ABSTRACT
Hollow vesicles made from a single or double layer of block-copolymer molecules, called polymersomes, represent an important technological platform for new developments in nano-medicine and nano-biotechnology. A central aspect in creating functional polymersomes is their combination with proteins, especially through encapsulation in the inner cavity of the vesicles. When producing polymersomes by techniques such as film rehydration, significant proportions of the proteins used are trapped in the vesicle lumen, resulting in high encapsulation efficiencies. However, because of the difficulty of scaling up, such methods are limited to laboratory experiments and are not suitable for industrial scale production. Recently, we developed a scalable polymersome production process in stirred-tank reactors, but the statistical encapsulation of proteins resulted in fairly low encapsulation efficiencies of around 0.5%. To increase encapsulation in this process, proteins were genetically fused with hydrophobic membrane anchoring peptides. This resulted in encapsulation efficiencies of up to 25.68%. Since proteins are deposited on the outside and inside of the polymer membrane in this process, two methods for the targeted removal of protein domains by proteolysis with tobacco etch virus protease and intein splicing were evaluated. This study demonstrates the proof-of-principle for production of protein-functionalized polymersomes in a scalable process.
Subject(s)
Cell Encapsulation/methods , Nanotechnology/methods , Peptides/chemistry , Polymers/chemistry , Proteins/chemistry , Hydrophobic and Hydrophilic Interactions , Membranes/chemistryABSTRACT
Parkinson's disease (PD) is the second most common neurological disorder, associated with decreased dopamine levels in the brain. The goal of this study was to assess the potential of a regenerative medicine-based cell therapy approach to increase dopamine levels. In this study, we used rat adrenal pheochromocytoma (PC12) cells that can produce, store, and secrete dopamine. These cells were microencapsulated in the selectively permeable polymer membrane to protect them from immune responses. For fabrication of the microcapsules, we used a modified Buchi spray dryer B-190 that allows for fast manufacturing of microcapsules and is industrially scalable. Size optimization of the microcapsules was performed by systematically varying key parameters of the spraying device. The short- and long-term stabilities of the microcapsules were assessed. In the in vitro study, the cells were found viable for a period of 30 days. Selective permeability of the microcapsules was confirmed via dopamine release assay and micro BCA protein assay. We found that the microcapsules were permeable to the small molecules including dopamine and were impermeable to the large molecules like BSA. Thus, they can provide the protection to the encapsulated cells from the immune cells. Griess's assay confirmed the non-immunogenicity of the microcapsules. These results demonstrate the effective fabrication of microcapsules encapsulating cells using an industrially scalable device. The microcapsules were stable, and the cells were viable inside the microcapsules and were found to release dopamine. Thus, these microcapsules have the potential to serve as the alternative or complementary treatment approach for PD.
Subject(s)
Aluminum Compounds/chemical synthesis , Capsules/chemical synthesis , Cell Encapsulation/methods , Cell- and Tissue-Based Therapy/methods , Parkinson Disease , Sodium Compounds/chemical synthesis , Aluminum Compounds/administration & dosage , Aluminum Compounds/metabolism , Animals , Brain/metabolism , Capsules/administration & dosage , Capsules/metabolism , Cell Survival/drug effects , Cell Survival/physiology , Dopamine/metabolism , Mice , PC12 Cells , Parkinson Disease/metabolism , Parkinson Disease/therapy , Polymers/administration & dosage , Polymers/chemical synthesis , Polymers/metabolism , Prospective Studies , RAW 264.7 Cells , Rats , Sodium Compounds/administration & dosage , Sodium Compounds/metabolism , Treatment OutcomeABSTRACT
BACKGROUND: Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic microorganisms can improve health. However, orally delivered probiotic microorganisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic microorganisms are still not effective enough. RESULTS: We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS), that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for at least 4 h without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by cellulase at concentrations found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract. CONCLUSION: Taken together, the data suggests that CS microencapsulation can protect bacteria and yeasts from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.
Subject(s)
Cell Encapsulation/methods , Cellulose/analogs & derivatives , Drug Compounding/methods , Drug Delivery Systems/methods , Escherichia coli/growth & development , Probiotics/administration & dosage , Animals , Cellulase/chemistry , Cellulose/chemistry , Gastric Juice , Gastrointestinal Microbiome , Humans , Hydrogen-Ion Concentration , Intestines/microbiology , Male , Mice , Mice, Nude , Microbial Viability , MicrospheresABSTRACT
Due to their excellent mechanical strength and biocompatibility, silk fibroin(SF) hydrogels can serve as ideal scaffolds. However, their slow rate of natural degradation limits the space available for cell proliferation, which hinders their application. In this study, litchi-like calcium carbonate@hydroxyapatite (CaCO3@HA) porous microspheres loaded with proteases from Streptomyces griseus (XIV) were used as drug carriers to regulate the biodegradation rate of SF hydrogels. The results showed that litchi-like CaCO3@HA microspheres with different phase compositions could be prepared by changing the hydrothermal reaction time. The CaCO3@HA microspheres controlled the release of Ca ions, which was beneficial for the osteogenic differentiation of mesenchymal stem cells (MSCs). The adsorption and release of protease XIV from the CaCO3@HA microcarriers indicated that the loading and release amount can be controlled with the initial drug concentration. The weight loss test and SEM observation showed that the degradation of the fibroin hydrogel could be controlled by altering the amount of protease XIV-loaded CaCO3@HA microspheres. A three-dimensional (3D) cell encapsulation experiment proved that incorporation of the SF hydrogel with protease XIV-loaded microspheres promoted cell dispersal and spreading, suggesting that the controlled release of protease XIV can regulate hydrogel degradation. SF hydrogels incorporated with protease XIV-loaded microspheres are suitable for cell growth and proliferation and are expected to serve as excellent bone tissue engineering scaffolds.
Subject(s)
Drug Carriers/chemical synthesis , Fibroins/chemistry , Pronase/administration & dosage , Tissue Scaffolds/chemistry , Animals , Biocompatible Materials/chemical synthesis , Biocompatible Materials/chemistry , Cell Differentiation/drug effects , Cell Encapsulation/instrumentation , Cell Encapsulation/methods , Cells, Cultured , Drug Carriers/chemistry , Durapatite/chemistry , Hydrogels/chemistry , Materials Testing , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/physiology , Mice , Microspheres , Microtechnology , Osteogenesis/drug effects , Pronase/chemistry , Pronase/pharmacokinetics , Silk/chemistry , Tissue Culture Techniques/methods , Tissue EngineeringABSTRACT
Droplet microfluidics-based single-cell encapsulation is a critical technology that enables large-scale parallel single-cell analysis by capturing and processing thousands of individual cells. As the efficiency of passive single-cell encapsulation is limited by Poisson distribution, active single-cell encapsulation has been developed to theoretically ensure that each droplet contains one cell. However, existing active single-cell encapsulation technologies still face issues related to fluorescence labeling and low throughput. Here, we present an active single-cell encapsulation technique by using microvalve-based drop-on-demand technology and real-time image processing to encapsulate single cells with high throughput in a label-free manner. Our experiments demonstrated that the single-cell encapsulation system can encapsulate individual polystyrene beads with 96.3 % efficiency and HeLa cells with 94.9 % efficiency. The flow speed of cells in this system can reach 150 mm/s, resulting in a corresponding theoretical encapsulation throughput of 150 Hz. This technology has significant potential in various biomedical applications, including single-cell omics, secretion detection, and drug screening.
Subject(s)
Single-Cell Analysis , Humans , Single-Cell Analysis/methods , HeLa Cells , Image Processing, Computer-Assisted , Polystyrenes/chemistry , Microfluidic Analytical Techniques/instrumentation , Lab-On-A-Chip Devices , Cell Encapsulation/methodsABSTRACT
Diabetes is a prevalent chronic disease affecting millions of people globally. To address this health challenge, advanced beta cell therapy using biomaterials-based macroscale, microscale, and nanoscale encapsulation devices must tackle various obstacles. First, overcoming foreign body responses is a major focus of research. Strategies such as immunomodulatory materials and physical immunoshielding are investigated to reduce the immune response and improve the longevity of the encapsulated cells. Furthermore, oxygenating strategies, such as the use of oxygen-releasing biomaterials, are developed to improve oxygen diffusion and promote cell survival. Finally, yet importantly, promoting vascularization through the use of angiogenic growth factors and the incorporation of pre-vascularized materials are also explored to enhance nutrient and oxygen supply to the encapsulated cells. This review seeks to specifically highlight the emerging research strategies developed to overcome these challenges using micro and nanoscale biomaterial encapsulation devices. Continuously improving and refining these strategies make an advance toward realizing the improved therapeutic potential of the encapsulated beta cells.
Subject(s)
Biocompatible Materials , Diabetes Mellitus, Type 1 , Insulin-Secreting Cells , Humans , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Diabetes Mellitus, Type 1/therapy , Animals , Biocompatible Materials/chemistry , Cell Encapsulation/methods , Oxygen/chemistryABSTRACT
Encapsulation of single cells is a powerful technique used in various fields, such as regenerative medicine, drug delivery, tissue regeneration, cell-based therapies, and biotechnology. It offers a method to protect cells by providing cytocompatible coatings to strengthen cells against mechanical and environmental perturbations. Silk fibroin, derived from the silkworm Bombyx mori, is a promising protein biomaterial for cell encapsulation due to the cytocompatibility and capacity to maintain cell functionality. Here, THP-1 cells, a human leukemia monocytic cell line, were encapsulated with chemically modified silk polyelectrolytes through electrostatic layer-by-layer deposition. The effectiveness of the silk nanocoating was assessed using scanning electron microscopy (SEM) and confocal microscopy and on cell viability and proliferation by Alamar Blue assay and live/dead staining. An analysis of the mechanical properties of the encapsulated cells was conducted using atomic force microscopy nanoindentation to measure elasticity maps and cellular stiffness. After the cells were encapsulated in silk, an increase in their stiffness was observed. Based on this observation, we developed a mechanical predictive model to estimate the variations in stiffness in relation to the thickness of the coating. By tuning the cellular assembly and biomechanics, these encapsulations promote systems that protect cells during biomaterial deposition or processing in general.
Subject(s)
Bombyx , Cell Survival , Humans , Cell Survival/drug effects , Animals , Silk/chemistry , THP-1 Cells , Fibroins/chemistry , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Cell Proliferation/drug effects , Cell Encapsulation/methodsABSTRACT
Tissue engineering at single-cell resolution has enhanced therapeutic efficacy. Droplet microfluidics offers a powerful platform that allows deterministic single-cell encapsulation into aqueous droplets, yet the direct encapsulation of cells into microgels remains challenging. Here, the design of a microfluidic device that is capable of single-cell encapsulation within polyethylene glycol norbornene (PEGNB) hydrogels on-chip is reported. Cells are first ordered in media within a straight microchannel via inertial focusing, followed by the introduction of PEGNB solution from two separate, converging channels. Droplets are thoroughly mixed by passage through a serpentine channel, and microgels are formed by photo-photopolymerization. This platform uniquely enables both single-cell encapsulation and excellent cell viability post-photo-polymerization. More than 90% of singly encapsulated mesenchymal stromal cells (MSCs) remain alive for 7 days. Notably, singly encapsulated MSCs have elevated expression levels in genes that code anti-inflammatory cytokines, for example, IL-10 and TGF-ß, thus enhancing the secretion of proteins of interest. Following injection into a mouse model with induced inflammation, singly encapsulated MSCs show a strong retention rate in vivo, reduce overall inflammation, and mitigate liver damage. These translational results indicate that deterministic single-cell encapsulation could find use in a broad spectrum of tissue engineering applications.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Norbornanes , Polyethylene Glycols , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Animals , Polyethylene Glycols/chemistry , Mice , Mesenchymal Stem Cell Transplantation/methods , Norbornanes/chemistry , Microgels/chemistry , Cell Encapsulation/methods , Hydrogels/chemistry , Hydrogels/pharmacology , Cell Survival/drug effects , HumansABSTRACT
This study presents the development and characterization of a novel double-network self-healing hydrogel based on N-carboxyethyl chitosan (CEC) and oxidized dextran (OD) with the incorporation of crosslinked collagen (CEC-OD/COL-GP) to enhance its biological and physicochemical properties. The hydrogel formed via dynamic imine bond formation exhibited efficient self-healing within 30 min, and a compressive modulus recovery of 92 % within 2 h. In addition to its self-healing ability, CEC-OD/COL-GP possesses unique physicochemical characteristics including transparency, injectability, and adhesiveness to various substrates and tissues. Cell encapsulation studies confirmed the biocompatibility and suitability of the hydrogel as a cell-culture scaffold, with the presence of a collagen network that enhances cell adhesion, spreading, long-term cell viability, and proliferation. Leveraging their unique properties, we engineered assemblies of self-healing hydrogel modules for controlled spatiotemporal drug delivery and constructed co-culture models that simulate angiogenesis in tumor microenvironments. Overall, the CEC-OD/COL-GP hydrogel is a versatile and promising material for biomedical applications, offering a bottom-up approach for constructing complex structures with self-healing capabilities, controlled drug release, and support for diverse cell types in 3D environments. This hydrogel platform has considerable potential for advancements in tissue engineering and therapeutic interventions.
Subject(s)
Cell Adhesion , Chitosan , Dextrans , Hydrogels , Hydrogels/chemistry , Hydrogels/pharmacology , Chitosan/chemistry , Dextrans/chemistry , Humans , Cell Adhesion/drug effects , Cell Survival/drug effects , Collagen/chemistry , Animals , Drug Liberation , Cell Proliferation/drug effects , Cell Encapsulation/methods , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Mice , Biomimetics/methods , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Tissue Scaffolds/chemistryABSTRACT
Microfluidic cell encapsulation has provided a platform for studying the behavior of individual cells and has become a turning point in single-cell analysis during the last decade. The engineered microenvironment, along with protecting the immune response, has led to increasingly presenting the results of practical and pre-clinical studies with the goals of disease treatment, tissue engineering, intelligent control of stem cell differentiation, and regenerative medicine. However, the significance of cell-substrate interaction versus cell-cell communications in the microgel is still unclear. In this study, monodisperse alginate microgels were generated using a flow-focusing microfluidic device to determine how the cell microenvironment can control human bone marrow-derived mesenchymal stem cells (hBMSCs) viability, proliferation, and biomechanical features in single-cell droplets versus multi-cell droplets. Collected results show insufficient cell proliferation (234 % and 329 %) in both single- and multi-cell alginate microgels. Alginate hydrogels supplemented with poly-l-lysine (PLL) showed a better proliferation rate (514 % and 780 %) in a comparison of free alginate hydrogels. Cell stiffness data illustrate that hBMSCs cultured in alginate hydrogels have higher membrane flexibility and migration potency (Young's modulus equal to 1.06 kPa), whereas PLL introduces more binding sites for cell attachment and causes lower flexibility and migration potency (Young's modulus equal to 1.83 kPa). Considering that cell adhesion is the most important parameter in tissue engineering, in which cells do not run away from a 3D substrate, PLL enhances cell stiffness and guarantees cell attachments. In conclusion, cell attachment to PLL-mediated alginate hydrogels is crucial for cell viability and proliferation. It suggests that cell-cell signaling is good enough for stem cell viability, but cell-PLL attachment alongside cell-cell signaling is crucial for stem cell proliferation and self-renewal.
Subject(s)
Alginates , Cell Adhesion , Cell Proliferation , Mesenchymal Stem Cells , Microgels , Polylysine , Alginates/chemistry , Alginates/pharmacology , Polylysine/chemistry , Polylysine/pharmacology , Humans , Cell Adhesion/drug effects , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Cell Proliferation/drug effects , Microgels/chemistry , Microfluidics/methods , Cell Communication/drug effects , Cell Survival/drug effects , Hydrogels/chemistry , Hydrogels/pharmacology , Cell Encapsulation/methods , Single-Cell Analysis , Cell Self Renewal/drug effects , Cell Differentiation/drug effectsABSTRACT
Smart hydrogels with versatile properties, including a tunable gelation time, nonswelling attributes, and biocompatibility, are in great need in the biomedical field. To meet this urgent demand, we explored novel biomaterials with the desired properties from sessile marine organisms. To this end, a novel protein, Sbp9, derived from scallop byssus was extensively investigated, which features typical epidermal growth factor-like (EGFL) multiple repetitive motifs. Our current work demonstrated that the key fragment of Sbp9 (calcium-binding domain (CBD) and 4 EGFL repeats (CE4)) was able to form a smart hydrogel driven by noncovalent interactions and facilitated by disulfide bonds. More importantly, this smart hydrogel demonstrates several desirable and beneficial features, which could offset the drawbacks of typical protein-based hydrogels, including (1) a redox-responsive gelation time (from <1 to 60 min); (2) tunable mechanical properties, nonswelling abilities, and an appropriate microstructure; and (3) good biocompatibility and degradability. Furthermore, proof-of-concept demonstrations showed that the newly discovered hydrogel could be used for anticancer drug delivery and cell encapsulation. Taken together, a smart hydrogel inspired by marine sessile organisms with desirable properties was generated and characterized and demonstrated to have extensive applicability potential in biomedical applications, including tissue engineering and drug release.
Subject(s)
Calcium-Binding Proteins/chemistry , Cell Encapsulation/methods , Drug Carriers/chemistry , Hydrogels/chemistry , Pectinidae/chemistry , Smart Materials/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Antineoplastic Agents/chemistry , Calcium-Binding Proteins/toxicity , Cell Line, Tumor , Doxorubicin/chemistry , Drug Carriers/toxicity , Drug Liberation , Humans , Hydrogels/toxicity , Hydrogen Peroxide/chemistry , Male , Mesenchymal Stem Cells/drug effects , Oxidation-Reduction , Porosity , Protein Domains , Rats, Sprague-Dawley , Smart Materials/toxicityABSTRACT
Live microbes such as lactobacilli have long been used as probiotic supplements and, more recently, have been explored as live biotherapeutic products with the potential to treat a range of conditions. Among these microbes is a category of anaerobes that possess therapeutic potential while exhibiting unique oxygen sensitivity and thus requiring careful considerations in the formulation and storage processes. Existing microbial formulation development has focused on facultative anaerobes with natural oxygen tolerance; a few strategies have been reported for anaerobes with demonstrated oxygen intolerance, warranting novel approaches toward addressing the challenges for these oxygen-sensitive anaerobes. Here, we develop a polymeric encapsulation system for the formulation and storage of Bifidobacterium adolescentis (B. adolescentis), a model anaerobe that loses viability in aerobic incubation at 37 °C within 1 day. We discover that this strain remains viable under aerobic conditions for 14 days at 4 °C, enabling formulation development such as solution casting and air drying in an aerobic environment. Next, through a systematic selection of polymer encapsulants and excipients, we show that encapsulation with poly(vinyl alcohol) (PVA) acts as an oxygen barrier and facilitates long-term storage of B. adolescentis, which is partially attributed to reduced generation of reactive oxygen species. Lastly, PVA-based formulations can produce oral capsule-loaded films and edible gummy bears, demonstrating its compatibility with both pharmaceutical and food dosage forms.
Subject(s)
Bifidobacterium adolescentis , Cell Encapsulation/methods , Polyvinyl Alcohol/chemistry , Probiotics/administration & dosage , Bifidobacterium adolescentis/metabolism , Capsules , Excipients/chemistry , Food Technology , Probiotics/metabolism , Reactive Oxygen Species/metabolismABSTRACT
In recent years, cell microencapsulation technology has advanced, mainly driven by recent developments in the use of stem cells or the optimization of biomaterials. Old challenges have been addressed from new perspectives, and systems developed and improved for decades are now being transferred to the market by novel start-ups and consolidated companies. These products are mainly intended for the treatment of diabetes mellitus (DM), but also cancer, central nervous system (CNS) disorders or lysosomal diseases, among others. In this review, we analyze the results obtained in clinical trials to date and define the global key players that will lead the cell microencapsulation market to bring this technology to the clinic in the future.
Subject(s)
Biocompatible Materials/administration & dosage , Cell Encapsulation/methods , Drug Delivery Systems , Biomedical Technology/methods , Capsules , Delayed-Action Preparations , Humans , Stem Cells/cytologyABSTRACT
The extracellular matrix (ECM) forms through hierarchical assembly of small and larger polymeric molecules into a transient, hydrogel-like fibrous network that provides mechanical support and biochemical cues to cells. Synthetic, fibrous supramolecular networks formed via non-covalent assembly of various molecules are therefore potential candidates as synthetic mimics of the natural ECM, provided that functionalization with biochemical cues is effective. Here, combinations of slow and fast exchanging molecules that self-assemble into supramolecular fibers are employed to form transient hydrogel networks with tunable dynamic behavior. Obtained results prove that modulating the ratio between these molecules dictates the extent of dynamic behavior of the hydrogels at both the molecular and the network level, which is proposed to enable effective incorporation of cell-adhesive functionalities in these materials. Excitingly, the dynamic nature of the supramolecular components in this system can be conveniently employed to formulate multicomponent supramolecular hydrogels for easy culturing and encapsulation of single cells, spheroids, and organoids. Importantly, these findings highlight the significance of molecular design and exchange dynamics for the application of supramolecular hydrogels as synthetic ECM mimics.
Subject(s)
Cell Encapsulation/methods , Hydrogels/chemistry , Blood Vessels/cytology , Cell Adhesion , Extracellular Matrix/chemistry , Fluorescence Recovery After Photobleaching , Fluorescent Dyes/chemistry , Humans , Polyethylene Glycols/chemistry , Pyrimidinones/blood , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
Conventional stem cell delivery typically utilize administration of directly injection of allogenic cells or domesticated autogenic cells. It may lead to immune clearance of these cells by the host immune systems. Alginate microgels have been demonstrated to improve the survival of encapsulated cells and overcome rapid immune clearance after transplantation. Moreover, alginate microgels can serve as three-dimensional extracellular matrix to support cell growth and protect allogenic cells from rapid immune clearance, with functions as delivery vehicles to achieve sustained release of therapeutic proteins and growth factors from the encapsulated cells. Besides, cell-loaded alginate microgels can potentially be applied in regenerative medicine by serving as injectable engineered scaffolds to support tissue regrowth. In this review, the properties of alginate and different methods to produce alginate microgels are introduced firstly. Then, we focus on diverse applications of alginate microgels for cell delivery in tissue engineering and regenerative medicine.
Subject(s)
Alginates/chemistry , Cell Transplantation/methods , Microgels/chemistry , Regenerative Medicine/methods , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Cell Encapsulation/methods , Cell Line, Tumor , HumansABSTRACT
We propose a compound interfacial shearing (CIS) process for versatile production of monodisperse Janus emulsions with controllable structural and topographic features. The process induces an active periodic force to decouple material and process parameters, enables independent control of compartmental features in Janus emulsions, and facilitates inline and on-demand generation of various geometric features for a large variety of process parameters and material properties. Janus emulsions of poly(ethylene glycol) diacrylate (PEGDA) with a controlled number of compartments are produced by CIS and photopolymerized to form micro-hydrogels with designated interfacial curvatures. PEGDA micro-hydrogels can be further modified to achieve anisotropy of surface or internal features by the content of an oily dispersed phase. MCF-7 human breast cancer cells are encapsulated in micro-hydrogels for cell proliferation with satisfactory viability. By modifying PEGDA micro-hydrogels with RGDS-conjugated polystyrene microspheres, we have demonstrated the controlled spatial adhesion of MCF-7 cells and human umbilical vein endothelial cells (HUVECs) on the substrates of different three-dimensional (3D) curvatures. Our pilot study suggests a simple and potentially scalable approach to produce 3D substrates with controllable structural and topographic features for 3D guided cell organization.
Subject(s)
Emulsions/chemistry , Hydrogels/chemistry , Anisotropy , Cell Adhesion/drug effects , Cell Encapsulation/methods , Cell Proliferation/drug effects , Cell Survival/drug effects , Human Umbilical Vein Endothelial Cells , Humans , MCF-7 Cells , Microspheres , Pilot Projects , Polyethylene Glycols/chemistry , Polystyrenes/chemistryABSTRACT
OBJECTIVE: Most acellular injectable biomaterials for vocal fold (VF) wound treatment have limited regenerative potential due to their fast enzymatic degradation and limited recruitment of native cells postinjection. The injection of cells as therapeutic treatment often results in apoptosis due to stresses within the needle and the immune response of the host. Degradable microspheres may improve treatment effectiveness by increasing cell residence time, shielding cells during injection, and offering early protection against the immune system response. The objective of the present study was to investigate the potential of human VF fibroblasts encapsulated in polymeric microspheres as an injectable therapeutic treatment in vitro. METHODS: Alginate, alginate-poly-L-lysine, and alginate-chitosan microspheres were fabricated using electrospraying and characterized in terms of biocompatibility, swelling, and mechanical properties as well as cytokine production. RESULTS: Alginate microspheres were found to have the most desirable properties for VF regeneration. They were resistant to mechanical challenges. They were found to have a stiffness similar to that reported for native VF-lamina propria. They were found to be biocompatible and increased the proliferation of fibroblasts. Human VF fibroblasts encapsulated in alginate microspheres induced the production of interleukin (IL)-8 and IL-4 at 24 hours. CONCLUSION: The alginate microspheres fabricated in this study were found to offer potential advantages, as cell delivery tool. This study highlights the importance of combining biomaterials and cells to expedite the wound-healing process through cytokine production. Future work is aimed to further analysis of the wound-healing properties the microspheres. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1828-1834, 2021.
Subject(s)
Biocompatible Materials/administration & dosage , Cell Encapsulation/methods , Fibroblasts/physiology , Guided Tissue Regeneration/methods , Vocal Cords/cytology , Alginates/administration & dosage , Cell Culture Techniques , Cell Proliferation/physiology , Chitosan/administration & dosage , Humans , Injections , Materials Testing , Microspheres , Mucous Membrane/cytology , Polylysine/administration & dosage , Polylysine/analogs & derivatives , Vocal Cords/injuries , Wound Healing/physiologyABSTRACT
A novel cell free protein synthesis (CFPS) system utilizing layer-by-layer (LbL) polymer assembly was developed to reduce the operational cost of conventional CFPS. This yielded an encapsulated cell system, dubbed "eCells", that successfully performs in vitro CFPS and allows cost-effective incorporation of noncanonical amino acids into proteins. The use of eCells in CFPS circumvents the need for traditional cell lysate preparation and purification of amino acyl-tRNA synthetases (aaRS) while still retaining the small scale of an in vitro reaction. eCells were found to be 55% as productive as standard dialysis CFPS at 13% of the cost. The reaction was shown to be scalable over a large range of reaction volumes, and the crowding environment in eCells confers a stabilizing effect on marginally stable proteins, such as the pyrrolysl tRNA synthetase (PylRS), providing a means for their application in in vitro protein expression. Photocaged-cysteine (PCC) and Nε-(tert-butoxycarbonyl)-l-lysine (Boc-lysine) were incorporated into Peptidyl-prolyl cis-trans isomerase B (PpiB) using small amounts of ncAA with an adequate yield of protein. Fluorescent activated cell sorting (FACS) was used to demonstrate the partition of the lysate within the eCells in contrast to standard one pot cell lysate-based methods.
Subject(s)
Artificial Cells/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Metabolic Engineering/methods , Protein Biosynthesis , Amino Acids/metabolism , Amino Acyl-tRNA Synthetases/metabolism , Bacterial Outer Membrane Proteins/metabolism , Cell Encapsulation/methods , Cell-Free System/metabolism , Cysteine/metabolism , Escherichia coli Proteins/genetics , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Lysine/analogs & derivatives , Lysine/metabolism , Transcription, Genetic/geneticsABSTRACT
Developmental Engineering aims to imitate natural tissue regeneration processes via an additive manufacturing approach. This research developes a technology to fabricate ready-made cell marbles (CMs) by wrapping cell suspension droplets of (3-15 µl) with electrospun hydrophobic nanofibers, as modular building blocks for developmental engineering. Human dermal fibroblasts and/or immortalised keratinocytes were suspended in the culture media cores of the CMs. The encapsulated cells were observed to precipitate at bottoms or up-inclined inner surfaces of the fibrous shells within 10 min. The CMs were mechanically strong enough to be handled as soft solids, thus easily and accurately delivered using forceps into three distinct culture systems, including tissue culture plastics, cellulosic scaffolds and in vitro fibrin wound models. The release of the cells, culture media and nanofibers into specific delivery points within the investigated culture systems was achieved via the controlled rupture of the CMs triggered by the simple hydrophobic-hydrophilic interaction between the nanofibers and the aqueous surroundings. Further cell and tissue culture studies indicated that the prominent traits of the skin cells were well preserved during cell encapsulation and delivery processes, suggesting the great potential of the CMs for additive tissue manufacturing in developmental engineering.
Subject(s)
Cell Encapsulation/methods , Nanofibers/chemistry , Tissue Engineering/methods , Biocompatible Materials/chemistry , Cell Culture Techniques , Culture Media , Fibrin , Fibroblasts/cytology , Humans , Keratinocytes , Polyesters , Skin , Suspensions , Tissue Scaffolds/chemistryABSTRACT
Encapsulated cell therapy has shown great potential in the treatment of several forms of cancer. Microencapsulation of these cancer cells can protect the core from the harmful effects of the neighboring cellular environment and can supply nutrients and oxygen. Such an encapsulation technique ensures cell viability and enables targeted drug delivery in cancer therapy. The cells immobilized with a biocompatible shell material can be isolated from the ambient and can move in constricted microcapillary. However, transportation of these cells through the narrow microcapillary may squeeze and mechanically damage the cells which threaten the cell viability. The cell type, conditions and the viscoelastic properties of the shell can dictate cell viability. A front-tracking numerical simulation shows that the engineered shell material with higher viscoelasticity improves the cell viability. It is also shown that low cortical tension of cells can contribute to lower cell viability.