JP2008178389A - Small-scaled cell culture container - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a small-scaled cell culture bioreactor easily controlling various process conditions such as temperature and degree of mixing. <P>SOLUTION: The bioreactor 10 has a filter 21, a perforated path 22 extending along the periphery of lower part of the bioreactor 10 and gas is introduced via an inlet 20 attached to a gas source. The cross-section of the inside volumetric part 26 along the periphery of the bioreactor 10 is constant, and the height of the effective volumetric part is kept substantially constant by added reactants or taken out products and after increasing the volume, the state of agitation as indicated by arrow marks 32 and 34 is substantially kept constant through the whole part around the bioreator 10. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  This application claims the benefit of US Provisional Patent Application No. 60 / 859,445, filed Nov. 16, 2006. The present invention relates to waste bioreactors for small-scale cell culture that can establish optimal conditions for bioreaction processes, and more particularly to such bioreactors that can easily control reaction temperature and reactant mixing.

Microbial cell culture (fermentation) or animal and plant cell culture (tissue culture) are commercially important chemical and biochemical processes. Such processes use live cells because they can economically synthesize commercially valuable chemicals, including proteins such as monoclonal antibodies or enzymes, vaccines or alcoholic beverages, generally using readily available starting materials. . In a commercial culturing process, the bioreaction is typically performed in a small culture shaker flask or roll bottle where the test reaction conditions can be precisely controlled to optimize the conditions.
Fermentation includes the growth or management of living cells in a nutrient medium. In a typical fermentation batch process, a desired microorganism or eukaryotic cell is placed in a medium consisting of water, nutrients, and dissolved gas and grown (or propagated) to a desired culture density. The nutrient medium should contain all chemicals that are necessary for the survival of living cells and that optimize the environmental conditions for continued growth and / or replication. Currently, in a typical microbial cell culture process, a group of microorganisms is suspended in a nutrient medium circulating in a stirred bed or gas fluidized bed continuous reactor. Similarly, in mammalian tube culture, cells are suspended in roll flasks, or in the case of cells that require surface attachment, cells are cultured in tissue culture flasks containing nutrient media on the adherent cells. Are densely cultured. The living cells so maintained metabolize the desired product or products from the precursor material introduced into the mixture of nutrients. The desired product or products are purified from the culture medium or extracted from the cells themselves.

  One system for a bioreactor uses a large table with a motor or hydraulic device with a bioreactor bag placed on top of it. The motor / hydraulic device locks the bioreactor bag and keeps the cells in constant motion. In addition, the bioreactor bag has a gas and nutrient substance supply tube and a waste gas and waste tube, and supplies air-like gas and nutrient substance for aerobic organic matter, exhaled gas, carbon dioxide and other Such waste can be removed. These tubes act to allow gas and fluid / solids to move uniformly as the bioreactor bag moves. See U.S. Patent No. 6,190,913. Such systems require capital intensive equipment to be used with wear-resistant components and the dimensions of the bioreactor bag that can be used with the table include the table dimensions and the motor / hydraulics that the table has. There are limitations due to the lift capacity of the equipment.

  Another system uses a long flexible tubular bag with both ends attached to a movable arm, suspends the filled bag from the movable arm in a U-shape, and then moves each arm up and down alternately, The fluid in the bag is swung and moved. If desired, a restriction can be provided in the central section of the bag to make the mixing operation more uniform. This system requires a specially shaped bag, requires hydraulic equipment and other lifting devices to move the liquid, and because of its weight, the size and volume of the bag will include the lifting capacity and strength of the equipment. There is a limitation by.

Improvements have been seen in using one or more bags that can be selectively pressurized and shrunk in combination with disposable biobags such as fermentation layers, mixing bags, storage bags, and the like. One or more pressure bags may be placed around the outer selected portion of the bag, or such pressure bag or bags may be housed within a small portion of the bag. Selectively pressurize and deflate the pressure bag or bags to move fluid within the bag, thus ensuring that the cells float in the bag without causing damaging shear or foam, Mixing and / or gas and / or nutrient / excrement can be transferred.
Alternatively, a stationary (non-moving) bag containing a sparger or other device that introduces gas into the bag may be selected. In this case, the gas moves the fluid in the bag and mixes and moves the gas, nutrients, and waste.

In US Pat. No. 5,565,015, a flat, inflatable, porous tube sealed in a plastic container is used. This tube expands under gas pressure and allows gas to flow into the bag. When no gas is added, the tube collapses to substantially close each hole to prevent liquid leakage from the bag.
In US Pat. No. 6,432,698, a tube is inserted and sealed through a gas diffuser in the bag. In this case, since there is no valve or other means for preventing backflow, it is necessary to maintain the gas under a constant positive pressure in order to prevent liquid in the bag from entering the air pump through the gas line from the diffuser. Seem.
Both of the structures disclosed in these two U.S. patents can leak liquid in the container, which can contaminate the contents of the bag of an upstream component of the system, such as a gas supply system. There is a fear. Furthermore, any invention introduces a separate component for gas dispensing.

US Provisional Patent Application No. 60/899445 US Pat. No. 6,190,913 US Pat. No. 5,565,015 US Pat. No. 6,432,698

  The problem to be solved is to provide a small-scale bioreactor apparatus that can easily control process conditions such as temperature and degree of mixing.

In accordance with the present invention, a disposable bioreactor is shaped such that the liquid reactant rises along the inner surface of the outer wall of the bioreactor and then descends in a direction away from the inner surface of the reaction volume. Bioreactors are provided.
The bioreactor includes a first inner surface that forms a closed volume with the second inner surface of the inner wall of the bioreactor on the first inner surface of the outer wall. The first inner surface and the second inner surface have at least a portion that converges or expands with each other to move the liquid reactant in the bioreactor in a natural spiral direction.
The bioreactor of the present invention includes a gas introduction unit and a gas elimination unit, and also includes a reactant addition unit and a unit for taking out one or more desired products.
The bioreactor of the present invention is formed from materials such as glass or polymeric organisms that do not contaminate the reactants or products.

  There is provided a small-scale bioreactor apparatus that can easily control process conditions such as temperature and degree of mixing.

In accordance with the present invention, a disposable bioreactor is provided having closed inner and outer walls that are spaced apart from each other to form a volume portion for conducting a bioreaction. At least a portion of the inner wall and a portion of the outer wall converge. The degree of convergence is such that the reactants are moved in a spiral direction within the bioreactor. Cultured cells, nutrients, and one or more gases are introduced into the bioreactor to cause a bioreaction within the bioreactor. Here, “effective volume” means the volume of the bioreactor where the reaction takes place. A portion of the volume of the bioreactor includes a gas-containing volume portion positioned above the effective volume portion. Gas is introduced into the bioreactor such that the reactants are mixed therein and contact the inner surface of the outer wall before the upper surface of the reactants is destroyed. By operating the gas in this way, the reactant mixture travels through the inherently spiral path across the entire circumference of the bioreactor.
The inner volume portion of the bioreactor is shaped so that the reactants are well mixed with each other in all portions within the effective volume. Thus, the occurrence of dead zones with little or no mixing is avoided. The bioreactor is provided with an external volume that houses a heater that controls its internal temperature. One or more inlets are provided for introducing reactants into or removing products from the bioreactor.

  The gas is introduced into the effective volume of the bioreactor by means of at least one perforated passage that can be locked together with the bioreactor, such as attached to the bioreactor along the length of the bioreactor, or Can be separated from the bioreactor like a sparger tube and can be progressively inserted into the bioreactor as the effective volume of the bioreactor increases. Conventional sealing means for preventing leakage from the portion of the bioreactor that is inserted through the perforated passage may be provided. Perforated passages do not contaminate flexible materials such as polymer assemblies that do not contaminate reactants or products, or glass such as ceramics, glass mats or sintered glass materials, or reactants or products It can be formed from sintered stainless steel.

  Suitable plastics can be hydrophilic or hydrophobic. However, if it is hydrophilic, the air pressure in the perforated passage is either constant at the liquid intrusion pressure or higher to keep the liquid outside the perforated passage, or like a valve or a hydrophobic filter. It should be provided to shut off the upstream side and ensure that there is no liquid overflow from the perforated passageway and / or upstream side of the gas supply. Plastics can be naturally hydrophilic or hydrophobic or can be surface treated to have the desired properties. The plastic can be a single layer or multiple layers if desired. One embodiment of the multi-layered passage is a pre-filter or depth filter with a high openness that allows the porous plastic layer to trap debris and prevent the trapped debris from clogging the perforated passage. A porous plastic layer covered with The pore size is selected based on the desired foam size of the gas, but can range from micropores (0.1-10 μm) to macropores (10 μm or more), such as microporous filters, woven Fibers or filters, porous non-woven materials such as Tyvek ™ sheet material, monoliths or pads commonly found in aquarium filters, and other nightly membranes or filters. The plastic chosen should be compatible with the bioreactor environment so that the cells growing inside it are not adversely affected. Suitable plastics include, but are not limited to, polyolefins such as polyethylene or polypropylene, polysulfones, or polysulfones such as polyethersulfone, nylon, PTFE resin, PEF, PVDF, PET and others. .

The introduced gas functions as a reactant and as a reactant stirring means.
With reference to FIGS. 1 and 2, the bioreactor 10 includes two legs 12 and 14, which are connected by a section 16 positioned above them. A gas volume 18 is provided above section 16 where unreacted gas is collected. Gas is introduced into the bioreactor 10 through an inlet 20 having a filter 21 and a perforated passage 22 extending along the lower peripheral portion of the bioreactor 10. The inlet 20 is connected to a gas source (not shown). The outer volume portion 24 is configured to store a temperature control heater (not shown) in the bioreactor 10. A gas outlet 23 having a filter 27 is also provided. A partition wall 25 for introducing a non-gaseous reactant is provided.
As shown in FIG. 3, the cross section of the inner volume portion 26 along the peripheral portion of the bioreactor 10 is constant. The height of the effective volume portion is essentially kept constant by the reactants added or the product removed. Thus, even after the volume is increased, the agitation situation as indicated by arrows 32 and 34 is inherently constant throughout the surrounding portion of bioreactor 10.

4 and 5, a bioreactor 11 according to another embodiment of the present invention is illustrated. The bioreactor 11 is configured such that an outer wall 36 positioned perpendicular to the bottom surface 38 forms legs 40 and 42 connected by a section 44. The surface 46 is converged towards the liquid and gas interface 48 so that the introduced gas moves in the direction indicated by arrows 50 and 52 before breaking the reactant surface 54. Gas is introduced into the perforated passageway 59 through an inlet 56 having a filter 58. Gas is removed from an outlet 62 having a filter 60. An outer volume 64 is provided for housing a heater (not shown). A partition wall 66 is provided for introducing the reactant into the bioreactor 11.
The bioreactor of the present invention can be formed from rigid plastic or glass. Preferred plastics include, but are not limited to, thermoplastic homopolymers such as polyethylene and polypropylene, polyolefin copolymers, polycarbonate, polystyrene and the like. The plastic is preferably transparent so that its internal action can be monitored by visual inspection.

It is a side view of the bioreactor of this invention. It is sectional drawing of the bioreactor of this invention shown in FIG. It is a side view which shows the movement of the liquid reactant in the bioreactor of this invention. It is a side view of the bioreactor of another aspect of this invention. It is sectional drawing in the other Example of the bioreactor of FIG.

Explanation of symbols

10, 11 Bioreactor 12, 14, 40, 42 Legs 16, 44 Section 18 Gas volume part 20 Inlet 21, 27, 60 Filter 22 Perforated passage 25, 59, 66 Septum 26 Inner volume part 36 Outer wall 38 Bottom face 54 Reaction Body surface 48 Liquid and gas interface 59 Inlet 62 Outlet 64 Outer volume

Claims (10)

A bioreactor comprising a first closed wall that forms a peripheral portion of the bioreactor, a first closed wall forming an outer surface of the bioreactor, and an inner peripheral portion of the bioreactor; A second closed wall spaced from the first closed wall; means for introducing gas into the bioreactor; and means for removing gas from the bioreactor; At least a portion of the height of the open wall extends toward the center of the bioreactor so that the gas introduced into the bioreactor contacts the first closed wall portion before contacting the top surface of the bioreactor Bioreactor. The bioreactor of claim 1, wherein the entire first closed wall extends toward the center of the bioreactor. The bioreactor of claim 1, comprising an outer volume adjacent to an outer surface of the second closed wall. The bioreactor of claim 2 including an outer volume adjacent to the outer surface of the second closed wall. The bioreactor of claim 1, wherein at least a portion of the wall of the inner peripheral portion and at least a portion of the wall of the outer peripheral portion converge. The at least a portion of the wall of the inner peripheral portion and at least a portion of the wall of the outer peripheral portion are converged such that the degree of convergence is such that the reactants are allowed to move in a spiral direction within the bioreactor. 1 bioreactor. The bioreactor of claim 1, wherein the inner volume portion has a constant cross-section along the peripheral portion of the bioreactor. Claims: Each cross section of the inner volume along the periphery of the bioreactor and the height of the effective volume is maintained essentially constant by the amount of nutrient added and product removed. 1 bioreactor. The bioreactor of claim 1, wherein the inner volume portion has a constant cross-section along the peripheral portion of the bioreactor, and mixing conditions throughout the bioreactor peripheral portion are inherently constant even after the effective volume portion has increased. The bioreactor of claim 1, wherein the means for introducing gas is a perforated passage formed from one or more porous materials.

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