WO2023218024A1

WO2023218024A1 - System for automated and dynamic cell culture

Info

Publication number: WO2023218024A1
Application number: PCT/EP2023/062744
Authority: WO
Inventors: Sebastian Josef MAERKL; Julia TISCHLER
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2022-05-13
Filing date: 2023-05-12
Publication date: 2023-11-16

Abstract

The present relates to a system for automated cell culture, the system comprising an inflow module, a cell culture plate and an outflow module, said inflow module being in fluid connection with the outflow module via the cell culture plate, the inflow module further comprising a microfluidic distribution module in fluid connection with the inflow module, the microfluidic distribution module being configured for controlling the distribution of a fluid flow from the inflow module to the cell culture plate, characterized in that the cell culture plate comprises at least one culture well configured for cell culture, and in that the system further comprises an adaptor coupled to the microfluidic distribution module, the adaptor being reversibly connectable to the cell culture plate so that when the adaptor is connected to the cell culture plate, the distribution of the fluid flow to said culture well is controlled by the microfluidic distribution module.

Description

System for automated and dynamic cell culture

Technical Field

[001] The present disclosure relates to a system for automated cell culture.

Background of the art

[002] In vitro cell culture technologies provide powerful tools for comprehensively exploring the principles underlying developmental processes during mammalian embryogenesis, as well as disease onset and progression.

[003] Conventionally, mammalian cell culture is performed in batch and involves predominantly manual media exchange and sub-culturing routines, conducted in daily intervals at best. Manual cell culture techniques, however, are cumbersome and prone to operator error, making it difficult to achieve precisely controlled processes. The laborintensity of manual cell culturing approaches restricts the scope and complexity of possible investigations such as the effect of different types, doses, and temporal stimulation profiles of cytokines, drugs, small-molecule modulators, or alike, or combinations thereof.

[004] Critically, the build-up of cell-secreted factors and excreted metabolic waste products in batch cultures adversely impacts the quality, robustness and reproducibility of cell culture conditions and presents a challenge to precisely controlling cellular behavior and cell fate decisions, and to quantitatively predicting cell fate outcomes. Well- defined, precisely controlled, and highly consistent cell culture conditions are extremely desirable for cell-based therapies and biomedical applications.

[005] Most notably, conventional batch culture techniques afford extremely limited temporal control over the media composition, and largely preclude investigations of cellular behavior in response to complex, dynamically changing cell culture conditions/environments.

[006] Currently, however, simple and accessible tools are lacking that enable fully automated media exchange routines and the delivery of complex, dynamic inputs to a broad array of biologically relevant in vitro cell culture models, ex vivo cell and tissue cultures, and other biological systems and living organisms.

[007] The latest developments of microfluidic technologies for cell biology applications present a major milestone. Sophisticated microfluidic devices have been engineered for i the distribution of combinatorial and time-varying signals to hundreds of individually addressable miniature cell culture chambers and enabled the exploration of complex cellular behaviors in response to dynamic modulation of the cell culture conditions and cellular environment, at an unparalleled precision and scale.

[008] However, the small size of microfluidic devices, and the challenge of cell loading and subsequent recovery restrict the number of cells available for analysis and downstream applications, particularly in regards to the investigation of emerging 3D cell and tissue culture models in vitro and ex vivo, such as embryoids, gastruloids, organoids, spheroids, tumoroids, tissue explants, and other living systems, such as embryos or small animals (model organisms).

[009] Therefore, there is a need to provide an improved system to overcome or at least minimize the drawbacks of the existing culturing technologies and systems.

[010] In particular, there is a need to facilitate the cell culture, notably to improve the reproducibility and robustness of cell culturing technologies, in particular for mammalian cells and tissue culture models in vitro and ex vivo, such as embryoids, gastruloids, organoids, spheroids, tumoroids, tissue explants, and other living systems, such as embryos or small animals (model organisms).

Summary of the invention

[011] The above problems are solved or at least minimized by the device and the method according to present invention.

[012] The invention concerns a system for automated and dynamic cell culture, the system comprising an inflow module, a cell culture plate and an outflow module, said inflow module being in fluid connection with the outflow module via the cell culture plate, the inflow module further comprising a microfluidic distribution module in fluid connection with the inflow module, the microfluidic distribution module being configured for controlling the distribution of a fluid flow from the inflow module to the cell culture plate, characterized in that the cell culture plate comprises at least one culture well configured for cell culture, preferably a plurality of culture wells, and in that the system further comprises an adaptor coupled to the microfluidic distribution module, the adaptor being reversibly connectable to the cell culture plate so that when the adaptor is connected to the cell culture plate, the distribution of the fluid flow to said culture well is controlled by the microfluidic distribution module.

Description of the invention

[013] The invention concerns a system for automated cell culture, the system comprising an inflow module, a cell culture plate and an outflow module, said inflow module being in fluid connection with the outflow module via the cell culture plate, the inflow module further comprising a microfluidic distribution module in fluid connection with the inflow module, the microfluidic distribution module being configured for controlling the distribution of a fluid flow from the inflow module to the cell culture plate, characterized in that the cell culture plate comprises at least one culture well configured for cell culture, preferably a plurality of culture wells, and in that the system further comprises an adaptor coupled to the microfluidic distribution module, the adaptor being reversibly connectable to the cell culture plate so that when the adaptor is connected to the cell culture plate, the distribution of the fluid flow to said culture well is controlled by the microfluidic distribution module.

[014] The present invention concerns an automated cell culture system comprising a cell culture plate with at least one well configured for cell culture.

[015] Cell culture includes herein culture of one or a plurality of any kind of biological entity that can be cultured. In the frame of the present disclosure, cell culture particularly refers to the culture of at least one cell, preferably a plurality of cells, adhering to the cell culture plate or floating freely (cell suspensions or suspension culture cells of animal or human origin) or within (micro-) structures. Such single or plurality of cells include, but are not limited to, various stem cell populations (embryonic and adult stem cells and germ cells, including embryonic and adult ‘stem cell-like’ cells, and ‘germ cell-like’ cells, any cells, cell types, and cell type intermediates differentiated from said cells, and any derived tissues, embryoids, organoids, embryos, organs, or organisms), bone marrow or adipose tissue derived adult stem cells, mesenchymal stem cells, cardiac stem cells, pancreatic stem cells, neuronal cells, glial cells, spermatozoids and ovocytes, endothelial progenitor cells, outgrowth endothelial cells, dendritic cells, hematopoietic stem cells, neural stem cells, satellite cells, side population cells. Such cells may further include but are not limited to, differentiated cell populations including osteoprogenitors and osteoblasts, chondrocytes, keratinocytes for skin, intestinal epithelial cells, smooth muscle cells, cardiac muscle cells, epithelial cells, endothelial cells, urothelial cells, fibroblasts, myoblasts, chondroblasts, osteoclasts, hepatocytes, bile duct cells, pancreatic islet cells, thyroid, parathyroid, adrenal, hypothalamic, pituitary, ovarian, testicular, salivary gland cells, adipocytes and combinations thereof. The cell culture system according to the present disclosure is suitably adaptable to further operate with grains, seed or pollens.

[016] The cell culture plate comprises at least one culture well, preferably a plurality of culture wells. For instance, the culture plate comprises between about 8 to 24 wells. Preferably, the present invention is also compatible with cell culture plates comprising larger numbers of culture wells (i.e. , 48- to 96-well plates, or 384-well plates).

[017] Preferably, the culture well is configured for the culture of adherent cells and cellular aggregates, for instance stem cells, 3D cell culture systems, complex mammalian 3D cell culture systems, tissue explants and embryos, preferably embryoids, gastruloids, organoids, spheroids, tumoroids, preferably embryos, larvae and/or zygotes of human and animal origin, including model organisms such as mice, rats, pigs, fish (e.g., D. rerio), insects (e.g., D. melanogaster), amphibians (e.g. Xenopus laevis) and nematodes, (e.g., C. elegans), more preferably the cell culture well is configured for the culture of mammalian cells and cellular aggregates.

[018] In a preferred embodiment, the culture well is compatible with the culture of at least all the following biological systems: adherent cells and cellular aggregates, for instance 3D cell culture systems, tissue explants and embryos. Thus, the present invention offers a unique broad scope of biological system contrary to the existing techniques that are largely limited to the culture of one, at most few, specific biological systems. In particular, the existing technologies are not compatible with the culture of adherent mammalian cells or 3D cellular aggregates.

[019] The existing cell culture systems and culture devices are largely not suitable for the culture, dynamic stimulation, and subsequent recovery for downstream application and assays of the above listed cellular systems, in particular of cellular aggregates and 3D cell culture systems, notably because of the culture chambers used in these cell culture systems. Importantly, existing microfluidic distribution techniques have not been integrated with standard (multi-) well tissue culture plates contrary to the present invention.

[020] In the present invention, the system comprises an adaptor which is reversibly connectable to the cell culture plate. The adaptor acts as an interface between on one hand a microfluidic distribution module and on the other hand the cell culture plate. The role of the adaptor is to connect the microfluidic distribution module(s) with the culture plate comprising the culture well(s).

[021] With the adaptor, the fluid flow, comprising a composition of media (i.e., a medium or a liquid or a composition of liquids), dispensed to the cell culture well of the cell culture plate is controlled by the microfluidic distribution module. In other words, the cell culture plate is connected to the microfluidic distribution module via the adaptor, and this is not possible in the existing cell culture systems.

[022] The present invention allows to mimic standard tissue culture techniques for the cells cultured in the cell culture well, such as gentle and controlled media addition via the microfluidic inflow module, and removal via aspiration, with media routing through microfluidic outflow modules. The present invention circumvents shear-induced effects, frequently observed in existing microfluidic cell culture systems.

[023] Advantageously, in the present invention, it is possible to use standard cell culture plates, for instance standard multi-well tissue culture plates routinely used for the culture of the above listed types of cells and/or cellular systems, notably for the fully automated culture, in particular of adherent mammalian cells, and complex mammalian systems, including 3D cell culture systems, embryos and tissue explants, under dynamically modulated media formulations.

[024] The use of standard culture wells enables simple cell loading and recovery for downstream cell culture applications and molecular characterization.

[025] It also assures compatibility with different cell types, cellular, or model systems, specific types and dimensions of culture wells, cell-specific substrates and/or specific surface coatings, internal structures (such as microwells), etc., to meet specific experimental needs.

[026] The present invention allows to improve the reproducibility and robustness of cell culturing technologies in particular for mammalian cells, thanks to the accuracy and unparalleled precision provided by microfluidic distribution technology.

[027] The present invention also offers solutions for temporally modulating media compositions, in order to enable investigations on the impact of complex dynamic stimulation profiles, such as timed solution exchanges, step-wise increases or decreases in concentrations of, for instance, cytokines, metabolites, pharmacological or smallmolecule modulators, pharmaceutical compounds and alike, and/or the dynamic ramping up, and down to specified concentrations, on cellular behavior. Such sophisticated dynamic fluidic routines could enable systematic investigations of cellular decisionmaking and commitment during cell fate specification programs, for instance the dependence of cell fate outcomes on the precise timing and duration of a stimulatory pulse, cell fate determination as a function of the speed and frequency of cytokine presentation, entrainment of signaling pathway dynamics, and defined perturbations of molecular clocks through the delivery of complex oscillatory inputs, and/or the emulation of intricate pharmacodynamic profiles for toxicology studies. [028] The present invention further comprises an inflow module, a cell culture plate, an outflow module, a microfluidic distribution module in fluid connection with the inflow module, the microfluidic distribution module being configured for controlling the distribution of a fluid flow from the inflow module to the cell culture plate. Advantageously, the microfluidic distribution module allows for independently addressing distinct cell culture wells, preferably culture wells, that are sequentially addressed with individual media formulations.

[029] Preferably the microfluidic distribution module is programmable so that the action executed by the microfluidic distribution module can be automated.

[030] Preferably, the microfluidic distribution module comprises a mixing unit for controlling the composition of the fluid flow, for instance when the fluid flow is a composition of various media, the mixing unit controls the ratio of the various media composing the fluid flow. For instance, when the fluid flow is a composition of various media, the mixing unit controls the ratio of the various media composing the fluid flow. Advantageously, the mixing unit may be embodied as a microfluidic pulse width modulation (PWM) module. The microfluidic mixing module enables complex real-time media formulation, thus the dynamic formulation of input solutions by alternately opening and closing of inflow channels drawing liquid from distinct media sources. Temporally modulating the flow times of specific media inputs generates dynamically varying media compositions and/or concentration profiles, enabling timed solution/media switches, and dynamic concentration changes.

[031] In a preferred embodiment the microfluidic distribution module comprises a multiplexing unit controlling the distribution of the fluid flow to said culture well, preferably the multiplexing unit is in fluid connection with the mixing unit. Advantageously, the multiplexing unit enables intricate fluidic operations with minimum control inputs.

[032] In a preferred embodiment the microfluidic distribution module is operated in a programmable manner to control the fluid flow, preferably the composition of the fluid flow and/or the dynamic modulation of the fluid composition, distributed to said culture well of the culture plate. In particular, this allows for independently addressing individual, parallel culture wells, each with individually customizable, time-varying media formulations, for instance by the programmable operation of microfluidic valves within the mixing and distribution modules.

[033] Preferably, the fluid flow from the inflow module to the outflow module is pressure driven, preferably at least one pressure unit is configured for dispensing the fluid flow to said culture well and/or removing media from said culture well. This supports facile setting and/or tuning of the flow rate of the fluid flow dispensed to the well of the cell culture plate. [034] Preferably, the fluid is driven by using a gas supply to drive the fluid flow. The gas supply is preferably coming from a pressurized gas container, acting as a pressure unit, to drive the fluid flow. For instance, the gas comprises 5% CO2 for the culture of mammalian cells. The gas container is connected to the inflow module, for instance to the container(s) of the inflow module to pressurize the container(s).

[035] The present invention comprises an adaptor coupled or connected to the microfluidic distribution module, the adaptor being reversibly connectable to the cell culture plate so that when the adaptor is connected to the cell culture plate, the distribution of the fluid flow to said culture well is controlled by the microfluidic distribution module.

[036] The adaptor is reversibly connectable to the cell culture plate, meaning the adaptor can be connected and disconnected from the cell culture plate. The reversible attachment of the adaptor to the cell culture plate enables the culture of cells and cellular aggregates in any standard tissue culture plate, and thus the cells’ accustomed cell culturing environment (i.e., a specific culture well format or type, with defined dimension, culture volume, substrate, surface coating, internal structures, etc.). This further allows for simple cell loading, and cell recovery at any given time, notably for subsequent downstream cell culture, in vivo applications, and functional and molecular characterization.

[037] In a preferred embodiment the adaptor is configured for maintaining an unpressurized atmosphere in said culture well, preferably equal or below atmospheric pressure. It is particularly advantageous to maintain the cells of the culture well in an unpressurized atmosphere, in order to mimic and recapitulate standard cell culturing protocols and techniques, in other words to be comparable to standard cell culture conditions.

[038] In a preferred embodiment the adaptor comprises a lid to cover said culture well and a tubing system comprising a plurality of tubing, said tubing being sealingly mounted through the lid. The tubing is sealed through the lid to maintain a sterile environment in the culture well.

[039] In another embodiment, said tubing is integrated within the lid. Alternatively, a plurality of channels can be embedded or integrated within the lid, with each tubing/channel entering an individual cell culture well from the top.

[040] In a preferred embodiment, access ports are drilled into the lid and fitted with the tubing from the tubing system, each tubing entering the individual cell culture well from the top. In another embodiment, fluidic channels directly integrated within the plate lid can substitute for tubing, at least partially.

[041] Preferably, said tubing system comprises for each culture well at least - a media inflow tubing in fluid connection with one individual channel for dispensing the fluid flow to said culture well, and

- an outflow tubing for removing the fluid from said culture well.

[042] This allows an individual control of the fluid flow dispensed to and removed from each culture well. Preferably, the outflow tubing is configured for removing completely the liquid of the culture well. Complete media removal from the culture well facilitates instantaneous media changes, and thus the discrete switching to newly formulated media compositions. This is preferably performed in conjunction with wash steps, implemented for clearing the inflow module notably the distribution module, and connecting tubing of media formulations from the directly preceding media composition. In another embodiment, the outflow tubing is configured to partial media exchanges only. This is relevant to remove only part of the media composition of the well, for instance, if potentially unknown cell-secreted factors are beneficial or essential for desired cellular behavior (i.e., growth and/or proliferation and/or differentiation of cells and cellular systems), or for the at least partial retention of cells cultured in suspension during liquid exchange cycles.

[043] Preferably, the tubing system further comprises a level setting tubing for adjusting the level of fluid in said culture well, preferably one level setting tubing per culture well. The level setting tubing allows to control and/or adjust the upper level of fluid, and thus the liquid volume in the culture well. This feature is advantageous as it enables the culture of cells in a precisely pre-defined and reproducibly established media volume, thus the recapitulation of, and the direct comparison to standard (manual) cell culturing approaches and protocols. This allows to maintain the culture volume as used for standard manual cell culturing approaches.

[044] In a preferred embodiment, the length of the level setting tubing determines the upper medium level, and thus the culture volume. In a preferred embodiment, the length of the level setting tubing, preferably in combination with the outflow tubing, defines the volume of medium that is replaced in each culture well, for instance during each liquid exchange cycle.

[045] In a preferred embodiment the tubing system further comprises a gas supply tubing for directly perfusing the media reservoir(s) and/or mammalian cell culture chamber with a gas, for instance CO2, for controlling the environment, preferably the gas composition environment, in said culture well. Pressurizing the media reservoirs with CO2, for instance 5 % CO2, pre-conditions the media and enables culturing of CO2-dependent cells, such as embryonic stem cells (ESCs), or ESC-based cellular aggregates and 3D cell culture models. [046] Preferably, additional supply tubing can be integrated to allow direct gas-, for instance 5 % CO2-perfusion into the head-space (above the media) of individual culture wells (demonstrated for the culture of mouse 3D gastruloids). The present invention thus directly integrates atmospheric (i.e., CO2-) control. In a preferred embodiment the gas supply tubing is routed through a humidifier assembly for directly supplying the cell culture chamber with pre-conditioned I humidified gas. Regarding the head space, for example, if a well is 1 cm deep, and it is only filled to half, i.e., to 0.5cm, the top half is considered as the 'head-space'.

[047] Preferably, the inflow module comprises one container with predefined media composition or a plurality of, preferably pressurized, media containers each comprising a predefined media composition.

[048] Preferably, the outflow module comprises a microfluidic outflow module in fluid connection with said culture well of the culture plate, preferably a programmable microfluidic outflow module, configured for controlling the collection of the fluid flow removed from said culture well. This allows to control the fluid outflow, and thus remove liquid from said culture well, for instance to control the media level setting, and the full or partial emptying of fluid from the culture well.

[049] In a preferred embodiment the outflow module further comprises at least one collection container to collect the fluid flow removed from the culture well.

[050] In a preferred embodiment, the system according to the invention comprises one or more sensors operatively connected thereto.

[051 ] In a still preferred embodiment, the system further comprises one or more sensors operatively connected thereto and to (a) measurement device(s) and controller system(s) enabling the implementation of a closed feed-back loop, wherein one or more parameters of the system can be modulated and/or adjusted in response to parameters measured by the sensor(s).

[052] A “closed-loop system”, also known as a feedback control system, refers herein to a control system which uses the concept of an open loop system (in which the output has no influence or effect on the control action of the input signal) as its forward path but has one or more feedback loops (hence its name) or paths between its output and its input. The reference to “feedback” means that some portion of the output is returned back to the input to form part of the system’s excitation. Closed-loop systems are usually designed to automatically achieve and maintain the desired output condition by comparing it with the actual condition. It does this by generating an “error” signal which is the difference between the output and the reference input. In other words, a closed-loop system is a fully automatic control system in which its control action is dependent on the output in some way.

[053] Preferably, the cell culture plate is placed on a microscope stage. Placing the setup onto a microscope stage allows real-time imaging, supporting validation of dynamically modulated media compositions through the real-time tracking of supplemented fluorescent dye tracers, and direct observation of cellular or organismal behavior. Preferably the microscope stage is a motorized microscope stage, preferably enclosed in a temperature-controlled chamber.

[054] Still preferably, an environmental control system or chamber is operatively coupled with the microscope stage, said environmental control system or chamber being equipped with a sensor or multiple sensors to measure and regulate environmental parameters central to the culture of biological systems (i.e., temperature, humidity, gas composition, etc.).

[055] Advantageously, in a further embodiment, sensors may be directly integrated within the adaptor. Sensors can include electrical sensors, reaching into individual culture chambers or positioned within the media outflow path, measuring critical biochemical and/or metabolic parameters, such as, for instance, but not limited to, glucose, lactate, or ammonia. ‘Externally’ positioned sensors, that is, sensors that are not in direct contact with the fluid flow I culture medium, can be embedded within the lid and record critical environmental parameters, such as temperature, humidity, or gas composition. Spectrophotometric sensors, for instance, can be attached to the lid and report on pH or liquid level changes.

[056] As used herein, the word "means" (singular or plural) preceded or followed by a function can be replaced by the word "unit" or "module". For instance, "pressure means" can be replaced by "pressure module " or "pressure unit".

Brief description of the drawings

[057] Further particular advantages and features of the invention will become more apparent from the following non-limitative description of at least one embodiment of the invention which will refer to the accompanying drawings, wherein

Figure 1 is an overview of a first embodiment of the system according to the present invention;

Figure 2 is a detailed view of the microfluidic distribution module of the system represented in figure 1 comprising a mixing unit and a multiplexing unit;

Figure 3 is a detailed view of the outflow module of the system represented in figure 1 ; Figure 4 illustrates views of the wells of the cell culture plate of the system represented in figure 1 equipped with an adaptor;

Figure 5 is a schematic representation of an embodiment of a closed-loop feedback system comprising a sensor.

Detailed description of the invention

[058] The present detailed description is intended to illustrate the invention in a non- limitative manner since any feature of an embodiment may be combined with any other feature of a different embodiment in an advantageous manner. The present invention is not limited to the embodiment represented in figures 1 to 5.

[059] Figure 1 is an overview of a system 1 according to the present invention. In the present example, the system 1 is dedicated to the culture of mammalian cells in an automated manner, namely mouse embryonic stem cells, their induced conversion into epiblast-like cells, and mouse 3D gastruloids, respectively (but the invention is not limited to these types of cells and cellular systems).

[060] The system 1 comprises an inflow module 2, a cell culture plate 3 and an outflow module 4. The inflow module 2, the cell culture plate 3 and the outflow module 4 are in fluid connection. The inflow module 2 further comprises a microfluidic distribution module 5 in fluid connection with the inflow module 2. In other words, the inflow module 2 dispenses a fluid flow to the microfluidic distribution module 5 which is in charge of distributing the fluid flow to the cell culture plate, the fluid flow being eventually collected by the outflow module 4.

[061] In the present non-limiting example, the fluid flow is switched between a composition of various media for the automated culture and timed stimulation of mouse 3D gastruloids. The inflow module 2 comprises a first media container 6 (i.e. , first solution) and a second media container 7 (i.e., second solution) as media sources which are in fluid connection with the microfluidic distribution module 5. In the present example, the first media container 6 comprises 3pM CHIR99021 (Merck/Millipore, 361559) in N2B27 supplemented with 10011 ml’¹ PS (N2B27/PS), and fluoresceinisothiocyanat (FITC)- dextran 10 kDa solution (Sigma, FD1 OS) at a final concentration of 2.5pM as a tracer and the second media container comprises N2B27/PS.

[062] In the present non-limiting example, the microfluidic distribution module 5 comprises a mixing unit 8 and a multiplexing unit 9, the mixing unit 8 being in fluid connection with the multiplexing unit 9, as illustrated in figure 2. The mixing unit 8 receives the media from the first container 6 and the second container 7 and regulates the inflow of solution contained in the first media container 6 versus the solution contained in the second media container 7. [063] In the present non-limiting example, the mixing unit 8 is a microfluidic pulse width modulation (PWM) module 10 capable of dispensing predetermined I preprogrammed pulses of the solution 1 and solution 2 in the fluid flow to control the composition of said fluid flow. In the present example, the mixing unit 10 comprises six inflow channels 11 branched on a main inflow channel 12, each inflow channel 11 drawing inflow solution from a solution container 6,7. In the present example, the PMW is connected to two solution containers 6,7, the first solution container 6 is branched on a first inflow channel 13 whereas the second solution container 7 is branched on a second inflow channel 14.

[064] In an alternative embodiment, to maximize the concentration range that can be generated or the complexity of the media composition(s), the exemplary six containers may each comprise individual solutions, so that a container with a distinct drug concentration or media composition is each routed through individual inflow channel.

[065] The PWM achieves the generation of time-varying media compositions via dynamic formulation of the fluid flow through alternating the opening and closing of inflow channels 11 drawing liquid from distinct media sources. The flow into the inflow channels 11 , which are converging into the main inflow channel 12, is controlled through the programmed operation (opening and closing) of the inflow valves 15 (or inflow control valves 15), one inflow control valve 15 per inflow channel 11.

[066] The multiplexing unit 9 is a microfluidic multiplexing (MUX) unit 16 downstream of the PWM module 10. The MUX 16 is an interface between the PMW 10 and the cell culture plate 3. The MUX 16 distributes the fluid flow from the main inflow channel 12 to each of the parallel culture wells 17 of the culture plate 3 via a plurality of individual channels 18 branched off from the main inflow channel 12. In the present example, the MUX 16 offers the ability to independently address up to eight culture wells 17 in parallel. Each individual channel 18 can be opened or closed by operating a unique series of three valves 19 (or control valves 19) in order to sequentially route the liquid flow addressed or directed to each individual culture well 17. For instance, operation of the microfluidic control valves is mediated through generic solenoid valves.

[067] In the present example, the microfluidic inflow module 2 is operated in a programmable manner to control the composition of the fluid flow distributed to each well 17 of the cell culture plate 3. In other words, a script or a program is supplied (for instance implemented in LabVIEW) in which instructions for dynamic media formulations are encoded through simple mathematical commands and/or operations. The user-defined program specifies which inflow channel 11 to draw liquid from and executes mathematically encoded pulse durations through operating the corresponding inflow control valves 15 in order to achieve the desired output concentrations or dispense the sought solutions. The total cycle time (the sum of alternating liquid pulses) can be set and/or adjusted in the user interface of the program. The program is configured for operating the opening/closing of all inflow control valves 15. In the present example, the program is configured for alternating the opening and closing of inflow channels 13 and 14, through individually operating the corresponding control valves 15 to adjust the ratio of first solution drawn from solution container 6 versus second solution drawn from solution container 7 in the composition of the fluid flow. The program is further configured for operating the individual control valves 19 in specific combinations so that each culture well 17 can be individually addressed and filled with a desired media composition or concentration (i.e., defined by the length of alternating pulses drawing liquid from the first solution and second solution).

[068] For instance, the present invention is configured for achieving two fully automated modes of operation. A ‘cell culture mode’ to perform complete media exchanges in the culture well(s) at user-defined intervals, and an integrated ‘dynamic mode’, to enable timed media switches, and complex real-time media formulation via the microfluidic pulse width modulation (PWM) module 16, with the capability to draw liquid from up to 6 different liquid containers. As it will be apparent for a person skilled in the art, and for the sake of clarity, the present examples can be generalized to envisage for instance more than six containers and/or input solutions without prejudice to the inventive concepts behind the present invention.

[069] The cell culture plate 3 in the present example comprises 8 culture wells 17. The culture plate 3 is a standard tissue culture plate, for instance, a standard 24-well polystyrene plate, or an imaging-compatible 24-well plate used for the culture of adherent mammalian cells, including mouse embryonic stem cells and differentiated derivatives, or a Gri3D hydrogel microwell array 24-well plate configured for mouse 3D gastruloid cultures. In another embodiment, the cell culture plate may comprise any plate (standard or custom-made) or any alternative device suitable for the culture of cells.

[070] The system 1 further comprises an adaptor 20, downstream of the MUX 16, reversibly connectable to the cell culture plate 3. When the adaptor is connected to the cell culture plate 3, the fluid flow from each individual channel 18 is distributed to a corresponding culture well 17. In the present example, the adaptor 20 comprises a lid 21 to cover said culture well 17 and a tubing system 22 comprising a plurality of tubing to control the media volume M present in each culture well 17.

[071] The tubing system 22 comprises

- a media inflow tubing 23 in fluid connection with one individual channel 18 of the MUX 16 for dispensing the composition of the fluid flow until a liquid volume above the target media volume M in the culture well 17 is achieved; - a (preferably media) outflow tubing 25 for removing part of, or, preferably, all of the fluid from the culture well 17;

- a (preferably level) setting tubing 24 for adjusting the level of fluid (i.e. , composition) in the culture well 17 to a pre-determined or pre-set culture volume.

[072] In the present example the lid 21 comprises substrate plate lid 26 and a sealing layer 27 covering said substrate plate lid 26 on the inside. For instance, the substrate plate lid 26 can be a polystyrene plate lid, and the sealing layer 27 can be made of PDMS. The sealing layer 27 could be any substance, preferably liquid, preferably viscous, that dries or cures into a dry layer, though that does not emit any cytotoxic compounds.

[073] Preferably, the PDMS layer is between about 0.1 mm to about 5 mm, more preferably between about 0.5 mm to 2.5 mm, preferably between about 0.5 mm and about 1.5 mm, for instance about 1 mm thick. The sealing layer 27 is used to slightly stabilize the tubing system and also contribute (even partially) to seal the lid when the drilled holes in the lid are larger than the outer diameter of the tubing.

[074] In other embodiment, the lid 21 comprises an alternative, or, preferably, additional sealing layer 27 on top of the plate lid, which is, or can be limited to the surrounding of the tubing system, meaning applied in the surrounding areas of a or each tubing where the tubing is inserted through the drilled holes in the plate lid.

[075] The sealing layer(s) ensure(s) the tubing system 22 is sealingly mounted through the lid 21. The top and bottom sealing layers together provide additional stability to the tubing system, and protect cell cultures within the plate from contamination through the surrounding environment. Access ports 28 are machined or prepared in the adaptor 20 so that each tube is mounted in a corresponding access port 28. In the present example, the access ports 28 are drilled into the lid 21 . The tubing system 22 within adaptor 20 can further comprise metal pins (i.e., small metal tubing, not represented in figures), preferably one pin per access port 28 at the I iquid-/m ed ia-Zcell cultures-facing side, for instance, for providing additional stability, and for facilitating straight-forward modifications of fluidic interconnects reaching into the cell culture wells, such as varying the insertion depth of the pin in order to readily re-adjust the culture volume. Media efflux tubing, for instance, can be fitted with a kinked metal pin, directed at the edge of the well, to minimize and/or prevent aspiration of cells during full emptying cycles. Preferably one additional pin per access port 28 was fitted for straightforwardly connecting to the microfluidic modules.

[076] Preferably, the culture volume M, and the volume of media that is replaced during each liquid exchange cycle, respectively, is set by the distance of the level setting and emptying outflow pins, respectively, from the bottom of the culture well. Tubing diameter, length and insertion depth, type, and shape of the metal pins can be tailored to specific experimental parameters and requirements.

[077] The level setting tubing 24 and the outflow tubing 25 are connected to outflow module 4 to collect the fluid (i.e., composition) removed from the culture well 17. Preferably, as shown in figure 3, the outflow module 4 comprises:

- a collection plate (preferably a microfluidic collection plate) connected to one type of tubing (setting or outflow) in other words in charge of routing of the outflow of one type of tubing; and

- an outflow container connected to the collection plate;

In the present example, the outflow module 4 comprises a setting outflow collection plate 29 branched to the setting tubing 24, the outflow collection plate 29 being in fluid connection with a setting outflow collection container 30 to collect the fluid removed from the setting tubing 24. Similarly, the outflow module 4 comprises an outflow collection plate 31 branched to the outflow tubing 25, the outflow collection plate 31 being in fluid connection with an outflow collection container 32 to collect the fluid removed from the outflow tubing 24. Preferably, individual channels within the setting outflow collection plate 29 are connected to individual setting tubing 24, with the outflow collection plate 29 being in fluid connection with at least one level setting outflow collection container 30 to collect the fluid removed from the cell culture plate via setting tubing 24. Likewise, individual channels within outflow collection plate 31 are connected to individual outflow tubing 25, with the microfluidic outflow collection plate 31 being in fluid connection with at least one media outflow collection container 32 to collect the fluid removed from the cell culture plate via the (i.e., emptying) outflow tubing 25.

[078] An example of a cycle is described below:

- a) filling: the culture well 17 is filled with the appropriate volume of media composition via the media inflow tubing 23 (filling times can be set in the userinterface of the program, for instance; duration: for instance, between about 75 s to about 270 s, depending on the desired culture volume and tubing diameter);

- b) the media volume M is adjusted via the level setting tubing 24 (level setting times can be set in the user-interface of the program, for instance; duration: for instance, between about 30 s to 60 s);

- c) incubation time state: the cells are cultured I incubated in the media composition for a user-defined length of time (culturing I incubation times can be set in the userinterface of the program, for instance; duration: for instance, between about 1 h to 6 h);

- d) media removal: the fluid is removed completely via the outflow tubing 25 (emptying times can be set in the user-interface of the program, for instance; duration: for instance, between about 45 s to 120 s, depending on the culture media volume and tubing diameter);

[079] In the present example, the fluid flow from the inflow module 2 into the cell culture plate 3 is pressure-driven. The system 1 comprises a vacuum unit 33 (vacuum pump) connected to the setting outflow collection container 30 and to the emptying outflow collection container 32 in order to remove the fluids from the culture wells via the corresponding tubing by suction.

[080] For instance, at an inflow pressure of 5psi, with inflow media routed through a single microfluidic channel, one complete cycle (steps a to d above) of a single 24-well culture well required less than three minutes.

[081] For instance, at an inflow pressure of 10psi, with inflow media routed through the microfluidic PWM-MUX module 9, one complete cycle (steps a to d above) of a single 24- well culture well required between four to seven minutes.

[082] Preferably, in order to prevent media formulations from preceding PWM cycles, remaining as ‘dead volume’ within the PWM-MUX module and connecting tubing, from contaminating the media compositions formulated by the active PWM cycle, half-wash cycles were integrated within the dynamic operation mode of the automated cell culture platform (ACCP). Half-wash cycles were achieved through the topping up of the culture well to 150% of its volume with the newly generated media formulation, followed by complete emptying, re-filling to its standard volume, and re-setting of the liquid level. In other embodiment, inflow modules, tubings, and/or channels can be purged through flowing ‘wash solutions’, such as phosphate buffered saline (PBS) or unconditioned medium via a designated channel, collection module and container, possibly even bypassing the culture wells.

[083] Media inflow is pressure-driven and can be easily adjusted by tuning the pressure. For media supply, tubings from pressurized media containers 6,7 were plugged into the inflow module, preferably into the inlets of the inflow module. Pressurizing mediacontaining bottles with pre-mixed 5% CO2 saturates the media and supports the culturing of CO2-dependent cells. Media is kept in reservoirs under CO2-pressure, at 37°C, for a maximum of 48h. This corresponds to standard culturing conditions in incubators, where, for instance, naive mouse embryonic stem cells are maintained at 37°C under CO2- atmosphere, routinely with media exchanges on alternate days. [084] In another embodiment, media reservoirs are placed within a cooling container or system.

[085] In another embodiment, which can be combined with any of the above embodiments, the system according to the invention comprises one or more sensors for measurement and analysis of parameters of the system itself.

[086] In this embodiment, one or more sensors 34 are operatively coupled or integrated within the lid 21 , first and/or second media containers 6, 7, culture plate 3, inflow/outflow channels, and preferably the adaptor 20, or any other suitable portions of the system in order to measure values or changes in one or more parameters such as pH, temperature, humidity and the like of the system.

[087] Accordingly, sensors suitable for operative coupling with the system of the invention can be selected from a non-limiting list including pH sensors, temperature sensors, humidity sensors, optical sensors to measure changes in turbidity or media colours, gas sensors or flow sensors, to cite a few.

[088] According to one characteristic, the adaptor 20 comprises at least one sensor 34.

[089] According to the previous characteristic, the adaptor 20 comprises at least one sensor located within the lid 21 .

[090] In some alternative or additional embodiments, sensors 34 include for instance electrical sensors reaching into individual culture wells 17 or positioned within the media outflow path for measuring critical biochemical and/or metabolic parameters, such as, for instance, glucose, lactate, or ammonia concentration.

[091] In some alternative, sensors 34 include spectrophotometric sensors attached to the lid 21 for measuring pH or liquid level changes.

[092] In another preferred embodiment, exemplarily depicted in figure 5, the system comprises a closed-loop feedback system.

[093] In this embodiment, sensors 34 are connected with one or more measurement device(s) and controller system(s) enabling the implementation of a closed feed-back loop

[094] According to the latter embodiment, the fluid and/or gas flow and composition can be modulated and adjusted in response to parameters measured by the sensor(s), and possibly in real time.

[095] According to another characteristic, a closed-loop feedback system comprises at least one sensor 34 operatively connected with at least one measuring device 35. [096] The measuring device 35 receives a first value of a fist parameter to be analyzed at time TO. The measuring device 35 then receives a second value of said fist parameter to be analyzed at time T1 and, through e.g. a computer device comprising a processor unit, determines a difference over time in said analyzed parameter.

[097] According to the latter characteristic, a controller unit 36 is operatively connected with the measuring device(s) 35 and with the system of the invention through any suitable portion thereof.

[098] The controller unit 36 modulates upon the system to re-set, modify (such as increase or decrease) or otherwise adjust the altered parameter to a pre-determined value, previously registered and stored in a memory unit.

[099] For instance, a temperature sensor 34 is coupled with (a) culture well(s) 17 to measure temperature of a medium located therein, the temperature sensor 34 being further connected to a temperature measurement device 35.

[0100] A plurality of sequential measurements can be pre-programmed at even timepoints, such as for instance every hour. The information registered by the temperature measurement device 35 are sent to a computer device acting as a controller unit 36. In case of changes or anomalies in the registered temperature, the controller unit 36 may operate on the inflow module 2 to inject fresh medium inside the culture well(s) 17 and eliminate old medium through the outflow module 4, until the sensed temperature goes back to normal, pre-set values.

[0101 ] Similar operations can be effected for each any every desired measurable parameter of the culture system, provided a sensor 34 is present. A skilled person would easily envisage sensors, parameters and operations to be adapted according to the need and circumstance.

[0102] While the embodiments have been described in conjunction with a number of embodiments, it is evident that many alternatives, modifications and variations would be or are apparent to those of ordinary skill in the applicable arts. Accordingly, this disclosure is intended to embrace all such alternatives, modifications, equivalents and variations that are within the scope of this disclosure. This, for example, is particularly the case regarding alternative systems and techniques for dynamic fluid I media formulation, liquid mixing and routing to the adaptor and to I from the culture wells, the different biological and cellular systems cultured, and the specific culturing protocols and techniques applied, in particular in regards to the culture and dynamic stimulation of cells and biological systems of any kind under temporally modulated media formulations, including, but not limited to, dynamically varying drug dosing schemes. REFERENCE NUMBERS

1 System according to the present invention

2 Inflow module

3 Culture plate

4 Outflow module

5 Distribution module

6 First media container

7 Second media container

8 Mixing unit

9 Multiplexing unit

10 Microfluidic pulse width modulation (PWM) module

11 Inflow channel

12 Main inflow channel

13 First inflow channel

14 Second inflow channel

15 Inflow valves

16 Microfluidic multiplexing unit (MUX)

17 Culture well

18 Individual channel

19 Individual valve

20 Adaptor

M Media volume

21 Lid

22 Tubing system

23 Media inflow tubing

24 Setting tubing

25 Outflow tubing Substrate plate

Sealing layer

Access port

Setting outflow collection plate

Setting outflow collection container

Outflow collection plate

Outflow collection container

Vacuum unit

Sensor

Measurement unit

Control unit

Claims

CLAIMS System (1 ) for automated cell culture, the system (1 ) comprising an inflow module (2), a cell culture plate (3) and an outflow module (4), said inflow module (2) being in fluid connection with the outflow module (4) via the cell culture plate (3), the inflow module (2) further comprising a microfluidic distribution module (5) in fluid connection with the inflow module (2), the microfluidic distribution module (5) being configured for controlling the distribution of a fluid flow from the inflow module

(2) to the cell culture plate (3), characterized in that the cell culture plate (3) comprises at least one culture well (17) configured for cell culture, and in that the system (1 ) further comprises an adaptor (20) coupled to the microfluidic distribution module (5), the adaptor (20) being reversibly connectable to the cell culture plate (3) so that when the adaptor (20) is connected to the cell culture plate

(3), the distribution of the fluid flow to said culture well (17) is controlled by the microfluidic distribution module (5). System (1 ) according to claim 1 , wherein when the adaptor (20) is configured for maintaining an unpressurized atmosphere in said culture well, preferably equal or below atmospheric pressure. System (1 ) according to claim 1 or 2, wherein said culture well (17) is configured for the culture of a single or a plurality of biological entities selected from a non-limiting list comprising embryonic stem cells differentiated into various cell types, bone marrow or adipose tissue derived adult stem cells, mesenchymal stem cells, cardiac stem cells, pancreatic stem cells, neuronal cells, glial cells, spermatozoids and ovocytes, endothelial progenitor cells, outgrowth endothelial cells, dendritic cells, hematopoietic stem cells, neural stem cells, satellite cells, side population cells, osteoprogenitors and osteoblasts, chondrocytes, keratinocytes for skin, intestinal epithelial cells, smooth muscle cells, cardiac muscle cells, epithelial cells, endothelial cells, urothelial cells, fibroblasts, myoblasts, chondroblasts, osteoclasts, hepatocytes, bile duct cells, pancreatic islet cells, thyroid, parathyroid, adrenal, hypothalamic, pituitary, ovarian, testicular, salivary gland cells, adipocytes and combinations thereof; grains, seed or pollens; of adherent cells and cellular aggregates, 3D cell culture systems, complex mammalian 3D cell culture systems, tissue explants and embryos, preferably embryoids, gastruloids, organoids, spheroids, tumoroids, preferably embryos, larvae and/or zygotes of human and animal origin, including model organisms such as mice, rats, pigs, fish (e.g., D. rerio), insects (e.g., D. melanogaster), amphibians (e.g. Xenopus laevis) and nematodes, (e.g., C. elegans).

4. System (1 ) according to any one of claims 1 to 3, wherein the microfluidic distribution module (5) comprises a mixing unit (8) for controlling the composition of the fluid flow, for instance when the fluid flow is a composition of various media, the mixing unit (8) controls the ratio of the various media composing the fluid flow.

5. System (1 ) according to claim 1 to 4, wherein the microfluidic distribution module (5) comprises a multiplexing unit (9) controlling the distribution of the fluid flow to said culture well (17), preferably the multiplexing unit (9) is in fluid connection with the mixing unit (8).

6. System (1 ) according to any one of claims 1 to 5, wherein the microfluidic distribution module (5) is operated in a programmable manner to control the fluid flow, preferably the composition of the fluid flow, and/or the dynamic modulation of the fluid composition, distributed to said culture well (17) of the cell culture plate (3).

7. System (1 ) according to any one of claims 1 to 6, wherein the fluid flow from the inflow module (2) to the outflow module (4) is pressure driven, preferably at least one pressure unit is configured for dispensing the fluid flow to said culture well (17) and/or removing media from said culture well (17).

8. System (1 ) according to any one of claims 1 to 7, wherein the adaptor (20) comprises a lid (21 ) to cover said culture well (17) and a tubing and/or channel system (22) comprising a plurality of tubing, said tubing being sealingly mounted through the lid (21 ), and/or a plurality of channels, embedded or integrated within the lid (21 ), with each tubing/channel entering an individual cell culture well from the top.

9. System (1 ) according to claim 8, wherein said tubing and/or channel system (22) comprises for each culture well (17) at least a media inflow tubing and/or channel (23) in fluid connection with one individual channel (18) for dispensing the fluid flow to said culture well (17), and an outflow tubing and/or channel (25) for removing the fluid from said culture well (17).

10. System (1 ) according to the preceding claim, wherein the tubing and/or channel system (22) further comprises a level setting tubing (24) for adjusting the level of fluid in said culture well (17).

11 . System (1 ) according to any one of claims 9 to 10, wherein the tubing and/or channel system (22) further comprises a gas supply tubing and/or channel for perfusing the mammalian cell culture chamber with a gas, for instance CO2, for controlling the environment, preferably the gas composition environment, in said culture well (17).

12. System (1 ) according to any one of claims 1 to 11 , wherein the inflow module (2) comprises one container (6,7) with predefined media composition or a plurality of media containers (6,7) each comprising a predefined media composition.

13. System (1 ) according to any one of claims 1 to 12, wherein the outflow module (4) comprises a microfluidic outflow module (29,31 ) in fluid connection with said culture well (17) of the cell culture plate (3), preferably a programmable microfluidic outflow module, configured for controlling the collection of the fluid flow removed from said culture well (17).

14. System (1 ) according to any one of claims 1 to 13, wherein the outflow module (4) further comprises at least one collection container (30,32) to collect the fluid flow removed from the cell culture well (17).

15. System (1 ) according to any one of claims 1 to 14, comprising one or more sensors (34) operatively connected to (a) measurement device(s) (35) and controller system(s) (36) enabling the implementation of a closed feed-back loop, wherein one or more parameters of the system can be modulated and/or adjusted in response to parameters measured by the sensor(s).