EP3794101A1

EP3794101A1 - Method for producing a cell culture insert having at least one membrane

Info

Publication number: EP3794101A1
Application number: EP19728879.8A
Authority: EP
Inventors: Lutz KLOKE; Alexander Thomas; Tobias GRIX; Benjamin NOICHL; Anna KREUDER
Original assignee: Cellbricks GmbH
Current assignee: Cellbricks GmbH
Priority date: 2018-05-16
Filing date: 2019-05-13
Publication date: 2021-03-24
Also published as: CN112424331A; US20210107212A1; DE102018111751A1; WO2019219605A1

Abstract

The present invention relates to a method for producing a cell culture insert having at least one membrane, in particular at least one biological membrane, comprising the following steps: providing at least one insert blank having at least one opening; and forming the membrane in the insert blank by means of a bioprinting method.

Description

Method for producing a cell culture insert with at least one membrane

The present invention relates to a method for producing a cell culture insert with at least one membrane, in particular a biological membrane, and a cell culture insert produced by this method.

description

Large scale cell culture assays and assays are performed by default in multiwell plates. For the cultivation of single-layered cell lawns (so-called monolayers) in such multi-well plates suitable inserts have been developed, which are generally referred to as cell culture use.

Cell culture inserts are practical, well-drained, or semipermeable support devices with ease of use for examining both anchorage-dependent (anchored) and anchorage-independent (non-adherent) cell lines. They are designed to create a cell culture environment that is more physiological than alternative two-dimensional culture systems.

According to the standardized sizes for multiwell plates, the diameters of the cell culture inserts are adjusted accordingly so that they are positioned in a multiwell in a suspended position.

The crucial part of the cell culture insert is a membrane on the bottom / bottom of the cell culture insert. This membrane can have a different porosity and consists of an inert plastic. On this membrane, cells are sown in everyday laboratory life. The seeded cells normally grow to a confluent monolayer. Following this, barrier or penetration assays can be performed.

In a conventional cell culture insert, when the plastic membrane is removed and a biopolymer is introduced, a meniscus is formed due to interfacial tension. When the polymer is cured, a cell culture insert with a concave membrane is formed. This membrane can not or only poorly colonize with cells, as they are pulled by the combination of gravity / meniscus in the middle of the cell culture insert. In this way, a cell agglomeration arises in the middle of the cell culture insert and no confluent monolayer. It is an object of the present invention to provide a cell culture insert which has a substantially flat and flat biological membrane without cone.

This object is achieved by a method having the features of claim 1 and a cell culture insert produced by this method having the features of claim 16.

Accordingly, a method is provided for producing a cell culture insert with at least one membrane, in particular at least one biological membrane, comprising the following steps:

- providing at least one hollow insert blank with at least one opening; and

- Forming the membrane in the insert blank by means of a Biodruckverfahrens.

According to a variant of the invention, the method comprises the following steps:

Providing at least one hollow insert blank with at least one opening,

Introducing the starting materials for producing the membrane into the insert blank,

Forming the membrane in a bio-printing process (e.g., by irradiation), and

- Removing the insert blank, wherein the formed membrane remains in the insert blank.

The introduction of the starting materials for membrane production in the insert blank, can be done in various ways. Thus, the insert blank can be introduced into a container containing the starting materials for membrane production. In this case, the insert blank is immersed in the starting material-containing liquid. But it is also possible to introduce the starting material-containing liquid in the insert blank (eg dropping). In this case, it is necessary to prevent the liquid from flowing out of the interior of the insert blank by suitable covering or sealing of the openings of the insert blank.

Accordingly, the method according to a further variant of the invention comprises the following steps:

Providing at least one hollow insert blank with at least one opening,

Introducing the insert blank into a container containing the starting materials for producing the membrane,

Forming the membrane in a bio-printing process (e.g., by irradiation), and

- Removing the insert blank from the container, wherein the formed membrane remains in the insert blank.

According to a further variant of the invention, the present method comprises the following steps:

Providing at least one hollow insert blank,

Covering an opening of the insert blank and introducing the starting materials for producing the membrane into the insert blank through the other opening of the insert blank,

Forming the membrane in a bio-printing process (e.g., by irradiation), and

- Removing the cover of the one opening, wherein the formed membrane remains in the insert blank. The insert blank used in this process is available in this variant as a separate, already prefabricated blank. The insert blank is preferably formed circular with a lower and an upper opening.

In a preferred embodiment, the method comprises the following steps:

- providing at least one circular, hollow insert blank having a lower and an upper opening;

- Imports and arranging at least one circular spacer in the insert blank at a predetermined distance h _m to the lower opening of the insert blank, wherein the distance h _{m of} the circular spacer from the lower opening of the insert blank by the thickness of the (inside) insert blank ( it) is determined to be generated membrane;

- introducing the starting materials for producing the membrane in the insert blank; e.g. by immersing the insert blank provided with at least one spacer into a container containing the starting materials for producing the membrane, or by introducing (dropping) the starting materials directly into the insert blank provided with at least one spacer;

Forming the membrane (e.g., by irradiation), and

- Removing the spacer from the insert blank, wherein the formed membrane remains in the insert blank.

Thus, there is provided a method by which a cone-free, planar biological membrane can be created in a culture insert using printer technology. This membrane is printed in an "empty" cell culture insert. For this purpose, cell culture insert blanks are provided, which can be finished ready by pressing the membrane after the biological membrane carrier. Upon completion, the culture insert is completed in place of a plastic membrane with a biopolymer membrane.

The advantage of a biopolymer is better growth and customization of the membrane to the cells to be sown, as not every cell type is on a plastic membrane cultivates and requires a very precise coordination of its extracellular environment. Amongst others, all biopolymers of the so-called extracellular matrix are used as polymers, including collagen, hyaluronic acid, gelatin, etc.

The culture inserts that can be produced by the present method have a number of improvements over conventional cell culture inserts:

There is provided a biological membrane consisting of one or more biopolymers. This provides the cells with a more physiological culture environment. Results obtained from biological experiments are much more meaningful than those that can be generated with conventional petri dishes or cell culture inserts.

The membrane can be modified because the physicochemical properties can be influenced by the composition of the matrix.

The membrane may e.g. be generated optically transparent, allowing a non-invasive control of the cell population in real time.

Detection substances can be coupled to the matrix to detect different phenomena during the experiment.

In addition, different architectures can be created on the matrix. In addition to a completely flat surface, different compartments, channels etc. can be created. Furthermore, z. B. conductive elements are printed in the membrane to give electrical stimuli or record electrical signals from cells.

Cells, bacteria, viruses and plant germs can already be introduced into the biomembrane.

It can be worked with more than one type of material in a plane to z. B to create areas with an altered matrix composition.

The membrane can consist of several printing layers, so that a different membrane composition can be produced not only in the X-Y plane but also in the Z plane.

The height of the membrane can be determined by the method.

In one embodiment of the present method, the insert blank used is designed in the shape of a hollow cylinder or a truncated cone.

In the case of a hollow, frusto-conical insert, the truncated cone has a diameter decreasing in the direction of the lower opening (conical taper), a base area G (upper opening) with radius R, a cover area D (lower opening) with radius r and a height h _k on. The material of the insert blank is a cell and biocompatible polymer, but especially PP, PE, PLA, PS, PC, PTFE, PVC, PMMA, PAA, PAN, PEG, PET, PU, silicones, etc. Orient the typical dimensions of the insert blank the height and width of normal multiwell plates. Table 1 gives the typical dimensions for conventionally used multiwell plates.

Table 1

The dimensions of the present cell culture insert are as follows (see Table 2):

Table 2

The dimensions of the present cultural inserts are not subject to exact classification, since the dimensions of individual multiwell plates vary from manufacturer to manufacturer. Therefore, in the present case, a mean value for the cultural inserts is given. Standard blanks or individually manufactured blanks can be used. The individual production allows an adaptation of the insert to its function. Its size and shape can be varied. This can be z. B. affect the suspension on the cell culture insert or the shape of the foot. Thus, the cell culture insert may be provided at its upper side or at its upper end with an outwardly directed projection, so that the cell culture insert can be cultivated hanging. In addition, the cell culture insert at its lower side or at its lower end (ie, the part of the cell culture insert that touches the bottom of the multiwell plate) may be provided with outwardly directed and angled projections (feet). This allows an automatic standing of the cell culture insert in the multiwell plate.

In addition, for example, recesses or grooves in the plane of the biomatrix can also be generated in order to achieve a better adhesion to the cell culture insert. Furthermore, conductive elements can also be introduced into the cell culture insert in order to obtain a signal derivation or a sensor system.

Furthermore, the edge of the insert blank may be formed continuously, so that a media exchange is only possible via the membrane itself.

But it is also possible to cut the edge. The cutout thus formed may have an arbitrarily adjustable height, e.g. the section may be below the filling level of the cell medium present in the multiwell plate. Accordingly, a supply of the interior of the culture insert and a flow through the membrane carrier is possible over the neckline. In the latter case, there is correspondingly no barrier function of the blank between the outer multiwell plate and the interior of the blank. Such a carrier serves pure culture and not a barrier function.

In a further embodiment of the present method, the spacer to be arranged in the insert blank is provided with at least one opening which allows a discharge of gas bubbles from the subsequently produced membrane and thus avoids accumulation of air bubbles in the membrane. The opening should be provided as possible at the edge of the spacer.

This opening can also be described as a "bubble trap". The Bubble Trap is a cutout in the spacer or a released spot in the below described Auxiliary membrane, which ensures an outflow of air. The air bubble which arises during insertion of the insert blank into the membrane polymer solution between spacers (or auxiliary membrane) can escape in this way. If the air remains under the spacer or the auxiliary membrane, this would be incorporated as air inclusion in the matrix, or the matrix would not have a flat surface at this point. In order to ensure a flat settlement area, inclusion of air must be prevented. The bubble trap should ideally be easily filled with membrane polymer solution to ensure that all air is displaced below the spacer or auxiliary membrane.

In a further embodiment of the present method, the at least one spacer is designed in the form of a stamp. Such a stamp is suitable for frustoconical or hollow cylindrical insert blanks. The height or length of the punch indicates the desired distance h _m to the lower opening of the insert blank and thus the thickness of the later membrane. It is advantageous if the stamp is coated on the outer surface in order to prevent adhesion of the membrane, in particular the biomembrane, during removal of the stamp.

The material of the stamp is made of a non-stick polymer. Non-adhesion in this case means adhesion between the placeholder / stamp and biomembrane during and after printing. The material of the stamp may in particular be PTFE, PEG or silicone.

The use of punches allows batch production of cell culture inserts with membranes.

In one embodiment variant, the method comprises the following steps:

Providing a single stamp or an array of multiple stamps, the stamp or stamps being vertically oriented;

Assigning at least one insert blank to each stamp, the stamp being arranged centrally in the respective insert blank;

Arranging a fixing mechanism to connect the insert blank to the respective punch;

- introducing the starting materials for membrane production, preferably a liquid containing the starting materials, via the free opening in the insert blank;

Covering the free openings of the insert blanks, preferably with a non-stick film (eg PDMS, FEP, PTFE), - Forming a polymer membrane by irradiation (irradiation preferably from above through the film);

- Remove the cover and the attachment mechanism, and

- Remove the stamp or the stamping array.

When assigning insert blank and punch, the insert blank may be e.g. put over the stamp or the stamp is inserted into the insert blank. The attachment mechanism for connecting the insert blank to the respective punch may be a clamping mechanism (eg made of springs) which effects a leak-tight seal from one of the openings of the insert blank, so that the subsequently introduced (liquid) starting materials for membrane production remain in the insert blank and do not leak ,

It is also possible to change the order of the process steps in the batch process. So the process can be done in the following order:

Providing a backing, in particular a film (e.g., PDMS film);

- Provide an aliquot of starting materials for membrane production at the point on the base, which is provided in each case for a blank insert (using a pipette or multipipette)

- placing a blank insert around the respective aliquot of starting materials for membrane production on the substrate;

- placing a fastening mechanism to secure the insert blank to the base;

Inserting a single stamp or an array of multiple stamps into the respective blank insert, the stamp or stamps being vertically oriented;

- Forming a polymer membrane by irradiation (preferably from below through the

Foil);

- Removing the stamp or stamping arrays from the insert blank; and

- Removing the insert blank with the polymer membrane formed therein, wherein the formed membrane remains in the insert blanks. In another embodiment, the at least one spacer is formed as a disc.

The material of the disc is according to the material of the stamp. The disc is made of PTFE, PEG or silicone. The diameter of the disc corresponds to the usual cell culture insert dimensions. The thickness of the disc corresponds to the desired membrane thickness between 10 gm and 1000 gm, preferably between 50 pm and 1000 pm, more preferably between 200 and 800 pm, even more preferably between 300 and 500 pm.

Such a disc is particularly suitable for frusto-conical insert blanks with decreasing diameter to the lower opening. Preferably, the disc bears against the inner wall of the insert blank. The diameter of the disc is individually adjustable so that the distance of the disc from the lower opening of the insert blank is determined by the diameter of the disc; that is, the closer the disk diameter is to the radius r of the lower opening, the smaller the distance h _m between the disk and the open top surface, wherein the distance h _m defines the thickness (or height) of the membrane to be produced.

In a further variant of the present method, the spacer, in particular a disk-shaped spacer, is used in combination with an auxiliary membrane.

Here, the auxiliary membrane is preferably on the top of the spacer (ie the pointing in the direction of the upper opening side of the spacer), in particular the disc-shaped spacer, by entries of a liquid composition containing starting materials suitable for forming the auxiliary membrane and subsequent curing (eg by irradiation with Light or a similar physicochemical process).

The auxiliary membrane is made of a material which can be dissolved by a physico-chemical or enzymatic process, in particular PEG, poloxamer, hyaluronic acid, chitosan, chitin, collagen etc.

The auxiliary membrane is produced by means of a printer in the insert blank. In this case, the insert blank is introduced into a printer, for example the Bioprinter from Cellbricks, and the auxiliary membrane is produced by stereolithography. Ie. the blank is placed in a liquid polymer and cured by irradiation from above and below the bed. In addition, the auxiliary membrane can be generated by means of a disk as a spacer. In this case, the insert blank is immersed in a carrier with a liquid auxiliary matrix, in the bottom of the spacer, eg disc is located. The diameter of the disc fills the interior of the blank flush. In this state, when the liquid matrix inside the insert blank is cured by irradiation from above or below the bed, a solid auxiliary membrane is formed in the insert blank. If the insert blank with auxiliary membrane is pulled out of the carrier with the liquid uncured auxiliary matrix, the spacer remains in the vessel with the liquid auxiliary matrix. In the insert blank with the auxiliary membrane, this creates a free space between the auxiliary matrix and the end of the insert blank, which in a next step can be lined with another membrane. The auxiliary membrane can be cured so that a structure is formed on the underside of the auxiliary membrane, which acts as a negative for shaping the membrane to be created subsequently.

After the curing of the auxiliary membrane, the spacer, in particular the disc-shaped spacer, is removed so that a stable auxiliary membrane / auxiliary layer remains in the insert blank. This auxiliary membrane contains an opening for the discharge of gas bubbles (see also "Bubble Trap" as described above).

Subsequently, the insert blank provided with the at least one auxiliary membrane can be introduced into a container which contains the starting materials for producing the membrane. The membrane is e.g. by irradiation (see also below), and after formation of the membrane, the auxiliary membrane is removed from the insert blank by suitable physicochemical methods (e.g., dissolution), leaving the formed (flat) membrane in the insert blank.

The auxiliary membrane is completely flat, since this layer was previously prepared by means of the spacer. In addition, the auxiliary membrane contains an incision that acts as a bubble trap. Potential air bubbles that form upon introduction of the biopolymer may flow out through this vent and will not be captured and incorporated into the biopolymer upon cure. After filling the biopolymer this is cured. Following the auxiliary membrane can be resolved by means of a physicochemical reaction, z. As temperature increase / decrease, pH change, resolution, etc., so that after completion of the resolution only a planar biopolymer membrane (apart from a small web due to the bubble trap) remains. As already mentioned several times, the membrane and here preferably the biological membrane after immersion of the culture insert with spacers in a container containing the starting materials for the production of the membrane is formed. The starting materials are in particular photopolymerizable substances.

The formation of the membrane is carried out using a bio-pressure method, e.g. in WO 2016/075103 A1.

In this case, after introduction of the culture insert with spacers into the container or the reaction vessel with the photopolymerizable liquid, a light radiation is focused on a first focal plane lying within a region of the reaction vessel filled with the liquid. By this light radiation, a polymerized structure is then produced in the reaction vessel. The polymerized structure is in a first layer.

Any additional layers can be applied to this first layer. For this purpose, a further photopolymerizable liquid is introduced into the reaction vessel, wherein the previously produced polymerized structure is at least partially covered with the further photopolymerizable liquid. Preferably, the previously produced polymerized structure is completely covered with the further photopolymerizable liquid. Now, a further light irradiation is carried out in a further focal plane, which lies within a region of the reaction vessel filled with the further liquid. This further focal plane thus differs from the first focal plane at least with respect to the already produced polymerized structure or with respect to the layer of this polymerized structure.

The abovementioned steps of introducing a further photopolymerizable liquid, focusing light onto a further focal plane and producing a further polymerized structure in another layer can now be repeated as desired with in each case further photopolymerizable liquids until the desired membrane has been produced.

The layer thickness of the membrane to be produced can be set as desired and adapted to the corresponding requirements. Thus, the layer thickness of the membrane between 10 gm and 1000 gm, preferably between 50 and 1000 pm, particularly preferred between 200 and 800 gm, more preferably between 300 and 500 gm, eg between 310 and 325 gm.

The photopolymerizable liquid may also contain biological cells or other substances. When polymerisation occurs as a result of the light irradiation, the cells contained in the liquid are embedded in a corresponding polymer.

With this printing process membranes of any polymers, preferably biopolymers, can be produced.

Accordingly, the membrane may be constructed of the following materials: engineering biopolymers such as gelatin; Alpha and beta polysaccharides such as pectins, chitin, callose and cellulose; Lipids, in particular membrane-forming lipids, such as phospholipids, sphingolipids, glycolipids and ether lipids; polyhydroxyalkanoates; bio-based polymers such as polylactide, polyhydroxybutyrate; petroleum-based polymers, such as polyesters, in particular polyethylene glycol, polyvinyl alcohol, polybutylene adipate terephthalate (PBAT, polybutylene succinate (PBS), polycaprolactones (PCL), polyglycolide (PGA), synthetic peptides, such as recombinantly produced amino acids, amides, components of the extracellular matrix, such as collagens, Fibrillin, elastin, glycosaminoglycans, especially hyaluronic acids, heparan sulfate, dermatan sulfate, chondroitin sulfate, and keratan sulfate Preferred membrane materials are collagens, hyaluronic acid, chitosan, gelatin, PLA, PEG, and combinations thereof.

In one example, the membrane consists of gelatin and collagen as an additive. For the preparation, the starting materials are a suitable photoinitiator, e.g. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) used.

In a further embodiment, a securing means for holding the membrane in use is provided. It is preferred if the at least one securing means in the form of a carrier consisting of different structures, which are adapted to the membrane material, in particular of lattice-shaped or spoke-like structures, is formed.

In a further variant of the method, the cell culture insert is made of insert blank and membrane in one piece using the printing method described above trained (one-pot synthesis). In analogy to the method described in WO 2016/075103 A1, the one-pot synthesis takes place additively with photopolymerizable materials. First, the base of the scaffold is printed from a first (cell repellent) biomaterial. Subsequently, a biocompatible layer of a second biomaterial is fabricated. In the last step, the scaffold is completed by the edges. Both materials are processed in a single printing process.

The insert blank formed thereby comprises an (upper) opening and may comprise a bottom. The insert blank is preferably made of a first cell repellent biomaterial or biopolymer, e.g. Polyethylene glycol (PEG) or one of the other possible for the insert blank above possible polymer materials prepared. At the bottom of the insert blank is another biocompatible and biofunctional biopolymer as a membrane, e.g. made of gelatin on which cell materials can grow. The combination of the two different materials creates a scaffold that can be selectively populated with cells on the biocompatible material.

In the course of a longer cultivation time, there may be a change in the biomembrane, z. B. shrinkage or change by cells. The securing means, e.g. Grid, in this case prevents slipping out or falling out of the biomembrane from the culture insert. In its simplest embodiment, the securing means may be applied to the underside of the cell culture insert, i. the side carrying the membrane can be placed. The mounting of the securing means, e.g. a grid, based on a simple press fit. In order subsequently to remove the securing agent (and also the membrane) from the culture insert, the press fit can be removed, for example. be removed again with a kind of bottle opener. Here, the press fit is simply levered.

As already indicated above, it is possible to produce a biological membrane in a blank of two or more polymeric materials, preferably biopolymers. The materials can be arranged in different architectures. Thus, the different polymer materials can be arranged one above the other, in layers, wherein the number of layers is freely selectable. In one variant, a layer of a first polymer and a layer of a second polymer are provided in each case. It is also conceivable that the polymer materials are arranged in a layer next to each other. Thus, in one variant, a membrane section made of a first polymer material may be provided, wherein the membrane section is integrated into the second polymer material (in the middle), ie Membrane portion of the first polymer material is surrounded by the second polymer material.

In a further embodiment, a three-dimensional architecture of the membrane is provided. For this, the membrane can be printed using a geometric shape. Thus, for example, villi, canals, hills, valleys, etc., - can be introduced - also from different materials. Thus, the membrane surface to be populated can be formed with regular or irregular structures (hills, valleys).

In one embodiment, at least one channel can be introduced into the membrane. The channel may e.g. be used to supply the inner compartment of the blank. The channel acts as a medium carrier with nutrient medium, which is flushed through the channel. Cells that are located inside the compartment on the membrane can be supplied from the channels through the membrane.

It is also possible to introduce functional material into the membrane. For example, an additional detector, dye, enzyme, chemokine, nanoparticles or the like can be integrated into the membrane during the printing process. Over time, this material can be used to monitor the cell culture online. For example, cell death can be detected by a fluorescent dye, or the current oxygen saturation or pH. The functional material may or may not be in contact with the inner and outer boundary layers. The functionalization can be observed by a color change, irradiation or other measurement to be detected. The functional material can be inserted point by point into the membrane material, or can also be provided in the membrane in a layered or layered manner.

It is also possible and conceivable to use the membrane surface e.g. divide into segments, imprint cell arrays, or introduce gradients. This makes it possible to structure the membrane in its thickness and surface exactly.

Furthermore, the optical transparency can be influenced by the formulation of the biomatrix. It can, for. As a transparent matrix can be generated, which allows an optical control of the cell population. Thus, the transparency allows a non-invasive control of the cell population, which is not possible with a conventional cell culture insert. In addition, a (non-polymeric) further material defined in the membrane and / or the blank can be introduced. In this way, for example, even more functions can be realized by introducing, for example, sensors, detection, conductive materials, chemicals, nanoparticles, etc.

Thus, in one embodiment, the blank is provided with at least one probe. The at least one probe, preferably two probes, can each be provided on the inside and the outside of the blank. Through the use of probes, the electrical resistance can be determined, whereby conclusions can be made about the density of the membrane and the cell lawn.

With the present method now a cell culture insert can be produced, which consists of a blank insert with at least one membrane arranged therein, in particular at least one biomembrane, wherein the at least one membrane is flat and has no cone; i.e. A cell culture insert with a confluent monolayer can be provided by this method.

The present cell culture insert can be used for culturing various cell lines and then performing barrier or penetration assays.

As a goal lines all cells and cell lines are suitable, which represent a barrier function, eg. Endothelial cells, trophoblasts, astrocytes, enterocytes, etc., or cells and cell lines representing metabolic function, e.g. As hepatocytes, cardiomyocytes, myocytes, etc. In general, all cell types are adherent.

The invention will be explained below with reference to several examples with reference to the figures. Show it:

FIG. 1 shows a comparison of a conventional culture insert with pores with a culture cell insert produced according to the invention;

Figure 2a shows a first embodiment of the method according to the invention;

FIG. 2b shows a second embodiment of the method according to the invention;

Figure 2c shows a third embodiment of the method according to the invention; FIG. 3 shows a fourth embodiment of the method according to the invention;

Figure 4A is a schematic representation of the fuse of the membrane produced in

Culture insert;

Figure 4B is a schematic representation of various embodiments of the

Securing means of Figure 4A;

Figure 5 shows a first embodiment of the invention produced

Cell culture insert;

FIG. 6A cell-cultivated membrane of a cell culture insert according to the invention;

FIG. 6B is a cross-sectional view of a cell-populated membrane in a cell culture insert according to the invention;

FIG. 6C micrographs of a cell-populated membrane in a cell culture insert according to the invention;

Figure 7 further embodiments of the invention produced

Cell culture insert;

FIG. 8 shows a further embodiment of the invention produced

Cell culture insert; and

9 shows a still further embodiment of the invention produced

Cell culture insert

FIG. 1 shows on the left side a) a conventional cell culture insert with pores.

The membrane formed at the bottom of the cell culture insert consists of an inert one

Plastic with adjustable porosity. On this membrane in the laboratory everyday cells are seeded, which normally grow to a monolayer and with which barrier or

Penetration assays can be performed.

On the right side b) of FIG. 1, a culture insert with a biological membrane produced according to the method according to the invention is shown. The formed biological membrane is flat and has no cone. The cone-free membrane allows a good colonization with cells.

In Figure 2a, a first embodiment of the method according to the invention is shown schematically, wherein a spacer, here in the form of a punch, is introduced into a frusto-conical cultural use. The punch is individually adjustable in height, so that after insertion of the punch in the culture insert, the distance between the punch and the lower opening of the culture insert is arbitrarily adjustable. The distance between the punch and the lower opening of the culture insert indicates the desired height or thickness of the biomembrane to be formed later. The stamper must also be coated with a suitable material to prevent adhesion of the formed biomembrane upon removal of the stamper from the culture insert.

In a second subsequent step, the culture insert with the stamp introduced therein is introduced into a reaction vessel with a polymerisable liquid, wherein the polymerisable liquid contains the starting components for the preparation of the desired biomembrane. The stamp should contain a "bubble trap" to avoid accumulation of air bubbles in the biomembrane

In the next, third step of the method, the formation of the biomembrane takes place in a printing process, wherein light is irradiated in a focal plane onto the polymerisable liquid, resulting in a polymerization to the biomembrane in the focal plane.

After hardening of the biomembrane, the stamp can be easily removed from the culture insert due to its material coating (step 4), leaving behind a planar biomembrane in culture use.

The embodiments of the present method illustrated in FIGS. 2 b and 2 c enable batch-wise membrane production.

In the embodiment of the method shown in FIG. 2b, the following steps are performed: 1) providing a single stamp or an array of several stamps, the stamp or stamps being oriented vertically; 2) associating at least one insert blank with each stamp, the stamp being centered in the respective insert blank; in the assignment of insert blank and stamp the insert blank can be put over the stamp, for example, or the stamp is in the Insert blank introduced; 3) placing a fastening mechanism (clamping mechanism eg with springs) to connect the insert blank with the respective punch; 4) introducing a quantity of pressure fluid (with the starting materials for membrane production) via the free opening in the insert blank (eg with a pipette); 5) covering the free openings of the insert blanks, preferably with a film (PDMS film), 6) forming a polymer membrane by irradiation (irradiation preferably from above through the film); 7) removing the PDMS film; 8) removing the attachment mechanism, and 9) removing the punch or stamping arrays.

In the embodiment of the method shown in Figure 2c, the order of process steps in the batch process is changed: 1) providing a film (e.g., PDMS film); 2) providing an aliquot of membrane production precursors at the location on the film, each one for a blank insert (using a pipette or multipipette); 3.) placing a blank insert around the respective aliquot of starting materials for membrane production on the foil; 4) placing a fastening mechanism to secure the insert blank to the foil; 5) inserting a single punch or an array of multiple punches into the respective insert blank, the punch or punches being vertically oriented; 6) forming a polymer membrane by irradiation (preferably from below through the film); 7) removing the punch or punching arrays from the insert blank; and 8) removing the insert blank with the polymer membrane formed therein. The insert blanks provided with the polymer membrane may then be e.g. be stored in multiwell plates. The fourth embodiment of the method according to the invention shown in FIG. 3 uses a disk instead of a stamp as a spacer. The disk is introduced into the culture insert in a first step and defines the thickness of the membrane to be formed via the distance to the lower opening of the culture insert. After the determination of the desired later membrane height by the disk as a placeholder or spacer a polymerizable liquid is introduced in a second step within the culture insert, which is then cured by light irradiation or a similar physico-chemical process to form an auxiliary membrane.

After the curing of the auxiliary membrane, the disc is removed as a spacer in a third step and leaves behind a stable auxiliary membrane having an opening as a "bubble trap". In the next, fourth step, the culture insert is introduced with the auxiliary membrane in a reaction vessel with the liquid biopolymer and then cured by irradiation in a printing process.

After curing of the biomembrane and removal of the culture insert from the reaction vessel, a double layer of biomembrane and auxiliary membrane remains in the culture insert. The auxiliary membrane is removed in a final step by a physico-chemical process or simple dissolution leaving behind a flat biomembrane.

In order to prevent the printed membrane layer from falling out, a plastic grid can be placed underneath the culture insert after the biomembrane has been fabricated (see FIG. 4A). The biomembrane is secured or secured by means of a grid, which is secured with a press fit at the foot of the culture insert. This safeguard of the biomembrane can be easily removed with a capsule lifter to ensure access to the biomembrane or their removal. The securing means may be lattice-shaped or spoke-shaped (see FIG. 4B).

An embodiment of a cell culture insert produced according to the invention is shown in FIG. Here, the cell culture insert is cylindrical. At the upper opening support webs are provided, which allow insertion and removal of the cell culture insert from a nutrient solution in a multiwell plate. At the lower opening, a grid can be seen as securing means for holding the biomembrane in the cylindrical housing.

Depending on the membrane material, different cells can settle on the biomembrane, forming monolayers (see FIGS. 6A, 6B).

The images of Figure 6A show microscopic transmitted light images of the top and bottom of a populated carrier. On the left, the underside is shown with a holder for the membrane, on the right the top of the carrier with populated surface. Caco-2 cells were used as cells.

The image of FIG. 6B shows a lateral cross section through a HUVEC cell-populated gelatin membrane, performed with a 2-photon microscope. The individual colors show the different markers investigated in this experiment: DAPI (cell nucleus), vWF and CD31 (vascular cell marker). It can be a polarization and thus a natural behavior of the cells are detected. The micrographs shown in FIG. 6C show a lateral section through the membrane. The upper side of the image represents the top of the membrane, the membrane populated with cells divides the image, and the lower half of the image represents the underside of the substrate. The individual colors indicate a polarization of the cells towards the membrane. In the case of a microscopic image, an increased collagen production of the cells towards the membrane can be shown. The cells are Vero cells. In addition, the nuclei were also stained with DAPI. With the help of the experiment a polarization and interaction of the cells should be shown. In comparison, this effect is less pronounced or not pronounced in a Petri dish

In the following two pictures the markers ITGB1 (right) and aPKC (bottom left) were examined, which also document a polarization towards or away from the membrane.

The present cell culture insert can be formed with a continuous edge (FIG. 7, a) for a barrier function or a cutout (FIG. 7, b). The cutout can be below the media level in the multiwell, so that a nutrient supply of the cells located on the membrane can take place.

The cell culture insert may be provided with two or more different materials as a membrane in a blank, which materials may be arranged in different architectures, e.g. B. side by side (Figure 7, c) or one above the other (Figure 7, d) or in other architectures. This architecture can be introduced in blanks for hanging or feet.

In another variant of the cell culture insert, the membrane is printed with a geometric shape. The membrane must not only be inserted straight into the blank, but may also have an architectural shape. Thus, for example, villi, canals, hills, valleys (FIG. 7, e) can be introduced, also from different materials (FIG. 7, f). This architecture can be placed in blanks for hanging or feet.

In the membrane of the cell culture insert, a channel can be introduced, which can be rinsed from outside the blank (Figure 7, g, h). In this case, the channel can be used to supply the inner compartment of the blank. The channel acts as a medium carrier with nutrient medium, which is flushed through the channel. Cells that are located on the membrane inside the compartment can be supplied by the membrane

Functional material can also be introduced into the membrane of the cell culture insert. For example, an additional detector, dye, enzyme, chemokine, nanoparticles or the like can be integrated into the membrane during the printing process. Over time, this material can be used to monitor the cell culture online. For example, cell death can be detected by a fluorescent dye, or the current oxygen saturation or pH. The functional material may or may not be in contact with the inner and outer boundary layers. The functionalization can be observed by a color change, irradiation or other measurement to be detected. The functional material can be introduced pointwise in the membrane (FIG. 7, i) or flat (FIG. 7, j).

The cell culture insert can also allow the measurement of the membrane density by the electrical resistance. In this case, a special blank can be used in which a probe is attached to the blank in each case inside and outside, in order to be able to determine the electrical resistance and thus conclusions about the density of the membrane and the cell lawn (FIG. 7, k).

Figure 8 shows a cell culture insert provided with outwardly directed and angled protrusions (feet) at its lower side and at its lower end (i.e., the part of the cell culture insert contacting the bottom of the multiwell plate). This allows an automatic standing of the cell culture insert in the multiwell plate.

FIG. 9 shows a further variant of the cell culture insert. In this variant of the cell culture insert two biomaterials are used which may differ in their properties. The cell culture insert consists of a bottomed cylinder (see Figure Biopolymer 2, blue). Another biopolymer (see Figure Biopolymer 1, green) is incorporated on the bottom. Material b (see figure) forms the framework that gives the construct stability. In the example, the biopolymer is based on polyethylene glycol (PEG), a biocompatible but cell repellent polymer. In material b, material a is incorporated, which in the example is based on gelatine. In contrast to PEG, gelatin is not only biocompatible, but biofunctional, so that cells grow on this material. The combination of the two materials creates a scaffold that is aligned with cells can be colonized. The area comprising biopolymer 1 (a) sets the maximum area at which cells can grow. In a colonization, the cells grow to the edge. Since the edge consists of the cell-repelling polymer 2 (b), they can not colonize the edge. Growth stops at this limit.

Example:

Cell culture inserts with a diameter of 12 mm were produced by means of the stamping process. In this case, a gelatin matrix with a concentration of 10% (w / v) was added, which was mixed with LAP as an initiator in a concentration of 0.1% (w / v). In addition, collagen I was used as an additive.

The blank was filled with a silicone stamp and placed in a basin in the printer containing the liquid gelatin matrix described above, so that the blank sits on the bottom of the basin in the printer. In order to produce a membrane of 500 pm, the distance was adjusted accordingly with the stamp before.

Subsequently, the gelatin matrix was cured by irradiation with a wavelength of 385 nm. After curing, the carrier was removed from the basin and the printer. In addition, the stamp was removed and left behind a cell culture insert with the previously defined height and with a flat surface for colonization.

Two different cell culture inserts were made. A first use with a membrane with collagen I as an additive and a second use with a membrane without this addition. The gelatin membrane was made transparent in this case and could be examined optically.

After preparation of the cell culture inserts with biological membrane they were populated with Vero cells. This is a kidney cell line of the green monkey. This cell line is often used for infection experiments. After colonization, the Vero cells formed a confluent monolayer over the entire area of the cell culture insert.

After colonization, a GFP-tagged cowpox strain was used to infect the Vero cells with the strain. The spread of infection could be due to the fluorescent viruses and the transparent cell culture insert are studied and observed over the course of the experiment of 28 days.

Following the experiment, the membrane was punched out and deep-frozen. In addition, the membrane could be cut and stained with the usual histological methods so that histological follow-up was possible.

Claims

claims

1. A method for producing a cell culture insert with at least one membrane, in particular at least one biological membrane, comprising the following steps:

- providing at least one hollow insert blank with at least one opening; and

- Forming the membrane in the insert blank by means of a Biodruckverfahrens.

2. The method of claim 1 with the following steps:

Providing at least one hollow insert blank with at least one opening,

- Forming the membrane in a Biodruckverfahren, and

3. The method according to any one of the preceding claims, characterized by the following steps:

Providing at least one hollow insert blank,

- Forming the membrane in a Biodruckverfahren and - Removing the cover of the one opening, wherein the formed membrane remains in the insert blank.

4. The method according to any one of claims 1 - 2, characterized by the following steps:

- providing at least one hollow insert blank with at least one opening;

- Forming the membrane in the insert blank by means of a Biodruckverfahrens, and

5. The method according to any one of the preceding claims with the following steps:

Inserting and arranging at least one circular spacer into the insert blank at a predetermined distance h _m from the bottom opening of the insert blank, the distance h _{m of} the circular spacer from the bottom opening of the insert blank being determined by the thickness of the membrane to be produced in the insert blank ;

Introducing the starting materials for producing the membrane into the insert blank provided with the at least one spacer,

- Forming the membrane by means of a Biodruckverfahrens, and - Removing the spacer from the insert blank, wherein the formed membrane remains in the insert blank.

6. The method according to claim 5, characterized in that the spacer is provided with at least one opening which allows a discharge of gas bubbles from the membrane.

7. The method according to claim 5 or 6, characterized in that the at least one spacer is formed in the form of a punch.

8. The method according to any one of claims 5-7, characterized in that the at least one spacer is formed as a disc.

9. The method according to any one of claims 5-8, characterized in that the spacer, in particular a disc-shaped spacer, is used in combination with an auxiliary membrane.

10. The method according to claim 9, characterized in that the auxiliary membrane on the top of the spacer, in particular the disc-shaped spacer, by entries of a liquid composition containing starting materials suitable for forming the auxiliary membrane and subsequent curing, is formed.

1 1. A method according to any one of claims 9-10, characterized in that the spacer, in particular the disc-shaped spacer, is removed after curing of the auxiliary membrane.

12. The method according to any one of claims 9-1 1, characterized in that the provided with the at least one auxiliary membrane insert blank is introduced into a container containing the starting materials for the production of the membrane, the membrane is formed, and the auxiliary membrane by suitable physiko chemical methods is removed from the insert blank, wherein the formed (flat) membrane remains in the insert blank.

13. The method according to any one of the preceding claims, characterized in that the membrane is constructed from the following materials or comprises: technical biopolymers, such as gelatin; Alpha and beta polysaccharides such as pectins, chitin, callose and cellulose; Lipids, in particular membrane-forming lipids, such as phospholipids, sphingolipids, glycolipids and ether lipids; polyhydroxyalkanoates; bio-based polymers such as polylactide, polyhydroxybutyrate; petroleum-based polymers, such as polyesters, in particular polyethylene glycol, polyvinyl alcohol, polybutylene adipate terephthalate (PBAT, polybutylene succinate (PBS), polycaprolactones (PCL), polyglycolide (PGA), synthetic peptides, such as recombinantly produced amino acids, amides, components of the extracellular matrix, such as collagens, Fibrillin, elastin, glycosaminoglycans, in particular hyaluronic acids, heparan sulfate, dermatan sulfate, chondroitin sulfate and keratan sulfate.

14. The method according to any one of the preceding claims, characterized in that a securing means for holding the membrane is provided in use.

15. The method according to claim 14, characterized in that the at least one securing means in the form of a carrier, consisting of different structures and adapted to the membrane material, in particular of latticed or spoke-like structures, is formed.

16. Cell culture insert producible in a method according to one of the preceding claims, consisting of a blank insert with at least one membrane arranged therein, in particular at least one biomembrane, wherein the at least one membrane has no cone.

17. Cell culture insert according to claim 16, characterized in that the membrane consists of two or more polymeric materials, preferably biopolymers.

18. Cell culture insert according to claim 16 or 17, characterized in that the membrane has a three-dimensional structure, preferably in the form of villi, canals, hills, valleys.

19. Cell culture insert according to one of claims 16-18, characterized in that the membrane comprises a functional material, preferably a detector, dye, enzymes, chemokines or nanoparticles.

20. Use of a cell culture insert according to any one of claims 16-18 for the cultivation of cells and performing barrier or penetration assays.