Cystic Fibrosis Mucus Model to Design More Efficient Drug Therapies.

Mucus represents a strong barrier to tackle for oral or pulmonary administered drugs, especially in mucus-related disorders. This study uses a pathological cystic fibrosis (CF) mucus model to investigate how mucus impacts the passive diffusion of 45 ad hoc commercial drugs selected to maximize physicochemical variability. An in vitro mucosal surface was recreated by coupling the mucus model to a 96-well permeable support precoated with structured layers of phospholipids (parallel artificial membrane permeability assay, PAMPA). Results show that the mucus model was not a mere physical barrier but it behaves like an interactive filter. In nearly one-half of the investigated compounds, the diffusion was reduced by mucus, while other drugs were not sensitive to the mucus barriers. We also found that permeability can be enhanced when drug-calcium salts are formed. This was confirmed with cystic fibrosis sputum as a rough ex vivo model of CF mucus. Since the drug discovery process is characterized by a high rate of failure, the mucus platform is expected to provide an efficient support to early reduce the number of poor-performing drug candidates.

Technological tools and strategies for culturing human gut microbiota in engineered in vitro models.

The gut microbiota directly impacts the pathophysiology of different human body districts. Consequently, microbiota investigation is an hot topic of research and its in vitro culture has gained extreme interest in different fields. However, the high sensitivity of microbiota to external stimuli, such as sampling procedure, and the physicochemical complexity of the gut environment make its in vitro culture a challenging task. New engineered microfluidic gut-on-a-chip devices have the potential to model some important features of the intestinal structure, but they are usually unable to sustain culture of microbiota over an extended period of time. The integration of gut-on-a-chip devices with bioreactors for continuous bacterial culture would lead to fast advances in the study of microbiota-host crosstalk. In this review, we summarize the main technologies for the continuous culture of microbiota as upstream systems to be coupled with microfluidic devices to study bacteria-host cells communication. The engineering of integrated microfluidic platforms, capable of sustaining both anaerobic and aerobic cultures, would be the starting point to unveil complex biological phenomena proper of the microbiota-host crosstalks, paving to way to multiple research and technological applications.

Engineered modular microphysiological models of the human airway clearance phenomena.

Mucociliary clearance is a crucial mechanism that supports the elimination of inhaled particles, bacteria, pollution, and hazardous agents from the human airways, and it also limits the diffusion of aerosolized drugs into the airway epithelium. In spite of its relevance, few in vitro models sufficiently address the cumulative effect of the steric and interactive barrier function of mucus on the one hand, and the dynamic mucus transport imposed by ciliary mucus propulsion on the other hand. Here, ad hoc mucus models of physiological and pathological mucus are combined with magnetic artificial cilia to model mucociliary transport in both physiological and pathological states. The modular concept adopted in this study enables the development of mucociliary clearance models with high versatility since these can be easily modified to reproduce phenomena characteristic of healthy and diseased human airways while allowing to determine the effect of each parameter and/or structure separately on the overall mucociliary transport. These modular airway models can be available off-the-shelf because they are exclusively made of readily available materials, thus ensuring reproducibility across different laboratories.

3D-Reactive printing of engineered alginate inks.

Alginate is a common component of bioinks due to its well-described ionic crosslinking mechanism and tunable viscoelastic properties. Extrusion-based 3D-printing of alginate inks requires additives, such as gelatin and Pluronic, pre- or post-printing crosslinking processes and/or coextrusion with crosslinkers. In this work, we aim to develop a different printing approach for alginate-based inks, introducing 3D-reactive printing. Indeed, the control over the crosslinking kinetics and the printing time allowed printing different inks while maintaining their final composition unaltered to identify a suitable formulation in terms of printability. Alginate solutions were crosslinked with insoluble calcium salts (CaCO3) inducing a dynamic modification of their microstructure and viscoelastic properties over time. The monitoring of fiber printability and internal microstructure, at different time points of ink gelation, was performed by means of a well-defined set of rheological tests to obtain a priori ink properties for the a posteriori 3D-printing process. This new perspective allowed 3D-reactive printing of alginate fibers with predetermined properties, without involving post-extrusion crosslinking steps and additives.

Cross-linked poly(acrylic acids) microgels and agarose as semi-interpenetrating networks for resveratrol release.

Carbomers, cross-linked poly(acrylic acid) microgels, have been widely used in pharmaceutical formulations as swollen hydrogels. Agarose, whose thermoreversibility may be exploited for drug loading, forms a gel with a mechanism involving coil-helix transition at about 36 °C. In this work carbomer microgels were combined with agarose networks in a semi-interpenetrating polymer network structure, aiming at obtaining suitable delivery systems for the loading and release of molecules with poor bioavailability but high therapeutic interest, like resveratrol. The rheological properties of the formulations and their in vitro cytocompatibility were studied and optimized acting on the neutralizing agent (triethylamine (N,N-diethylethanamine), triethanolamine (tris(2-hydroxyethyl)amine) and sodium hydroxide) and amount of OH donors (1,2-propanediol and glycerol). As a preparation method, autoclaving was introduced to simultaneously obtain heating and sterilising. Among the different neutralizing agents, NaOH was chosen to avoid the use of amines, considering the final application. Without the addition of alcohols as typical OH donors to induce Carbomer gelification, gels with appropriate rheological properties and stability were produced. For this formulation, the release of resveratrol after 7 days was about 80 % of the loaded mass, suggesting it is an interesting approach to be exploited for the development of innovative resveratrol delivery systems.

Bioinspired Bioinks for the Fabrication of Chemomechanically Relevant Standalone Disease Models of Hepatic Steatosis.

Hepatotoxicity-related issues are poorly predicted during preclinical experimentation, as its relevance is limited by the inadequacy to screen all the non-physiological subclasses of the population. These pitfalls can be solved by implementing complex in vitro models of hepatic physiology and pathologies in the preclinical phase. To produce these platforms, extrusion-based bioprinting is focused on, since it allows to manufacture tridimensional cell-laden constructs with controlled geometries, in a high-throughput manner. Different bioinks, whose formulation is tailored to mimic the chemomechanical environment of hepatic steatosis, the most prevalent hepatic disorder worldwide, are proposed. Internally crosslinked alginate hydrogels are chosen as structural components of the inks. Their viscoelastic properties (G' = 512-730 Pa and G″ = 94-276 Pa, depending on frequency) are tuned to mimic those of steatotic liver tissue. Porcine hepatic ECM is introduced as a relevant biochemical cue. Sodium oleate is added to recall the accumulation of lipids in the tissue. Downstream analyses on 14-layered bioprinted structures cultured for 10 days reveal the establishment of steatotic-like features (intracellular lipid vesicles, viability decrease up to ≈50%) without needing external conditionings. The presented bioinks are thus suitable to fabricate complex models of hepatic steatosis to be implemented in a high-throughput experimental frame.

Growing Role of 3D In Vitro Cell Cultures in the Study of Cellular and Molecular Mechanisms: Short Focus on Breast Cancer, Endometriosis, Liver and Infectious Diseases.

Over the past decade, the development of three-dimensional (3D) models has increased exponentially, facilitating the unravelling of fundamental and essential cellular mechanisms by which cells communicate with each other, assemble into tissues and organs and respond to biochemical and biophysical stimuli under both physiological and pathological conditions. This section presents a concise overview of the most recent updates on the significant contribution of different types of 3D cell cultures including spheroids, organoids and organ-on-chip and bio-printed tissues in advancing our understanding of cellular and molecular mechanisms. The case studies presented include the 3D cultures of breast cancer (BC), endometriosis, the liver microenvironment and infections. In BC, the establishment of 3D culture models has permitted the visualization of the role of cancer-associated fibroblasts in the delivery of exosomes, as well as the significance of the physical properties of the extracellular matrix in promoting cell proliferation and invasion. This approach has also become a valuable tool in gaining insight into general and specific mechanisms of drug resistance. Given the considerable heterogeneity of endometriosis, 3D models offer a more accurate representation of the in vivo microenvironment, thereby facilitating the identification and translation of novel targeted therapeutic strategies. The advantages provided by 3D models of the hepatic environment, in conjunction with the high throughput characterizing various platforms, have enabled the elucidation of complex molecular mechanisms underlying various threatening hepatic diseases. A limited number of 3D models for gut and skin infections have been developed. However, a more profound comprehension of the spatial and temporal interactions between microbes, the host and their environment may facilitate the advancement of in vitro, ex vivo and in vivo disease models. Additionally, it may pave the way for the development of novel therapeutic approaches in diverse research fields. The interested reader will also find concluding remarks on the challenges and prospects of using 3D cell cultures for discovering cellular and molecular mechanisms in the research areas covered in this review.

Permeability Assessment of a High-Throughput Mucosal Platform.

Permeability across cellular membranes is a key factor that influences absorption and distribution. Before absorption, many drugs must pass through the mucus barrier that covers all the wet surfaces of the human body. Cell-free in vitro tools currently used to evaluate permeability fail to effectively model the complexity of mucosal barriers. Here, we present an in vitro mucosal platform as a possible strategy for assessing permeability in a high-throughput setup. The PermeaPad 96-well plate was used as a permeability system and further coupled to a pathological, tridimensional mucus model. The physicochemical determinants predicting passive diffusion were determined by combining experimental and computational approaches. Drug solubility, size, and shape were found to be the critical properties governing permeability, while the charge of the drug was found to be influential on the interaction with mucus. Overall, the proposed mucosal platform could be a promising in vitro tool to model the complexity of mucosal tissues and could therefore be adopted for drug-permeability profiling.

Internally crosslinked alginate-based bioinks for the fabrication ofin vitrohepatic tissue models.

Bioprinting is a key technique to fabricate cell-laden volumetric constructs with controlled geometry. It can be used not only to replicate the architecture of a target organ but also to produce shapes that allow for the mimicry,in vitro,of specific desired features. Among the various materials suitable to be processed with this technique, sodium alginate is currently considered one of the most appealing because of its versatility. To date, the most widespread strategies to print alginate-based bioinks exploit external gelation as a primary process, by directly extruding the hydrogel-precursor solution into a crosslinking bath or within a sacrificial crosslinking hydrogel, where the gelation takes place. In this work, we describe the print optimization and the processing of Hep3Gel: an internally crosslinked alginate and ECM-based bioink for the production of volumetric hepatic tissue models. We adopted an unconventional strategy, by moving from the reproduction of the geometry and the architecture of liver tissue to the use of bioprinting to fabricate structures that can promote a high degree of oxygenation, as is the case with hepatic tissue. To this end, the design of structures was optimized by employing computational methods. The printability of the bioink was then studied and optimized through a combination of differenta priorianda posteriorianalyses. We produced 14-layered constructs, thus highlighting the possibility to exploit internal gelation alone to directly print self-standing structures with finely controlled viscoelastic properties. Constructs loaded with HepG2 cells were successfully printed and cultured in static conditions for up to 12 d, underlining the suitability of Hep3Gel to support mid/long-term cultures.

Hep3Gel: A Shape-Shifting Extracellular Matrix-Based, Three-Dimensional Liver Model Adaptable to Different Culture Systems.

Drug-induced hepatotoxicity is a leading cause of clinical trial withdrawal. Therefore, in vitro modeling the hepatic behavior and functionalities is not only crucial to better understand physiological and pathological processes but also to support drug development with reliable high-throughput platforms. Different physiological and pathological models are currently under development and are commonly implemented both within platforms for standard 2D cultures and within tailor-made chambers. This paper introduces Hep3Gel: a hybrid alginate-extracellular matrix (ECM) hydrogel to produce 3D in vitro models of the liver, aiming to reproduce the hepatic chemomechanical niche, with the possibility of adapting its shape to different manufacturing techniques. The ECM, extracted and powdered from porcine livers by a specifically set-up procedure, preserved its crucial biological macromolecules and was embedded within alginate hydrogels prior to crosslinking. The viscoelastic behavior of Hep3Gel was tuned, reproducing the properties of a physiological organ, according to the available knowledge about hepatic biomechanics. By finely tuning the crosslinking kinetics of Hep3Gel, its dualistic nature can be exploited either by self-spreading or adapting its shape to different culture supports or retaining the imposed fiber shape during an extrusion-based 3D-bioprinting process, thus being a shape-shifter hydrogel. The self-spreading ability of Hep3Gel was characterized by combining empirical and numerical procedures, while its use as a bioink was experimentally characterized through rheological a priori printability evaluations and 3D printing tests. The effect of the addition of the ECM was evident after 4 days, doubling the survival rate of cells embedded within control hydrogels. This study represents a proof of concept of the applicability of Hep3Gel as a tool to develop 3D in vitro models of the liver.

Bioinspired in vitro intestinal mucus model for 3D-dynamic culture of bacteria.

The intestinal mucus is a biological barrier that supports the intestinal microbiota growth and filters molecules. To perform these functions, mucus possesses optimized microstructure and viscoelastic properties and it is steadily replenished thus flowing along the gut. The available in vitro intestinal mucus models are useful tools in investigating the microbiota-human cells interaction, and are used as matrices for bacterial culture or as static component of microfluidic devices like gut-on-chips. The aim of this work is to engineer an in vitro mucus models (I-Bac3Gel) addressing in a single system physiological viscoelastic properties (i.e., 2-200 Pa), 3D structure and suitability for dynamic bacterial culture. Homogeneously crosslinked alginate hydrogels are optimized in composition to obtain target viscoelastic and microstructural properties. Then, rheological tests are exploited to assess a priori the hydrogels capability to withstand the flow dynamic condition. We experimentally assess the suitability of I-Bac3Gels in the evolving field of microfluidics by applying a dynamic flow to a bacterial-loaded mucus model and by monitoring E. coli growth and survival. The engineered models represent a step forward in the modelling of the mucus, since they can answer to different urgent needs such as a 3D structure, bioinspired properties and compatibility with dynamic system.

Toward 3D-Bioprinted Models of the Liver to Boost Drug Development.

The main problems in drug development are connected to enormous costs related to the paltry success rate. The current situation empowered the development of high-throughput and reliable instruments, in addition to the current golden standards, able to predict the failures in the early preclinical phase. Being hepatotoxicity responsible for the failure of 30% of clinical trials, and the 21% of withdrawal of marketed drugs, the development of complex in vitro models (CIVMs) of liver is currently one of the hottest topics in the field. Among the different fabrication techniques, 3D-bioprinting is emerging as a powerful ally for their production, allowing the manufacture of three-dimensional constructs characterized by computer-controlled and customized geometry, and inter-batches reproducibility. Thanks to these, it is possible to rapidly produce tailored cell-laden constructs, to be cultured within static and dynamic systems, thus reaching a further degree of personalization when designing in vitro models. This review highlights and prioritizes the most recent advances related to the development of CIVMs of the hepatic environment to be specifically applied to pharmaceutical research, with a special focus on 3D-bioprinting, since the liver is primarily involved in the metabolism of drugs.

3D bioprinting of multi-layered segments of a vessel-like structure with ECM and novel derived bioink.

3D-Bioprinting leads to the realization of tridimensional customized constructs to reproduce the biological structural complexity. The new technological challenge focuses on obtaining a 3D structure with several distinct layers to replicate the hierarchical organization of natural tissues. This work aims to reproduce large blood vessel substitutes compliant with the original tissue, combining the advantages of the 3D bioprinting, decellularization, and accounting for the presence of different cells. The decellularization process was performed on porcine aortas. Various decellularization protocols were tested and evaluated through DNA extraction, quantification, and amplification by PCR to define the adequate one. The decellularized extracellular matrix (dECM), lyophilized and solubilized, was combined with gelatin, alginate, and cells to obtain a novel bioink. Several solutions were tested, tuning the percentage of the components to obtain the adequate structural properties. The geometrical model of the large blood vessel constructs was designed with SolidWorks, and the construct slicing was done using the HeartWare software, which allowed generating the G-Code. The final constructs were 3D bioprinted with the Inkredible + using dual print heads. The composition of the bioink was tuned so that it could withstand the printing of a segment of a tubular construct up to 10 mm and reproduce the multicellular complexity. Among the several compositions tested, the suspension resulting from 8% w/v gelatin, 7% w/v alginate, and 3% w/v dECM, and cells successfully produced the designed structures. With this bioink, it was possible to print structures made up of 20 layers. The dimensions of the printed structures were consistent with the designed ones. We were able to avoid the double bioink overlap in the thickness, despite the increase in the number of layers during the printing process. The optimization of the parameters allowed the production of structures with a height of 20 layers corresponding to 9 mm. Theoretical and real structures were very close. The differences were 14% in height, 20% internal diameter, and 9% thickness. By tailoring the printing parameters and the amount of dECM, adequate mechanical properties could be met. In this study, we developed an innovative printable bioink able to finely reproduce the native complex structure of the large blood vessel.

Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics.

Introduction: In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods: The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion: The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture.

Mucosomes: Intrinsically Mucoadhesive Glycosylated Mucin Nanoparticles as Multi-Drug Delivery Platform.

Mucus is a complex barrier for pharmacological treatments and overcoming it is one of the major challenges faced during transmucosal drug delivery. To tackle this issue, a novel class of glycosylated nanoparticles, named "mucosomes," which are based on the most important protein constituting mucus, the mucin, is introduced. Mucosomes are designed to improve drug absorption and residence time on the mucosal tissues. Mucosomes are produced (150-300 nm), functionalized with glycans, and loaded with the desired drug in a single one-pot synthetic process and, with this method, a wide range of small and macro molecules can be loaded with different physicochemical properties. Various in vitro models are used to test the mucoadhesive properties of mucosomes. The presence of functional glycans is indicated by the interaction with lectins. Mucosomes are proven to be storable at 4 °C after lyophilization, and administration through a nasal spray does not modify the morphology of the mucosomes. In vitro and in vivo tests indicate mucosomes do not induce adverse effects under the investigated conditions. This study proposes mucosomes as a ground-breaking nanosystem that can be applied in several pathological contexts, especially in mucus-related disorders.

Enzymatic cross-linking of human recombinant elastin (HELP) as biomimetic approach in vascular tissue engineering.

The use of polymers naturally occurring in the extracellular matrix (ECM) is a promising strategy in regenerative medicine. If compared to natural ECM proteins, proteins obtained by recombinant DNA technology have intrinsic advantages including reproducible macromolecular composition, sequence and molecular mass, and overcoming the potential pathogens transmission related to polymers of animal origin. Among ECM-mimicking materials, the family of recombinant elastin-like polymers is proposed for drug delivery applications and for the repair of damaged elastic tissues. This work aims to evaluate the potentiality of a recombinant human elastin-like polypeptide (HELP) as a base material of cross-linked matrices for regenerative medicine. The cross-linking of HELP was accomplished by the insertion of cross-linking sites, glutamine and lysine, in the recombinant polymer and generating ε-(γ-glutamyl) lysine links through the enzyme transglutaminase. The cross-linking efficacy was estimated by infrared spectroscopy. Freeze-dried cross-linked matrices showed swelling ratios in deionized water (≈2500%) with good structural stability up to 24 h. Mechanical compression tests, performed at 37°C in wet conditions, in a frequency sweep mode, indicated a storage modulus of 2/3 kPa, with no significant changes when increasing number of cycles or frequency. These results demonstrate the possibility to obtain mechanically resistant hydrogels via enzymatic crosslinking of HELP. Cytotoxicity tests of cross-linked HELP were performed with human umbilical vein endothelial cells, by use of transwell filter chambers for 1-7 days, or with its extracts in the opportune culture medium for 24 h. In both cases no cytotoxic effects were observed in comparison with the control cultures. On the whole, the results suggest the potentiality of this genetically engineered HELP for regenerative medicine applications, particularly for vascular tissue regeneration.

Trends in biomedical engineering: focus on Regenerative Medicine.

Regenerative medicine is a critical frontier in biomedical and clinical research. The major progresses in the last few years were driven by a strong clinical need which could benefit from regenerative medicine outcomes for the treatment of a large number of conditions including birth defects, degenerative and neoplastic diseases, and traumatic injuries. Regenerative medicine applies the principles of engineering and life sciences to enhance the comprehension of the fundamental biological mechanisms underlying the structure-function relationships in physiologic and pathologic tissues and to accomplish alternative strategies for developing in vitro biological substitutes which are able to restore, maintain, or improve tissue, and organ function. This paper reviews selected approaches currently being investigated at Politecnico di Milano in the field of regenerative medicine. Specific tissue-oriented topics are divided in three sections according to each developmental stage: in vitro study, pre-clinical study, and clinical application. In vitro studies investigate the basic phenomena related to gene delivery, stem cell behavior, tissue regeneration, and to explore dynamic culture potentiality in different applications: cardiac and skeletal muscle, cartilage, hematopoietic system, peripheral nerve, and gene delivery. Specific fields of regenerative medicine, i.e., bone, blood vessels, and ligaments engineering have already reached the preclinical stage providing promising insights for further research towards clinical applications. The translation of the results obtained during in vitro and preclinical steps into clinical organ replacement is a very challenging issue, which can offer a valid alternative to fight morbidity, organ shortage, and ethical-social problems associated with allotransplantation as shown in the clinical case reported in this review.

Trends in biomedical engineering: focus on Smart Bio-Materials and Drug Delivery.

The present article reviews on different research lines, namely: drug and gene delivery, surface modification/modeling, design of advanced materials (shape memory polymers and biodegradable stents), presently developed at Politecnico di Milano, Italy. For gene delivery, non-viral polycationic-branched polyethylenimine (b-PEI) polyplexes are coated with pectin, an anionic polysaccharide, to enhance the polyplex stability and decrease b-PEI cytotoxicity. Perfluorinated materials, specifically perfluoroether, and perfluoro-polyether fluids are proposed as ultrasound contrast agents and smart agents for drug delivery. Non-fouling, self-assembled PEG-based monolayers are developed on titanium surfaces with the aim of drastically reducing cariogenic bacteria adhesion on dental implants. Femtosecond laser microfabrication is used for selectively and spatially tuning the wettability of polymeric biomaterials and the effects of femtosecond laser ablation on the surface properties of polymethylmethacrylate are studied. Innovative functionally graded Alumina-Ti coatings for wear resistant articulating surfaces are deposited with PLD and characterized by means of a combined experimental and computational approach. Protein adsorption on biomaterials surfaces with an unlike wettability and surface-modification induced by pre-adsorbed proteins are studied by atomistic computer simulations. A study was performed on the fabrication of porous Shape Memory Polymeric structures and on the assessment of their potential application in minimally invasive surgical procedures. A model of magnesium (alloys) degradation, in a finite element framework analysis, and a bottom-up multiscale analysis for modeling the degradation mechanism of PLA matrices was developed, with the aim of providing valuable tools for the design of bioresorbable stents.

From tissue engineering to engineering tissues: the role and application of in vitro models.

Engineered models have emerged as relevant in vitro tools to foresee the translational potential of new therapies from the bench to the bedside in a fast and cost-effective fashion. The principles applied to the development of tissue-engineered constructs bring the foundation concepts to engineer relevant in vitro models. Engineered models often face scepticism, because regularly these do not include the extreme complexity of nature, but rather a simplification of a phenomenon. While engineering in vitro models, a hypothesis is imposed towards which defined parameters are included to assess the degree of similarity between the in vitro model and the native phenomenon, keeping in mind their intrinsic limitations. The development of in vitro models has been highly supported and disseminated by different regulatory agencies. This review aims at defining and exploring the multifaceted potential of tangible, not theoretical, models within the biomedical field to represent physiological tissues and organ-related phenomena.

Microbiological-Chemical Sourced Chondroitin Sulfates Protect Neuroblastoma SH-SY5Y Cells against Oxidative Stress and Are Suitable for Hydrogel-Based Controlled Release.

Chondroitin sulfates (CS) are a class of sulfated glycosaminoglycans involved in many biological processes. Several studies reported their protective effect against neurodegenerative conditions like Alzheimer's disease. CS are commonly derived from animal sources, but ethical concerns, the risk of contamination with animal proteins, and the difficulty in controlling the sulfation pattern have prompted research towards non-animal sources. Here we exploited two microbiological-chemical sourced CS (i.e., CS-A,C and CS-A,C,K,L) and Carbopol 974P NF/agarose semi-interpenetrating polymer networks (i.e., P.NaOH.0 and P.Ethanol.0) to set up a release system, and tested the neuroprotective role of released CS against H2O2-induced oxidative stress. After assessing that our CS (1-100 µM) require a 3 h pre-treatment for neuroprotection with SH-SY5Y cells, we evaluated whether the autoclave type (i.e., N- or B-type) affects hydrogel viscoelastic properties. We selected B-type autoclaves and repeated the study after loading CS (1 or 0.1 mg CS/0.5 mL gel). After loading 1 mg CS/0.5 mL gel, we evaluated CS release up to 7 days by 1,9-dimethylmethylene blue (DMMB) assay and verified the neuroprotective role of CS-A,C (1 µM) in the supernatants. We observed that CS-A,C exhibits a broader neuroprotective effect than CS-A,C,K,L. Moreover, sulfation pattern affects not only neuroprotection, but also drug release.