of tube at any desired depth within the said hollow body when the said action ceases.

The cross section of the said hollow body is preferably circular. Advantageously, the shape of the said closed bottom end of the said hollow body is hemispherical or tapering. However, it can have any

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other shape suitable for centrifuging.

The said filtering device is preferably of any known type suitable for filtering and sterilizing the air passing through it.

In a preferred embodiment, the said filtering device is fixed to the said stopper by means of any known type of adhesive material compatible with the biological materials which are to be treated in the device according to the present invention.

Advantageously, the said open top end of the said hollow body is joined to the said stopper by screwing. The shape of the said chamber is not critical. It can extend over the whole surface of the said stopper or only over part of it. The sole critical condition regarding the said chamber of this invention is that it must have the said second opening passing through it.

Typically, the cross section of the said chamber is circular or takes the form of an asymmetrical ellipse in which the two radii of curvature are different from each other.

In one embodiment of the present invention, the said stopper also comprises a third opening which allows a hollow needle to pass through it and enter the said hollow body to transfer a fluid biological material into or out of the said hollow body.

Advantageously, the said third opening also passes through the said chamber housing the said resilient element, and the said hollow needle enters the said hollow body after perforating the said resilient element. The said substantially rigid tube is preferably provided, in an upper part which does not enter the said second opening, with a connection device of a known type for the sterile transfer of biological fluids into and out of the said hollow body. Typical examples of such connectors are Luer connectors and pierceable (spikable) membranes.

Advantageously, a lower part of the said tube is provided with a stop to prevent the said tube from passing out of the said second opening

when a lifting action is performed on it.

In the present description and in the claims, the expression "pierceable membrane" refers to a resilient membrane which can be pierced and passed through by a pointed body, such as a pointed hollow connector (spike coupler), and can reseal itself hermetically when the said pointed hollow connector is pulled out. Membranes of this type have been well known for many years in the pharmaceutical field. They are usually made from an elastomeric polymer such as a natural rubber or a synthetic rubber, for example a styrene- ethylene/butylene copolymer having the trade name Cawiton™.

Typically, the said resilient element housed in the said chamber is of annular shape or is a pierceable membrane.

Advantageously, the materials used to produce the device according to the present invention conform to the specifications of health authorities for materials used for medical purposes.

The present invention will now be described more fully with the aid of the following description and the attached figures, provided solely by way of example and without any restrictive intent, in which:

Figure 1 shows a front view of a hollow body according to a first embodiment of the present invention;

Figure 2 shows a view from above of a stopper according to a first embodiment of the present invention;

Figure 3 shows a front view of a tube according to a first embodiment of the present invention; Figure 4 shows a view in longitudinal axial section of the tube of Figure 3;

Figure 5 shows a view from above of the tube of Figure 3;

Figure 6 shows a front view of a stop flange housed in the stopper of Figure 2; Figure 7a is a partial view of the subsequent Figure 7b;

Figure 7b shows a view in longitudinal section, taken along the line A-A' of Figure 2, of a disposable device according to a first embodiment of the present invention;

Figure 8 shows a view from above of a stopper according to a second embodiment of the present invention;

Figure 9 shows a view in longitudinal section, taken along the line B- B' of Figure 8, of a disposable device according to a second embodiment of the present invention.

As shown in Figures 1-7b, a disposable device (1 ) for centrifuging and treating a fluid biological material comprises (i) a hollow body (2) provided with an open top end (21 ) and with a closed bottom end (22) of hemispherical shape and (ii) a stopper (3) which can be joined to the said open top end (21 ).

The hollow body (2) can be made from rigid or semi-flexible material. In the specific embodiment shown in Figures 1-7b, the hollow body (2) has a capacity of about 50 ml, a diameter of about 29 mm and a length of about 115 mm. In a preferred embodiment (not shown), the outer wall of the hollow body (2) is graduated to facilitate the reading of the volume of material contained. Additionally, the hollow body of the specific embodiment illustrated is rigid and is made from Eastar

Copolyester MN058™, produced by the Eastman Chemical Company.

The stopper (3) includes a substantially flat surface (31 ) and a substantially circular crown (32) provided with an internal thread (not shown) so that it can be joined by screwing to the thread (23) of the hollow body (2).

The stopper (3) can also be made of rigid or semi-flexible material.

Two openings (33, 34) are provided in the substantially plane surface (31 ) of the stopper (3).

A filtering device (11 , 12), provided with a porous diaphragm (12) capable of sterilizing the air which passes from the external

environment to the hollow body (2), is inserted into the first opening (33).

The second opening (34) extends towards the inside of the hollow body (2), forming a substantially cylindrical chamber (39) delimited by walls (35) fixed to the stopper (3). The chamber (39) is about 12 mm deep and its diameter is about 5 mm, making the ratio of depth to diameter 2.4.

A resilient ring (37) is fitted into the chamber (39) and is retained there by a stop flange (36). The disposable device (1 ) also comprises a tube (4) having a rigid portion (L) which passes through the chamber (39) and extends into the hollow body (2) to form a passage (41 ) for transferring a fluid into and out of the said hollow body (2).

In the specific embodiment shown in Figs. 1-7b, the length of the portion (L) of the tube (4) is about 118 mm.

A ring (45) is mounted on the tube (4) in a fixed way to act as a stop. This prevents the tube (4) from being pulled out of the stopper (3).

The resilient ring (37) hermetically seals the second aperture around the tube (4). The ring (37) also allows the tube (4) to slide with a degree of friction within it when the operator performs a pushing or lifting action on the tube (4), and also enables the tube to be kept at any desired depth within the hollow body (2) when the said action ceases.

The upper part of the tube (4) has a first projecting element (42a) which has a first half-housing and can be joined to the lower part of a second projecting element (42b) which has a second half-housing. The said first and second half-housings come into contact with each other when the said first (42a) and second (42b) projecting elements are joined together, forming a single housing (46) for a pierceable membrane (43). Above the said second projecting element (42b), the tube (4) has a

cylindrical element (44) which houses a seat (47) for a pointed hollow connector (spike coupler).

Figures 8-9 show a second embodiment of a disposable device (100) according to the present invention. In Figures 8-9, reference numbers identical to those of Figures 1-7b indicate identical parts. Additionally, the parts not shown in Figures 8-9 and/or not described below are also identical to those of Figures 1-7b.

Three openings (33, 134, 138) are provided in the substantially plane surface (31 ) of the stopper (130). The stopper (130) is also provided with a chamber (139) through which both the second opening (134) and the third opening (138) pass.

The chamber (139) is delimited by walls (135) fixed to the stopper (130).

The cross section of the chamber (139) takes the form of an asymmetrical ellipse in which the two radii of curvature are different from each other.

The chamber (139) houses a pierceable membrane (137) which hermetically seals the second opening (134) around the tube (4). The pierceable membrane (137) also allows the tube (4) to slide with a degree of friction when the operator performs a pushing or lifting action on the tube (4), and also enables the tube to be kept at any desired depth within the hollow body (2) when the said action ceases.

The pierceable membrane (137) also hermetically seals the third opening (138). A hollow needle (not shown, because it is of a conventional type) can pass through the third opening (138), this needle initially penetrating the pierceable membrane (137) and then entering the hollow body (2) to transfer a fluid biological material into or out of the hollow body (2).

When the hollow needle is withdrawn, the membrane (137) reseals itself, thus hermetically sealing the third opening (138).

By way of example, a description will now be given of the application of the method described in international patent application WO 01/043787, using the disposable device (1 ) of Figures 1 -7.

A sterile disposable device (1 ) is prepared, and in this device the hollow body (2) is screwed firmly on to the stopper (3) provided with a filtering device (11 , 12) and with a tube (4).

40-50 ml of venous blood is collected from a patient with a first sterile injection syringe provided with a needle (these are not shown, because they are of conventional types). The conventional injection needle is replaced with a sterile pointed hollow connector of the spike coupler type (not shown, because it is of a conventional type).

The pointed hollow connector is fitted into the corresponding seat (47) located at the top of the tube (4). A degree of pressure is exerted on the body of the said first syringe until the pointed hollow connector penetrates through the pierceable membrane (43).

The plunger of the said first syringe is pushed to transfer the patient's blood in sterile conditions from the syringe to the hollow body (2). During this operation, the blood enters the hollow body (2) through the passage (41 ) and an equal volume of air flows out of the hollow body (2) through the filtering device (11 , 12).

After the patient's blood has been transferred to the device (1 ) in sterile conditions, the device (1 ) is placed in a centrifuge (not shown) and is centrifuged at a specified speed and for a specified time to obtain two phases, namely a dark bottom phase formed by red and white cells and a light top phase formed by platelet-rich plasma. Advantageously, the centrifuge is operated at 180 g for 20 minutes.

A second conventional sterile syringe is prepared, this syringe being provided with a second sterile pointed hollow connector and having its

plunger in the lowered position.

The second pointed hollow connector is fitted into the corresponding seat (47) located at the top of the tube (4), the tube being in a lowered position so that it nearly touches the bottom end (22) of the hollow body (2).

A degree of pressure is exerted on the body of the said second syringe until the pointed hollow connector penetrates through the pierceable membrane (43).

The plunger of the second syringe is raised, in order to slowly draw the said dark bottom phase, formed by red and white cells, from the bottom (22) of the hollow body (2), through the passage (41 ). During this drawing procedure, the vacuum created in the hollow body (2) is compensated for by the entry of sterilized ambient air through the filtering device (11 , 12). When the dark bottom phase has been drawn off, the pointed hollow connector is removed from its seat (47).

The device (1 ) is again placed in a centrifuge (not shown) and is centrifuged at a specified speed and for a specified time, to obtain two phases, namely a platelet sediment and a supernatant liquid formed by platelet-poor plasma. Advantageously, the centrifuge is operated at 580 g for 20 minutes.

A third conventional sterile syringe is prepared, this syringe being provided with a third sterile pointed hollow connector and having its plunger in the lowered position. The tube (4) is positioned in the supernatant liquid.

The third pointed hollow connector is fitted into the corresponding seat (47) located at the top of the tube (4).

A degree of pressure is exerted on the body of the said third syringe until the pointed hollow connector penetrates through the pierceable membrane (43).

The plunger of the third syringe is raised in order to slowly transfer most of the supernatant liquid from the hollow body (2) to the third syringe through the passage (41 ). During this operation, the lower end of the tube (4) is positioned in such a way that about 6 ml of a material formed by platelet sediment and by some of the supernatant liquid formed by platelet-poor plasma is left on the bottom (22) of the hollow body (2) of the device (1 ). Additionally, during this operation, the vacuum created in the said hollow body (2) is compensated for by the entry of ambient air through the filtering device (11 , 12). A fourth conventional sterile syringe is prepared, this syringe being provided with a sterile pointed hollow connector and containing a suitable dose of a known activating solution.

The fourth pointed hollow connector is fitted into the corresponding seat (47) located at the top of the tube (4). A degree of pressure is exerted on the body of the said fourth syringe until the pointed hollow connector penetrates through the pierceable membrane (43).

The plunger of the fourth syringe is pushed down to slowly transfer the activating solution into the hollow body (2) containing the platelet sediment.

The device (1 ) is shaken slowly for about 30 seconds.

This forms an autologous platelet gel, of the type described in international patent application WO 01/043787, which can be used for accelerating wound healing. The autologous platelet gel obtained in this way is easily removed from the hollow body (2) of the device (1 ) once the cover (3) has been unscrewed and removed.

The said activating solution is typically formed by an aqueous solution of a suitable enzyme and an organic or inorganic calcium salt. Typical examples of suitable enzymes are thrombin, batroxobin and

fibrin. Typical examples of suitable calcium salts are chloride, gluconate and lactate.

It should be noted that the device (1 ) according to the present invention has the advantage of producing an autologous platelet gel in the device (1 ) into which the venous blood collected from the patient has been transferred, without the need for any further transfer. This provides the major advantage of facilitating the maintenance of sterile conditions and facilitating the operator's work.

Although the present description has been specifically illustrated with reference to the preparation of an autologous platelet gel of the type described in international patent application WO 01/043787, a person skilled in the art will realize that the sterile disposable device (1 ) according to the present invention is suitable for the application of many other methods which include various operations of centrifuging and/or treatment of biological fluids in a sterile environment.