Cell-stretching device, method for performing cell-stretching experiments, and system therefor

Abstract

The invention relates to a cell-stretching device which is suitable for performing in vitro stretching experiments by means of automated high-resolution live cell imaging on living cells or the tissues thereof, by applying the cells to be examined onto the cell culture surface of an elastic membrane. The invention also relates to a method for performing in vitro cell-stretching experiments by means of such a cell-stretching device in a multimodal manner (i.e. in various stretching modes including radial / equibiaxial, uniaxial, biaxial, and in different orientations of the main stretching directions).

Description

[0001] Cell elongation device, method for performing cell elongation tests and system therefor

[0002] The invention relates to a cell stretching device for conducting in vitro stretching studies on living cell cultures, comprising an elastic membrane having a cell culture surface onto which the cell culture to be examined is to be applied. The invention also relates to a method and a system for conducting multimodal in vitro cell stretching studies using such a cell stretching device.

[0003] In vitro modeling of physiological processes such as respiration, digestion, muscle contraction, heartbeat, and brain development requires that cells are cyclically stretched and / or compressed. Since mechanical stretch is ubiquitous in tissues and the cells that form them, it is crucial to consider it in in vitro experiments (Constantinou & Bastounis (2023), Trends in Biotechnology 41 (7): 939-950).

[0004] Cell stretch devices with transparent, elastic membranes are used for biomimetic (near-v / vo) modeling of cell cultures in vitro and for their imaging. The following sections ("Relevance of Mechanical Stretches in Biomedical Research" and "The Role of Mechanical Stretches in Healthy and Diseased Tissues / Cells") reproduce content from the publication by Constantinou & Bastounis (2023), Trends in Biotechnology 41 (7): 939-950 and serve as an introduction to the topic of cell stretch devices and the role of mechanical stretching in biomedical and pharmaceutical research, to provide a theoretical background, and to clarify the relevance of the present invention to these research fields. Relevance of Mechanical Stretches in Biomedical Research:

[0005] Many physiological functions such as respiration, digestion, muscle contraction, heart rate, and brain development require cells to be (cyclically) stretched and / or compressed. Cells in vivo respond to mechanical forces emanating from neighboring cells, their environment, and the changing properties of their extracellular matrix. By perceiving (sensing) and transducing (transducing) these mechanical stimuli, cells respond biochemically and biomechanically, regulating cellular processes such as motility and cell differentiation, thus critically influencing human (patho)physiology. Because mechanical stretch is ubiquitous in tissues and the cells that form them, it is crucial to consider it in in vitro experiments.To capture the spatiotemporal changes that occur at the level of tissues, cells, and molecules due to stretching, cell stretching devices for in vitro cell culture are used together with microscopic imaging.

[0006] The role of mechanical stretch in healthy and diseased tissues / cells: Various tissues and their constituent cells are exposed to stretch in vivo. Changes in physiological stretch can be either the cause or the consequence of disease. In cardiac muscle, for example, stretch controls the development and regulation of periodic cardiac contractions. Dysregulation of the response of cardiac muscle cells to stretch is associated with various cardiac diseases. Conditioning cells through mechanical stretch directs stem cell differentiation in vitro and, in regenerative medicine, improves the strength and functionality of engineered cardiac muscle tissue. In diseases such as chronic hypertension, cardiac hypertrophy develops, which is associated with remodeling of myocardial tissue, cardiomyocyte enlargement, and greater stretch amplitude.

[0007] In the intestine, mechanical stress can reprogram intestinal epithelial cells (IECs) by altering their gene expression and generating signals to regulate food intake. With mechanical stretch, a coculture of IECs with commensal microbes could be cultured for longer periods (>1 week) without compromising IE cell viability compared to static cultures due to bacterial overgrowth. Consistent with this, patients with irritable bowel syndrome, in whom mechanical stretch of IECs is impaired, experienced bacterial overgrowth. During infections, mechanical stretch and coculture with commensal microbes protect the epithelium from the spread of infection and injury. However, the precise contributions of each species and the mechanisms involved in regulating infection have not been elucidated, possibly due to incompatibility of the cell stretch system with video microscopy and live cell imaging.Decoupling the effect of extracellular physical stimuli from the effect of the microbiome could allow a clear determination of their respective contributions.

[0008] Airway epithelial cells (AECs) are also subjected to stretching during respiration, with the magnitude being an order of magnitude higher in pathological conditions such as asthma-induced bronchospasm. Acute static stretching (during lung injury) and chronic cyclic stretching have different effects on the functions and developmental behavior of lung epithelial cells. This underscores the importance of cell stretching devices that allow independent adjustment of the magnitude, frequency, and direction of stretching.

[0009] Similar to AECs, endothelial cells lining the inner lumen of blood vessels align perpendicular to the direction of stretch and alter the extracellular matrix and cell-cell adhesions in a load- and time-dependent manner. Pathologically high stretch (18%, as seen in bronchospasms of asthma patients) compared to normal physiological stretch (5%) compromises the integrity of the endothelial barrier due to activation of extracellular signal-regulated kinase (ERK) and changes in cell contractility, which also differ when stretch is applied to the cells statically or cyclically. High strain compared to normal physiological stretch can also alter the transcriptional profile of endothelial cells and contribute to atherosclerotic plaque formation.Investigating the underlying mechanisms in real time would enable a better understanding of how blood vessels are damaged and how atherosclerosis develops. A recent in vivo study showed that endocytic regulatory pathways are controlled by stretch-activated channels and can be inhibited by mechanical stretch. This could be important for developing better methods for drug delivery and fighting infections, as many pathogens exploit vesicular-directed transport to infect cells. Furthermore, mechanical stretch induces reprogramming of endothelial cell memory, which is maintained even when the cells are placed on inelastic matrices after stretching.In connection with the concept of mechanical memory, preconditioning endothelial cells through mechanical stretch improves their barrier integrity—a property exploited for the production of readily implantable vascular grafts. However, for a better understanding of tissue and cell (patho)physiology, it is crucial to microscopically image living cells during cyclic stretch.

[0010] Cell stretching platforms for studying the biological responses of multicellular assemblies to mechanical stress have been used in research laboratories for decades. Since the development of the first systems in the 1970s, a variety of cell stretching platforms have been developed, including commercially available systems, notably the Flexcell® (from Flexcell International) and StrexCell® (from STREX Inc.) bioreactors.

[0011] Flexcell®^ devices are pneumatically actuated systems that, according to the manufacturer, are compatible with inverted 40x objectives when combined with the Inverted StageFlexer®. The Inverted StageFlexer® contains a ring that tensions the membrane from below and keeps the membrane movement planar. No high-resolution temporal and spatial live cell imaging data during cyclic stretching have been published for this system to date.

[0012] EP 3 663 388 A1 describes a pneumatic system for the simultaneous radial stretching of multiple cell culture membranes in a multi-well plate. However, the system is not designed for microscopy during the stretching cycles because components block the optical path during the stretching. The membranes also bend during stretching, causing the cell culture surface to be out of focus.

[0013] Description of the invention:

[0014] The invention is based on the object of providing an improved cell stretching device as well as a method and a system formed therewith.

[0015] This object is achieved in a cell stretching device of the type mentioned at the outset in that the cell stretching device has a plurality of controllable actuators which are each mechanically coupled to the membrane in a ring shape around the cell culture surface, wherein the actuators are each set up for the stretching deflection of the membrane in the region of the cell culture surface in different spatial directions parallel to the cell culture surface, wherein the cell culture surface can be deflected in different stretching directions by individual actuation of the actuators during the examination of the same cell culture applied to the cell culture surface.With such a cell stretching device, an automated execution of multimodal in vitro cell stretching tests can be carried out particularly efficiently, in particular in such a way that during the course of a continuous cell stretching test cycle, the same cell culture on the cell culture area is stretched by the actuators in several different stretching modes.

[0016] As a cell culture, cells can be applied in all possible arrangements on the cell culture area, e.g. single cells and / or cell monolayers and / or other cell tissues.

[0017] Depending on the design of the cell stretching device, stretching can be carried out in the following different stretching directions or different stretching modes: uniaxial stretching in different orientations in the plane of the cell culture surface (by activating an actuator pair consisting of two opposite actuators) biaxial stretching in different orientations in the plane of the cell culture surface (by activating two orthogonal actuator pairs or by superimposing two uniaxial stretchings) radial stretching, i.e. uniform stretching in all available axes by activating all ring-shaped actuators combinations or superimpositions of the above-mentioned stretching configurations.

[0018] The elastic membrane can be made of an elastomer, particularly a polymer from the silicone class, e.g., polydimethylsiloxane (PDMS). PDMS can be cross-linked, which then imparts elastomeric properties. For compatibility with microscopy (primarily inverted fluorescence or confocal microscopes), a transparent, thin, stretchable membrane is required. Such membranes are made of elastomeric materials, primarily PDMS or other silicones. The membrane can be actuated by a motor or pneumatically, e.g., using a vacuum. Other drive types (e.g., piezoelectric, electromagnetic) are also conceivable.

[0019] A cell culture surface is defined as a specific, usually central, area of ​​the membrane on which the cell culture to be examined is cultivated. Typically, the cell culture surface is located on only one side of the membrane. A microscope is typically used to conduct the examinations, which may, for example, be equipped with a camera for recording and documenting cell stretching experiments. In an advantageous embodiment of the cell stretching device, the microscope lens is then positioned on the side of the membrane facing away from the cell culture surface.

[0020] According to the invention, the controllable actuators are each mechanically coupled to the membrane in a ring around the cell culture surface in order to deflect the membrane, i.e., to stretch and relax it again. The actuators themselves do not necessarily have to be arranged in a ring around the cell culture surface, but their position of influence on the cell culture surface should be arranged in a ring around it. In an advantageous embodiment, the actuators themselves can also be arranged in a ring around the cell culture surface.

[0021] According to an advantageous embodiment of the invention, the cell stretching device has at least four controllable actuators. Such a number of actuators already allows for several different stretching modes to be realized. The higher the number of actuators, especially if they are individually controllable actuators, the more differentiable stretching states can be realized, and the higher the spatial resolution of the stretch deflections fed into the cell culture surface.

[0022] According to an advantageous embodiment of the invention, several or all actuators can be individually controlled individually and / or in groups. This allows individual control of the actuators and, accordingly, the automatic implementation of a variety of expansion modes without reconfiguring the cell expansion device.

[0023] The actuators can be designed as electromechanical actuators, e.g. as electric motors with or without gears, e.g. in the form of stepper motors, as electroactive polymers and / or as piezo actuators.

[0024] According to an advantageous embodiment of the invention, it is provided that several or all of the actuators are designed as pneumatic actuators, in particular as vacuum actuators. This has the advantage that the actuators can be realized with a particularly simple design and small space requirement, so that the entire cell expansion device can be designed to be particularly compact. In addition, pneumatic actuators allow for high actuating speeds. The actuators can be connected to pneumatic valves, e.g. in the form of switchable directional control valves, to adjust the respective pressure in their pressure chamber. Pneumatic actuators can be designed, for example, as pneumatic actuating cylinders. In an advantageous embodiment, the pneumatic actuators can be designed as vacuum actuators. Vacuum actuators are actuated by negative pressure in a pressure chamber (vacuum chamber) of the actuator.

[0025] According to an advantageous embodiment of the invention, the vacuum actuators each have a vacuum chamber, with the vacuum chambers of the vacuum actuators being arranged in a ring around the cell culture surface of the membrane. This allows the actuators to be positioned directly at the location where the mechanical deflection of the membrane is to occur. This allows for direct force transmission to the membrane while maintaining a compact design of the cell expansion device.

[0026] According to an advantageous embodiment of the invention, the vacuum chambers each have at least one elastically deformable wall that is mechanically coupled to the membrane. The elastically deformable wall can thus directly stretch the membrane and thus the cell culture surface. Any actuation or deflection mechanisms can be eliminated. The elastically deformable wall can thus replace a piston used in conventional pneumatic actuators. This allows the vacuum actuators to be realized with particularly compact dimensions.

[0027] According to an advantageous embodiment of the invention, the vacuum chambers are rigid and have an open side, which is closed off by the adjacent (not permanently attached) membrane. The membrane is then drawn through the adjacent vacuum chambers and thus expanded.

[0028] According to an advantageous embodiment of the invention, the vacuum chambers and / or their elastically deformable walls are arranged on the side of the membrane where the cell culture surface is located. This also contributes to a space-saving design of the cell expansion device.

[0029] According to an advantageous embodiment of the invention, the cell expansion device has a spacer by means of which at least the cell culture surface is held in a constant spatial plane, independent of the actuation of the actuators. In this way, the cell culture surface can be held in a constant focal plane relative to the microscope objective, regardless of the currently set expansion state. In particular, the cell culture surface is held in a constant spatial plane relative to the housing of the cell expansion device. The spacer can be formed integrally with a housing part of the cell expansion device. According to an advantageous embodiment, the spacer is formed as a separate component that can be fastened to a housing part of the cell expansion device. This simplifies the structural design and manufacture of the parts of the cell expansion device.In addition, the spacer can be removed or replaced if necessary.

[0030] According to an advantageous embodiment of the invention, the spacer rests against the membrane on the side where the elastically deformable walls of the vacuum chambers are located. In this way, the objective lens can be arranged relatively close to the membrane on the rear side of the cell culture surface. The spacer can have an annular sleeve. The annular sleeve can be arranged concentrically within the elastically deformable walls, e.g., parallel to the elastically deformable walls.

[0031] According to an advantageous embodiment of the invention, the elastic membrane is openly accessible at least temporarily. In this way, unlike in closed systems, the membrane is directly accessible, for example, for a pipette. The membrane can always be openly accessible. In an advantageous embodiment of the invention, the membrane is covered or can be covered by a lid, with the lid being removable.

[0032] The cell stretching device according to the invention can be used to answer increasingly complex biological questions, e.g., how stretching affects a variety of cell functions. Depending on the biological questions posed, various parameters must be considered regarding the design, capabilities, and automation of the cell stretching device, which can be met by the cell stretching device according to the invention. The cell stretching device according to the invention, in combination, allows:

[0033] (i) Compatibility with Live Cell Imaging

[0034] (ii) biomimetic capabilities (e.g. direction(s) of applied stretch)

[0035] (iii) different strain modes (e.g. uniaxial, (equi-)biaxial, radial) as well as an automated switch between different strain modes, ie multimodal operation without reconfiguration of the experiment

[0036] (iv) Compatibility with high-resolution inverted microscopy

[0037] (v) Consistent position of the membrane in the focal plane during stretching (vi) Compatibility with specific assays due to open design, which is accessible for pipetting (relevant for controlled cell seeding and introduction of pathogens in infection assays)

[0038] (vii) Compatibility with the integration of hydrogels to control substrate stiffness (mimicking extracellular matrix stiffness) and to perform (bio)mechanical characterization methods (AFM, TFM, MSM)

[0039] (viii) Possibility of simultaneous integration of other physical stimuli (e.g. shear flow)

[0040] (ix) automated live cell imaging during stretching over extended periods (from minutes to days) with images in the stretched and relaxed state in high temporal and spatial resolution

[0041] Automated live cell imaging with high spatial and temporal resolution: In vitro cell cultures can be observed during mechanical stretch cycles in a microscope (inverted or upright), enabling automated, spatially and temporally high-resolution live cell imaging during mechanical stimulation of the cells over long observation periods (hours / days). "Automated" means that no manual readjustment of the system (e.g., the focal plane) is necessary during the experiment. Spatially high resolution means that cells can be imaged with an objective lens, e.g., with a magnification of at least 40x. Temporally high resolution means that images alternating between the stretched and unstretched states can be captured at intervals of seconds. This has not been demonstrated with any system to date.

[0042] Multimodal actuation within an experiment: The cell culture system is capable of switching between uniaxial, radial / equibiaxial, and biaxial stretching in various stretching orientations during the experiment without changing the experimental setup. This enables new investigations on cell cultures that involve automatically alternating stretching modes and directions. Only one cell stretcher design is required to produce different stretching modes and directions, since the variation of the stretching modes and directions is determined purely by controlling the individual actuators, e.g., by differently branched vacuum channels in the upper half of the housing. This has not been achieved with any system to date.

[0043] Focus-stable membrane stretching: Membrane movement perpendicular to the focal plane can be prevented, for example, by using a hollow spacer sleeve, which keeps the membrane in focus during stretching cycles. Compatibility with high-resolution inverted microscopy with a short working distance: The system offers unobstructed access to the membrane from below, with sufficient space for moving an objective lens close to the cell culture membrane. This enables very short working distances, which is a prerequisite for high-resolution imaging.

[0044] Open-top cell culture chamber: Pipetting for seeding and manipulating cells and for treating the cells or membrane (e.g., with adhesion promoters, collagen, fluorescent particles, dyes, pathogens, etc.) is possible, as with protocols using standard Petri dishes. The open access enables mechanical investigations such as atomic force microscopy (AFM), traction force microscopy (TFM), and monolayer stress microscopy (MSM), as well as the integration of hydrogels, which can mimic different stiffnesses of the extracellular matrix (and are also necessary for performing TFM and MSM).

[0045] The system design also offers the following additional advantages: Simple design with few components: Pneumatic actuation reduces the complexity of assembly compared to motorized actuators. Despite its multimodal functionality, the design is simple, and there is a low risk of contamination, as it contains only a few, easily sterilized components. Compared to existing systems, the present invention offers research laboratories a low-threshold option for implementing near-v / o expansion in their cell cultures, as it does not require complex retrofitting and the assembly comprises few components.

[0046] Compact design: The pneumatic design eliminates the need for a motor unit or gearbox, saving installation space.

[0047] The aforementioned object is further achieved by a method for conducting multimodal in-front-of-front stretching studies using a cell stretching device of the type described above, wherein, during a continuous cell stretching study cycle, the same cell culture is stretched in the cell culture area by the actuators in several different stretching modes. This also allows the previously described advantages to be realized. Automated live cell imaging of the cell culture can be performed.

[0048] The invention is therefore compatible with an automated process, although this is not mandatory. Automated implementation of the process has the advantage that no manual adjustments are necessary.

[0049] According to an advantageous embodiment of the invention, time-lapse recordings are carried out by means of automated live cell imaging (or video microscopy) of the cell culture with previously unattained temporal and spatial resolution, without the need for manual readjustment of the focal plane.

[0050] According to an advantageous embodiment of the invention, it is provided that the continuous cell elongation test cycle of the same cell culture is carried out over several minutes, hours or days.

[0051] According to an advantageous embodiment of the invention, the membrane is pre-stretched during the seeding of the cell culture onto the cell culture surface. After a waiting period, the membrane's stretching is reduced, thereby compressing the cell culture onto the cell culture surface. The invention thus enables a method by which the cells are compressed by pre-stretching the membrane during the seeding of the cells onto the cell culture surface. Relaxing the membrane then results in compression / shrinkage of the cells.

[0052] Further advantageous embodiments of the invention: the elastic membrane can be coated with different ligands / extracellular matrix proteins, depending on which cell types are seeded or which integrins one wants to activate.

[0053] Hydrogels with adjustable stiffness can also be applied to the elastic membrane to imitate the native (patho-)physiological stiffness of the extracellular matrix of cells / tissues.

[0054] By integrating hydrogels, traction force microscopy (TFM) and monolayer stress microscopy (MSM) can also be performed. These methods allow the determination of the spatiotemporal dynamics of the forces exerted by cells on their matrix and on each other. These forces are a proxy measure of the barrier integrity of the cell monolayer or epithelium.

[0055] The open design of the invention allows additional functions to be added through attached modules.

[0056] The invention allows fluorescent particles to be applied to the membrane, which are used to determine the displacements / strains through image correlation (comparing the relaxed and stretched configurations). This allows monitoring data on the strains in the membrane to be collected. Live analysis of this data makes it possible to track the strains in the membrane during the experiment and to recalibrate them during the experiment using control loops.

[0057] The object mentioned above is also achieved by a system for carrying out multimodal in front-to-front cell stretching examinations, comprising a cell stretching device of the type explained above and a control device for controlling the actuators of the cell stretching device, wherein the system is set up to carry out a method of the type explained above by automatically controlling the actuators of the cell stretching device by the control device. This also makes it possible to realize the advantages explained above. The system can also have further components, such as the aforementioned microscope or a pneumatic overpressure or negative pressure source. The control device can, for example, have a programmable computer which can be coupled to the actuators via appropriate interfaces for converting between digital signals and analog signals.

[0058] The invention is suitable for investigating fundamental human functions such as respiration and digestion through mechanical stretching of cells and tissues. In laboratory experiments, the mechanical stretching experienced by cells in the body can be significantly more accurately replicated, enhancing the biomimetic nature of the studies and the relevance of the results. Stretchable membranes, such as silicone-based membranes, are used to replicate the in-vivo mechanical conditions in in vitro cell cultures and to study the response of cells to mechanical stimuli.

[0059] Compatibility with high-resolution optical microscopy requires a highly transparent, thin membrane accessible for imaging with an inverted microscope. Using an upright microscope, which requires immersion of the objective lens in the cell medium, would not be ideal, especially for infection assays.

[0060] In order to obtain meaningful data on the mechanisms of biomechanical processes in cells and tissues, it is also crucial to enable automated, high-resolution, time-lapse imaging of cyclically stretched cell cultures over a longer period (several hours) during the stretching process at short time intervals. The invention allows the cells to be stretched in various axial configurations (stretch modes), e.g., uniaxially (along one axis), biaxially (along two orthogonal axes), or radially (uniformly in all directions available on the circumference of the actuator ring). Any superposition of these configurations is also possible. Depending on the nature of the biological question, different stretch modes are of interest.In order to investigate the influence of varying stretch directions and modes on biomedical processes in tissues and cells, it is interesting to use a stretch system that allows for multiple stretch modes. In addition, a system design that combines multiple stretch modes offers more flexibility and is relevant to a broader target group than designs that specialize in one stretch mode. The system according to the invention allows for arbitrary switching between multiple stretch modes and stretch orientations. Varying the stretch directions requires no manual adjustment and thus no interruption of the experiment. This makes it practical to observe one and the same cell under the microscope under varying stretch modes. If the experiment were to be adjusted, however, there would be no guarantee that the reference cell could be found again with reasonable effort after a change.

[0061] Conducting infection assays ideally requires a system that allows top-loading access similar to a well plate or Petri dish. While the introduction of microorganisms through a fluid stream is feasible in organ-on-chip systems, this method lacks reproducibility and control compared to established infection protocols performed in conventional well plates. Furthermore, top-loading ensures compatibility with other biomechanical measurements such as atomic force microscopy (AFM; for measuring cell stiffness), traction force microscopy (TFM; for measuring stresses exerted by cells on their ECM), and monolayer stress microscopy (MSM; for determining intracellular stresses within a cell layer).These techniques are used to evaluate the mechanical properties of cells and the forces they generate, which is of utmost importance as they play a key role in cellular (patho)physiology.

[0062] In order to conduct meaningful studies on cyclically stretched cell cultures, the invention also meets further technical requirements:

[0063] 1. Objective access from below must be ensured to enable high-resolution images with an inverted microscope at a short working distance. 2. The membrane must remain in focus during the stretch cycles. Planar membrane movement must be ensured, i.e., movement of the cell culture surface perpendicular to the focal plane (lifting / lowering) must be prevented.

[0064] 3. The system must be open on the top or freely accessible from above for

[0065] • Pipetting for controlled and reproducible seeding and treatment / infection of cells as well as modification of the membrane (e.g. by fluorescent particles)

[0066] • mechanical investigations such as atomic force microscopy (AFM), traction force microscopy (TFM) and monolayer stress microscopy (MSM)

[0067] The invention is explained in more detail below using exemplary embodiments and drawings.

[0068] It shows

[0069] Figure 1 shows a cell stretching device in perspective view,

[0070] Figure 2 shows the cell expansion device according to Figure 1 in an exploded view, Figure 3 shows a cell expander of the cell expansion device according to Figure 1 in plan view, Figure 4 shows the cell expander according to Figure 3 in the sectional plane AA,

[0071] Figure 5 shows the cell expander according to Figure 3 in the section plane shown in Figure 4

[0072] BB,

[0073] Figure 6 is an enlarged detail C from Figure 4,

[0074] Figure 7 shows another cell stretching device in a side sectional view,

[0075] Fig. 8-11 schematic sectional views of a cell stretching device in different operating states,

[0076] Figure 12 exemplary stretching configurations of differently designed cell stretching devices,

[0077] Figure 13 shows a system for performing multimodal cell strain studies.

[0078] Figure 1 shows a cell expansion device 1 having a housing 2. The housing can, for example, be designed in several parts, e.g. with an upper housing part 20 and a lower housing part 21. The housing can be open at an upper side. This open upper side can be covered by a lid 3, e.g. a Petri dish lid. A plurality of pneumatic connections 4 are provided on the housing 2. The housing 2, in particular the upper housing part 20, can have one or more ventilation structures 29 through which an air gap is provided between the housing 2 and the lid 3. As Figure 2 illustrates, a cell expander 5 and a spacer 6, e.g. in the form of a spacer sleeve, are arranged within the housing as a further assembly.

[0079] Figures 3 to 6 illustrate an advantageous design of the cell expander 5 in various views. The cell expander 5 has a body 53 that is ring-shaped, so that a central, essentially cylindrical through-opening 50 is formed therein. Formed in the body 53 are a plurality of vacuum chambers 54 arranged in a ring around the opening 50 as pneumatic actuators 9, each of which adjoins the opening 50 with an elastically deformable wall 55. The vacuum chambers 54 can each be coupled to a pneumatic connection 4 via a vacuum inlet 51. The vacuum inlets 51 do not necessarily have to be coupled to a pneumatic connection 4 each; a pneumatic connection 4 can also be assigned to several vacuum inlets 51, as can be seen in the embodiment in Figure 1. The elastically deformable walls 55 can also be individually mechanically deflected by individually applying negative pressure to the respective vacuum chamber 54.

[0080] An elastic membrane 7 for conducting cell expansion tests is attached to the body 53 on the underside shown in the figures. The membrane 7 has a cell culture surface 70 on one side facing the top, i.e., the interior of the opening 50, onto which the cell culture to be tested is to be deposited. The membrane 7 is connected to the elastically deformable walls 55 and can be deflected by these walls 55.

[0081] The body 53 may have a coding element 52, e.g., in the form of a projection or a recess. The coding 52 ensures that the body 53 is inserted in a structurally defined position in the parts 20, 21 of the housing 2.

[0082] The membrane 7 can be formed as a thin, elastic, and highly transparent PDMS membrane (<100 pm) that is connected to the body 53. The body 53 can be formed as a transparent and elastic, round PDMS casting. However, other elastomers can potentially also be used. The casting has the coding element 52, which serves as an aid for removing the casting from the mold and for aligning the cell expander in the housing. The central cell culture chamber with the expandable membrane is surrounded by the annularly arranged vacuum chambers 54. The vacuum inlets 51, which are connected to the vacuum chambers 54, lead vertically to the top of the cell expander 5 and form the interface for the housing upper part 20, which distributes the vacuum to the vacuum inlets 51.

[0083] The upper housing part 20 and the lower housing part 21 can be designed as rigid housing halves, which are connected by four screws, for example, and enclose the cell expander 5 made of PDMS. Depending on the design, the upper housing part 20 has several (multimodal operation) or one (radial operation) vacuum connections, which draws suction into the vacuum chambers of the cell expander via a channel system. By screwing it to the lower housing part 21, the vacuum interface is pressed against the upper side of the cell expander. This tightly seals the vacuum channels of the upper housing part 20 with the vacuum inlets of the cell expander. The lower housing part 21 can have a viewing window to keep the optical path clear and enable lens access with a short working distance to the membrane in the inverted microscope.

[0084] Figure 7 shows a sectional view of a cell expansion device 1, which may correspond to the cell expansion device already described or may be constructed slightly differently. The cell expansion device 1 according to Figure 7 again has a housing 2 with the upper housing part 20 and the lower housing part 21. The housing 2 is covered at the open top by a lid 3. Located in the housing are a cell expander 5 and a spacer 6, e.g. in the form of a spacer sleeve. The spacer 6 can be screwed to the body 53 of the cell expander 5, e.g. by means of screws. It can be seen in Figure 7, particularly in the enlarged detail shown below, that a sleeve-shaped section 60 of the spacer 6 rests with its free end against the membrane 7. A lubricated contact 8 can be formed at the contact point.

[0085] It can also be seen that the housing 2 is open or made of transparent material on the underside, ie at the bottom of the housing lower part 21, in the central area so that this area can be captured by the lens of a microscope.

[0086] The spacer 6 can be mounted on the upper housing section 20 using four additional screws, for example, and is in annular contact with the upper side of the central cell culture membrane. The spacer 6 can be designed as a hollow die and screwed onto the upper housing section 20 via a flange so that it lies concentrically in the cell culture chamber. The contact between the spacer 6 and the membrane 7 can be lubricated with silicone-free laboratory grease (e.g., Glisseal N) to allow the membrane 7 to slide over the spacer 6. To protect the cell culture from contamination, the upper housing section 20 is covered with a commercially available plastic Petri dish lid, leaving at least a thin gap to ensure air exchange in the cell culture. The ventilation structures 29 serve this purpose.

[0087] Possible manufacturing method:

[0088] The production of the system is divided into the following steps:

[0089] (i) The upper part of the PDMS cell expander is manufactured by molding PDMS in a 3D-printed mold and curing it under temperature.

[0090] (ii) The PDMS membrane is spin-coated onto a glass wafer and cured under temperature.

[0091] (iii) The vacuum accesses are punched (by a biopsy punch).

[0092] (iv) Membrane and casting are bonded by treatment with O2 plasma.

[0093] (v) The finished part is cut out with a scalpel and detached from the glass wafer.

[0094] Other parts such as the housing halves, spacer sleeve, and microscope mount can be manufactured using 3D printing (Keyence Agilista 3200-W, material M2). The lid can be a standard commercially available Petri dish lid.

[0095] With reference to Figures 8 and 9, the behavior of the cell expansion device 1 will first be explained in an embodiment in which no spacer 6 is present. In Figure 8, the vacuum chambers 54 are initially not subjected to negative pressure, so that the elastic walls 55 are in their initial state and are not yet deformed. Accordingly, the membrane 7 is also still in its initial state and not yet stretched.

[0096] Figure 9 shows a state in which a vacuum is created in the vacuum chambers 54. The vacuum draws the walls 55 inward and deforms them accordingly, causing the membrane 7 to stretch in the area of ​​the cell culture surface 70. However, the deformation of the walls 55 also slightly displaces the cell culture surface 70 in the vertical direction by the amount dz. As a result, the cell culture surface 70 moves slightly out of the original focal plane F. Accordingly, the microscope objective must be refocused. Refocusing can occur automatically, for example, by sensing the amount dz using a sensor device or by determining it mathematically based on stored characteristics of the vacuum chambers 54, and the objective is automatically refocused by an actuator. Figures 10 and 11 show an embodiment of the cell stretching device 1 with a built-in spacer 6.Figure 10 again shows the initial state without vacuum application of the vacuum chambers 54, Figure 11 the state of the vacuum chambers 54 with vacuum application. It can be seen that the spacer 6 keeps the membrane in the area of ​​the cell culture surface 70 constantly in the same plane, namely the originally set focal plane F. Refocusing of the lens can therefore be omitted.

[0097] Figure 12 shows examples of various expansion configurations with possible expansion orientations for differently designed cell expanders, particularly for designs with different numbers of vacuum chambers. The first row of the table shows an embodiment with ten vacuum chambers. Possible expansion modes are radial expansion and uniaxial expansion with two, four, or six chambers, each using opposing groups of chambers. By selecting different vacuum chamber configurations, different angular positions of uniaxial expansion can be realized.

[0098] The second row shows a design with eight vacuum chambers. For example, radial expansion, biaxial expansion, and uniaxial expansion can be performed with four or two chambers, each at different angular positions.

[0099] The third row shows a design with six vacuum chambers. The expansion modes possible here are radial expansion and various uniaxial expansions, each with two opposing chambers, again at different angular positions.

[0100] An equibiaxial stretch state (i.e., a stretch state equally pronounced in two principal stretch directions) can be created in the center of the cell culture surface using both a radial and a uniform biaxial stretch configuration.

[0101] The system shown in Figure 13 for conducting multimodal in-front cell stretching examinations comprises a control device 10, a digital / analog interface device 11, a vacuum source 12, an electrical energy source 13, a pressure control device 14, and a microscope 15, e.g., an epifluorescence microscope, with one or more objectives 16. A cell stretching device 1 of the type described above is arranged in an examination area of ​​the microscope 15.

[0102] The control device 10 can be designed as a computer of various types. The control device 10 generates target values ​​30 for the respective expansion modes to be set on the cell expansion device 1, e.g., in the form of digital target values, which are transmitted to the digital / analog interface device 11. The digital / analog interface device 11 converts the digital target values ​​30 into analog control signals 35, which are transmitted to the pressure control device 14. The pressure control device 14 can have switchable valves or proportional pressure control valves. A negative pressure from the vacuum source 12 is supplied to the pressure control device 14 via a line 32. The pressure control device 14 also receives electrical energy 33 from the energy supply device 13.

[0103] The pressure signals emitted by the valves of the pressure control device 14, which are each routed separately via pressure lines 36 to different vacuum chambers or pneumatic connections 4 of the cell expansion device 1, are also recorded as analog actual values ​​34 and transmitted to the digital / analog interface device 11. The digital / analog interface device 11 digitizes the analog pressure values ​​and makes them available to the control device 10 as digital actual values ​​31.

[0104] The control device 10 can also be connected to the microscope 15 via connecting lines 37, e.g. for automatic control of the microscope 15 and for recording the image data recorded via the lens 16 and a camera arranged there, with which images of the cell culture on the cell culture surface 70 of the cell stretching device 1 are captured.

[0105] The vacuum source can be a house vacuum connection, a local vacuum pump, or a compressed air supply with a connected vacuum nozzle. The cell expansion device 1 can be connected to pneumatic tubes, which transition into microfluidic tubes via adapters. The microfluidic tubes are tightly screwed to the upper housing section 20 of the cell expansion device 1 using microfluidic fittings and ferrules. The cell expansion device 1 is placed in the microscope holder via a suitable adapter.

[0106] The stretch in membrane 7 is created pneumatically by contracting the elastic walls 55 adjacent to the cell culture chamber. Activating the vacuum in a vacuum chamber 54 contracts the vacuum chamber, particularly the wall 55 adjacent to the cell culture membrane. This pulls the edge of the cell culture membrane toward the outside of the device, resulting in membrane stretch.

[0107] The vacuum pressures are controlled, for example, by proportional pressure control valves, with a stronger vacuum leading to greater expansion. The proportional pressure control valves are controlled by a setpoint value and output a measured actual value. The setpoint and actual values ​​are generated or measured as analog voltage signals, for example, via a computer-controlled data acquisition device with analog inputs and outputs. LabVIEW or MATLAB, for example, is used as software to control the signals. The shape of the periodic setpoint vacuum pressure signal can be defined using any desired function (sine, square, triangle, trapezoid, etc.). It is also possible to set different frequencies for the expansion cycles.

[0108] By individually controlling the pressures in the vacuum chambers, different strain modes and orientations can be achieved, as shown in Figure 12.

[0109] The invention was tested in cell culture experiments at the University of Tübingen. During the experiments, it was possible, for example, to automatically acquire live cell imaging data from stretched MDCK cells in both radial and uniaxial stretch over a test duration of 12 hours. An inverted epifluorescence microscope (Nikon Eclipse Ti-2) with a 40x objective was used. When radial stretch was applied, characteristic parameters of the cells could be evaluated both in the stretched and in the relaxed state at different times within a period of 1.4 hours (within the total test duration of 12 hours). The parameters cell area, circularity and form factor could be evaluated and displayed for individual cells and the entire cell layer. The enlargement of the cells in the stretched state can be clearly seen.Over time, the area and form factor increase while circularity decreases, indicating a response of the cells to the mechanical stretch cycles.

[0110] The design of the cell expansion device is potentially compatible with a continuous production process, enabling scale-up of production to higher volumes. Through the use of injection molding tools and continuous plasma treatment (corona treatment), the production of cell expanders based on silicone or other elastomers can be expanded to an industrial scale. Due to the integrated channel systems, the housing halves are manufactured using 3D printing and can be produced on a larger scale using multiple and / or larger 3D printing systems. By modularly dividing the upper housing half, it could also be manufactured using other processes, such as injection molding.

Claims

Patent claims:

1. Cell stretching device (1) for carrying out in vitro stretching tests on living cell cultures, comprising an elastic membrane (7) which has a cell culture surface (70) onto which the cell culture to be examined is to be applied, characterized in that the cell stretching device (1) has a plurality of controllable actuators (9) which are each mechanically coupled to the membrane (7) in a ring shape around the cell culture surface (70), wherein the actuators (9) are each configured for stretching deflection of the membrane (7) in the region of the cell culture surface (70) in different spatial directions parallel to the cell culture surface (70), wherein the cell culture surface (70) can be deflected in different stretching directions by individually actuating the actuators (9) during the examination of the same cell culture applied to the cell culture surface (70).

2. Cell expansion device according to claim 1, characterized in that the cell expansion device (1) has at least four controllable actuators (9).

3. Cell expansion device according to one of the preceding claims, characterized in that several or all actuators (9) can be individually controlled individually and / or in groups.

4. Cell expansion device according to one of the preceding claims, characterized in that several or all actuators (9) are designed as pneumatic actuators, in particular as vacuum actuators.

5. Cell expansion device according to claim 4, characterized in that the vacuum actuators each have a vacuum chamber (54), wherein the vacuum chambers (54) of the vacuum actuators are arranged in a ring around the cell culture surface (70) of the membrane (7). are arranged.

6. Cell expansion device according to one of claims 4 to 5, characterized in that the vacuum chambers (54) each have at least one elastically deformable wall (55) which is mechanically coupled to the membrane (7).

7. Cell expansion device according to one of claims 4 to 6, characterized in that the vacuum chambers (54) and / or their elastically deformable walls (55) are arranged on the side of the membrane (7) on which the cell culture surface (70) is located.

8. Cell expansion device according to one of the preceding claims, characterized in that the cell expansion device (1) has a spacer (6) by means of which at least the cell culture surface (70) is held in a constant spatial plane independently of the actuation of the actuators (9).

9. Cell expansion device according to claim 8, characterized in that the spacer (6) rests against the membrane (7) on the side on which the elastically deformable walls (55) of the vacuum chambers (54) are located.

10. Cell expansion device according to one of the preceding claims, characterized in that the elastic membrane (7) is at least temporarily openly accessible.

11. A method for carrying out multimodal in vitro stretching tests by means of a cell stretching device (1) according to one of the preceding claims, characterized in that in the course of a continuous cell stretching test cycle, the same cell culture on the cell culture surface (70) is stretched by the actuators (9) in several different stretching modes.

12. The method according to claim 11, characterized in that automated live cell imaging of the cell culture is carried out.

13. Method according to one of claims 11 to 12, characterized in that the continuous cell elongation test cycle of the same cell culture is carried out over several minutes, hours or days.

14. Method according to one of claims 11 to 13, characterized in that the membrane (7) is pre-stretched during the seeding of the cell culture on the cell culture surface (70) and after a waiting time the stretching of the membrane is reduced, whereby the cell culture is compressed on the cell culture surface (70).

15. System for carrying out multimodal in front of / from cell stretching examinations, comprising a cell stretching device (1) according to one of claims 1 to 10 and a control device (10) for controlling the actuators (9) of the cell stretching device (1), characterized in that the system is set up to carry out a method according to one of claims 11 to 14 by automatic control of the actuators (9) of the cell stretching device (1) by the control device (10).

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