Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Summary

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Abstract

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 µm in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Introduction

Hydrogels are materials that form insoluble 3D networks capable of binding large amounts of water. Such materials can provide excellent environmental conditions for living cells. Currently, there is increasing interest in the generation of three-dimensional hydrogel structures and in the development of processes to tailor their chemical and physical properties. Once this is achieved, a template for the growth of cells and the manipulation of cellular behavior can be generated^1,2,3,4. These 3D structures not only create a more natural and realistic environment than conventional two-dimensional approaches, but they also reveal new possibilities for the growth of stem cells or tumor models⁵. Different materials possess a range of characteristics that mainly depend upon the pore size of the gel⁶. The pores play a crucial role in cell culture applications, tissue engineering, and the directed growth of stem cells. For example, oxygen and nutrients diffuse through the matrix, and adequate amounts must be able to reach the cells⁷. On the other hand, harmful metabolites must be removed as quickly as possible, and sufficient space for cell growth must be available⁷. Consequently, the properties of the material, and thus the pore size, severely influence the potential benefit and possible applications of the matrix. Depending upon the properties of the material, different cell-growth processes can occur in 3D cell culture, including the formation of neuronal structures; the growth and differentiation of skin or bone cells; and the directed growth of special stem cell lines, like hepatocytes or fibroblasts^{2,3,8,9,10,11}. Another crucial point influencing the possible application of a material is its stability towards external stimuli¹². For example, the hydrogel must maintain its mechanical integrity in cell culture media or the human body.

In recent years, research on 3D cell culture hydrogels intensified, and many studies were carried out to resolve the 3D architectures of the systems¹³. Hydrogels composed of chemically synthesized components are most commonly investigated because they can be easily synthesized and chemically modified and they exhibit high stability (see Zhu et al., 2011 for a review)⁵. However, proteins have many beneficial properties: as so-called "precision polymers," they are biocompatible; they have a defined length; they are relatively easy to modify; and they have a large number of target sites^14,15. In this regard, highly specific, innovative structures can be generated for application in many fields. In this study, a protein-based hydrogel¹⁶ was used to demonstrate the ability of well-established methods to influence the 3D architecture of the material. Furthermore, the capability of and applicability to pore generation was also investigated.

Many different techniques are available to modify 3D structures, including both simple methods and sophisticated, highly specialized techniques from different fields of material science. A widespread technique is the use of electrospinning to generate well-defined structures¹⁷. Charged fibers are pulled from a solution by an electric field and then solidify upon exposure to oxygen. In this way, fibers in the range of several nanometers up to several microns can be produced. Additional techniques to tune the size, structure, and distribution of the pores within the matrix are soft lithography, photolithography, hydrodynamic focusing, electro-spraying, and bio-printing^18,19,20. A significant drawback of these techniques is their dependency upon specific, expensive equipment and special chemicals or materials. Furthermore, experience with these techniques is often not directly transferrable to protein-based materials, and many of the chemicals and methods are not cell compatible.

On the other hand, many techniques do not rely upon special equipment, making them easier and cheaper to apply and to reproduce. A widespread method for structure manipulation is solvent casting^21,22,23. Particles are added prior to the polymerization reaction and are distributed homogenously to saturate the solution. After the polymerization, a change of conditions, such as a dilution or a pH change, leads to the solvation of the particles, while the pores remain within the material. The chemicals used in these techniques, such as salt, sugar, paraffin, gelatin, and chalk, are cheap and readily available. In freeze-drying, swollen hydrogels are frozen. The subsequent sublimation of the liquid phases under a vacuum is then performed^23,24,25. Water sublimation from the network is gentle enough to maintain the specific 3D structures of the material. In gas foaming, a solution is streamed with a gas while the polymerization takes place, leaving pores within the gel²¹. The size and distribution of the pores can be adjusted depending upon the gas stream.

To form the protein hydrogel, BSA is reacted with tetrakis (hydroxymethyl) phosphonium chloride (THPC) in a Mannich-type reaction to allow for the formation of covalent bonds between primary amines and the hydroxy groups of the four-armed linker molecule²⁶. Possible harmful intermediates are removed by excessive washing of the material after the reaction occurs.

This study demonstrates the possibility of treating a BSA-based material with different techniques to manipulate and tailor the size of the pores. Each of the techniques can be used in any laboratory worldwide, as no special equipment is necessary. In addition, different parameters, such as swelling ratio, enzymatic degradability, pH stability, and temperature sensitivity, were examined and compared to each other, especially respect to the influence of the different techniques on the generation of 3D architectures. Finally, the materials were functionalized with cell-adhesive peptides to investigate the possible application of the materials to cell culture. Two different model cell lines were used: A549 and MCF7.

Protocol

1. Hydrogel Preparation

1. Mix 200 mg of BSA with 1 mL of deionized H₂O to create 20% (w/v) BSA stock (stock solution A).
2. Mix 165 µL of THPC solution (134 mg/mL) with 4.835 mL of deionized water to create THPC stock solution (stock solution B).
3. Weigh 1 mg of KCSSGKSRGDS (1,111.1 g/mol) peptide (or an equivalent cell-adhesive peptide) and dilute it in 100 µL of sterile H₂O to obtain a 10 mg/mL solution (stock solution C).
   NOTE: This step is optional and only needs to be included if the hydrogel is meant for cell culture application.
4. Remove the bottom of a 96-well plate and replace it with removable plastic wrap.
   NOTE: For the plates used here, the bottom can easily be removed by applying pressure to the bottom of each well; the thin plastic bottom will simply fall out.
5. Mix 100 µL of BSA stock solution (A) and 100 µL of THPC stock solution (B) (optional: 4 µL of stock solution C for functionalized, cell-adhesive hydrogels) in the 96-well plate to obtain 200 µL of hydrogel. Mix the components by pipetting up and down at least 5 times to guarantee a uniform hydrogel after polymerization.
6. Place the 96-well plate at room temperature (RT) for about 10 min until all hydrogels are properly polymerized.
7. Carefully and slowly remove the plastic wrap from the bottom of the plate.
8. Press the hydrogels out of the 96-well plate using a small stamp and transfer them to 1.5 mL tubes with sterile PBS, pH 7.4.
   NOTE: The hydrogels have a cylindrical shape, with a diameter of approximately 4 mm and a height of 8 mm.
9. Store the hydrogels in phosphate-buffered saline (PBS) at 4 °C for up to several months.

2. Freeze-drying the Hydrogels

1. Fill 1.5 mL tubes with 500 µL of sterile deionized H₂O and remove the cap. Transfer the hydrogels to the 1.5 mL reaction tubes using a spatula.
2. Wrap the 1.5 mL tubes tightly with paraffin film-at least three layers for each vial. Use a needle to pierce small holes in the film to enable gas release from the tube.
3. Proceed to one of the following steps:
   1. Transfer the vial to liquid nitrogen solution for 5 min to guarantee that the water and hydrogel completely freeze. Immediately after removal from the liquid nitrogen, transfer the vials to the freeze dryer to prevent the material from thawing.
      NOTE: This procedure results in pores about 10-15 µm in radius.
   2. Keep the vials at -20 °C overnight to slowly freeze the hydrogels. Immediately after removal from -20 °C, transfer the vials to the freeze dryer to prevent the material from thawing.
      NOTE: This procedure results in pores about 50-60 µm in radius.
4. After 24 h (and the complete evaporation of the water in and around the hydrogel), thaw the material by removing it from the freeze dryer.
   NOTE: The pore sizes can be analyzed with confocal laser scanning microscopy (step 5, "Hydrogel Visualization").

3. Particle Leaching

1. Prepare the hydrogel as described in steps 1.1-1.5.
2. Directly after mixing the components, add NaCl until saturation occurs (36 mg/mL). Add salt until a salt crystal can be seen in the solution as a white precipitate.
3. Transfer the 96-well plate to a shaker and shake until polymerization takes place (about 10 min). Remove the hydrogels from the plate, as described in steps 1.7-1.8.
4. Incubate the hydrogels for at least 24 h at RT in sterile water on a shaker (20 rpm) to elute all salt from the hydrogel template. Store the hydrogels at 4 °C in PBS for up to several months.

4. Channel Formation

1. Prepare hydrogels as described in step 1. Remove a hydrogel from solution using a pincer, remove the excess water with highly absorbent paper, and place it on top of a block of dry ice.
2. Freeze the hydrogel for 30 s and carefully remove it from the block. Do not damage the hydrogel; carefully use a spatula to scrape it off the block. Transfer the hydrogel to a 1.5 mL tube and dry it overnight at 37 °C.

5. Hydrogel Visualization

1. Prepare a rhodamine B stock solution by diluting 1 mg of rhodamine B in 10 mL of PBS. Prepare a serial dilution by diluting the rhodamine stock solution in PBS until a concentration of 0.001 mg/mL is reached (dilution factor: 100)
2. Remove the hydrogel from the storage solution (e.g., from step 2.4.) and transfer it to a 1.5 mL tube with 1 mL of 0.01 mg/mL rhodamine B solution. Stain the hydrogel overnight in rhodamine B solution at RT.
3. The next day, transfer the hydrogel that is to be visualized to 10 mL of PBS (pH 7.4) and wash for at least 3 h.
4. Transfer the hydrogel onto a µ-slide 8 well and cover it with PBS.
5. Cut small slices out of the hydrogel (about 0.5 mm in height) using a blade.
6. Using a confocal laser scanning microscope, visualize the hydrogel at a wavelength of 514 nm (objective: EC Plan-Neofluar 40x/1.30 Oil DIC M27, plane scan mode: 514 nm, and zoom: 1X).

6. Cell Culture Feasibility

1. Sterile-filter all stock solutions (A, B, and C) with a 0.45 µm filter.
2. Prepare the hydrogel as described in steps 1.1-1.4, including the cell-adhesive peptide prior to hydrogel polymerization.
3. After mixing the components, immediately pipette the mixture into a µ-slide 8 well until the bottom is completely covered.
   NOTE: This step must be performed quickly and directly after mixing the components, as polymerization takes place within minutes in the slide.
4. Culture adhesion cells and passage to obtain a single-cell suspension using standard cell culture techniques. Transfer the cells into pre-warmed, sterile DMEM cell culture medium supplemented with fetal bovine serum (FBS, 10% (w/v)), penicillin-streptomycin (1% (w/v)), and nonessential amino acid solution (MEM, 1% (w/v)).
5. Count the cells with a Neubauer counting chamber and carefully pipette 200 µL of the desired number of cells (2 x 10⁵ cells/cm²) onto the hydrogel surface.
6. Cover the µ-slide 8 well with the lid and transfer it to an incubator (37 °C, 5% CO₂). Incubate for at least 4 h at 37 °C.
7. After at least 4 h of cellular attachment, wash the cells twice with 200 µL of sterile cell culture PBS.
8. Fix the cells with 200 µL of 3.7% (v/v) formaldehyde for 10 min at RT and wash twice with PBS (~200 µL).
   NOTE: Wear appropriate personal protective equipment when handling formaldehyde.
9. Permeabilize the cells with 200 µL of 0.1 % Triton X for 5 min. Wash twice with PBS.
10. Stain the cells with phalloidin-rhodamine by mixing 5 µL of methanolic stock with 195 µL of PBS and adding it to the cells at RT. Stain the cells for 20 min in the dark. Wash twice with PBS.
11. Investigate the cell adhesion properties using a confocal microscope at 514 nm (objective: EC Plan-Neofluar 40x/1.30 Oil DIC M27, plane scan mode: 514 nm, and zoom: 1X). Analyze the images using appropriate software (see the Table of Materials).

7. Hydrogel Properties

1. Swelling ratio.
   1. Completely dry the hydrogels at 37 °C for at least one day.
   2. Weigh each hydrogel and note the exact weight.
   3. Fill a 2 mL reaction tube with 1.5 mL of PBS.
   4. Transfer the hydrogel into this 2 mL reaction tube and completely immerse it in PBS.
   5. Leave the hydrogel for at least two days in PBS at RT to reach equilibrium with the PBS.
   6. After three days, remove the hydrogel from the solution and dry it with a paper tissue to remove excess water from the hydrogel surface.
   7. Weigh the hydrogel.
   8. Calculate the swelling ratio using the following formula:
      
      where W_d is the weight of the dried gel (step 7.1.2.) and W_s is the weight of the wet gel (step 7.1.7). Multiply with 100 to get the swelling ratio percent.
2. pH and temperature stability.
   1. Transfer 5 mL of PBS to a 15-mL reaction tube.
   2. Adjust the solution to the appropriate pH (e.g., pH 2, 7, or 10) with NaOH and HCl. Bring the solution to the appropriate temperature (e.g., RT, 37 °C, or 80 °C).
   3. Transfer the hydrogel that is to be investigated for its stability into the appropriate solution. Readjust the pH if necessary.
      NOTE: All hydrogels should be completely swollen at this point (see the swelling ratio) to prevent the incorrect interpretation of the weight due to the swelling of the material.
   4. After certain time intervals, remove the hydrogel from the solution (e.g., each hour for up to 2 days), dry it with a highly absorbent paper tissue to remove excess water, and weigh it.
3. Enzymatic degradation.
   1. Prepare a stock enzyme solution of 300 U trypsin and pepsin, as per the manufacturer's instructions.
   2. Transfer the hydrogel (e.g., from step 1.8) to the appropriate solution.
      NOTE: All hydrogels should be completely swollen at this point to prevent the incorrect interpretation of the weight.
   3. After certain time intervals, remove the hydrogel from the solution (e.g., each hour), dry it with a highly absorbent paper tissue to remove excess water, and weigh it.

Results

Hydrogel development has become one of the most prominent fields in material research-related biological studies, with thousands of entries indexed in scientific research archives. Although the behavior of many systems is well studied, the manipulation of 3D networks, especially of sensitive protein-based materials, is often a major issue in material science. Another commonly underestimated challenge is the correct measurement of the native structure of a material using cryo electron micr...

Discussion

The production of macroporous matrices can be beneficial to many different fields. It has high technical and economic potential due to the defined structure of the hydrogel and the ability to control and tune specific material properties. However, the introduction of supramolecular structural elements, such as pores or channels, to a 3D template might influence the overall properties of a material, such as the swelling ratio or the stiffness. This can result in the undesired decomposition, degradation, or breakdown of th...

Materials

Name	Company	Catalog Number	Comments
Phosphate Buffered Saline (PBS)	Thermo Fisher Scientific	10010023
Dulbecco’s modified Eagle’s medium (high glucose)	Life Technologies / Thermo Fisher	11140-050
Fetal Bovine Serum (FBS)	Life Technologies / Thermo Fisher	10270-106
Penicillin-Streptomycin	Life Technologies / Thermo Fisher	15140122
MEM Nonessential Amino Acid Solution	Sigma Aldrich	M7145-100ML
Trypsin EDTA 0.05% Phenol Red	Thermo Fisher Scientific	25300062
Ethanol 99.8%, vergällt	Ölfabrik Schmidt	2133
NaCl	Carl Roth	9265.1
Albumin Fraction V	Carl Roth	3854.2
THPC	Sigma Aldrich	404861-100ML	Toxic
0.1% Triton X-100	Sigma Aldrich	X100-100ML	Slightly toxic
Phalloidin-rhodamine	Life Technologies / Thermo Fisher	R415
3.7% Formaldehyde	Life Technologies / Thermo Fisher	F8775-25ML	Toxic
Rhodamine B	Sigma Aldrich	81-88-9
Filtropur S 0.2	Sarsted Ag und Co.	2 83.1826.001
µ slide 8 well	Ibidi GmbH	80826
KCSSGKSRGDS peptide	UPEP Ulm	Custom sysnthesis
Ethanol 99.8%, vergällt	Carl Roth	K928.5
Falcon 5 ml Polysterene Round-Bottom Tube	Sarsted Ag und Co.	62.547.254
Tubes 50 ml	Sarsted Ag und Co.	62.547.254
Tubes 1.5 ml	Sarsted Ag und Co.	72,690,001
Tubes 2 ml	Sarsted Ag und Co.	72,691
CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM	Greiner	655073
FreezeDryer Epsilon 1-6D,	Christ, Osterode am Harz, Germany
Confocal Laser Scanning Microscope	Carl Zeiss AG, Oberkochen, Germany
Zen Software Version 2012 Sp1, black edition, 407 version 8,1,0,484	Carl Zeiss AG, Oberkochen, Germany
GSA Imaga Analyzer Software, GSA Image Analyzer, GSA, Version 419 3.8.7	GSA GmbH