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![Principle of electrostatic (left) and coaxial air flow (right) bead generators [80].](publication/277574329/figure/fig6/AS:393312138743812@1470784329283/Principle-of-electrostatic-left-and-coaxial-air-flow-right-bead-generators-80.png)
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This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the typ...
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Context 1
... Coaxial air or liquid flow: The coaxial air jet system is a simple way of generating small beads (down to around 400 µm), although the size distribution will normally be larger as compared to an electrostatic system. In this system, a coaxial air stream is used to pull droplets from a needle tip into a gelling bath (Figure 7). ...
Citations
... 16 Meanwhile, the sodium salt of alginate is known to take on a soft and sponge-like structure when freeze-dried, and studies have attempted to employ a sponge in cell culture and transplant therapy. 17 Although alginate has a limited capacity for cell adhesion and cellular interaction in the case of animal cells, 18 the high biocompatibility, lack of toxicity, and interesting properties of this compound make alginate a promising new biomaterial. 19 At the same time, a composite of calcium phosphate crystals and alginate gel has been a subject of research, given that calcium phosphate can compensate for the poor mechanical strength and bioactivity of alginate alone. ...
Ex vivo tissue engineering is an effective therapeutic approach for the treatment of severe cartilage diseases that require tissue replenishment or replacement. This strategy demands scaffolds that are durable enough for long‐term cell culture to form artificial tissue. Additionally, such scaffolds must be biocompatible to prevent the transplanted matrix from taking a toll on the patient's body. From the viewpoint of structure and bio‐absorbability, a β‐tricalcium phosphate (β‐TCP) fiber scaffold (βTFS) is expected to serve as a good scaffold for tissue engineering. However, the fragility and high solubility of β‐TCP fibers make this matrix unsuitable for long‐term cell culture. To solve this problem, we developed an alginate‐coated β‐TCP fiber scaffold (βTFS‐Alg). To assess cell proliferation and differentiation in the presence of βTFS‐Alg, we characterized ATDC5 cells, a chondrocyte‐like cell line, when grown in this matrix. We found that alginate coated the surface of βTFS fiber and suppressed the elution of Ca²⁺ from β‐TCP fibers. Due to the decreased solubility of βTFS‐Alg compared with β‐TCP, the former provided an improved scaffold for long‐term cell culture. Additionally, we observed superior cell proliferation and upregulation of chondrogenesis marker genes in ATDC5 cells cultured in βTFS‐Alg. These results suggest that βTFS‐Alg is suitable for application in tissue culture.
... 93 An important feature of alginate is its inherent cell adhesion properties and limited cell-to-cell interactions, which favor encapsulating cells (Figure 3(d)). 94 Alginate can also be used in cancer treatment (Figure 3(a)). 95 In addition, various materials can also be incorporated into the alginate to improve its printability or bioactivity and make it a suitable bioink (Figure 3(c)). ...
... (d) Encapsulation of cells with alginate. Adapted from Andersen et al.94 ...
Digestive system tumors are the leading cause of cancer-related deaths worldwide. Despite ongoing research, our understanding of their mechanisms and treatment remain inadequate. One promising tool for clinical applications is the use of gastrointestinal tract tumor organoids, which serve as an important in vitro model. Tumor organoids exhibit a genotype similar to the patient’s tumor and effectively mimic various biological processes, including tissue renewal, stem cell, and ecological niche functions, and tissue response to drugs, mutations, or injury. As such, they are valuable for drug screening, developing novel drugs, assessing patient outcomes, and supporting immunotherapy. In addition, innovative materials and techniques can be used to optimize tumor organoid culture systems. Several applications of digestive system tumor organoids have been described and have shown promising results in related aspects. In this review, we discuss the current progress, limitations, and prospects of this model for digestive system tumors.
... Herein, we report a method for their fabrication. Alginate gels are used in many cell culture devices [30][31][32][33][34][35][36] and can be used without affecting the cell cultures [36,37]. ...
Three-dimensional cell culture spheroids are commonly used for drug evaluation studies because they can produce large quantities of homogeneous cell aggregates. As the spheroids grow, nutrients supplied from outer spheroid regions render the inner spheroid areas hypoxic and hyponutrient, which makes them unobservable through confocal microscopy. In this study, we fabricated a cancer cell aggregate culture device that facilitates the observation of nutrient and oxygen gradients. An alginate gel fiber was created in the cell culture chamber to ensure a flow path for supplying the culture medium. A gradient of nutrients and oxygen was generated by positioning the flow channel close to the edge of the chamber. We devised a fabrication method that uses calcium carbonate as a source of Ca2+ for the gelation of sodium alginate, which has a slow reaction rate. We then cultured a spheroid of HCT116 cells, which were derived from human colorectal carcinoma using a fluorescent ubiquitination-based cell cycle indicator. Fluorescence observation suggested the formation of a hypoxic and hyponutrient region within an area approximately 500 µm away from the alginate gel fiber. This indicates the development of a cancer cell aggregate culture device that enables the observation of different nutrition and oxygen states.
... Undoubtedly, hydrogels based on alginate and its derivatives constituted a group very frequently used in the last decade. Nevertheless, most of those in vitro studies were made on a micro scale rather than on constructs capable to be used in clinical practice [6,73,74]. Hence, presented in this study, the hydrogel was enriched with GelMa with the concentration of 20% w/v. GelMa has a great potential as a bioink, but it has been reported that the high concentration (>15% w/v) needed for maintaining structural fidelity may lead to a decline in cell viability [18,75,76]. ...
There is a growing interest in the production of bioinks that on the one hand, are biocompatible and, on the other hand, have mechanical properties that allow for the production of stable constructs that can survive for a long time after transplantation. While the selection of the right material is crucial for bioprinting, there is another equally important issue that is currently being extensively researched—the incorporation of the vascular system into the fabricated scaffolds. Therefore, in the following manuscript, we present the results of research on bioink with unique physico-chemical and biological properties. In this article, two methods of seeding cells were tested using bioink B and seeding after bioprinting the whole model. After 2, 5, 8, or 24 h of incubation, the flow medium was used in the tested systems. At the end of the experimental trial, for each time variant, the canals were stored in formaldehyde, and immunohistochemical staining was performed to examine the presence of cells on the canal walls and roof. Cells adhered to both ways of fiber arrangement; however, a parallel bioprint with the 5 h incubation and the intermediate plating of cells resulted in better adhesion efficiency. For this test variant, the percentage of cells that adhered was at least 20% higher than in the other analyzed variants. In addition, it was for this variant that the lowest percentage of viable cells was found that were washed out of the tested model. Importantly, hematoxylin and eosin staining showed that after 8 days of culture, the cells were evenly distributed throughout the canal roof. Our study clearly shows that neovascularization-promoting cells effectively adhere to ECM-based pancreatic bioink. Summarizing the presented results, it was demonstrated that the proposed bioink compositions can be used for bioprinting bionic organs with a vascular system formed by endothelial cells and fibroblasts.
... 48,49 Moreover, stiffer terpolymer matrix could result in lower diffusion coefficients for oxygen, nutrients, and metabolic by products, thus affecting viability. 50,51 Similar results, were observed with other stiff matrices used for T cell encapsulation, including chitosan 30and alginate-based 32 scaffolds. ...
Suspension cell culture and rigid commercial substrates are the most common methods to clinically manufacture therapeutic CAR-T cells ex vivo. However, suspension culture and nano/micro-scale commercial substrates poorly mimic the microenvironment where T cells naturally develop, leading to profound impacts on cell proliferation and phenotype. To overcome this major challenge, macro-scale substrates can be used to emulate that environment with higher precision. This work employed a biocompatible thermo-responsive material with tailored mechanical properties as a potential synthetic macro-scale scaffold to support T cell encapsulation and culture. Cell viability, expansion, and phenotype changes were assessed to study the effect of two thermo-responsive hydrogel materials with stiffnesses of 0.5 and 17 kPa. Encapsulated Pan-T and CAR-T cells were able to grow and physically behave similar to the suspension control. Furthermore, matrix stiffness influenced T cell behavior. In the softer polymer, T cells had higher activation, differentiation, and maturation after encapsulation obtaining significant cell numbers. Even when terpolymer encapsulation affected the CAR-T cell viability and expansion, CAR T cells expressed favorable phenotypical profiles, which was supported with cytokines and lactate production. These results confirmed the biocompatibility of the thermo-responsive hydrogels and their feasibility as a promising 3D macro-scale scaffold for in vitro T cell expansion that could potentially be used for cell manufacturing process.
... Furthermore, AL hydrogels are able to prevent the death of loaded cells from the host immune response, i.e., they act as a barrier or protection, preventing the host's immune system from detecting and attacking the loaded cells, allowing the cells to survive [28]. They have also shown great applicability as structures for cell immobilization [29]. Lee et al. [19] described in their research the development of bioinks from hyaluronic acid and sodium alginate at different ratios and the bioprinting 3D constructs through extrusion techniques using all the bionks. ...
Three-dimensional (3D) bioprinting is considered one of the most advanced tools to build up materials for tissue engineering. The aim of this work was the design, development and characterization of a bioink composed of human mesenchymal stromal cells (hMSC) for extrusion through nozzles to create these 3D structures that might potentially be apply to replace the function of damaged natural tissue. In this study, we focused on the advantages and the wide potential of biocompatible biomaterials, such as hyaluronic acid and alginate for the inclusion of hMSC. The bioink was characterized for its physical (pH, osmolality, degradation, swelling, porosity, surface electrical properties, conductivity, and surface structure), mechanical (rheology and printability) and biological (viability and proliferation) properties. The developed bioink showed high porosity and high swelling capacity, while the degradation rate was dependent on the temperature. The bioink also showed negative electrical surface and appropriate rheological properties required for bioprinting. Moreover, stress-stability studies did not show any sign of physical instability. The developed bioink provided an excellent environment for the promotion of the viability and growth of hMSC cells. Our work reports the first-time study of the effect of storage temperature on the cell viability of bioinks, besides showing that our bioink promoted a high cell viability after being extruded by the bioprinter. These results support the suggestion that the developed hMSC-composed bioink fulfills all the requirements for tissue engineering and can be proposed as a biological tool with potential applications in regenerative medicine and tissue engineering.
Graphical abstract
... Alginate's ability to form hydrogels in the presence of divalent cations, such as Ca 2+ and Ba 2+ , is attributed to the carboxylate groups present in its structure [37,38]. The crosslinking network of alginate hydrogels enables them to simulate ECM-like characteristics such as high water content, porosity, permeability, and viscoelasticity [36,[38][39][40][41][42]. The advantages of alginate hydrogels, including ease of chemical modification (addition of bioactive cues [43-49] and therapeutic agents), support of cell viability (biocompatibility [43,50], mild gelation, and soft-tissue-like mechanical properties), and ease of transplantation (biodegradability [32], malleability [39,40], and high ionic conductivity [51]), have made it a popular biomaterial for drug delivery systems [52], tissue engineering scaffolds [33], and encapsulation of living cells [39]. ...
Mesenchymal stromal cells (MSCs) have displayed potential in regenerating organ function due to their anti-fibrotic, anti-inflammatory, and regenerative properties. However, there is a need for delivery systems to enhance MSC retention while maintaining their anti-fibrotic characteristics. This study investigates the feasibility of using alginate hydrogel microstrands as a cell delivery vehicle to maintain MSC viability and phenotype. To accommodate cell implantation needs, we invented a Syringe-in-Syringe approach to reproducibly fabricate microstrands in small numbers with a diameter of around 200 µm and a porous structure, which would allow for transporting nutrients to cells by diffusion. Using murine NIH 3T3 fibroblasts and primary embryonic 16 (E16) salivary mesenchyme cells as primary stromal cell models, we assessed cell viability, growth, and expression of mesenchymal and fibrotic markers in microstrands. Cell viability remained higher than 90% for both cell types. To determine cell number within the microstrands prior to in vivo implantation, we have further optimized the alamarBlue assay to measure viable cell growth in microstrands. We have shown the effect of initial cell seeding density and culture period on cell viability and growth to accommodate future stromal cell delivery and implantation. Additionally, we confirmed homeostatic phenotype maintenance for E16 mesenchyme cells in microstrands.
... Based on this feedback, the flow rates of the syringe pumps were updated, and this closed-loop control system led to the formation of controlled concentration gradients. We validated the platform by studying the growth of HEK293FT cells trapped into alginate microbeads (Figure 1ii) (Andersen et al., 2015). Following the formation of the alginate concentration gradient, corresponding to different gel stiffnesses, beads were subsequently cultured in 24-well plates and regularly imaged by a high-content screener to obtain a time-lapse evaluation of cellular growth across all beads. ...
The study of dose-response relationships underpins analytical biosciences. Droplet microfluidics platforms can automate the generation of microreactors encapsulating varying concentrations of an assay component, providing datasets across a large chemical space in a single experiment. A classical method consists in varying the flow rate of multiple solutions co-flowing into a single microchannel (producing different volume fractions) before encapsulating the contents into water-in-oil droplets. This process can be automated through controlling the pumping elements but lacks the ability to adapt to unpredictable experimental scenarios, often requiring constant human supervision. In this paper, we introduce an image-based, closed-loop control system for assessing and adjusting volume fractions, thereby generating unsupervised, uniform concentration gradients. We trained a shallow convolutional neural network to assess the position of the laminar flow interface between two co-flowing fluids and used this model to adjust flow rates in real-time. We apply the method to generate alginate microbeads in which HEK293FT cells could grow in three dimensions. The stiffnesses ranged from 50 Pa to close to 1 kPa in Young modulus and were encoded with a fluorescent marker. We trained deep learning models based on the YOLOv4 object detector to efficiently detect both microbeads and multicellular spheroids from high-content screening images. This allowed us to map relationships between hydrogel stiffness and multicellular spheroid growth.
... A hydrogel that has been covalently bonded is chemically stable and offers a variety of ways to relieve tension. The degree of methacrylation of alginate can be changed to alter the mechanical characteristics and biodegradation rates of a hydrogel (Andersen et al., 2015;Li et al., 2022). ...
Biotechnology is one of the emerging fields that can add new and better application in a wide range of sectors like health care, service sector, agriculture, and processing industry to name some. This book will provide an excellent opportunity to focus on recent developments in the frontier areas of Biotechnology and establish new collaborations in these areas. The book will highlight multidisciplinary perspectives to interested biotechnologists, microbiologists, pharmaceutical experts, bioprocess engineers, agronomists, medical professionals, sustainability researchers and academicians. This technical publication will provide a platform for potential knowledge exhibition on recent trends, theories and practices in the field of Biotechnology.
... This relevant architecture further benefited from the hydrogel composition by taking advantage of alginate native ionic exchange in the absence of calcium supplementation [50,51], creating an biolabile . Parallel Testing of Anti-cancer Drugs in the 3D Biofabricated models: (a) Schematic representation of the rationale behind parallel drug testing using prostate cancer fiber segments, which can be easily exposed to distinct drugs and quickly imaged together with native RFP expression for fast quantification of cancer growth. ...
Glandular cancers are amongst the most prevalent types of cancer, which can develop in many different organs, presenting challenges in their detection as well as high treatment variability and failure rates. For that purpose, anticancer drugs are commonly tested in cancer cell lines grown in 2D tissue culture on plastic dishes in vitro, or in animal models in vivo. However, 2D culture models diverge significantly from the 3D characteristics of living tissues and animal models require extensive animal use and time. Glandular cancers, such as prostate cancer - the second leading cause of male cancer death - typically exist in co-centrical architectures where a cell layer surrounds an acellular lumen. Herein, this spatial cellular position and 3D architecture, containing dual compartments with different hydrogel materials, is engineered using a simple co-axial nozzle setup, in a single step utilizing prostate as a model of glandular cancer. The resulting hydrogel soft structures support viable prostate cancer cells of different cell lines and enable over-time maturation into cancer-mimicking aggregates surrounding the acellular core. The biofabricated cancer mimicking structures are then used as a model to predict the inhibitory efficacy of the poly ADP ribose polymerase (PARP) inhibitor, Talazoparib, and the antiandrogen drug, Enzalutamide, in the growth of the cancer cell layer. Our results show that the obtained hydrogel constructs can be adapted to quickly obtain 3D cancer models which combine 3D physiological architectures with high-throughput screening to detect and optimize anti-cancer drugs in prostate and potentially other glandular cancer types.
