Cell transport device

Abstract

The invention relates to a device (1; 1 ') for storing and / or transporting cells, in particular cell aggregates; 10; 15), wherein the device comprises a base (5; 11) and a top (6; 12), wherein the top (6; 12) is located on the base (5; 11), wherein the base (5; 11) is formed from a substrate comprising at least one groove (7) for collecting a fluid, wherein the base (5; 11) and the top (6; 12) is less than the width (9) of the at least one groove (7), and wherein the width (9) of the at least one groove (7) is the smallest line segment between two opposite points passing through the center of the base region of the at least one groove (7).

Description

Technical Field

[0001] The present invention relates to a device for storing and / or transporting cells. Background Art

[0002] The processing, cultivation and analysis of living cell cultures are common methods in biomedical research and development and the pharmaceutical industry. Primary cell cultures and continuous cell lines are widely used in virus culture, biopharmaceutical production, efficacy research and toxicity testing. For example, US2020 / 095526 A1 discloses a method for culturing and analyzing spheroid cells using a microfluidic array. The same device is also described in WO 2022 / 086926 Al and Eilenberger et al. (2021, Advanced Science, 8, 11, 2198-3844). Therefore, worldwide, researchers need to use cell cultures of various cells according to different research topics. To meet the growing demand for living cell cultures, cells are transported from cell banks to research and production institutions around the world. However, the transportation of living cells is challenging because cell viability must be maintained during transportation. Another important aspect of cell transportation involves high costs. For example, Germany's second largest cell bank (Cell Line Service; CLS) spends 40,000 to 50,000 euros annually on cell transportation. The cost depends mainly on the type of transport and the customs regulations of the receiving country (e.g., EU country, non-EU country).

[0003] There are currently two commonly used shipping options, including live cell shipping or dry ice frozen cell shipping.

[0004] When transporting live cells, the cells are placed in a thawed cell culture flask for transportation. Different flask sizes can be selected based on the cell type and transportation time. Example of a standard transport flask size: The surface area of ​​the flask (relative to the growth surface) is 25 cm 2 or 75cm 2 During transport, cells remain viable and capable of division. The conditions during live cell transport differ significantly from optimal cell culture conditions (such as a suitable temperature of 37°C, optimal CO2 supply, and an appropriate amount of culture medium), resulting in limited cell growth behavior during transport.

[0005] Cultured cells can also be shipped on dry ice (a solid form of carbon dioxide), which keeps them frozen at -80°C. However, shipping dry ice is expensive, bulky, and hazardous, so most shippers refuse to handle it. Furthermore, if the dry ice sublimates before reaching its destination, cell recovery will be compromised because the cells will rupture, and the cryoprotectants required for low-temperature storage are toxic to cells at ambient temperatures. Therefore, dry ice must be handled with care, and clear safety procedures must be implemented. It is common for cells shipped on dry ice to have a viability reduction of up to 30% by the time they reach the recipient. Differences in the success rate of adherent versus suspension cultures can also be observed in receiving laboratories.

[0006] It is therefore an object of the present invention to provide devices and methods for enhancing the efficiency and ease of transport of cells and cell aggregates. Summary of the Invention

[0007] The present invention thus relates to a device for storing and / or transporting cells, particularly cell aggregates, wherein the device comprises at least one chamber having a base and a top, wherein the top is located on opposite sides of the base, wherein the base is formed by a substrate containing at least one recess for collecting a fluid, wherein the distance between the base and the top of the at least one chamber is less than the width of the at least one recess, and wherein the width of the at least one recess is the smallest line segment between two opposite points passing through the center of the base region of the at least one recess. Surprisingly, the present invention has been shown to exhibit excellent performance for transporting and / or storing cells, particularly cell aggregates. Cells and cell aggregates can be safely stored in the at least one recess of the device according to the present invention. Thus, using the device according to the present invention, cells and cell aggregates can be transported and / or stored in a reliable, compact, and cost-effective manner. Transporting and / or storing cells in aggregate form is advantageous because aggregated cells require only a limited amount of space for storage and / or transport due to their compact form and exhibit increased resistance to harmful influences. Furthermore, cell aggregates typically have a volume that is larger than the dimensions of the chamber of the microfluidic device, for example, making it impossible for the cell aggregates to move out of the recess. Even if they are subjected to severe vibrations and shocks during storage and / or transportation, the cell aggregates remain contained in the one or more recesses of the device of the present invention. This is particularly advantageous because living cells are often sensitive.

[0008] The size (e.g., diameter) of the cell aggregate can be limited by the width of at least one groove. For example, the size of the cell aggregate is limited by the groove width, wherein the chamber height is less than the groove width. Therefore, the cell aggregate is larger than the chamber height and cannot enter the chamber due to geometric limitations.

[0009] Furthermore, due to the limited volume of the device of the present invention, only a small amount of fluid is required for storage and / or transportation of the cell aggregates. For example, compared to existing transportation methods, the cell aggregates can be shipped at significantly lower costs due to the limited volume of fluid required and the reduced packaging weight.

[0010] Another aspect of the present invention relates to a method for storing and / or transporting cells, in particular cell aggregates, comprising the following steps:

[0011] a) applying a fluid comprising cells to at least one chamber of a device as defined herein, thereby providing said fluid to at least one recess, and

[0012] b) incubating the cells until at least one aggregate is formed in at least one of the recesses.

[0013] It has been demonstrated that the method of the present invention can efficiently store and / or transport cell aggregates. Cell aggregates can be formed by simply adding a fluid containing individual cells to at least one chamber, particularly at least one groove of the device of the present invention. After formation, the cell aggregates are hindered from being flushed out of the at least one groove because the chamber height is lower than the groove width. This feature has the following beneficial effects: a) after the fluid containing individual cells is applied to the at least one chamber, the cells settle in the at least one groove, and b) the cell aggregates are safely formed and stored in the at least one groove.

[0014] Another aspect of the invention relates to the use of a device as defined herein for the storage and / or transport of cells, in particular cell aggregates.

[0015] Yet another aspect of the present invention relates to a method for screening the effect of a compound on cell aggregates, the method comprising the steps of:

[0016] a) providing a solution comprising cells,

[0017] b) applying the solution of step a) to at least one chamber of a device as defined herein, thereby providing said fluid to at least one recess,

[0018] c) incubating the cells until at least one cell aggregate is formed in at least one recess,

[0019] d) treating at least one cell aggregate with at least one compound, and

[0020] d) measuring the effect of said at least one compound on said at least one cell aggregate.

[0021] Specifically, the method of the present invention has practical applications in biomedical research, drug screening, and personalized medicine. For example, the present invention can be used to analyze a patient's physiological state to identify the preclinical stage of a disease and make a comprehensive differential diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1a A schematic top view of an embodiment of the device according to the invention is shown.

[0023] Figure 1b Show the basis Figure 1a A cross-sectional side view of an embodiment of a device having a hemispherical recess and a frustoconical hollow body.

[0024] Figure 1c Show the basis Figure 1a A cross-sectional side view of an embodiment of a device comprising a fluid chamber, a hemispherical recess, and a frustoconical hollow body.

[0025] Figure 2a A schematic plan view of an embodiment of the device according to the invention is shown.

[0026] Figure 2b Show the basis Figure 2a A cross-sectional side view of an embodiment of a microfluidic device having a hemispherical recess and a frustoconical hollow body.

[0027] Figure 2c Show the basis Figure 2a A cross-sectional side view of an embodiment of a device comprising a fluid chamber, a hemispherical recess, and a frustoconical hollow body.

[0028] Figure 3a A schematic plan view of another embodiment of the device according to the invention is shown.

[0029] Figure 3b Show the basis Figure 3a A cross-sectional side view of an embodiment of a device comprising a fluid chamber, a hemispherical recess, and a frustoconical hollow body.

[0030] Figure 4a Show the basis Figure 2a Schematic plan view of a base embodiment of the device.

[0031] Figure 4b Show the basis Figure 3a Schematic plan view of a base embodiment of the device.

[0032] Figure 5a and Figure 5b Show respectively according to Figure 2a Schematic top view and schematic side view of a top layer of a microfluidic device embodiment.

[0033] Figure 6 A schematic workflow of the method according to the invention for storing and / or transporting cells, in particular cell aggregates, is shown.

[0034] Figure 7 A schematic workflow of the method for screening the effects of compounds on cell aggregates according to the present invention is shown.

[0035] Figure 8a Schematically shown are diagrams of different chamber heights (50 μm, 100 μm, 150 μm) at a constant groove width.

[0036] Figure 8b The flow pattern distribution of the microfluid flow in the chamber of the device according to the present invention is shown at different chamber heights.

[0037] Figure 9a Schematically, a diagram of different hollow body widths (500 μm, 600 μm, 700 μm, 800 μm, 9000 μm) at a constant groove width (500 μm) is shown.

[0038] Figure 9b The flow pattern distribution of the microfluid flow in the chamber of the device according to the present invention is shown at different hollow body widths.

[0039] Figure 10 Shown are the cell viabilities of cell aggregates after overnight storage at 37° C. and room temperature (RT) in a device according to the invention.

[0040] Figure 11 Fluorescence micrographs of cells harvested from a device according to the present invention after seeding onto a cell culture plate are shown. Cells were applied to the device at six different densities.

[0041] Figure 12 Shown are the cell viabilities after cells were stored at -196°C for three days using various concentrations of cryopreservation medium. DETAILED DESCRIPTION

[0042] As used herein, "cell" refers to a particle surrounded by a membrane, which may contain proteins, nucleic acids, lipids, metabolites and / or organelles. Cells can grow and / or divide into other cells, or release other cells. According to another preferred embodiment of the present invention, the cell is selected from the group consisting of: eukaryotic cells, prokaryotic cells, extracellular vesicles, and combinations thereof. In particular, the eukaryotic cell may be a mammalian cell, preferably an epithelial cell, a neural cell, a muscle cell, a connective tissue cell, a stem cell, preferably a tumor cell. The prokaryotic cell may be a bacterial cell or an archaeal cell. In particular, the extracellular vesicle may be an exosome, a microvesicle or an apoptotic body. The cell size according to the present invention may be between 80 nm and 100 μm, preferably between 90 nm and 80 μm, more preferably between 100 nm and 60 μm.

[0043] The term "cell aggregate" refers to a three-dimensional micro-aggregate formed by individual cells suspended in a fluid. The cell aggregates according to the present invention are formed by cell aggregation. Individual cells can adhere to, bind, attach or connect to each other to form aggregates. Individual cells can also fuse together to form cell aggregates. The aggregate size can be between 60 μm and 3 mm, preferably between 80 μm and 2 mm, and more preferably between 100 μm and 1 mm. Cell aggregation can be a reversible or irreversible process.

[0044] The "size" of cells can be determined by methods known in the art (eg, electron microscopy).

[0045] During the aggregation process, the size of the cell aggregates can increase and sedimentation can occur. The reverse process of redispersing the cell aggregates into individual cells hardly occurs spontaneously, but can occur under mechanical and / or chemical depolymerization. Specifically, the cell aggregates composed of eukaryotic cells are three-dimensional cell microclusters in the form of spheroids or with a spheroid-like form. Such spheroids can also be regarded as "organoids". "Cell spheroids" can be formed by eukaryotic cells, particularly mammalian cells (such as human cells), wherein particularly preferred cells are cells present in mammalian organs and tissues. These spheroids can contain one or more types of cells. Using different types of cells can produce more complex "organoids" or tissue-like structures. Cell aggregates can also be three-dimensional microclusters of extracellular vesicles, or spheroid-like structures formed by the combination of eukaryotic cells or prokaryotic cells with extracellular vesicles.

[0046] As used herein, "base" refers to the inner bottom surface of at least one chamber, which is substantially below the fluid introduced into the chamber and parallel to the direction of fluid flow. As used herein, "top" refers to the inner top surface of at least one chamber, which is located on the opposite side of the inner bottom surface. As used herein, "base region" refers to the two-dimensional cross-sectional area of ​​a three-dimensional geometric body. Preferably, the base region of at least one groove has a circular or elliptical shape.

[0047] Cells and / or cell aggregates according to the present invention can be stored and / or transported in a fluid. A fluid can be introduced into at least one chamber of a device of the present invention via at least one opening. As used herein, "fluid" refers to a liquid.

[0048] According to another preferred embodiment of the present invention, the base of at least one chamber further comprises a hollow body, wherein the hollow body is in fluid communication with the interior of the at least one chamber and the at least one groove, and wherein the width of the base region of the hollow body is at least partially the width of the at least one groove. Specifically, the hollow body is located between the at least one groove and the interior of the at least one chamber, wherein the at least one groove is located below the hollow body and the interior of the at least one chamber is located above the hollow body. An advantage of the hollow body of the present invention is that the applied individual cells are effectively captured in each groove of the device according to the present invention. Surprisingly, it was found that the hollow body of the present invention promotes cell aggregation because the cells can automatically fall into the grooves due to gravity rather than being stored in the chamber. Therefore, the presence of the hollow body of the present invention can effectively increase the cell aggregation in the at least one groove. Another advantage relates to an increased supply of fluid to the cell aggregates in the at least one groove. It has been demonstrated that by introducing a hollow body located between the at least one groove and the interior of the at least one chamber, more fluid can be collected in the at least one groove and the cell aggregates can be continuously supplied with fluid. At least one chamber, at least one hollow body and at least one groove are fluidically connected so that the fluid can flow through the chamber and fill the hollow body and the groove where the cell aggregates are stored. As mentioned above, the device of the present invention may be subjected to severe vibration and impact during storage and / or transportation, and often tilts. Specifically, tilting the device of the present invention will cause fluid flow in at least one chamber. As shown in the "Examples" section, the presence of the hollow body ensures that the fluid flow in the groove can be enhanced, thereby optimizing the fluid supply of the cell aggregates through nutrients and improving the cell capture effect. Therefore, the cell aggregates can obtain a uniform supply of fluid during storage and / or transportation, while being safely stored in the groove of the device of the present invention. The fluid supply to the cell aggregates in at least one groove can also be adjusted by the shape and / or width of the hollow body.

[0049] As used herein, "the base region of the hollow body" refers to the two-dimensional cross-sectional area of ​​the three-dimensional hollow body that contacts the base region of the at least one groove and is located on the opposite side of the base of the at least one chamber. Preferably, the base region of the hollow body has a circular or oval shape. The width of the base region of the hollow body is at least partially equal to, and preferably equal to, the width of the at least one groove.

[0050] As used herein, "top region of a hollow body" refers to the two-dimensional cross-sectional region of a three-dimensional hollow body that contacts the base of at least one chamber and is located on the opposite side of the base region of at least one groove.

[0051] According to another preferred embodiment of the present invention, the width of the top region of the hollow body is between 500 μm and 3 mm, preferably between 1 mm and 2 mm, wherein the width of the hollow body is defined as the smallest line segment between two opposite points passing through the center of the top region of the at least one recess. The width of the hollow body can be adjusted to enhance fluid flow into the at least one recess and cell capture.

[0052] According to another preferred embodiment of the present invention, the hollow body has a frustoconical shape, and wherein the base region and the top region of the frustoconical shape have a circular or elliptical shape.

[0053] To prevent cell aggregates from being lost and washed out of at least one groove of the present invention during storage and / or transportation, the distance between the base and the top of at least one chamber is less than the width of the at least one groove. Due to this geometric limitation, cell aggregates are blocked and cannot escape from at least one groove of the present invention and are stored in the groove. The height of at least one chamber can be adapted to the width of at least one groove. According to another preferred embodiment of the present invention, the ratio of the distance between the base and the top of at least one chamber to the width of at least one groove is between 0.5:1 and 0.01:1, preferably between 0.2:1 and 0.02:1, and preferably between 0.1:1 and 0.04:1.

[0054] According to another preferred embodiment of the present invention, the substrate comprises, consists of or is coated with a biocompatible material.

[0055] According to another preferred embodiment of the present invention, the biocompatible material is silicone, plastic material or glass. Glass has optical transparency, electrical insulation and chemical inertness. Silicone has optical transparency, oxygen permeability, low cost and resistance to organic solvents. Plastic-based devices are also of great significance in industrial applications. The diversity of plastic materials provides great flexibility in selecting suitable materials with specific properties. Compared with inorganic materials, polymers are easy to obtain and low in cost, and therefore have become a common material for devices suitable for cell storage and / or transportation. Preferably, the biocompatible material is a plastic material. The manufacturing and / or molding processes of plastic substrates are known in the art, such as injection molding or 3D printing.

[0056] According to another preferred embodiment of the present invention, the biocompatible material is selected from the group consisting of polystyrene, cycloolefin copolymer, polymethyl methacrylate, cycloolefin polymer, polydimethylsiloxane, polycarbonate (PC), polypropylene (PP), polyvinyl chloride (PVC), perfluoropolyether (PFPE), polyurethane, polyethylene terephthalate (PET), polyester and thiol-ene. Preferably, the biocompatible material is suitable for use at temperatures between -196°C and 121°C and can withstand repeated temperature changes. The biocompatible material of the device of the present invention can also withstand sterilization procedures known in the art, such as gamma irradiation, steam sterilization or ethylene oxide sterilization.

[0057] According to another preferred embodiment of the present invention, at least one recess and / or substrate is at least partially coated with an anti-adhesion layer. The anti-fouling layer prevents cells and proteins from adhering to the surface of the at least one recess. In another preferred embodiment of the device according to the present invention, at least one chamber is also at least partially coated with an anti-fouling layer.

[0058] According to another preferred embodiment of the present invention, the anti-adhesion layer is selected from the group consisting of: polyethylene glycol (PEG)-based polymers (preferably PLL-g-PEG or PEG with different lengths, having about 22 to 450 repeating units and / or corresponding molecular weights ranging from 1000 Da to 20000 Da), polybetaines (such as polysulfobetaine (PSB) or polycarboxybetaine (PCB)), polyampholytes, fluorinated polymers, polysaccharides (such as agar or agarose), polyhydroxy polymers (such as poly-2-hydroxyethyl methacrylate (poly-HEMA) or polyhydroxypropyl methacrylate (poly-HPMA)), polyethylene oxide, hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol ( The present invention relates to a novel surface-active agent for the prevention of surface-induced catalytic activity of a novel nanostructured film, wherein the surface of the nanostructured film is oxidized to form a new type of surface-active agent. The present invention relates to a novel surface-active agent for the prevention of surface-induced catalytic activity of a novel nanostructured film, wherein the surface of the nanostructured film is oxidized to form a new type of surface-active agent for the prevention of surface-induced catalytic activity of a novel nanostructured film, wherein the surface of the nanostructured film is oxidized to form a new type of surface-active agent for the prevention of surface-induced catalytic activity of a novel nanostructured film, wherein the surface of the nanostructured film is oxidized to form a new type of surface-active agent for the prevention of surface-induced catalytic activity of a novel nanostructured film, wherein the surface of the nanostructured film is oxidized to form a new type of surface-active agent for the prevention of surface-induced catalytic activity of a novel nanostructured film,

[0059] According to another preferred embodiment of the present invention, at least one groove has the shape of an elliptical parabola or a hemisphere.

[0060] According to another preferred embodiment of the present invention, in the case where at least one groove has an elliptical paraboloid shape, at least part of the shell of the parabola is given by the formula y=A*x B A parabola is formed by rotating in space, wherein A is between 1 and 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and B is 2, 4 or 6. By having at least one groove in the shape of an elliptical parabola or a hemisphere according to the configuration described above, a single circular cell aggregate can be formed in at least one groove.

[0061] According to another preferred embodiment of the present invention, the width of the at least one groove is from 100 μm to 3 mm, preferably from 200 μm to 2 mm, more preferably from 500 μm to 1.5 mm.

[0062] Figures 1a to 1cA schematic plan view of one embodiment of a device 1 according to the present invention is shown. The chamber 2 of the device 1 includes a base 5 formed of a substrate, the base including eighteen grooves 7. A top 6 is located on opposite sides of the base 5, wherein the distance 8 between the base 5 and the top 6 is less than the width 9 of at least one groove 7. A hollow body 14 is located between the chamber 2 and the at least one groove 7, wherein the width of the hollow body 14 is equal to the width 9 of the at least one groove 7. The device 1 may further include a spacer arranged between the top 6 and the base 5 of the chamber 2, wherein the height of the spacer corresponds to the distance 8 between the base 5 and the top 6 of the chamber 2.

[0063] According to another preferred embodiment of the present invention, the base body comprises a base of at least one chamber, the top body comprises a top of at least one chamber, and wherein the base body and the top body are reversibly sealed to each other. As used herein, "reversibly sealed" means that the base body and the top body of the present invention can be attached and detached from each other at least once. After attaching the base body and the top body, the device can be leak-free. The attachment of the base body and the top body can be achieved by mechanical, physical and / or chemical methods. For example, the base body and the top body can be mechanically attached by a locking mechanism.

[0064] Preferably, the base body and the top body are attached to each other via an adhesive or a locking mechanism. For example, the base body and the top body of the present invention can be detached from each other by simply opening the locking mechanism or lifting the top body from the base body, allowing both to be peeled away from the adhesive without damage. After the top body is detached from the base body, the open side of the at least one groove of the present invention can be accessed, and a fluid containing cells can be applied to the at least one chamber via the at least one opening of the device.

[0065] According to another preferred embodiment of the present invention, the device is a microfluidic device, and the chamber comprises at least one opening for introducing a fluid into the chamber and at least one opening for discharging the fluid from the chamber. Surprisingly, it has been demonstrated that the microfluidic device of the present invention exhibits excellent performance for transporting and / or storing cells, in particular cell aggregates. The use of the microfluidic device according to the present invention enables the transport and / or storage of cells and cell aggregates in a reliable, compact and cost-effective manner. As described above, the size of the cell aggregates can be limited by the width of at least one groove, thereby limiting the chance of their backflow into the chamber. In addition, due to the limited volume of the microfluidic device of the present invention, the amount of fluid required to store and / or transport the cell aggregates is even less. For example, compared to existing transportation methods, the transportation costs of cell aggregates can be significantly reduced, thanks to the limited volume of fluid required and the reduced packaging weight.

[0066] Thus, the microfluidic device of the present invention comprises at least one chamber comprising at least one opening for introducing a fluid into the chamber and at least one opening for draining the fluid from the chamber, wherein the at least one chamber comprises a base and a top, wherein the top is located on opposite sides of the base, wherein the base is formed from a substrate comprising at least one groove for collecting the fluid, wherein the distance between the base and the top of the at least one chamber is less than a width of the at least one groove, and wherein the width of the at least one groove is the smallest line segment between two opposite points passing through the center of its base area.

[0067] The fluid can also be applied to the microfluidic device by introducing it into at least one chamber via at least one fluid reservoir. According to another preferred embodiment of the present invention, at least one opening is connected to at least one fluid reservoir. The advantage of at least one reservoir is that it can be filled with sufficient fluid to meet the needs of storage and / or transportation of cell aggregates. The fluid reservoir of the microfluidic device of the present invention can have any shape and can have a limited volume to accommodate (for example) enough fluid to supply a certain level of fluid to at least one chamber. The volume of at least one reservoir can be adjusted according to the expected storage and / or transportation time of the cell aggregates and the target field of use. A major advantage of the present invention is that the volume of fluid required to transport and / or store cell aggregates is significantly reduced compared to, for example, the volume of a culture bottle.

[0068] According to another preferred embodiment of the present invention, the volume of at least one reservoir is 1 mm 3 Up to 5000mm 3 , preferably 2 mm 3 Up to 4000mm 3 , preferably 5mm 3 Up to 2000mm 3 , preferably 10 mm 3 Up to 1000mm 3 , preferably 20 mm 3 Up to 500mm 3 .

[0069] Figures 2a to 2c A schematic plan view of one embodiment of a microfluidic device 10 according to the present invention is shown. The microfluidic device 10 comprises a chamber 2 having a fluid inlet 3 for introducing a fluid into the chamber 2 and a fluid outlet 4 for discharging the fluid from the chamber 2. Furthermore, the chamber 2 comprises a base 5 formed of a substrate, the base comprising fifteen grooves 7. A top 6 is located on opposite sides of the base 5, wherein a distance 8 between the base 5 and the top 6 is less than a width 9 of at least one groove 7. A hollow body 14 is located between the chamber 2 and the at least one groove 7, wherein the width of the hollow body 14 is equal to the width 9 of the at least one groove 7.

[0070] Figure 3a and Figure 3b A schematic plan view of another embodiment of a microfluidic device 15 according to the present invention is shown. The microfluidic device 15 is distinguished from the microfluidic device according to Figures 2a to 2c The microfluidic device 10 of the present invention is characterized in that the chamber 2 of the microfluidic device 10 is essentially embedded in the substrate forming the base 5 of the chamber 2. In contrast, the microfluidic device 15 according to the present invention has a planar base 11 comprising fifteen recesses 7, wherein the chamber 2 is embedded in the top layer of the device 15. Figure 3a and Figure 3b A microfluidic device 15 according to the invention is shown, the base 11 of which is attached to a top layer forming a top portion 12 of the chamber 2. A hollow body 14 is located between the chamber 2 and the at least one groove 7, wherein the width of the hollow body 14 is equal to the width 9 of the at least one groove 7. In addition, the microfluidic device 15 is distinguished from the device according to Figures 2a to 2c The microfluidic device 10 is also different in that the grooves 7 of the microfluidic device 10 are arranged in the footprint of a 1536-well plate. Therefore, the microfluidic device 15 of the present invention is suitable for standardized laboratory processes such as microplate reader detection, automated liquid handling procedures, automated imaging, etc.

[0071] The microfluidic device 15 is Figures 2a to 2c Similar elements of the microfluidic device 10 are denoted by the same reference numerals. The base 5; 11 may also comprise a different number of recesses 7.

[0072] Figure 4a Shown to contain six Figures 2a to 2c Schematic plan view of an embodiment of a microfluidic device 10. Each chamber 2 of the microfluidic device 10 is embedded in a base body.

[0073] Figure 4b Shown to include six Figure 3a and Figure 3b Schematic plan view of one embodiment of a microfluidic device 15. Fifteen wells 7 arranged in a 1536-well plate footprint are housed in a base body.

[0074] Figure 5a and Figure 5b Show the basis Figures 2a to 2c Schematic plan view of the top layer of the microfluidic device 10, which includes six pairs of fluid reservoirs 13. The fluid reservoirs 13 are connected to an opening 3 for introducing fluid into the chamber 2 and an opening 4 for draining fluid from the chamber 2. The top layer can also include various chamber structures adapted to the planar base 11, for example, according to Figure 3a and Figure 3b One embodiment of a microfluidic device 15 is shown.

[0075] The substrate of the microfluidic device can comprise, consist of, or be coated with a biocompatible material, wherein the biocompatible material is silicone, a plastic material, or glass. Silicone and glass are typically processed using standard photolithographic methods and allow for rapid and easy fabrication of microfluidic devices. The microfluidic devices of the present invention can be produced using microfabrication methods known in the art, such as casting, 3D printing, photolithography, hot embossing, or microinjection molding.

[0076] As discussed above, the base body includes the base of at least one chamber, and the top body includes the top of at least one chamber. According to another embodiment of the present invention, the top of the chamber can be detached from the base. At least one chamber of the device of the present invention can also be closed by attaching the top and the base to each other, wherein the distance between the base and the top of the closed chamber is less than the width of the at least one groove. Therefore, after attaching the base and the top, the size of the cell aggregate can be limited to the width of the at least one groove.

[0077] According to another preferred embodiment of the present invention, the device includes a spacer disposed between the top and base of the chamber, wherein the height of the spacer corresponds to the distance between the base and top of the chamber when the top of the chamber is attached to the base. Since the distance between the base and top of the chamber is crucial, as described above, the spacer of the present invention is able to maintain this distance. The presence of the spacer is also user-friendly, as it automatically ensures the proper chamber height according to the present invention, eliminating the need for manual adjustment of the critical distance between the base and top.

[0078] According to another preferred embodiment of the present invention, the base is circular and the spacers are arranged along the circumference of the circular base.

[0079] According to another further preferred embodiment of the present invention, the spacers are arranged equidistantly along the circumference of the base.

[0080] The base and the top may be attached to each other by a locking mechanism, such as a threaded connection. According to another preferred embodiment, the top is connectable to the base via a threaded connection.

[0081] According to another preferred embodiment, the device comprises a base body having a base with a chamber, and a top body having a top with a chamber, wherein the base body comprises a cylindrical receiving volume, the base being arranged at the bottom of the receiving volume, and the top body comprising an extension adapted to extend into the receiving volume when the threaded connection is closed, the top being arranged at one end of the extension, and the threaded connection being arranged at the opposite end region of the extension. In this case, the distance between the base and the top of at least one closed chamber can be less than the width of at least one groove. The cells in the device can form aggregates that are stored in at least one groove of the device for transport and / or storage.

[0082] Another aspect of the present invention relates to a method for storing and / or transporting cell aggregates. A fluid containing cells is applied to at least one chamber of a device as defined above, and the cells are incubated until at least one aggregate is formed in at least one groove. According to the present invention, the cell aggregates can be stored and / or transported for a period of time between 1 day and 10 days, preferably between 2 days and 8 days.

[0083] In particular, the present invention provides a method for storing and / or transporting cells, preferably eukaryotic cells, preferably mammalian cells, preferably stem cells and / or tumor cells.

[0084] Surprisingly, it has been demonstrated that extracellular vesicles can also be stored and / or transported by the methods of the present invention. Extracellular vesicles can have a diameter of 20 nm to 5000 nm and can be secreted by a wide variety of cell types. In general, extracellular vesicles (such as exosomes, microvesicles, and apoptotic bodies) are membrane-bound structures and can be loaded with, for example, therapeutic substances. Exosomes are a type of extracellular vesicle that can be secreted by most eukaryotic cells. Microvesicles are another type of extracellular vesicle that buds outward from the cell surface membrane. On the other hand, apoptotic bodies are extracellular vesicles formed by dead cell fragments. Exosomes, microvesicles, and apoptotic bodies can be released in vivo or in vitro (such as in cell culture). It is known that extracellular vesicles secreted from various cells act as intercellular communication media in vivo and play an important role in the physiological phenomena of various diseases such as cancer.

[0085] Preferably, these cells are stored and / or transported in the form of aggregates. It has been demonstrated that the method of the present invention can transport and / or store cells in the form of aggregates in a compact and cost-effective manner. Therefore, due to the reduction in the required packaging size, the transportation process is simple, efficient and inexpensive. The method of the present invention requires much fewer reagents than other live cell transport technologies. Preferably, cells are stored and transported under different conditions. For example, cell aggregates can be stored at a temperature between -196°C and 40°C. Cell aggregates can also be stored at low temperatures (e.g., -80°C) and transported at room temperature or 37°C. Figure 6 A schematic workflow illustrating a method according to one embodiment of the present invention includes applying a cell suspension to a device according to the present invention, incubating the device to form cell aggregates, freezing the cell aggregates within the device according to the present invention in liquid nitrogen (N2) for storage, thawing the aggregates in a cell culture incubator after storage, transporting the device, disaggregating the cell aggregates into individual cells, and seeding the resulting cell suspension into a cell culture flask. It has been found that cells exhibit good survival after storage and / or transport.

[0086] As used herein, the term "cell viability" refers to the number of viable eukaryotic and / or prokaryotic cells in a cell population.

[0087] Surprisingly, the inventive method is proven to be a reliable and cost-effective method for transporting and / or storing cell aggregates, while ensuring good cell viability and natural cell environment during the whole storage and / or transportation of preferably eukaryotic cells and / or prokaryotic cells by three-dimensional cell culture. Preferably, the fluid comprising the cells also comprises nutrients to supply cells, such as cell culture medium. If the cells are stored and / or transported under freezing conditions, the fluid comprising the cells can comprise cryopreservation medium. The freezing and thawing of biological samples (such as cells) may be a key step in the cryopreservation process, which can affect the recovery rate of living cells after storage and / or transportation. Cryopreservation medium can achieve the cryopreservation of various source cells, and highly maintains the survival rate and the recovery rate of cells and tissues after thawing. Cryopreservation medium is known in the art, and can comprise serum, dimethyl sulfoxide (DMSO), glycerol or a combination thereof. In addition, cryopreservation medium can be substantially free of serum, and comprises DMSO, glycerol or a combination thereof. Preferably, cryopreservation medium is free of animal components, serum-free and protein-free. It has been found that by mixing cryopreservation medium and CO2 independent culture medium, good cell viability is achieved in the whole storage and / or transportation process of frozen cells.CO2 independent culture medium is also known in the art, and can be used for supporting cell growth under the condition of not having CO2 cell culture box.The fluid comprising cell can comprise at least one cryopreservation medium of 5 volume % to 95 volume %, preferably 10 volume % to 90 volume %, preferably 10 volume % to 60 volume %, preferably 10 volume % to 40 volume %, preferably 10 volume % to 40 volume %, preferably 15 volume % to 30 volume %.Preferably, fluid comprises at least one cryopreservation medium and at least one CO2 independent culture medium, its ratio is between 1:1 to 1:10 (cryopreservation medium: CO2 independent culture medium), preferably between 1:2 to 1:5.

[0088] In order to use extracellular vesicles for analysis and diagnosis, they must first be concentrated and recovered from biological samples such as blood, urine, or saliva. The concentrated extracellular vesicles can be stored and / or transported simply and safely using the methods of the present invention.

[0089] Another advantage of the present invention relates to the possibility of storing and / or transporting cells of different types, sources and / or patients. An advantage of the method of the present invention is that a single device can store and / or transport different samples without increasing the packaging size. Different samples in the form of cells, in particular cell aggregates, can be stored and / or transported in different chambers of the device (in particular in a microfluidic device), so that each cell type, patient, etc. can be directly classified into each chamber. The method of the present invention requires much less reagents than other live cell transport technologies. According to the method of the present invention, at least one device, preferably at least two devices, preferably at least three devices, preferably at least four devices, preferably at least five devices, preferably at least ten devices, preferably at least twenty devices, preferably at least thirty devices, preferably at least forty devices, preferably at least fifty devices, preferably at least sixty devices, preferably at least seventy devices, preferably at least eighty devices, preferably at least ninety devices, preferably at least one hundred devices can be stored and / or transported simultaneously.

[0090] According to another preferred embodiment of the present invention, the device is centrifuged at a speed of 50 rcf to 500 rcf, preferably 150 rcf to 300 rcf after step a) to promote cell aggregation. It has been confirmed that the centrifugation of the device can effectively optimize and improve cell aggregation time and cell aggregate morphology.

[0091] According to another preferred embodiment of the present invention, the applied fluid comprises 1.0×10 5 cells / mL, preferably 2.5×10 5 cells / mL, preferably 5.0×10 5 cells / mL, 7.5×10 5 cells / mL, 1.0×10 6 cells / mL, preferably 2.0×10 6 cells / mL, preferably 3.0×10 6 The cell density of a fluid can be determined by methods known in the art, such as using a hemocytometer.

[0092] To simulate the natural environment of cell aggregates, preferably cell aggregates within the device of the present invention, it is particularly preferred to apply a hydrogel to at least one chamber of the device. For example, the hydrogel can form a matrix for producing spheroids.

[0093] According to another preferred embodiment of the present invention, a hydrogel is introduced into at least one chamber after step a) or b), wherein the hydrogel consists of at least one compound selected from the group consisting of polyethylene glycol, polyacrylamide, dextran, collagen, fibrin, fibronectin, laminin, hyaluronic acid, silk fibroin, alginate, cellulose or a combination thereof.

[0094] Preferably, the cell aggregates can be harvested from the device according to the present invention after storage and / or transportation. After harvest, the cell aggregates can be further used for example in biomedical research, drug production, personalized medicine programs, etc. In order to harvest cell aggregates from the device, it may be necessary to digest the hydrogel to reach the corresponding cell aggregates. According to another preferred embodiment of the present invention, the hydrogel is digested by an enzyme selected from the group consisting of collagenase, hyaluronidase, cellulase, ethylenediaminetetraacetic acid (EDTA), sodium citrate, dispase, α-chymotrypsin, N-acetylcysteine, glutathione, dithiothreitol, and combinations thereof.

[0095] According to another preferred embodiment of the present invention, at least one aggregate and / or cell is collected from the device.

[0096] According to another preferred embodiment of the present invention, at least one cell aggregate and / or cell is collected from at least one groove by separating the top body and the bottom body as mentioned above. Specifically, the top body and the bottom body of the device of the present invention are disassembled from each other, and then the cell aggregates are collected from the device by, for example, pipetting. Alternatively, after separating the top body and the bottom body, the hydrogel can also be digested by enzymes. In this case, the top body and the bottom body can be carefully disassembled, and then the enzyme can be added to the hydrogel containing the cell aggregates. After digesting the hydrogel, the cell aggregates can be collected from the device.

[0097] Cell aggregates can also be disaggregated into individual cells. Specifically, spheroids or organoids can be disaggregated into individual cells, or aggregates composed of extracellular vesicles can be disaggregated into individual extracellular vesicles. This has the advantage that, for example, in the case of eukaryotic cells, individual cells can be further passaged as a monolayer culture in a cell culture flask. According to another preferred embodiment of the present invention, cells are collected by disaggregating at least one aggregate into cells, and wherein the cells are collected through at least one opening and / or collected from at least one chamber by separating the top body and the bottom body as defined above.

[0098] According to another preferred embodiment of the present invention, disaggregation is achieved by applying a fluid containing trypsin, collagenase, Accutase, dispase, elastase, ethylenediaminetetraacetic acid (EDTA), sodium citrate or a combination thereof to at least one aggregate.

[0099] Another aspect of the present invention relates to the use of a device as defined above for the storage and / or transport of cells and / or cell aggregates. In particular, the device of the present invention can be used to transport living cells in a compact, reliable and cost-effective manner. In addition, the device can be used for personalized medical solutions. For example, eukaryotic cells, preferably tumor cells, or extracellular vesicles from a patient's blood or urine can be collected and transported to a desired destination using the device of the present invention for screening, evaluation and selection of appropriate treatment. A major advantage of the present invention relates to the fact that the amount of patient sample required is minimal compared to standard transport solutions.

[0100] Another aspect of the present invention relates to a method for screening compounds in cell aggregates. Specifically, the method of the present invention can be used to screen compound libraries of synthetic molecules, natural products or extracts, traditional small molecule drugs (usually derived from chemical synthesis) and biopharmaceuticals, wherein biopharmaceuticals include recombinant proteins, vaccines, therapeutic blood products (such as intravenous immunoglobulins), gene therapy, monoclonal antibodies and cell therapy (such as stem cell therapy). Different concentrations of compounds are applied to cell aggregates to determine whether the effect of the substance on the cells in the cell spheroids is beneficial or negative. This method can be used, for example, to identify compound doses that may have beneficial, toxic, inhibitory and / or stimulatory effects on cell aggregates.

[0101] Figure 7A schematic workflow of a method according to one embodiment of the present invention is shown, comprising: applying a cell suspension to a device according to the present invention (particularly a microfluidic device), incubating the device to form cell aggregates, applying to at least one compound by, for example, a pipetting robot to treat the cell aggregates, and measuring the effect of the compound on the aggregates by high content and / or high throughput screening. Cell aggregates can be treated in the device according to the present invention, or the aggregates can be harvested from the device before being treated with the compound. In the latter case, the aggregates can be treated and analyzed in a separate cell culture platform (as known in the art). Preferably, at least one compound is applied to at least one cell aggregate stored in the microfluidic device according to the present invention, and then the effect of the compound on the aggregates is measured. In addition, cell aggregates can be treated and analyzed on a microfluidic device. Preferably, cell aggregates can be generated and analyzed in a reliable and reproducible manner on a single microfluidic device by the method according to the present invention. In addition, the microfluidic device can be compatible with standardized laboratory equipment. For example, fluids, cells, compounds, etc. can be applied to the microfluidic device by a microchannel pipette and a pipetting robot, and the effect of the compound on the cell aggregates can be measured by an advanced microplate reader. Therefore, the present invention can analyze the patient's physiological state in a multi-parameter and multi-modal manner.

[0102] Preferably, the cells are obtained from a mammalian source, preferably a human.By means of the present invention, physiological parameters and / or disease states can be determined "from bench to bedside", meaning that the results obtained using the methods of the present invention can be used directly to formulate a treatment plan for a patient.

[0103] According to another preferred embodiment of the present invention, the cell is a tumor cell. Preferably, the tumor cell is selected from the group consisting of benign adenoma tumor cells, angiofibroma tumor cells, hemangioma tumor cells, leiomyoma tumor cells (fibroma), benign chorioangioma tumor cells, benign colon tumor cells, cystadenoma tumor cells, dermoid tumor cells, desmoid tumor cells, ductal carcinoma in situ (DCIS) tumor cells, fibroadenoma tumor cells, fibroma tumor cells, benign ganglioneuroma tumor cells, lipoma tumor cells, meningioma tumor cells, myxoma tumor cells, neurofibroma tumor cells, nevus tumor cells, osteochondroma tumor cells, pheochromocytoma tumor cells, polyposis tumor cells, schwannoma tumor cells, prostatic intraepithelial neoplasia tumor cells, benign prostatic hyperplasia (hypertrophic) tumor cells, benign teratoma tumor cells, benign thymoma tumor cells and Brenner tumor tumor cells.

[0104] According to another preferred embodiment of the present invention, tumor cells are obtained by dissociating solid tumors. As used herein, "solid tumor" refers to an abnormal tissue mass that generally does not contain cysts or fluid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Different types of solid tumors are named according to the type of cells they form. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.

[0105] Example

[0106] Materials and methods

[0107] Microfluidic device preparation:

[0108] The microfluidic device according to the present invention was prepared by double-molding of polydimethylsiloxane (PDMS).The master mold containing the groove and chamber structure was made of polymethyl methacrylate (PMMA) by CNC micro-milling (Denz-Biomedical, Austria).

[0109] PDMS (Sylgard 184 silicone elastomer, Farnell, Austria) was mixed with a curing agent in a 10:1 weight ratio. The polymer was degassed in a vacuum chamber for 1 hour, cast onto the PMMA structure, and then baked at 80°C for 2 hours. The structure was then peeled from the PMMA substrate and hard-baked at 90°C for 48 hours. This resulted in the final PDMS mold for base layer fabrication. To ensure sufficient separation of the PDMS structure from the mold, the PDMS mold surface was plasma activated and silanized with trichloro(1H,1H,2H,2H-perfluorooctyl)silane (Sigma-Aldrich, Austria) under vacuum for 10 minutes, followed by baking at 80°C for 1 hour. The top mold containing the reservoir was 3D-printed by iMaterialise (Denmark). The PDMS master mix was poured into the 3D-printed mold and baked at 70°C for 2 hours. Before bonding, each chamber was coated with a 0.5 wt% antifouling Lipidure-CM5206 solution (AMSbio, UK) at 80°C for 1 hour. A 1.5 mm diameter hole was punched in the reservoir layer using a biopsy needle to connect the reservoir to the chamber. The two PDMS layers (base layer and top layer containing the reservoir) were bonded by O plasma activation (30 s, 0.9 mbar, 200 W (Diener, Germany)) and then baked at 80°C overnight.

[0110] Cell culture:

[0111] HeLa and HEK cells were expanded in T75 culture flasks (Greiner, Austria) containing 10 mL of DMEM cell culture medium (Sigma-Aldrich, Austria) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Austria), 2 mM L-glutamine (Sigma-Aldrich, Austria), and 1% penicillin / streptomycin at 37°C and 5% CO2. Cells were expanded to 80%-90% confluency in preparation for inoculation into the microfluidic device. Prior to inoculation, cells were washed once with PBS (Sigma-Aldrich, Austria) and detached from the expansion flasks with 2 mL of trypsin-EDTA solution (Sigma-Aldrich, Austria) for 5 minutes. Detached cells were collected, counted, and the concentration adjusted accordingly in complete growth medium.

[0112] Cell loading:

[0113] Before cell inoculation, the chamber was filled with 70% ethanol and placed in an ultrasonic bath to remove air bubbles. The device was washed three times with 200 μL of 70% ethanol and then sterilized by washing three times with 200 μL of 1× PBS (Sigma-Aldrich, Austria) supplemented with 1% penicillin / streptomycin to remove ethanol from the chamber. The device was maintained and incubated in a quadriPERM chamber (Sarstedt, Austria) filled with 2 mL of 1× PBS supplemented with 1% antibiotic / antimycotic solution (Sigma-Aldrich, Austria) to avoid liquid evaporation. Before cell inoculation, PBS was removed from all reservoirs and pretreated with 200 μL of cell culture medium. After removing the culture medium, 100 μL of cell suspension was added to each chamber. The next day, the chamber was rinsed with 200 μL of growth medium to remove excess cells.

[0114] Cell viability assessment:

[0115] Cell viability was determined by live-dead cell staining. Therefore, 10 μg / mL Hoechst 33342 (Sigma-Aldrich, Austria), 2 μM calcein-AM (Invitrogen, Austria) and 4 μM EtBr (Invitrogen, Austria) were mixed in complete growth medium and then applied to the cell aggregates in the microfluidic device. The microfluidic device was placed in 37°C, 5% CO2 and incubated under a fluorescence microscope (Olympus IX83) for 30 hours to collect images. ImageJ was used to measure the fluorescence intensity of different channels (blue / Hoechst 33342 / total cell count; green / calcein-AM / living cells; red / EtBr / dead cells) of cell aggregates.

[0116] CFD simulation:

[0117] All CFD simulations were performed using AutoCAD software (Autodesk). The generated SAT files were loaded into Autodesk CFD software (Autodesk). The model was assigned water properties (Autodesk default parameters), with the simulated flow rate of the microfluidic device set to 10 μL / min and the outlet pressure set to atmospheric pressure. All simulations were performed using these settings.

[0118] result

[0119] To estimate the microfluidic flow pattern distribution of the device according to the present invention under different operating conditions, computational fluid dynamics (CFD) simulations were performed. Velocity measurements were determined by computer simulation to evaluate the effects of three different channel heights at a fixed simulated flow rate of 10 μL / min. Figure 8a and Figure 8b Results in the study showed that the fluid flow distribution was more uniform at a lower chamber height of 50 μm compared to a higher chamber height of 150 μm. Surprisingly, these findings indicate that the lower chamber height resulted in fluid streamlines that uniformly wrapped around the entire groove array of the microfluidic device without generating turbulence, indicating that the culture medium within the grooves was more efficiently refreshed at the lower chamber height compared to the higher chamber height.

[0120] Furthermore, it has been observed that devices with chamber heights smaller than the groove width can avoid spheroid loss and / or damage compared to microfluidic devices with chamber heights greater than the groove width.

[0121] To evaluate the effect of the hollow body width on the microfluidic flow pattern distribution within the device, (CFD) simulations were performed at a constant simulated flow rate of 10 μL / min. Figure 9a and Figure 9b Results in the study showed that increasing the width of the hollow body enhanced fluid flow within the grooves. This effect resulted in better fluid supply (e.g., of cells) with nutrients and improved cell capture within the grooves during application of a cell suspension.

[0122] To evaluate the optimal storage conditions and / or storage conditions for cells in a microfluidic device according to the present invention, HeLa cells were applied to two different devices: one device was stored under cell culture conditions (37°C, 5% CO2 atmosphere) and the second device was stored at room temperature. After overnight storage in both devices, the survival rate of the cells in the form of spheroids was assessed. Figure 10 The results in

[14] showed that HeLa cell aggregates cultured in the microfluidic device did not significantly lose viability after 24 h compared to culture conditions at 37°C, 5% CO2 and room temperature.

[0123] In addition, cell aggregates cultured at room temperature for 48 hours can be harvested from the microfluidic device by digesting the aggregates and removing the cell-containing liquid. Figure 11 As shown, the cell concentration trend of the harvested cell suspension when seeded in a cell culture plate is the same as that observed when seeded in a microfluidic device. These results indicate that a wide range of viable HeLa cell seeding densities can be achieved in culture flasks after storage as cell aggregates in the microfluidic device of the present invention.

[0124] In addition, the effect of storage medium was tested in more detail. Aggregates of HeLa and Hek cells were stored in cryopreservation medium containing different concentrations of Sartorius) CO2-independent medium (Gibco TM The cells were incubated for three days in CO2-independent culture medium (Fisher Scientific). After incubation, the cell aggregates were harvested by digestion and the cell viability was determined in 96-well cell culture plates. Figure 12 The results showed that HeLa and Hek cell lines showed good cell survival rates after three days of culture at a cryopreservation medium concentration below 30% by volume. Furthermore, when the cryopreservation medium concentration was 15% by volume, the cells showed good survival rates, especially for HeLa cells.

[0125] in conclusion:

[0126] The design of the microfluidic device has been optimized to achieve a streamlined (e.g., no voids and dead volume) flow pattern distribution by adjusting the channel height and groove shape. CFD simulation results confirm that reducing the channel height has a significant impact on the flow pattern distribution within the microfluidic device. The optimized flow pattern distribution improves the supply of nutrients to the captured cell aggregates while enhancing the capture effect of the cells in the grooves. The microfluidic device is capable of forming cell aggregates from a solution containing individual cells and maintaining a similar number of cells in each groove. In summary, the device according to the present invention is a simple, cost-effective tool suitable for the storage and / or transportation of cell aggregates and has a wide range of applications.

Claims

1. A device (1; 10; 15) for storing and / or transporting cells, in particular cell aggregates, wherein the device comprises at least one chamber (2) having a base (5; 11) and a top (6; 12), wherein the top (6; 12) is located on opposite sides of the base (5; 11), wherein the base (5; 11) is formed by a substrate comprising at least one groove (7) for collecting fluid, wherein the distance (8) between the base (5; 11) and the top (6; 12) of the at least one chamber (2) is smaller than the width (9) of the at least one groove (7), and wherein the width (9) of the at least one groove (7) is the smallest line segment between two opposite points passing through the center of the base area of ​​the at least one groove (7).

2. The device (1; 10; 15) according to claim 1, wherein the base (5; 11) of the at least one chamber (2) further comprises a hollow body (14), wherein the hollow body (14) is fluidically connected to the interior of the at least one chamber (2) and the at least one groove (7), and wherein the width of the base region of the hollow body (14) is at least partially the width (9) of the at least one groove (7).

3. A device (1; 10; 15) according to claim 2, wherein the width of the top area of ​​the hollow body (14) is between 500 μm and 3 mm, preferably between 1 mm and 2 mm, wherein the width of the hollow body (14) is the smallest line segment between two opposite points passing through the center of the top area of ​​the at least one groove (7).

4. Device (1; 10; 15) according to claim 2 or 3, wherein the hollow body (14) has a frustoconical shape, and wherein the base area and the top area of ​​the frustoconical shape have a circular or elliptical shape.

5. Device (1; 10; 15) according to any one of claims 1 to 4, wherein the ratio of the distance (8) between the base (5; 11) and the top (6; 12) of the at least one chamber (2) to the width (9) of the at least one groove (7) is between 0.5:1 and 0.01:1, preferably between 0.2:1 and 0.02:1, preferably between 0.1:1 and 0.04:

1.

6. Device (1; 10; 15) according to any one of claims 1 to 5, wherein the at least one groove (7) has the shape of an elliptical parabola or a hemisphere.

7. The device (1; 10; 15) according to any one of claims 1 to 6, wherein the width (9) of the at least one groove (7) is 100 μm to 3 mm, preferably 200 μm to 2 mm, more preferably 500 μm to 1.5 mm.

8. A device (1; 10; 15) according to any one of claims 1 to 7, wherein the base body comprises the base (5; 11) of the at least one chamber (2), and the top body comprises the top (6; 12) of the at least one chamber (2), and wherein the base body and the top body are reversibly sealed to each other.

9. The device (10; 15) according to any one of claims 1 to 8, wherein the device is a microfluidic device and the chamber (2) comprises at least one opening (3) for introducing a fluid into the chamber (2) and at least one opening (4) for discharging a fluid from the chamber (2).

10. Device (10; 15) according to claim 9, wherein the at least one opening (3, 4) is connected to at least one fluid reservoir (13).

11. The device (10; 15) according to any one of claims 1 to 8, wherein the top (6; 12) of the chamber (2) is detachable from the base (5; 11).

12. The device (1) according to claim 11, wherein the device comprises a spacer arranged between the top (6) and the base (5) of the chamber (2), wherein the height of the spacer corresponds to the distance (8) between the base (5) and the top (6) of the chamber (2) when the top (6) of the chamber (2) is attached to the base (5).

13. The device (1) according to claim 12, wherein the base (5) is circular and the spacers are arranged along the circumference of the circular base (5).

14. The device (1) according to claim 13, wherein the spacers are arranged equidistantly along the circumference of the base (5).

15. The device (1) according to any one of claims 11 to 14, wherein the top (6) is connectable to the base (5) via a threaded connection.

16. A device (1) according to any one of claims 15, wherein the device (1) comprises a base body having the base (5) having the chamber (2), and a top body having the top (6; 12) having the chamber (2), and wherein the base body comprises a cylindrical receiving volume, the base (5) being arranged at the bottom of the receiving volume, and the top body comprising an extension portion, which is suitable for extending into the receiving volume when the threaded connection is closed, the top portion (6) being arranged at one end of the extension portion, and the threaded connection being arranged at the opposite end region of the extension portion.

17. A method for storing and / or transporting cells, in particular cell aggregates, comprising the following steps: a) applying a fluid containing cells to the at least one chamber (2) of the device (1; 10; 15) according to any one of claims 1 to 16, thereby providing the fluid to the at least one recess (7), and b) incubating the cells until at least one aggregate is formed in the at least one recess (7).

18. The method according to claim 17, wherein after step a) or b) a hydrogel is introduced into the at least one chamber (2), wherein the hydrogel consists of at least one compound selected from the group consisting of: Polyethylene glycol, polyacrylamide, dextran, collagen, fibrin, fibronectin, laminin, hyaluronic acid, silk fibroin, alginate, cellulose or a combination thereof.

19. The method of claim 18, wherein the hydrogel is digested by an enzyme selected from the group consisting of collagenase, hyaluronidase, cellulase, ethylenediaminetetraacetic acid (EDTA), sodium citrate, dispase, α-chymotrypsin, N-acetylcysteine, glutathione, dithiothreitol, and combinations thereof.

20. The method according to any one of claims 17 to 19, wherein the at least one cell aggregate and / or the cells are collected from the device (1; 10; 15) after step b).

21. Use of the device (1; 10; 15) according to any one of claims 1 to 16 for the storage and / or transport of cells, in particular cell aggregates.

22. A method for screening the effect of a compound on cell aggregates, comprising the steps of: a) providing a solution comprising cells, b) applying the solution of step a) to the at least one chamber (2) of the device (1; 10; 15) according to any one of claims 1 to 16, thereby providing the fluid to the at least one groove (7), c) incubating the cells until at least one cell aggregate is formed in the at least one recess (7), d) treating at least one cell aggregate with at least one compound, and d) measuring the effect of said at least one compound on said at least one cell aggregate.

Images