US-12618034-B2 - Device and method for cell cultivation

US12618034B2US 12618034 B2US12618034 B2US 12618034B2US-12618034-B2

Abstract

A carrier suitable for the cultivation of cells includes a plate-shaped carrier, e.g. made of glass, silicon or plastic or a combination thereof. At least one first recess is formed in the carrier and extends over a first thickness section of the carrier. An arrangement of second recesses extends from the first recess into a second thickness section of the carrier adjacent to the first thickness section. The first thickness section can have a thickness of a few micrometers to a few centimeters. The second recesses extending from a first recess into the second thickness section, each forming a second arrangement, form wells suitable for receiving cells and/or synthetic particles, e.g. made of plastic or glass.

Inventors

Robin A. Krüger
Antonia Heine-Dank
Oktavia Ostermann
Bernd Rösener
Malte Schultz-Ruhtenberg

Assignees

LPKF LASER & ELECTRONICS AG

Dates

Publication Date: 20260505
Application Date: 20220506
Priority Date: 20210507

Claims (19)

1 . A carrier for cultivating cells, comprising at least one first recess extending over a first thickness section from a first surface up to a second thickness section adjacent to the first thickness section, an arrangement of second recesses in the second thickness section and extending from the first recess, wherein the second recesses comprise a bottom, wherein the second recesses comprise an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, the carrier comprising an outlet whose cross-section opens in or adjacent to the first thickness section, the carrier comprising a connection conduit connected to the outlet and extending to a second surface that lies opposite the first surface.
2 . The carrier according to claim 1 , wherein the outlet comprises a cross-section projecting into the carrier and open to the plane of the first surface.
3 . The carrier according to claim 1 , wherein the connection conduit extends along an end wall of the carrier o through the carrier.
4 . The carrier according to claim 1 , comprising a supply conduit in the first surface or in the first thickness section, the supply conduit opening into the first recess.
5 . A carrier for cultivating cells, comprising at least one first recess extending over a first thickness section from a first surface up to a second thickness section adjacent to the first thickness section, an arrangement of second recesses in the second thickness section and extending from the first recess, wherein the second recesses comprise an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, the carrier comprising an outlet whose cross-section opens in or adjacent to the first thickness section, the carrier comprising a connection conduit connected to the outlet and extending to a second surface that lies opposite the first surface, wherein the outlet and the connection conduit are adjacent each other and comprise a hydrophilic surface, and wherein regions of the first surface and/or regions of the second surface are hydrophobic.
6 . A carrier for cultivating cells, comprising at least one first recess extending over a first thickness section from a first surface up to a second thickness section adjacent to the first thickness section, an arrangement of second recesses in the second thickness section and extending from the first recess, wherein the second recesses comprise an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, the carrier comprising an outlet whose cross-section opens in or adjacent to the first thickness section, wherein the carrier is defined in a single piece of glass.
7 . A carrier for cultivating cells, comprising at least one first recess extending over a first thickness section from a first surface up to a second thickness section adjacent to the first thickness section, an arrangement of second recesses in the second thickness section and extending from the first recess, wherein the second recesses comprise an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, the carrier comprising an outlet whose cross-section opens in or adjacent to the first thickness section, consisting of a second partial carrier extending over the second thickness section and of a first partial carrier extending over the first thickness section, the first and second partial carriers being arranged in a liquid-tight manner relative to one another.
8 . The carrier according to claim 7 , wherein the first and the second partial carriers are arranged displaceably against one another.
9 . The carrier according to claim 7 , wherein one or both of the first and second partial carriers consists of glass, plastic and/or silicon.
10 . The carrier according to claim 7 , wherein the second partial carrier is arranged at least partially within the first recess of the first partial carrier, the second recesses each having at least one through hole boring in their bottoms, so that each second recess is connected to the first recess through at least one through hole boring.
11 . The carrier according to claim 7 , wherein the first recesses and second recesses are formed as spaced-apart through holes in the first partial carrier and are connected to one another by transition conduits formed by a bottom recess, which is formed as a region-wise spacing between the first partial carrier and the second partial carrier.
12 . The carrier according to claim 11 , wherein the first thickness section is equal to the second thickness section and the second partial carrier has a bottom recess extending into the second partial carrier by the height of the cross-section of the transition conduit, the first partial carrier being connected to the second partial carrier along regions of the second partial carrier projecting beyond the bottom recess.
13 . A method for cultivating cells, comprising introducing liquid in which cells are suspended into second recesses of the carrier according to claim 7 , and subsequent remaining of liquid from the first thickness section along the outlet and the connection conduit.
14 . The method according to claim 13 , wherein the first partial carrier is formed only by the surface of the second partial carrier by which the second recesses are spaced apart, and the first recess adjacent to the arrangement of second recesses is formed solely by a liquid layer generated by surface tension.
15 . The method according to claim 13 , wherein the carrier has only second recesses and medium forms a layer projecting above the plane of the cross-sectional openings of the second recesses, which layer forms the volume of the first recess.
16 . The method according to claim 13 , wherein the carrier comprises recesses whose cross-sectional openings are spaced apart by surface regions of the carrier, these surface regions and the cross-sectional openings of the recesses lying in a common plane, wherein liquid is applied onto this plane and the liquid is moved along the carrier by tilting the carrier against the horizontal, by vibrating the carrier, and/or by applying pressurized gas to the plane.
17 . A carrier for cultivating cells, comprising at least one first recess extending over a first thickness section from a first surface up to a second thickness section adjacent to the first thickness section, an arrangement of second recesses in the second thickness section and extending from the first recess, wherein the second recesses comprise an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, the carrier comprising an outlet whose cross-section opens in or adjacent to the first thickness section, comprising at least two first recesses, each of which is enclosed by circumferentially arranged connection conduits extending through the carrier.
18 . A carrier for cultivating cells, comprising at least one first recess extending over a first thickness section from a first surface up to a second thickness section adjacent to the first thickness section, an arrangement of second recesses in the second thickness section and extending from the first recess, wherein the second recesses comprise an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, the carrier comprising an outlet whose cross-section opens in or adjacent to the first thickness section, comprising at least two first recesses, at least one of which is enclosed by an outlet in the form of a trench that opens into another one of the at least two first recesses, which is enclosed by circumferentially arranged connection conduits extending through the carrier.
19 . A carrier for cultivating cells, comprising at least one first recess extending over a first thickness section from a first surface up to a second thickness section adjacent to the first thickness section, an arrangement of second recesses in the second thickness section and extending from the first recess, wherein the second recesses comprise an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, the carrier comprising an outlet whose cross-section opens in or adjacent to the first thickness section, comprising a controlled valve is arranged in an outlet, in a connection conduit, and/or in a supply conduit.

Description

PRIORITY CLAIM This application is a 35 U.S.C. 371 US National Phase and claims priority under 35 U.S.C. § 119, 35 U.S.C. 365(b) and all applicable statutes and treaties from prior PCT Application PCT/EP2022/062301, which was filed May 6, 2022, which application claimed priority from German application 10 2021 204 675.4, which was filed May 7, 2021. FIELD OF THE INVENTION A filed of the invention concerns cell cultivation devices, use of such devices and methods of cell cultivation. BACKGROUND Conventionally, for such methods, so-called microtiter plates are used as devices for cell cultivation and for conducting biological experiments. Microtiter plates have wells with a volume of e.g. approx. 300 μl. One biological experiment can be performed per well, and a large amount of reagents is therefore required per well. In addition, microtiter plates are comparatively large and only provide space for typically 96 and up to 1536 parallel experiments. Locating individual cells in these wells, e.g. using a light microscope, is laborious. This poses major challenges for high-throughput experiments in which specific cell types are to be cultured and studied, e.g. cell line development, monoclonal antibody development, synthetic biology, cell therapy, immunotherapy, stem cell research. The use of highly miniaturized wells, e.g. volumes reduced by a factor of 100,000, is technically possible, but brings new challenges, in particular evaporation, feeding of nutrients, concentration of metabolic products and cell migration. The latter arises from the movement of the cells under their own power or from turbulence when refilling reagents or media, when moving carriers, from heating and convection. For most biomedical developments, constant conditions must be ensured in each individual experiment. For the approval of medical products, monoclonality must be demonstrated. DE 10 2020 209 825 A1 describes a method for manufacturing a plastic part having a glass insert from a prefabricated plastic part having a circumferential weld web and a glass insert, which has through-holes spaced apart in a circumferentially closed abutment region, and heating the weld web and pressing the glass insert with its abutment region against the weld web of the plastic part to produce a joint region in which plastic rests in the abutment region and has passed through the through-holes of the glass insert. DE 10 2019 217 466 A1 describes glass reaction vessels formed as recesses of at least 30 μm depth in a one-piece glass plate or in two interconnected glass plates. The recesses can have a section extending into the glass plate only up to a first thickness section, from which further smaller recesses can extend up to a deeper second thickness section. WO 2016/041544 A1 generally describes a method preferred for making recesses in glass. SUMMARY OF THE INVENTION The invention provides an alternative method for the cultivation of cells and a carrier suitable for use in the method, the carrier having recesses for cells, in order to hold cells in their separate wells and yet to contact several wells with a common volume of medium forming a common supply of medium. Preferably, the carrier is adapted to selectively drain or withdraw liquid, in particular cell culture medium, from recesses. An embodiment of the invention provides a carrier for cultivating cells. The carrier includes at least one first recess) extending over a first thickness section from a first surface of the carrier up to a second thickness section which is adjacent to the first thickness section and in which an arrangement of second recesses extends from the first recess. The second recesses have an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm. The carrier has an outlet whose cross-section opens in or adjacent to the first thickness section. BRIEF DESCRIPTION OF THE DRAWINGS The invention is now described in more detail by means of examples with reference to the figures, which show schematically in FIG. 1 an embodiment of the invention, FIG. 2 an embodiment of the invention with supply conduit from the second surface of the carrier, FIG. 3 an embodiment of the invention with supply conduit and outlet from the second surface of the carrier, FIG. 4 embodiments of the invention in top view, FIG. 5 an embodiment of the invention with supply conduit and outlet, FIG. 6 a further embodiment of the invention with supply conduit and outlet, FIG. 7 a further embodiment of the invention, FIG. 8 a further embodiment of the invention, FIGS. 9A in cross-section and 9B in plan view on a carrier an embodiment, FIG. 9C a first partial carrier in plan view, FIG. 10 a further embodiment of the invention, FIGS. 11A and 11B a further embodiment of the invention and FIG. 12 a further embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to a device, the use of the device for cell cultivation, and a method of cell cultivation using