CELL CULTURE SUBSTRATE FOR CULTIVATING ADHERENT CELLS

Abstract

A cell culture substrate for cultivating adherent cells, including: a substrate (S), a polymer (P) comprising amino groups, which is bonded to the substrate, and a saccharide (Z) having at least two monosaccharide units for attaching the adherent cells, wherein the saccharide (Z) is covalently bonded to the polymer (P) via the amino groups.

Such a cell culture substrate is suitable for cultivating adherent cells and allows the cells to be detached from the cell culture substrate in a gentle manner by adding a saccharide.

Claims

1. A cell culture substrate for cultivating adherent cells, comprising a substrate (S), a polymer (P) comprising amino groups, which is bonded to the substrate, and a saccharide (Z) having at least two monosaccharide units for attaching the adherent cells, wherein the saccharide (Z) is covalently bonded to the polymer (P) via the amino groups.

2. The cell culture substrate according to claim 1, wherein the saccharide comprises an open-chain monosaccharide unit and is bonded to a secondary amine group of the polymer (P) via this monosaccharide unit.

3. The cell culture substrate according to claim 1, wherein the saccharide is an oligosaccharide or polysaccharide having 2 to 500 monosaccharide repeat units.

4. The cell culture substrate according to claim 1, wherein the substrate comprises glass or plastic.

5. The cell culture substrate according to claim 4, wherein the substrate is covalently bonded to the polymer comprising amino groups.

6. The cell culture substrate according to claim 1, wherein the polymer comprises poly(ethyleneimine) (PEI) or poly(amidoamine) (PAMAM).

7. The cell culture substrate according to claim 1, comprising the following structure of the general formula I: ##STR00005## wherein S represents the substrate, P represents the polymer with saccharide bonded thereto and A represents a group linking the polymer P and the substrate S, wherein x is a number of repeat units in the polymer P, wherein the polymer P comprises repeat units which are selected from a group of repeat units having the general formulae A to F: ##STR00006## wherein Z represents the saccharide, and a, b, c, d, e and f are a number of respective repeat units in the polymer P, wherein a, b, c, d, and e, are independently selected integers, a+b+c+d+e+f=x, and “*” represents attachment sites of the repeat units to further repeat units in the polymer P or, when the repeat units are terminal repeat units, the attachment to the group A or to a terminal amino group —NH.sub.2 in the general formula I.

8. The cell culture substrate according to claim 7, wherein the group A linking the polymer P and the substrate S is selected from a group consisting of the following groups: ##STR00007## wherein “*” represents the attachment sites of the group A to the polymer P and the substrate S.

9. The cell culture substrate according to claim 1, wherein the saccharide Z is selected from a group consisting of the following groups having the formulae G to J: ##STR00008## wherein parameters g, i and j are, independently of one another, a natural integer between 0 and 400, and wherein “*” represents attachment sites of the saccharide Z to the polymer P.

10. The cell culture substrate according to claim 1, formed as a cell culture vessel or as a particle.

11. A process for producing a cell culture substrate comprising: A) providing a polymer (P) comprising amino groups and a saccharide (Z) having at least two monosaccharide units, B) forming a covalent conjugate between the polymer (P) and the saccharide (Z), and C) linking the conjugate to a substrate (S).

12. The process according to claim 11, wherein in step A) the saccharide (Z) is a reducing saccharide, and in step B) the covalent conjugate is formed by means of reductive amination.

13. The process according to claim 11, wherein the polymer (P) in step A) is a synthetic polymer.

14. The process according to claim 11, wherein in step C) the substrate has been activated by means of an oxygen plasma.

15. The process according to claim 14, wherein in step C) covalent bonds are formed between the substrate and the polymer saccharide conjugate via the amino groups of the polymer (P).

16. The process for cultivating adherent cells using the cell culture substrate according to claim 1 comprising: 1) bringing the adherent cells into contact with the cell culture substrate, wherein the cells attach to the cell culture substrate via the saccharide, 2) cultivating the cells with the cell culture substrate in a cell culture medium, 3) detaching the cells from the cell culture substrate by bringing a saccharide into contact with the cells.

17. The process according to claim 16, wherein in process step 3) the saccharide is a component of the cell culture substrate.

18. The process according to claim 16, wherein in process step 3) the saccharide is used in a concentration of from 5 to 25 mM.

19. (canceled)

20. A kit for cultivating adherent cells, comprising: a cell culture substrate according to claim 1, and a saccharide for detaching the cells from the cell culture substrate.

21. (canceled)

22. The process according to claim 13, wherein the synthetic polymer is poly(ethyleneimine) (PEI) or poly(amidoamine) (PAMAM), which may also be branched.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0054] In the following, the disclosure will be described in more detail with reference to figures and embodiment examples. There are shown in:

[0055] FIG. 1 depicts an example of a synthesis scheme of a polymer saccharide conjugate by means of a reductive amination using NaBH.sub.3CN.

[0056] FIG. 2 provides examples of functional groups that can be formed on a polystyrene surface by means of an oxygen plasma treatment.

[0057] FIG. 3 depicts a direct covalent bond between the polymer containing amino groups and polystyrene as the substrate.

[0058] FIG. 4 is a bar chart showing the results of a lactate dehydrogenase assay for determining cell integrity in adherent cells which have been detached from cell culture plates either by means of a process according to the disclosure or by means of a trypsin treatment.

[0059] FIG. 5 is a graph showing the results of a fluorescence test for determining a caspase as evidence of cell integrity in adherent cells which have been detached from cell culture plates either by means of a process according to the disclosure or by means of a trypsin treatment.

[0060] FIG. 6A-C are the results of protein SDS gels (A and C) and western blots (B) for determining the surface protein E-cadherin on the surface of adherent cells which have been detached from cell culture plates either by means of a trypsin treatment or by means of a process according to the disclosure.

[0061] FIG. 7 is a graph showing growth curves for adherent cells that have been cultivated on different cell culture plates.

DETAILED DESCRIPTION

[0062] FIG. 1 shows an example of a synthesis of a polymer saccharide conjugate based on the polymer PEI. The saccharide lactose can be linked directly to the amino groups of the PEG by means of reductive amination with NaBH.sub.3CN, wherein one of the cyclic hemiacetal units of the lactose opens and only the galactose remains as a cyclic hemiacetal form and is available for bonding the adherent cells.

[0063] FIG. 2 shows the functional groups formed by means of an oxygen plasma on the surface of polystyrene. For instance, carboxylates, hydroxide groups, and keto groups can be formed. Furthermore, radical oxygen species can also be formed. These groups can react with the amino groups of the polymer and form a permanent covalent bond.

[0064] FIG. 3 shows such covalent bonds between an example of a polystyrene substrate and the amino groups of the polymer. For example, imine groups or amine groups can be formed on the amide groups.

Embodiment Examples

[0065] In the following, examples of syntheses of some polymer saccharide conjugates will be presented.

Lactose PEI:

[0066] Polyethylene imine solution (Mn ˜60,000 GPC (gel permeation chromatography), Mw ˜750,000 LS (light scattering spectroscopy), 50 wt. % in water) (PEI, 20 g, 0.1667 mmol) and D-lactose monohydrate (38.028 g, 105.5 mmol, 5 eq. per ideal repeat unit of PEI) are dissolved in MeOH (70 mL) and 50 mM sodium tetraborate solution (aq) (100 mL) and heated to 60° C. Once the components had completely dissolved, the system was cooled down again and the pH was set to a value of 3 with formic acid. NaBH.sub.3CN (33.148 g, 527.5 mmol, 5 eq. based on lactose) was dissolved in MeOH (30 mL) and added to the system in 6 portions. The reaction took place for 2 h at 60° C. Subsequently, the MeOH was removed in vacuo and the solution was dialyzed via a cellulose membrane (exclusion limit 10 kDa) and freeze-dried.

Maltose PEI:

[0067] PEI (10.286 g; see above) and maltose monohydrate (27.124 g, 75.28 mmol; 7.5 eq. per ideal repeat unit of PEI) are completely dissolved in 35 mL MeOH and 5 mM sodium tetraborate solution (aq) at 60° C. After cooling and setting the pH (see above), a suspension of NaBH.sub.3CN (16.5870 g, 263.96 mmol) in 15 mL MeOH was added in portions. The reaction took place for 17 h at 60° C. Subsequently, the MeOH was removed in vacuo and the solution was dialyzed via a cellulose membrane (exclusion limit 10 kDa) and freeze-dried.

Mannan PEI:

[0068] 500 mg galactomannan (e.g. locust bean gum or guar gum) is incompletely dissolved in 49 mL H.sub.2O and added to 1 mL 1 M H.sub.2SO.sub.4. The solution is hydrolyzed in the microwave at 600 W for 60 s and then filtered. This results in a hydrolysis of the galactomannan, wherein polysaccharides with 200 to 300 mannose repeat units are formed.

[0069] The filtrate is mixed with 1 g PEI, 6 mL glacial acetic acid and 664 mg NaBH.sub.3CN and stirred for 3 h at 60° C. Then, the solution is dialyzed via a cellulose membrane (exclusion limit 10 kDa) and freeze-dried.

Plasma Treatment of the Substrate and Formation of the Cell Culture Substrate:

[0070] The cell culture substrate, for example cell culture plates, can be treated with an oxygen plasma as follows:

[0071] Untreated 24-well polystyrene plates are activated with oxygen plasma at 150 W for 90 seconds in vacuo (0.2 mbar) and then 500 μL of a 1 mg/mL aqueous solution of the PEI derivatives (polymer saccharide conjugates) is added to each well. The plates were incubated for 2 h at RT, washed with water and heated, covered, for an hour at 80° C. The plates are then suitable for direct use in the cell culture as cell culture substrates according to the disclosure.

Cell Culture on Glycopolymer-Coated 24-Well Plates as Cell Culture Substrate According to the Disclosure:

Media:

[0072] CHO-K1 cell lines: Ham's F12 with 10% (v/v) FCS (FCS=foetal calf serum)

[0073] HEK293 and HeLa cell lines: DMEM with 10% (v/v) FCS and 2% glutamine

[0074] The incubation took place at 37° C. and with a CO.sub.2 content of 5% in the atmosphere. Normally, 0.05-1×10.sup.6 cells/mL (500 μL) are added to the wells. Passaging is effected by replacing the medium with 500 μL of a 5-50 mM solution of the respective sugar in PBS or medium and incubating for 15-30 minutes at room temperature or 37° C. Depending on the PEI derivative, cell line specificity can be achieved. PEI lactose surfaces are well suited to CHO cell lines, for example. HeLa cells prefer mannan PEI or maltose PEI.

Tests for Determining Cell Integrity:

[0075] In the following, the results of some tests for determining cell integrity after detaching the adherent cells from the cell substrate will be shown. For this, adherent cells which have been detached from a cell substrate according to the disclosure by means of the process according to the disclosure are compared with adherent cells which have been detached by means of a trypsin treatment from conventional cell culture plates that have been surface-modified by means of a plasma. The cells are CHO-1 cells which have been cultivated on polystyrene Petri dishes with PEI lactose.

Lactate Dehydrogenase Assay for Determining Cell Integrity:

[0076] In the event of damage to the plasma membrane, lactate dehydrogenase (LDH) is released from various adherent cells into the cell culture media. The released LDH can be quantified by a coupled enzymatic reaction. First, LDH catalyzes the conversion of lactate into pyruvate by reducing NAD.sup.+ to NADH. Subsequently, the NADH is used to reduce a tetrazolium salt to a red formazan product by means of the enzyme diaphorase. The quantity of formazan formed, which is determined at a wavelength of 490 nm, is directly proportional to the quantity of released LDH in the medium. The lactate dehydrogenase assay was carried out with a commercially available kit, the “Pierce LDH Cytotoxicity Assay Kit” (Thermo Fisher Scientific, USA).

[0077] FIG. 4 shows a bar chart in which the difference between the absorption at 490 nm (absorption of formazan) and the absorption at 680 nm (background signal of the instrument) is plotted on the y axis. Listed on the x axis are various ways of detaching adherent cells from the cell substrate using different concentrations of lactose (10 mM, 25 mM, 50 mM and 200 mM) compared with a conventional process, the trypsin treatment.

[0078] It is clear to see in FIG. 4 that the lactate dehydrogenase activity in the cell culture medium is much greater in the case of the cells which were treated with trypsin than in the case of the adherent cells which were treated with lactose. This clearly shows that with the present disclosure, cell integrity is less impaired than with the trypsin treatment.

Caspase Assay for Determining Cell Integrity:

[0079] With a fluorescence-based caspase 3/7 assay, apoptosis (programmed cell death) is measured by detecting a caspase in cell cultures. For this, a caspase 3/7 green detection reagent is used, which is a peptide with four amino acids (DEVD) with a cleavage site for caspase 3/7, which is conjugated to a nucleic acid-binding dye. The dye is not fluorescent as long as it is conjugated to the peptide. Once the peptide has been cleaved by the caspase, the dye is activated, binds to DNA and can be detected using fluorescence at an excitation/emission maximum of approx. 502/530 nm.

[0080] FIG. 5 shows a bar chart in which the fluorescence of the caspase 3/7 green detection reagent is plotted for HeLa cells that have been subjected to different treatments for 30 minutes (black bars) and 24 hours (light bars), respectively. The assay was carried out with the “CellEvent™ Caspase-3/7 Green Detection Reagent” kit (Thermo Fisher Scientific, USA).

[0081] In the chart, the bars labelled “Medium” and “PBS” show the caspase-mediated fluorescence of cells that were only washed with the cell culture medium or PBS (phosphate buffered saline), respectively and thus were not exposed to any cell stress at all. The bars labelled “Staurosporine [10 μM]” show the caspase-mediated fluorescence of cells that were treated with staurosporine, a broad-spectrum kinase inhibitor, which triggers apoptosis in many cells. The caspase-mediated fluorescence of HEK cells which were subjected to a trypsin treatment is labelled “Trypsin-EDTA [0.5 and 20%/0.02%]”. Furthermore, the caspase-mediated fluorescence of cells that were cultivated on a cell culture substrate according to the disclosure and then detached by adding a saccharide, namely 10 mM mannose, 25 mM or 50 mM mannose, is shown.

[0082] The caspase-mediated activity of the cells not exposed to any cell stress is comparable to the activity of the caspase in adherent cells which were cultivated and detached using a process according to the disclosure. In contrast thereto, the caspase-mediated activity in cells which were subjected to a trypsin treatment is considerably increased. The experimental data of FIG. 5 clearly show that with a cultivation process according to the disclosure, or with the use of a cell culture substrate according to the disclosure, cell integrity is improved compared with a trypsin treatment.

[0083] FIG. 6 shows the detection of the protein E-cadherin, a transmembrane glycoprotein, in cells which were exposed to the same conditions as represented in FIG. 5. During the trypsin treatment, E-cadherin was also digested, with the result that this protein is no longer detectable in the SDS protein gel (FIGS. 6A and C). In the case of cells detached using lactose, the band for E-cadherin is still clearly visible, similar to the cells treated with the medium or PBS, which indicates high cell integrity (FIG. 6B). This shows that trypsin digestion also impairs surface proteins that are important for cell metabolism, whereas with the present disclosure this is not the case.

[0084] FIG. 7 shows the growth curve of CHO-1 cells on various cell culture vessels. The CHO cells which were cultivated on a cell culture substrate according to the disclosure (polystyrene Petri dishes with PEI lactose) show similar growth curves to cells which were cultivated in conventional cell culture vessels (with growth curves labelled “Standard”). The growth curve labelled “untreated” shows the growth of the cells on untreated cell culture plates which were also used as substrate for the cell culture substrate according to the disclosure in this experiment. The curve labelled “O.sub.2 plasma” shows the growth on cell culture plates which were plasma-treated, but to which the polymer saccharide conjugate was not bonded.

[0085] The disclosure is not limited by the description based on the embodiment examples. Rather, the disclosure comprises each new feature and every combination of features, which includes, for instance, every combination of features in the claims, even if this feature or combination is itself not explicitly specified in the claims or embodiment.