Source: http://patents.com/us-6632658.html
Timestamp: 2018-01-17 13:21:38
Document Index: 6864397

Matched Legal Cases: ['art 65', 'arts 62', 'art 65', 'art 65', 'art 65', 'art 65', 'art 65', 'art 65', 'art 65']

US Patent # 6,632,658. Bioreactor and method for fluidly suspending a product - Patents.com
United States Patent 6,632,658
Schoeb October 14, 2003
The method for the holding in flotation of a substance such as a tissue part in a bioreactor (61) is characterized in that the substance (73) is acted upon with a fluid; and in that the flow of the fluid acts counter to gravity in such a manner that the substance (73) is held in flotation. The bioreactor (61) with a container (62) for a substance (73) which is to be acted upon with fluid comprises a first flow chamber (66a) to which a flowing fluid can be supplied, with the first flow chamber (66a) being designed in such a manner that the fluid which flows upwardly therein has a lower speed with increasing height.
Inventors: Schoeb; Reto (Volketswil, CH)
Appl. No.: 09/655,203
Aug 18, 2000 [EP] 00810734
Current U.S. Class: 435/293.1 ; 435/1.2; 435/286.5; 435/295.1; 435/302.1; 435/403
Current International Class: C12M 1/04 (20060101); C12M 1/08 (20060101); C12M 003/02 (); C12N 005/00 ()
Field of Search: 435/3,383,393,403,286.5,289.1,293.1,295.1,295.2,299.1,1.1,1.2,302.1
4978616 December 1990 Dean
5445073 August 1995 Gilwood
5501971 March 1996 Freedman et al.
6100618 August 2000 Schoeb et al.
0472223 Feb., 1992 EP
WO 86/00636 Jan., 1986 WO
Ver, Leah May B., "Design Criteria of a Fluidized Bed Oyster Nursery", Aquacultural Engineering, vol. 14, No. 3, pp. 229-249, (1995). .
Maruyama, Toshiro, et al., "Liquid fluidization in conical vessels", The Chemical Engineering Journal, 46, 15-21 (1991)..
1. A bioreactor comprising: a container comprising a first flow chamber; at least one scaffold having cells deposited on it for growing a tissue part, wherein the scaffold is acted upon with fluid; and an apparatus for conveying the fluid, wherein the scaffold is arranged in the first flow chamber in such a manner that the fluid holds the scaffold in free flotation.
2. A bioreactor in accordance with claim 1 further comprising: a sensor; and a regulation apparatus, wherein the fluid conveying apparatus is connected to the first flow chamber, and wherein the regulation apparatus is connected to the fluid conveying apparatus and to the sensor in such a manner that the position of the scaffold may be measured and regulated.
3. A bioreactor in accordance with claim 1, wherein the first flow chamber widens upwardly.
25. A method for floating a scaffold for growing a tissue part, in a bioreactor, the method comprising: providing at least one scaffold having cells deposited on it; acting upon said scaffold with fluid, wherein the fluid holds the scaffold in free flotation and wherein the fluid flows in a direction counter to gravity when a density of said scaffold is greater then a density of the fluid, and in a direction counter to buoyancy when a density of said scaffold is less then a density of the fluid.
26. A method in accordance with claim 25, wherein the fluid has an increasingly lower flow speed in the direction counter to gravitation.
33. A method for floating a scaffold for growing a tissue part in a bioreactor, the method comprising: providing one or more tissue parts on at least one scaffold; and acting upon said scaffold and tissue parts with fluid, wherein the fluid holds the scaffold and tissue parts in free flotation and wherein the fluid flows in a direction counter to gravity when a density of said scaffold including tissue parts is greater then a density of the fluid, and in a direction counter to buoyancy when a density of said scaffold including tissue parts is less then a density of the fluid.
34. A method in accordance with claim 33, wherein the scaffold and tissue parts are acted upon with at least one fluid jet.
The artificial production of tissue material, designated in English as "tissue engineering", is increasingly gaining in importance in order to produce biological substitutes for damaged tissue or damaged organs. Artificial tissue material can be produced in that cell cultures in vitro are deposited at or in a tissue carrier, also termed a matrix. The tissue carrier consists for example of a synthetic polymer or of a biological material such as collagen. A tissue carrier of this kind is also designated as a "scaffold". The cells are sown out onto the tissue carrier and begin to multiply if the environmental parameters are physiologically favorable. The tissue carrier can be designed in such a manner that the latter disintegrates with time, so that after a certain time only the tissue part which is formed from the cells is present. The tissue carrier and/or the tissue carrier which is formed on it is designated as "substance" in the following. The conditions which are required for the cell growth are produced in a bioreactor, within which the required oxygen and a nutrient medium are supplied to the substance and within which the substance remains from several days to weeks until the desired size has been reached. The geometrical shape which the artificially produced tissue material assumes during growth is substantially influenced through the measures by means of which the substance is held in the bioreactor.
Thus in the following the term "substance" will be understood to mean both the tissue carrier per se and the tissue carrier with cells deposited on it, or, if the tissue carrier is designed to be decomposable, the artificially produced cell culture or the artificially produced tissue part respectively.
FIGS. 2a, 2b are longitudinal sections through further exemplary embodiments of bioreactors;
FIGS. 3a-3d are longitudinal sections through further exemplary embodiments of bioreactors;
FIG. 4 is a longitudinal section along the line B--B through a further bioreactor with a magnetically coupled vaned wheel;
FIG. 5 is a section through FIG. 4 along the line A--A;
FIG. 7 is a longitudinal section along the line D--D through a further bioreactor with a magnetically coupled vaned wheel;
FIG. 8 is a section through FIG. 7 along the line C--C;
The bioreactor 61 which is illustrated in FIG. 1 comprises a container 62 which has an opening 62c at the top, which can be closed by a closure 63. Arranged in the inner space of the container 62 is a flow guiding means 66 having the shape of a truncated cone which is formed as a hollow body, the cross-sectional area of which increases upwardly. The inner space of the container 62 is largely filled with a liquid 64, which is set into a circulation flow by the vaned wheel 65c of the motor 65, so that the liquid 64 has the flow direction which is illustrated by the arrows 64a, 64b, 64c. The liquid which flows in the direction of the arrows 64a enters from below with relatively high flow speed via the entry opening 66d into the inner space 66a of the flow guiding means 66, flows upwards in the inner space 66a with decreasing speed, and leaves the inner space 66a at the top again with relatively low flow speed through the outlet opening 66e, as illustrated by the arrows 64b. In the inner space 66a the flow speed decreases as a result of the upwardly widening cross-section. The inner space 66a forms the first flow chamber. If the diameter of the outlet surface 66e is for example twice as great as the diameter of the inlet surface 66d, then the speed at the outlet surface 66e corresponds to one-fourth of the speed at the inlet surface 66d. The buoyancy force which is caused by the flow speed still amounts at the outlet surface 66e to one-sixteenth of that at the inlet surface 66d. The substance 73, which is arranged in the inner space 66a, is held in an equilibrium position through the upwardly flowing liquid, with the suspension level, i.e. the equilibrium position of buoyancy force and gravitation, setting in by itself as a result of the weight and the working surface of the substance.
Arranged at the bottom in the inner space 62e is a ring-shaped distributor 67, through which air or oxygen is led in for the gasification of the liquid 64, which forms air bubbles 68 within the liquid 64 which have the tendency to rise. Through the liquid, which flows downwardly in the direction 64c, the rising of the air bubbles 68 is delayed or prevented, which furthers the gas exchange to the liquid 64.
FIG. 1a shows a perspective illustration of the flow guiding means 66 with inner space 66a.
FIG. 2a shows schematically a longitudinal section through a further exemplary embodiment of a bioreactor 61, which differs with respect to the example which is illustrated in FIG. 1 in that the flow guiding means 66 is arranged in reverse, which means with a downwardly widening cross-section. The pump 65 comprising the iron stator 65a and the rotatable part 65b with vaned wheel 65c causes a flow in the direction 64a, 64b in the liquid 64. The inner space 66a, in which the liquid flows upwards, and in which the substance 73 is held, is located between the flow guiding means 66 and the outer wall of the container 62.
FIG. 2b shows schematically a longitudinal section through a further exemplary embodiment of a bioreactor 61, which differs with respect to the example which is illustrated in FIG. 2a in that the flow guiding means 66 is designed to be seated at the top and that the fluid pump 74 is arranged outside the container 62, with the pump 74 being connected in a fluid conducting manner to the inner space of the container 62 via lines 76a, 76b. The fluid which flows in the direction 64a enters from below into the inner space 66a and flows around the substance 73.
FIG. 3a shows schematically a longitudinal section through a further exemplary embodiment of a bioreactor 61, which likewise has a fluid pump 74 which is arranged outside the container 62 and which is connected to the inner space in a fluid guiding manner via lines 76a, 76b. The flow guiding means 66 is designed to be upwardly widening only on the one inner side of the container 62. The substance 73 is held in suspension through the liquid which circulates in the direction 64a, 64b, 64c in the inner space 66a.
FIG. 3b shows schematically a longitudinal section through a further exemplary embodiment of a bioreactor 61, which likewise has a fluid pump 74 which is arranged outside the container 62 and which is connected to the inner space in a fluid guiding manner via lines 76a, 76b. Along a section 62f the container 62 has an upwardly widening container wall 62d. Along this section 62f a flow develops with a flow speed which decreases upwardly, so that the inner space 66a is formed to hold the substance 73 in suspension along this section 62f.
FIG. 3c shows schematically a longitudinal section through a further exemplary embodiment of a bioreactor 61, which likewise has a fluid pump 74 which is arranged outside the container 62 and which is connected to the inner space in a fluid guiding manner via lines 76a, 76b. The line 76a opens into a section 62f in the container 62 which widens upwardly. A cylindrically designed container section 62 is arranged afterwards, within which a linear flow 64a develops and within which the substance 73 is arranged. The vertical position of the substance 73 is monitored by a sensor 85. A regulation apparatus 86 is connected in a signal conducting manner via an electrical line 85a, 86a to the sensor 85 and to the pump 74. The speed of rotation of the pump 74 is regulated in such a manner that the substance 73 remains in the region of the sensor 85.
FIG. 3d shows schematically a longitudinal section through a further exemplary embodiment of a bioreactor 61, which likewise has a fluid pump 74 which is arranged outside the container 62 and which is connected in a fluid guiding manner via lines 76a, 76b to the inner space. A plurality of, for example three, nozzles 70a, 70b open with orientation onto the substance 73 inside the container 62, with the flow direction which is illustrated by 64a having a flow speed which reduces in the upward direction, so that the substance 73 is supported by this flow and automatically finds an equilibrium position.
In all bioreactors 61 which are illustrated in FIGS. 1 to 3d the substance 73 is held in a state of suspension by means of the same method, namely in that the substance 73 is acted upon with a fluid, the flow of which acts counter to the gravitational force acting on the substance 73 in such a manner that the substance 73 is held in suspension. In the exemplary embodiments in accordance with FIGS. 1, 2a, 2b, 3a, 3b and 3d the fluid has a lower flow speed in the inner space 66a with increasing height. In the exemplary embodiment in accordance with FIG. 3c the speed of the fluid is regulated with a sensor 85 in dependence on the position of the substance 73.
In the exemplary embodiment in accordance with FIG. 1 a downwardly flowing flow 64c is produced within the container 62, with a gaseous fluid such as air or oxygen being led into this flow 64c. The flow speed of the flow 64c can be chosen in such a manner that the gaseous fluid which is introduced is slowed down or even no longer rises in the container 62.
FIG. 4 shows a further exemplary embodiment of a bioreactor 61 in a longitudinal section along the line B--B in accordance with FIG. 5. Although otherwise designed similarly to the bioreactor 61 illustrated in FIG. 1, in the bioreactor 61 in accordance with FIG. 4, the pump 65 is arranged at the bottom in the region of the entry opening 66d of the flow guiding means 66. A vaned wheel 65c is rotatably arranged within the container 62 on a track bearing 65i, with the track bearing 65i lying on the container wall 62d. A plurality of permanent magnets 65h which are distributed around the periphery is cast into the vaned wheel 65c, which consists of a plastic. Arranged outside the container 62 is a magnetic coupling which is journalled so as to be rotatable in the direction 65e and which comprises two bearings 65f and a ring-shaped permanent magnet 65g. The rotatable shaft 65d is driven by a non-illustrated motor. A stand apparatus 75 forms a gap pot 75a which is designed to be cylindrical and which is arranged to extend between the two permanent magnets 65g, 65h. The container wall 62d forms a gap pot section 62a at the gap pot 75a. The magnetic coupling, which comprises the permanent magnets 65h, 65i, causes the rotational motion of the rotatable shaft 65d to be transmitted to the vaned wheel 65c and the vaned wheel 65c to be held with respect to a tilting motion. The vaned wheel 65c is thus passively held magnetically.
The container 62 can, as illustrated in FIG. 4, have additional openings 63a, 63b, for example for measurement probes.
FIG. 5 shows a cross-section along the line A--A in accordance with FIG. 4. Arranged in the center is the rotatable shaft 65d to which four spaced-apart permanent magnets 65g are secured. The container wall 62d of the container 62 forms a gap pot section 62a. The gap pot 75a is arranged between the gap pot section 62a and the rotatable shaft 65d with permanent magnet 65g. The gap pot section 62a is surrounded by the vaned wheel 65c, within which four permanent magnets 65h are arranged, with their polarization, illustrated by arrows, being oriented to be matched to that of the permanent magnets 65g. The flow guiding means 66 is connected via fluid guiding parts 62d to the outer wall of the container 62. The flow chamber 62e, which widens downwardly, is arranged between the flow guiding means 66 and the outer wall of the container 62. In addition the ring-shaped distributor 67 is shown.
FIG. 6 shows a longitudinal section through a further exemplary embodiment of a bioreactor 61. In contrast to the bioreactor 61 which is illustrated in FIG. 1, in the bioreactor 61 in accordance with FIG. 6 the pump 65 is arranged in the closure 63 and is designed as a centrifugal pump. The pump 65 is designed as a split tube or canned motor and comprises the firmly arranged iron stator 65a and the contact-free, rotatably journalled, rotatable part 65b, which is designed as a permanent magnet and which is firmly connected to the vaned wheel 65c. The iron stator 65a comprises a soft iron 65k which is surrounded by a plurality of coils 65l. The coils 65l are arranged and can be excited in such a manner that the rotatable part 65b is driven and held without contact. The closure 63 has a gap pot section 63e, which is arranged in the gap between the iron stator 65a and the permanent magnet 65b.
An arrangement of this kind comprising a stator and a rotor which is held and driven with magnetically acting forces is also termed a temple motor and is known to the skilled person, for example from the specification WO 96/31934, in particular from its FIG. 12.
FIG. 7 shows a further exemplary embodiment of a bioreactor 61 in a longitudinal section along the line D--D in accordance with FIG. 8. In contrast with the bioreactor 61 which is illustrated in FIG. 4 the pump 65 has a completely magnetically journalled and driven rotatable part 65b with vaned wheel 65c. The bearingless drive of the pump 65 is illustrated in detail in cross-section along the section line C--C which is illustrated in FIG. 8. The method of functioning of a drive of this kind is for example disclosed in the specification WO 98/59406. The iron stator 65a is designed as a cross-shaped sheet metal package 65k, at the arms of which coil 65l are arranged. Through a corresponding excitation of the coils 65l a rotating magnetic field can thereby be produced. The rotatable part 65b comprises four permanent magnets 65h which are arranged in the peripheral direction, with two adjacent permanent magnets 65h in each case being polarized in opposite directions. These permanent magnets 65h are cast in or encapsulated in the vaned wheel 65c or in the pump blades 65c respectively. Sensors 65m are arranged in the stator which measure the position of the permanent magnets 65h. Electronic components 75b are arranged in the stand apparatus 75, comprising an electrical lead 75d for the coils 65l of the motor and with an electrical lead 75c for the heater 69. In addition electrical lines are arranged which connect the sensors 65m to the electronic components 75b. The coils 65l are excited in such a manner that the rotatable part 65 with pump blades 65c is held and driven without contact. The pump 65 forms an axial pump. The gap pot 75a and the gap pot section 62a of the container wall 62d are arranged between the iron stator 65a and the rotatable part 65b.
The stand apparatus 75 and the heater 69 form a firm support and holder into which the container 62 can be introduced. This arrangement has the advantage that the container 62 can be placed very simply onto the stand apparatus 75 with the heater 69, and the axial pump 65 can subsequently be operated immediately without the need for additional manipulations. The container 62 with rotatable part 65b and pump blades 65c is designed as a once-only (disposable) product, whereas the expensive components of the stand apparatus 75 and the heater 69 can be used as often as desired. In addition the stand apparatus 75 and the heater 69 need not be sterile, so that no laborious cleaning process is required. Advantages of this arrangement are the facts that the inner space of the container 62 can be kept sterile without problem and that the stand apparatus 75 can be operated without a laborious cleaning process and thus economically.
In the container 62 in accordance with FIG. 7 the inlet and outlet lines 67a, 67b for gases such as O.sub.2, CO.sub.2, N.sub.2, pass through the closure 63b, with the inlet line 67a being connected in a fluid guiding manner to the ring-shaped distributor 67. The inlet and outlet lines 77a, 77b for the nutrient medium pass through the closure 63a. In addition, probes with probe heads 72a, for example for the measurement of temperature or pH value, pass through the closure 63d.
FIG. 9 shows schematically a longitudinal section through a further exemplary embodiment of a bioreactor 61, which likewise has a fluid pump 74 which is arranged outside the container 62 and which is connected in a fluid guiding manner to the inner space via lines 76a, 76b. The line 76b opens into the section of the flow guiding means 66 which widens upwardly. The fluid is conducted to the fluid pump 74 via the lines 76a which are arranged in the base region of the container 62, so that the fluid has the flow behavior which is indicated by the arrows 64a, 64b, 64c.
In FIG. 10 a further exemplary embodiment of the bioreactor 61 in accordance with the invention is illustrated in which cells can be cultivated. One recognizes the reaction container 62, which is surrounded here by a further vessel 84 which can, for example, contain water in order, for example, to be able to hold the reaction container 62 at a desired temperature. Arranged in the reaction container is a hollow body 66 in the shape of truncated circular cone which forms the flow guiding means 66 and which subdivides the container 62 into an upper chamber 79a and a lower chamber 79b. The jacket of the hollow body 66, in the shape of a truncated circular cone, is connected at its upper end to the wall of the reaction container 62 and tapers towards the lower end of the reaction container. The upper and lower end surfaces of the hollow body 66 are made permeable to gas and liquid, and indeed in such a manner that a membrane 80a or 80b which is designed to be permeable to gas and liquid is respectively arranged in the region of the upper and lower end surfaces. Cell carriers, for example consisting of plastic or ceramics, with cells 73, for which the membranes 80a and 80b are impermeable, can be arranged in the cavity which is enclosed between the membranes 80a and 80b. The infeed line 70 for the nutrient solution N opens in the lower chamber 79b into a ring-shaped distributor 68, which surrounds the hollow body 66. In the upper chamber 79a a suction device 81 is provided which is connected to an outlet line 71 which leads to the reservoir 82, where the nutrient solution N which is led off can be renewed or enriched with nutrients respectively. For the conveying of the nutrient solution N an expendable pump 65 or a pump with expendable parts is provided, which can for example be formed as a gear pump or as a centrifugal pump.
The nutrient solution N which is conveyed by the pump 65 out of the reservoir 82 enters into an oxygenator 83, where a gas such as for example oxygen can be admixed to the nutrient solution N or carbon dioxide removed from it. The nutrient solution N which is thus blended with oxygen or freed from carbon dioxide respectively then enters in the further course into the ring-shaped distributor 68, which is arranged in the lower chamber 79b. With the help of the expendable pump 65 and the suction device 81 a liquid flow is produced which is indicated by the arrows 64a, 64b in FIG. 5. In the region of the membrane 80b the flow speed is comparatively high; it then decreases upwardly as a result of the hollow body 66, which widens in the manner of a truncated cone. Through a suitable choice of the flow parameters or of the geometry of the hollow body 66 respectively a situation can be achieved in which the cells 73 or the substance 73 respectively are held in suspension in the region between the membranes 80b and 80a. This can favor the formation of a three-dimensional cells assembly of cells or tissue part respectively. In this exemplary embodiment the supply of nutrient solution N on the one hand and of gases such as e.g. oxygen on the other hand does not take place separately, but rather the nutrient solution N is blended with oxygen before it is introduced with the help of the infeed line 70 and the distributor 68 into the container 62.
FIG. 11 shows schematically, in a longitudinal section, a further embodiment of a bioreactor 61. This has a container 62 with a first flow chamber 66a and a second flow chamber 66f arranged above it. These two flow chambers 66a, 66f form a common inner space, which has a respective inlet opening 66d for the fluid at the top and at the bottom. A ring-like outlet opening 66e is arranged between the upper and lower inlet openings 66d by which the fluid can be supplied by means of a ring-like discharge passage 66g and the fluid line 76a to the pump 74. After the pump 74 the fluid line 76b leads through an oxygenator 83, whereupon the fluid line 76b divides into two branches which supply the fluid to the upper and/or lower inlet openings 66d. The quantity of fluid flowing in these branches can be set or controlled via the first and second clamping devices 87a, 87b. The clamping devices 87a, 87b permit the diameter of the fluid line 76b to be changed. The clamping devices 87a, 87b can for example be actuated by hand or can have electrical drive devices which are connected via non-illustrated control lines to a higher level regulating device. In an advantageous setting approximately the same quantity of fluid flows through the two branches of the fluid line 76a, so that approximately the same flow conditions arise in the first and second flow chambers 66a, 66f, but in opposite directions, so that the material or substance 63 is reliably kept in suspension within the container 62, both when buoyancy forces are acting and when gravity is acting. The substance 73 can thus be kept in suspension without an automatic regulation. The position of the material 73 can also be monitored and influenced with the aid of an automatic regulating system by detecting the position of the substance 73 with a non-illustrated sensor. Should the specific weight of the substance 73 be lighter than that of the fluid, i.e. of the nutrient solution, then the material 73 is subject to buoyancy. In this case the fluid will increasingly flow into the container 62 via the upper inlet opening 66d in order to bring about a downwardly directed fluid flow in the second flow chamber 66f, so that the substance 73 is kept in suspension by the fluid flow acting against the buoyancy. Should the specific weight of the substance 73 change in the course of time and become smaller than that of the nutrient solution, so that a downwardly acting gravitational force now acts on the substance 73, then the fluid is increasingly supplied to the lower inlet opening 66d, in order to produce an upwardly directed fluid flow in the first flow chamber 66a and thereby a buoyancy force on the substance 73. The required quantity of fluid per unit of time and the division of the partial quantities to the upper and/or lower inlet openings 66d takes place manually or with a non-illustrated regulating apparatus in such a way that the substance 73 is continually kept in suspension by appropriately selected fluid flows, both with respect to a buoyancy force that is acting and also with respect to the gravitational force that is acting.
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