To date, cell cultures have been created either on 2-dimensional (2D) polystyrene surfaces or in 3-dimensional (3D) systems, which do not offer a controlled chemical composition, and which lack the soft environment encountered in vivo and the chemical stimuli that promote cell proliferation and allow complex cellular behavior. In this study, pectin-based hydrogels were developed and are proposed as versatile cell culture systems.
Pectin-based hydrogels were produced by internally crosslinking pectin with calcium carbonate at different initial pH, aiming to control crosslinking kinetics and degree. Additionally, glucose and glutamine were added as additives, and their effects on the viscoelastic properties of the hydrogels and on cell viability were investigated.
Pectin hydrogels showed in high cell viability and shear-thinning behavior. Independently of hydrogel composition, an initial swelling was observed, followed by a low percentage of weight variation and a steady-state stage. The addition of glucose and glutamine to pectin-based hydrogels rendered higher cell viability up to 90%-98% after 1 hour of incubation, and these hydrogels were maintained for up to 7 days of culture, yet no effect on viscoelastic properties was detected.
Pectin-based hydrogels that offer tunable composition were developed successfully. They are envisioned as synthetic extracellular matrix (ECM) either to study complex cellular behaviors or to be applied as tissue engineering substitutes.
Post author correction
Article Type: ORIGINAL RESEARCH ARTICLE
Article Subject: Biomaterials and nanotechnology
AuthorsRoberta Gentilini, Fabiola Munarin, Nora Bloise, Eleonora Secchi, Livia Visai, Maria Cristina Tanzi, Paola Petrini
- • Accepted on 08/12/2017
- • Available online on 21/12/2017
This article is available as full text PDF.
Until recently, cell line and primary cell cultures have conventionally been created on 2-dimensional (2D) substrates. Although 2D cell culture systems have provided important mechanistic insights, they lack the 3-dimensional (3D) complexity of the in vivo environment, as provided by the morphological and topographical features of the extracellular matrix (ECM). Properties of the substrates, such as matrix stiffness and porosity, as well as surface topography, affect cellular responses including those directing cell fate (1-2-3-4-5). Three-dimensional substrates better mimic the natural in vivo setting compared with traditional 2D cell cultures in polystyrene.
The need for 3D environments for cell cultures is shared by different fields of application, such as in the development of constructs for regenerative medicine and in vitro models, a recent field of application extending the know-how derived from extensive research in regenerative medicine, to systems that mimic specific issues for different tissues (6, 7). In vitro models can be employed as fast screening tools for new therapeutics, selectively filtering active compounds before application in in vivo models (8, 9). Alternatively, they can be designed to study biological mechanisms underlying different pathologies (7, 10). Three-dimensional environments have been obtained with porous scaffolds (11, 12), as soft hydrogel (13-14-15-16) or microspheres (16-17-18).
Biophysical characteristics include the mechanical forces between adhering cells and their surroundings, which differ in 2D and 3D environments. The polymeric or glass substrates for 2D in vitro cultures are usually stiffer compared with the soft hydrated ECM, which is more closely mimicked by hydrogels. First of all, hydrogels, as 3D soft environments, overcome spatial limitations and allow cell mobility and proliferation. In addition, it is highly desirable to provide the appropriate mechanical cues for the different physiological cell environments, with stiffness ranging from brain mimicking (0.1-1 kPa) to muscle-like and bone-like (8-10 and 25-40 kPa, respectively). The quest for hydrogels as a tunable mechanical environment is an advanced and recent topic of research, and an increasing range of hydrogels are reported to modify only the mechanical properties, independently from the biochemical cues (3, 20-21-22-23-24-25-26-27). Among the different substrates, polysaccharide hydrogels based on pectin can be produced with a range of stiffnesses by controlling the mechanism of crosslinking in mild conditions compatible with cell encapsulation (17, 28-29-30).
The issue of recreating the ECM’s complex chemical environment might possibly be addressed by the tunable and controlled release of encapsulated bioactive agents from hydrogel matrixes (6, 9, 14, 19, 31-32-33-34-35-36-37-38-39-40), thus providing the necessary stimuli for cell migration, differentiation and production of neo-tissue growth or regeneration. The release of active molecules is limited by the difficulty of retaining their bioactivity and achieving in situ, optimal doses, gradients and timing.
Furthermore, the optimization of both density and 3D distribution of such molecules in the hydrogel structure needs to be tailored to the specific tissue to be regenerated. As an example, when delivered without stabilization, growth factors diffuse rapidly, undergo denaturation and consequently lose bioactivity under normal physiological conditions (30-31-32). The suboptimal release of growth factors, at high concentration or over a prolonged time, may lead to serious side effects, including the growth of tumors (41, 42). Thus, it is necessary to sustain bioactivity by developing controlled release systems (33, 34, 37-38-39-40) that can mimic the in vivo environment and the ECM’s complex signaling.
In this work, we put forward the possibility of using simple, nonimmunogenic and non-patient-specific molecules, such as glucose (Glc) and glutamine (Gln), present in extracellular fluids and typically included in cell culture media, as universal additives for the production of 3D cell culture systems based on pectin hydrogels. These systems can represent a basic model to be implemented with more specific biomolecules for different purposes, and may be a cost-effective solution for in vitro 3D cell growth either for tissue engineering or for the development of in vitro tissue models.
Materials and methods
Pectin CU701 powder (lot 01011968) was kindly provided by Herbstreith & Fox (Neuenbürg, Germany). NaCl, NaHCO3, CaCO3 and Glc were supplied by Sigma Aldrich and used as received. Gln and all other reagents used for cell culture were purchased from Lonza, unless otherwise specified.
Preparation of the injectable hydrogels
Pectin solutions were obtained by dissolving 2.4% (w/v) pectin powders in 0.9% (w/v) NaCl, adjusting the pH with 23 mM NaHCO3 as previously optimized (32). Pectin solutions were also prepared at the native pH – i.e., without NaHCO3 – to be used as positive control (Pe0Ca). Pectin solutions were enriched with Glc or Gln, or with both additives in varying concentrations, up to 50 and 4 mM, respectively, prior to gel formation (
Hydrogel formulations (2% w/v pectin with 0.9% NaCl), crosslinked with 50 mM CaCO3, employed for cell immobilization
|NaHCO3 (mM)||Glucose (mM)||Glutamine (mM)||Pectin solution||Hydrogel, as prepared||Hydrogels incubated in medium|
|The presence of 25 mM glucose and 2 mM glutamine is indicated as Glc and Gln, respectively, in the names of the samples, while Gln/2 and Gln/2 and 2Glc and 2Gln indicate half or double the said amounts, respectively. The pH and gel point of the different gel formulations are also reported.|
|PeCa Glc/2 + Gln/2||23||12.5||1.0||3.64||5.93||7.39|
|PeCa Glc + Gln||23||25||2||3.65||5.91||7.36|
|PeCa 2Glc + 2Gln||23||50||4.0||3.68||5.88||7.31|
To form pectin hydrogels, 50 mM CaCO3 was suspended in deionized water and sonicated at a frequency of 35 kHz, temperature 30°C for 60 minutes, then gently mixed with pectin solutions using a dual-syringe system (see supplementary figure 1, available online as supplementary material at www.artificial-organs.com – Procedure to form pectin hydrogels).
The gelling mixture was poured into 24-well plates to obtain size-controlled crosslinked samples (diameter 1 cm), and it was allowed to gel at 23°C ± 1°C for 10 minutes before adding the culture medium for incubation.
Characterization of the produced hydrogels
Measurements of pH
The pH of pectin solutions and hydrogels was measured with a Cyberscan pH 110219 pH meter (Eutech Instruments), using a specific electrode for soft hydrogels (Hamilton double pore slim). The pH measurements were performed on pectin solutions, before gelling, and on pectin hydrogels, immediately after preparation and after 1 hour of immersion in RPMI 1640 culture medium (Gibco).
Optical microanalysis of pectin hydrogels
The optical setup consisted of a standard Olympus BX61 microscope equipped with long working distance objectives. Sample images were acquired with a simple CCD camera (12 bit, 640 × 480 pixels, 7.4 × 7.4 μm in size; Pike Allied Vision).
For the optical measurements, a volume of about 0.6 mL of sample was confined between 2 optical glass slides. The final sample thickness was 1 mm, and in the observation plane, the sample area was 2 × 3 cm. The observation window was 860 × 645 μm in size. The gel sample was kept in conditions of controlled humidity to prevent evaporation.
A typical measurement consisted of a sequence of 200 images, acquired with a frame rate of 0.2 frames/s, an exposure time of 300 milliseconds, and a condenser numerical aperture (NA) = 0.3.
Rheological analysis was performed using an AR 1500ex rheometer (TA Instruments, Italy), equipped with parallel-plate geometry (diameter = 20 mm, working gap = 1,000 μm). Time sweeps (temperature 25°C, frequency 0.7 Hz, σ = 5 Pa) and frequency sweep (temperature 25°C, frequency 0.1-10 Hz, σ = 5 Pa) were conducted to evaluate the rheological parameters (storage modulus, tan δ and complex viscosity) of the pectin hydrogels.
Pectin hydrogel samples with an initial wet weight of 0.50 ± 0.05 g were immersed in culture medium (RPMI 1640) at room temperature for up to 24 hours. The percentage of weight variation was calculated [
where Wt is the wet weight of gels at time point t and W0 is the initial weight of the gels, measured immediately after their preparation.
Stability was determined by incubating pectin hydrogels (initial wet weight = 0.50 ± 0.05 g) in RPMI 1640 culture medium at 37°C and measuring their percentage weight loss (WL%) for up to 7 days [
where Wf is the final dry weight and W0 is the initial dry weight of the samples.
A L929 cell line (mouse fibroblasts) purchased from the American Type Culture Collection (Rockville, MO, USA) was cultured in RPMI 1640 culture medium (pH 8.2), supplemented with 10% (v/v) fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate and 1% penicillin/streptomycin. Cells were used within 7 to 9 passages after defrosting. Cells were incubated at 37°C in a 5% CO2 atmosphere, until 70% confluence, and then trypsinized to be passaged.
L929 cells were immobilized in the different hydrogel formulations (
L929 cells were suspended in pectin solutions with a density of 2.5 × 106 cells/mL. The cell-loaded solutions were crosslinked with 50 mM CaCO3. Then, the crosslinking hydrogel (0.4 mL) was extruded from the dual-syringe system (see supplementary figure 1B, available online at www.artificial-organs.com – Procedure to form pectin hydrogels) into each well of a 24-well cell culture plate. The hydrogel was allowed to set for 5 minutes before the addition of 1 mL of culture medium, and the plates were incubated in controlled atmosphere, at 37°C in 5% CO2.
Cell viability and metabolic activity
Trypan blue assay was performed after 1, 2, 4 and 24 hours of incubation. Cell pellets were recovered after dissolving the hydrogels with 1 mL of 0.9% (w/v) NaCl and centrifuging the samples at 1,300 rpm for 3 minutes. Then, the pellet was resuspended with 80 μL of trypan blue solution and 20 μL of fresh culture medium. Then 6.6 μL of the sample was drawn into a KOVA Slide2 chamber (Glasstic Slide 10; KOVA International), and the cells were counted under an optical microscope (Olympus IX50). Cell viability (%) was expressed as percentage of the live cell number/the total cell number.
Metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 4 and 24 hours of incubation of the cell-loaded gels, gels were dissolved, and the recovered cells were removed from the well with the gels and left overnight in new wells to proliferate. Then, MTT solution (5 mg MTT/1.5 mL phosphate-buffered saline) was added, and the cells were incubated at 37°C. After 3 hours, the MTT solution was removed and replaced with 0.5 mL/well of isopropanol in 0.04 N HCl. The wells were stirred for 5 minutes until their color was uniform. Absorbance was evaluated at 595 nm. The analyses were performed in triplicate, using cells seeded on thermoresponsive tissue culture polystyrene (TCPS) as control.
To determine the viability of L929 cells during the first hour of immobilization, without dissolving the pectin hydrogels, a LIVE/DEAD® Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA) was used. The assay was performed according to the manufacturer’s instructions. Stained cells were examined under a Leica confocal laser scanning microscope (CLSM, model TCS SPII; Leica, Heidelberg, Germany) using a × 20 oil immersion objective. The excitation and emission wavelengths used for detecting calcein-AM were 494 nm and 517 nm, respectively. Ethidium homodimer-1 was excited at 527 nm, and its emission was monitored at 617 nm. Optical sections of 0.9 mm were collected from the complete thickness of the samples, and for each sample, images from 3 randomly selected positions were acquired. The resulting stacks of images were analyzed using Leica confocal software.
All measurements were made in triplicate, the results are presented as average weight variation and errors were calculated, with standard deviation. One-way analysis of variance (ANOVA) with Bonferroni post hoc analysis was used to determine any significant difference between the means of the samples. A difference was considered significant when the value of p was <0.05.
Pectin hydrogels were obtained by internal gelation with calcium carbonate by mixing pectin and an aqueous suspension of the insoluble calcium (Ca2+) salt (23) in a dual-syringe system (see supplementary figure 1, available online at www.artificial-organs.com – Procedure to form pectin hydrogels). The gelling mixture was extruded while the homogeneous dispersion was still behaving as a viscous liquid (<2 min from the beginning of the mixing), as determined by rheological measurements (data not shown). With this straightforward method, samples with reproducible size, shape and rheological properties were obtained.
Measurements of pH
The pH of pectin solutions increased during the crosslinking process for all of the formulations (
Optical microanalysis of pectin hydrogels
CaCO3 grain distribution and dimensions did not vary with aging (1, 4 and 24 hours). The use of pectin with higher pH led to the formation of hydrogels with a more homogeneous structure, having undissolved CaCO3 grains equally distributed within the gel volume (
Images of PeCa solution (
When the initial pH of pectin solutions increased (PeCa formulations, initial pH 3.69), the microstructure of the hydrogel was homogeneous within the whole sample volume, though CaCO3 grains were not completely dissolved. The amount of CO2 bubbles nucleated in these formulations was significantly lower than in the Pe0Ca hydrogels.
Both storage modulus G’ and tan δ exhibited a frequency-independent behavior in the frequency ranges considered (
Storage modulus and complex viscosity were significantly higher for Pe0Ca than for hydrogels produced at increased pH of pectin solutions. No substantial effects over rheological properties were observed when comparing hydrogels enriched with Glc and Gln with PeCa hydrogels (
The presence of a high density of L929 cells in PeCa and PeCa 2Glc + 2Gln (i.e., twice 25 mM Glc and twice 2 mM Gln) formulations did not induce significant changes in the rheological behavior of pectin hydrogels (
Storage modulus and complex viscosity (inset (
Swelling and stability
Hydrogel swelling increased with time due to water absorption until equilibrium was reached after 4 hours of incubation, with average swelling in the range of 35%-70%, depending on the gel formulation. No clear trends could be found related to the amounts of additives (Glc and Gln) used in the gels (
All of the formulations tested lost between 8% to 20% their initial dry weight after 24 hours of incubation (
Cell immobilization and viability
To evaluate separately the contribution of each additive, L929 cell viability assessed in the hydrogels without additives (Pe0Ca and PeCa) was compared with that in hydrogels enriched with either Glc or Gln. Poor L929 viability was observed in Pe0Ca samples from 1 hour up to 24 hours of incubation, due to the acidic pH of the hydrogels (
The results demonstrated that the presence of Glc and Gln improved cell viability in the short-term period, yet no differences were found in the long term, even when additives were used with high concentrations (
Confocal microscopy performed on the 3D hydrogels loaded with fibroblasts, confirmed that for all PeCa formulations, most of the cells were viable (green fluorescence) and homogeneously dispersed in the 3D pectin network, and only a very few dead (red) cells were randomly distributed in the hydrogels (
The current study proposed that a pectin-based hydrogel could provide a 3D platform for cell growth and proliferation. This platform could provide a base with a chemical composition that can be further tailored by the appropriate addition of different molecules, to serve as a synthetic ECM.
To achieve this, pectin hydrogels were obtained by internal gelation, mixing pectin and L929 cells with a poorly soluble calcium salt (i.e., calcium carbonate). The pH of pectin solutions was increased to reduce the release of the calcium (Ca2+) ions involved in the gelation process, as well as to create a suitable environment for cell hosting. During crosslinking, only a thin superficial layer of the CaCO3 grains was dissolved, as grains with comparable size and shape were measured before and after gelation (
By controlling the kinetics of the crosslinking reaction, it was possible to obtain a homogeneous distribution of the cells along the 3 axes of the 3D hydrogel structure while retaining their viability upon extrusion (
Pectin solutions with lower pH (Pe0Ca) promoted the rapid dissolution of CaCO3 powders (
In 3D networks, the viability of the immobilized cells can be reduced in the first hours of immobilization, due to the reduced perfusion and availability of nutrients. We therefore aimed to enrich the produced hydrogels with nonspecific additives to support the viability and metabolic activity of any cell type that could possibly be immobilized for tissue regeneration.
It has been generally accepted that Glc and Gln are the main carbon and energy sources of cell culture media (47-48-49-50). Glc consumption is one of the key indicators of cell metabolic activity, and it tightly correlates with the cell growth rate or the cell population dynamics. In 2D in vitro models, when the cell population increases rapidly (up to 3 or 4 days), a large amount of Glc is consumed for cell proliferation (about 25 fmol/cell per day). Then, the cell population comes into steady phase, and Glc is mainly consumed for cell survival (48). Gln is naturally involved in a wide variety of processes, including energy production, nitrogen metabolism, ammonia detoxification and amino acid and protein metabolism (47), and under specific conditions, it can contribute significantly to cellular metabolism (51, 52).
According to the literature, high doses of Glc (about 20 mM) and Gln (2 mM) maintain cell viability and proliferation even in hypoxic conditions (47).
Glc and Gln appeared to maintain cell viability, even when used separately and at low concentrations (
Regarding soft tissue regeneration and the development of in vitro tissue models, the soft hydrogels developed, enriched with Glc and Gln, represent a valid system, showing high viability of the immobilized cells, even in the short-term period.
In this work, pectin-based injectable hydrogels enriched with Glc and Gln were proposed as 3D microenvironments for cell encapsulation. The selected additives preserved the fibroblast cell viability even in the short-term period, which is known to be the most critical phase of cell immobilization. These additives can be used in different hydrogels to improve the viability of different cell types for the regeneration of diverse tissues. The possibility of tuning the composition of the pectin hydrogels, as shown in this work with nonspecific additives such as Glc and Gln, can be explored further and expanded to incremental levels of complexity for in vitro models or for tissue engineering, by addition of parameters to be implemented and controlled one by one.
The authors would like to acknowledge Herbstreith & Fox (Neuenbürg, Germany) for kindly providing the low methoxyl pectin used in this study. The authors also wish to thank Drs. Stefano Buzzaccaro and Matteo David Alaimo for their generous support in the optical analysis.
- Gentilini, Roberta [PubMed] [Google Scholar] 1, 2
- Munarin, Fabiola [PubMed] [Google Scholar] 1
- Bloise, Nora [PubMed] [Google Scholar] 3
- Secchi, Eleonora [PubMed] [Google Scholar] 4
- Visai, Livia [PubMed] [Google Scholar] 3, 5
- Cristina Tanzi, Maria [PubMed] [Google Scholar] 1
- Petrini, Paola [PubMed] [Google Scholar] 1, * Corresponding Author (firstname.lastname@example.org)
Department of Chemistry, Materials and Chemical Engineering, Unità di Ricerca Consorzio INSTM, Politecnico di Milano, Milan - Italy
Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan - Italy
Department of Molecular Medicine, Unità di Ricerca Consorzio INSTM and Center for Tissue Engineering (C.I.T.), University of Pavia, Pavia - Italy
Department of Chemistry, Materials and Chemical Engineering, Politecnico di Milano, Milan - Italy
Salvatore Maugeri Foundation, IRCCS Scientific Institutes for Research and Care, Pavia - Italy
Roberta Gentilini and Fabiola Munarin contributed equally to this work.
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