DE102020131894B4 - Cultivation insert for a culture dish, flow insert for a culture dish and corresponding kit for studying cell dynamics under fluid shear stresses - Google Patents

Abstract

Cultivation insert for a culture dish, comprising: a base body which has a bottom and a top, a through opening running through the base body and extending between the bottom and the top; wherein a first edge of the through opening, which is arranged on the underside, a Cultivation surface is defined and flat, so that when the cultivation insert is placed on any flat surface, the first edge lies fully against this surface.

Description

The present invention relates to a cultivation insert for a culture dish, a flow insert for a culture dish and a corresponding kit which includes these two and the culture dish for the purpose of studying cell dynamics under fluid shear stresses.

The interaction of inflammatory cells, tumor cells and pathogens (e.g. bacteria, viruses, parasites) with epithelial and endothelial tissues play an important role in basic research and in elucidating the pathogenesis of diseases. For example, leukocytes can initially adhere to the cells in areas of inflammation and then migrate through the cell layers in a complex process (diapedesis or transmigration). There are numerous findings that show that fluid flows such as: B. generated by blood flow have a significant influence on the endothelium and epithelium both under physiological conditions and under pathological conditions. In addition, suspended cells also react to currents and thereby change their properties. This applies to almost all forms of inflammatory cells and also appears to have a significant influence on tumor cells and the interaction of cells with pathogens. Numerous systems have been developed to investigate the influence of shear stresses on endothelial cells. These include, for example, cone-plate systems or parallel plate systems. For the interaction of leukocytes with endothelial cell systems, flow chambers are predominantly used, which are based on the principle of parallel plates and are commercially available. Parallel plate flow chambers are characterized by the fact that two plates are glued to one another in parallel or otherwise connected to one another using a spacer, which typically has a thickness between 0.3 and 1 mm. A three-dimensional shear gap is created between the plates, through which a liquid (e.g. nutrient medium or buffer) flows through connection pieces using a pressure difference (through hydrostatic pressure or a pump) in the experiment. Cells can be added into the flow chamber via the ports, where they then attach to one of the plates and are cultured. By adding cell suspensions to the flow chamber, the adhesion of suspended cells (e.g. leukocytes, tumor cells or other cells) to the cell layer can then also be determined under currents. The flow chambers can also be used to study shear stress effects on endothelial cells.

• 1) Die Platte, auf der die Zellen wachsen sollen, kann bei den kommerziell erhältlichen Systemen nicht separat oberflächenbeschichtet werden. Durch die feste Konstruktion der Kammern wird dann die obere Platte immer mit beschichtet. Dadurch kann sich das Strömungsverhalten ändern und muss ggf. immer neu bestimmt werden, was einen erheblichen Aufwand bedeutet.
• 2) Da die Versorgung der Zellen während der Wachstumsphase über Diffusion Nährstoffen und Gasen von den Anschlussstücken her in den Scherspalt hinein erfolgen muss, stellt dieser während der Wachstumsphase ein Nadelöhr dar und führt ungünstiger Weise zu einem Gradienten für Nährstoffe, Sauerstoff, Kohlenstoffdioxid und Metaboliten. Abhilfe dieses erheblichen Problems kann bei den auf dem Markt erhältlichen Kammern durch eine permanente Perfusion erfolgen, die unmittelbar nach der Aussaat der Zellen beginnen muss. Dieses Vorgehen bedeutet jedoch einen erheblichen zusätzlichen Aufwand, insbesondere wenn mehrere Proben untersucht werden sollen.
• 3) Aufgrund der in der Natur der Sache liegenden Kompaktheit der Parallel-Strömungssysteme gestalten sich experimentelle Manipulationen des Zellrasens, z.B. ein „scratch assay“, sehr schwierig oder sind sogar unmöglich.
• 4) Die Zuläufe zum Scherspalt am Ein- und am Auslass führen zu einer gestörten Strömung, so dass das Strömungsprofil quer zur Längenausdehnung des Scherspaltes als auch entlang der Längsausdehnung des Scherspaltes nicht gleichmäßig ist. Insbesondere liegt kein laminares Strömungsprofil vor.
• 5) Bei den derzeit auf dem Markt verfügbaren Strömungssystemen handelt es sich entweder um aufwendig hergestellte und damit teure Einmalartikel oder um zusammenzusetzende Selbstbauten, die aufwendig zusammengebaut werden müssen.

The structure of the previously available parallel flow systems involves significant problems due to the principle of fixed structure, which will be discussed below.

• 1) The plate on which the cells are to grow cannot be surface-coated separately in commercially available systems. Due to the solid construction of the chambers, the top plate is always coated. As a result, the flow behavior can change and may have to be determined again and again, which means considerable effort.
• 2) Since the cells must be supplied with nutrients and gases during the growth phase via diffusion from the connecting pieces into the shear gap, this represents a bottleneck during the growth phase and unfortunately leads to a gradient for nutrients, oxygen, carbon dioxide and metabolites. This significant problem can be remedied in the chambers available on the market through permanent perfusion, which must begin immediately after the cells have been seeded. However, this procedure involves considerable additional effort, especially if several samples are to be examined.
• 3) Due to the inherent compactness of the parallel flow systems, experimental manipulations of the cell layer, e.g. a “scratch assay”, are very difficult or even impossible.
• 4) The inlets to the shear gap at the inlet and outlet lead to a disturbed flow, so that the flow profile is not uniform across the longitudinal extent of the shear gap or along the longitudinal extent of the shear gap. In particular, there is no laminar flow profile.
• 5) The flow systems currently available on the market are either elaborately manufactured and therefore expensive disposable items or self-assembled items that have to be laboriously assembled.

Against this background, the object of the present invention can be seen as, starting from the poor handling and the culture and flow conditions of the flow chambers known from the prior art, not being defined precisely enough, to provide a corresponding system in which these problems are eliminated or at least can be partially avoided.

The present invention is based on the approach for the phase in which the cell lawn is formed, i.e. in which the cells are sown on a surface and cultivated, and for the usually subsequent phase in which experiments are carried out on the cultivated cell lawn, a modified device is to be provided that is specifically adapted to the requirements of the respective phase. The special feature according to the invention is that both devices are designed as attachments that can be inserted into a culture dish, for example into a commercially available culture dish, and that the result achieved by means of a first device in the cultivation phase (hereinafter referred to as the cultivation insert) - the cell lawn - is used as part of the experimental phase within a second device (hereinafter referred to as flow insert). For this purpose, both the cultivation insert and the flow insert have a designated area, which each functions as an interface area to the culture dish used. Using the interface area, each of the two devices can be coupled to the culture dish. The two interface areas have essentially the same size in both devices, so that the dimension of the cell lawn formed by means of the cultivation device is adapted to the cell lawn size that can be used by means of the flow insert, in particular to a bottom of a flow channel.

For the purposes of the present invention, basically any culture dish can be used, for example round, commercially available disposable culture dishes, which are either made entirely of plastic (e.g. polycarbonate) or are available as so-called “glass bottom dishes”, i.e. bowls with a glass bottom, whereby the glass bottom corresponds to a microscopy cover glass in terms of its optical properties and is therefore suitable for high-resolution microscopic image analysis. If necessary, differently shaped culture dishes, for example with a rectangular basic shape, can of course also be used. In principle, the shape and dimension of the cultivation and flow insert can be freely chosen, provided that their shape and dimension is adapted to the culture dish used with it.

The present invention is particularly suitable for studying heterogeneous cell interactions and cell transmigration through cell lawns using light microscopic techniques.

According to the invention, in various embodiments, a cultivation insert for a culture dish is provided with a base body which has a bottom and a top and with a through opening running through the base body and extending between the bottom and the top. The cultivation insert is designed in such a way that a first edge of the through opening, which is arranged on the underside, defines a cultivation surface and is flat, so that when the cultivation insert is placed on any flat surface, the first edge lies fully against this surface.

The cultivation insert is a device that can be inserted into a matching culture dish. The cultivation insert is designed in such a way that after it has been inserted into a suitable culture dish, its underside lies in a sealing manner against the bottom of the culture dish. The first edge of the through-opening can therefore correspond to a lower edge of the through-opening. The lower edge of the through opening and the (outer) edge of the underside of the cultivation insert can be arranged in one plane, with this plane being arranged parallel to the bottom surface of the culture dish after the cultivation insert has been inserted into the culture dish. After inserting the cultivation insert into the culture dish, the lower edge of the through opening defines an area of the bottom of the culture dish, which is exposed and on which a cell lawn can be formed. The area delimited by the lower edge of the passage opening is freely accessible during the cultivation period. The cultivation insert can have a total base area whose dimensions do not exceed the size of the bottom area of the culture dish, so that there is no play after the cultivation insert has been inserted into the culture dish. Through this precise insertion of the cultivation insert into the culture dish, the position of the cultivation area within the culture dish is determined precisely and invariantly over time. The cultivation area can in principle have any desired geometric shape, but for practical purposes it can have a rectangular or square shape.

The cultivation insert according to the invention can be used in particular for cell cultivation under standard cell culture conditions. For this purpose, cells (e.g. endothelial, epithelial cells or other cells) are seeded in the area of the cultivation area and covered with a standard cell culture dish lid. The generously dimensioned through-opening provided, which is particularly open at the top, allows the cells to grow under standard cell culture conditions since, as in normal cell culture dishes, these cells are adequately supplied with medium and gases during their proliferation time.

According to further embodiments of the cultivation insert, a second edge of the through opening, which is arranged on the top of the base body, can describe or define a second area, wherein the second area can be larger or the same size as the cultivation area. In other words, the lower opening area of the through-hole can be smaller than the upper opening area of the through-opening. At least two inner walls of the through opening, for example two opposing inner walls, can be bevelled, so that in a side cross-sectional view an inclined surface runs between the upper edge and the lower edge of the through opening. This allows access to the cultivation area to be further simplified. The second edge can - based on the designation of the first edge as the lower edge - be understood as the upper edge of the through opening.

According to further embodiments of the cultivation insert, the cultivation area can be arranged within the second area in the top view of the cultivation insert. In particular, the first surface can be arranged centrally within the second surface, with at least one edge of the first surface being able to coincide with an edge of the second surface in the top view of the cultivation insert.

According to further embodiments of the cultivation insert, the first edge of the through opening can act as a seal or have one, so that after inserting the cultivation insert into the corresponding culture dish, the cultivation surface exposed towards the top is laterally sealed. This allows the growth of the cells sown within the passage opening on the bottom of the culture dish to be limited to exactly this area, which a fluid flows over in a subsequent experimental phase. In this way it can be ensured that the nutrients provided to the cell layer are only consumed by those cells that are also in the flow channel in the later experimental phase and therefore take part in the experiment. At the same time, no cells that later do not take part in the experiment are involved in the gas exchange in or through the nutrient medium, so that the gas exchange without saturation effects is only carried out by the cells of the cell layer, which later contribute to it during the subsequent experiment.

According to further embodiments of the cultivation insert, the base body can have a biochemically inert material, which is also preferably autoclavable. The cultivation use can, for example, plastics such as polyetheretherketone (PEEK), metals such as. B. stainless steel alloys, or mixtures thereof. A biochemically inert and autoclavable cultivation insert has the advantage that it can be cleaned after an experiment and made sterile and can be used multiple times, which is advantageous from the perspective of the sustainable use of laboratory equipment.

According to further embodiments of the cultivation insert, after inserting the cultivation insert into the culture dish, the area of the cultivation dish enclosed by the first edge of the through opening (and preferably also laterally sealed) can correspond to the cultivation area. For this purpose, the underside of the cultivation insert can rest on the bottom of the culture dish, so that the lower edge of the through opening encloses an area on the bottom of the culture dish on which cell proliferation can take place after sowing. Consequently, after completion of the cultivation phase, the shape of the cell lawn formed essentially corresponds to the shape of the cultivation area.

Furthermore, according to the invention, in various embodiments, a flow insert for a culture dish is provided, which has a base body with a bottom and a top. In principle, the external shape of the flow insert can correspond to the external shape of the cultivation insert. The flow insert further has a substantially cuboid-shaped flow channel, which is formed in the base body and has an inlet at one end and an outlet at the corresponding other end, the flow channel having a ceiling and two side walls and being open towards the underside the open bottom of the flow channel corresponds to its base area. The flow insert further has a first chamber which has an inlet and an outlet, the outlet being fluid-mechanically coupled to the inlet of the flow channel, and a second chamber which has an inlet and an outlet, the inlet being fluid-mechanically coupled to the outlet of the Flow channel is coupled. The first chamber can act as a kind of calming chamber for the fluid to be admitted into the flow channel as part of flow experiments. This means that the first chamber, which can be designed in the shape of a shaft, acts as a retention space for any turbulence that may occur on the surface of the fluid located in the first chamber due to its introduction, so that this turbulence cannot extend to the flow channel or be largely decoupled from this.

The flow channel is set up in such a way that at the transition between the outlet of the first chamber and the inlet of the flow channel, a preferably essentially vertical side wall of the first chamber merges continuously into the ceiling of the flow channel without kinks. For example, a rounded edge can be arranged on the kink-free transition between the side wall of the first chamber and the ceiling of the flow channel, so that the side wall, which runs, for example, essentially perpendicular to the ceiling of the flow channel, has a curvature connecting it to the ceiling of the flow channel towards its lower end having. The ceiling of the flow channel can, for example, be arranged horizontally and thus parallel to the bottom of the culture dish into which the flow insert is inserted. The shape of the kink-free transition between the side wall of the first chamber and the ceiling of the flow channel can, for example, be described in cross section along the flow channel by a continuously differentiable function.

According to further embodiments of the flow insert, the base area of the first chamber can be larger than, preferably at least twice as large as, the maximum cross section of the transition between the outlet of the first chamber and the inlet of the flow channel. In general, the transition between the outlet of the first chamber and the inlet of the flow channel can have a rectangular shape. The height of the rectangular shape can gradually decrease over the transition between the side wall of the first chamber and the ceiling of the flow channel in accordance with the curved transition between the two.

According to further embodiments of the flow insert, the first chamber can be open to the underside of the base body. In other words, the level of the floor of the first chamber can correspond to the level of the floor of the flow channel, whereby the floor in both areas can be formed by the surface of the culture dish floor (in the case of the floor of the flow channel, this is then covered with the cell lawn). In particular, the bottom-side transition between the first chamber and the flow channel can be free of edges or steps.

According to further embodiments of the flow insert, the first chamber can have a bottom, wherein a lower edge of the outlet of the first chamber can be arranged at the level of the lower edge at the inlet of the flow channel. The bottom of the first chamber can be formed by a separate material layer which, for example, slopes from one bottom side to the opposite bottom side on which the transition to the flow channel is arranged. By means of a suitably shaped floor, which can in particular be uneven, the flow properties of the flow insert can be optimized with regard to a laminar flow in the flow channel.

According to further embodiments of the flow insert, the ratio of channel width to channel height can be at least twenty in the cross section along the flow direction through the channel. Consequently, the flow channel can correspond to a flat cuboid, so that its fluid mechanical properties, e.g. the resistance to a fluid flowing through it, are largely determined by the ceiling and the floor covered with the cell turf.

According to further embodiments of the flow insert, the inlet of the first chamber can be arranged above the outlet of the first chamber. As a result, fluid-mechanical disturbances that can occur on the surface of the flow insert when a fluid flows into the first chamber can be dampened on the surface of the fluid. In particular, their spread to the lower area of the first chamber, in particular the area in which the fluid passes from the first chamber into the flow channel, can be prevented.

According to further embodiments of the flow insert, after inserting the flow insert into the culture dish, the bottom of the flow channel can correspond to a corresponding area of the surface of the culture dish, against which the underside of the base body rests. In particular, this area can correspond to the cultivation area on which culture cells were previously grown using the cultivation insert. The statement that a corresponding surface of the bottom of the culture dish corresponds to the bottom of the flow channel refers to the basic structural design of the flow channel and can in particular include that this area of the bottom of the culture dish is lined with a cell culture turf.

According to further embodiments of the flow insert, the transition between the outlet of the flow channel and the inlet of the second chamber can have a structure that is symmetrical to the transition between the outlet of the first chamber and the inlet of the flow channel. The second chamber can therefore in particular have an inlet in the lower region, preferably at the bottom of the second chamber, through which the fluid flows during operation of the flow insert Flows through the flow channel. The second chamber can also have an outlet which is arranged above the inlet. Inlet and outlet of the second chamber can, for example, be arranged in opposite side walls of the second chamber, analogous to the first chamber. Depending on the needs and design of the flow insert, the inlet of the first chamber can be arranged in height or level above the outlet of the second chamber. As a result, during operation of the flow insert, a higher fluid column can form in the first chamber than in the second chamber. The increase in the fluid column creates a hydrostatic pressure, which can influence the flow speed of the fluid through the flow channel. Likewise, in further embodiments, the height relationship can be reversed and a pump can be used with which the first chamber is filled with the fluid against the hydrostatic pressure of the additional fluid column in the second chamber. In even further embodiments, the outlet of the first chamber and the inlet of the second chamber can be arranged at the same height when viewed vertically and also preferably have the same dimensions.

According to further embodiments of the flow insert, the width of the first chamber can correspond to the width of the flow channel and the width of the second chamber can correspond to the width of the flow channel. This means that the side walls of the first and second chambers can merge in a straight line into the flow channel. This allows the formation of turbulence in the area of the transition between the chambers and the flow channel to be prevented or at least reduced.

Furthermore, according to the invention, in various embodiments, a kit for examining cell dynamics under liquid shear stresses is provided, which comprises a culture dish, a previously described cultivation insert and a previously described flow insert, wherein the cultivation insert and the flow insert are designed such that the cultivation area essentially corresponds to the base area of the flow channel corresponds.

According to further embodiments of the kit, the base body of the cultivation insert and the base body of the flow insert can be dimensioned such that they can be inserted into the culture dish with a precise fit. In other words, the base bodies of both inserts can essentially have the same base area in terms of circumference.

According to further embodiments, the kit can further have a stabilization dish, which is set up to hold the culture dish and is dimensioned such that after the culture dish has been inserted into the stabilization dish, it rests tightly on the floor and at least on part of the side wall of the culture dish. The shape of the stabilization dish can be adapted to the shape of the culture dish in such a way that it fits very closely to the culture dish and exerts pressure on the floor as well as on at least part of the side wall of the culture dish. The stabilization dish can be used to prevent possible vibration of the culture dish, which can be excited by the fluid flowing through the flow channel during operation of the device. The stabilization dish can therefore be viewed as a support corset, which includes or surrounds at least the lower region of the culture dish, but if necessary also the entire outer surface of the culture dish, and thus suppresses vibration of the culture dish.

According to further embodiments of the kit, the stabilization dish can have at least one opening in the base, the size and position of which is selected such that the opening can be arranged under the cultivation area, if necessary by rotating the stabilization dish relative to the culture dish. The size of the opening can be in the range of the size of the cultivation area or the bottom of the flow channel, the bottom of which is formed by the bottom of the culture dish, which is covered with the cultivated cell lawn. Through the opening, the behavior of the cell culture at the bottom of the flow channel can be observed through a microscope. The motivation for the smallest possible dimension of the opening comes from the fact that it is advantageous if the stabilization dish rests on the outer surface of the culture dish over as large an area as possible in order to develop its stabilizing or vibration-damping properties.

According to further embodiments of the kit, the stabilization shell can be made of a rigid material, preferably stainless steel. If necessary, a viscous, adhesive medium can be arranged between the inside of the stabilization dish and the outside of the culture dish in order to effect adhesion between the culture dish and the stabilization dish. This measure can achieve a further dampening effect on vibrations in the culture dish wall.

• i) Bereitstellen eine Kulturschale,
• ii) Einsetzen des Kultivierungseinsatzes in die Kulturschale,
• iii) Heranzüchten von Zellen am Boden der Kulturschale auf der Kultivierungsfläche,
• iv) Herausnehmen des Kultivierungseinsatzes aus der Kulturschale,
• v) Einsetzen des Strömungseinsatzes in die Kulturschale derart, dass die Grundfläche des Strömungskanals über der mit den herangezüchteten Zellen belegte Kultivierungsfläche positioniert wird, und
• vi) Ausbilden einer Flüssigkeitsströmung durch den Strömungskanal durch Einleiten einer Flüssigkeit in die erste Kammer.

In further embodiments, a method for studying cell dynamics under fluid shear stresses is further used of the kit described herein. The procedure has the following steps:

• i) provide a culture dish,
• ii) inserting the cultivation insert into the culture dish,
• iii) growing cells at the bottom of the culture dish on the cultivation area,
• iv) removing the cultivation insert from the culture dish,
• v) inserting the flow insert into the culture dish in such a way that the base area of the flow channel is positioned above the cultivation area occupied by the grown cells, and
• vi) Forming a liquid flow through the flow channel by introducing a liquid into the first chamber.

It should be mentioned here that when using the cultivation insert to grow the cells, cell attachment and cell growth are automatically limited to the area of the cultivation area, since only this part of the bottom of the culture dish is available for colonization by the cells. In the second phase, i.e. after the cell layer has formed, all that remains is to insert the flow insert into the same culture dish. As already explained, the big advantage is that by using different inserts, the structural restrictions resulting from the optimal design of the experimental environment for the investigation of cell dynamics under fluid shear stresses do not affect the optimal design of the environment, which is designed for optimal cultivation of the cell lawn is.

In further embodiments of the method, this may further include observing the cell dynamics in the flow channel using a microscope through the opening in the bottom of the stabilization shell.

In further embodiments of the method, this may further include placing the culture dish in the stabilization dish before or after inserting the flow insert into the culture dish.

• 1A und 1B zeigen jeweils eine Ausführungsbeispiel eines erfindungsgemäßen Kultivierungseinsatzes (perspektivische Ansicht und Querschnittsansicht).
• 2A und 2B zeigen ein Ausführungsbeispiel eines erfindungsgemäßen Strömungseinsatzes (perspektivische Ansicht und Querschnittsansicht).
• 3 zeigt eine perspektivische Explosionsansicht einer Vorrichtung, welche eine Ausführungsform des erfindungsgemäßen Strömungseinsatzes und eine dazu passende Kulturschale aufweist.
• 4 zeigt ein das Ergebnis einer Strömungssimulation zur Untersuchung der Strömung in einem Modell des Strömungskanals des erfindungsgemäßen Strömungseinsatzes.
• 5 zeigt eine graphische Auswertung der auftretenden Wandschubspannung der sich im Strömungskanal des erfindungsgemäßen Strömungseinsatzes ausbildenden Strömung.
• 6 zeigt eine graphische Auswertung der Abweichung der auf Basis einer dreidimensionalen Strömungssimulation des Strömungskanals berechneten Wandschubspannungsverteilung von einer entsprechenden zweidimensionalen Auslegung.

Some exemplary embodiments of the invention are explained below with reference to the accompanying drawings, from which advantages and further refinements of the invention can be derived. It is understood that the features mentioned above and those to be explained below can be used not only in the combination specified in each case, but also in other combinations or alone, without departing from the scope of the present invention.

• 1A and 1B each show an exemplary embodiment of a cultivation insert according to the invention (perspective view and cross-sectional view).
• 2A and 2 B show an exemplary embodiment of a flow insert according to the invention (perspective view and cross-sectional view).
• 3 shows an exploded perspective view of a device which has an embodiment of the flow insert according to the invention and a matching culture dish.
• 4 shows the result of a flow simulation to examine the flow in a model of the flow channel of the flow insert according to the invention.
• 5 shows a graphical evaluation of the wall shear stress occurring in the flow forming in the flow channel of the flow insert according to the invention.
• 6 shows a graphical evaluation of the deviation of the wall shear stress distribution calculated based on a three-dimensional flow simulation of the flow channel from a corresponding two-dimensional design.

In the 1A and 1B An exemplary embodiment of a cultivation insert 1 according to the invention is shown in each case, where 1A a perspective three-dimensional top view and 1B shows a cross-sectional view. The cultivation insert 1 has a base body 11, which has a bottom 13 and a top 12. A through opening 15 running through the base body 11 is provided, which extends between the bottom 13 and the top 12. The through opening 15 has a first edge 17, which is arranged on the underside 13 and a cultivation surface 18 is defined and flat, so that when the cultivation insert 1 is placed on any flat surface, the first edge 17 lies fully against this surface. In the present case, the first edge 17 also acts as a seal, which prevents fluid flow and/or cell migration under the underside 13 of the cultivation insert 1.

The through opening 15 has a second edge which defines an upper opening surface 19. The upper opening area 19 in the example shown is larger than the cultivation area che 18, whereby a dimension (depending on the opinion, for example the width) of the opening surface 19 and the corresponding dimension of the cultivation surface 18 are the same. Two side walls of the through opening 15 are straight and two further side walls 14 are designed so that they run obliquely in the upper area and then merge into side wall sections arranged perpendicular to the cultivation surface 18. However, this configuration is optional, so that, in particular depending on the size and position of the cultivation area relative to the upper opening area 19 of the through opening 15, the side walls 14 (or all four side walls or a different number of side walls according to other embodiments) can have a different shape , for example can have several oblique segments or can run in a straight line between the first edge 17 and the second edge. In the outer wall of the cultivation insert 1, a full sealing lip 16 is also arranged in the lower area.

In the 2A and 2 B An exemplary embodiment of a flow insert 2 according to the invention is shown in each case, where 2A a perspective three-dimensional top view and 2 B shows a cross-sectional view. The flow insert 2 has a base body 21, which has a bottom 23 and a top 22. The flow insert further has a substantially cuboid-shaped flow channel 25, which is formed in the base body 21 and has an inlet at one end and an outlet at the other end, the flow channel 29 having a ceiling and two side walls and being open towards the underside , whereby the open bottom of the flow channel corresponds to its base area. In the 2A and 2 B the flow channel 29 is shown without a ceiling. This can be realized, for example, by means of a plane-parallel glass pane, which can be inserted into the through opening 25 in the base body and seals the flow channel 29 at the top. At least one side wall 24 of the through opening 25 can, as in the case of the cultivation insert 1, run obliquely, which can in particular simplify the insertion of the glass pane to form the ceiling of the flow channel 29. For example, a step can be formed between the oblique side wall section and the side wall section of the side wall 24 arranged perpendicular to the flow channel 29, which can serve to fix the inserted glass pane. The opposite side wall can then have a groove (particularly in the case of a straight side wall) or it can also, as in 2A side wall 24 shown, have a step on which the glass pane can be placed and thus fixed.

The flow insert 2 further has a first chamber 27, which has an inlet 271 and an outlet 272, the outlet 272 being fluid-mechanically coupled to the inlet of the flow channel 272 or can essentially correspond to this. Furthermore, the flow insert 2 has a second chamber 28, which has an inlet 281 and an outlet 282, wherein the inlet 281 is fluid-mechanically coupled to the outlet of the flow channel 29 or can essentially correspond to this. The flow insert 2 is designed in such a way that at the transition between the outlet 272 of the first chamber 27 and the inlet of the flow channel, a side wall of the first chamber 27 continuously merges into the ceiling of the flow channel 29 without kinks. This aspect is in the 2A and 2 B not shown in detail, but can 3 be removed. The transition between the flow channel 29 and the inlet 281 of the second chamber 28 can be designed analogously, i.e. in such a way that the ceiling of the flow channel 29 continuously merges into the corresponding side wall of the second chamber 28 without kinks. Analogous to the design of the cultivation insert 1 according to the invention, a full-circumferential sealing lip 26 can be arranged in the outer wall of the flow insert 2 in the lower region.

In 3 is a perspective exploded view of a device 3 illustrated, which has an embodiment of the flow insert 2 according to the invention and a matching culture dish 30. The culture bowl 30 is designed in two parts and has a bottomless bowl 36 in which a glass base 35 is inserted. In particular, the culture dish can be designed as a “glass bottom dish”.

The flow insert 2 has a first cover 31, which is placed on the inlet 271 of the first chamber, with a filter structure 32, for example a sieve or a filter membrane, being located between these two. Furthermore, a hose connection can be formed on the first cover 31 (in 3 not explicitly shown), to which a hose can be connected in order to supply the first chamber 27 with a fluid. Furthermore, a second cover 32 is provided, which can be placed on the second chamber 28 and functions as a lid. The drain 282 of the second chamber 28 is formed in the side wall of the base body of the flow insert 2. At the in 3 In the embodiment of the flow insert 2 shown, the drain 282 is arranged in the side wall in such a way that, after the flow insert 2 has been inserted into the culture dish, it is arranged above its edge, so that a hose can be connected to it, through which the fluid can be removed from the second chamber 28 can.

In 3 An additional optional element of the device 3 is also sketched, which is a stabilization bowl 37, into which the culture bowl 30 is inserted and which tightly encloses or encompasses it. At least one opening 38 (breakthrough) is provided in the bottom of the stabilization shell 37, through which the bottom of the flow channel in the flow insert 2 can be observed, for example using a microscope. The cell culture dish 30 is clamped into the stabilization dish 37 in order to ensure the mechanical stability of the entire flow insert 2 using a stainless steel device.

The cultivation insert 1, the flow insert 2, the matching culture dish 30 and optionally the stabilization dish 37 represent the kit according to the invention, which can be used to investigate cell dynamics under liquid shear stresses.

Before the flow experiment begins, the cultivation insert 1 can be removed from the culture dish 30 after the formation of the cell lawn to be examined and replaced with the flow insert 2. The flow insert 2 has an opening in its bottom surface, the dimension of which corresponds to the cultivation area of the cultivation insert 1. This opening is open to the underside of the flow insert and corresponds to the open bottom of the flow channel. Towards the top of the flow insert, this opening is covered and sealed, for example by a plane-parallel glass pane, the covering corresponding to the ceiling of the flow channel. The height of the flow channel can usually be in the range of a few millimeters, for example 1 mm, 2 mm or more. is sealed and fixed at the top. A separate seal is not absolutely necessary on the underside of the flow insert 2 - outside the flow channel - since only a capillary gap remains here, which immediately fills with the fluid flowing through the flow channel and does not influence the flow properties within the flow channel.

In 4 the result of a flow simulation is illustrated in a model of the flow channel 4 for examining the flow in the flow channel of the flow insert according to the invention, a half model of the flow insert 2 being reproduced here. In the model 4 used, the core of the flow insert 2 is modeled, having the first chamber 27, which can also be referred to as a pre-flood chamber, the second chamber 28, which can also be referred to as a calming chamber, and the flow channel 29 arranged between them, in which the gap flow forms. The flow insert 2 also has an access, for example a hose attachment (in 4 not explicitly shown), which opens into the spacious first chamber 27. In the bottom area, the first chamber 27 merges into the flow channel 29, in which the desired shear stresses for the experimental investigations of the cells occur. The outlet of the flow channel 29 opens into the second chamber 28, which is also large and is provided with an outlet 282 in the form of a hose attachment.

Between the first chamber 27 and the flow channel there is a first transition region 41, which corresponds to the transition region between the outlet of the first chamber 27 and the inlet of the flow channel 29. Analogously, at the outlet of the flow channel 29 there is a second transition region 42 between the outlet of the flow channel 29 and the inlet of the second chamber 28. The first transition region 41 can basically correspond to the second transition region 42 under the aspect that both transition regions are each continuous , have a kink-free transition, which means that the formation of turbulence can be suppressed. In this regard, the actual transitions in a real embodiment of the flow insert 2 can be as shown in FIG 4 Model 4 shown can be formed.

This in 4 Model 4 shown is at the same time provided with a continuous contour plot, which illustrates the flow velocity in the flow insert, the scaling associated with the contour plot being indicated in the subdiagram 40. During the simulation, a pressure boundary condition was specified at the inlet of the flow channel 29. In terms of effect, this corresponds to a fixed water column or a pressure-controlled pump. The first chamber 27 is designed upstream of the first transition region 41 in such a way that there is a comparatively low flow velocity based on the flow velocity in the gap in order to avoid the formation of a highly profiled velocity distribution in the inflow. The simulation data indicates that the fluid in the first chamber 27 sinks laminarly to the floor and only experiences an acceleration in the first transition region 41. The flow velocity over the length of the flow channel 29 is essentially constant.

In order to generate this well-defined and uniform liquid shear stress required for carrying out the experiments, the first transition region 41 from the first chamber 27 into the flow channel 29 is designed so that the Fluid flow can follow the wall contour or sink evenly along it without causing local speed increases or flow separations and the associated recirculation areas in the immediate vicinity of the inlet into the flow channel 29. The rapid change in cross-section results in a rapid acceleration of the fluid, whereby the formation of a boundary layer in front of the inlet into the flow channel 29 is suppressed. In the flow channel itself, the desired wall shear stress and the associated shear force on the cells to be examined are set via the flow velocity or, for a given cross-sectional area, via the mass flow.

wobei m = Massenstrom [kg/s], ρ = Dichte des Fluids [kg/m³], t = Kanaltiefe [m], h = Kanalhöhe [m], µ = dynamische Viskosität [Pa s] und τ_W = Wandschubspannung [N/m²]. Die angegebene Formel, welch die gewünschte Wandschubspannung in Relation zum Massenstrom setzt, ist das Resultat einer analytischen 2D-Betrachtung des strömungsmechanischen Problems. Durch Vergleich mit den Ergebnissen der Strömungssimulationen auf Basis eines dreidimensionalen Kanalmodells hat sich gezeigt, dass die zweidimensionale Abschätzung sehr gut die Simulationsdaten wiedergibt. Dieser Aspekt wird weiter unten mit Bezug auf 6 genauer erläutert.The mass flow to be set can be estimated/determined depending on the desired shear stress as follows: $$\dot{m}=\frac{\rho t{H}^2{\tau }_W}{6\mu }$$

where m = mass flow [kg/s], ρ = density of the fluid [kg/m ³ ], t = channel depth [m], h = channel height [m], µ = dynamic viscosity [Pa s] and τ _W = wall shear stress [ N/m ² ]. The formula given, which relates the desired wall shear stress to the mass flow, is the result of an analytical 2D consideration of the fluid mechanics problem. Comparison with the results of flow simulations based on a three-dimensional channel model showed that the two-dimensional estimation reproduces the simulation data very well. This aspect will be discussed further below with reference to 6 explained in more detail.

mit dem hydraulischen Durchmesser $D_h=\frac{2ht}{h+t}$

und der mittleren Strömungsgeschwindigkeit $\overline{u}=\frac{{\tau }_{W}h}{6\mu }.$

The maximum flow velocity for a laminar flow, which at the same time advantageously results in a constant wall shear stress, is limited by the critical Reynolds number (Re _max ≈2000) and depends on the geometry of the gap, the density and the viscosity of the flowing fluid. The Reynolds number is calculated as: $Re=\frac{\rho \overline{u}{D}_H}{\mu }$

with the hydraulic diameter $D_H=\frac{2Ht}{H+t}$

and the average flow velocity $\overline{u}=\frac{{\tau }_{W}H}{6\mu }.$

A high ratio of channel depth to channel height, preferably in the range of 20 or more, ensures that the influence of the boundary layer on the side walls of the flow channel 29 is low and the shear stress on almost the entire area (≥90%) of the flow channel 29 gap is constant. In 5 is the one wall shear stress distribution 5 on or along the floor within an exemplary “flat” flow channel 29 shown, the associated color scaling being indicated in the subdiagram 50. The flow channel 29 on which the data is based has a length of 43 mm, a width of 10 mm and a height of 0.5 mm. The boundary conditions of the simulation, for example the specification of a pressure boundary condition at the inlet of the flow channel, which causes a mass flow, were determined on the basis of an analytical two-dimensional observation of the flow channel 29. It can be seen that, on the one hand, the wall shear distribution within the flow channel 29, apart from the entrance area of the flow channel 29, is constant over its entire length and that a reduction in the wall shear stress in the range of 5% only occurs on the side walls of the flow channel 29 boundary layer that forms there. The arrow f indicates the fluid flow direction.

6 shows a graphical evaluation of the deviation of the wall shear stress distribution calculated based on a three-dimensional simulation of the flow channel from a corresponding wall shear stress analytically calculated according to the formula given above in a simplified two-dimensional channel (with the same width and length and virtually infinite depth). The first deviation distribution 61 corresponds to the deviation on or along the ceiling of the flow channel 29 and the second deviation distribution 62 corresponds to the deviation on or along the bottom of the flow channel 29, whereby the arrow f also indicates the fluid flow direction. The associated color scaling is indicated in subdiagram 60.

In the 6 The deviation evaluation shown suggests that the wall shear stress calculated using CFD simulation based on a three-dimensional model of the flow channel 29 deviates only slightly from the two-dimensional analytical design described above. Like the two in 6 can be seen from the deviation distributions 61, 62 shown, the displacement of the flow through the boundary layer on the sides of the flow channel 29 leads to a minimally increased average flow velocity in the channel and thus to an approximately 3% excessive wall shear stress. If necessary, this effect can be counteracted by a corresponding reduction in the mass flow. Furthermore, one can see from the deviation distributions 61, 62 that within the flow channel 29 the deviation is provided with a relatively small (approx. 3%) error, which, apart from the inlet and outlet of the flow channel 29, is constant over its length.

The cultivation insert and flow insert according to the invention presented here can be used as part of the kit also described with a variety of “basic vessels”, mostly in the form of culture dishes. They can be used in parallel in Petri dishes of different geometries and sizes as well as in “multi-well dishes” (e.g. 6-well dishes).

This allows several flow experiments to be carried out at the same time and time-lapse images to be created with an automated and motorized microscope. The simple and quick exchange of the inserts also allows the lege artis extraction of cells to obtain DNA and RNA as well as (depending on the size of the culture dish) for protein biochemical studies (e.g. Western Blots, IP). Cells such as: B. leukocytes or tumor cells, can be injected through an access.

• 1) Die Zellen werden unter Standardkulturbedingungen gezüchtet und sind keinen Gradienten von Gasen, Nährstoffen und Metaboliten ausgesetzt. Dadurch kann eine gute homogene Zellqualität über den gesamten angezüchteten Zellrasen erreicht werden.
• 2) Die Einsätze sind bevorzugt autoklavierbar und relativ kostengünstig herstellbar. Damit sind die Einsätze zugleich wiederverwendbar, was ihren Einsatz umweltfreundlich und kostengünstig macht.
• 3) Das Einsetzten und die Entnahme der Einsätze wird durch einfache und sterile Werkzeuge vorgenommen, so dass Kontaminationen weitgehend ausgeschlossen sind. Bei Bedarf kann auf beiden Einsetzen mindestens eine Markierung vorgesehen sein, beispielsweise eine kleine Materialaussparung, etwa eine Kerbe, welche mit jeweils einer entsprechenden auf der Kulturschale angebrachten Markierung, z.B. ebenfalls einer Materialaussparung oder einer Farbmarkierung, in Überlapp gebracht werden. Dadurch kann eine genaue Ausrichtung des Strömungskanals in dem Strömungseinsatz relativ zum ausgebildeten Zellrasen erreicht werden.
• 5) Es können nach Bedarf durch eine einfache Skalierungsoperation Kultivierungs- und Strömungseinsätze unterschiedlicher Größe (nach Bedarf) hergestellt werden.
• 6) Der Einsatz kommerziell erhältlicher Zellkulturschalen macht die Verwendung der Einsätze ebenfalls kostengünstig.

Overall, the following advantages can be mentioned in connection with the invention described herein:

• 1) Cells are grown under standard culture conditions and are not exposed to gradients of gases, nutrients and metabolites. This allows good, homogeneous cell quality to be achieved across the entire cultured cell layer.
• 2) The inserts are preferably autoclavable and can be produced relatively inexpensively. This means that the inserts are also reusable, which makes their use environmentally friendly and cost-effective.
• 3) The inserts are inserted and removed using simple and sterile tools, so that contamination is largely excluded. If necessary, at least one marking can be provided on both inserts, for example a small material recess, such as a notch, which is brought into overlap with a corresponding marking made on the culture dish, for example also a material recess or a color marking. As a result, a precise alignment of the flow channel in the flow insert relative to the formed cell layer can be achieved.
• 5) Cultivation and flow inserts of different sizes (as needed) can be manufactured as needed through a simple scaling operation.
• 6) The use of commercially available cell culture dishes also makes the use of the inserts cost-effective.

Claims (15)

Cultivation insert for a culture dish, comprising: a base body which has a bottom and a top, a through opening running through the base body and extending between the bottom and the top; wherein a first edge of the through opening, which is arranged on the underside, defines a cultivation surface and is flat, so that when the cultivation insert is placed on any flat surface, the first edge lies fully against this surface. Cultivation use according to Claim 1 , wherein a second edge of the through opening, which is arranged on the top of the base body, describes a second surface; and wherein the second area is larger than or equal to the cultivation area; wherein the cultivation area is further preferably arranged within the second area in the top view of the cultivation insert. Cultivation use according to Claim 1 or 2 , wherein the first edge of the through opening acts as a seal, so that after inserting the cultivation insert into the corresponding culture dish, the cultivation surface exposed towards the top is laterally sealed. Cultivation use according to one of the Claims 1 until 3 , wherein the base body has a biochemically inert material, which is also preferably autoclavable; wherein preferably after inserting the cultivation insert into the culture dish, the area of the cultivation dish enclosed by the first edge of the through opening corresponds to the cultivation area. Flow insert for a culture dish, comprising: a base body which has a bottom and a top; a substantially cuboid-shaped flow channel, which is formed in the base body and has an inlet at one end and an outlet at the other end, the flow channel having a ceiling and two side walls and being open towards the underside, the open underside of the flow channel being base area corresponds; a first chamber having an inlet and an outlet, the outlet being fluid-mechanically coupled to the inlet of the flow channel; a second chamber, which has an inlet and a Has outlet, wherein the inlet is fluid-mechanically coupled to the outlet of the flow channel; wherein at the transition between the outlet of the first chamber and the inlet of the flow channel, a side wall of the first chamber continuously merges into the ceiling of the flow channel without kinks. Flow application according to Claim 5 , wherein the base area of the first chamber is larger than, preferably at least twice as large as, the maximum cross section of the transition between the outlet of the first chamber and the inlet of the flow channel; wherein the first chamber is open to the underside of the base body. Flow application according to Claim 5 or 6 , wherein the first chamber has a floor; and wherein a lower edge of the outlet of the first chamber is arranged at the level of the lower edge at the inlet of the flow channel. Flow use according to one of the Claims 5 until 7 , where in the cross section along the direction of flow through the channel the ratio of channel width to channel height is at least twenty. Flow use according to one of the Claims 5 until 8th , wherein the inlet of the first chamber is arranged above the outlet of the first chamber; furthermore, preferably after inserting the flow insert into the culture dish, the bottom of the flow channel corresponds to a corresponding area of the surface of the culture dish, against which the underside of the base body rests. Flow use according to one of the Claims 5 until 9 , wherein the transition between the outlet of the flow channel and the inlet of the second chamber has a structure that is symmetrical to the transition between the outlet of the first chamber and the inlet of the flow channel; wherein further preferably the width of the first chamber corresponds to the width of the flow channel and the width of the second chamber corresponds to the width of the flow channel. Kit for studying cell dynamics under fluid shear stresses, comprising: a culture dish; a cultivation use according to one of the Claims 1 until 4 , a flow insert according to one of the Claims 5 until 10 , whereby the cultivation area essentially corresponds to the base area of the flow channel. Kit according to Claim 11 , wherein the base body of the cultivation insert and the base body of the flow insert are dimensioned such that they can be inserted into the culture dish. Kit according to Claim 12 , further comprising: a stabilization dish, which is set up to hold the culture dish and is dimensioned such that after the culture dish has been inserted into the stabilization dish, it rests closely on the floor and at least on a part of the side wall of the culture dish; wherein the stabilization dish preferably has at least one opening in the base, the size and position of which is selected such that the opening can be arranged under the cultivation area, if necessary by rotating the stabilization dish relative to the culture dish; wherein the stabilization shell is also preferably made of a rigid material, preferably stainless steel. Method for studying cell dynamics under fluid shear stresses using the kit according to one of Claims 11 until 13 , comprising: providing a culture dish; Inserting the cultivation insert into the culture dish; Growing cells at the bottom of the culture dish in the area of the cultivation area; removing the cultivation insert; Inserting the flow insert into the culture dish in such a way that the base area of the flow channel is positioned above the cultivation area occupied by the grown cells; Forming a liquid flow through the flow channel by introducing a liquid into the first chamber. Procedure according to Claim 14 , further comprising: observing the cell dynamics in the flow channel using a microscope through the opening in the bottom of the stabilization shell; wherein the method further optionally comprises placing the culture dish in the stabilization dish before or after inserting the flow insert into the culture dish.