Patent ID: 12233192

Referring toFIG.1, a simple example embodiment of a cell washing apparatus10according to the first or second aspect of the present invention is shown. In the embodiment shown, the cell washing apparatus10comprises a substantially cuboidal first fluid conduit12, forming a substantially planar sheet, having a longitudinal axis arranged in a horizontal orientation such that a first end of the first fluid conduit12is affixed to a first fluid inlet manifold14having a first fluid inlet aperture permitting inflow26of a cell-containing first fluid into a cavity13of the first fluid conduit12. At a second end of the first fluid conduit12is affixed a first fluid outlet manifold16having a first fluid outlet aperture arranged to permit outflow30of the cell-containing first fluid from the cavity13of the first fluid conduit12.

Positioned adjacent the first fluid conduit12and having the same orientation is a substantially cuboidal second fluid conduit18, forming a second substantially planar sheet. The second fluid conduit18comprises a first end having affixed thereto a second fluid inlet manifold20comprising a second fluid inlet aperture arrange to permit inflow32of a second fluid into a cavity19of the second fluid conduit18. At a second end of the second fluid conduit18and affixed thereto is a second fluid outlet manifold22having a second fluid outlet aperture arranged to permit outflow36of the second fluid from the cavity19of the second fluid conduit18.

The apparatus10further comprises a semi-permeable membrane24positioned between the first fluid conduit cavity13and the second fluid conduit cavity19and providing a semipermeable interface therebetween. The semi-permeable membrane24comprises a plurality of pores arranged to permit diffusion of substances between the first fluid conduit cavity13and the second fluid conduit cavity19.

In use, the first fluid inlet manifold14is in fluid communication with a first fluid container (not shown) positioned above the apparatus10and oriented such that a cell-containing first fluid flows out of the first fluid container by force of gravity. The first fluid outlet manifold16is in fluid communication with a first fluid collection container (not shown) arranged to collect the cell-containing first fluid from the first fluid outlet manifold16. Under force of gravity, the cell-containing first fluid exits the first fluid container26and flows in a horizontal first direction28through the first fluid conduit12and out30to the first fluid collection container. The first fluid collection container is positioned below the first fluid outlet manifold16to aid continued flow of the first fluid by force of gravity alone.

Further in use, the second fluid inlet manifold20is in fluid communication with a reservoir (not shown) of a second fluid, the reservoir positioned above the second fluid inlet manifold20. Positioned between the reservoir and the second fluid inlet manifold20is a pump (not shown) arranged to pump the second fluid such that it flows from the reservoir32toward the second fluid inlet manifold20. The second fluid subsequently passes in a horizontal second direction34, diametrically opposed to the first direction28, along the second fluid conduit18and out of the second fluid outlet manifold22to a waste container (not shown)36, the waste container being positioned below the second fluid outlet manifold22. In the embodiment shown, the second fluid may also travel from the second fluid reservoir, into and out of the apparatus by force of gravity alone.

The flow of the cell-containing first fluid26,28,30and the flow of the second fluid32,34,36occurs simultaneously. Transfer of species between the cell-containing first fluid and the second fluid occur by way of the semi-permeable membrane24during counterflow of the cell-containing first fluid and the second fluid. The cuboidal first conduit12and second conduit18are each in the form of substantially planar sheets providing a large surface area of contact between the first fluid conduit12and the second fluid conduit18. In the embodiment shown, the entire surface of contact between the first fluid conduit12and the second fluid conduit18is comprised of the semi-permeable membrane24.

In the embodiment shown, the cell-containing first fluid is stored transfusion blood containing red blood cells, which is stored in a blood bag. Handling and storage of the transfusion blood over time causes degradation of red blood cells, resulting in leaking of free haemoglobin into the surrounding blood. Said free haemoglobin has been implicated in post-transfusion morbidity. Accumulation of such toxic species over time is common not only in transfusion blood, but in any cell-containing fluid. Cells grown in laboratory culture generate toxic waste products which are required to be periodically “washed” from the surrounding cell culture medium.

In the example embodiment shown inFIG.1, the blood bag is positioned above the apparatus10and oriented such that the blood flows downward toward the first fluid inlet manifold by force of gravity. The pores of the semi-permeable membrane24of the example shown are sized at a minimum of 0.2 μm to a maximum of 2.0 μm, and are therefore sized to permit passage of toxic metabolites and free haemoglobin from the first fluid to the second fluid, but also to prevent passage of red blood cells out of the first fluid across the membrane24.

A more complex example of a cell washing apparatus40of the first or second aspect of the present invention is shown inFIG.2, which shows a substantially cylindrical elongate housing defining a first fluid conduit having an uppermost first entry portion41defining a first entry cavity43, and a lowermost second exit portion42defining a second exit cavity44, wherein each of the first entry cavity43and the second exit cavity44are in fluid communication by way of a plurality of elongate cylindrical channels45. The elongate housing further comprises a second fluid conduit46positioned between the first entry cavity43and the second exit cavity44of the first fluid conduit. The plurality of channels45extend through a cavity47of the second fluid conduit46, and wherein said channels45are secured within the second fluid conduit cavity47at either end thereof by potting55.

Extending from the uppermost first entry cavity43of the first fluid conduit is a first fluid inlet48, and extending from the lowermost second exit cavity44is a first fluid outlet49. Extending from a lowermost portion of the second fluid conduit46is a second fluid inlet50, and extending from an uppermost portion of the second fluid conduit46is a second fluid outlet52. The second fluid outlet52is positioned above the second fluid inlet50each being oriented such that they are diametrically opposed. As such, in use, a second fluid such as that described hereinbefore is arranged to flow51into the second fluid conduit46by way of the second fluid inlet50. Flow of the second fluid subsequently proceeds in an upwardly oriented direction toward the second fluid outlet52. The first fluid flows into the first fluid inlet48and in a downward direction into the first fluid entry cavity43, through the elongate channels45, and toward the first fluid exit cavity44where the first fluid converges and exits56the first fluid conduit by way of the first fluid outlet49.

In the embodiment shown, the elongate channels45comprise walls separating the elongate channels from the second fluid conduit46, wherein the walls comprise a semi-permeable membrane comprising pores arranged to permit the diffusion of substances between the two fluids.

FIG.3shows a perspective view of the apparatus described and shown inFIG.2.

In the example embodiment shown inFIG.2andFIG.3, the first fluid conduit comprises a plurality of channels45arranged to extend through the cavity47of the second fluid conduit46. Alternate embodiments will be appreciated wherein the elongate channels are of rectangular cross-section, rather than the cylindrical channels shown, and embodiments wherein the inlet and outlet ports are shaped to give a more uniform distribution of the fluid between the channels. The cylindrical (hollow fibre) membranes may also be replaced by planar (flat sheet) membranes.

FIG.4, shows such an embodiment70which incorporates both flat sheet membranes and entry and exit ports designed to give a more uniform flow of the fluids between the channels. The illustration shows flat-sheet membranes wherein the second fluid conduit90is arranged to extend through the first fluid conduit72. In the embodiment70ofFIG.4, the elongate channels92of the second fluid conduit90are formed of substantially planar sheets. The first fluid enters as stream94and flows into the narrowing headspace74to give a uniform flow between the channels90. The fluid exits as stream96through broadening space82again to promote uniform flow distribution between the channels90. The second fluid inlet and outlet ports distributing the fluid to flow within the channels90is not illustrated. In counter-current flow, the second fluid enters at the bottom of the device and exits at the top.

The embodiments described and shown are in a vertical orientation but the apparatus according to the present invention operates equally well at any orientation. In another manifestation, the apparatus could have a rectangular cross-section. In yet another manifestation, the first fluid could enter and leave by the sides of the entry and exit spaces and the spaces may be angled so that the velocity of the first fluid remains approximately constant as it flows across the top of the channels (so that the depth is zero opposite the first fluid entry and exit). Similarly, the space at the bottom may be angled so that the depth opposite the exit is zero.

FIG.5shows a method in accordance with the third or fourth aspects of the present invention, the method comprising the steps:i. providing the apparatus shown inFIG.3100;ii. inserting transfusion blood through the first fluid inlet and subsequently passing said inserted transfusion blood through the first fluid conduit; and simultaneously102, and simultaneouslyiii. passing a second fluid through the second fluid inlet and subsequently through the second fluid conduit104; andiv. retrieving the transfusion blood from the first fluid outlet, the retrieved transfusion blood or packed red blood cells comprising a lower concentration of free haemoglobin than present in the inserted transfusion blood106.

A suitable membrane for use with the present invention would include MicroPES available from 3M/Membrana. The membrane is available in both hollow fibre and flat sheet versions.

Optionally, in order to achieve very low concentrations of the undesired species, several apparatuses of the present invention may be used in series.

It will be appreciated that the above described embodiments are given by way of example only and that various modifications thereto may be made without departing from the scope of the invention as defined in the appended claims.

It will also be appreciated that, although the invention is described using (human) blood as the first fluid. The present invention could be employed for treating any type of cell-containing first fluid, wherein said fluid comprises one or more “contaminants” and thus the cells require “washing”. Additional first fluid may, for example, include mammalian or reptilian blood, or cell culture medium, among others well-known in the art. The invention could also be employed to wash any type of biological cells, such as those that may be used during fermentation and other microbiological processes. In one example, cells cultured in a lab suffer build-up of toxic substances, which may include metabolites such as lactic acid, or a harmful secretome of proteins and non-protein components such as non-protein components, such as lipids, micro-RNA and messenger-RNA, in their cell culture media. Said media therefore requires regular changing to permit continued culture of the cells. Said toxic substances could optionally be transported out of said cell culture media using the present invention. In a further example, biological cells, such as yeast, may be used for production of industrially applicable substance or metabolite, such as an antibiotic. In the example case of antibiotic production, the concentration of antibiotic over time could become toxic to the antibiotic-producing cells and therefore require cell washing. The present invention can preferably be used to remove said antibiotic and/or other toxic substance from the surrounding media. In examples using yeast, depending on the strain, yeast cells may be 3.0 μm to 4.0 μm in size up to 30.0 μm to 40.0 μm in size. For treating cells of differing sizes membranes of differing pore size may be needed.

The present invention may be further understood with reference to the following paragraphs:

A red blood cell washing apparatus arranged to exchange a contaminated first fluid containing red blood cells with an uncontaminated second fluid such that the red blood cells are now contained in an uncontaminated fluid, the apparatus comprising,a first fluid conduit and a second fluid conduit, the second fluid conduit being separated from the first fluid conduit by a semi-permeable membrane disposed therebetween;the first fluid conduit having a first fluid inlet and a first fluid outlet, the first fluid conduit arranged to transport the first fluid in a first direction between the first fluid inlet and the first fluid outlet;the second fluid conduit being arranged to house a second fluid;wherein the semi-permeable membrane comprises a plurality of pores arranged to permit transport of said one or more exchangeable entities from the first fluid to the second fluid;wherein the contaminated first fluid diffuses through the membrane and may be replaced with uncontaminated second fluid that counter-diffuses through the membrane. The first fluid may be SAGM or other solution used to suspend red blood cells in packed cells. The second fluid may be SAGM or other solution used in making up packed cells as widely used for blood transfusion and may contain desirable species.

A red blood cell washing apparatus arranged to separate blood cells from whole blood to produce packed cells and a separate blood plasma stream. The whole blood consists of blood cells suspended in blood plasma (first fluid) and the second fluid consists of SAGM or another solution suitable for constituting the liquid phase of whole blood. The apparatus comprises, a first fluid conduit and a second fluid conduit, the second fluid conduit being separated from the first fluid conduit by a semi-permeable membrane disposed therebetween;the first fluid conduit having a first fluid inlet and a first fluid outlet, the first fluid conduit arranged to transport the first fluid in a first direction between the first fluid inlet and the first fluid outlet;the second fluid conduit being arranged to house a second fluid;wherein the semi-permeable membrane comprises a plurality of pores arranged to permit transport of said one or more exchangeable entities from the first fluid to the second fluid; wherein the plasma from the whole blood diffuses through the membrane and may be replaced with second fluid that counter-diffuses through the membrane. The process may be conducted in more than one stage to separate red blood cells from white blood cells. The first fluid may be blood plasma (so that the mixture constitutes whole blood) or may be SAGM or other solution used to suspend red blood cells in packed cells.