GB2477140A

GB2477140A - Hollow porous fibre perfusion bioreactor

Info

Publication number: GB2477140A
Application number: GB1001111A
Authority: GB
Inventors: Stephen Owen
Original assignee: Automation Partnership Cambridge Ltd
Current assignee: Automation Partnership Cambridge Ltd
Priority date: 2010-01-25
Filing date: 2010-01-25
Publication date: 2011-07-27
Also published as: GB201001111D0

Abstract

A hollow fibre perfusion bioreactor is disclosed including bundles of hollow fibres 142 (122, 132) for supplying nutrients and gases to a cell development chamber (114), the fibres including a lumen 124 defined by membrane material 126. The membrane material has pores 128 with diameters in the range of 3.0 to 8.0 pm which connect the lumen 124 to the cell development chamber (114). Cell development materials (e.g. stem cells (170)) develop in the chamber (114) into developed cells 172 (e.g. red blood cells), which are smaller and/or more deformable than the development materials. Developed cells 172 are thus able to pass through pores 128 into the lumen (124) for collection. Independent claims to the hollow fibres comprising pores with diameters in the range of 3.0 to 8.0 µm, methods of manufacturing these hollow fibres, and methods of harvesting cells, are included.

Description

HOLLOW FIBRE PERFUSION BIOREACTOR

Field of the Invention

The invention relates to hollow fibre perfusion bioreactors for culturing and using S metabolisms and/or for maintaining micro-organisms, particularly cells, viruses or bacteria. In particular, the invention relates to the hollow fibres used within such bioreactors. The invention further relates to methods of harvesting cultured micro-organisms through the use of such fibres.

Background to the Invention

A typical hollow fibre perfusion bioreactor system 10, as disclosed in US 5,516,691, is illustrated in FIGS 2, 3A and 3B. FIG 1 shows another typical system including nutrient and gas supplies. A housing 12 defines a bioreactor chamber 14 within which is a tightly-packed interwoven network 16 of three independent capillary systems 20, 30, 40, as described below.

A first independent hollow fibre membrane system 20 is formed by a first bundle of hollow fibres 22. A second independent hollow fibre membrane system 30 is formed by a second bundle of hollow fibres 32. A third independent hollow fibre membrane system 40 is formed by a third bundle of hollow fibres 42. Each of the fibres 22, 32, 42 is formed of a tubular membrane 24 having a lumen 26 therethrough.

Various arrangements of the three membrane systems 20, 30, 40 are known, including the respective bundled fibres being interwoven at 60° to each other along a common plane (as alluded to in FIG 1) or orthogonally (such as shown in cross-section in FIG 2 and schematically in FIG 3).

FIGS. 3A and 3B show, in detail, individual hollow fibres 22, 32, 42 of the three independent hollow fibre membrane systems 20, 30, 40. As indicated by the arrows, medium 60 is supplied by perfusion through the membrane 24 of the hollow fibre 22 of the first hollow fibre membrane system 20 from the lumen 26 through the fibre to the exterior of the fibre inside the chamber 14. The cells 70 are located in the cavities of the close packed network 16 and/or adhere to the hollow fibres 22, 32, 42. A material exchange takes place between the cells 70 and the medium 60.

Gases, such as oxygen, are supplied through the second independent hollow fibre membrane system 30 in a similar manner.

The third independent hollow fibre membrane system 40, represented by an individual fibre 42, is used for the medium outflow. Thus, the medium 60 is removed after exchange with the cells 70. FIG 3B shows the perfusion of the cells 70 and the medium flow along the hollow fibres 42.

The individual hollow fibres 22, 32, 42 are components of the close packed network 16 in the bioreactor chamber 14 and are interwoven in such a way that at each point within the chamber 14, there are similar supply conditions. At each point of the chamber 14, the cells 70 are uniformly supplied and consequently material exchange is optimum and controllability thereof is ensured.

Thus, for the operation of the bioreactor 10, medium 60 is supplied through a medium inflow head 62 into the lumens 26 of the hollow fibres 22 of the first independent hollow fibre membrane system 20. This medium 60 now passes through the membranes of the hollow fibres 22 and there is a material exchange with the cells 70, which are either located in the cavities and/or adhere to the fibres. The cells 70 are supplied with oxygen during the material exchange. The oxygen passes through the oxygen inflow head 64 into the lumens of the hollow fibres 32 of the second independent hollow fibre membrane system 30 and is removed via the oxygen outflow head 66. The hollow fibres 32 of the independent system 30 for the oxygen supply may be formed by U-shaped hollow fibres 32, as shown in FIG 2. The conveying away of the material takes place via the third independent hollow fibre membrane system 40 through the fibres 42 indicated by the circles in FIG 2. The lumens 26 of these hollow fibres 42 then issue into a medium outflow head 68, from where the medium can be removed.

Cells 70 can be supplied through the access 72 into the bioreactor chamber 14.

Whereas the bioreactor is suitable for culturing cells, and for the exchange of materials with cells within the chamber under the supply of suitable gases, it is not especially suitable for the harvesting of cultured cells. The primary objective of the system of FIG 2 is the material exchange, which is facilitated by the presence of the cells; the development of those cells is not of primary importance and harvesting of the cells once developed is not particularly contemplated.

It would be advantageous to be able to harvest the developed cells in a simple and efficient manner. One option would be to evacuate the whole contents of the bioreactor chamber 14 once the cells 70 had developed and to filter those contents so as to separate the desired developed cells from the remainder of the contents. This would require a separate evacuation and filtration system.

Membranes having precisely defined pore diameters are known, for example, from US 3,303,085 and US 4,491,012, relating to track etched membrane and, specifically, HemafilTM. It is known that such membranes can be used to determine the deformability of red blood cells, by drawing a sample of blood across the membrane by capillary action.

Summary of the Invention

The invention relates to the concept of fabricating at least some of the hollow fibres within a hollow fibre perfusion bioreactor from such a membrane material so as to enable the passage of developed cells from the bioreactor chamber into the lumens of those fibres for extraction. It is particularly applicable to the harvesting of red blood cells which are smaller in size (at 7-8 pm in diameter) than the stem cells from which they develop (10-15 pm). As mentioned above, red blood cells are also known to be deformable, assisting in their passage through pores having a relatively small diameter.

According to a first aspect of the invention, there is provided a hollow fibre comprising a lumen defined by surrounding membrane material, the membrane material having pores of a predetermined maximum diameter therethrough connecting the lumen with the exterior of the fibre, wherein said predetermined pore diameter is in the range of 3.0 to 8.0 pm.

Such a fibre could be used to filter developed cells, such as red blood cells, from the larger materials from which they develop.

The pores are preferably substantially uniformly sized. This would ensure that the filtration is even across the surface area of the fibre.

Optionally, the membrane material is track etched membrane (TEM). This, as disclosed in US 3,303,085, known to have pores of the correct size and distribution.

However, it is anticipated that other membranes and methods of producing membranes could be employed.

The predetermined maximum pore diameter may be 4.7±0.2 pm. Alternatively, the predetermined maximum pore diameter may be 3.0±0.2 pm. 4.7±0.2 pm is the known pore size of Hema-filTM membrane material and is suitable for the filtration of developed red blood cells. It is anticipated that other cell types may be developed and harvested and that the maximum pore diameter of 3.0±0.2 pm would be appropriate.

According to a second aspect of the invention, there is provided a bioreactor including at least one hollow fibre according to the first aspect.

The bioreactor may further include a cell development chamber in which the at least one hollow fibre is housed. Although it is anticipated that a cell development chamber would be desirable, it is not thought to be necessary. For example, cells may develop directly on the exterior surface of a hollow fibre. Moreover, there may be lab-on-a-chip applications that do not require a chamber as such.

The bioreactor may further include means for urging developed cells of a predetermined size and/or deformability through the pores and into the lumen. The urging means may comprise pressurisation means to assert a pressure differential across the membrane material. The pressurisation means may comprise a negative pressure source connected to the lumen. Alternatively, or additionally, the pressurisation means may comprise means to raise the pressure of the exterior of the fibre. Whereas the passage of developed cells from the exterior of the fibre to the lumen within might be achieved by diffusion or other unassisted mechanism, it is preferable to assist the passage, particularly where deformation of the developed cells is required. To that end, these urging means may be used.

The urging means may be adapted to apply a pulsatile pressure differential across the membrane material. Such a pulsatile pressure differential might imitate the natural mechanism at work in the passage of red blood cells into the blood supply.

The bioreactor may further include a cell collection device to which the lumen is connected. This provides a dedicated repository for the developed cells to be delivered and stored.

The bioreactor may further include a nutrient source and/or a gas source for respectively supplying nutrients and/or gases to the exterior of the fibre. The lumen of the hollow fibre may be connected to the nutrient source. Where nutrients and or gases are supplied to the bioreactor, these may conveniently be supplied through the lumen, much as described with reference to the prior art arrangement of US 5,516,691.

The bioreactor may include bundles of said hollow fibres. A large number of fibres would be needed to process significant quantities of cells. Smaller scale, experimental applications can be envisaged in which individual fibres are instead used.

The bundles of said hollow fibres may be formed by sealing a corrugated sheet of the membrane material to another sheet of the membrane material. This is one expedient way to manufacture the bundles of fibres.

According to a third aspect of the invention, there is provided a method of manufacturing hollow fibres, comprising the steps of: providing a first, corrugated sheet of membrane material having a series of peaks and troughs; providing a second sheet of the membrane material; sealingly connecting said first sheet along each trough thereof to said second sheet, thereby defining a series of parallel lumens between the troughs; wherein the membrane material has pores of a predetermined maximum diameter formed therethrough, wherein said predetermined pore diameter is in the range of 3.0 to 8.0 pm.

Preferably, the pores are substantially uniformly sized.

Optionally, the membrane material is track etched membrane (TEM).

The predetermined maximum pore diameter may be 4.7±0.2 pm. Alternatively, the predetermined maximum pore diameter may be 3.0±0.2 pm.

The second sheet may be substantially flat.

The method according to the third aspect may further comprise the step of separating along the sealed troughs to form individual fibres.

According to a fourth aspect of the invention, there is provided a method of harvesting developed cells from a bioreactor, comprising the steps of: providing within the bioreactor a hollow fibre comprising a lumen defined by surrounding membrane material, the membrane material having pores of a predetermined maximum diameter therethrough connecting the lumen with the exterior of the fibre, wherein said predetermined pore diameter is in the range of 3.0 to 8.0 pm; providing cell development materials in the bioreactor, exterior to the fibre; developing cells from those materials; and filtering the developed cells from the development materials, wherein the filtration step comprises urging the developed cells from the exterior of the hollow fibre to the lumen through the pores.

Advantages of this method have been covered by the above discussion of the associated bioreactor.

Preferably, the pores are substantially uniformly sized.

Optionally, the membrane material is track etched membrane (TEM).

The method may further comprise the step of passing the developed cells through the lumen to a cell collection device.

The developed cells may comprise red blood cells. As discussed above, this invention has particular application to red blood cells, but other cells are also though to be suitable for development and harvesting in this manner.

The development materials may comprise stem cells. The development materials may further comprise nutrients and/or gas(es). The method may further comprise the step of supplying said nutrients and/or gas through the lumen of the hollow fibre.

The bioreactor may be provided with multiple said hollow fibres. Optionally, different fibres may have different functions, selected from one or more of: supplying said development materials; and filtering the developed cells from the development materials. Where the method includes the step of supplying said nutrients and/or gas through the lumen of the hollow fibre, selected fibres may supply said nutrients and selected other fibres may supply said gas(es). In one arrangement, it is anticipated that a first bundle of fibres would supply nutrients, a second bundle of fibres would supply gas(es) and a third bundle of fibres would be used to extract the developed cells. Only the third bundle of fibres would need to have the defined maximum pore diameter. In other arrangements, the function of each fibre may be dual: such as both supplying nutrients and extracting developed cells.

The method according to the fourth aspect may further comprise the step of asserting a pressure differential across the membrane material. The pressure differential may be pulsatile. As discussed above, this could be beneficial as imitating the natural process of, for example, red blood cells passing into the bloodstream.

Brief Description of the Drawings

The invention will be described, by way of example, with reference to the accompanying drawings, in which: FIG. I is a schematic diagram of a known hollow fibre bioreactor and associated equipment: Fig. 2 is a cross-sectional view through a known hollow fibre bioreactor chamber, showing interwoven bundles of fibres; Fig. 3A is a detail view of interwoven fibres and cell development materials within the known bioreactor chamber; Fig. 36 is a corresponding detail view of the interwoven fibres, showing medium perfusing through the fibres; Fig. 4 corresponds to Figure 3A, but shows the known fibres replaced by fibres having pores of a predetermined maximum diameter according to the invention; Fig. 5 is a cross-sectional view through a single fibre, showing the passage of red blood cells through the pores and into the lumen of the fibre; and Fig. 6 shows one method of forming a bundle of hollow fibres according to the invention.

Detailed Description

With reference to FIGS 4 and 5 in particular, in order to develop and harvest red blood cells 172, in a simple and efficient manner, without the need for external bioreactor chamber evacuation and filtration equipment, at least some of the hollow fibres 122, 132, 142 within a hollow fibre perfusion bioreactor chamber 114 are fabricated from a membrane material 126 that has pores 128 of a predetermined maximum diameter therethrough connecting the lumen 124 with the exterior of the fibre (i.e. the interior of the bioreactor chamber 114 in which the cells 172 are developed). That predetermined diameter is in the range of 3.0 to 8.0 pm. Those precisely defined pores 128 enable the passage of developed cells 172 from the bioreactor chamber 114 into the lumens 124 of those fibres for extraction.

Red blood cells 172 are developed within the bioreactor chamber 114 through the use of suitable development materials. Those materials include stem cells, nutrients and gases such as oxygen and carbon dioxide. Developed red blood cells 172 are smaller in size (at 7-8 pm in diameter) than the stem cells from which they develop (10-15 pm). As mentioned above, red blood cells 172 are also known to be deformable, assisting in their passage through pores 128 having a relatively small diameter.

FIG 4 corresponds to FIG 3A, but in which the hollow fibres of the prior art system are replaced by hollow fibres 122, 132, 142 fabricated in accordance with the invention. The fibres 122, 132, 142 each comprise a lumen 124 defined by surrounding membrane material 126 through which are defined the pores 128 of maximum predetermined diameter.

FIG 5 shows an enlarged cross-sectional view of one such fibre 142. In the description which follows, the fibre 142 is taken as representative of any of the fibres within the bioreactor chamber 114. As described with reference to the prior art, it will be appreciated that different fibres or bundles of fibres may perform different functions or combinations of functions. One such function is to extract the developed cells 172 from the chamber 114. The fibres 122 and 132 could each also carry out the cell extraction function if fabricated in the same manner.

The pores 128 are preferably substantially uniformly sized so as to ensure that filtration is even across the surface area of the fibre 142.

One known way of defining pores having a precise predetermined maximum diameter within a membrane is the track etching technique, as disclosed for example in US 3,303,085. The resultant membrane material is track etched membrane (TEM). TEM is known to have pores of the correct size and distribution.

The predetermined maximum pore diameter may be 4.7±0.2 pm, which is the known pore size of Hema-fiITM membrane material and is suitable for the filtration of developed red blood cells. The density of the pores through the material is in the range 4.0x105 (±15%) pores/cm2. Alternatively, the predetermined maximum pore diameter may be 3.0±0.2 pm, which is another known pore size and which may be suitable for the filtration of other types of developed cells. There need not be a minimum required pore diameter.

The bioreactor includes means for urging the developed cells of a predetermined size and/or deformability through the pores and into the lumen 124. The urging means (not shown) may comprise pressurisation means to assert a pressure differential across the membrane material 126. The pressurisation means may comprise a negative pressure source connected to the lumen 124. Alternatively, or additionally, the pressurisation means may comprise means to raise the pressure of the exterior of the fibre 142 (i.e. the interior of the bioreactor chamber surrounding the fibres). Whereas the passage of developed cells from the exterior of the fibre 142 to the lumen 124 within might be achieved by diffusion or other unassisted mechanism, it is preferable to assist the passage, particularly where deformation of the developed cells 172 is required. The skilled person will appreciate that any suitable pump or vacuum source may be employed as the urging means. Another possibility could be to employ a chemical gradient across the membrane material 126 to urge the developed cells 172 through the pores 128 and into the lumen 124.

The urging means may be adapted to apply a pulsatile pressure differential across the membrane material 126. Such a pulsatile pressure differential might imitate the natural mechanism at work in the passage of red blood cells into the blood supply.

The bioreactor includes a cell collection device (not shown) to which the lumen 124 is connected. This provides a dedicated repository for the developed cells 172 to be delivered and stored.

The bioreactor further includes a nutrient source and a gas source for respectively supplying nutrients and gases to the exterior of the fibre 142. These, together with the stem cells, which are supplied separately to the exterior of the fibre 142, comprise the cell development materials. The lumen 124 of the hollow fibre 142 may be connected to the nutrient source and/or the gas source as well as to the cell collection device.

The bioreactor includes bundles of such hollow fibres 122, 132, 142. The bundles may each perform a different function or a combination of functions. For example, one bundle, comprising the fibres 142 may be for cell extraction only, with a second bundle of the fibres 132 being for nutrient supply only and with a third bundle being for gas supply only. The individual bundles may be connected via separate manifolds to respective inputs and outputs, e.g. a manifold connecting a single nutrient source to each of the nutrient-supplying fibres 132, or a manifold connecting each cell extraction fibre 142 to the cell collection device. A large number of fibres would be needed to process significant quantities of cells 172. However, smaller scale, experimental applications can be envisaged in which individual fibres are instead used.

Those bundles of hollow fibres may be formed by sealing a corrugated sheet of the membrane material 126a to another sheet of the membrane material 126b, as shown in FIG 6. This is one expedient way to manufacture the bundles of fibres.

The method of manufacturing hollow fibres thus comprises the steps of: providing a first, corrugated sheet 126a of membrane material having a series of peaks 127 and troughs 129; providing a second sheet 126b of the membrane material; and sealingly connecting said first sheet 126a along each trough 129 thereof to said second sheet 126b, thereby defining a series of parallel lumens 124 between the troughs.

The membrane material 126a, 126b has pores 128 of a predetermined maximum diameter in the range of 3.0 to 8.0 pm formed therethrough, as described above.

The second sheet 126b may be substantially flat. This would facilitate manufacture.

The method may further comprise a step of separating along the sealed troughs 129 to form individual fibres 142.

Although the invention has been described in terms of the development and harvesting of red blood cells, it is anticipated that the concepts are equally applicable to the development and harvesting of other types of cell or micro-organism, provided that the developed cell or micro-organism is smaller than and/or more deformable than the initial development material (i.e. stem cells), since it is this which enables the discriminatory filtration of developed cells over the development materials.

A cell development chamber 114 is not thought to be necessary. For example, cells 172 may develop directly on the exterior surface of a hollow fibre 142. Moreover, there may be lab-on-a-chip applications that do not require a chamber as such.

Whereas the invention has been described in terms of hollow fibres, it will be understood that the principles and functionality may equally be provided by other hollow forms, provided that the forms include a lumen bounded by the membrane material with the pores of the maximum predetermined diameter. For example, the lumen may be defined between two sheets of such membrane material.

Claims

CLAIMS1. A hollow fibre comprising a lumen defined by surrounding membrane material, the membrane material having pores of a predetermined maximum diameter therethrough connecting the lumen with the exterior of the fibre, wherein said predetermined pore diameter is in the range of 3.0 to 8.0 pm.
2. The fibre according to claim 1, wherein the pores are substantially uniformly sized.
3. The fibre according to claim 1 or claim 2, wherein the membrane material is track etched membrane (TEM).
4. The fibre according to claim 1, 2 or 3, in which the predetermined maximum pore diameter is 4.7±0.2 pm.
5. The fibre according to any of claims 1, 2 or 3, in which the predetermined maximum pore diameter is 3.0±0.2 pm.
6 A bioreactor including at least one hollow fibre according to any preceding claim.
7 The bioreactor according to claim 6, further including a cell development chamber in which the at least one hollow fibre is housed.
8. The bioreactor according to claim 6 or claim 7, further including means for urging developed cells of a predetermined size and/or deformability through the pores and into the lumen.
9. The bioreactor according to claim 8, wherein the urging means comprises pressurisation means to assert a pressure differential across the membrane material.
10. The bioreactor according to claim 9, wherein the pressurisation means comprises a negative pressure source connected to the lumen.
11. The bioreactor according to claim 9 or claim 10, wherein the pressurisation means comprises means to raise the pressure of the exterior of the fibre.
12. The bioreactor according to any of claims 8 to 11, wherein the urging means is adapted to apply a pulsatile pressure differential across the membrane material.
13. The bioreactor according to any of claims 6 to 12, further including a cell collection device to which the lumen is connected.
14. The bioreactor according to any of claims 6 to 13, further including a nutrient source and/or a gas source for respectively supplying nutrients and/or gases to the exterior of the fibre.
15. The bioreactor according to claim 14, wherein the lumen of the hollow fibre is connected to the nutrient source.
16. The bioreactor according to any of claims 6 to 15, including bundles of said hollow fibres.
17. The bioreactor according to claim 16, wherein the bundles of said hollow fibres are formed by sealing a corrugated sheet of the membrane material to another sheet of the membrane material.
18. A method of manufacturing hollow fibres, comprising the steps of: providing a first, corrugated sheet of membrane material having a series of peaks and troughs; providing a second sheet of the membrane material; sealingly connecting said first sheet along each trough thereof to said second sheet, thereby defining a series of parallel lumens between the troughs; wherein the membrane material has pores of a predetermined maximum diameter formed therethrough, wherein said predetermined pore diameter is in the range of 3.0 to 8.0 pm.
19. The method according to claim 18, wherein the pores are substantially uniformly sized.
20. The method of claim 18 or claim 19, wherein the membrane material comprises track etched membrane (TEM).
21. The method according to claim 18, 19 or 20, in which the predetermined maximum pore diameter is 4.7±0.2 pm.
22. The method according to claim 18, 19 or 20, in which the predetermined maximum pore diameter is 3.0±0.2 pm.
23. The method according to any of claims 18 to 22, wherein the second sheet is substantially flat.
24. The method according to any of claims 18 to 23, further comprising the step of separating along the sealed troughs to form individual fibres.
25. A method of harvesting developed cells from a bioreactor, comprising the steps of: providing within the bioreactor a hollow fibre comprising a lumen defined by surrounding membrane material, the membrane material having pores of a predetermined maximum diameter therethrough connecting the lumen with the exterior of the fibre, wherein said predetermined pore diameter is in the range of 3.0 to 8.0 pm; providing cell development materials in the bioreactor, exterior to the fibre; developing cells from those materials; and filtering the developed cells from the development materials, wherein the filtration step comprises urging the developed cells from the exterior of the hollow fibre to the lumen through the pores.
26. The method according to claim 25, wherein the pores are substantially uniformly sized.
27. The method according to claim 25 or claim 26, in which the predetermined maximum pore diameter is 4.7±0.2 pm.
28. The method according to claim 25 or claim 26, in which the predetermined maximum pore diameter is 3.0±0.2 pm.
29. The method according to any of claims 25 to 28, further comprising the step of passing the developed cells through the lumen to a cell collection device.
30. The method according to any of claims 25 to 30, wherein the developed cells comprise red blood cells.
31. The method according to any of claims 25 to 30, wherein the development materials comprise stem cells.
32. The method according to claim 31, wherein the development materials further comprise nutrients and/or gas(es).
33. The method according to claim 32, further comprising the step of supplying said nutrients and/or gas through the lumen of the hollow fibre.
34. The method according to any of claims 25 to 33, wherein the bioreactor is provided with multiple said hollow fibres.
35. The method according to claim 34, wherein different fibres have different functions, selected from one or more of: supplying said development materials; and filtering the developed cells from the development materials.
36. The method according to claim 35, when dependent on claim 33, wherein selected fibres supply said nutrients and selected other fibres supply said gas(es).
37. The method according to any of claims 25 to 36, further comprising the step of asserting a pressure differential across the membrane material.
38. The method according to claim 37, wherein the pressure differential is pulsatile.
39. The method according to any of claims 25 to 38, wherein the cells comprise viruses.