SAMPLE HOLDER DEVICE FOR BIOLOGICAL SAMPLES, COMPRISING A SAMPLE HOLDER MADE OF A CARBON-BASED MATERIAL

Abstract

A sample holder device 100, 101 which is designed to hold biological samples 1 includes a base body 10 having at least one wall 11 which is arranged to delimit a sample receptacle 12, wherein the at least one wall 11 includes, at least on a surface facing the sample receptacle 12, a planar, carbon-based material which is impermeable to a liquid in sample receptacle 12, wherein the carbon-based material has such a high carbon content that the carbon-based material is opaque and electrically conductive. The sample holder device includes, e.g., a dish, in particular petri dish 101, a planar substrate, a multiwell plate, a sample beaker, in particular in the form of a beaker glass, a sample tube, in particular in the form of a test tube or a tube for cryopreservation (cryovial), and/or a hollow fiber. Methods for using the sample holder device are also described.

Claims

1. A sample holder device which is configured to hold biological samples, comprising a base body having at least one wall which is arranged to delimit a sample receptacle, wherein the at least one wall comprises, at least on a surface facing the sample receptacle, a planar, carbon-based material which is impermeable to a liquid in the sample receptacle, wherein the carbon-based material has such a high carbon content that the carbon-based material is opaque and electrically conductive.

2. The sample holder device according to claim 1, wherein the at least one wall consists of the carbon-based material.

3. The sample holder device according to claim 2, wherein the at least one wall consisting of the carbon-based material has a thickness in a range from 150 μm to 1 mm.

4. The sample holder device according to claim 2, wherein the entire base body consists of the carbon-based material.

5. The sample holder device according to claim 1, wherein the at least one wall has, on the surface facing the sample receptacle, a coating which consists of the carbon-based material.

6. The sample holder device according to claim 5, wherein the coating consisting of the carbon-based material has a thickness in a range from 2 nm to 500 μm.

7. The sample holder device according to claim 1, wherein the carbon-based material has, on the surface facing the sample receptacle, a surface structure which promotes a mechanical interaction of biological samples with the carbon-based material.

8. The sample holder device according to claim 7, wherein the surface structure comprises at least one of a predetermined roughness of the carbon-based material and a plurality of projections of the carbon-based material.

9. The sample holder device according to claim 8, wherein the surface structure comprises the plurality of projections of the carbon-based material, wherein the projections are dimensioned and arranged such that several projections are provided in a region of a contact area of a biological cell.

10. The sample holder device according to claim 1, wherein the carbon-based material consists of at least one of pure carbon, carbon fiber-reinforced plastic and silicon carbide.

11. The sample holder device according to claim 1, further comprising at least one contact section which is arranged for a connection of the at least one wall to at least one of a voltage source and a measuring device.

12. The sample holder device according to claim 1, wherein the base body comprises several walls which enclose a volume of the sample receptacle, wherein the carbon-based material of the walls is formed in one piece.

13. The sample holder device according to claim 1, comprising at least one of a dish, a flat substrate, a multiwell plate, a sample beaker, a sample tube, and a hollow fiber.

14. A method of using the sample holder device according to claim 1, said method comprising carrying out at least one of the following steps: processing of cell or tissue samples, cultivation of cell cultures, differentiation of cell cultures, optical measurement fluorescence measurement, electrophysiological measurement, derivation of electric potentials or currents, transport of biological samples transport of biological samples in a frozen state, storage of biological samples storage of biological samples in a frozen state cryogenic treatment of biological samples, high-throughput testing, and high-throughput testing for diagnostic or regenerative medicine tasks.

15. The sample holder device according to claim 1, comprising at least one of: a petri dish, a beaker glass; a test tube or a tube for cryopreservation, and a hollow fiber configured for adherent holding of biological cells.

Description

[0028] Further details and advantages of the invention are described below with reference to the enclosed drawings, which show schematically:

[0029] FIG. 1: a perspective view of an embodiment of the sample holder device according to the invention in the form of a petri dish;

[0030] FIGS. 2A and 2B: side views of an embodiment of the sample holder device according to the invention in the form of a cryotube;

[0031] FIGS. 3 and 4: perspective views of an embodiment of the sample holder device according to the invention in the form of a multiwell plate;

[0032] FIG. 5: an illustration of an electrophysiological measurement using an embodiment of the sample holder device according to the invention;

[0033] FIG. 6: an illustration of an optical measurement using an embodiment of the sample holder device according to the invention; and

[0034] FIG. 7: an embodiment of the invention, in the case of which a plurality of sample holder devices in the form of hollow fibers are arranged in a bioreactor.

[0035] Embodiments of the invention are described below with exemplary reference to embodiments of the sample holder device according to the invention in the form of a petri dish, a cryotube and a multiwell plate. It is emphasized that the implementation of the invention is not restricted to these variants, but rather can be correspondingly used with other vessel forms, such as e.g. a beaker, a flask, a hollow tube reactor or the like, or a sample holder device in the form of a flat substrate. Moreover, modifications of the dimensions and/or forms of the sample holder device and/or the individual sample receptacles, in particular for an adjustment to a special application, are possible. Details of the processing and/or investigating of biological samples are not described here since they are known per se from conventional technology.

[0036] FIG. 1 shows an embodiment of sample holder device 100 according to the invention in the form of a petri dish 101. The shape and size of petri dish 101 can be selected as is known from conventional petri dishes. It can have in particular a height of 1 cm and a diameter of 3 to 12 cm. The petri dish 101 comprises a base body 10 in the form of a dish part which forms the sample receptacle 12 for the biological sample 1. Sample receptacle 12 is delimited by walls 11 which comprise the dish base and the laterally circumferential dish wall, e.g. made of glass or plastic. A coating 13 composed of carbon fiber-reinforced plastic is provided on the inner side of the walls 11. A solid, artificial cultivation ground for the culture of e.g. cells or cell tissue can be arranged on the dish base.

[0037] The petri dish 101 is furthermore preferably provided with a closing cover part 14. Cover part 14 is shown to be transparent in order to illustrate the inside of petri dish 101, but is composed like the dish part of plastic or glass with an inner coating composed made of carbon fiber-reinforced plastic. Cover part 14 can particularly preferably be coupled in a liquid-impervious manner to the base body 10 (dish part).

[0038] FIG. 2 shows two variants of an embodiment of the sample holder device 100 according to the invention in the form of a cryotube 102. According to FIG. 2A, cryotube 102 comprises externally plastic or glass and internally a coating 13 made of the carbon-based material, e.g. carbon fiber-reinforced plastic, while according to FIG. 2B the entire cryotube 102 is manufactured from the carbon-based material. In detail, cryotube 102 comprises a base body 10 in the form of a sample tube closed on one side, having a cylindrical wall 11 closed at the lower end (base). The inside of the sample tube forms sample receptacle 12. A cover part 14 which closes in a liquid-impervious manner is fastened to the upper end of the sample tube. The cryotube 102 has e.g. an inner diameter of 11 mm and an axial length of 4.1 cm.

[0039] Further embodiments of the sample holder device 100 according to the invention in the form of a multiwell plate 103 are shown schematically in FIGS. 3 and 4. An arrangement of sample receptacles 12 (wells) is provided in a base body 10 which forms a base plate of multiwell plate 103. The number and size of the sample receptacles 12 is selected as is known per se from conventional micro- or nanotiter plates. The multiwell plate 103 furthermore has a cover part 14 with which the sample receptacles 12 are covered and optionally sealed off in a liquid-impervious manner. According to FIG. 3, the entire multiwell plate 103 is manufactured from the carbon-based material, e.g. from pyrolytic carbon or silicon carbide. According to FIG. 4, only the sample receptacles 12 of the multiwell plate 103 and the side of the cover part facing the sample receptacles 12 are provided with the carbon-based material, e.g. a layer of carbon fiber-reinforced plastic, while the remaining base plate and the remaining cover part are manufactured from plastic or glass. In order to isolate the sample receptacles 12 electrically from one another even when using the multiwell plate 103 with closed cover part 14, the cover part 14 can be provided with a structured coating restricted to the openings of sample receptacles 12 and made of the carbon-based material.

[0040] FIG. 4 furthermore illustrates contact sections 30 which comprise metallic conductor strips on the surface of the holding body 10. The conductor strips are electrically connected separately from one another in each case to one of the sample receptacles 12. Although FIG. 4 only shows for the first row of sample receptacles 12 on the grounds of clarity, each sample receptacle 12 preferably can be provided with an associated contact section 30 for connection to a voltage source and/or a measuring device 40 (see FIG. 5). Specific electrical measurements and/or stimulations in individual sample receptacles 12 are thus advantageously enabled. Alternatively, the sample receptacles 12 of multiwell plate 103 can be coupled to the voltage source and/or measuring device in groups or all jointly via several or a single contact section 30.

[0041] Further features of preferred embodiments of the invention which can be realized individually or in combination in the case of the various variants of sample holder device 100 are shown in the schematic sectional view of sample holder device 100 according to FIG. 5. A biological sample with at least one biological cell 2 in a liquid medium 3, e.g. cultivation medium and/or medium with differentiation factors, is located in the sample receptacle 12, of which only the lower wall 11 (base section) is shown.

[0042] The carbon-based material of wall 11 has, on its inner surface facing the sample receptacle 12, a surface structure 20 with column-shaped projections 21 of the carbon-based material. The projections 21 have, for example, a height of 2 μm, a cross-sectional dimension, e.g. diameter, of 5 μm, and a mutual center-center spacing of 20 μm. In FIG. 5, all projections 21 are dimensioned with an identical height such that the free ends of projections 21 span a planar carrier surface for adherent holding of the biological sample, such as e.g. the adherent cell 2. Alternatively, the projections 21 can have different heights, as a result of which an adherence of cells to the surface can be increased. The biological cell 2 touches the projections 21 in the lateral direction along the surface over a contact area with a typical extent of e.g. 40 μm and is as a result supported by several projections 21.

[0043] The free ends of projections 21 or their tips or edges form geometrical surface features (coupling points), on which the adherent coupling of biological cells is promoted. The adherence can be further increased in that projections 21 are provided with a functional coating in order to increase adherence, e.g. made of fibronectin, laminin or synthetic RGD peptide sequences.

[0044] FIG. 5 furthermore schematically shows a measuring device 40 for electrical measurements which are connected via connecting lines 41 on one hand to the carbon based material of wall 11 and on the other hand to the interior of sample receptacle 12, e.g. directly to biological cell 2 or to liquid medium 3. Contact with the carbon-based material can be realized via a contact section (not represented, see FIG. 4). The measuring device 40 comprises e.g. a voltage measuring device for the derivation of membrane potentials or membrane potentials currents from cell 2. Deviating from FIG. 5, other arrangements of one or more measuring devices and one or more connecting lines can be provided.

[0045] FIG. 6 schematically illustrates a measuring device 40 for optical measurement on a biological sample in the form of a cell culture 4 in sample receptacle 12 according to a further embodiment of a sample holder device 100 according to the invention. The measuring device 40 comprises one or more excitation light sources 42, such as e.g. laser diodes, and one or more sensor devices 43, such as e.g. photodiodes, spectrally resolving detectors and/or sensor cameras. The excitation light sources 42 and the sensor devices 43 are optically coupled via optical fibers to the interior of sample receptacles 12. Disruptive external light is excluded in the interior of sample receptacles 12 as a result of the formation of wall 11 and cover 14 with the opaque carbon-based material. The excitation light sources 42 and the sensor devices 43 are furthermore connected to a control device (not shown) which is configured to control the excitation light sources 42 and to record and evaluate sensor signals. For example, fluorescence measurements in the sample receptacle can be executed with the measuring device 40 for optical measurement.

[0046] According to the schematic partial view in FIG. 7, a further embodiment of the invention comprises a plurality of hollow fibers 104 which are arranged in a bioreactor 200. The hollow fibers 104 are manufactured at least on their surfaces e.g. from plastic reinforced with carbon fibers and/or coated with carbon, and they have an inner diameter in the range from e.g. 0.1 mm to 5 mm. The bioreactor 200 comprises in a manner known per se a container, e.g. in the form of a hollow cylinder, with a container wall closed on all sides (shown open here). The container wall is provided with fluidic and sensor connections and optionally with windows and/or further access openings. The hollow fibers 104 extend in axial direction of the bioreactor 200. For example, 10000 hollow fibers 104 are arranged in the bioreactor, and it is filled with a cultivation medium which washes around hollow fibers 104. It is preferably provided that the cultivation medium flows through bioreactor 200.

[0047] Applications of the sample holder device according to the invention were tested during the vitrification of biological samples. With the vitrification e.g. of Drosophila melanogaster embryos (DM embryos), human stem cells (embryonal, adult, induced), differentiated cells, in particular those which can be tested electrophysiologically (cardiomyocytes, neuronal cells), proteins, sperm cells and tissue (e.g. biopsy samples), in particular an SiC substrate has been shown to be advantageous due to the rapid exchange of heat with a cooling device coupled to the sample holder device.

[0048] Further applications of the sample holder device according to the invention in the case of electrophysiological measurements were likewise successful. Electrophysiological measurements are often preceded by protracted cultivation and differentiation protocols lasting from weeks to months until the cells have the required degree of maturity which is characterized by the formation of particular channels or contacts. The sample holder device offers various possibilities for deriving electrophysiological signals over a larger surface area than is possible in the case of the current prior art. For example, in the case of derivations according to the patch-clamp method, electrophysiological signals are typically measured with only one cell. The technology according to the invention enables parallel measurement at several cells. Moreover, cells which grow adherently in the sample holder device can be manipulated via electrical signals, and as a result differentiating steps can be influenced. As a result of the opaqueness of the sample holder device, fluorescence-based measurements of the calcium efflux can be recorded without background noise. In particular for the patch-clamp method, cells are initially cultivated and then measured in the same cultivation vessel, such as e.g. a petri dish with 35 mm diameter. In particular walls of the sample receptacles made of pyrolytic carbon have been shown to be advantageous for electrophysiological measurements.

[0049] The features of the invention disclosed in the above description, the drawings and the claims can be of importance both individually and in combination or sub-combination in order to carry out the invention in its various configurations.