IT201900005820A1 - Cell culture device - Google Patents

Description

Description of industrial invention

Cell culture device

[0001] Field of the invention

[0002] The invention relates to a device that can be used to cultivate cells, in particular human or animal eukaryotic cells, and to allow microscopic observation of the cultured cells, in particular by means of a transmission electron microscope.

[0003] The invention can be applied industrially in the field of manufacturing laboratory material for cell cultures and for microscopic observation of cells using transmission electron microscopy.

[0004] State of the art

Transmission electron microscopy is a microscopic observation method that plays a key role in studying the ultrastructural characteristics of cells. The aforementioned observation method is in fact able to provide details on morphology, on the various cell organelles, as well as on cell-cell interactions. These details are not obtainable using traditional light microscopy, which allows to observe and select cells in culture (for example, cells included in a cell monolayer) but cannot provide information on their ultrastructure. In this context, numerous methods have been studied to examine specific areas of monolayers of cultured cells by combining optical microscopy (suitable for identifying single cells) with transmission electron microscopy (suitable for providing details on cellular ultrastructure).

In the first methods used to include the adhered cell monolayer in situ, plastic flasks for cell cultures were employed. Despite the numerous advantages described by the Authors, to recover the thin resin layer containing the included cellular monolayer it is necessary to remove the resin by cutting the plastic envelope, select and recover a small area to be glued later on a solidified resin capsule (Brinkley BR, Murphy P, Richardson LC. Procedure for embedding in situ selected cells cultured in vitro. J Cell Biol. 1967; 35 (1): 279-83). The procedure described above therefore has the drawback of substantial laboriousness.

[0007] A further drawback related to the use of flasks or plates of known plastics is incompatibility with solvents, such as propylene oxide used to prepare resins, or with some types of resins. This required changes or adaptations of known and used protocols for resin inclusion (Kuhn H. A simple method for the preparation of cell cultures for ultrastructural investigation. J Histochem Cytochem. 1981 Jan; 29 (1): 84-6; Hanson HH, Reilly JE, Lee R, Janssen WG, Phillips GR. Streamlined embedding of cell monolayers on gridded glass bottom imaging dishes for correlative light and electron microscop. Microsc Microanal 2010; 16 (6): 747–754. Doi: 10.1017 / S1431927610094092; Spoerri PE, Dresp W, Heyder E. A simple embedding technique for monolayer neuronal cultures grown in plastic flasks. Acta Anat. (Basel), 1980; 107 (2): 221-3).

[0008] The use of specimen slides or coverslips has been proposed as supports for culturing and including a monolayer of cells intended to be observed in transmission electron microscopy. A drawback connected to the aforementioned use consists in the extreme difficulty, or even impossibility, of separating the slide (cover-object or object-holder) from the polymerized resin (Chang JP. A new technique for separation of coverglass substrate from epoxy-embedded specimens for electron microscopy J. Ultrastruct. Res.

1971 Nov; 37 (3): 370).

[0009] To overcome this drawback, plastic coverslips have been used in other protocols (Trusal LR, Baker CJ, Guzman AW. Transmission and scanning electron microscopy of cell monolayers grown on polymethylpentene coverslips Stain Technol. 1977; 52 (4 ): 240-2) or glass slides pre-treated with different compounds (Chang JP. A new technique for separation of coverglass substrate from epoxy-embedded specimens for electron microscopy J. Ultrastruct. Res. 1971 Nov; 37 (3): 370-7; Heyner S. In situ embedding of cultured cells or tissue, grown on glass, in epoxy resins for electron microscopy. Stain Technol. 1963 Nov; 38: 335-6) to facilitate the separation and recovery of the specimen to be examined .

[0010] In order to facilitate the in situ inclusion procedure of the cell monolayer grown on the coverslips, further methods have been developed which use BEEM <®> capsules of known type. The BEEM <®> capsule is used in electron microscopy to embed tissues and comprises a cylindrical hollow body made of plastic (eg, polyethylene), provided with a conical or flat bottom and a hinged cap. According to a protocol based on the use of BEEM <®> capsules, the latter are filled with resin and superimposed on specific areas of the cell monolayer adhered to the cover slide, so as to allow the subsequent preparation of ultra-thin sections suitable for electron microscopy transmission. After resin polymerization, the capsules are removed by liquid nitrogen vapors (Sawaguchi A, Aoyama F, Ide S, Suganuma T. Capsule-supporting ring: a new device for resin embedding of glass-mounted specimens. Journal of Microscopy 2009; 234 (2): 113-7) or elevated temperatures (Hanson HH, Reilly JE, Lee R, Janssen WG, Phillips GR. Streamlined embedding of cell monolayers on gridded glass bottom imaging dishes for correlative light and electron microscop. Microsc Microanal 2010; 16 (6): 747–754. Doi: 10.1017 / S1431927610094092).

[0011] A drawback of the procedure described above consists in the fact that, once the polymerization of the embedding material (resin) is completed, the slide may fail to detach or break with consequent loss of the cell sample. A further drawback of the aforementioned protocol is that it allows you to observe only some areas of the entire sample.

[0012] According to another procedure, the cells are cultured on the cap surface of BEEM <®> capsules pre-treated with solutions (Asafo-Adjei E, Bernard EF, Hase T. Growing and embedding monolayer cells in situ BEEM capsule caps for light and electron microscopy. Journal of electron microscopy technique. 1987; 5: 367-372) or carbon coated (Eppig JJ, Leiter EH, Waymouth C. Culture of cells in BEEM capsules: a new technique for electron microscopic study of monolayer cultures. In Vitro, 1976 Jan; 12 (1): 65-73), processed and included directly in the capsule. A drawback of the procedure described above is that the cap must be removed from the capsule before proceeding with the resin impregnation, processed separately and attached to a new capsule for the final step of polymerization of the resin and formation of the block in which the cell sample will be included (Asafo-Adjei E, Bernard EF, Hase T. Growing and embedding monolayer cells in situ BEEM capsule caps for light and electron microscopy. Journal of electron microscopy technique. 1987; 5: 367-372). Additionally, the conical portion of the BEEM <®> capsule must be cut (e.g., by scalpel) at the end of cell culture before proceeding with the fixation, dehydration, and resin impregnation steps (Eppig JJ, Leiter EH, Waymouth C. Culture of cells in BEEM capsules: a new technique for electron microscopic study of monolayer cultures. In Vitro, 1976 Jan; 12 (1): 65-73).

[0013] According to a further procedure, the conical bottom of the pretreated BEEM <®> capsules is also used as a cell growth support before proceeding to the resin embedding step (Wang F, Ledford LB, Head JF, Elliott RL. A novel cell culture technique for electron microscopy. Microsc. Res. Tech. 1993 Dec 15; 26 (6): 517-22). A drawback of this procedure is the impossibility of observing the cell monolayer under an inverted optical microscope during culture and identifying a particular cellular detail.

[0014] Another protocol foresees the execution of the ultrastructural analysis on cellular aggregates (pellets) obtained after centrifugation of fixed cells, which are detached from the plastic growth support through the use of mechanical means (scraper) or through a rapid treatment in propylene oxide, possibly incorporated in solidified agarose, before proceeding with the resin inclusion phase (Graham L, Orenstein JM. Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research. Nat. Protoc. 2007; 2 (10): 2439-50). Although the protocol described above is easy to use, a not negligible drawback consists in the risk of losing morphological details of the cells grown on the support or morphological details that appear in the same cells after specific experimental treatments. In case of reduced visibility of the tiny cell pellet, the latter is fixed, stained and incorporated in solidified agarose before processing for transmission electron microscopy (Kumar S, Ciraolo G, Hinge A, Filippi MD. An efficient and reproducible process for transmission electron microscopy (TEM) of rare cell populations. J. Immunol. Methods 2014 Feb; 404: 87-90. doi: 10.1016 / j.jim.2013.11.025).

[0015] In light of the foregoing, there is a strong need among those skilled in the art to overcome the many drawbacks that substantially limit the applicability of the known procedures and devices.

[0016] Aims of the invention

[0017] An object of the invention is to improve the protocols and devices of the known type, which can be used to examine cells in culture by combining optical microscopy with transmission electron microscopy.

Another object is to provide a device for microscopically examining cells in culture avoiding the risk of losing the cell sample and / or any cellular morphological details deriving from experimental treatments, such as for example cell differentiation assays.

A further object is to provide a device for microscopically examining cells in culture which allows specific areas of the cell sample to be observed.

[0020] Yet another object is to provide a device for microscopically examining cells in culture that is substantially simple to use, in particular a device that does not require the use of liquid nitrogen or high temperature heat treatments to recover the cell sample.

Still a further object is to provide a device in which cells can be cultured, processed and included in situ and examined microscopically.

[0022] Brief description of the invention

[0023] According to the invention, a device for cultivating cells is provided, as defined in claim 1.

[0024] Thanks to the invention, the various drawbacks associated with the use of known devices and protocols are overcome. In fact, the device according to the invention comprises at least one culture well having two opposite open ends and provided with a closing member, which is flat, transparent and removable. The closing member of the well is applied (by pressure or through an adhesive compound) on one end of the body of the well corresponding to the bottom (or base) of the latter and can be removed if necessary. An internal surface (or face) of the closure member of the well can be suitably used as a solid support on which to seed and grow cells, so as to produce a cell monolayer. Since the closure member of the well is transparent and corresponds to the base (or bottom) of the well, it is possible to place the base of the well on the object tray of an inverted light microscope and observe the cells forming the monolayer under the microscope.

The same cells observed under the light microscope can subsequently be examined under the transmission electron microscope. In fact, using known methods and materials inside the well, it is possible to prepare a block of polymerized resin, which comes into contact with (and gradually incorporates) the cells forming the monolayer and adhering to the closing member. By manually removing the closing member, the cell monolayer is transferred from the closing member to the adjacent surface of the polymerized resin block and remains included therein. The cellular monolayer included in the resin block can then be dissected by means of an ultramicrotome, so as to obtain ultra-thin sections observable under the transmission electron microscope.

[0026] As will become clear to those skilled in the art, to recover the resin block (in which the cell sample is included) from the device according to the invention it is not necessary to carry out drastic treatments, for example using liquid nitrogen or high temperatures. Therefore, the device according to the invention avoids all unwanted phenomena of cell sample alterations, which can be caused by the chemical, mechanical and / or enzymatic treatments necessary to remove the sample from a well-known culture well. In particular, thanks to the invention, a single device is made available in which cells can be cultured, processed and included in situ, and observed microscopically (both in optical and electron microscopy).

[0027] Brief description of the drawings

[0028] The invention can be better understood and implemented with reference to the attached drawings which illustrate an exemplary and non-limiting form of implementation, in which:

[0029] Figure 1 is a perspective schematic view of an embodiment of the device according to the invention, in a first operating configuration;

[0030] Figure 2 is a schematic perspective view of the device of Figure 1, in a second operating configuration;

[0031] Figure 3 is a perspective schematic view of another embodiment of the device according to the invention, in a first operating configuration;

[0032] Figure 4 is a schematic perspective view of the device of Figure 3, in a second operating configuration;

[0033] Figure 5 is a schematic view, partly lateral and partly perspective, of a further embodiment of the device according to the invention;

[0034] Figure 6 is a schematic view, partly lateral and partly perspective, of a still further embodiment of the device according to the invention;

[0035] Figure 7 is a schematic plan and top view of the device of Figure 5, showing the internal cavity of the device;

[0036] Figure 8 is a schematic plan and top view of the device of Figure 5, showing the device closed by the corresponding lid;

[0037] Figure 9 is a schematic plan and top view of the device of Figure 6, showing the device closed by the corresponding lid;

[0038] Figure 10 is a schematic side view, partially sectioned, of a further embodiment of the device according to the invention;

[0039] Figure 11 is a schematic side view, partially sectioned, of a still further embodiment of the device according to the invention.

[0040] Detailed description of the invention

[0041] Figures 1 and 2 show an embodiment of the invention constituted by a device 1 for cell culture, constructed in a suitable material of known type (for example polystyrene or polyethylene) and maintained in sterile conditions (by means of use of apparatuses and methods known to those skilled in the art) up to the moment of use. The device 1 comprises a single culture well 2, in turn comprising a hollow and cylinder-shaped body 2a and a transparent closing member 3. The well 2 can be placed inside an incubator (for cell cultures) of a known type. Well 2 can have a length of 1.5 cm and an internal diameter of 8 mm. In one embodiment, the body 2a of the culture well 2 is also transparent. The culture well 2 can be housed in a culture plate of a known type, having a transparent base (bottom) wall.

[0042] The aforesaid dimensions of the well 2 are exemplary and can be suitably changed during the manufacturing phase, if this is deemed appropriate or necessary. Furthermore, in embodiments not shown, the hollow body 2a can have a non-cylindrical shape and be provided with a polygon-shaped cross section, for example a quadrilateral-shaped one.

[0043] The body 2a communicates with the outside through a first opening 2b and a second opening 2c, mutually opposite. The second opening 2c is closed by applying a transparent closing member 3, comprising a cap portion 4 and a flap portion 5. The cap portion 4 is made in form coupling with the second opening 2c and can be applied by pressure ( manually or mechanically) on the second opening 2c. The plug portion 4 comprises an internal face 4a, which faces the second opening 2c when the plug portion 4 is connected to the body 2a. The flap portion 5 is constructed in the form of a lamina and protrudes from one face of the cap portion 4 opposite the inner face 4a. In use, the flap portion 5 can be grasped by an operator to manually remove the cap portion 4 (and therefore all the transparent closing member 3) from the well 2.

[0044] During the manufacturing phase of the device 1, the transparent closing member 3 (comprising the cap portion 4 and the flap portion 5) can be positioned on a suitable resting surface, so that the cap portion 4 has its internal face 4a facing upwards (i.e., facing in the opposite direction to the supporting surface), and the body 2a can be moved according to the direction indicated by the pair of arrows F1 (i.e., according to a substantially vertical direction) in Figure 2, so as to bring the body 2a closer to the plug portion 4 until the body 2a is pressed into the plug portion 4.

[0045] In use, on the inner face 4a it is possible to seed, adhere and grow cells (for example, human or animal eukaryotic cells) until a cellular monolayer is obtained. The inner face 4a thus acts as a solid growth support for the cells (ie, a solid support on which it is possible to seed and grow cells), which are introduced into the well 2 through the first opening 2b. Since the cap portion 4 is transparent (and the bottom of any culture plate in which the culture well 2 is housed is also transparent), by positioning the device 1 on the object holder plate of an inverted optical microscope it is possible to observe the cells of the monolayer and identify any areas of interest in the latter, for example for the particular morphology of the cells.

[0046] Thanks to the structure of the device 1, the cells of the monolayer can be the subject of a subsequent ultrastructural investigation. In fact, by acting through the first opening 2b (which allows the passage of fixatives and alcohols necessary for the dehydration process) it is possible to fix, dehydrate and include the cell monolayer directly inside the well 2. In particular, after fixing and dehydrating (according to methods known to those skilled in the art) the cellular monolayer, the latter can be included inside the body 2a, by pouring into the body 2a a suitable inclusion material (of a known type) in the fluid state (i.e., not polymerized) with increasing concentration, such as for example resin.

[0047] After completing the polymerization of the resin a solid block is obtained and the cell monolayer is included at the interface between the polymerized resin and the inner face 4a of the cap portion 4. The operator can then remove the well 2 from the corresponding culture plate, grasp the flap portion 5 and, acting on the latter (in the direction indicated by the pair of arrows F1), remove the plug portion 4 from the body 2a. In this phase, due to the effect of this maneuver or of chemical and / or physical interactions that presumably occur during the processing of the sample, the cellular monolayer has already been transferred from the internal face 4a to the adjacent surface of the resin block and the latter can be easily extracted from the body 2a. This can be achieved, for example, by blocking the well 2 with a clamp and laterally cutting the body 2a. In an embodiment not shown, small pre-cuts are made in the side wall of the well, ie in the body 2a, in correspondence with the first opening 2b, so as to facilitate the breaking, at least partially, of the body 2a. The successful transfer can be verified by observation through the eyepieces of an ultramicrotome of known type and / or by preparing semi-thin sections from the resin block and observing the sections (appropriately stained) under an optical microscope, so as to confirm the presence of included cells in the resin block. Subsequently, the ultra-thin sections necessary for observation under the transmission electron microscope can be obtained from the resin block.

[0048] Figures 3 and 4 show another embodiment of the invention consisting of a device 1a for cell culture, comprising a single culture well 2. The elements of the device 1a equal to corresponding elements of the device 1 (Figure 1; Figure 2) will be designated with the same reference numbers and will not be described in detail below.

[0049] The device 1a differs from the device 1 previously described only for the structure of the transparent closing member 3, in which the cap portion 4 and the flap portion 5 are replaced by a peelable flap 5a, made of a material (of known type) transparent and flexible. The peelable flap 5a is connected by chemical adhesive means of a known type to the body 2a, and in particular it is connected to the edge (defined by the body 2a) of the second opening 2c. Therefore, the cells can be seeded on an inner face 5b of the peelable flap 5a. The inner face 5b faces the second opening 2c when the peelable flap 5a is connected to the body 2a. The inner face 5b thus acts as a solid growth support for the cells, which are introduced into the well 2 through the first opening 2b. The remaining structural and use characteristics of the device 1a are the same as described with reference to the device 1.

[0050] In embodiments not shown, the device 1 and / or the device 1a comprise a box-shaped container (constructed using known plastic, for example polystyrene or polyethylene), inside which the well 2 is housed and which can be placed inside a known type of incubator (for cell cultures). For example, the aforementioned container can be constructed in a similar way to the containers 10a and 100a which will be described below with reference to Figures 5 to 9. The container comprises a containing part and a removable lid. The well 2 is positioned inside the containing portion in such a way that the transparent closing member 3 is in contact with the transparent base (bottom) wall of the containing portion, which allows the observation with an optical microscope of the cells that can be grown on the internal face 4a of the cap portion 4 or on the internal face 5b of the peelable flap 5a. Once the observation under the optical microscope has been completed, to fix, dehydrate and include the cells, simply remove the lid of the container and proceed as described above. In one embodiment, the entire container is constructed of transparent material.

[0051] Figures 5, 7 and 8 show a further embodiment of the invention consisting of a device 10 for cell culture, comprising a plurality of culture wells 2 positioned inside a container 10a. The device 10 can be placed inside a known type of incubator (for cell cultures). The elements of the device 10 equal to corresponding elements of the device 1 (Figure 1; Figure 2) or of the device 1a (Figure 3; Figure 4) will be designated with the same reference numbers and will not be described in detail below. Furthermore, although in Figures 5, 7, 8 wells 2 provided with the cap portions 4 and the flap portions 5 are shown, these wells can be replaced by, or used in combination with, the wells 2 provided with the peelable flaps 5a.

[0052] The container 10a is built in a suitable material of a known type (for example polystyrene or polyethylene) and kept in sterile conditions (through the use of equipment and methods known to technicians in the field) until the moment of use. The container 10a is box-shaped having a rectangular shape in plan and comprises a containing portion 10c, in which a cavity 10e is defined, and a lid portion 10b. The lid portion 10b is positioned on the containing portion 10c, so as to protect the wells 2 from the environment outside the cavity 10e and at the same time allow gas exchanges during the culture in the incubator.

[0053] Inside the cavity 10e a plurality of culture wells 2 are housed, having the same structural and use characteristics described with reference to the device 1. In an embodiment not shown, the wells 2 have the same characteristics structurally described with reference to the device 1a, ie they are provided with a transparent closing member 3 in which the cap portion 4 and the flap portion 5 are replaced by the peelable flap 5a. In a further embodiment not shown, the device 10 comprises both wells 2 having the same structural characteristics described with reference to the device 1, and wells 2 having the same structural characteristics described with reference to the device 1a.

[0054] As can be seen in Figures 5, 7 and 8, four wells 2 are housed inside the containing portion 10c, kept in position, ie connected to each other and to the internal walls of the containing portion 10c, by means of connection 11. connection means 11 comprise a plurality of connection elements 12, 13, of which (as visible for example in Figure 7) the connection elements 12 have an approximately elliptical plan shape, while the connection elements 13 have a plan shape approximately "butterfly". In embodiments not shown, the connection elements 12, 13 can have different forms from those described with reference to Figures 5, 7 and 8.

[0055] The four wells 2 are mutually aligned along a longitudinal axis (not shown) of the containing portion 10c and are positioned in the latter in such a way that the corresponding transparent closing members 3 are in contact with, and therefore face, a transparent base wall 10d of the container 10a (this detail is not visible in the Figures as the latter schematically illustrate the invention, as will become clear to those skilled in the art). As shown in Figure 7, each of the two end wells is interposed between an adjacent end zone 14a, 14b of the internal wall of the containing portion 10c and an adjacent well 2, while each of the two remaining wells 2 is interposed between two adjacent wells 2. Since the base wall 10d is transparent, it can be placed on the object-holder plate of an inverted optical microscope and allow the microscopic observation of cell monolayers grown on the transparent closing members 3, i.e. on the internal faces 4a of the portions a cap 4 or on the inner faces 5b of the peelable flap portions 5a.

[0056] To remove the wells 2 from the cavity 10e it is sufficient to cut the connecting elements 12, 13 using a suitable tool (for example a pair of scissors or a scalpel). To facilitate the cutting of the connecting elements 12, 13, the latter can be manufactured using a soft plastic, for example polyethylene.

[0057] The procedure to be used to include the cell monolayer grown on (more exactly, inside the) transparent closure members 3 and to obtain sections of the cell monolayer suitable for observation under the transmission electron microscope is the same as described with reference to device 1 and device 1a.

[0058] In one embodiment of the container 10a, all of the containing portion 10c and / or all of the lid portion 10b are constructed of a transparent material.

[0059] Figures 6 and 9 show a further embodiment of the invention consisting of a device 100 for cell culture, comprising a plurality of culture wells 2 positioned inside a container 100a. The device 100 can be placed inside a known type of incubator (for cell cultures). The elements of the device 100 equal to corresponding elements of the device 1 (Figure 1; Figure 2), of the device 1a (Figure 3; Figure 4) or of the device 10 (Figure 5; Figure 7; Figure 8) will be designated with the same numbers reference and will not be described in detail below. Furthermore, although wells 2 provided with cap portions 4 and flap portions 5 are shown in Figures 6 and 9, these wells can be replaced by, or used in combination with, the wells 2 provided with peelable flaps 5a.

[0060] The container 100a is made of a suitable transparent material of a known type (for example polystyrene or polyethylene) and kept in sterile conditions (through the use of apparatuses and methods known to those skilled in the art) until the moment of use . The container 100a is shaped in the form of a box having a rectangular shape in plan and comprises a containing portion 100c, in which a cavity 100e is defined, and a lid portion 100b. The lid portion 100b is positioned on the containing portion 100c, so as to protect the wells 2 from the environment outside the cavity 100e and at the same time allow gas exchanges during the culture in the incubator.

[0061] A plurality of cell culture wells 2 are housed inside the cavity 100e, having the same structural and use characteristics described with reference to the device 1. In an embodiment not shown, the wells 2 have the same structural characteristics described with reference to the device 1a, ie they are provided with a transparent closing member 3 in which the cap portion 4 and the flap portion 5 are replaced by the peelable flap 5a. In a further embodiment not shown, the device 100 comprises both wells 2 having the same structural characteristics described with reference to the device 1, and wells 2 having the same structural characteristics described with reference to the device 1a.

[0062] Unlike what has been described with reference to the device 10, and as can be seen from Figures 6 and 9, six wells 2 are housed inside the containing portion 100c. The six wells 2 are kept in position, i.e. connected to each other and to the internal walls of the containing portion 100c, through the connection means 11, comprising the connecting elements 12, 13. The six wells 2 are arranged to form two parallel rows along a longitudinal axis (not shown) of the containing portion 100c, so such that the corresponding transparent closing members 3 are in contact with and therefore face a transparent base wall 100d of the container 100a. Since it is transparent, the base wall 100d allows the observation - through an inverted optical microscope - of cell monolayers grown on the () transparent closure members 3 of the wells 2.

[0063] The procedure to be used to remove the wells 2 from the container 100a of the device 100 is the same as described with reference to the container 10a of the device 10.

[0064] The procedure to be used to include the cell monolayer grown on (more exactly, inside the) transparent closure members 3 and to obtain sections of the cell monolayer suitable for observation under the transmission electron microscope is the same as described with reference to device 1 and device 1a.

In one embodiment of the container 100a, all of the containing portion 100c and / or all of the lid portion 100b are constructed of a transparent material.

[0066] As will become clear to those skilled in the art, both the device 10 and the device 100 can be modified so as to be able to contain a number of wells 2 respectively different from four or six, in particular a number of wells 2 greater than six. Furthermore, the plan shape of the device 10 and / or of the device 100 can be non-rectangular, for example it can be square or round.

[0067] Figure 10 describes a further embodiment of the invention, consisting of a device 200 for cell culture. The device 200 can be placed inside a known type of incubator (for cell cultures) and comprises a plurality of culture wells 2 positioned inside a container 200a. The container 200a is built in a suitable transparent material of a known type and kept in sterile conditions (through the use of equipment and methods known to the technicians of the sector) until the moment of use. The container 200a is shaped in the form of a box having a rectangular shape in plan and comprises a containing portion 200c and a lid portion 200b. The lid portion 200b is positioned on the containing portion 200c, so as to protect the wells 2 from the environment outside the containing portion 200c and at the same time allow gas exchanges during the culture in the incubator. The containing portion 200c comprises a transparent base wall 200d, in which a plurality of seats 200e - in particular four seats 200e - mutually aligned along a longitudinal axis (not shown) of the containing portion 200c are formed.

[0068] Each seat 200e comprises a side wall 200g, which projects from the transparent base wall 200d towards the lid portion 200b (Figure 10), and an internal face 200f. The side wall 200g and the inner face 200f are mutually connected. The side wall 200g is made in shape coupling with the end of the body 2a (of the well 2) close to the second opening 2c, while the internal face 200f corresponds to the portion of the transparent base wall 200d enclosed inside the side wall 200g .

[0069] During the manufacturing phase of the device 200, the body 2a of each well 2 can be moved in a direction similar to that indicated by the pair of arrows F1 in Figure 2 or in Figure 4, so as to bring the body 2a closer to a corresponding seat 200e until the body 2a is pressed into the side wall 200g of seat 200e. When the body 2a is thus positioned, the second opening 2c is closed by the adjacent internal face 200f of the seat 200e.

[0070] Therefore, in the device 200, the seats 200e overall define a single transparent closing member 3, capable of simultaneously closing the second opening 2c of all the wells 2 housed in the containing portion 200c. In other words, in the device 200 the transparent closing member 3 comprises the plurality of seats 200e.

[0071] The device 200 shown in Figure 10 is provided with four seats 200e and can therefore contain up to four wells 2, mutually aligned along the longitudinal axis of the containing portion 200c. However, as will become clear to those skilled in the art, the device 200 can be modified to be able to contain a number of wells 2 greater than four, for example six, or more than six, wells 2. Furthermore, the shape in plan of the device 200 can be non-rectangular, for example it can be square or round. In one embodiment of the container 200a, all of the containing portion 200c and / or all of the lid portion 200b are constructed of a transparent material.

[0072] In use, on the inner face 200f it is possible to seed, adhere and grow cells (for example, human or animal eukaryotic cells) until a cellular monolayer is obtained. The inner face 200f thus acts as a solid growth support for the cells, which are introduced into the well 2 through the first opening 2b. Since the internal face 200f is transparent, by positioning the device 200 on the object holder plate of an inverted optical microscope it is possible to observe the cells of the monolayer and identify any areas of interest in the latter, for example for the particular morphology of the cells.

[0073] The procedure to be used to include the cell monolayer grown on the internal faces 200f and to obtain sections of the cell monolayer suitable for observation under the transmission electron microscope is the same as described with reference to the device 1 and the device 1a. As previously described, the cellular monolayer is included at the interface between the polymerized resin and the internal face 200f of each seat 200e. To remove the wells 2 from the container 200a, it is sufficient to grasp the body 2a of each well 2 and gently move the body 2a until the latter disengages from the side wall 200g of the corresponding seat 200e.

[0074] Figure 11 describes a still further embodiment of the invention, consisting of a device 300 for cell culture. The device 300 comprises a plurality of culture wells 2 positioned inside a container 300a. The device 300 can be placed inside a known type of incubator (for cell cultures). The container 300a is built in a suitable transparent material of a known type (for example polystyrene or polyethylene) and kept in sterile conditions (through the use of equipment and methods known to technicians in the field) until the moment of use. The container 300a is shaped in the form of a box having a rectangular shape in plan and comprises a containing portion 300c and a lid portion 300b. The lid portion 300b is positioned on the containing portion 300c, so as to protect the wells 2 from the environment outside the containing portion 300c and at the same time allow gaseous exchanges during the culture in the incubator. The containing portion 300c comprises a transparent base wall 300d, in which a plurality of seats 300e - in particular four seats 300e - mutually aligned along a longitudinal axis (not shown) of the containing portion 300c is formed.

[0075] Each seat 300e comprises a side wall 300g, which protrudes from the transparent base wall 300d in the opposite direction with respect to the lid portion 300b (Figure 11), and an internal face 300f. The side wall 300g and the inner face 300f are mutually connected. The side wall 300g is made in shape coupling with the end of the body 2a (of the well 2) next to the second opening 2c, while the internal face 300f is made in shape coupling with the second opening 2c. Therefore, the seats 200e (of the device 200) differ from the seats 300e (of the device 300) in that the latter are made in the form of depressed areas, ie recesses, of the transparent base wall 300d.

[0076] During the manufacturing phase of the device 300, the body 2a of each well 2 can therefore be moved in a direction similar to that indicated by the pair of arrows F1 in Figure 2 or in Figure 4, so as to bring the body 2a closer to a corresponding seat 300e until the body 2a is pressed into the side wall 300g of the seat 300e, ie inside the recess defined as a whole by the side wall 300g and by the internal face 300f. As previously described with reference to the device 200, when the body 2a is pressed into the seat 300 and the second opening 2c is closed by the adjacent internal face 300f.

[0077] Therefore, in the device 300 the seats 300e define as a whole a single transparent closing member 3, capable of simultaneously closing the second opening 2c of all the wells 2 housed in the containing portion 300c. In other words, in the device 300 the transparent closing member 3 comprises the plurality of seats 300e.

[0078] Although the device 300 shown in Figure 11 is provided with four seats 300e and can therefore contain up to four wells 2, mutually aligned along the longitudinal axis of the containing portion 300c, it will appear clear to those skilled in the art that the device 300 can be modified to be able to contain a number of wells 2 greater than four, for example six, or more than six, wells 2. Furthermore, the plan shape of the device 300 can be non-rectangular, for example it can be square or roundabout. In one embodiment of the container 300a, all of the containing portion 300c and / or all of the lid portion 300b are constructed of a transparent material.

[0079] In use, on the inner face 300f it is possible to seed, adhere and grow cells (for example, human or animal eukaryotic cells) until a cellular monolayer is obtained. The inner face 300f thus acts as a solid growth support for the cells, which are introduced into the well 2 through the first opening 2b. Since the internal face 300f is transparent, by positioning the device 300 on the object holder plate of an inverted optical microscope it is possible to observe the cells of the monolayer and identify any areas of interest in the latter, for example for the particular morphology of the cells.

[0080] The procedure to be used to include the cell monolayer grown on the internal faces 300f and to obtain sections of the cell monolayer suitable for observation under the transmission electron microscope is the same as described with reference to the device 1 and the device 1a. As previously described, the cellular monolayer is included at the interface between the polymerized resin and the internal face 300f of each seat 300e. To remove the wells 2 from the container 300a, it is sufficient to grasp the body 2a of each well 2 and gently move the body 2a until the latter disengages from the side wall 300g of the corresponding seat 300e.

[0081] By way of example, but not limiting, of the invention, an experimental test performed by the Inventor is described below to verify the technical feasibility of the device according to the invention.

[0082] Example 1 - Realization of a prototype of a device according to the invention [0083] To make a prototype of the culture well 2, which is the simplest embodiment of the device according to the invention, a test tube was used plastic (transparent) with flat bottom and closing cap (transparent). The tube was turned upside down to rest on the cap, and the flat bottom was removed mechanically. A sample of 6 x 10 <4> cells was seeded and left to grow inside the cap, so as to form a cell monolayer. After observing the morphological characteristics of the monolayer by means of an inverted optical microscope, the cell monolayer was fixed, dehydrated and embedded in resin directly in the same tube, using the end of the tube corresponding to the bottom, previously removed, as a way through which to introduce the various reagents and inclusion material. After polymerization of the resin at 60 ° C, a resin block was obtained having the cell monolayer included at the resin-cap interface. The cap was removed, the side wall of the tube was cut to the side - allowing the resin block to be extracted - and semi-thin sections were obtained from the resin block. The latter were observed under an optical microscope and confirmed the presence of the cell monolayer (previously included at the resinated interface). Ultra-thin sections were then obtained from the resin block, which were observed under a transmission electron microscope. The observation of the ultra-thin sections with a transmission electron microscope has shown that the prototype of the device according to the invention allows to preserve the original ultrastructure of the cells, as well as the cell-cell interactions, and also allows to obtain high quality images at high magnification and high resolution.

[0084] From what has been described above, the many advantages and various possible applications of the device according to the invention clearly emerge.

[0085] First of all, the device according to the invention allows the cultivation of cell monolayers in vitro (human or animal cells, primary cell cultures, stem cells, cell or tumor lines, rare cell populations, cells of pathological origin) under different experimental conditions , observe through an optical microscope (in particular, through an inverted optical microscope) the cells grown in vitro, select any cells (contained in specific areas of the monolayer) to be subjected to an ultrastructural examination, process in situ (i.e. fix, dehydrate, include) the cells and obtain suitable samples (ultra-thin sections) for observation by electron microscope (in particular, transmission electron microscope).

[0086] The device according to the invention therefore allows to observe in transmission electron microscopy the same cells observed in optical microscopy, obtaining high quality images at high magnification and high resolution, as well as to preserve the original ultrastructure of the cells without losing details or alter the cell morphology due to chemical, mechanical and / or enzymatic treatments.

[0087] The device according to the invention can be used to define the phenotype of cells and / or the co-expression of different surface, cytoplasmic and nuclear antigens by means of immunoelectromicroscopy.

[0088] The device according to the invention can also be used to prepare in vitro cultures of organ or tissue fragments in order to examine their cells and could also be applied in the field of microbiology to study viruses, bacteria, parasitic protozoa and the way in which these microorganisms interact with human and / or animal eukaryotic cells.

Claims (14)

CLAIMS 1. Device (1; 1a; 10; 100; 200; 300) for culturing cells, called device (1; 1a; 10; 100; 200; 300) allowing said cells to be processed and included in situ and to be observed both under the optical microscope and the electron microscope, said device (1; 1a; 10; 100; 200; 300) comprising: - at least one culture well (2), provided with a first opening (2b) and a second opening (2c), said first opening (2b) and said second opening (2c) being mutually opposite; - at least one transparent closing member (3) applied to said second opening (2c) so as to close said second opening (2c), said at least one transparent closing member (3) comprising at least one internal face (4a; 5b; 200f ; 300f) facing said second opening (2c) and arranged to act as a solid support on which said cells can grow. Device (1; 1a; 10; 100; 200; 300) for culturing cells according to claim 1, wherein said at least one transparent closing member (3) is removable from said second opening (2c) or wherein said well (2) of culture is removable from said at least one transparent closure member (3). Device (1; 10; 100) for culturing cells according to claim 2, wherein said at least one transparent closing member (3) comprises a plug portion (4) in which said inner face (4a) is included. Device (1; 10; 100) for culturing cells according to claim 3, wherein said at least one transparent closing member (3) further comprises a flap portion (5) projecting from said plug portion (4). Device (1a; 10; 100) for culturing cells according to claim 2, wherein said at least one transparent closing member (3) comprises a peelable flap (5a), said inner face (5b) being included in said peelable flap (5a). Device (10; 100; 200; 300) for culturing cells according to one of claims 1 to 5, wherein said at least one culture well (2) is housed in a container (10a; 100a; 200a; 300a) at least partially transparent. Device (10; 100; 200; 300) for culturing cells according to claim 6, wherein said at least partially transparent container (10a; 100a; 200a; 300a) comprises a containing portion (10c; 100c; 200c; 300c) and a lid portion (10b; 100b; 200b; 300b), said containing portion (10c; 100c; 200c; 300c) being arranged to house said at least one culture well (2) and said lid portion (10b; 100b; 200b) ; 300b) being arranged to be positioned on said containing portion (10c; 100c; 200c; 300c). Device (10; 100) for culturing cells according to claim 7, wherein said at least one culture well (2) is positioned inside said containing portion (10c; 100c) so that said at least one member of transparent closure (3) faces a transparent base wall (10d; 100d) of said container (10a; 100a). Device (200; 300) for culturing cells according to claim 7, wherein said at least one transparent closing member (3) comprises at least one seat (200e; 300e) formed in a transparent base wall (200d; 300d) of said container (200a; 300a), said inner face (200f; 300f) being included in said at least one seat (200e; 300e), and in which said at least one culture well (2) is positioned inside said containing portion (200c; 300c) in such a way that a body (2a) of said at least one culture well (2) is received at least partially inside said at least one seat (200e; 300e) and said second opening (2c) is closed by said at least one seat (200e; 300e). Device (200; 300) for culturing cells according to claim 9, wherein, when said body (2a) of said at least one culture well (2) is received at least in part inside said at least one seat (200e ; 300e), said body (2a) is inserted inside a side wall (200g; 300g) of said at least one seat (200e; 300e). Device (300) for culturing cells according to claim 9, or 10, in which said at least one seat (300e) is formed in the form of a recess in said transparent base wall (300d). Device (10; 100; 200; 300) for culturing cells according to one of claims 7 to 11, comprising a plurality of said culture wells (2). Device (10; 100) for culturing cells according to claim 12, in which said culture wells (2) of said plurality are connected to each other and / or to internal walls of said containing portion (10c; 100c) by means of connection (11). Device (10; 100) for cultivating cells according to claim 13, wherein said connecting means (11) comprise a plurality of connecting elements (12, 13), said connecting elements (12, 13) being interposed between said culture wells (2) or being interposed between said culture wells (2) and said internal walls of said containing portion (10c; 100c).