Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/38—Caps; Covers; Plugs; Pouring means
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
  • B01F31/20—Mixing the contents of independent containers, e.g. test tubes
  • B01F31/22—Mixing the contents of independent containers, e.g. test tubes with supporting means moving in a horizontal plane, e.g. describing an orbital path for moving the containers about an axis which intersects the receptacle axis at an angle
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F35/00—Accessories for mixers; Auxiliary operations or auxiliary devices; Parts or details of general application
  • B01F35/50—Mixing receptacles
  • B01F35/53—Mixing receptacles characterised by the configuration of the interior, e.g. baffles for facilitating the mixing of components
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
  • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/12—Well or multiwell plates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/24—Gas permeable parts
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00306—Reactor vessels in a multiple arrangement
  • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
  • B01J2219/00315—Microtiter plates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00479—Means for mixing reactants or products in the reaction vessels
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0829—Multi-well plates; Microtitration plates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0848—Specific forms of parts of containers
  • B01L2300/0858—Side walls

Definitions

• the invention relates to a microreactor having at least one cavity, which has a bottom, a side wall and an opening opposite the bottom.
• microreactor arrays e.g. 6, 24, 48, 96, 384 or more individual microreactors realize.
• volume of the individual reactors may also be different. While it is spoken at scales below 10 ml of microreactors, a further reduction of the volume to less than 1 ml, less than 500 ul, less than 100 ul or even less than 10 ul take place.
• a microreactor serves as a reaction vessel for biochemical, chemical or enzymatic reactions as well as microbial fermentations.
• a reactor array allows the study of cell cultures in high parallelism with low work volume, high information gain and the possibility for simplified automation. Such arrays are particularly useful for automating screening assays with improved mixing and mass transfer conditions, and allow for externally isolated or sterile, aseptic or monoseptic operation.
• Microreactor arrays such as microtiter plates, provide an ideal platform for achieving a high level of parallelization. Due to the small reaction volumes (eg> 10 ⁇ l to ⁇ 10 ml per chamber), the high degree of parallelization (eg 6 to 1536 chambers per plate) and the possibility of automating the cultivation processes (robot-manageable form), microreactor arrays are the most cost-effective and cost-effective most promising bioreactor.
• Microtiter plates are currently being used to screen biological systems. For this purpose, the individual reaction chambers are filled, inoculated and incubated on a rotary shaker. The most orbital shaking movement improves the entry of oxygen into the reaction liquids and results in a thorough mixing of the reaction mixture. To keep the system sterile, the microtiter plates are covered by an air-permeable membrane (pore size ⁇ 0.2 ⁇ m) or an airtight foil or lid construction, or cultured open in a sterile environment.
• an air-permeable membrane pore size ⁇ 0.2 ⁇ m
• an airtight foil or lid construction or cultured open in a sterile environment.
• microtiter plates used for the applications described are offered by various manufacturers in two basic versions: with circular or rectangular cavities.
• the first microtiter plates were made in 1951 by Dr. med. G. Takatsky and had a round cross Liermann-Castell PQ 2 7 2 3WO
• microtiter plates of square and rectangular cross-section were introduced.
• microtiter plates are used in many fields of chemistry, medicine, biotechnology and biology, so that almost no development has taken place specifically for the cultivation of cells.
• baffles can contribute to severe dripping / splashing, resulting in inhomogeneities, increased wall growth, and wetting and occlusion of the gas permeable cover of the plates (Büchs J., Introductory to Advantages and Problems of Shake Cultures, Biochem. Eng J. 7 (2), 91-98, 2001). Furthermore, no further reference to the realization and examination of a microtiter plate with a variation of the cavity geometry (outside of the circular and the rectangular cross-section) can be found in the market and in the specialist literature.
• microtiter plates Various systems are available to cover such microtiter plates. On the one hand, most manufacturers of microtiter plates include a plastic lid which is loosely placed on the microtiter plate.
• mats made of flexible plastic are distributed, whose knob-like protuberances reach into each individual cavity and thus close it.
• US Pat. No. 6,889,848 discloses a cover which completely engages around the microtiter plate, thus finding a hold on the microtiter plate.
• special devices for application or when applying the lid are necessary. Both systems can not be automated without additional holders or applicators.
• the object of the invention is to overcome the above-described disadvantages of conventional microtiter plates and thus expand the established concept of a microtiter plate as a vessel mainly for chemical and biochemical reaction mixtures in principle to a full-fledged and universally operational reaction and cultivation system, preferably by the following points be fulfilled.
• the solution of the described objects is achieved by changing the geometry of a cavity, away from the established geometries of a circular cylindrical shape or a rectangular cross section.
• the object is achieved by modifying the round or square cavities known from the prior art in such a way that the positive properties of a current disturbance result from the introduction of protrusions or indentations into the cavity as well as the positive properties of a round cavity and thus ideally complement an undisturbed flow as possible for the described application.
• the design-relevant length of the basic side of a polygon can be calculated for a given area of 112.16 mm 2 also on the construction of a triangle between a base side and two adjacent radii of the polygon. Therefore, it is alternatively suggested that the cross-section has fewer than four corners.
• the shape can be described by the fact that a cross-section which crosses the side wall parallel to the bottom has at least one concave and / or convex circular segment which protrudes with a radius into or out of the cross section, this radius being between 0.067 and 0.49 times the diagonal of the cross section.
• the basic shape of the cross section is any polygon or circle that has several concave or convex circle segments. Liermann-Castell P ⁇ 2723WO
• the cross section may have an arc forming a circle segment of more than 90 °, or the cross section may have more than 3, preferably more than 4, arcs each forming a circle segment of more than 90 °.
• a pentagon was chosen as the initial shape, and gradually converted over the rounding of the corners in a circle.
• a fourth approach which is to change the originally circular basic form of the cavity, involves the introduction of baffles of different shape and size.
• the resulting base area is not readily calculable in many cases.
• Ver. 14.01 of the company Autodesk Inc. measured and then scaled accordingly.
• the area is a rectangle or a circle segment.
• the respective cross-section of the geometry of the cavities used can be widened in the height direction, e.g. to ensure better demoulding during injection molding, or to narrow in the vertical direction, e.g. the filling volume can be further increased at a corresponding shaking frequency without the liquid spilling over.
• the above-mentioned Kavticiansgeometrien can be used, which then go in the height direction up or down in a different Kavticiansgeometrie.
• the transition can take place between one of the Kavticiansgeometrien described here or go into a round, square or rectangular Kavticiansgeometrie.
• a transition between a round, square or rectangular Kavticiansgeometrie done.
• At least one component which changes the cross-section is introduced through the bottom or a cover into the cavity.
• the floor is formed of an optically transparent material.
• the microreactor has a plurality of cavities, which are particularly preferably arranged in the form of an array. Liermann-Castell P ⁇ 27 23 WO
• This cover preferably has a gas-permeable surface in order, in particular in the case of an array, to seal each individual cavity with respect to solids and liquids from the environment.
• a gas-permeable surface in order, in particular in the case of an array, to seal each individual cavity with respect to solids and liquids from the environment.
• an opening is provided, which is designed in its shape and size, as well as the material occluding it so that the evaporation of reaction liquid is greatly reduced and a transfer of material from the surrounding gas phase in the Fluids. sigphase in the cavity and in the opposite direction is not affected.
• the lid has a resealable surface. It is particularly preferred if the cover is formed integrally with the wall and / or the bottom except for a gas-permeable and / or resealable surface.
• the lid has a sandwich material made of a solid, a flexible and / or a gas-permeable material.
• An embodiment provides that the lid has a stable frame with a seal.
• the lid can be fastened under prestress to the wall and it can be attached to the wall according to the Luer principle. It is preferred that the male part of a luer lock is attached to the lid and the wall forms the female counterpart.
• a variant provides that the lid is connected to the wall via mutually displaceable inclined planes. This can be achieved, for example, by releasing the cover from the wall into a gap in which a wedge can be inserted in order to detach the cover from the wall.
• holes are provided in the lid for releasing the lid, into which a gripper can engage.
• the grippers for releasing the lid have an arrangement for applying a mechanical or pneumatic counter-pressure.
• holes may be provided in openings through which pins or hollow needles of a gripper arm for releasing the cover from the reaction vessel assembly apply a mechanical or pneumatic pressure to the cavity.
• the cover be glued to the wall.
• the lid can be secured to the wall by latching or it can have a device for generating a negative pressure in the cavity.
• microreactor forms part of a microreactor array having a plurality of identical cavities and preferably has a shaking device.
• the drawing shows measurement results and design variants for microreactors, reactor arrays, and various covers for reactors and reactor arrays.
• FIG. 1 variations of the number of corners of a cavity
• FIG. 2 shows variations of the formation of corners on a square cavity
• FIG. 3 shows variations of the formation of the corners on a pentagonal cavity, Liermann-Castell P02723WO
• FIG. 4 shows schematic illustrations of cavities with rectangular and semicircular baffles
• FIG. 5 schematically shows the construction of a pentagonal base with asymmetric semicircular baffles
• FIG. 6 schematically shows pentagonal and hexagonal bases with asymmetrical semi-circular baffles
• Figure 7 schematically four, five and hexagonal bases with rounded
• FIG. 8 is a photograph of various microreactor geometries arranged as an array
• Figure 9 is a photograph of the prototypes of the cover with Luer principle for
• FIG. 10 shows schematic representations for explaining the Luer principle
• FIG. 11 schematically shows an arrangement for holding the cover on a micro-reactor array by negative pressure
• FIG. 12 schematically shows the representation of the hook for holding the lid on a microreactor array
• FIG. 13 shows schematically possibilities for releasing the lid from the microreactor array
• FIG. 14 schematically shows a multi-functional lid placed on a microreactor array, Liermann-CasteU P ⁇ 2723WO
• FIG. 15 shows graphic representations of measurement results for the maximum oxygen transfer rate in different example geometries
• FIG. 16 shows graphical representations of measurement results for the maximum oxygen transfer rate in further exemplary geometries that have been implemented
• FIG. 17 graphically shows measurement results for the maximum oxygen transfer rate in example geometries with baffles
• FIG. 18 Measurement results for the maximum filling volume in realized example geometries
• FIG. 19 Measurement results for the measurable fill level in a cavity in realized example geometries with orbital shaking motion.
• Figure 1 shows how the design-relevant length of the base side of a polygon can be calculated for a given area - in the present example of 112.16 mm - on the construction of a triangle between a base side and two adjacent radii of the polygon.
• FIG. 4 shows how an originally circular basic shape of a cavity can be modified by incorporating baffles of different shape and size.
• Figures 5 to 7 show basic surface geometries, which are the result of theoretical considerations for meaningful implementation in various geometric shapes. For all of these basic shapes circles of defined size were introduced whose radius was changed in 1 mm increments. The selection of the resulting geometries was based on a purely theoretical assessment of their influence on the flow in the cavity. Thus, forms with extremely strong or very weak buffers were excluded from consideration.
• Figures 6 and 7 show baffles starting from a five- or hexagonal base. Their construction is illustrated in Figures 6 and 7 by way of example. The corner of these bases were rounded off, taking as a radius for the corner circles one or two millimeters. At each corner, a semicircular chicane with a radius of 1 mm is created. This design dictates a direction of rotation for shaking due to the lack of symmetry for the cavities.
• Figure 7 shows further basic forms of cavities. Starting from four-, five-, six- or seven-cornered basic shapes, the corners are again rounded, in which case the area between these corners is not flat, but has an inwardly reaching point. These peaks form the harassment in these cavities. _ _.
• the prototype cover of a microreactor array shown in FIG. 9 seals each individual cavity tightly against the environment and has, above each cavity, an opening designed to greatly reduce the evaporation of the reaction liquid and a mass transfer the surrounding gas phase is not affected in the liquid in the cavity and in the opposite direction.
• single or all reactors of a microreactor array are designed as luer sleeves in relation to lids designed with luer cores.
• the Luer principle shown in Figure 10 has proven to be very advantageous in this prototype. It is able to tightly seal the individual reaction chambers from the environment.
• the cavity 1 with a cover 2, which serves as a cover, closed.
• the lid 2 has conical fitting elements 3, which abut against the cavity wall and seal the lid 2 to the cavity 1.
• the lid 2 has a gas-permeable film 4, which is glued to the lid or welded to it. This film provides the necessary gas exchange, reduced evaporation and monoseptic operation. It is envisaged to pre-sterilize the microreactor array and fit lid components with final gas-permeable foil to the user.
• FIG. 10B Another embodiment is shown in FIG. 10B.
• a flexible sealing layer 7 is provided on the cavities 5, 6, on which a gas-permeable film 8 is located.
• the microreactor array has a conical cavity in the body, which serves as a female Luer sleeve 9.
• a cover 10 for the microreactor array has a Luer core 11, which cooperates as a male part with the sleeve 9 and holds the cover on the array.
• the flexible layer 7 is placed over the cavities. It seals by corresponding contact pressure of the lid, which seals all wells held by the Luer connection.
• the luer sleeves can be mounted, for example, between the individual wells and on the frame of the microreactor array. In Figure IOC a possible order is shown. Herein, the possible positions for luer sleeves are shown as attachment points 12 on the microreactor array 13 for sealing the cavities 14.
• FIG. 10D shows how the Luer principle can be applied over the whole of the microreactor array.
• the array frame 15 are bevelled. They thus serve as Luer core for the lid 16, which either forms a circumferential Luer sleeve or is slipped over the sides of the array frame 15 only on the opposite sides.
• the array cover 16 is thus formed as a Luer sleeve and enters into a frictional connection with the array frame 15. Between the cover 16 and the microreactor array frame in turn a gas-permeable film 17 and a flexible cover layer 18 is provided.
• FIG. 11 shows a variant in which the cover 22 is sucked onto the microreactor array 23 with vacuum or negative pressure.
• vacuum 23 is sucked through a bore 24 in the body of the microreactor array and Liermann-Castell P ⁇ 2 723WO
• FIG 12A it is shown how the lid 30 is fixed to the plate geometry by a barb 31.
• the barb 31 hooks on a web 32.
• the lid 30 is held so close to the microreactor array 33 that it presses the flexible layer 34 onto the cavities (not shown) and thus seals.
• FIG. 12B shows a variant in which the barb 35 hooks into a groove 36 in the microreactor array 37.
• FIG. 12D shows an alternative solution for attaching the cover 40 to the microreactor array 41 by applying a spring tension to the barb 43 by means of the spring 42. Only by applying an external force 44 by means of a gripping arm of a pipetting robot or manually can the tension of the spring Liermann-Castell P02723WO
• the overpressure can be controlled by a compressed air line attached to the gripper arm of a pipetting robot
• Compressed air line 51 are guided.
• Figure 13B shows how pins 52 are mounted on a plate 52, which release the luer cores 54 from below.
• the plate is simply placed on the pins 53 and the pins 53, guided by bores 55 in the microreactor array main body 56, press the cover 57 against the luer cores 54 upwards.
• FIG. 13D A slight modification of this principle is shown in FIG. 13D.
• the shoe 66 is chamfered upwards.
• the cover 67 moves upwards via a slope 68, the microreactor array 69 remaining in its fixed position.
• FIG. 13E Another release mechanism is shown in Figure 13E.
• This release mechanism does not require a special beveled shoe on the fingers of a gripper arm.
• sterile sampling is extremely important in order to prevent contamination of the mostly monoseptic culture.
• FIG. 14 has a cannula 80 which is guided through an opening 82 in a rigid lid back 83 through a gas-permeable foil 84 and a flexible covering layer 85 into a cavity 86 in order to remove reaction fluid from the cavity 86 or to insert into the cavity. Holes 87 are provided in the flexible covering layer 85 for material transport between inside and outside, which holes are aligned with openings 88 in the rigid cover back 83.
• microtiter plate in particular when used as a Zeilkultiv istssystem fundamentally improved. Due to the fact that a significantly higher degree of sintering can be achieved with sufficiently homogeneous hydrodynamics without droplet and spatter formation, a broad range of unlimited fermentation of microorganisms and higher cells (plant, animal and human cells) is possible. By preventing the freewheeling of the bottom of the cavity, a sufficiently high liquid column remains at the bottom of the cavity even at high shaking speeds. As a result, the liquid of a measurement at the bottom of the cavity is made much more accessible. Sensors attached here do not run the risk of losing contact with the reaction mixture.
• microtiter plate cover overcomes a serious drawback that normally occurs during culture in microtiter plates.
• the loss of fluid from the cavity occurring in particular at higher cultivation temperatures is markedly reduced.
• a septum keeps each cavity accessible for sampling.

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• 2008
  • 2008-02-08 DE DE102008008256A patent/DE102008008256A1/de not_active Withdrawn
  • 2008-10-08 WO PCT/DE2008/001623 patent/WO2009046697A2/de not_active Ceased
  • 2008-10-08 DK DK08837177.8T patent/DK2205716T3/en active
  • 2008-10-08 EP EP08837177.8A patent/EP2205716B1/de active Active
  • 2008-10-08 DE DE112008002590.8T patent/DE112008002590B4/de active Active
  • 2008-10-08 JP JP2010527321A patent/JP2011501689A/ja active Pending
  • 2008-10-08 CN CN2008801192016A patent/CN101932693B/zh active Active
  • 2008-10-08 US US12/734,054 patent/US8828337B2/en active Active

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