WO1996026264A1

WO1996026264A1 - A bioreactor

Info

Publication number: WO1996026264A1
Application number: PCT/GB1996/000251
Authority: WO
Inventors: Ian Michael Reed
Original assignee: United Kingdom Atomic Energy Authority
Priority date: 1995-02-18
Filing date: 1996-02-05
Publication date: 1996-08-29
Also published as: GB9503197D0; GB2297980A; GB2297980B; GB9602270D0; AU4629096A

Abstract

A bioreactor (10) suitable for cell lines such as hepatocytes which function best when attached to a surface consists of a stack of flexible membranes (22), which define several flow channels (25) between pairs of adjacent membranes (22). The channel width can therefore be adjusted, for example by fluid pressure, wide channels (25) aiding the loading of cells into the bioreactor (10), and narrower channels (25) providing less hold-up during use. Nutrient channels (20) may be defined by the same membranes (22), so nutrient channels (20) and flow channels (25) alternate up the stack. Hollow fibres (26) may be provided within the flow channels (25) for in situ oxygenation.

Description

A Bioreactor

This invention relates to a bioreactor within which cells, for example from multicellular organisms, may be grown, and to a method of growing cells by use of such a bioreactor.

Many cell types, particularly those from multicellular organisms, function most efficiently when attached to a surface rather than being in free suspension. However the presence of surfaces in a bioreactor may make it more difficult to supply nutrients and oxygen to the cells. It is also usually desirable to have a large number of cells per unit volume; but if the surfaces are arranged close together with the aim of increasing the cell density, this may lead to problems in seeding the bioreactor with cells, and may also lead to poor oxygen supply.

According to the present invention there is provided a bioreactor comprising a plurality of flexible membranes stacked together to define a multiplicity of cell channels between pairs of membranes, the bioreactor being arranged such that the separation of the pairs of membranes defining the cell channels is adjustable.

Because the membrane separation is adjustable, the channel thickness may be made larger while the bioreactor is being seeded with cells, and then be made narrow while the cells are cultured. The membrane separation may be adjustable by adjusting the fluid pressure within the cell channels. A mechanical clamp may also be provided to constrain the distance between the endmost membranes of the stack.

Preferably means are provided to supply oxygen to the cell channels, and to supply nutrients. For example oxygen may be supplied through oxygen permeable tubular fibres within the cell channels. Nutrients may be supplied by transport through the membrane at at least one side of a cell channel, if that membrane is sufficiently permeable and a nutrient flow channel is provided at the other side of that membrane. For example the membranes might be microporous, for example of polypropylene; nutrients can diffuse through the membrane into the cell channel, and waste products from the cells may diffuse out of the cell channels into the adjacent nutrient flow channel.

The bioreactor is particularly suited for use with liver cells (hepatocytes) and can enable high cell densities to be achieved during culturing. It is equally applicable to any cell line which functions best when attached to a surface.

The invention also provides a method of use of such a bioreactor involving the steps of seeding the bioreactor with cells while separating the pairs of membranes defining the cell channels, and then culturing the cells while arranging the membranes to be closer together.

The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings in which:

Figure 1 shows a diagrammatic plan view of a bioreactor;

Figure 2 shows a cross-sectional view on the line II-II of Figure 1;

Figure 3 shows a cross-sectional view of an alternative bioreactor; and Figure 4 shows a cross-sectional view of another alternative bioreactor.

Referring to Figure 1 there is shown a bioreactor 10, square in plan view, for use with hepatocytes (liver cells) . The bioreactor 10 enables a flow of blood plasma to contact the cells, the plasma flow passing through manifolds 12 on two opposite faces of the bioreactor 10. The bioreactor 10 is also provided with an inlet 14 and an outlet 15 for a nutrient stream between two opposite corners, and headers 16 on the other pair of opposed faces for a flow of oxygen.

Referring also to Figure 2, the bioreactor 10 has four flow channels 20 for the nutrient stream. Each channel 20 is defined by two square sheets 22 of microporous polypropylene sealed to each other by a bond 23 around their entire perimeter except at two opposite corners where the channel 20 communicates with the inlet 14 and the outlet 15 (indicated diagrammatically in Figure 2) . The pores through the microporous sheets 22 are of size about 0.2 μm. Three plasma flow channels 25 are defined in between these nutrient flow channels 20. Five porous- walled tubular oxygenation fibres 26 extend transverse to the flow direction within each channel 25, their ends extending into the oxygen headers 16. The fibres 26 are of polypropylene, of diameter 0.3 mm and wall thickness 0.1 mm. Along the sides of the flow channel 25 through which the ends of the oxygenation fibres 26 extend, the edges of the sheets 22 are bonded to each other. The channels 25 thus connect the opposed manifolds 12. At the top and bottom of the bioreactor 10 are square, flexible, but impermeable protective membranes 28, sealed around their edges to the adjacent membranes 22. In use the bioreactor 10 is first loaded with cells, by supplying a suspension of cells through the plasma flow path, via the manifolds 12 into the plasma flow channels 25. The fluid pressure within the channels 25 is maintained sufficiently high that the channels 25 are at least 1 mm wide, for example 2 mm or 3 mm wide. Oxygen is supplied through the fibres 26, and nutrients are supplied to the flow channels 20. The channels 25 are sufficiently wide that the cells can flow freely, and become attached substantially uniformly over the entire surface of the membranes 22 which they contact. Once the bioreactor 10 is sufficiently loaded with cells, the supply of the cell suspension is terminated. The cell density typically is in

5 2 the range 0.5 to 4.0 x 10 cells/cm .

Blood plasma can then be caused to flow through the bioreactor 10 so as to contact the cells on the membranes 22. The plasma flow channels 25 are not pressurised, and are typically no more than 0.5 mm wide. The width of the flow channels 25 may be constrained by clamping the bioreactor 10 between rigid plates (not shown) outside the protective outer membranes 28. The flows of oxygen and of nutrients are as described above. The cells consequently receive a plentiful supply of oxygen, and of nutrients (which may include glucose, amino-acids, vitamins, organic salts, hormones and growth factors) which diffuse through the membranes 22 from the channels 20. The liver cells remove toxins from the plasma. Waste products from the cells may diffuse back through the membranes 22 into the channels 20. The plasma is thus purified and can be returned to a patient, who may be suffering from liver failure.

It will be appreciated that the bioreactor 10 may be modified in a variety of ways while remaining within the scope of the invention. It may be used with other types of primary cell or established cell lines. The number of plasma flow channels 25 may differ, and might for example be ten, sandwiched between eleven nutrient flow channels 20. The number of oxygenation fibres 26 may differ from that described above, and might for example be a hundred or more in each flow channel 25; they may be spaced about 1 mm or 2 mm apart throughout the whole length of the channel 25. They may be of diameter in the range 200-350 μm, and wall thickness in the range 50-100 μm, and might be of a different material such as a polyethene/polyurethane/poly- ethene composite. For some purposes the oxygenation fibres 26 may be omitted, and instead either the plasma or the nutrient stream be oxygenated before being supplied to the bioreactor 10. For some purposes the nutrient flow channels 20 may be omitted, if there are sufficient nutrients present in the liquid in the flow channels 25.

Referring to Figure 3 there is shown a cross-sectional view of an alternative bioreactor 30, which has many features in common with the bioreactor 10 of Figures 1 and

2. It differs in that each plasma flow channel 25 is about

50% wider than in the bioreactor 10 and is divided in two by a corrugated polypropylene membrane 32. The corrugations extend perpendicular to the direction of flow.

The membrane 32 is substantially non-porous, and about 0.2 mm thick, and it provides an additional substrate for cell growth. Oxygenation fibres 26 are provided at both sides of each membrane 32, aligned with the corrugations, and locating below the peaks and above the troughs.

This bioreactor 30 provides a larger substrate area for cell growth, as cells can grow both on the membranes 22 and the membranes 32. There is also a more uniform oxygen concentration over the surface of the membrane 32. In other respects the bioreactor 30 is used in the same way as described above.

Referring now to Figure 4 there is shown a cross- sectional view of another alternative bioreactor 40. It differs from the bioreactor 30 in that each corrugated membrane 32 is replaced by a corrugated nutrient flow channel 42 defined between two microporous polypropylene membranes 44 sealed to each other by a bond 23 around their perimeter except at two opposite corners where the channel 42 communicates with the inlet 14 and outlet 15 (as indicated diagrammatically) . Thus the bioreactor 40 defines six plasma flow channels 45 each defined between a flat nutrient flow channel 20 and a corrugated nutrient flow channel 42. The bioreactor 40 is used in substantially the same manner as described earlier.

It will again be appreciated that a bioreactor may differ from those shown in the figures while remaining within the scope of the invention. In particular the number of corrugations of each membrane 32 or of each channel 42, and consequently the number of oxygenation fibres 26, may differ from those shown, as may the shape of the corrugations.

Claims

1. A bioreactor (10,30,40) comprising a plurality of flexible membranes (22,32,44) stacked together to define a multiplicity of cell channels between pairs of membranes (22,32,44), the bioreactor (10,30,40) being arranged such that the separation of the pairs of membranes (22,32,44) defining the cell channels (25,45) is adjustable.

2. A bioreactor as claimed in Claim 1 also comprising oxygen permeable tubular fibres (26) within the cell channels (25,45) .

3. A bioreactor as claimed in Claim 1 or Claim 2 comprising headers (12) to supply a first liquid medium to flow through the cell channels (25,45).

4. A bioreactor as claimed in any one of the preceding claims also comprising a nutrient channel (20,42) defined at at least one side of each cell channel (25,45), the nutrient channel (20,42) being separated from the cell channel (25,45) by a flexible microporous membrane (22,44).

5. A bioreactor as claimed in any one of the preceding Claims wherein at least some of the membranes (32,44) which define the cell channels (25,45) are of corrugated form.

6. A bioreactor as claimed in Claim 5 wherein each cell channel (25,45) is defined between a substantially flat membrane (22) and a corrugated membrane (32,44).

7. A method of growing cells in a bioreactor (10,30,40) as claimed in any one of the preceding Claims, the method involving the steps of seeding the bioreactor with cells while separating the pairs of membranes (22,32,44) defining the cell channels (25,45), and then culturing the cells while arranging the membranes (22,32,44) to be closer together.

8. A method as claimed in Claim 7 wherein the cells are hepatocytes.

9. A method as claimed in Claim 7 or Claim 8 wherein, during the step of culturing the cells, blood plasma is caused to flow through the cell channels (25,45).