ALGINATE DIALDEHYDE-COLLAGEN HYDROGELS AND THEIR USE IN 3D CELL CULTURE

Abstract

The present invention relates to a method of generating a hydrogel comprising alginate dialdehyde (ADA) and collagen, which are covalently cross-linked, and optionally, further component(s), and to uses of such hydrogel. The present invention further relates to using the hydrogel for culturing cells, in particular neuronal cells, and for further uses, such as 3D bioprinting. The present invention furthermore relates to a cell culture system comprising a hydrogel of alginate dialdehyde (ADA) and collagen, which are covalently cross-linked, and, optionally, further components. Furthermore, the present invention relates to a method of generating a three-dimensional (3D) cell culture using a hydrogel according to the invention.

Claims

1. A cell culture system comprising: (i) a hydrogel, wherein said hydrogel comprises alginate dialdehyde (ADA) and collagen, wherein the ADA and the collagen are covalently cross-linked, and (ii) optionally, further component(s).

2. (canceled)

3. The cell culture system of claim 1, wherein the hydrogel is obtained by dissolving ADA in a cell culture medium before adding the collagen to said cell culture medium, wherein the pH of the cell culture medium is from 8.0 to 8.4, before the addition of ADA and/or collagen, and/or wherein the temperature is from 0 to 4° C.

4. The cell culture system according to claim 1, wherein the collagen is collagen type I.

5. The cell culture system according to claim 1, wherein the hydrogel has adjustable: hydrogel stiffness, crosslinking density, crosslinking degree, diffusity, porosity, swelling kinetics, degradation kinetics, scaffold geometry, hydrogel stress relaxation, and/or controllable adhesion.

6. The cell culture system according to claim 1, comprising one or more further components selected from: growth factor(s), antibiotic(s), cytokine(s), nutrient(s), blood serum(s), cell fragments, saline containing divalent cations, and/or buffer containing physiological concentrations of calcium, glycosaminoglycan(s) supplements, and/or further components of native extracellular matrix.

7. The cell culture system according to claim 1, further comprising cells that are embedded in said hydrogel.

8. The cell culture system of claim 7, wherein said cells form a three-dimensional (3D) cell culture in said hydrogel.

9. The cell culture system according to claim 7, wherein said cells are selected from neuronal cells, bone cells, stem cells, immortal cell lines, muscle cells, cartilage cells, cells forming blood vessels, and cancer cells.

10. A method for culturing cells, wherein said method comprises the use of the cell culture system of claim 1 and wherein the cells are selected from neuronal cells, bone cells, stem cells, immortal cell lines, muscle cells, cartilage cells, cells forming blood vessels, and cancer cells.

11. A method for 3D bioprinting, wherein said method comprises the use of a cell culture system of claim 1.

12. Use of the cell culture system of claim 1 as an in vitro 3D cell culture platform.

13. A method for creating a tumor, wherein said method comprises use of the cell culture system of claim 1.

14. The cell culture system of claim 1 used to create a “lab on a chip” device.

15. A method of generating a hydrogel of oxidized alginate covalently crosslinked with collagen (ADA-Col), the method comprising: (1) providing alginate dialdehyde (ADA), which is obtained by controlled oxidation of sodium alginate from brown algae with an oxidizing agent, in the absence of light, over a time period of about 2 to 10 hours, (2) dissolving the ADA of step (1) in a cell culture medium, (3) adding collagen to the dissolved ADA of step (2), and furthermore adding sodium bicarbonate to said cell culture medium, (4) obtaining the ADA-Col hydrogel.

16. The method of claim 15, wherein during obtaining the ADA provided in step (1), the reaction is in a mixture of ethanol and water (50/50 volume/volume), and/or supplemented with radical scavengers during the synthesis, and/or wherein the reaction is quenched by the addition of ethylene glycol.

17. The method of claim 15, wherein the pH of the cell culture medium is about 7.8 to 8.6, before the addition of ADA and/or collagen.

18. The method of claim 15, wherein the temperature of step (3) is from 0 to about 4° C.

19. A method of generating a three-dimensional (3D) cell culture, said method comprising the steps: performing the method of generating a hydrogel according to claim 15, adding cells after step (3), and prior to, or concomitantly with, step (4), such that said cells become embedded in said hydrogel, optionally further comprising, incubating said cells embedded in said hydrogel for a period of from 1 h to 10 days.

20. The method according to claim 19, wherein said cells are selected from: neuronal cells, bone cells, stem cells, immortal cell lines, muscle cells, cartilage cells, cells forming blood vessels, and cancer cells.

21. The method according to claim 15, wherein the oxidizing agent is selected from sodium metaperiodate, potassium permanganate, and 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0168] FIG. 1. Culturing neuronal cells in 2D versus 3D culture.

[0169] A) 2D cell culture of neuronal cells results in forced basal-apical cell polarity, wrong stiffness and porosity.

[0170] B) 3D cell culture system allows free cell polarity.

[0171] FIG. 2. Preparation of dorsal root ganglion (DRG) cells.

[0172] a) from sacrificed mice. b) Dousing fur with 70% ethanol. c) Foreceps were used for removing skin by using scissors with one big cut. d,e) Removing spinal cord with two long incisions along the spinal cord, then cutting hips and neck. f) Spinal cord washed in PBS then muscle, fat and other soft tissues were cut from the spinal cord. g,h) Thick forceps were used to secure the spinal column dorsal side up, before cutting it into two equal halves along the midline. i) The spinal cord was peeled from the pinned column in a rostral to caudal direction. j) Individual ganglia were extricated by clasping and lifting with forceps the distally projecting axon bundles found on the lateral side of the DRG. k,l) Care must be taken not to damage the DRG with the forceps DRG were then pinned out via their axons, and any residual meninges removed before cutting the axons close to the DRG.

[0173] FIG. 3. DRG cells grow in the ADA-Col hydrogel of the invention and show 3D self-organization within the hydrogel.

[0174] A) Live-dead staining of DRG from baby C57/b6 mice in ADA collagen hydrogel after three days of incubation at 37° C. and 5% CO2 in the incubator (top). Live-dead staining of DRG cells from baby C57BL/6 mice in ADA collagen hydrogel after seven days of incubation (bottom). Living cells stained in green with Calcein and dead cells stained in red with Propidium iodide. The scale bar is 50

[0175] B) Live-dead staining of DRG cells from baby C57/b6 mice in matrix hydrogel C57/b6 after three days incubation at 37° Cs and 5% CO2 in incubator (top). Live dead staining of DRG cells from baby C57BL/6 mice in matrix hydrogel after seven days of incubation (bottom). Living cells stained in green with Calcein and dead cells stained in red with Propidium iodide. The scale bar is 50

[0176] FIG. 4. Neuronal cells grow in the ADA-Col hydrogel of the invention.

[0177] Immunhistochemistry staining of DRG cells from baby C57/b6 mice in matrix hydrogel C57/b6 after three days incubation at 37° Cs and 5% CO2 in incubator (top) and after seven days of incubation (bottom). The scale bar is 50

EXAMPLES

[0178] Example 1 Alginate Dialdehyde (ADA) Synthesis Sodium alginate (sodium alginate (E401) from brown algae, DuPont GRINDSTED Alginate (PH 124) was obtained from Sweet Ingredients GmbH, Germany (material number: 60516). Sodium metaperiodate and calcium chloride di-hydrate (CaCl.sub.2×2H.sub.2O) were purchased from Sigma Aldrich, Germany.

[0179] Alginate di-aldehyde (ADA) was synthesized by controlled oxidation of sodium alginate in a mixture of equal volumes of ethanol and water. Briefly, 10 g of sodium alginate PH 124 were dispersed in 50 ml of ethanol (Sigma Aldrich, Germany) and 2.674 g of sodium metaperiodate were dissolved in 50 ml of ultrapure water (Direct-Q, Merck Millipore, Germany) in the absence of light to get a 12.5 mmol sodium metaperiodate solution. The periodate solution was slowly added to the sodium alginate dispersion, which was continuously stirred at 250 300 rpm in the dark at 22° C. (room temperature) for 6 hours. The reaction was quenched after 6 hours by adding 10 ml of ethylene glycol (density 1.113 g.Math.ml.sup.−1 at 25° C.) (Sigma Aldrich, Germany) under continuous stirring for 30 minutes. The resultant suspension was dialyzed against ultrapure water (Direct-Q®, Merck Millipore, Germany) using a dialysis membrane with a molecular weight cut off (MWCO) of 6000-8000 Da (Repligen Biotech, Spectrumlabs, USA) for 5 days with water changes twice a day. The absence of periodate was confirmed by adding 0.5 ml of 1% (w/v) silver nitrate (Sigma Aldrich, Germany) solution to 0.5 ml of ADA ensuring the absence of any precipitate. The final ADA solution was frozen at −21° C. for a minimum of 24 hours and lyophilized using a freeze dryer (Alpha 1-2 LD plus, Martin Christ, Germany) for one week.

Example 2 Cell Preparation

[0180] Dorsal root ganglion (DRG) cells were obtained from three to seven days old wildtype C57BL/6 mice sacrificed in carbon dioxide atmosphere to prevent damage of cervical DRGs (Sleigh, Weir, & Schiavo, 2016). The spinal cord was dissected and DRGs (20-35 of each animal) were collected in phosphate buffered saline (PBS). The cell preparation is shown in FIG. 2. Briefly, DRGs were placed into Dulbecco's Modified Eagle Medium 4.5 g/L (DMEM, Gibco, Germany), where nerve trunks and connective tissue were dissected. DMEM was removed and Enzyme mix (see Table 1) was added. Following a 30 min incubation in a humidified incubator (37° C., 5% CO2), DRG were washed with DMEM twice and once with TNB100 basal medium (TNB, Biochrom, Germany). The cell suspension was spun for 3 minutes at 1000 rpm. By triturating DRG through a glass pipette the ganglion cells were dissociated and the cell pellet was resuspended.

TABLE-US-00001 TABLE 1 List of chemicals and medium DMEM 500 ml DMEM (Gibco, Germany) + 2.5 ml Gentamycin (Sigma, Germany) Enzyme mix 50 ml DMEM + 50 mg Collagenase (Sigma, Germany) + 25 mg Protease (Sigma, Germany) TNB (Biochrom, +2 ml Proteincomplex (Biochom, Germany) + Germany) 1 ml Penicillin Steptomycin (PenStrep, Sigmal, Germany) + 500 μlnerve growth factor (NGF, Alomone Labs Nr. 130, Germany)

Example 3 Hydrogel Preparation

[0181] For the 4× Opti-MEM medium, 13.6 g Opti-MEM reduced serum medium powder (ThermoFisher, Germany) were dissolved in 200 ml aqua dest, stirring for 20 minutes. Subsequently 2.4 g sodium hydrogen carbonate (Roth, Germany) was added. A pH value of 8.2 was adjusted finally in 250 ml aqua dest. Finally, the 4× Opti-MEM was sterilely filtered through a 0.22 μm filter (Roth, Germany).

[0182] 0.1 g ADA (PH 124, Sweet Ingredients GmbH, Germany) were dissolved in 2500 μl 4× Opti-MEM under continuous stirring for 1 hour. The ADA dissolved in Optimem was filtered sterile by a 0.22 μm filter (Roth, Germany). In a 15 ml falkon (VWR, Germany) 75 μl ADA dissolved in 4× Opti-MEM, 164.4 μl Collagen type I (Corning, Germany), 4 μl sodium bicarbonate (Roth, Germany) 3 μl penicillin/streptomycin (Sigma, Germany), 53.5 μl aqua dest. and 3 μl NGF (Alomone Labs Nr. 130, Germany) were mixed on ice to a total stock solution of 300 μl in a 15 ml falkon.

[0183] The prepared DRG cells (see Example 2) were taken up in 150 μl TNB medium and vortexted with 300 μl total stock solution. 225 μl each were seeded in one Ibidi vessel (Ibidi, Germany). The hydrogel was then incubated with the cells for one hour at 37 degrees Celsius and 5% CO2 in the incubator. On each well 150 μl FCS (Gibco, Germany) with 30 μl NGF were added and then incubated at 37 degrees Celsius, 5% CO2 for 3 and 7 days.

Example 4 Live-Dead Staining

[0184] Calcein/propidium iodide (PI, Thermofisher, Germany) iodide assay was used to estimate the ratio of live/dead cells. Using the following protocol, living cells were stained with green fluorescent marker calcein and dead cells with red propium iodide (PI). (Non-fluorescent calcein is taken up by living cells and cut intracellularly by an esterase. Afterwards, calcein is green fluorescent and impermeable for cell membrane. PI is a red fluorescent dye for nuclei, which is impermeable for cell membrane of living cells but binds diploid DNA).

[0185] Hydrogel was washed with Hank's balanced salt solution (HBSS, Sigma, Germany), followed by adding staining solution to the sample at a final concentration of 4μl/ml calcein/HBSS and 1μl/ml PI/HBSS. After 45 minutes of incubation of the sample in the dark. Before imaging the hydrogel was washed with HBSS. For imaging, live and dead cell fluorescence microscopy (Axio, Zeiss, Germany) was used.

[0186] Live-dead staining using calcein and propidium iodide showed that >99% of neurons were living. Results are shown in FIGS. 3A and B.

Example 5 Immunohistochemistry

[0187] Immunohistochemistry was made after checking the dendrite growth with light microscopy after three and seven days of incubation. Manufacturer, details and dilutions of primary and secondary antibody are shown in Tables 2 to 4.

TABLE-US-00002 TABLE 2 List of chemicals 4% Paraformaldehyde (PFA) 40 g Paraformaldehyde + 500 ml Aqua dest + 14.42 g NA.sub.2HPO.sub.4x2H.sub.2O PBS-Bovine serum albumin (PBS-BSA) 50 ml PBS + 0.5 g BSA (Sigma, Germany) 0.5% TritonX-100 (PBS-BSA-TX) 100 ml PBS-BSA + 0.5 g TritonX (Sigma, Germany) 4′,6′-Diamidino-2-phenylindole hydrochloride (DAPI)

TABLE-US-00003 TABLE 3 List of primary antibodies (dissolved in PBS/BSA/TX) Antigen Name Host Characteristics Source Dilution Guinea Pig Guinea pig Soluble Chemicon 1:50 Anti-Protein cytoplasmic international, gene product human PGP 9.5 USA 9.5 (GP PGP 9.5) Anti- Rabbit Phosporylated Sigma, 1:200 Neurofilament H tail of Germany 200 Neurofilament

TABLE-US-00004 TABLE 4 List of secondary antibodies (dissolved in PBS/BSA/TX) Antigen Name Host Characteristics Source Dilution Cy3-AffiniPure Donkey Anti- GP PGP 9.5 Jackson guinea pig IgG Immuno (H + L) Research, USA Donkey anti- Donkey anti Alexa 488 Thermo goat IgG goat IgG Fisher, USA

[0188] Hydrogel was fixed with 4% (w/v) paraformaldehyde (PFA, pH 7.4, Sigma, Germany) for 10 minutes, followed by washing two times for 10 minutes in PBS and incubated for “blocking” with 5% donkey normal serum in PBS-BSA-TX overnight. (Triton X100 is increasing the antibody permeability and blocking serum is used to minimize nonspecific bindings to the surfaces).

[0189] After blocking, hydrogel was washed for 10 minutes in PBS followed by incubation with guinea pig anti-protein gene product 9.5 (GP PGP 9.5, Chemicon International, USA) antibody or Anti-Neurofilament200 (Sigma, Germany) in PBS-BSA-TX. After overnight incubation of the primary antibody at room temperature, 3 washes with PBS (15 minutes each) were performed, followed by addition of the secondary antibody Cy3-AffinPure donkey anti-guinea pig (Chemicon international, USA) and 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI, Sigma-Aldrich, USA). After 4 h of incubation with the secondary antibodies, hydrogel was finally washed three times in PBS.

[0190] Confocal microscopy was used for imaging of both live and fixed samples. The immunostained samples were analysed using a LSM 780 light and confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) mounted on an inverted Axio Observer Z1. Three dry objective lenses (10×, 20× and 40×) were used. Fluorescent structures were observed in the light path mode using red and green filters. Confocal images were taken using filter settings for Alexa 488 and 555 with a resolution of 1024×1024 or 512×512 pixels. Z-stacks of images were taken to approve the 3D-growth of ganglion cells. Pictures were converted to a 12-bit RGB tiff-file using confocal assistant software ZEN 2010. After 72 and 168 hours (three to seven days) the cultured ganglion cells showed 2-5 extensions that formed a dense three-dimensional network after three days (FIG. 4, top) and seven days (FIG. 4, bottom). The cells consisted mainly of neurons, glial cells could not clearly be identified. Live-dead staining using calcein and propidium iodide showed that >99% of neurons were living (Example 4).

REFERENCES

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