A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation.

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets. Human islets were embedded in polysaccharide 3D-hydrogel in cell culture inserts or gas-permeable chambers with serum-free CMRL 1066 supplemented media for up to 8 weeks. The long-term cultured islets maintained better morphology, cell mass, and viability at 4 weeks than islets in conventional suspension culture. In fact, islets cultured in the 3D-hydrogel retained ß cell mass and function on par with freshly isolated islets in vitro and, when transplanted into diabetic mice, restored glucose balance similar to fresh islets. Using gas-permeable chambers, the 3D-hydrogel culture method was scaled up over 10-fold and maintained islet viability and function, although the cell mass recovery rate was 50%. Additional optimization of scale-up methods continues. If successful, this technology could afford flexibility and expand access to islet transplantation, especially single-donor islet-after-kidney transplantation.

Tumor necrosis factor-α promotes and exacerbates calcification in heart valve myofibroblast populations.

Pro-inflammatory cytokines play critical roles in regulating valvular interstitial cell (VIC) phenotypic changes that can cause heart valve fibrosis and calcification. Tumor necrosis factor alpha (TNF-α) is a cytokine known to influence VIC behavior and has been reported at high levels in calcified valves ex vivo. We sought to understand the specific effects of TNF-α on VIC phenotypes (eg, fibroblast, profibrotic activated myofibroblasts) and its link with heart valve disorders. We characterize human aortic valve tissue from patients with valve disorders and identify a high variability of fibrotic and calcific markers between tissues. These results motivated in vitro studies to explore the effects of TNF-α on defined VIC fibroblasts and profibrotic activated myofibroblasts, induced via FGF-2 and TGF-ß1 treatment. Using 3D hydrogels to culture VICs, we measure the effect of TNF-α (0.1-10 ng/mL) on key markers of fibrosis (eg, αSMA, COL1A1) and calcification (eg, RUNX2, BMP2, and calcium deposits). We observe calcification in TNF-α-treated VIC activated myofibroblasts and identify the MAPK/ERK signaling cascade as a potential pathway for TNF-α mediated calcification. Conversely, VIC fibroblasts respond to TNF-α with decreased calcification. Treatment of VIC profibrotic activated myofibroblast populations with TNF-α leads to increased calcification. Our in vitro findings correlate with findings in diseased human valves and highlight the importance of understanding the effect of cytokines and signaling pathways on specific VIC phenotypes. Finally, we reveal MAPK/ERK as a potential pathway involved in VIC-mediated matrix calcification with TNF-α treatment, suggesting this pathway as a potential pharmaceutical target for aortic valve disease.

Protein Isolation from 3D Hydrogel Scaffolds.

Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell culture or tissue sections, and have been adapted to particular downstream analyses (e.g., western blotting or mass spectrometry). An increasing trend in bioengineering and cell biology is to use three-dimensional (3D) hydrogel-based scaffolds for cell culture. In principle, the same protocols can be used to extract protein from hydrogel-based cell and tissue constructs. However, in practice the yield and quality of the recovered protein pellet is often substantially lower when using standard protocols and requires tuning of multiple steps, including the selected lysis buffer and the scaffold homogenization strategy, as well as the methods for protein purification and reconstitution. We present here specific protocols tailored to common 3D hydrogels to help researchers using hydrogel-based 3D cell culture improve the quantity and quality of their extracted protein. We focus on three materials: protease-degradable PEG-based hydrogels, collagen hydrogels, and alginate hydrogels. We discuss how the protein extraction procedure can be adapted to the scaffold of interest (degradable or non-degradable gels), proteins of interests (soluble, matrix-bound, or phosphoproteins), and downstream biochemical assays (western blotting or mass spectrometry). With the growing interest in 3D cell culture, the protocols presented should be useful to many researchers in cell biology, protein science, biomaterials, and bioengineering communities. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolating proteins from PEG-based hydrogels Basic Protocol 2: Isolating proteins from collagen hydrogels Basic Protocol 3: Isolating proteins from alginate hydrogels Alternate Protocol: Isolating protein from alginate gels using EDTA to dissolve the gel Support Protocol: Isolating protein and RNA simultaneously from the same samples.

Integration of Hydrogels and 3D Bioprinting Technologies for Chronic Wound Healing Management.

The integration of hydrogel-based bioinks with 3D bioprinting technologies presents an innovative approach to chronic wound management, which is particularly challenging to treat because of its multifactorial nature and high risk of complications. Using precise deposition techniques, 3D bioprinting significantly alters traditional wound care paradigms by enabling the fabrication of patient-specific wound dressings that imitate natural tissue properties. Hydrogels are notably beneficial for these applications because of their abundant water content and mechanical properties, which promote cell viability and pathophysiological processes of wound healing, such as re-epithelialization and angiogenesis. This article reviews key 3D printing technologies and their significance in enhancing the structural and functional outcomes of wound-care solutions. Challenges in bioink viscosity, cell viability, and printability are addressed, along with discussions on the cross-linking and mechanical stability of the constructs. The potential of 3D bioprinting to revolutionize chronic wound management rests on its capacity to generate remedies that expedite healing and minimize infection risks. Nevertheless, further studies and clinical trials are necessary to advance these therapies from laboratory to clinical use.

Human Minor Salivary Glands: A Readily Available Source of Salivary Stem/Progenitor Cells for Regenerative Applications.

Resident stem/progenitor cells within the secretory salivary glands offer a potential therapeutic resource for use in the regeneration of salivary glands needed to restore saliva production in patients with chronic xerostomia, or dry mouth. Methods were developed previously to isolate human stem/progenitor cells (hS/PCs) from major salivary glands (parotid/submandibular). Abundant minor salivary glands located in readily accessible locations in the oral cavity and lip could provide an additional valuable therapeutic resource. An advantage of this cell resource is that these minor glands about the size of grape seeds can be harvested from healthy donors using minimally invasive surgical procedures. The disadvantage of using minor glands is that they contain many fewer cells than do major glands, and thus harvested cells need to be expanded in the lab to create a therapeutic resource. While earlier work has described isolation of proliferative cell populations from minor salivary glands that could be used in regenerative medicine, most of these expanded cells possess properties of mesenchymal cells rather than the epithelial population that secretes salivary products.Here, we describe in detail our recently established methods to isolate and expand hS/PCs isolated from human labial minor salivary glands. Expanded hS/PC populations are epithelial assessed by their expression of epithelial progenitor markers K5 and K14. Like expandable cell populations previously isolated from the major salivary glands, these cells also express nuclear p63, consistent with their ability to be expanded after explant culture. When hS/PCs with these properties are encapsulated into a customized 3D biomimetic hyaluronic acid-based hydrogel, they will assemble into microstructures that retain some progenitor markers while also beginning to differentiate. The increased expression of secreted mucin MUC-7 was used to demonstrate differentiation and secretory potential in assembled hS/PC microstructures. Compared to hS/PCs from major glands, those from minor salivary glands tend to be more heterogeneous in early passage; thus, use of K5/K14/p63 as an early quality assessment tool is highly recommended. Additionally, hS/PCs from minor glands are sensitive to stress and if mishandled will demonstrate a stress response that leads to their transitioning to a flat, squamous cell-like appearance that is of limited utility in regenerative medicine applications. We conclude that properly handled hS/PCs from minor salivary glands represent a powerful new source of therapeutic cells for applications including treating patients with chronic xerostomia.

Biofabricated poly (γ-glutamic acid) bio-ink reinforced with calcium silicate exhibiting superior mechanical properties and biocompatibility for bone regeneration.

Background/purpose: The modification in 3D hydrogels, tissue engineering, and biomaterials science has enabled us to fabricate novel substitutes for bone regeneration. This study aimed to combine different biomaterials by 3D technique to fabricate a promising all-rounded hydrogel for bone regeneration. Materials and methods: In this study, glycidyl methacrylate (GMA)-modified poly γ-glutamic acid (γ-PGA-GMA) hydrogels with calcium silicate (CS) hydrogel of different concentrations were fabricated by a 3D printing technique, and their biocompatibility and capability in bone regeneration were also evaluated. Results: The results showed that CS γ-PGA-GMA could be successfully fabricated, and the presence of CS enhanced the rheological and mechanical properties of γ-PGA-GMA hydrogels, thus making them more adept at 3D printing and implantations. SEM images of the surface structure showed that higher CS concentrations (5% and 10%) contributed to denser surface architectures, thus achieving improved cellular adhesion and stem cell proliferation. Furthermore, higher concentrations of CS resulted in elevated expressions of osteogenic-related markers such as alkaline phosphatase (ALP) and osteocalcin (OC), as well as enhanced calcium deposition represented by the increased Alizarin Red S staining. In vivo studies referring to critical defects of rabbit femur further showed that the existence of hydrogels alone was able to induce partial bone regeneration, demonstrated by the results from quantitative and qualitative analysis of micro-CT scans. However, CS alterations caused significant increases in bone regeneration, as indicated by micro-CT and histological staining. Conclusion: These results robustly suggest combining different biomaterials is crucial to producing a well-rounded hydrogel for tissue regeneration. We hope this study could be applied as a platform for others to brainstorm potential out-of-the-box solutions, contributing to developing high-potential biomaterials for bone regeneration.

A bioengineered tumor matrix-based scaffold for the evaluation of melatonin efficacy on head and neck squamous cancer stem cells.

Head and neck squamous cell carcinoma (HNSCC) presents a significant challenge worldwide due to its aggressiveness and high recurrence rates post-treatment, often linked to cancer stem cells (CSCs). Melatonin shows promise as a potent tumor suppressor; however, the effects of melatonin on CSCs remain unclear, and the development of models that closely resemble tumor heterogeneity could help to better understand the effects of this molecule. This study developed a tumor scaffold based on patient fibroblast-derived decellularized extracellular matrix that mimics the HNSCC microenvironment. Our study investigates the antitumoral effects of melatonin within this context. We validated its strong antiproliferative effect on HNSCC CSCs and the reduction of tumor invasion and migration markers, even in a strongly chemoprotective environment, as it is required to increase the minimum doses necessary to impact tumor viability compared to the non-scaffolded tumorspheres culture. Moreover, melatonin exhibited no cytotoxic effects on healthy cells co-cultured in the tumor hydrogel. This scaffold-based platform allows an in vitro study closer to HNSCC tumor reality, including CSCs, stromal component, and a biomimetic matrix, providing a new valuable research tool in precision oncology.

Nondestructive ultrasound evaluation of microstructure-related material parameters of skeletal muscle: an in silico and in vitro study.

Direct and nondestructive assessment of material properties of skeletal muscle in vivo shall advance our understanding of intact muscle mechanics and facilitate personalized interventions. However, this is challenged by intricate hierarchical microstructure of the skeletal muscle. We have previously regarded the skeletal muscle as a composite of myofibers and extracellular matrix (ECM), formulated shear wave propagation in the undeformed muscle using the acoustoelastic theory, and preliminarily demonstrated that ultrasound-based shear wave elastography (SWE) could estimate microstructure-related material parameters (MRMPs): myofiber stiffness µf, ECM stiffness µm, and myofiber volume ratio Vf. The proposed method warrants further validation but is hampered by the lack of ground truth values of MRMPs. In this study, we presented analytical and experimental validations of the proposed method using finite-element (FE) simulations and 3D-printed hydrogel phantoms, respectively. Three combinations of different physiologically relevant MRMPs were used in the FE simulations where shear wave propagations in the corresponding composite media were simulated. Two 3D-printed hydrogel phantoms with the MRMPs close to those of a real skeletal muscle (i.e., µf=2.02kPa, µm=52.42kPa, and Vf=0.675,0.832) for ultrasound imaging were fabricated by an alginate-based hydrogel printing protocol that we modified and optimized from the freeform reversible embedding of suspended hydrogels (FRESH) method in literature. Average percent errors of (µf,µm,Vf) estimates were found to be (2.7%,7.3%,2.4%)in silico and (3.0%,8.0%,9.9%)in vitro. This quantitative study corroborated the potential of our proposed theoretical model along with ultrasound SWE for uncovering microstructural characteristics of the skeletal muscle in an entirely nondestructive way.

22 nm Resolution Achieved by Femtosecond Laser Two-Photon Polymerization of a Hyaluronic Acid Vinyl Ester Hydrogel.

Three-dimensional (3D) bioinspired hydrogels have played an important role in tissue engineering, owing to their advantage of excellent biocompatibility. Here, the two-photon polymerization (TPP) of a 3D hydrogel with high precision has been investigated, using the precursor with hyaluronic acid vinyl ester (HAVE) as the biocompatibility hydrogel monomer, 3,3'-((((1E,1'E)-(2-oxocyclopentane-1,3-diylidene) bis(methanylylidene)) bis(4,1-phenylene)) bis(methylazanediyl))dipropanoate as the water-soluble initiator, and dl-dithiothreitol (DTT) as the click-chemistry cross-linker. The TPP properties of the HAVE precursors have been comprehensively investigated by adjusting the solubility and the formulation of the photoresist. The feature line width of 22 nm has been obtained at a processing laser threshold of 3.67 mW, and the 3D hydrogel scaffold structures have been fabricated. Furthermore, the average value of Young's modulus is 94 kPa for the 3D hydrogel, and cell biocompatibility has been demonstrated. This study would provide high potential for achieving a 3D hydrogel scaffold with highly precise configuration in tissue engineering and biomedicine.

A Three-Dimensional Engineered Cardiac In Vitro Model: Controlled Alignment of Cardiomyocytes in 3D Microphysiological Systems.

Cardiomyocyte alignment in myocardium tissue plays a significant role in the physiological, electrical, and mechanical functions of the myocardium. It remains, however, difficult to align cardiac cells in a 3D in vitro heart model. This paper proposes a simple method to align cells using microfabricated Polydimethylsiloxane (PDMS) grooves with large dimensions (of up to 350 µm in width), similar to the dimensions of trabeculae carneae, the smallest functional unit of the myocardium. Two cell groups were used in this work; first, H9c2 cells in combination with Nor10 cells for proof of concept, and second, neonatal cardiac cells to investigate the functionality of the 3D model. This model compared the patterned and nonpatterned 3D constructs, as well as the 2D cell cultures, with and without patterns. In addition to alignment, we assessed the functionality of our proposed 3D model by comparing beating rates between aligned and non-aligned structures. In order to assess the practicality of the model, the 3D aligned structures should be demonstrated to be detachable and alignable. This evaluation is crucial to the use of this 3D functional model in future studies related to drug screening, building blocks for tissue engineering, and as a heart-on-chip by integrating microfluidics.

Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels.

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix are reorganized by biophysical forces into aligned domains extending several millimeters and with varying degrees of fiber alignment. These aligned fibers can transmit traction forces, guide tumor cell migration, facilitate angiogenesis, and influence tissue morphogenesis. To create aligned COL1 domains in microfluidic cell culture models, shear flows have been used to align thin COL1 matrices (<50µm in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130µm). Here, we show that pure shear flows do not induce fiber alignment in 3D atelo COL1 hydrogels, but the simple addition of local extensional flow promotes alignment that is maintained across several millimeters, with a degree of alignment directly related to the extensional strain rate. We further advance experimental capabilities by addressing the practical challenge of accessing a 3D hydrogel formed within a microchannel by introducing a magnetically coupled modular platform that can be released to expose the microengineered hydrogel. We demonstrate the platform's capability to pattern cells and fabricate multi-layered COL1 matrices using layer-by-layer fabrication and specialized modules. Our approach provides an easy-to-use fabrication method to achieve advanced hydrogel microengineering capabilities that combine fiber alignment with biofabrication capabilities.

Generation of functional fat organoid from rat superficial fascia.

The organoid is a 3D cell architecture formed by self-organized tissues or cells in vitro with similar cell types, histological structures, and biological functions of the native organ. Depending on the unique organ structures and cell types, producing organoids requires individualized design and is still challenging. Organoids of some tissues, including adipose tissue, remain to generate to be more faithful to their original organ in structure and function. We previously established a new model of the origin of adipose cells originating from non-adipose fascia tissue. Here, we investigated superficial fascia fragments in 3D hydrogel and found they were able to transform into relatively large adipocyte aggregates containing mature unilocular adipocytes, which were virtually "fat organoids". Such fascia-originated fat organoids had a typical structure of adipose tissues and possessed the principal function of adipose cells in the synthesis, storage, hydrolysis of triglycerides and adipokines secretion. Producing fat organoids from superficial fascia can provide a new approach for adipocyte research and strongly evidences that both adipose tissues and cells originate from fascia. Our findings give insights into metabolic regulation by the crosstalk between different organs and tissues and provide new knowledge for investigating novel treatments for obesity, diabetes and other metabolic diseases.Abbreviations: 3D: three dimensional; ASC: adipose-derived stromal cells; C/EBP: CCAAT-enhancer-binding protein; EdU: 5-ethynyl-2-deoxyuridine; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; FSCs: fascia-derived stromal cells; Plin1: perilipin-1; Plin2: perilipin-2; PPARγ: peroxisome proliferator-activated receptor γ; WAT: white adipose tissue.

Modulation of Cell Behavior by 3D Biocompatible Hydrogel Microscaffolds with Precise Configuration.

Three-dimensional (3D) micronano structures have attracted much attention in tissue engineering since they can better simulate the microenvironment in vivo. Two-photon polymerization (TPP) technique provides a powerful tool for printing arbitrary 3D structures with high precision. Here, the desired 3D biocompatible hydrogel microscaffolds (3D microscaffold) with structure design referring to fibroblasts L929 have been fabricated by TPP technology, particularly considering the relative size of cell seed (cell suspension), spread cell, strut and strut spacing of scaffold. Modulation of the cell behavior has been studied by adjusting the porosity from 69.7% to 89.3%. The cell culture experiment results reveal that the obvious modulation of F-actin can be achieved by using the 3D microscaffold. Moreover, cells on 3D microscaffolds exhibit more lamellipodia than those on 2D substrates, and thus resulting in a more complicated 3D shape of single cell and increased cell surface. 3D distribution can be also achieved by employing the designed 3D microscaffold, which would effectively improve the efficiency of information exchange and material transfer. The proposed protocol enables us to better understand the cell behavior in vivo, which would provide high prospects for the further application in tissue engineering.

Synergistic control of mechanics and microarchitecture of 3D bioactive hydrogel platform to promote the regenerative potential of engineered hepatic tissue.

Culturing autologous cells with therapeutic potential derived from a patient within a bioactive scaffold to induce functioning tissue formation is considered the ideal methodology towards realizing patient-specific regenerative medicine. Hydrogels are often employed as the scaffold material for this purpose mainly for their tunable mechanical and diffusional properties as well as presenting cell-responsive moieties. Herein, a two-fold strategy was employed to control the physicomechanical properties and microarchitecture of hydrogels to maximize the efficacy of engineered hepatic tissues. First, a hydrophilic polymeric crosslinker with a tunable degree of reactive functional groups was employed to control the mechanical properties in a wide range while minimizing the change in diffusional properties. Second, photolithography technique was utilized to introduce microchannels into hydrogels to overcome the critical diffusional limit of bulk hydrogels. Encapsulating hepatic progenitor cells derived via direct reprogramming of tissue-harvested fibroblasts, the application of this strategy to control the mechanics, diffusion, and architecture of hydrogels in a combinatorial manner could allow the optimization of their hepatic functions. The regenerative capacity of this engineered hepatic tissue was further demonstrated using an in vivo acute liver injury model.

Self-decoration of N-doped graphene oxide 3-D hydrogel onto magnetic shrimp shell biochar for enhanced removal of hexavalent chromium.

In this work, a novel decorated and combined N-doped graphene oxide hydrogel with shrimp shell magnetic biochar (NGO3DH-MSSB) biosorbent was fabricated as an effective material for Cr(VI) removal. Three-dimensional self-assembled graphene oxide hydrogel was synthesized using nitrogen source, ethylenediamine (EDA). Characterizations of NGO3DH-MSSB biosorbent were established by FT-IR, TGA, SEM and BET, where high surface area (398.05 m2/g) compared with that of MSSB (138.64 m2/g) was characterized. The maximum achieved swelling ratio (800%) was only after 300 min. The binding mechanisms between Cr(VI) ions and NGO3DH-MSSB biosorbent were controlled by electrostatic adsorption (ion-pair), pore filling, and reduction-coordination reaction. Adsorption was described by the pseudo-second order kinetic (R2 =0.9994, 0.9983 and 0.9992) at 10, 50 and 100 mg/L and Langmuir isotherm model (R2 =0.9997, 0.9957 and 0.9912) at 25, 40 and 50 °C. The adsorption capacity (350.42 mg/g) was achieved at pH 1.0, using initial Cr(VI) concentration (100 mg/L) and contact time (180 min) at room temperature. NGO3DH-MSSB biosorbent could be successfully reused after eight cycles. The percentage removal of Cr(VI) were confirmed as 99.79%, 99.20% and 98.00% from tap water, sea water and wastewater, respectively.

Immunomodulatory Scaffolds Derived from Lymph Node Extracellular Matrices.

Immunomodulation in the local tissue microenvironment is pivotal for the determination of macrophage phenotypes and regulation of functions necessary for pro-healing effects. Herein, we demonstrate that a lymph node extracellular matrix (LNEM) prepared by the decellularization of lymph node tissues can mimic lymph node microenvironments for immunomodulation in two-dimensional (2D) and three-dimensional (3D) formats. The LNEM exhibits strengthened immunomodulatory effects in comparison to conventional collagen-based platforms. A 3D LNEM hydrogel is more effective than the 2D LNEM coating in inducing M2 macrophage polarization. The 3D LNEM induces macrophage elongation and enhances the M2-type marker expression and the secretion of anti-inflammatory cytokines. Additionally, the phagocytic function of macrophages is improved upon exposure to the intricate 3D LNEM environment. We demonstrate the reduced susceptibility of liver organoids to a hepatotoxic drug when co-cultured with macrophages in a 3D LNEM. This effect could be attributed to the enhanced anti-inflammatory functions and indicates its potential as a drug-testing platform that enables drug responses similar to those observed in vivo. Finally, the implantation of an LNEM hydrogel in a mouse volumetric muscle loss model facilitates the recruitment of host macrophages to the site of injury and enhances macrophage polarization toward the M2 phenotype for tissue healing in vivo. Therefore, 3D immune system-mimicking biomaterials could serve as useful platforms for tissue modeling and regenerative medicine development.

Ionic Carbazole-Based Water-Soluble Two-Photon Photoinitiator and the Fabrication of Biocompatible 3D Hydrogel Scaffold.

Two-photon polymerization of a three-dimensional (3D) hydrogel structure has been widely applied in biological tissue engineering. For improving the biocompatibility of hydrogel structures, a new kind of ionic carbazole water-soluble photoinitiator was prepared to realize the fabrication of a 3D hydrogel structure in aqueous phase. 3,6-Bis[2-(1-methyl-pyridinium)vinyl]-9-methyl-carbazole diiodide (BMVMC) and cucurbit[7]uril (CB7) have been employed to generate a complex with better water solubility by host-guest interactions. The binding ratio of the complex was demonstrated to be 1:1 through the characterization of isothermal titration calorimetry (ITC). The two-photon absorption (TPA) cross section of the complex increases to 2500 GM compared with the 750 GM of the BMVMC molecule. Then, an aqueous-phase photoresist was obtained using the CB7/BMVMC complex as the photoinitiator and poly(ethylene glycol) diacrylate (PEGda) as the hydrogel monomer. Two-photon fabrication capability in aqueous phase has been studied using the as-prepared photoresist. A low laser threshold of 3.7 mW as well as a high resolution of 180 nm are achieved. Benefiting from the fluorescence properties of the photoinitiator, we can achieve the confocal fluorescence images without any assistance of fluorescent probes. Subsequently, a 3D engineered hydrogel scaffold microstructure was fabricated by the two-photon polymerization technology, whose biocompatibility was demonstrated by culturing the structure with living cells of L929. The BMVMC-CB7 complex and the as-prepared photoresist are demonstrated to have good biocompatibility, which is prospective for further application in tissue engineering.

Perfusion Flow Enhances Viability and Migratory Phenotype in 3D-Cultured Breast Cancer Cells.

Conventional 2D cell culture, a traditional tool in pre-clinical studies, can hardly be regarded as a representation of a natural cell microenvironment. In this respect, it might result in altered cellular behaviors. To overcome such a limitation, different approaches have been tested to conduct more representative in vitro studies. In particular, the use of 3D cell culture introduces variables, such as cell-cell and cell-extracellular matrix interactions; cell features such as survival, proliferation and migration are consequently influenced. For an example, an enhanced drug resistance and increased invasiveness are shown by cancer cells when cultured in 3D versus 2D conventional culture models. In this setting however, non-uniform cell distribution and biological behaviors appear throughout the scaffold, due to reduced diffusion of oxygen and nutrients. Perfusion in bioreactor systems can be used to improve medium transport. In this line of reasoning, this study proposes a breast cancer cell culture model sustained by an integrated approach that couples a 3D environment and a fluid perfusion. This model improves viability and uniformness of cell distribution, while inducing morphological, functional and molecular cancer cell remodeling.

Quantitatively Designed Cross-Linker-Clustered Maleimide-Dextran Hydrogels for Rationally Regulating the Behaviors of Cells in a 3D Matrix.

Biomaterials can serve as the cornerstone of extracellular matrices (ECMs) in three-dimensional (3D) cell culture. Designing of diverse ECM materials with adjustable inner structures exhibits growing superiority in inducing varied cellular behaviors, such as spreading, proliferation, migration, differentiation, and apoptosis. However, a rational and easy-to-implement strategy for regulating cell behaviors during cellular self-organization in 3D hydrogels is still lacking. In this study, we propose that maleimide-functionalized dextran hydrogels are cross-linked with quantitatively predesigned nanoscaled heterogeneity. The heterogeneity is obtained via mixing two parts of a dextran polymer with different amounts of available cross-linking sites. We experimentally demonstrate that this rational strategy has a noteworthy impact on cell morphology and fate. The cross-linker-clustered hydrogels are found to contain more microscopic heterogeneous pore structures and show larger heterogeneity in local stiffness, compared to the homogeneously cross-linked hydrogels. Further, 3D cell culture results show that myoblast cells exhibit better spreading, lower circularity, and perceptible changes in the expression of a differentiation marker in cross-linker-clustered hydrogels, which can also be tuned with the cross-linker-clustering degree. The possible mechanisms are discussed by considering the cellular durotaxis in nanoscales, mechanical cues, and biological cues in extracellular and intracellular networks. It is inferred that the effect of nanoscaled durotaxis is triggered during the tentative protrusion of the C2C12 cells, which thus repeatedly and continuously enhances the cellular elongation during their oriented polarization. In conclusion, the predesigned 3D cross-linker-clustered maleimide-dextran hydrogels are able to affect the cell morphology and fate in a rational way by modulating physical, chemical, and biological cues related to the cell-hydrogel interactions. This study provides a promising strategy for quantitative designing and fabrication of functional biomaterials, and theoretical study of mechanobiological mechanisms underlying the cellular behavior.

Fabrication of three-dimensional calcium alginate hydrogels using sacrificial templates of sugar.

Hydrogels are receiving increasing attention in bioapplications. Among hydrogels, calcium alginate (Ca-alginate) hydrogels are widely used for their biocompatibility, low toxicity, low cost, and rapid fabrication by simple mixing of Ca2+ and sodium alginate (Na-alginate). For bioapplications using hydrogels, it is necessary to construct designed hydrogel structures. Although several methods have been proposed for fabricating designed hydrogels, a simple and low-cost method is desirable. Therefore, we developed a new method using sacrificial templates of sugar structures to fabricate three-dimensional (3D) designed Ca-alginate hydrogels. In this method, Na-alginate solution is mixed with molten sugar, and the resulting highly viscous material used to mold 3D sugar structures as sacrificial templates. Since sugar constructs are easily handled compared to hydrogels, sugar templates are useful for preparing 3D constructs. Finally, the sugar and Na-alginate structure is immersed in a CaCl2 solution to simultaneously dissolve the template and form the Ca-alginate hydrogel. The resulting hydrogel takes the shape of the sugar template. By stacking and fusing various sugar structures, such as fibers and blocks, 3D designed Ca-alginate hydrogels can be successfully fabricated. This simple and low-cost method shows excellent potential for application to a variety of bioapplications.