WO2020182646A1 - Système de traitement et procédé de traitement pour ensembles sous forme tridimensionnelle de cellules cultivées et programme informatique - Google Patents

Système de traitement et procédé de traitement pour ensembles sous forme tridimensionnelle de cellules cultivées et programme informatique Download PDF

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Publication number
WO2020182646A1
WO2020182646A1 PCT/EP2020/055984 EP2020055984W WO2020182646A1 WO 2020182646 A1 WO2020182646 A1 WO 2020182646A1 EP 2020055984 W EP2020055984 W EP 2020055984W WO 2020182646 A1 WO2020182646 A1 WO 2020182646A1
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WIPO (PCT)
Prior art keywords
culture
processing system
cells
medium
dimensional
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Ceased
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PCT/EP2020/055984
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German (de)
English (en)
Inventor
Werner Wittke
Nariman ANSARI
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Leica Microsystems CMS GmbH
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Leica Microsystems CMS GmbH
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Publication of WO2020182646A1 publication Critical patent/WO2020182646A1/fr
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    • 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—Rigid containers without fluid transport within
    • B01L3/5085—Rigid containers without fluid transport within 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • 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
    • B01L2300/02—Identification, exchange or storage of information
    • B01L2300/023—Sending and receiving of information, e.g. using Bluetooth®
    • 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
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • 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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules

Definitions

  • the present invention relates to a processing system and a
  • the cells are enabled to grow and interact in all spatial directions in vitro, and thus at least partially to grow and interact in vivo
  • Three-dimensional cell cultures are usually cultivated in bioreactors, small capsules in which the cells can grow into spheroids, or in the form of three-dimensional cell colonies. For example, a large number of corresponding spheroids can be cultivated in bioreactors.
  • Spheroids on the other hand, resemble in vivo tissue in terms of cellular
  • extracellular matrix allows toes to move in their spheroid, much like cells would move in living tissue.
  • three-dimensional cell cultures improved models for the Zehmigration that Differentiation of cells, cell survival and cell growth are created.
  • three-dimensional cell cultures reproduce the natural cell polarization better, since cells in two-dimensional cell cultures can only be partially polarized.
  • Cells cultivated in three dimensions can also have a different gene expression than cells cultivated in two dimensions.
  • the three-dimensional cultivation of cells can in principle be carried out using structural frameworks, for example using hydrogels
  • Cells are cultivated, for example, in the form of the spheroids already mentioned, i.e. essentially spherical aggregates.
  • spheroids for example, healthy cells and
  • Tumor cells were co-cultured to simulate how the tumor cells interact with the healthy cells.
  • Corresponding spheroids can be generated using different methods.
  • a common method which can also be used in particular in connection with the present invention, is the use of multi-chamber culture plates with low cell adhesion, typically in the known 96-well form, for the mass production of spheroid cultures in which the aggregates are typically rounded bottom of the chambers.
  • Spheroids can also be grown in pendent drops that hang from the surface of a culture plate.
  • Other methods include the use of rotating-walled bioreactors that rotate the cells in constant free fall and form layered aggregates.
  • Bioreactors used for three-dimensional cell cultures are typically designed in the form of small cylindrical plastic chambers. Here will be described
  • bioactive materials such as polyethylene terephthalate membranes are used to keep the spheroid cells in an environment that ensures a high nutrient content.
  • the chambers can be opened and closed so that the spheroids can be removed for testing.
  • the chambers are part of a larger arrangement that can be rotated to ensure even cell growth in every direction.
  • three-dimensional cell assemblies which are produced in vitro and at least partially living organs with regard to the tissue structure or morphology
  • Organoids Correspond to organisms. Organoids therefore have, in particular, a realistic micro-anatomy. Organoids are obtained from one or a few toes of tissue, from embryonic stem cells or induced pluripotent stem cells, which can organize themselves in three-dimensional culture due to their ability to self-renew and differentiate.
  • the production of organoids typically involves the cultivation of
  • Stem or progenitor cells in a medium or a corresponding matrix can also be used to produce organoids.
  • the organoids can be produced by embedding stem cells in an appropriate matrix, using suitable inductors for differentiation.
  • Organoids can also be made using adult stem toes derived from the
  • Target organ can be extracted and cultured in a medium.
  • Hydrogels which do not necessarily have to have an organ-like microstructure, as well as organoids, are each cells that are or have been cultivated in the form of three-dimensional assemblies. Therefore, in the context of the present application, the collective term “in the form of three-dimensional
  • a composite can be a cell-cell composite in which the cells adhere to one another or have grown together, but also a composite provided by a framework such as a hydrogel.
  • the cells cultivated in the form of three-dimensional assemblies are arranged in aggregates, have several cell layers in each spatial direction, and the toes adhere to one another through mutual adhesion or through a natural or artificial matrix.
  • the present invention has in particular the task of handling cells cultivated in the form of three-dimensional assemblies, in particular cells
  • the present invention proposes to solve this problem
  • the generation of cells cultivated in three-dimensional networks is intended to mean, in particular, the formation of corresponding aggregates from one or more individual cells, in particular through aggregation of several cells or the division of one or more output lines, the daughter cells in
  • Cells cultivated in three-dimensional assemblies typically require an exchange, advantageously a continuous exchange, of a nutrient medium or a fluid in which the cells cultivated in three-dimensional assemblies grow, or one or more components thereof.
  • an exchange advantageously a continuous exchange, of a nutrient medium or a fluid in which the cells cultivated in three-dimensional assemblies grow, or one or more components thereof.
  • the staining of cells cultivated in three-dimensional assemblies is generally known, so that reference can be made to the corresponding specialist literature. It is carried out in particular by adding one or more dyes detectable in the visible, infrared or ultraviolet wavelength range, in particular also fluorescent dyes which emit light of a different wavelength when excited with light of one wavelength. Especially when using several dyes detectable in the visible, infrared or ultraviolet wavelength range, in particular also fluorescent dyes which emit light of a different wavelength when excited with light of one wavelength. Especially when using several
  • the stimulation carried out within the scope of the present invention can in particular be a mechanical chemical, temperature or radiation stimulation which causes a defined, known, unknown or to be examined reaction in the cells cultivated in three-dimensional networks, for example a release of certain signal substances, a certain gene expression , a division, a differentiation or the like.
  • the present invention proves to be particularly advantageous in connection with a chemical stimulation which is accompanied by the addition of one or more stimulation substances, since this no longer has to be carried out manually or no longer exclusively within the scope of the present invention.
  • Control unit be set up, for example to control one or more pumps for metering or feeding in one or more fluids for
  • Control of one or more heating or cooling devices for thermal stimulation for controlling one or more motors for positioning, for controlling one or more cameras and the like.
  • Cultivation, stimulation and sample preparation are conventionally carried out independently of one another and in separate manual steps.
  • the present invention integrates and automates at least some of these components
  • Steps in a processing system as proposed according to the invention and which is set up to carry out a corresponding method Steps in a processing system as proposed according to the invention and which is set up to carry out a corresponding method.
  • the present invention creates, in particular, a coherent workflow or work stream that is largely automated or can be performed without or without significant user intervention after the start.
  • the entire sequence of sample preprocessing and sample processing of thick, three-dimensional cellular samples such as tumor spheroids and organoids can be partially or completely automated.
  • the present invention proposes a processing system for toes cultivated in the form of three-dimensional composites (that is to say in particular the three-dimensional cell cultures and organoids explained).
  • a culture unit is provided, which can in particular be exchangeable and designed in the form of a culture plate.
  • the culture unit has a number of
  • Culture chambers which are designed as explained in detail below and are set up to accommodate the cells cultivated in the form of three-dimensional composites and a culture medium.
  • the culture chambers can in particular be present in a number and arrangement that corresponds to the number of so-called woes in known multi-well plates in order to ensure compatibility, for example, with commercially available multi-pipettes or pipetting devices and detectors such as fluorescence readers.
  • the processing system further comprises a microscope, wherein the culture unit is arranged or can be arranged in the processing system in such a way that the culture chambers and their contents can be viewed through a base of the culture unit.
  • Positioning means can be provided which
  • the processing system proposed according to the invention has a control unit, the control unit being set up to use the processing system for generating, processing, stimulating and / or documenting the shape of three-dimensional composites, cells cultivated under the microscope at least partially in accordance with a procedure made before the generation, processing, stimulation and / or documentation, for example by a user
  • control specification can take place, for example, in accordance with a predetermined processing protocol, with, for example, a time sequence or metering of
  • the metered addition can in particular also take place for differentiation, that is to say for example for the generation of organoids, or for stimulation. Also an automated or partially automated observation or documentation that takes place over time by means of the microscope in a predetermined sequence and / or using predetermined ones
  • Detection modalities can be the subject of the present invention.
  • the use of the present invention significantly simplifies the generation, processing and / or stimulation as well as the documentation of corresponding toes, in particular because there are no or a reduced number of toes
  • the present invention also creates a higher level of reproducibility and the method can be carried out in a skill-neutral manner.
  • the state of the art the state of the art
  • the present invention goes beyond pure automation, since with it the steps mentioned can be carried out in particular in a single structural unit and in particular there is also no need to replace the culture units used in this process and to change pipetting or the like.
  • the culture unit can in particular be designed as an exchangeable culture plate, the
  • Culture chambers are arranged side by side in the culture unit. In this way, by shifting in the horizontal plane, each of the
  • Culture chambers are brought into the observation beam path of the microscope and the contents can be observed in this way.
  • the culture unit as an exchangeable culture plate, it can after processing in the
  • Culture chambers in particular have a cell-repellent coating. In this way, any interaction between the surface of the
  • Coatings are known to the person skilled in the art and are commercially available.
  • the at least one culture chamber is connected to a supply channel for supplying one or more fluids into the at least one culture chamber and / or to a discharge channel for discharging one or more fluids from the at least one culture chamber fluidic coupling, but not necessarily a fixed mechanical connection, should be understood.
  • a supply channel for supplying one or more fluids into the at least one culture chamber and / or to a discharge channel for discharging one or more fluids from the at least one culture chamber fluidic coupling
  • Feed channel for feeding the fluid or fluids into the at least one culture chamber and / or the discharge channel can be used for discharging the fluid or fluids, for example by means of a corresponding mechanical or spatial arrangement.
  • a discharge channel can, for example, also be designed as an immersion tube which is merely immersed in the culture chamber and in this way can draw off medium from the floor of the culture chamber or near the floor.
  • a supply channel can be designed as a feed tube which opens into the culture chamber or above the culture chamber.
  • a system of supply and discharge channels can also be used as a exchangeable or movable unit are provided separately from the culture unit or a culture plate.
  • the cells cultivated in the form of three-dimensional composites can be left in the respective culture chambers, for example to induce differentiation into different cell types, for stimulation, coloring, lightening or the like, and a costly transfer is not required. In this way, the implementation of corresponding experiments is simplified.
  • the supply channels of several culture chambers are connected to a common feed line and / or the discharge channels of several culture chambers are connected to a common removal line.
  • the feed line can in particular be set up on a reservoir for the one or more fluids, it being possible to provide a pump by means of which, in particular according to the control device, the fluid or fluids can be conveyed.
  • the extraction line which can be connected to a waste container in particular via a corresponding controllable pump.
  • a single pump with different fluid channels for example a peristaltic pump, can also be used.
  • the supply channel (s) and / or the discharge channel (s) can be set up for a flow that is based on a physiological
  • Flow rate is, in particular, a rate at which a fluid exchange would also take place in vivo, at which certain components are converted, or the like.
  • the setting can take place within the scope of the present invention in particular by a corresponding design of the supply channel or channels and / or the discharge channel or channels or by suitable dimensioning and / or control of a pump.
  • At least one of the supply channels and / or the discharge channels can be designed as a microfluidic channel in order to enable particularly careful metering in this way. It can also corresponding microfluidic mixing or metering devices are provided in order to be able to mix or meter fluids in a targeted manner, for example.
  • Culture chambers free of supply channels and / or discharge channels, so that in this way unimpeded observation through the microscope is made possible.
  • free of refers in particular to an arrangement in which the
  • the supply channel or channels of the at least one culture chamber, which has a supply channel is / are led laterally to the at least one culture chamber, the discharge channel or channels of the at least one culture chamber, which has a discharge channel, in particular opposite the supply channel or channels at least another of the culture chambers is or are brought in.
  • the discharge channels are advantageously each below the supply channels
  • a bottom of the at least one culture chamber has a thickness in a range from 5 to 300 ⁇ m, in particular in a range from 25 to 170 ⁇ m. In this way, sufficient stability can be achieved at the same time as low enough for higher magnifications
  • the bottom of the at least one culture chamber is advantageously designed to be transparent, with “transparent” being understood to mean a permeability for at least one wavelength of visible, infrared or ultraviolet light of at least 50%, 60%, 70%, 80% or 90%.
  • the transparency therefore does not have to be complete and for all wavelengths.
  • the bottom of the at least one culture chamber can in particular be made of glass, quartz or a cycloolefin copolymer.
  • Other alternatives are known and commercially available.
  • polytetrafluoroethylene materials in particular in transparent form, can be used.
  • Cells cultivated in three-dimensional assemblies are generated, processed, stimulated and / or documented in a culture unit which has a number of culture chambers in which the cells cultivated in the form of three-dimensional assemblies and a culture medium are accommodated.
  • the cells cultivated in the form of three-dimensional assemblies are stimulated under a microscope and, according to the invention, this is at least partially according to a prior to generation, processing, stimulation and / or
  • the method proposed according to the invention advantageously comprises an introduction or generation of the cells cultivated in the form of three-dimensional assemblies into at least one or one of the culture chambers.
  • the cells can therefore be cultivated there or introduced after cultivation.
  • Processing, stimulation and / or documentation can in particular
  • the cells can continuously receive the nutrients and nutrients required for growth
  • the at least one first medium can in particular have a growth medium which in particular contains one or more active ingredients, one or more dyes, one or more
  • At the second medium can in particular be used up, ie first medium changed due to the physiological activity of the cells.
  • the cells cultivated in the form of three-dimensional assemblies can advantageously remain permanently in the same culture chamber during processing, so that transfer is unnecessary.
  • the supply of the at least one first medium and / or the removal of the at least one first medium or the at least one second medium can, as mentioned,
  • the present invention also relates to a computer program that executes a method as explained above in any configurations when it is executed on a computer, in particular in a control unit of a processing system, as explained above in different configurations.
  • a hardware device such as a processor, a microprocessor, a
  • one or more of the most important method steps can be carried out by such a device.
  • Embodiments of the invention can be implemented in hardware or software.
  • the implementation can be carried out with a non-volatile storage medium such as a digital storage medium, for example a floppy disk, a DVD, a Blu-Ray, a CD, a ROM, a PROM and EPROM, an EEPROM or a FLASH memory, on which electronically readable
  • a non-volatile storage medium such as a digital storage medium, for example a floppy disk, a DVD, a Blu-Ray, a CD, a ROM, a PROM and EPROM, an EEPROM or a FLASH memory, on which electronically readable
  • Control signals are stored, which interact (or can interact) with a programmable computer system so that the respective method is carried out. Therefore, the digital storage medium can be computer readable.
  • Some exemplary embodiments according to the invention comprise a data carrier with electronically readable control signals, which with a programmable Computer system can interact so that one of the methods described herein is carried out.
  • exemplary embodiments of the present invention can be implemented as a computer program product with a program code, the program code being effective for executing one of the methods when the computer program product is running on a computer.
  • the program code can be stored, for example, on a machine-readable carrier.
  • machine-readable carrier is stored.
  • an embodiment of the invention is therefore a
  • Computer program with a program code for performing one of the methods described herein when the computer program runs on a computer is Another embodiment of the present invention.
  • a storage medium (or a data carrier or a computer-readable medium) having a computer program stored thereon for executing any of the herein
  • the data carrier, the digital storage medium or the recorded medium are usually tangible and / or not seamless.
  • Another embodiment of the present invention is an apparatus as described herein that includes a processor and the storage medium.
  • Another embodiment of the invention is a data stream or a
  • Signal sequence that represents the computer program for performing one of the methods described herein.
  • the data stream or the signal sequence can be configured in such a way that they are transmitted over a data communication connection, for example over the Internet.
  • Another embodiment comprises a processing means, for example a computer or a programmable logic device, which is configured or adapted to carry out one of the methods described herein.
  • a processing means for example a computer or a programmable logic device, which is configured or adapted to carry out one of the methods described herein.
  • Another embodiment of the present invention comprises a computer, on which the computer program for carrying out one of the methods described herein is installed.
  • Another embodiment according to the invention comprises an apparatus or a system that is configured to transmit (for example electronically or optically) a computer program for carrying out one of the methods described herein to a receiver.
  • the receiver can be, for example, a computer, a mobile device, a storage device, or the like.
  • the device or the system can for example comprise a file server for transmitting the computer program to the recipient.
  • a programmable logic device e.g., a field programmable gate array, FPGA
  • FPGA field programmable gate array
  • a field programmable gate arrangement can cooperate with a microprocessor to perform any of the methods described herein.
  • the methods are preferably performed by any hardware device.
  • Embodiments of the present invention can each also be used as an organoid
  • OMS Organoid Management System
  • organoid processing system contain an overall concept for the formation, staining, stimulation and the subsequent checking of three-dimensional cell cultures. This is achieved through the use of standardized and established consumables as well as already published and established protocols. Embodiments of the present invention reduce the effort for the individual manual
  • Steps during the work strand to a minimum and standardize the process. This primarily refers to the time-consuming sample transfer and pipetting steps that are affected by many sources of error. Failure to follow these steps not only reduces the workload but also leads to
  • One embodiment of the present invention is based on the use and modification of already existing multiwell plates (96 to 384 well format) WO 2020/182646 - 16 - PCT / EP2020 / 055984 a 25 to 170 mih thin base made of cycloolefin copolymer.
  • Multiwell plate formats are also possible.
  • the surface of the individual wells in the plate is cell-repellent due to chemical treatment or a corresponding coating and is both U-shaped and flat
  • the plate Due to its optical and chemical properties, the plate is resistant to organic solvents (e.g. BABB) and can also be used in fluorescence microscopy due to the low level of autofluorescence.
  • Organic solvents e.g. BABB
  • Media and chemicals for sample preparation are introduced into and removed from the multiwell plates via supply and discharge channels. These steps follow defined protocols that are stored in the system software and that control the procedure. This includes the feed and
  • the ID of the plate used can be recorded automatically (barcode, RFID or similar) and the data of the cells used can be saved and documented in the software together with the preparation protocol.
  • Embodiments of the present invention relate to the automation of an entire sample preparation work string of thick, three-dimensional cellular samples (cellular spheroids and organoids). There are widely published works for the individual steps in the sample preparation strand (see also Pubmed). This mainly affects the steps for that
  • Embodiments of the present invention also referred to as an organoid management system (OMS) or an organoid processing system, include a
  • One embodiment of the invention is based on a multiwell plate (96 to 384 well format) with a 25 to 170 ⁇ m thick base made of glass or cycloolefin copolymer.
  • Other multiwell plate formats are also possible.
  • the surface of the individual wells in the plate is cell-repellent due to chemical treatment or an appropriate coating and can be available in both a U-shape and a flat version. Due to its optical and chemical properties, the plate is suitable for microscopy as well as being resistant to organic solvents. Incoming and outgoing channels can enter the sample space
  • Media and chemicals for the preparation are supplied. These steps follow defined protocols that are stored in the system software and that control the procedure. This includes the supply and residence time of chemicals in the sample space for staining, blocking and washing steps.
  • the ID of the plate used is automatically recorded (barcode, RFID or similar) and the data of the cells used WO 2020/182646-18-PCT / EP2020 / 055984 are used, for example, in the software together with the
  • the first well has a supply channel for supplying media and a discharge channel for discharging media.
  • the bottom of the organoid processing system having a thickness in a range from 5 to 300 ⁇ m, preferably from 25 to 170 ⁇ m.
  • organoid processing system according to one of the preceding paragraphs A) to D), wherein at least one second well of the plurality of wells has a supply channel for supplying media and a discharge channel for discharging media, the supply channel of the first well with the supply channel of the second well is connected and wherein the discharge channel of the first well is connected to the discharge channel of the second well.
  • Feed channel of the first well is brought up.
  • organoid processing system wherein the organoid processing system is a multiwell plate.
  • a second medium is discharged through a discharge channel of the well in which the organoid is located.
  • FIG. 1 shows a processing system according to an embodiment of the present invention
  • FIGS. 2A to 2C illustrate a processing system according to an embodiment of the present invention in partial schematic representations
  • Figure 3 illustrates a method according to an embodiment of the invention in the form of a schematic flow chart.
  • elements that correspond to one another are possibly identical
  • FIG. 1 a processing system according to an embodiment of the present invention is schematically illustrated and illustrated overall at 100.
  • FIG. 2A shows a perspective view of part of the processing system 200
  • FIGS. 1 to 2B show an enlarged side view in a detail
  • FIG. 2A shows a perspective view of part of the processing system 200
  • FIGS. 1 to 2B show an enlarged side view in a detail
  • FIG. 2C is an enlarged plan view in detail. Figure 1 and Figures 2A to 2C are explained together below.
  • the culture unit 110 has a number of culture chambers 112 which are each set up to receive the cells 10 cultivated in the form of three-dimensional assemblies and a culture medium 20.
  • the culture chambers are arranged next to one another in the culture unit in the culture unit 110, which is in particular designed as an exchangeable culture plate.
  • a microscope 120 (shown greatly simplified and shown in Figure 2A as a block with a microscope objective 22) and a control unit 130 (shown in Figure 2A as a computer and otherwise very simplified) are also part of the processing system 100, the control unit 130 being set up to control the processing system 100 for generating, processing and / or stimulating the shape of three-dimensional assemblies cultivated cells 10 under the microscope 120 at least partially in accordance with a control specification made before the generation, processing and / or stimulation and without manual intervention.
  • wired or wireless communication links 132 are provided for the exchange between the control unit 130, which can also be integrated in other devices, and the rest of the processing system 100.
  • the microscope 120 can be an integral part of the system 100 or connected to the same.
  • the microscope 120 can be designed in particular to record images and is connected to the control unit 130, in particular designed as a computer system and described below as such.
  • the control unit 130 which, as mentioned, can in particular be designed as a computer system, is designed for
  • control unit 130 can execute a machine learning algorithm
  • the control unit 130 and the microscope 120 can be separate units, but can also be integrated together in a common housing.
  • the control unit 130 could be part of a central The processing system of the microscope 120 and / or the control unit 130 could be part of a subcomponent of the microscope 120, such as a sensor, an actuator, a camera or a lighting unit, etc. of the microscope 120.
  • the control unit 130 can be a local computer device (e.g. personal computer , Laptop, tablet computer or mobile phone) with one or more processors and one or more storage devices or can be a distributed
  • the controller 130 may include any circuit or combination of circuits.
  • the controller 130 may include one or more processors, which may be of any type.
  • processor can mean any type of computing circuit such as, but not limited to, a microprocessor, a microcontroller, a complex instruction set microprocessor (CISC), a reduced instruction set microprocessor (RISC), a very long instruction word - (Very Long Instruction Word; VLIW) microprocessor, a graphics processor, a digital signal processor (DSP), a multi-core processor, a field-programmable gate array (FPGA), e.g. one
  • control unit 130 may be a custom built circuit, an application specific integrated circuit (ASIC), or the like, such as one or more circuits (e.g., a communication circuit) for use in wireless devices such as a wireless device .
  • ASIC application specific integrated circuit
  • the control unit 130 can comprise one or more storage devices, which can comprise one or more storage elements suitable for the respective application, such as, for example, a main memory in the
  • the control unit 130 can also comprise a display device, one or more loudspeakers, and a keyboard and / or controller that includes a mouse, trackball, touchscreen, May include voice recognition device or any other device that allows a system user to input information into and receive information from control unit 130.
  • several culture chambers 112 are connected to supply channels 114 for supplying one or more fluids into the at least one culture chamber 112 and to discharge channels 116 for discharging one or more fluids from the at least one culture chamber 112. As illustrated in particular in the side view of FIG. 2B, the discharge channels 116 are arranged below the supply channels 114 or dip into the culture chambers 112.
  • the supply channels 114 of several culture chambers 112 are connected to a common one
  • Feed line 115 and the discharge channels 116 of a plurality of culture chambers 112 are connected to a common extraction line 117.
  • the feed line 115 is in turn connected to a reservoir system 150 via a (peristaltic) pump 140, which can be controlled by means of the control device 130, or in the latter to a fluid reservoir 152.
  • the extraction line 117 is connected via the pump 140, which is controlled by means of the
  • Control device 130 is controllable to a waste container 154 in the
  • FIG. 3 a method is illustrated in the form of a simplified process flow diagram and denoted by 200, which method is partially configured in accordance with an embodiment of the present invention.
  • a user carries out preliminary work and fills, for example, a fluid reservoir, for example the fluid reservoir 152 of the system 100, with a desired solution,
  • a waste container for example the waste container 154
  • a pump 140 is connected.
  • the system 100 After switching on the control unit 130, starting appropriate software and selecting a desired protocol in the software, the system 100 for example three times with a suitable washing buffer such as
  • PBS phosphate buffered saline
  • a parallel or subsequent step 202 that is also not yet
  • Culture chambers 122 pipetted, for example centrifuged and in a
  • step 203 a method is carried out in accordance with an embodiment of the invention. If not already done, the samples are washed, for example, three times with PBS after the protocol selected and processed in step 201 and a corresponding exposure on the microscope. The can be preserved via a fixation protocol and used for further workflows (e.g. clearing).
  • step 204 which again is not part of the
  • the system 100 is dismantled, for example, hoses are removed, containers dismantled and autoclaved, and the software is ended and the pump 140 is stopped.

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Abstract

L'invention concerne un système de traitement (100) pour des ensembles sous forme tridimensionnelle de cellules cultivées (10), pourvu d'une unité (110) de culture, qui présente un certain nombre de chambres (112) de culture, qui sont conçues pour recevoir les ensembles sous forme tridimensionnelle de cellules cultivées (10) et un milieu (20) de culture, d'un microscope (120) et d'une unité de commande (130), l'unité de commande (130) étant conçue pour commander le système de traitement (100) pour générer, traiter et/ou stimuler la forme d'ensembles tridimensionnels de cellules cultivées (10) au microscope (120) au moins en partie conformément à une spécification de commande définie avant la génération, le traitement et/ou la stimulation et sans intervention manuelle. L'objet de l'invention est également un procédé (200) correspondant.
PCT/EP2020/055984 2019-03-08 2020-03-06 Système de traitement et procédé de traitement pour ensembles sous forme tridimensionnelle de cellules cultivées et programme informatique Ceased WO2020182646A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6548263B1 (en) * 1997-05-29 2003-04-15 Cellomics, Inc. Miniaturized cell array methods and apparatus for cell-based screening
US20030186217A1 (en) * 2000-09-19 2003-10-02 Augustinus Bader Method and device for growing and/or treating cells
US20150247112A1 (en) * 2014-03-03 2015-09-03 Kiyatec Inc. 3D Tissue Culture Devices and Systems

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6548263B1 (en) * 1997-05-29 2003-04-15 Cellomics, Inc. Miniaturized cell array methods and apparatus for cell-based screening
US20030186217A1 (en) * 2000-09-19 2003-10-02 Augustinus Bader Method and device for growing and/or treating cells
US20150247112A1 (en) * 2014-03-03 2015-09-03 Kiyatec Inc. 3D Tissue Culture Devices and Systems

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