Field of the invention The invention relates generally to modeling a neurovascular unit in vitro. More particularly, the invention relates to a cell culturing platform, to a cell culture system, and to a method for modeling a neurovascular unit in vitro.

Background

The blood-brain barrier "BBB" is an endothelial interface that controls interaction between the bloodstream and the brain interstitial space. During development, the BBB arises as a result of complex multicellular interactions between immature endothelial cells and neural progenitors, neuronal cells, radial glia, and pericytic cells. When the brains develop, astrocytic cells and pericytic cells further contribute to the BBB induction and maintenance of the BBB phenotype. Because BBB disease states are difficult and time-consuming to study in vivo, researchers are interested in utilizing in vitro models for simplified analyses and higher throughput. However, many in vitro BBB models are hampered by uncertain model reliability. Furthermore, it is quite challenging to isolate the BBB endothelium from animal or human brains. Publication Lippmann et al. : Modelling the blood-brain barrier using stem cell sources, Fluids and Barriers of the central nervous system "CNS" 2013, 10:2, Dept. of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Dr., Madison, Wl 53706, USA describes recent efforts in differentiating human pluripotent stem cells "hPSCs" to endothelial cells with robust BBB characteristics. Furthermore, Lippmann et al. describe how these cells could ultimately be used to study BBB development and maintenance, to model neurological disease, and to screen neuropharmaceuticals. The physical arrangement of the in vitro model is however such that the endothelial cells are located on semipermeable membrane above the neural cell cultures. Due to the physical arrangement described above, it may be in some cases quite challenging to model, detect, monitor, and/or analyze the interactions between the cells.

Summary

The following presents a simplified summary in order to provide a basic understanding of some aspects of various invention embodiments. The summary is not an extensive overview of the invention. It is neither intended to identify key or critical elements of the invention nor to delineate the scope of the invention. The following summary merely presents some concepts of the invention in a simplified form as a prelude to a more detailed description of exemplifying and non-limiting embodiments of the invention.

In accordance with the invention, there is provided a new cell culturing platform suitable for culturing e.g. endothelial cells, astrocytic cells, and neuronal cells so as to model a neurovascular unit in vitro. The astrocytic cells are cells that take care of homeostasis, nutrient and ion stability, and a part of signaling in the brain tissue, and the endothelial cells are cells that form blood vessels. Here, the neurovascular unit determinates the simplest unit which includes endothelial cells, astrocytes, and neurons and which is simpler that above-described BBB structure. A cell culturing platform according to the invention comprises solid material adapted to constitute: - one or more first cell compartments for containing somas of neuronal cells,

- a second cell compartment for containing astrocytic cells and being adjacent to each of the one or more first cell compartments, and

- a third cell compartment for containing endothelial cells and being adjacent to the second cell compartment, The wall between each of the one or more first cell compartments and the second cell compartment comprises one or more first channels and the wall between the second and third cell compartments comprises one or more second channels. The one or more first channels are suitable for guiding growth of neuronal cell processes of the neuronal cells from the one or more first cell compartments to the second cell compartment and for inhibiting somas of the neuronal cells from moving from the one or more first cell compartments to the second cell compartment and somas of the astrocytic cells from moving from the second cell compartment to the one or more first cell compartments. The above-mentioned one or more second channels are suitable for guiding growth of cell processes of the astrocytic cells from the second cell compartment to the third cell compartment and for inhibiting the somas of the astrocytic cells from moving from the second cell compartment to the third cell compartment and the endothelial cells from moving from the third cell compartment to the second cell compartment. With the aid of the above-presented cell culturing platform, a novel in vitro model of a functional neurovascular unit which contains endothelial cells connected to astrocytic cells connected, in turn, to neuronal cells can be created. The above- presented cell culturing platform is used advantageously in horizontal position so that the endothelial cells, astrocytic cells, and neuronal cells are cultured in a planar manner in separate compartments which allow 1 ) the astrocytic cells to connect to the endothelial cells via the endfeet, i.e. via the cell processes of the astrocytic cells, and 2) astrocytic cells to connect to the functional neuronal network constituted by the neuronal cells.

A cell culturing platform according to an embodiment of the invention makes it possible to provide a horizontal in vitro model where:

1 ) Relevant cell types, e.g. endothelial cells, astrocytic cells, and neuronal cells, are able to create physiologically relevant controlled contacts between each other similarly to the in vivo situation.

2) Contacts between endothelial and astrocytic cells can be monitored optically. As these contacts are in major role in propagating insult from blood compartment to the brain parenchyma, their monitoring is important.

3) Separate compartments exist for different cell types. This allows monitoring for example an insult caused damage propagation mimicking a situation in vivo. The separate compartments enable propagation of insult from the endothelial compartment that models a blood vessel to the astrocytic and neuronal cells compartments that model the brain parenchyma in vivo.

In accordance with the invention, there is provided also a new cell culture system for modeling a neurovascular unit in vitro. A cell culture system according to the invention comprises a cell culturing platform according to the invention, wherein:

- each of the one or more first cell compartments contains somas of neuronal cells,

- the second cell compartment contains somas of astrocytic cells,

- the third cell compartment contains endothelial cells, - neuronal cell processes of the neuronal cells are capable of entering the second cell compartment through the one or more first channels and of forming contacts with the astrocytic cells, and

- cell processes of the astrocytic cells are capable of entering the third cell compartment through the one or more second channels and of forming contacts with the endothelial cells.

In accordance with the invention, there is provided also a new method for modeling a neurovascular unit in vitro. A method according to the invention comprises culturing neuronal cells, astrocytic cells, and endothelial cells in a cell culturing platform according to the invention, wherein: - each of the one or more first cell compartments contains somas of the neuronal cells,

- the second cell compartment contains somas of the astrocytic cells,

- the third cell compartment contains the endothelial cells,

- neuronal cell processes of the neuronal cells enter the second cell compartment through the one or more first channels and form contacts with the astrocytic cells, and - cell processes of the astrocytic cells enter the third cell compartment through the one or more second channels and form contacts with the endothelial cells.

A number of exemplifying and non-limiting embodiments of the invention are described in accompanied dependent claims.

Various exemplifying and non-limiting embodiments of the invention both as to constructions and to methods of operation, together with additional objects and advantages thereof, will be best understood from the following description of specific exemplifying embodiments when read in connection with the accompanying drawings.

The verbs "to comprise" and "to include" are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in dependent claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality.

Brief description of figures

The exemplifying and non-limiting embodiments of the invention and their advantages are explained in greater detail below with reference to the accompanying drawings, in which: figure 1 a illustrates a cell culturing platform according to an exemplifying and non- limiting embodiment of the invention, figure 1 b shows a magnification of a part of figure 1 a, figure 1 c shows a section view of the cell culturing platform, figure 2 illustrates a cell culture system according to an exemplifying and non- limiting embodiment of the invention for modeling a neurovascular unit in vitro, and figure 3 shows a chart of a method according to an exemplifying and non-limiting embodiment of the invention for modeling a neurovascular unit in vitro.

Description of exemplifying embodiments

Figure 1 a illustrates a cell culturing platform 101 according to an exemplifying and non-limiting embodiment of the invention, and figure 1 b shows a magnification of a part 1 15 of figure 1 a. The cell culturing platform 101 comprises solid material that is adapted to constitute first cell compartments 102a, 102b, and 102c for containing somas of neuronal cells, a second cell compartment 103 for containing astrocytic cells and being adjacent to each of the one or more first cell compartments, and a third cell compartment 104 for containing endothelial cells and being adjacent to the second cell compartment. The above-mentioned solid material can be transparent material, for example, polystyrene or polyvinyl chloride with or without copolymers, polyethylenes, polystyrene-acrylonitrile, polypropylene, polyvinylidine chloride, silicone eleastomers and similar materials. Each wall between one of the first cell compartments and the second cell compartment comprises first channels. In figure 1 a, three of the first channels are denoted with reference numbers 106a, 106b, and 106c, and the walls comprising the first channels are denoted with reference numbers 105a, 105b, and 105c. The wall 107 between the second and third cell compartments comprises second channels. In figures 1 a and 1 b, one of the second channels is denoted with a reference number 108. The above-mentioned first channels are suitable for guiding growth of neuronal cell processes of the neuronal cells from the first cell compartments 102a-102c to the second cell compartment 103 and for inhibiting somas of the neuronal cells from moving from the first cell compartments to the second cell compartment and somas of the astrocytic cells from moving from the second cell compartment to the first cell compartments. The above-mentioned second channels are suitable for guiding growth of cell processes of the astrocytic cells, i.e. astrocytic endfeet, from the second cell compartment 103 to the third cell compartment 104 and for inhibiting the somas of the astrocytic cells from moving from the second cell compartment to the third cell compartment and the endothelial cells from moving from the third cell compartment to the second cell compartment. As there are separate compartments for different cell types, it is possible for example to monitor an insult caused damage propagation mimicking a situation in vivo. The cell culturing platform 101 is suitable for providing a horizontal in vitro model. Therefore, the walls 105a-105c comprising the first channels and the wall 107 comprising the second channels are substantially vertical when the cell culturing platform is in its advantageous operating position.

The dimensions of the first cell compartment 102a shown in figure 1 a can be for example:

- the width W1 on the range from 1 mm to 5 mm,

- the length L1 on the range from 1 mm to 5 mm, and - the height on the range from 1 μιη to 10 mm, where the height is substantially vertical when the cell culturing platform is in its operating position, i.e. the heights are parallel with the z-direction of a coordinate system 199.

The dimensions of the first cell compartments 102b and 102c shown in figure 1 a can be for example:

- the width W2 on the range from 1 mm to 5 mm,

- the length L2 on the range from 1 mm to 5 mm, and

- the heights on the range from 1 μιη to 10 mm, where the height are substantially vertical when the cell culturing platform is in its operating position, i.e. the heights are parallel with the z-direction of the coordinate system 199.

The dimensions of the second cell compartment 103 shown in figure 1 a can be for example:

- the width W3 on the range from 0.2 mm to 5 mm, - the length L3 on the range from 0.2 mm to 5 mm, and - the height on the range from 1 μιη to 10 mm, where the height is substantially vertical when the cell culturing platform is in its operating position, i.e. the heights are parallel with the z-direction of the coordinate system 199. The dimensions of the third cell compartment 104 shown in figure 1 a can be for example:

- the width W4 on the range from 1 mm to 5 mm,

- the length L4 on the range from 1 mm to 5 mm, and

- the height on the range from 1 μιη to 10 mm, where the height is substantially vertical when the cell culturing platform is in its operating position, i.e. the heights are parallel with the z-direction of the coordinate system 199.

The dimensions of the first channel 106b shown in figure 1 b and of the other first channels can be for example: - the width W5 on the range from 0.5 μιτι to 15 μιτι,

- the length L5 on the range from 50 μιτι to 500 μιτι, and

- the height on the range from 0.5 μιτι to 15 μιτι, where the height is substantially vertical when the cell culturing platform is in its operating position, i.e. the heights are parallel with the z-direction of the coordinate system 199.

The dimensions of the second channel 108 shown in figure 1 b and of the other second channels can be for example:

- the width W6 on the range from 0.5 μιτι to 15 μιτι,

- the length L6 on the range from 20 μιτι to 500 μιτι, and - the height on the range from 0.5 μιτι to 15 μιτι, where the height is substantially vertical when the cell culturing platform is in its operating position, i.e. the heights are parallel with the z-direction of the coordinate system 199. Figure 1 c shows a view of a section of the cell culturing platform 101 in a case where the cell culturing platform is provided with a cover portion 1 1 1 . The cover portion 1 1 1 is not shown in figure 1 a. The section is taken along the line A-A shown in figure 1 a. The cover portion 1 1 1 is adapted to constitute an inlet channel 1 12 connected to a first end of the third cell compartment 104 and an outlet channel 1 13 connected to a second end of the third cell compartment 104 as illustrated in figure 1 c. The inlet and outlet channels enable fluid to flow through the third cell compartment so that the fluid flows substantially parallel with the wall between the second and third cell compartments. The flow of the fluid is schematically depicted with a dashed line arrow 1 16 shown in figure 1 c. The flow of the fluid can be used for driving the endothelial cells towards the wall between the second and third cell compartments. The endothelial cells gathered against the wall mimic a wall a blood vessel.

A cell culturing platform according to an exemplifying and non-limiting embodiment of the invention comprises electrodes and wirings for directing electrical signals to the neuronal cells and for receiving electrical signals from the neuronal cells. Advantageously, there are electrodes at least in the first cell compartments 102a- 102c. In figure 1 a, three electrodes of the first cell compartments are denoted with reference numbers 109a, 109b and 109c. Furthermore, there can be electrodes in the second cell compartment 103. One of the electrodes of the second cell compartment is denoted with a reference number 1 10 in figures 1 a and 1 b. Yet furthermore, there can be electrodes in the third cell compartment 104.

A cell culturing platform according to an exemplifying and non-limiting embodiment of the invention comprises a circuitry connected to the above-mentioned wirings and adapted to measure electrical signals from one or more of the electrodes and record the measurement results. The above-mentioned circuitry is denoted with a reference number 1 14 in figure 1 c. The measurements of the electrical signals enable monitoring of the activity of the modeled neurovascular unit on the functional level.

The exemplifying cell culturing platform illustrated in figures 1 a-1 c comprises the three first cell compartments 102a, 102b and 102c for the somas of the neuronal cells. The three first cell compartments are adjacent to three sides of the second cell compartment 103 which is surrounded by the three first cell compartments and the third cell compartment 104 as illustrated in figure 1 a. All the cell compartments are substantially on a same plane. It is, however, also possible that there is only one or two first cell compartments for the somas of the neuronal cells. Furthermore, a cell culture platform according to an exemplifying and non-limiting embodiment of the invention includes drug/medium application inlets in the first cell compartments 102a, 102b and 102c, in the second cell compartment 103, and/or in the third cell compartment 104 that facilitate providing drug/medium changes only to desired/dedicated areas of the cell culture platform. Cell culturing platforms of the kind described above can be fabricated by using a slightly modified, i.e. different masktype and slower but more accurate process, version of rapid prototyping method which is commonly used in fabrication of Polydimethylsiloxane "PDMS" structures. In this method, the PDMS structure is molded by using an SU-8 mold. SU-8 is a commonly used epoxy-based negative photoresist. It is a very viscous polymer that can be spun or spread over a thickness ranging from below 1 micrometer up to above 300 micrometers and still be processed with standard contact lithography. Thus, the SU-8 mold can be fabricated by using standard lithography methods.

The SU-8 mold is fabricated by spin-coating SU-8 photoresist on top of e.g. silicon wafer, the height of the layer can be controlled by changing the spinning speed or viscosity of used SU-8. SU-8 is then hard baked and exposed to UV-light through a lithography mask. During the exposure, the features in the mask are transferred to the SU-8. SU-8 is then baked again and developed. This process is repeated multiple times as each height in the mold requires its own SU-8 layer. Once the SU-8 mold is completed, the PDMS is molded in it. The PDMS components are mixed together by using 1 :10 curing agent - base polymer ratio and poured onto the mold. The PDMS is then exposed to vacuum in order to remove air bubbles. After the vacuum treatment, the PDMS is baked in e.g. 60 degrees for 10 hours. After the bake, the PDMS is cut out of the mold and the necessary inlets for fluids are punched into it by using punching tools. Before using the PDMS structures, they are exposed to oxygen plasma in order to make them hydrophilic.

Figure 2 illustrates a cell culture system according to an exemplifying and non- limiting embodiment of the invention for modeling a neurovascular unit in vitro. The cell culture system comprises a cell culturing platform 101 according to an embodiment of the invention. Each of the first cell compartments 102b contains somas of neuronal cells. In figure 2, one of the somas of the neuronal cells is denoted with a reference number 220. The second cell compartment 103 contains somas of astrocytic cells. In figure 2, one of the somas of the astrocytic cells is denoted with a reference number 221 . The third cell compartment 104 contains endothelial cells which mimic a wall of a blood vessel. In figure 2, one of the endothelial cells is denoted with a reference number 222. Neuronal cell processes of the neuronal cells are capable of entering the second cell compartment 103 through the first channels 106b and of forming contacts with the astrocytic cells. One of the neuronal cell processes of the neuronal cells is denoted with a reference number 223. Cell processes, i.e. astrocytic endfeet, of the astrocytic cells are capable of entering the third cell compartment 104 through the second channels 108 and of forming contacts with the endothelial cells. One of the cell processes of the astrocytic cells is denoted with a reference number 224.

In a cell culture system according to an exemplifying and non-limiting embodiment of the invention, the third cell compartment 104 contains insult for causing operational disturbance in the contacts between the endothelial cells and the cell processes of the astrocytic cells so as to mimic a damage situation in vivo. In a cell culture system according to an exemplifying and non-limiting embodiment of the invention, the cell culturing platform comprises an inlet channel and an outlet channel for enabling fluid to flow through the third cell compartment so that the fluid flows substantially parallel with the wall 107 between the second and third cell compartments and drives the endothelial cells to gather against the wall 107. The fluid may comprise cell culturing medium. Instead of or in addition to the cell culturing medium, the fluid may comprise the above-mentioned insult. In figure 2, the flow of the fluid is schematically depicted with a dashed line arrow 216.

Figure 3 shows a chart of a method according to an exemplifying and non-limiting embodiment of the invention for modeling a neurovascular unit in vitro. The method comprises culturing 301 neuronal cells, astrocytic cells, and endothelial cells in a cell culturing platform according to an embodiment of the invention, wherein:

- each of the one or more first cell compartments contains somas of the neuronal cells,

- the second cell compartment contains somas of the astrocytic cells, - the third cell compartment contains the endothelial cells,

- neuronal cell processes of the neuronal cells enter the second cell compartment through the one or more first channels and form contacts with the astrocytic cells, and

- cell processes of the astrocytic cells enter the third cell compartment through the one or more second channels and form contacts with the endothelial cells.

A method according to an exemplifying and non-limiting embodiment of the invention comprises supplying insult to the third cell compartment. The insult causes operational disturbance in the contacts between the endothelial cells and the cell processes of the astrocytic cells so as to mimic a damage situation in vivo.

A method according to an exemplifying and non-limiting embodiment of the invention comprises directing fluid to flow through the third cell compartment so that the fluid flows substantially parallel with the wall between the second and third cell compartments so as to gather the endothelial cells on the wall between the second and third cell compartments.

A method according to an exemplifying and non-limiting embodiment of the invention comprises optically inspecting, with the aid of a microscope, the contacts formed between the endothelial cells and the cell processes of the astrocytic cells.

In a method according to an exemplifying and non-limiting embodiment of the invention, the cell culturing platform comprises electrodes for directing electrical signals to the neuronal cells and for receiving electrical signals from the neuronal cells. The method according to this embodiment of the invention comprises measuring one or more electrical signals from one or more of the electrodes so as to monitor activity of the neuronal cells.

The non-limiting, specific examples provided in the description given above should not be construed as limiting the scope and/or the applicability of the appended claims.