Cell Culture Applications Research Articles

Fiber Thickness and Porosity Control in a Biopolymer Scaffold 3D Printed through a Converted Commercial FDM Device.

3D printing has opened exciting new opportunities for the in vitro fabrication of biocompatible hybrid pseudo-tissues. Technologies based on additive manufacturing herald a near future when patients will receive therapies delivering functional tissue substitutes for the repair of their musculoskeletal tissue defects. In particular, bone tissue engineering (BTE) might extensively benefit from such an approach. However, designing an optimal 3D scaffold with adequate stiffness and biodegradability properties also guaranteeing the correct cell adhesion, proliferation, and differentiation, is still a challenge. The aim of this work was the rewiring of a commercial fuse deposition modeling (FDM) 3D printer into a 3D bioplotter, aiming at obtaining scaffold fiber thickness and porosity control during its manufacturing. Although it is well-established that FDM is a fast and low-price technology, the high temperatures required for printing lead to limitations in the biomaterials that can be used. In our hands, modifying the printing head of the FDM device with a custom-made holder has allowed to print hydrogels commonly used for embedding living cells. The results highlight a good resolution, reproducibility and repeatability of alginate/gelatin scaffolds obtained via our custom 3D bioplotter prototype, showing a viable strategy to equip a small-medium laboratory with an instrument for manufacturing good-quality 3D scaffolds for cell culture and tissue engineering applications.

3D printing: Economical and supply chain independent single-use plasticware for cell culture

3D printing represents a democratization of manufacturing processes, and inexpensive 3D printed parts for cell culture have been tested as replacements for single-use plastics currently unavailable due to worldwide supply chain issues. In addition, such distributed manufacturing of cell culture laboratory materials helps remote areas and developing countries with limited resources. HEK293 cells were used to test printed shake flasks for cell culture applications and their ease of manufacture. Recorded growth curves showed that renewable biodegradable poly(lactic acid) (PLA) thermoplastic is an excellent and economical replacement for single-use plastic shake flasks, which have shipment lead times during pandemic situations or other supply chain disruptions of over 6 months. With a price of 0.60 € in materials, and printing machines with prices lower than one box of single-use pre-sterilized plastic shake flasks (<350€), the use of PLA is very affordable. Low-cost photopolymerization resins were also tested, but the inherent cytotoxicity of these materials prevented cell growth. This was also true for plant-based resins marketed as having low volatile organic compounds (VOC). Treatment of parts to reduce VOC content was partially successful, but not sufficient to sustain prolonged cell growth. A high-cost medical device IIa-class material showed no improved cell growth. Nevertheless, with PLA a low-cost printing material was identified and the use as cell culture compatible material was demonstrated, providing low-cost supply chain independence. In the future, the printing of pilot-scale bioreactors with PLA as a green sustainable material at the point of its use will be possible.

Polymeric Hydrogels for In Vitro 3D Ovarian Cancer Modeling.

Ovarian cancer (OC) grows and interacts constantly with a complex microenvironment, in which immune cells, fibroblasts, blood vessels, signal molecules and the extracellular matrix (ECM) coexist. This heterogeneous environment provides structural and biochemical support to the surrounding cells and undergoes constant and dynamic remodeling that actively promotes tumor initiation, progression, and metastasis. Despite the fact that traditional 2D cell culture systems have led to relevant medical advances in cancer research, 3D cell culture models could open new possibilities for the development of an in vitro tumor microenvironment more closely reproducing that observed in vivo. The implementation of materials science and technology into cancer research has enabled significant progress in the study of cancer progression and drug screening, through the development of polymeric scaffold-based 3D models closely recapitulating the physiopathological features of native tumor tissue. This article provides an overview of state-of-the-art in vitro tumor models with a particular focus on 3D OC cell culture in pre-clinical studies. The most representative OC models described in the literature are presented with a focus on hydrogel-based scaffolds, which guarantee soft tissue-like physical properties as well as a suitable 3D microenvironment for cell growth. Hydrogel-forming polymers of either natural or synthetic origin investigated in this context are described by highlighting their source of extraction, physical-chemical properties, and application for 3D ovarian cancer cell culture.

Synthesis of Poly(acrylic acid)-Cysteine-Based Hydrogels with Highly Customizable Mechanical Properties for Advanced Cell Culture Applications.

The fabrication of highly customizable scaffolds is a key enabling technology in the development of predictive in vitro cell models for applications in drug discovery, cancer research, and regenerative medicine. Naturally derived and synthetic hydrogels are good candidates for in vitro cell growth studies, owing to their soft and biocompatible nature; however, they are often hindered by limited ranges of stiffness and the requirement to modify the gel with additional extracellular matrix (ECM) proteins for cell adherence. Here, we report on the synthesis of a printable synthetic hydrogel based on cysteine-modified poly(acrylic acid) (PAA-Cys) with tuneable mechanical and swelling properties by incorporating acrylic acid into the PAA-Cys network and subsequent photoinitiated thiol-acrylate cross-linking. Control of the acrylic acid concentration and UV curing time produces a series of hydrogels with swelling ratios in excess of 100% and Young’s modulus values ranging from ∼2 to ∼35 kPa, of which most soft tissues fall within. Biocompatibility studies with RPE1 cells showed excellent cell adhesion and cell viability without the need for further modification with ECM proteins, but still can be modified as needed. The versatility of the hydrogel tuneable properties is demonstrated by culturing with RPE1 cells, which in vivo perform an important function in the visual process and the dysfunction of which may lead to various retinal abnormalities, such as glaucoma.

Macroscopic membranes self‐assembled by alginate and a cationic and amphiphilic peptide for cell culture

AbstractPeptides self‐assembly systems have been explored over the past years in the context of various biological applications, such as tissue engineering and drug delivery. Here we present the development of hierarchically ordered planar and spherical macroscopic membranes constructed by an amphiphilic and cationic β‐sheet peptide that spontaneously assembles with the negatively charged alginate biopolymer. These two components generate a condensed film that developed to exhibit perpendicularly oriented fibers under specific conditioning over days. Here we examined the suitability of these spherical and planar membranes to serve as substrates for cell culture applications. Planar and spherical membranes were stable in the cell culture Dulbecco's modified eagle medium, at 37°C, over days. Breast‐cancer cells seeded on the peptide and alginate sides of fully developed and non‐fully developed planar membrane were viable for more than 2 weeks. In addition, cells were injected into spherical membranes and detected viable close to the rim even at 3 weeks. These results demonstrate the suitability of planar and spherical membranes to serve as a platform, for example, in studying cancer cells in unique niches and alternatively in potential allogeneic cell therapies.

Desymmetrization via Activated Esters Enables Rapid Synthesis of Multifunctional Benzene-1,3,5-tricarboxamides and Creation of Supramolecular Hydrogelators.

Supramolecular materials based on the self-assembly of benzene-1,3,5-tricarboxamide (BTA) offer an approach to mimic fibrous self-assembled proteins found in numerous natural systems. Yet, synthetic methods to rapidly build complexity, scalability, and multifunctionality into BTA-based materials are needed. The diversity of BTA structures is often hampered by the limited flexibility of existing desymmetrization routes and the purification of multifunctional BTAs. To alleviate this bottleneck, we have developed a desymmetrization method based on activated ester coupling of a symmetric synthon. We created a small library of activated ester synthons and found that a pentafluorophenol benzene triester (BTE) enabled effective desymmetrization and creation of multifunctional BTAs in good yield with high reaction fidelity. This new methodology enabled the rapid synthesis of a small library of BTA monomers with hydrophobic and/or orthogonal reactive handles and could be extended to create polymeric BTA hydrogelators. These BTA hydrogelators self-assembled in water to create fiber and fibrous sheet-like structures as observed by cryo-TEM, and the identity of the BTA conjugated can tune the mechanical properties of the hydrogel. These hydrogelators display high cytocompatibility for chondrocytes, indicating potential for the use of these systems in 3D cell culture and tissue engineering applications. This newly developed synthetic strategy facilitates the simple and rapid creation of chemically diverse BTA supramolecular polymers, and the newly developed and scalable hydrogels can unlock exploration of BTA based materials in a wider variety of tissue engineering applications.

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies.

Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

Recent Advances in Stimuli-Responsive DNA-Based Hydrogels.

Stimuli-responsive DNA-based hydrogels are attracting growing interest because of their smart responsiveness, excellent biocompatibility, regulated biodegradability, and programmable design properties. Integration of reconfigurable DNA architectures and switchable supramolecular moieties (as cross-linkers) in hydrogels by responding to external stimuli provides an ideal approach for the reversible tuning structural and mechanical properties of the hydrogels, which can be exploited in the development of intelligent DNA-based materials. This review highlights recent advances in the design of responsive pure DNA hydrogels, DNA-polymer hybrid hydrogels, and autonomous DNA-based hydrogels with transient behaviors. A variety of chemically and physically triggered DNA-based stimuli-responsive hydrogels and their versatile applications in biosensing, biocatalysis, cell culture and separation, drug delivery, shape memory, self-healing, and robotic actuators are summarized. Finally, we address the key challenges that the field will face in the coming years, and future prospects are identified.

Compatibility of Popular Three-Dimensional Printed Microfluidics Materials with In Vitro Enzymatic Reactions.

3D printed microfluidics offer several advantages over conventional planar microfabrication techniques including fabrication of 3D microstructures, rapid prototyping, and inertness. While 3D printed materials have been studied for their biocompatibility in cell and tissue culture applications, their compatibility for in vitro biochemistry and molecular biology has not been systematically investigated. Here, we evaluate the compatibility of several common enzymatic reactions in the context of 3D-printed microfluidics: (1) polymerase chain reaction (PCR), (2) T7 in vitro transcription, (3) mammalian in vitro translation, and (4) reverse transcription. Surprisingly, all the materials tested significantly inhibit one or more of these in vitro enzymatic reactions. Inclusion of BSA mitigates only some of these inhibitory effects. Overall, inhibition appears to be due to a combination of the surface properties of the resins as well as soluble components (leachate) originating in the matrix.

Organoid-based expansion of patient-derived primary alveolar type 2 cells for establishment of alveolus epithelial Lung-Chip cultures.

Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.

Decellularized In Vitro Capillaries for Studies of Metastatic Tendency and Selection of Treatment.

Vascularization plays an important role in the microenvironment of the tumor. Therefore, it should be a key element to be considered in the development of in vitro cancer assays. In this study, we decellularized in vitro capillaries to remove genetic material and optimized the medium used to increase the robustness and versatility of applications. The growth pattern and drug responses of cancer cell lines and patient-derived primary cells were studied on decellularized capillaries. Interestingly, two distinct growth patterns were seen when cancer cells were grown on decellularized capillaries: “network” and “cluster”. Network formation correlated with the metastatic properties of the cells and cluster formation was observed in non-metastatic cells. Drug responses of patient-derived cells correlated better with clinical findings when cells were cultured on decellularized capillaries compared with those cultured on plastic. Decellularized capillaries provide a novel method for cancer cell culture applications. It bridges the gap between complex 3D culture methods and traditional 2D culture methods by providing the ease and robustness of 2D culture as well as an in vivo-like microenvironment and scaffolding for 3D cultures.

Investigation and comparison of resin materials in transparent DLP-printing for application in cell culture and organs-on-a-chip.

Organs-on-a-Chip (OOCs) have recently led to major discoveries and a better understanding of 3D cell organization, cell-cell interactions and tissue response to drugs and biological cues. However, their complexity and variability are still limited by the available fabrication technology. Transparent, cytocompatible and high-resolution 3D-printing could overcome these limitations, offering a flexible and low-cost alternative to soft lithography. Many advances have been made in stereolithography printing regarding resin formulation and the general printing process, but a systematic analysis of the printing process steps, employed resins and post-treatment procedures with a strong focus on the requirements in OOCs is missing. To fill this gap, this work provides an in-depth analysis of three different resin systems in comparison to polystyrene (PS) and poly(dimethylsiloxane) (PDMS), which can be considered the gold-standards in cell culture and microfluidics. The resins were characterized with respect to transparency, cytocompatibility and print resolution. These properties are not only governed by the resin composition, but additionally by the post-treatment procedure. The investigation of the mechanical (elastic modulus ∼2.2 GPa) and wetting properties (∼60° native / 20° plasma treated) showed a behavior very similar to PS. In addition, the absorbance of small molecules was two orders of magnitude lower in the applied resins (diffusion constant ∼0.01 μm2 s-1) than for PDMS (2.5 μm2 s-1), demonstrating the intrinsic suitability of these materials for OOCs. Raman spectroscopy and UV/VIS spectrophotometry revealed that post-treatment increased monomer conversion up to 2 times and removed photo initiator residues, leading to an increased transparency of up to 50% and up to 10-times higher cell viability. High magnification fluorescence imaging of HUVECs and L929 cells cultivated on printed dishes shows the high optical qualities of prints fabricated by the Digital Light Processing (DLP) printer. Finally, components of microfluidic chips such as high-aspect ratio pillars and holes with a diameter of 50 μm were printed. Concluding, the suitability of DLP-printing for OOCs was demonstrated by filling a printed chip with a cell-hydrogel mixture using a microvalve bioprinter, followed by the successful cultivation under perfusion. Our results highlight that DLP-printing has matured into a robust fabrication technology ready for application in extensive and versatile OOC research.

Functional amyloid-chitin hybrid ink coupled with flexible fabrication approaches for diverse macro and micro-structures.

The precise fabrication of artificially designed molecular complexes into ordered structures resembling their natural counterparts would find broad applications but remains a major challenge in the field. Here we genetically design chitin-binding domain (CBD)-containing amyloid proteins, and rationally fabricate well-ordered CBD-containing functional amyloid-chitin complex structures by coupling a top-down manufacturing process with a bottom-up self-assembly. Our fabrication approach starts with the dissolution of both CBD-containing functional amyloid and chitin in hexafluoroisopropanol (HFIP) to make a hybrid ink. This hybrid ink platform, coupled with multiple fabrication methods including airbrushing, electrospinning and soft-lithography, produces a series of unique freestanding structures. The structural features of the products, such as the ability to direct the light path and mimicking of the extracellular matrix enable applications in functional light gratings and cell culture, respectively. Further genetic engineering of the protein component allowed tunable functionalization of these materials, including nanoparticle immobilization and protein conjugation, resulting in broad applications in electronic devices and enzyme immobilization. Our technological platform can drive new advances in biocatalysis, tissue engineering, biomedicine, photonics and electronics.

Application of Monolayer Cell Cultures for Investigating Basic Mechanisms of Photodynamic Therapy.

Conventional monolayer cell cultures continue to be an inexpensive and highly accessible model of human disease that can be easily harnessed to study the molecular and cellular mechanisms of photodynamic therapy (PDT). In this communication, a collection of informative assays for conventional cell cultures are provided to determine (1) the photosensitizer uptake kinetics and localization, (2) the efficacy of PDT using metabolism- or protein-based quantification methods, (3) the effects of PDT and combination treatments on the cell cycle, (4) the cell death pathways induced by PDT, and (5) the extent of mitochondrial membrane permeabilization of PDT and photochemotherapy combinations. For each type of assay, examples from the recent literature are provided in which novel photosensitizers, their nanocarriers, and various PDT-based combination therapies are investigated. Together, these assays are examples of approaches by which monolayer cell cultures can be used as a simple yet robust and versatile model to investigate PDT.

Methods for Processing Protein Aggregates into Surfaces.

The processing of inclusion bodies (IBs) into surfaces is of great interest for cell culture applications due to the combined physical and biological cues these particles provide. The arrangement of these IBs into defined and tunable micropatterns can be useful for basic research purposes regarding the mechanical properties needed for cell adhesion and migration, among other responses. There are several approaches that can be used when functionalizing a substrate with IBs, regarding both the strategy used and also the kind of surface-particle interaction. The interaction between surface and IB can be mainly of three types: physisorption, electrostatic or covalent. This interaction can be controlled by depositing an appropriate self-assembled monolayer (SAM) on top of a substrate as an interface. Furthermore, several strategies can be used to immobilize IBs on surfaces in various configurations, like random deposition, micrometric printed geometries or gradient patterns.

3D Printing of Biocompatible Scaffolds for Eye Tissue Engineering

Eye tissue engineering is becoming a promising field in the study and treatment of vitreoretinal diseases, and novel hydrogels are under investigation for vitreous body replacement and 3D cell culture applications. In order to handle a not self-standing hydrogel and to provide it with eye shape, a rigid scaffold that is transparent, non-toxic, and easy to move is needed. To this purpose, we produced bowl-shaped samples by stereolithography (SLA) starting from different commercial transparent resins. We optimized the 3D printing protocol by tuning parameters such as exposure time, layer thickness, and light blocker content added to the resins. The specimens that showed the best dimensional accuracy were chosen to perform biocompatibility tests at different time-point and transparency tests under an optical microscope. Preliminary results showed that the cell culture medium conditioned by the first tested resin formulations caused a massive cell death in the MS5 cell line tested, whereas different formulations did not impact on cell viability. Furthermore, the as built resin samples did not allow cell observation under inverted microscope. It was demonstrated that post-processing progressive grinding remarkably improved cell visualization.

Profile of Flavonoid and Antioxidant Activity in Cell Suspension Culture of Elaeocarpus grandiflorus

Cell suspension cultures of Elaeocarpus grandiflorus produce flavonoids and various secondary metabolites. Flavonoid profile and antioxidant activity of the suspension culture extract of E. grandiflorus cells have not been studied. Therefore, this research aimed to analyze the antioxidant activity and flavonoid profile of the suspension culture extract of E. grandiflorus cells. Cell suspension cultures of E. grandiflorus were produced from leaf stalk callus that grew on WPM medium with a growth regulator 2.5 ppm 2,4-D. Cells were harvested at 30 days old and then extracted for profile analysis of bioactive compounds using the LC-MS method. The antioxidant activity analysis was conceded out using the DPPH method. The results showed that there were 32 types of flavonoids, of which 11 compounds had a concentration of more than 1% of the total bioactive compounds and had the potential to have antioxidant activity. The analysis results also showed that flavonoids type, composition, and antioxidant activity were not significantly different between the ages of E. grandiflorus cell suspension cultures. In addition, there was no correlation between flavonoid concentration and antioxidant activity. This study enriches information about secondary metabolites production in the E. grandiflorus cell culture which is still very rarely studied because of its rarity. The study also provides potential antioxidant as a scientific basis evident for E. grandifloras cell culture application in medical purpose.

Conductive Bioimprint Using Soft Lithography Technique Based on PEDOT:PSS for Biosensing.

Culture platform surface topography plays an important role in the regulation of biological cell behaviour. Understanding the mechanisms behind the roles of surface topography in cell response are central to many developments in a Lab on a Chip, medical implants and biosensors. In this work, we report on a novel development of a biocompatible conductive hydrogel (CH) made of poly (3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) and gelatin with bioimprinted surface features. The bioimprinted CH offers high conductivity, biocompatibility and high replication fidelity suitable for cell culture applications. The bioimprinted conductive hydrogel is developed to investigate biological cells’ response to their morphological footprint and study their growth, adhesion, cell–cell interactions and proliferation as a function of conductivity. Moreover, optimization of the conductive hydrogel mixture plays an important role in achieving high imprinting resolution and conductivity. The reason behind choosing a conducive hydrogel with high resolution surface bioimprints is to improve cell monitoring while mimicking cells’ natural physical environment. Bioimprints which are a 3D replication of cellular morphology have previously been shown to promote cell attachment, proliferation, differentiation and even cell response to drugs. The conductive substrate, on the other hand, enables cell impedance to be measured and monitored, which is indicative of cell viability and spread. Two dimensional profiles of the cross section of a single cell taken via Atomic Force Microscopy (AFM) from the fixed cell on glass, and its replicas on polydimethylsiloxane (PDMS) and conductive hydrogel (CH) show unprecedented replication of cellular features with an average replication fidelity of more than 90%. Furthermore, crosslinking CH films demonstrated a significant increase in electrical conductivity from 10−6 S/cm to 1 S/cm. Conductive bioimprints can provide a suitable platform for biosensing applications and potentially for monitoring implant-tissue reactions in medical devices.

Development of a mechanically stable human hair keratin film for cell culture

An easy-to-handle keratin film was successfully fabricated using solely purified hair keratins. Keratin was extracted from human hair by an existing protocol. The extracted keratin was made into a mechanically stable film by solution casting and air-drying at room temperature. The films obtained were characterized for surface morphology, wettability, protein secondary structures, mechanical properties, permeability, and thermal properties. Interestingly, the keratin film showed distinct surface and cross-sectional morphology, and protein secondary structure transformation. In addition, the keratin film exhibited Young’s modulus of 1.05 ± 0.09 GPa when it was dry. In the wet state, the keratin film behaved as viscoelastic material and was highly stretchable at 179 ± 17% strain at break. Permeability test was conducted using 20 kDa-FITC dextran which revealed an anomalous diffusion mechanism through the keratin film. Additionally, the keratin film elicited positive cellular responses by human epidermal keratinocytes (HEKs) in terms of enhanced cell proliferation, viability, keratin 14 expression, and IL-1α secretion, in comparison to collagen I. Taken together, a human hair keratin-based film with its mechanical and thermal stability, and cytocompatibility, presents a promising platform for cell culture applications.

Squarate Cross-Linked Gelatin Hydrogels as Three-Dimensional Scaffolds for Biomedical Applications.

Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.