WO2016166282A1

WO2016166282A1 - Substrate for cultivating cells

Info

Publication number: WO2016166282A1
Application number: PCT/EP2016/058342
Authority: WO
Inventors: Thomas Knott
Original assignee: Cytocentrics Bioscience Gmbh
Priority date: 2015-04-15
Filing date: 2016-04-15
Publication date: 2016-10-20

Abstract

The invention relates to a substrate for cultivating at least one cell, in particular for cultivating an accumulation or an aggregate of cells in order to detect biologically relevant measurement variables in the cells or cell membranes. The substrate has at least one surface which has an affinity for adhering to the cells in certain positions, preferably in certain addressable positions. The invention also relates to a method for cultivating at least one cell, in particular for cultivating an accumulation or an aggregate of cells, wherein the aforementioned substrate is introduced into a cell-repellent hydrophobic cultivation device, and cells are cultivated on the substrate. The described substrate and the described method are suitable in particular for analytical methods which are used in patch-clamp technology.

Description

description

Substrate for cultivating cells

The invention relates, inter alia. a substrate for cultivating cells according to the preamble of claim 1. This substrate according to the invention is intended in particular for culturing a collection or an aggregate of cells for the determination of preferably biologically relevant measured variables on these cells or their cell membranes. The accumulation or aggregate of cells may also be referred to as tissue. In particular, the substrate serves to culture so-called adherent cells, such as fibroblasts, endothelial cells, neurons, muscle cells, and the like, which grow on surfaces, as opposed to cells that grow freely in the nutrient medium.

According to the invention, the substrate is characterized in that it has at least one surface which, at certain positions, preferably at certain addressable positions, has an affinity, in particular an improved affinity, for adhering the cells to the substrate surface.

The term "addressable" is to be understood according to the invention that the corresponding position on the surface of the substrate (again) can be found, and so can be controlled, for example, in a subsequent measurement method to a cell adhering to this position / position specifically to the appropriate measurement or investigation.

Advantageously, the substrate according to the invention is suspendable in at least one solvent or at least one solvent mixture, i. the corresponding substrates can be converted into a suspension. In particular, these are a buffered solvent or a buffered solvent mixture, i. a solvent or solvent system that does not (strongly) change its pH within certain limits on addition of an acid or a base.

In a further development, the substrate according to the invention is preferably planar or planar. In the context of the present invention, this should mean that the substrate has a smaller, in particular significantly smaller, dimension in one spatial direction than in the two other spatial directions. In these cases, the substrate according to the invention is, in particular, a disk-shaped or annular substrate, wherein the substrate may preferably be composed of at least two disks or rings connected to one another. These embodiments will be explained in more detail in connection with the figures.

The already mentioned features of the substrate according to the invention and further preferred features are mentioned in the subclaims dependent on claim 1. The wording of all of these claims is hereby expressly incorporated by reference into this specification. The same applies to the methods claimed in the independent method claims 12 and 14, with their respective subclaims, and for the claimed in claim 20 kit with its subclaims.

In the invention, individual cells or aggregates and aggregates of 2, 3 or up to several 100 cells, preferably up to about 200 cells on the substrate according to the invention, also called "floating substrate" or miniaturized microstructured substrates (MMS), spatially organized The density of the MMS in this case controls the lowering, floating or rising in a suspension of the substrates in a solvent or solvent mixture, preferably in buffer solution The relative density of the MMS compared to culture medium, washing and measuring buffers and compared to the respective cells an adjustable parameter for optimizing the cell culture processes (sinking or hovering or ascending) and the measuring processes, in particular the patch-clamp processes (MMS acts as a kind of sinker or parachute or hot-air balloon for the cells) from the perspective of the present invention.

Through the targeted growth of the cells and thus the exact positioning of the cells, and by the possibility of temporarily suspending the MMS by force to overcome gravity, buoyancy and viscosity (force by flow, magnetic force, electrostatics, ultrasound, heat, light, etc. ), in order to transport the tissues relative to the measuring device, in particular relative to the patch position or into the patch chip, it is possible, in particular the patch clamp technique, for the first time also to perform tissue automatically.

The patch-clamp technique is an electrophysiological method of measuring the current flowing through individual ion channels in the cell membrane of a cell. can make. The term "patch" refers to the small membrane section under the so-called patch pipette, which also serves as a measuring electrode, so that it is possible with this method to measure the function of cells in their natural environment in the living organism -Clamp technique according to the so-called cytocentering method (EP 1 31 1 655), cell suspension is pipetted onto a chip having microstructured openings By negative pressure, a cell of the cell suspension is selectively positioned on the opening of the chip and electrically Compared to the conventional manual patch-clamp method, the method offers a number of advantages: In addition to the significantly higher throughput rate, the integration of microfluidic channels, for example, enables automated drug addition, as is the case in the Pharmafor The perfusion is also the intracellular egg This makes it much easier to investigate drugs that act intracellularly.

The disclosure of EP 131 1655, as far as it is relevant in combination with the features of the present invention, is hereby expressly incorporated by reference into the content of this description.

However, with automated patch clamping, the cells must be isolated to transport them to the patch position, so far no measurements on tissue sections are possible.

According to the present invention, in order to cultivate spatially organized cells in specific, in particular addressable positions, the "floating substrate" according to the invention for cultivation is introduced into a cell-repellent and hydrophobic culture dish or a device of comparable functionality, in particular growth of the cells on the cell Bottom of the culture dish excluded.

A concave culture dish is preferred to collect the substrates and the cells to be cultured at the lowest point of the dish. A dish with many concave (micro) wells is more preferred.

It is also conceivable to place a plurality of substrates in a culture dish, wherein these should preferably not touch, in order to avoid a growing together of the cells. The substrate according to the invention may vary in size, shape as well as in the coating. In addition, these parameters (size, shape and coating) are crucial for the growth or the amount of growing cells on the substrate. In particular be provided by an appropriate coating at certain points of the surface of the substrate affinity for adherence of the cells to be cultivated and / or improved. Furthermore, it has proved to be advantageous to provide the substrates with a microstructure as an alternative or in addition to a coating. The microstructuring can take place by means of geometric shape (provision of depressions, elevations and the like in the surface, possibly also in regular structures) and / or structured coating (possibly also as regularly repeating structures). The coating can be carried out by physical, chemical, biochemical, molecular biological or biological methods. The composition of the coating can be defined chemically or biochemically. This allows further control of cell growth. Microstructured surfaces have already been used with cells in recent years. Especially for the implantation of implants, this technology opens up new possibilities in the targeted control of cell growth. This can be used, for example, for the treatment of peripheral nerve lesions in that the artificial nerve implant has microstructured surfaces that allow sequential growth of the severed nerve endings. Another application is the targeted differentiation of endothelial and smooth muscle cells for use in vascular implants. Here, too, an extracellular environment is created by the microstructured surface, which should favor the formation of artificial vessels. The present invention applies such microstructured surfaces in a novel way to the growth of susceptible micro-tissues for analytical purposes.

Furthermore, the growth of the cells is possible without having to give the substrate in suspension. Thus, inter alia, the adverse influence of shear forces, which occurs in a cultivation of cells in a moving suspension avoided. Only for the measurement, the substrate is transferred into suspension. For cultivation, however, the substrate is gently rinsed only with the appropriate nutrient medium. In the process, cells which have "lost their way" in the culture dish or on the substrate are rinsed off, so that optimal and targeted cultivation is possible.The surface of the substrate is such that the cells are only in the positions defined for them With the help of optionally integrated racks, through levels, or generally through a three-dimensional architectural structure, the MMS allow fouling on all sides of the room.When cultivating cells in suspension, there is often the problem that these are very This is prevented by the substrate according to the invention and the method according to the invention, but rather a spatially organized growth of the cells at certain, in particular addressable, positions is possible. In preferred embodiments of the invention, the MMS or "floating substrate" according to the invention has a density which is smaller than the density of the cells to be cultivated, then the cells can settle on the substrate according to the invention without problems, for example the typical density of cells at 1 .1 g / mL, so that in these cases a density of the "floating substrate" of 1.05 g / mL is preferred. It has also proven to be particularly advantageous if the density of the "floating substrate" has a density which is greater than the density of the buffer, then the substrate according to the invention can settle out in this solvent / solvent mixture.

The substrate according to the invention is preferably "soft", but stable against cellular forces and / or not sharp-edged Ideally, the Shore hardness of the substrate is adjustable since the substrate hardness is a mechanical parameter that influences the differentiation of stem cells.

Both resorbable, biocompatible, biosynthetic material and non-resorbable material can be used as material, as is known from the prior art for so-called microcarriers for adherent cells. Known materials are DEAE (diethylaminoethyl) -dextran, glass, polystyrene plastics, acrylamide plastics, collagens, alginates and the like. Also combinations of materials are included. Furthermore, gases or vacuum may also be sealed into the material, in particular in order to vary the density of the substrate. Weight of the substrate divided by total volume (including inclusion volume) is referred to as the resulting density of the substrate.

In addition, the structuring or coating of the substrates according to the invention can be carried out such that both a 2-dimensional and a 3-dimensional cell growth is possible.

The size of the micro-substrates lies within the size ranges specified in the claims, advantageously between 1 μm to a few 100 μm.

By the targeted positioning of the cells on the substrate according to the invention, it is possible to produce correspondingly adequate "patch chips" for the planar patch clamping, which at the points at which the cells are grown on the substrate according to the invention, for the patch Clamping required microstructured openings, so that specifically several cells or tissue can be measured simultaneously.Since the cells are already separated on the "floating substrate" according to the invention, they can be targeted docked for measurement. On the part of the MMS and on the side of the patch chips, ana- mesh structures to convey an exact positioning of cells to patch positions. These anchor structures may include mechanical, magnetic and / or optical means.

The above and further advantages of the invention will become apparent from the following description taken in conjunction with the figures, in addition in conjunction with the claims. In this case, the individual features can be implemented individually or in combination with each other.

In the figures show:

1 is a schematic representation of a culture dish with various substrates according to the invention, on which cells are already partly cultivated, and

2 shows the schematic representation of an arrangement for a patch clamp measurement, in which cells to be examined are cultivated on a substrate according to the invention, and assigned to a patch clamp chip.

FIG. 1 shows a schematic plan view of a culture dish 6 into which various substrates 1, 2, 3 and 4 according to the invention have been introduced. Some of these substrates already have cells 5 cultured, i. these cells 5 are adhesively anchored to the respective substrates 2, 3 and 4.

According to FIG. 1, the substrate 1 is a disk-shaped substrate which is not (yet) covered with a cell. The substrate 2 is also disk-shaped and has a slightly larger diameter than the substrate. 1 On the surface of this substrate 2, a cell 5 is fixed.

The substrate 3 according to FIG. 1 is an annular substrate on which a cell 5 is likewise cultivated.

The substrate 4 according to the invention according to FIG. 1 is constructed from annular elements, specifically from five annular elements in the manner of the olympic rings. At this substrate 4, three cells 5 are already cultured, which are joined together at the corresponding location of the substrate 4 to a cell collection or a cell composite. The respective surfaces of the substrates 1, 2, 3 and 4 are modified by coatings and / or microstructures so that the substrate has sufficient affinity to allow good adhesion of the cells 5. As can be seen from FIG. 1, corresponding coatings and / or microstructures are already sufficient on annular substrates in order to allow cultivation of the cells on the substrates according to the invention.

As already explained in the description, the inner surface of the culture dish 6 is cell-repellent and hydrophobic, so that the growth of the cells takes place on the substrates and not on the inner surface of the culture dish 6. In the cultivation of the cells 5 in the culture dish 6 on Substrates 1, 2, 3 and 4, it is not necessary to keep the substrates in suspension. The cultivation of the cells takes place in the appropriate culture medium, and only after the cultivation of the cells 5 on the corresponding substrates 1, 2, 3 and 4 are the substrates cultured with the cells transferred into suspension in order to provide them in a suitable manner for the subsequent analysis. The corresponding methods are generally carried out in such a way that the substrates according to the invention are introduced into the (cell) culture dish 6, in particular are sprinkled in, before the sowing of the cells takes place. Excess cells are carefully pipetted off as part of the change of medium, while the cells 1, 2, 3 and 4 occupied with cells 5 remain at the bottom of the culture dish 6. Possibly. For this purpose, the substrates may be magnetically equipped to optimize the fixation at the bottom of the culture dish 6 accordingly. For the subsequent measurement method, for example with the aid of an automated patch-clamp device, the substrates according to the invention are transferred into suspension, for example by whirling, and, for example, into the corresponding patch clamp chip, for example the CytoPatch ™ chip of US Pat Applicant, transferred. FIG. 2 shows, in a schematic sectional view, how cells 5, which are cultivated on an annular substrate 2 according to the invention, can be supplied to a patch clamp measurement.

In the upper part of FIG. 2, the annular substrate 2 is shown with two cells 5 cultured on this substrate 2. In this case, the two cells 5 in the substrate 2 are cultured together to form a cell collection consisting of two cells. The method described below can be carried out in the same way according to the invention with individual cells, as shown for example in Figure 1. In the upper part of Figure 2 is also schematically an anchoring element 7 is shown, by means of which the substrate 2 can be handled, and transferred in the desired manner in the patch-clamp device and can be positioned on or in the patch clamp chip. This transfer and positioning is indicated by the two arrows in Figure 2 graphically. Such a patch clamp chip 8 is shown schematically in sectional view in the lower part of FIG. As already explained in the introduction, this patch clamp chip in addition to the actual patch clamp openings 9, 9 'suction openings 10, 10' with their schematically indicated suction channels 1 1, 1 1 'for sucking the cells to be examined and cell membranes to the patch clamp openings 9, 9 '. The channels 12, 12 'connected to the patch clamp openings 9, 9' are also indicated in the drawing. About these channels 12, 12 'takes place necessary for the measurement contact of the cell membranes with the measuring devices. Not shown in FIG. 2 are optical windows on the underside of the patch clamp chip 8, with the aid of which the docking of the cells 5 to the openings 9, 9 'can be monitored and, if necessary, documented and possibly optically analyzed. Possibly. the patch clamp chip 8 has devices which cooperate with anchor elements 7 of the substrate 2 in order to ensure an exact positioning of the substrate 2 in or on the patch clamp chip 8.

As shown in Figure 1 and Figure 2, it is possible by the invention to detect both single cells and aggregates or aggregates of cells, i. Cultivating tissue on a substrate / carrier, in suspension on this carrier and then analyze, in particular by means of the patch-clamp method.

Claims

claims

1 . Substrate (1, 2, 3, 4) for cultivating at least one cell (5), in particular for culturing a collection or an aggregate of cells for the determination of preferably biologically relevant parameters on these cells or their cell membranes, characterized in that the substrate has at least one surface which has an affinity for adherence of the cells at certain positions, preferably at certain addressable positions.

2. Substrate according to claim 1, characterized in that the substrate is suspendable in at least one solvent or at least one solvent mixture, which is preferably a buffered solvent or a buffered solvent mixture.

3. Substrate according to claim 1 or claim 2, characterized in that it is formed flat.

4. Substrate according to one of the preceding claims, characterized in that it is disc-shaped (1, 2) or annular (3, 4) is formed, wherein preferably the substrate (4) is composed of at least two interconnected discs or rings.

5. Substrate according to one of the preceding claims, characterized in that the dimensions of the substrate between 0.1 μηη and 10 mm, in particular between 1 μηη and 500 μηη.

6. Substrate according to one of the preceding claims, characterized in that the substrate has at least one microstructured surface.

7. Substrate according to one of the preceding claims, characterized in that the density of the substrate is smaller than the density of the cells to be cultivated.

8. A substrate according to any one of claims 2 to 7, characterized in that the density of the substrate is greater than the density of the solvent or the Gemischge- solvent mixture.

9. Substrate according to one of the preceding claims, characterized in that the surface of the substrate is designed for a two-dimensional cell growth in the cultivation.

10. Substrate according to one of the preceding claims, characterized in that the surface of the substrate is designed for a three-dimensional cell growth.

1 1. Substrate according to one of the preceding claims, characterized in that means (7) for spatial alignment of the substrate (2), in the manner of anchor devices (7), are provided which in particular have an orientation of the substrate in all three spatial coordinates and in all three solid angles enable.

12. A method for culturing at least one cell, in particular for culturing a collection or an aggregate of cells for the determination of preferably biologically relevant parameters to cells or their cell membranes, characterized in that at least one substrate (1, 2, 3, 4) according to one of the preceding claims in a cell repellent, hydrophobic culture device, preferably culture dish (6) is introduced, and cells (5) are cultivated on this substrate.

13. The method according to claim 12, characterized in that at least two substrates (1, 2, 3, 4) in the spatial distance from each other in the culture dish (6) are introduced.

14. A method for determining preferably biologically relevant parameters on membranes and cells, in particular cell membranes, characterized in that at least one substrate according to one of claims 1 to 1 1, were cultivated on the cells, in particular according to a method of claim 12 or 13, be suspended in at least one solvent or at least one solvent mixture, and then with the aid of this suspension, the determination of the measured quantities of individual cells or on a collection or on an aggregate of cells prepared and in particular also carried out.

15. The method according to claim 14, characterized in that the substrate (2) with the cultured cells (5) by means of existing alignment means, in particular by means of anchor devices (7), is positioned in a desired manner for determining the measured variables.

16. The method according to claim 14 or 15, characterized in that the measured variables using an electrophysiological measurement method, in particular using the so-called patch-clamp technique are determined.

17. The method according to claim 16, characterized in that the determination of the measured variables on a planar patch clamp substrate, in particular a so-called patch clamp chip (8) is carried out, wherein on the substrate (2) cultured cells (5 ) are positioned over the corresponding patch clamp opening (9, 9 ') in the patch clamp substrate (8), in particular by means of alignment means (7).

18. The method according to claim 17, characterized in that the determination of the measured variables is carried out on a patch clamp chip according to EP 131 1655.

19. The method according to claim 17 or claim 18, characterized in that the patch clamp substrate, in particular the patch clamp chip, on its side remote from the patch clamp opening side has at least one optical window, with the aid of the positioning The cultured cells can be monitored, documented and optically analyzed.

20. Kit for the determination of preferably biologically relevant parameters on membranes and cells, in particular on cell membranes, characterized in that it comprises at least one substrate according to one of claims 1 to 1 1.

21. Kit according to claim 20, characterized in that it additionally comprises at least one device for cultivating cells, in particular at least one culture dish.

22. Kit according to claim 20 or claim 21, characterized in that it additionally comprises at least one device on which a patch clamp measurement can be carried out, in particular at least one patch clamp chip.