Method for the Preparation of a Cell Culture Insert with at Least One Membrane

Abstract

A method for producing a cell culture insert with at least one membrane, in particular at least one biological membrane, may include the following steps: providing at least one insert blank with at least one opening; and forming the at least one membrane in the insert blank by means of a bio-printing method.

Claims

1. A method for the preparation of a cell culture insert having at least one membrane, the method comprising the following steps: providing at least one hollow insert blank with at least one opening; and forming at least one biological membrane in the insert blank using a bio-printing method.

2. The method according to claim 1, further comprising the following steps: introducing starting materials to produce the membrane in the at least one hollow insert blank; removing the insert blank, leaving the formed at least one biological membrane in the insert blank.

3. The method according to claim 1, further comprising the following steps: covering the at least one one opening of the at least one hollow insert blank and introducing starting materials for producing the at least one biological membrane into the at least one hollow insert blank through a different opening of the at least one hollow insert blank; and removing a cover from at least one opening, leaving the formed at least one membrane in the at least one hollow insert blank.

4. The method according to claim 1, further comprising the following steps: placing the at least one hollow insert blank in a container containing starting materials for producing the at least one membrane; removing the at least one hollow insert blank from a tank, leaving the formed at least one biological membrane in the at least one hollow insert blank.

5. The method according to claim 1, further comprising the following steps: providing at least one circular, hollow insert blank with a lower and an upper opening; inserting and arranging at least one circular spacer into the at least one circular, hollow insert blank at a predetermined distance from the lower opening of the at least one circular, hollow insert blank, wherein the distance of the circular spacer from the lower opening of the at least one circular, hollow insert blank is determined by a thickness of the at least one biological membrane to be produced in the at least one circular, hollow insert blank; introducing starting materials for the production of the at least one biological membrane into the at least one circular, hollow insert blank provided with at least one spacer; and removing the at least one circular spacer from the at least one circular, hollow insert blank, leaving the formed at least one biological membrane in the at least one circular, hollow insert blank.

6. The method according to claim 5, wherein the at least one circular spacer is provided with at least one opening which allows gas bubbles to escape from the at least one biological membrane.

7. The method according to claim 5, wherein the at least one circular spacer is in the form of a stamp.

8. The method according to claim 5, wherein the at least one circular spacer is formed as a disc.

9. The method according to claim 5, wherein the at least one circular spacer is used in combination with an auxiliary membrane.

10. The method according to claim 9, wherein the auxiliary membrane is formed on an upper side of the at least one circular spacer by introducing a liquid composition containing starting materials suitable for forming the auxiliary membrane and subsequent curing.

11. The method according to claim 9, the spacer is removed after the auxiliary membrane has cured.

12. The method according to claim 9, wherein the at least one hollow insert blank provided with the auxiliary membrane is placed in a container containing the starting materials for producing the at least one biological membrane, the at least one biological membrane is formed, and the auxiliary membrane is removed from the at least one hollow insert blank by suitable physicochemical methods, the formed at least one biological membrane remaining in the at least one hollow insert blank.

13. The method according to claim 1, wherein the at least one biological membrane comprises: technical biopolymers; alpha- and beta-polysaccharides; lipids; polyhydroxyalkanoates; bio-based polymers; petroleum-based polymers; and components of the extracellular matrix.

14. The method according to the claim 1, wherein a securing means is provided for holding the at least one biological membrane in place in the at least one hollow insert blank.

15. The method according to claim 14, wherein the securing means is in the form of a carrier consisting of different structures and adapted to the at least one biological membrane material.

16. A cell culture insert producible in a method according to claim 1, comprising an insert blank with at least one membrane arranged therein, wherein the at least one membrane does not have a cone.

17. The cell culture insert according to claim 16, wherein the at least one membrane comprises of two or more polymeric materials.

18. The cell culture insert according to claim 16, wherein the at least one membrane has a three-dimensional structure.

19. The cell culture insert according to claim 16, wherein the at least one membrane comprises a functional material.

20. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] The proposed solution is explained below with reference to the FIGS. by means of several examples. It shows:

[0121] FIG. 1 shows a comparison of a conventional culture insert with pores with a culture cell insert manufactured according to the solution;

[0122] FIG. 2a shows a first embodiment of the method according to the solution;

[0123] FIG. 2b shows a second embodiment of the procedure according to the solution;

[0124] FIG. 2c a third embodiment of the method according to the solution;

[0125] FIG. 3 shows a fourth embodiment of the method according to the solution;

[0126] FIG. 4A shows a schematic illustration of how the manufactured membrane is secured in the culture insert;

[0127] FIG. 4B shows a schematic representation of the different types of the securing device in FIG. 4A;

[0128] FIG. 5 shows a first embodiment of the cell culture insert manufactured according to the solution;

[0129] FIG. 6A shows membrane of a cell culture insert cultivated with cells according to the solution;

[0130] FIG. 6B shows a cross-sectional image of a membrane populated with cells in a cell culture insert according to the solution;

[0131] FIG. 6C shows microscopic images of a membrane populated with cells in a cell culture insert according to the solution;

[0132] FIG. 7 shows a further embodiment of the cell culture insert manufactured according to the solution;

[0133] FIG. 8 shows a further embodiment of the cell culture insert manufactured according to the solution; and

[0134] FIG. 9 shows an even further embodiment of the cell culture insert manufactured according to the solution.

DESCRIPTION OF THE INVENTION

[0135] 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 plastic with adjustable porosity. Cells are seeded on this membrane in the daily laboratory routine. These cells normally grow into a monolayer and can be used for barrier or penetration assays.

[0136] On the right side b) of FIG. 1, a culture insert with a biological membrane produced according to the method according to the solution is shown. The formed biological membrane is flat and has no cone. The cone-free membrane allows a good colonization with cells.

[0137] FIG. 2a schematically illustrates a first embodiment of the procedure according to the solution, whereby a spacer, here in the form of a stamp, is inserted into a frustoconical culture insert. The height of the stamp can be adjusted individually, so that after inserting the stamp into the culture insert, the distance between the stamp and the lower opening of the culture insert can be adjusted as desired. The distance between the stamp and the lower opening of the culture insert determines the desired height or thickness of the biomembrane to be formed later. Furthermore, the stamp must be coated with a suitable material to prevent the formed biomembrane from sticking when removing the stamp from the culture insert.

[0138] In a second subsequent step, the culture insert with the stamp inserted therein is placed in a reaction vessel containing a polymerizable liquid, the polymerizable liquid containing the starting components for the production of the desired biomembrane. The stamp should contain an opening (“bubble trap”) to prevent the accumulation of air bubbles in the biomembrane.

[0139] In the next, third step of the method, the biomembrane is formed in a printing method, whereby light is irradiated onto the polymerizable liquid in a focal plane, resulting in polymerization to the biomembrane in the focal plane.

[0140] After curing of the biomembrane, the stamp can be easily removed from the culture insert due to its material coating (step 4), leaving a flat biomembrane in the culture insert.

[0141] FIGS. 2b and 2c illustrate the embodiment of this method, which allows batch membrane production.

[0142] In the embodiment of the method shown in FIG. 2b, the following steps are performed: 1) providing a single stamp or an array of multiple stamps, the stamp or stamps being vertically oriented; 2) assigning at least one insert blank to each stamp, the stamp being positioned centrally in the respective insert blank; in assigning the insert blank and stamp, the insert blank may be z. When assigning the insert blank and stamp, the insert blank can be put over the stamp or the stamp is inserted into the insert blank; 3) Arrangement of a fastening mechanism (clamping mechanism, e.g.e.g. with springs) to connect the insert blank with the respective stamp; 4) introducing a quantity of printing fluid (with the starting materials for membrane production) into the insert blank via the free opening (e.g. with a pipette); 5) covering the free openings of the insert blanks, preferably with a foil (PDMS foil), 6) forming a polymer membrane by irradiation (irradiation preferably from above through the foil); 7) removing the PDMS foil; 8) Remove the mounting mechanism, and 9) Remove the stamp or stamp array.

[0143] In the method embodiment shown in FIG. 2c, the sequence of method steps in the batch method is changed: 1) providing a film (e.g. PDMS film); 2) providing an aliquot of starting materials for membrane production at the position on the film which is intended for one feed blank at a time (using a pipette or multipipette); 3). arranging an insert blank around the respective aliquot of membrane forming starting materials on the film; 4) arranging a fastening mechanism to fasten the insert blank to the film; 5) inserting a single die or an array of multiple dies into the respective insert blank, the die or dies being vertically oriented; 6) forming a polymer membrane by irradiation (preferably from below through the film); 7) removing the stamp or stamp array from the insert blank; and 8) removing the insert blank with the polymer membrane formed therein. The insert blanks provided with the polymer membrane can then be stored e.g. in multiwell plates.

[0144] The fourth embodiment of the method shown in FIG. 3 uses a disc instead of a stamp as spacer. In a first step, the disc is inserted into the culture insert and defines the thickness of the membrane to be formed by the distance to the lower opening of the culture insert. In a second step, after the desired membrane height has been determined by the disc as a spacer, a polymerizable liquid is introduced into the culture insert. This liquid is then cured by light irradiation or a similar physicochemical method to form an auxiliary membrane.

[0145] After the auxiliary membrane has cured, the disc is removed again as a spacer in a third step, leaving behind a stable auxiliary membrane with an opening as a “bubble trap”.

[0146] In the next, fourth step, the culture insert with the auxiliary membrane is placed in a reaction vessel containing the liquid biopolymer and then cured by irradiation in a printing method.

[0147] After hardening of the biomembrane and removal of the culture insert from the reaction vessel, a bilayer of biomembrane and auxiliary membrane remains in the culture insert. In a final step, the auxiliary membrane is removed by a physicochemical method or simple dissolution, leaving behind a flat biomembrane.

[0148] To prevent the printed membrane layer from falling out, a plastic grid can be placed under the culture insert after the biomembrane has been manufactured (see FIG. 4A). The biomembrane is held or secured by a grid that is secured with an interference fit at the base of the culture insert. The biomembrane retainer can be easily removed with a cap lifter to allow access to or removal of the biomembrane. The securing device can be either a grid or a spoke-shaped device (see FIG. 4B).

[0149] An embodiment of a cell culture insert manufactured according to the solution is shown in FIG. 5. Here the cell culture insert is cylindrical in shape. The upper opening is provided with support bars, which allow the cell culture insert to be inserted into and removed from a nutrient solution in a multiwell plate. At the lower opening, a grid is visible as a safety device to hold the biomembrane in the cylindrical housing.

[0150] Depending on the membrane material, different cells can colonize the biomembrane and form monolayers (see FIGS. 6A and 6B).

[0151] The illustrations of FIG. 6A show microscopic transmitted light images of the top and bottom side of a colonized carrier. On the left, the bottom side is shown with a holder for the membrane, on the right the top side of the carrier with colonized surface. The cells used were Caco-2 cells.

[0152] FIG. 6B shows a lateral cross-section of a gelatine membrane populated with HUVEC cells, performed with a 2-photon microscope. The individual colors show the different markers that were examined in this experiment: DAPI (cell nucleus), vWF and CD31 (vascular cell marker). A polarization and thus a natural behavior of the cells can be detected.

[0153] The microscopic images shown in FIG. 6C show a lateral section through the membrane. The upper side of the image represents the top side of the membrane, the cell-populated membrane divides the image, and the lower half of the image represents the bottom side of the carrier. The individual stains indicate a polarization of the cells towards the membrane. In the case of the microscopic image an increased collagen production of the cells towards the membrane can be shown. The cells are Vero cells. In addition, the cell nuclei were also stained with DAPI. The aim of the experiment was to show a polarization and interaction of the cells. In comparison, this effect is less pronounced in a Petri dish or not at all.

[0154] In the following two images, the markers ITGB1 (right) and aPKC (bottom left) were examined, which also document a polarization towards or away from the membrane.

[0155] The present cell culture insert can be designed with a continuous rim (FIG. 7, a) for a barrier function or a cut-out (FIG. 7, b). The cutout can be located below the media level in the multiwell, so that the cells on the membrane can be supplied with nutrients.

[0156] The cell culture insert can be provided with two or more different materials as a membrane in a blank, whereby the materials can be arranged in different architectures, e.g. side by side (FIG. 7, c) or on top of each other (FIG. 7, d) or in other architectures. This architecture can be placed in blanks for hanging or with feet.

[0157] 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 can also have an architectural shape. For example, villi, channels, hills, valleys (FIG. 7, e) can be inserted, even if they are made of different materials (FIG. 7, f). This architecture can be inserted in blanks for hanging or with feet.

[0158] A channel may be introduced into the membrane of the cell culture insert, which can be flushed from outside the blank (FIG. 7, g, h). The channel can be used to supply the inner compartment of the blank. The channel acts as a medium carrier with culture medium, which is flushed through the channel. Cells that are located on the membrane inside the compartment can be supplied through the membrane.

[0159] Functional material may also be incorporated into the membrane of the cell culture insert. For example, an additional detector, dye, enzyme, chemokine, nanoparticles or similar can be integrated into the membrane during the printing method. Over time, this material can be used for online monitoring of the cell culture. For example, cell death can be detected by a fluorescent dye or the current oxygen saturation or pH value. The functional material may or may not have contact to the inner and outer boundary layer. The functionalization can be observed by a color change, irradiation or other detectable measurement. The functional material can be introduced pointwise into the membrane (FIG. 7, i) or flat (FIG. 7, j).

[0160] The cell culture insert can also enable the measurement of membrane density by electrical resistance. In this case, a special blank can be used in which a probe is attached to the inside and outside of the blank in order to measure the electrical resistance and thus draw conclusions about the density of the membrane and the cell layer (FIG. 7, k).

[0161] FIG. 8 shows a cell culture insert with outwardly directed and angled projections (feet) on its lower side or end (i.e. the part of the cell culture insert that contacts the bottom of the multiwell plate). This allows the cell culture insert to stand upright in the multiwell plate automatically.

[0162] FIG. 9 shows another variation of the cell culture insert. In this variant of the cell culture insert two biomaterials are used, which can differ in their properties. The cell culture insert consists of a cylinder with a bottom (see figure Biopolymer 2, blue). On the bottom another biopolymer is incorporated (see figure Biopolymer 1, green). 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. Material b incorporates material a, which in the example is based on gelatine. In contrast to PEG, gelatine is not only biocompatible but also biofunctional, so that cells grow on this material. The combination of both materials creates a scaffold that can be colonized with cells in a directed manner. The area comprising biopolymer 1 (a) determines the maximum area on which cells can grow. During colonization, the cells grow to the edge. Since the border consists of the cell-repellent polymer 2 (b), they cannot colonize the border. The growth stops at this border.

Example

[0163] Cell culture inserts with a diameter of 12 mm were produced using the stamping method. A gelatine matrix with a concentration of 10% (W/V) was used, mixed with LAP as initiator in a concentration of 0.1% (W/V). Furthermore, collagen I was used as an additive.

[0164] The blank was filled with a silicone stamp and placed in a basin in the printer containing the liquid gelatine matrix described above, so that the blank rests on the bottom of the basin in the printer. To create a membrane of 500 μm, the distance was adjusted accordingly with the stamp before.

[0165] Subsequently, the gelatine matrix was cured by irradiation with a wavelength of 385 nm. After hardening, the carrier was removed from the basin and the printer. In addition, the stamp was removed, leaving behind a cell culture insert with the previously defined height and with a flat surface for colonization.

[0166] Two different cell culture inserts were produced. A first insert with a membrane with collagen I as an additive and a second insert with a membrane without this additive. In this case the gelatine membrane was made transparent and could be examined optically.

[0167] After production of the cell culture inserts with biological membrane, they were colonized 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 surface of the cell culture insert.

[0168] After colonization, a GFP-tagged cowpox strain was used to infect the Vero cells with the strain. The spread of the infection could be studied and observed by the fluorescent viruses and the transparent cell culture insert over the course of the experiment of 28 days.

[0169] After the experiment, the membrane was stamped out and deep-frozen. In addition, the membrane could be cut and stained with the usual histological methods, so that a histological follow-up was possible.