METHOD FOR THE CULTURING AND DIFFERENTIATION OF CELLS

Abstract

The present invention relates to a method for the culturing of cells on a cell culture substrate, wherein the cell culture substrate comprises a cell culture substrate made of glass and at least a part of the cell culture substrate made of glass has a surface with a nanoporous structure with an average pore diameter of 2 to 150 nm, and the use of a cell culture substrate for the culturing or differentiation of cells, as bottom of a cell culture vessel or bioreactor, as removable insert for cell culture vessels or bioreactors and/or as perfusive membrane for 3D cell culture reactors, whereby the cell culture substrate comprises a cell culture substrate made of glass and at least a part of the cell culture substrate made of glass has a surface with a nanoporous structure with an average pore diameter of 2 to 150 nm.

Claims

1. A method for the culturing of cells, the method comprising: a) providing at least one cell that is present in a cell culture medium, and a cell culture substrate; b) contacting the at least one cell that is present in the cell culture medium with the cell culture substrate; c) incubating the at least one cell that is present in the cell culture medium on the cell culture substrate; wherein the cell culture substrate comprises a cell culture substrate made of glass and at least a part of the cell culture substrate made of glass has a surface with a nanoporous structure with an average pore diameter of 2 to 150 nm.

2. The method according to claim 1, wherein the at least one cell that is present in the cell culture medium provided in step a) is a stem cell, and the method is a method for the differentiation of stem cells.

3. The method claim 1, wherein the surface with a nanoporous structure has an average pore diameter of 40 to 150 nm.

4. The method according to claim 1, wherein no additives are added to the cell culture medium.

5. The method according to claim 1, wherein the cell culture substrate has a thickness of 10 to 500 m.

6. The method according to claim 1, wherein the cell culture substrate is transparent.

7. The method according to claim 1, wherein the cell culture substrate has at least one of a surface functionalization and surface coating.

8. The method according to claim 1, wherein the cell culture substrate is a part of a cell culture vessel or a bioreactor.

9. The method according to claim 1, wherein the cell culture substrate is an insert for cell culture vessels or bioreactors.

10. The method according to claim 1, wherein the cell culture substrate is a removable insert for cell culture vessels or bioreactors.

11. The method according to claim 1, wherein the cell culture substrate is a bottom of a cell culture vessel or bioreactor.

12. The method according to claim 1, wherein the cell culture substrate is a perfusive membrane for 3D cell culture reactors.

13. The method claim 1, wherein the surface with a nanoporous structure has an average pore diameter of 80 to 150 nm.

14. The method according to claim 1, wherein the cell culture substrate made of glass has at least one of a surface functionalization and surface coating.

Description

[0050] The present invention shall be illustrated based on the following examples and related figures.

[0051] FIG. 1 shows the relative expression of the cartilage-specific genes Col1a1 (FIG. 1a), Col10 (FIG. 1b), and Sox9 (FIG. 1c) in primary human mesenchymal stem cells (hMSCs) of two patients on two control surfaces (TCPS=tissue culture polystyrene, FG=flat cover glass) after 7 to 12 days as compared to the growth of the cells on the cell culture substrate according to the present invention (average pore diameter 17 nm, bars represent the means of the two patients).

[0052] FIG. 2 shows a phalloidin staining of the actin cytoskeleton of primary human mesenchymal stem cells (hMSCs) grown on a nanoporous glass membrane with an average pore diameter of 17 nm (left) and of cells grown on the two control substrates (middle, right) after 1, 2, 5, and 7 days.

[0053] FIG. 3 shows proliferation rates of L929 fibroblasts in defined periods of time on cell culture substrates according to the present invention with different average pore diameters and on two control surfaces, each under standard cell culture conditions (TCPS=tissue culture polystyrene, FG=flat cover glass).

[0054] FIG. 4a shows the development of the relative cell count of SK-MEL-28 melanoma cells in overhead culture on cell culture substrates according to the present invention with an average pore diameter of 20 nm and on flat cover glasses (FG).

[0055] FIG. 4b shows the adhesion of SK-MEL-28 melanoma cells on a nanoporous glass membrane with an average pore diameter of 20 nm and on a flat non-porous glass surface (FG) after 3 hours of incubation on the respective substrate.

[0056] FIG. 5 schematically shows the morphology and adhesion of SK-MEL-28 melanoma cells grown on a nanoporous glass membrane versus SK-MEL-28 melanoma cells grown on a flat non-porous glass surface (FG) in overhead culture at different points in time.

[0057] FIG. 6 shows the analysis of the mRNA expression of L929 cells after 48 h of culturing on the different nanoporous glass membranes (17 nm, 45 nm, 81 nm, 124 nm) and on the two control surfaces (FG, TCPS).

[0058] FIG. 7 shows the development of the relative cell count of MDA-MB-231 breast cancer cells in overhead culture on cell culture substrates according to the present invention with different average pore diameters (17 nm, 26 nm, 46 nm, 81 nm, 124 nm) and on flat cover glasses (FG) without active agent (CONTROL) and exposed to 500 nM paclitaxel in each case (TREATMENT).

[0059] FIG. 8 shows a scanning electron micrograph of a lamellopodium of a human mesenchymal stem cell (hMSC) with many small filopodia after two days of incubation on a cell culture substrate according to the invention with an average pore diameter of 17 nm.

[0060] FIG. 9 shows a scanning electron micrograph of human mesenchymal stem cells (hMSCs) incubated for two days on a cell culture substrate according to the present invention with an average pore diameter of 17 nm.

[0061] FIG. 10 shows a scanning electron micrograph of a lamellopodium of an L929 fibroblast with many small filopodia after two days of incubation on a cell culture substrate according to the invention with an average pore diameter of 124 nm.

[0062] FIG. 11 shows four different nanoporous glass membranes according to the present invention (top) and scanning electron micrographs of the nanoporous surface structure of the individual membranes.

EXAMPLES

1. Production and Physical Properties of Nanoporous Glass Membranes of Different Pore Size

[0063] In order to test the influence of nanoporous glass on the behavior of viable cells and the dependence on the pore diameter, a modified VYCOR process was used to produce glass membranes with different average pore diameter for Examples 2 to 7. It was evident that the membranes became increasingly opaque with increasing temperature during leaching, which indicates that the average pore diameter was increased (FIG. 11). This macroscopic observation was confirmed by UV/VIS experiments from which it was clearly evident that increasing temperature during phase separation is associated with a broadening of the range of the wavelengths absorbed by the nanoporous glass. Controlling the temperature during the phase separation, cooling process, and controlled leaching, enables to produce nanoporous glass membranes with an average pore diameter between 17 and 124 nm and a thickness of only 250 m.

2. Culture and Induction of Chondrogenic Differentiation of hMSCs

[0064] In order to test the ability of the cell culture substrates according to the present invention to induce a chondrogenic differentiation, primary hMSCs of two patients were incubated on two control surfaces (TCPS=tissue culture polystyrene, FG=flat cover glass) and on a cell culture substrate according to the present invention, namely a cell culture substrate comprising a VYCOR membrane with a nanoporous structure with an average pore size of 17 nm, and the relative expression of the cartilage-specific genes Col1a1, Col10, and SOX9 was determined by means of qPCR.

[0065] Compared to the two control surfaces, a clear increase of the relative expression of Col1a1 (FIG. 1a), Col10 (FIG. 1b), and SOX9 (FIG. 1c) was evident upon incubation of the cells on a cell culture substrate according to the present invention.

[0066] In addition, the actin cytoskeleton of cells grown on the nanoporous glass membrane with an average pore diameter of 17 nm and of the cells grown on the two control substrates was stained with phalloidin. It was evident that the actin filaments in the cells cultured on the 2D control surfaces were significantly more well-ordered than the actin filaments of the cells cultured on nanoporous glass membranes (FIG. 2).

[0067] Said induction of a chondrogenic differentiation without the addition of external media additives on a cell culture substrate according to the present invention as early as after the first week advantageously allows for the utilization of cell culture substrates according to the present invention as surface for rapid and inexpensive differentiation of hMSCs.

3. Comparison of the Proliferation Rates of L929 Fibroblasts on Cell Culture Substrates According to the Present Invention Versus Proliferation Rates on Control Surfaces

[0068] The cell proliferation on standard 2D surfaces often differs strongly from the proliferation inside the human body since the cells in the body are situated inside 3D tissues and often proliferate individually, whereas a usually uncontrolled growth of the cells is possible on a standard 2D surface.

[0069] In the present experiment, L929 fibroblasts were seeded and incubated under standard 2D culture conditions on two control surfaces (TCPS=tissue culture polystyrene, FG=flat cover glass) and on different cell culture substrates according to the present invention, namely cell culture substrates, each of which having a VYCOR membrane with a nanoporous structure with different average pore diameters (17 nm, 45 nm, 81 nm, 124 nm). After just a few days, the L929 fibroblasts reached similar proliferation rates on the cell culture substrates according to the present invention as on the smooth control surfaces (FIG. 3).

[0070] Accordingly, similar proliferation rates as upon the growth of cells on standard 2D surfaces can be attained on the cell culture substrates according to the present invention with topographic stimulation of the cells by the surface with a nanoporous structure.

4. Proliferation of SK-MEL-28 Melanoma Cells in Overhead Culture on Cell Culture Substrates According to the Present Invention Versus Proliferation on Smooth Glass Surfaces

[0071] For investigation of the proliferation of SK-MEL-28 melanoma cells in overhead culture on the surfaces of the cell culture substrates according to the present invention with a nanoporous structure versus the growth of cells on smooth glass surfaces, SK-MEL-28 melanoma cells were seeded on the different substrates and incubated in overhead culture for a period of 9 days. In this context, the cells that had been incubated on the cell culture substrates according to the present invention (cell culture substrate with nanoporous VYCOR membrane) with an average pore diameter of 20 nm were detected to show strong proliferation in overhead culture, whereas the cell count on the smooth glass surfaces decreases steadily under the same conditions (FIG. 4a).

[0072] In particular, it was evident that as early as after 3 hours of incubation on a flat non-porous glass surface, the adhesion of SK-MEL-28 melanoma cells with a relative cell count of 0.530.07 was clearly lower than the adhesion of SK-MEL-28 melanoma cells on a nanoporous glass membrane with an average pore diameter of 20 nm (FIG. 4b).

[0073] In addition, scanning electron micrographs showed that the cells grown on a flat non-porous glass surface significantly more often comprise a circularity and a higher solidity, which is indicative of a rather passive spreading process with a more circular morphology and fewer filopodia. In contrast thereto, the cells grown on nanoporous glass membranes had more filopodia and occupied a larger area of the substrate surface, which is indicative of an active spreading process with strong focal adhesion of the cells to the topographic surface in overhead culture (FIG. 5).

[0074] Thus, the cell culture substrates according to the present invention advantageously allow the cell adhesion to be improved by simulating a three-dimensional environment even under the effect of gravity and without additional functionalization/coating. Accordingly, the surface of the cell culture substrates according to the present invention resembles the natural environment in the human body more closely than smooth 2D surfaces.

5. Different mRNA Expression on Nanoporous Glass Membranes with Different Average Pore Diameter

[0075] The mRNA expression of L929 cells on the different nanoporous glass membranes (17 nm, 45 nm, 81 nm, 124 nm) was analyzed by means of qPCR after 48 h of culturing, i.e. during the initial resting phase, in which the cells settle on the surface of the membranes (FIG. 6). It is evident that in particular cells that are being cultured on nanoporous glass membranes with an average pore diameter of 81 nm or 124 nm show an mRNA expression profile that is very similar to the one of cells cultured on a flat non-porous glass surface. This shows a positive interaction between the cells and the surface, although no extensive proliferation of the cells has commenced at this point in time. Moreover, the induction of cell proliferation is significantly increased in the presence of the nanoporous glass membranes with an average pore diameter of 81 nm or 124 nm as compared to the other nanoporous glass membranes. This is evident from the increased expression of proliferation-specific proteins (MKI67, MCM2). In addition, genes regulating other cell functions, such as cell adhesion (FAK, Itgb1), matrix production (COL1A1, FN1), and contraction (ACTA2), were also analyzed. There is a notable reduced expression of ACTA2 by the cells cultured on the nanoporous glass membranes as compared to cells cultured on the flat non-porous glass surface. A drastic change of the expression profile is detectable below an average pore diameter of 80 nm, wherein cells cultured on these nanoporous glass membranes have a clearly increased expression of PTK2/FAK (focal adhesion kinase), whereas other essential genes are strongly down-regulated.

6. Simulation of the Physiological Adhesion Mechanism of Cells to Demonstrate the Effectiveness of Cytoskeleton-Effective Agents

[0076] In the present experiment, MDA-MB-231 breast cancer cells were initially seeded on cell culture substrates according to the present invention, in particular nanoporous glass membranes with average pore diameters of 17 nm, 26 nm, 46 nm, 81 nm, and 124 nm, and on a smooth non-porous glass surface and cultured for 24 h in order to obtain homogeneous cell colonization on all substrates. Subsequently, the samples were inverted and divided into two groups: one control group and one test group, wherein the culturing took place in overhead culture for 48 h. In this context, the control group was cultured in normal culture medium and 500 nM paclitaxel was added to the culture medium of the test group. During culturing for 48 h in overhead culture, a reduction of the relative cell count on the substrates according to the invention by approximately 35-55% in the test group as compared to the control group was observed (FIG. 7). Interestingly, the reduction of the relative cell count within the 48 h period was considerably lower on the smooth non-porous glass surface (approximately 5%).

[0077] The present result shows the feasibility of simulating the physiological adhesion mechanism on the cell culture substrates according to the present invention and indicates the suitability of the cell culture substrates for demonstration of the effectiveness of agents that intervene in cytoskeletal processes.

7. Proliferation of Primary Human Mesenchymal Stem Cells (hMSC) on Nanoporous Glass Membranes of Different Pore Size

[0078] Primary hMSC were seeded on nanoporous glass membranes having three different average pore diameters and two control substrates (TCPS=tissue culture polystyrene, FG=flat cover glass). The samples were fixated with glutaraldehyde at different points in time and prepared for scanning electron microscopy. All tested samples showed good cell adhesion and cell proliferation. During the first days of culturing on the nanoporous glass membranes, the formation of cell clumps was observed. These were no longer present after day 3, which indicated full spreading of the cells.