Evaluation of Alginate Hydrogel Microstrands for Stromal Cell Encapsulation and Maintenance.

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.

Co-immobilized multi-enzyme biocatalytic system on reversible and soluble carrier for saccharification of corn straw cellulose.

Herein, three enzymes (cellulase, ß-glucosidase, and pectinase) with synergistic effects were co-immobilized on the Eudragit L-100, and the recovery of co-immobilized enzymes from solid substrates were achieved through the reversible and soluble property of the carrier. The optimization of enzyme ratio overcomed the problem of inappropriate enzyme activity ratio caused by different immobilization efficiencies among enzymes during the preparation process of co-immobilized enzymes. The co-immobilized enzymes were utilized to catalytically hydrolyze cellulose from corn straw into glucose, achieving a cellulose conversion rate of 74.45% under conditions optimized for their enzymatic characteristics and hydrolytic reaction conditions. As a result of the reversibility and solubility of the carrier, the co-immobilized enzymes were recovered from the solid substrate after five cycles, retaining 54.67% of the enzyme activity. The aim of this study is to investigate the potential of co-immobilizing multiple enzymes onto the Eudragit L-100 carrier for the synergistic degradation of straw cellulose.

Interfacing neural cells with typical microelectronics materials for future manufacturing.

The biocompatibility of materials used in electronic devices is critical for the development of implantable devices like pacemakers and neuroprosthetics, as well as in future biomanufacturing. Biocompatibility refers to the ability of these materials to interact with living cells and tissues without causing an adverse response. Therefore, it is essential to evaluate the biocompatibility of metals and semiconductor materials used in electronic devices to ensure their safe use in medical applications. Here, we evaluated the biocompatibility of a collection of diced silicon chips coated with a variety of metal thin films, interfacing them with different cell types, including murine mastocytoma cells in suspension culture, adherent NIH 3T3 fibroblasts, and human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs). All materials tested were biocompatible and showed the potential to support neural differentiation of iPSC-NPCs, creating an opportunity to use these materials in a scalable production of a range of biohybrid devices such as electronic devices to study neural behaviors and neuropathies.

Recreating the Trabecular Outflow Tissue on Implantable, Micropatterned, Ultrathin, Porous Polycaprolactone Scaffolds.

Glaucoma, where increased intraocular pressure (IOP) leads to damage to the optic nerve and loss of sight, is amongst the foremost causes of irreversible blindness worldwide. In primary open angle glaucoma, the increased IOP is a result of the malfunctioning human trabecular meshwork (HTM) cells' inability to properly regulate the outflow of aqueous humor from the eye. A potential future treatment for glaucoma is to replace damaged HTM cells with a tissue-engineered substitute, thus restoring proper fluid outflow. Polycaprolactone (PCL) is a versatile, biodegradable, and implantable material that is widely used for cell culture and tissue engineering. In this work, PCL scaffolds were lithographically fabricated using a sacrificial process to produce submicron-thick scaffolds with openings of specific sizes and shapes (e.g., grid, hexagonal pattern). The HTM cell growth on gelatin-coated PCL scaffolds was assessed by scanning electron microscopy, tetrazolium metabolic activity assay, and cytoskeletal organization of F-actin. Expression of HTM-specific markers and ECM deposition were assessed by immunocytochemistry and qPCR analysis. Gelatin-coated, micropatterned, ultrathin, porous PCL scaffolds with a grid pattern supported proper HTM cell growth, cytoskeleton organization, HTM-marker expression, and ECM deposition, demonstrating the feasibility of using these PCL scaffolds to tissue-engineer implantable, healthy ocular outflow tissue.

Salivary Gland Bioengineering.

Salivary gland dysfunction affects millions globally, and tissue engineering may provide a promising therapeutic avenue. This review delves into the current state of salivary gland tissue engineering research, starting with a study of normal salivary gland development and function. It discusses the impact of fibrosis and cellular senescence on salivary gland pathologies. A diverse range of cells suitable for tissue engineering including cell lines, primary salivary gland cells, and stem cells are examined. Moreover, the paper explores various supportive biomaterials and scaffold fabrication methodologies that enhance salivary gland cell survival, differentiation, and engraftment. Innovative engineering strategies for the improvement of vascularization, innervation, and engraftment of engineered salivary gland tissue, including bioprinting, microfluidic hydrogels, mesh electronics, and nanoparticles, are also evaluated. This review underscores the promising potential of this research field for the treatment of salivary gland dysfunction and suggests directions for future exploration.

Alginate microfibers as therapeutic delivery scaffolds and tissue mimics.

Alginate, a naturally occurring polysaccharide, has been widely used in cell encapsulation, 3D culture, cell therapy, tissue engineering, and regenerative medicine. Alginate's frequent use comes from its biocompatibility and ability to easily form hydrogel in a variety of forms (e.g. microcapsules, microfibers, and porous scaffolds), which can provide immunoprotection for cell therapy and mimic the extracellular matrix for tissue engineering. During the past 15 years, alginate hydrogel microfibers have attracted more and more attention due to its continuous thin tubular structures (diameter or shell thickness ⩽ 200 µm), high-density cell growth, high handleability and retrievability, and scalability. This review article provides a concise overview of alginate and its resultant hydrogel microfibers for the purpose of promoting multidisciplinary, collaborative, and convergent research in the field. It starts with a historical review of alginate as biomaterials and provides basics about alginate structure, properties, and mechanisms of hydrogel formation, followed by current challenges in effective cell delivery and functional tissue engineering. In particular, this work discusses how alginate microfiber technology could provide solutions to unmet needs with a focus on the current state of the art of alginate microfiber technology and its applications in 3D cell culture, cell delivery, and tissue engineering. At last, we discuss future directions in the perspective of alginate-based advanced technology development in biology and medicine.

Engineering cryoelectrospun elastin-alginate scaffolds to serve as stromal extracellular matrices.

Scaffold-based regenerative strategies that emulate physical, biochemical, and mechanical properties of the native extracellular matrix (ECM) of the region of interest can influence cell growth and function. Existing ECM-mimicking scaffolds, including nanofiber (NF) mats, sponges, hydrogels, and NF-hydrogel composites are unable to simultaneously mimic typical composition, topography, pore size, porosity, and viscoelastic properties of healthy soft-tissue ECM. In this work, we used cryoelectrospinning to fabricate 3D porous scaffolds with minimal fibrous backbone, pore size and mechanical properties similar to soft-tissue connective tissue ECM. We used salivary glands as our soft tissue model and found the decellularized adult salivary gland (DSG) matrix to have a fibrous backbone, 10-30µm pores, 120 Pa indentation modulus, and ∼200 s relaxation half time. We used elastin and alginate as natural, compliant biomaterials and water as the solvent for cryoelectrospinning scaffolds to mimic the structure and viscoelasticity of the connective tissue ECM of the DSG. Process parameters were optimized to produce scaffolds with desirable topography and compliance similar to DSG, with a high yield of >100 scaffolds/run. Using water as solvent, rather than organic solvents, was critical to generate biocompatible scaffolds with desirable topography; further, it permitted a green chemistry fabrication process. Here, we demonstrate that cryoelectrospun scaffolds (CESs) support penetration of NIH 3T3 fibroblasts 250-450µm into the scaffold, cell survival, and maintenance of a stromal cell phenotype. Thus, we demonstrate that elastin-alginate CESs mimic many structural and functional properties of ECM and have potential for future use in regenerative medicine applications.

Alginate Hydrogel Microtubes for Salivary Gland Cell Organization and Cavitation.

Understanding the different regulatory functions of epithelial and mesenchymal cell types in salivary gland development and cellular organization is essential for proper organoid formation and salivary gland tissue regeneration. Here, we demonstrate a biocompatible platform using pre-formed alginate hydrogel microtubes to facilitate direct epithelial-mesenchymal cell interaction for 3D salivary gland cell organization, which allows for monitoring cellular organization while providing a protective barrier from cell-cluster loss during medium changes. Using mouse salivary gland ductal epithelial SIMS cells as the epithelial model cell type and NIH 3T3 fibroblasts or primary E16 salivary mesenchyme cells as the stromal model cell types, self-organization from epithelial-mesenchymal interaction was examined. We observed that epithelial and mesenchymal cells undergo aggregation on day 1, cavitation by day 4, and generation of an EpCAM-expressing epithelial cell layer as early as day 7 of the co-culture in hydrogel microtubes, demonstrating the utility of hydrogel microtubes to facilitate heterotypic cell-cell interactions to form cavitated organoids. Thus, pre-formed alginate microtubes are a promising co-culture method for further understanding epithelial and mesenchymal interaction during tissue morphogenesis and for future practical applications in regenerative medicine.

Evaluation of Preparation and Detoxification of Hemicellulose Hydrolysate for Improved Xylitol Production from Quinoa Straw.

Quinoa straw is rich in hemicellulose, and it could be hydrolyzed into xylose. It is a promising energy resource alternative that acts as a potential low-cost material for producing xylitol. In this study, quinoa straw was used as a substrate subjected to the hydrolysis of dilute sulfuric acid solution. Based on the production of xylose and inhibitors during hydrolysis, the optimal conditions for the hydrolysis of hemicellulose in quinoa straw were determined. Detoxification was performed via activated carbon adsorption. The optimal detoxification conditions were determined on the basis of major inhibitor concentrations in the hydrolysate. When the addition of activated carbon was 3% at 30 °C for 40 min, the removal of formic acid, acetic acid, furfural, and 5-HMF could reach 66.52%, 64.54%, 88.31%, and 89.44%, respectively. In addition to activated carbon adsorption, vacuum evaporation was further conducted to perform two-step detoxification. Subsequently, the detoxified hydrolysate was used for xylitol fermentation. The yield of xylitol reached 0.50 g/g after 96 h of fermentation by Candida tropicalis (CICC 1779). It is 1.2-fold higher than that obtained through the sole vacuum evaporation method. This study validated the feasibility of xylitol production from quinoa straw via a biorefinery process.

Predictable fabrication of pre-made alginate hydrogel microtubes for stem cell aggregation using needle-in-needle devices.

Alginate hydrogels in microtubular structures have great potential to advance three-dimensional (3D) culture, organoid formation, tissue engineering, and cell therapy. To address the need of fabricating consistent, stable hydrogel microtubes for efficient large organoid generation in a simple and quick manner, we have designed needle-in-needle devices to fabricate alginate hydrogel microtubes without any dead volume of the cell-alginate mixture and demonstrated the feasibility of injecting and culturing embryoid bodies in these pre-made hydrogel microtubes. We further used a reverse engineering approach to find out the optimal flow rates and alginate concentration for fabricating pre-made hydrogel microtubes with desired diameter using particular sets of needle-in-needle devices. We established the relationship of the alginate flow rate with diameter and wall thickness of the microtube using mathematic modeling. It offers a way to determine the flow rate for making microtubes with the desired dimension. Additionally, we evaluated the effect of CaCl2concentration on the diameter as well as stem cell viability. At last, we demonstrated the capacity of fabricating hydrogel microtubes of varying diameters using three sets of needle-in-needle devices and evaluated stem cell growth in these hydrogel microtubes. It provides a new avenue to accessible, repeatable, scalable, and easy to use pre-made 'off-the-shelf' hydrogel microtubes for 3D cell culture including, but not limiting to stem cells.

Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery.

Cell therapy for the injured spinal cord will rely on combined advances in human stem cell technologies and delivery strategies. Here we encapsulate homotypic spinal cord neural stem cells (scNSCs) in an alginate-based neural ribbon delivery platform. We perform a comprehensive in vitro analysis and qualitatively demonstrate graft survival and injury site retention using a rat C4 hemi-contusion model. Pre-configured neural ribbons are transport-stable modules that enable site-ready injection, and can support scNSC survival and retention in vivo. Neural ribbons offer multifunctionality in vitro including co-encapsulation of the injury site extracellular matrix modifier chondroitinase ABC (chABC), tested here in glial scar models, and ability of cervically-patterned scNSCs to differentiate within neural ribbons and project axons for integration with 3-D external matrices. This is the first extensive in vitro characterization of neural ribbon technology, and constitutes a plausible method for reproducible delivery, placement, and retention of viable neural cells in vivo.

A Biomimetic, Stem Cell-Derived In Vitro Ocular Outflow Model.

Age-related human trabecular meshwork (HTM) cell loss is suggested to affect its ability to regulate aqueous humor outflow in the eye. In addition, disease-related HTM cell loss is suggested to lead to elevated intraocular pressure in glaucoma. Induced pluripotent stem cell (iPSC)-derived trabecular meshwork (TM) cells are promising autologous cell sources that can be used to restore the declining TM cell population and function. Previously, an in vitro HTM model is bioengineered for understanding HTM cell biology and screening of pharmacological or biological agents that affect trabecular outflow facility. In this study, it is demonstrated that human iPSC-derived TM cells cultured on SU-8 scaffolds exhibit HTM-like cell morphology, extracellular matrix deposition, and drug responsiveness to dexamethasone treatment. These findings suggest that iPSC-derived TM cells behave like primary HTM cells and can thus serve as reproducible and scalable cell sources when using this in vitro system for glaucoma drug screening and further understanding of outflow pathway physiology, leading to personalized medicine.

Two-step induction of trabecular meshwork cells from induced pluripotent stem cells for glaucoma.

Glaucoma is a leading cause of irreversible blindness worldwide. Reducing intraocular pressure is currently the only effective treatment. Elevated intraocular pressure is associated with increased resistance of the outflow pathway, mainly the trabecular meshwork (TM). Despite great progress in the field, the development of novel and effective treatment for glaucoma is still challenging. In this study, we reported that human induced pluripotent stem cells (iPSCs) can be cultured as colonies and monolayer cells expressing OCT4, alkaline phosphatase, SSEA4 and SSEA1. After induction to neural crest cells (NCCs) positive to NGFR and HNK1, the iPSCs can differentiate into TM cells. The induced iPSC-TM cells expressed TM cell marker CHI3L1, were responsive to dexamethasone treatment with increased expression of myocilin, ANGPTL7, and formed CLANs, comparable to primary TM cells. To the best of our knowledge, this is the first study that induces iPSCs to TM cells through a middle neural crest stage, which ensures a stable NCC pool and ensures the high output of the same TM cells. This system can be used to develop personalized treatments using patient-derived iPSCs, explore high throughput screening of new drugs focusing on TM response for controlling intraocular pressure, and investigate stem cell-based therapy for TM regeneration.

Insulin production from hiPSC-derived pancreatic cells in a novel wicking matrix bioreactor.

Clinical use of pancreatic ß islets for regenerative medicine applications requires mass production of functional cells. Current technologies are insufficient for large-scale production in a cost-efficient manner. Here, we evaluate advantages of a porous cellulose scaffold and demonstrate scale-up to a wicking matrix bioreactor as a platform for culture of human endocrine cells. Scaffold modifications were evaluated in a multiwell platform to find the optimum surface condition for pancreatic cell expansion followed by bioreactor culture to confirm suitability. Preceding scale-up, cell morphology, viability, and proliferation of primary pancreatic cells were evaluated. Two optimal surface modifications were chosen and evaluated further for insulin secretion, cell morphology, and viable cell density for human-induced pluripotent stem cell-derived pancreatic cells at different stages of differentiation. Scale-up was accomplished with uncoated, amine-modified cellulose in a miniature bioreactor, and insulin secretion and cell metabolic profiles were determined for 13 days. We achieved 10-fold cell expansion in the bioreactor along with a significant increase in insulin secretion compared with cultures on tissue culture plastic. Our findings define a new method for expansion of pancreatic cells a on wicking matrix cellulose platform to advance cell therapy biomanufacturing for diabetes.

A bioengineering approach to Schlemm's canal-like stem cell differentiation for in vitro glaucoma drug screening.

Human Schlemm's canal (HSC) cells are critical for understanding outflow physiology and glaucoma etiology. However, primary donor cells frequently used in research are difficult to isolate. HSC cells exhibit both vascular and lymphatic markers. Human adipose-derived stem cells (ADSCs) represent a potential source of HSC due to their capacity to differentiate into both vascular and lymphatic endothelial cells, via VEGF-A and VEGF-C. Shear stress plays a critical role in maintaining HSC integrity, function, and PROX1 expression. Additionally, the human trabecular meshwork (HTM) microenvironment could provide cues for HSC-like differentiation. We hypothesize that subjecting ADSCs to VEGF-A or VEGF-C, shear stress, and co-culture with HTM cells could provide biological, mechanical, and cellular cues necessary for HSC-like differentiation. To test this hypothesis, effects of VEGF-A, VEGF-C, and shear stress on ADSC differentiation were examined and compared to primary HSC cells in terms of cell morphology, and HSC marker expression using qPCR, immunoblotting, and immunocytochemistry analysis. Furthermore, the effect of co-culture with HTM cells on porous scaffolds on ADSC differentiation was studied. Treatment with VEGF-C under shear stress is effective in differentiating ADSCs into PROX1-expressing HSC-like cells. Co-culture with HTM cells on porous scaffolds leads to HTM/ADSC-derived HSC-like constructs that regulate through-flow and respond as expected to dexamethasone. STATEMENT OF SIGNIFICANCE: We successfully generated human Schlemm's canal (HSC) like cells from adipocyte-derived stem cells induced by biochemical and biomechanical cues as well as bioengineered human trabecular meshwork (HTM) on micropatterned, porous SU8 scaffolds. These stem cell-derived HSC-like cells co-cultured with HTM cells on SU8 scaffolds can regulate through-flow, and in particular, are responsive to steroid treatment as expected. These findings show that ADSC-derived HSC-like cells have the potential to recreate the ocular outflow pathway for in vitro glaucoma drug screening. To the best of our knowledge, it is the very first time to demonstrate derivation of Schlemm's canal-like cells from stem cells. It provides an important alternative source to primary Schlemm's canal cells that are very difficult to be isolated and cultured from human donors.

Screening and optimization of potential injection vehicles for storage of retinal pigment epithelial stem cell before transplantation.

Retinal pigment epithelial (RPE) cells are highly specialized neural cells that have several functions essential for vision. Progressive deterioration of RPE cells in elderly individuals can result in visual impairment and ultimately the blinding disease age-related macular degeneration. Subretinal transplantation of stem cell-derived RPE cell suspensions is being explored as a strategy to recover the damaged retina and improve vision. This approach may be improved by developing a vehicle that increases postinjection cell viability and distribution and integration of RPE cells. In this study, Food and Drug Administration-approved natural polymers, including alginate, methylcellulose, and hyaluronic acid (HA), were examined for performance as cell vehicles for adult human RPE stem cells (RPESCs). We compared the effect of RPESC storage as a cell suspension in these delivery vehicles for 1-96 hr at different temperatures on subsequent cell performance in a cell culture model. RPESC survival, attachment, distribution, proliferation, and differentiation into RPE cells were monitored by microscopy over the course of 8 weeks. Our in vitro results demonstrate that RPESC suspension in a 0.2% HA solution promotes better initial cell distribution, proliferation, cobblestone formation, and expression of RPE cell markers (microphthalmia-associated transcription factor and orthodenticle homeobox 2) after 96 hr of storage. These data suggest that HA addition to the vehicle can significantly enhance RPESC performance in vitro and is a promising strategy to pursue an improved delivery vehicle supporting in vivo RPE cell transplantation.

Regenerating Eye Tissues to Preserve and Restore Vision.

Ocular regenerative therapies are on track to revolutionize treatment of numerous blinding disorders, including corneal disease, cataract, glaucoma, retinitis pigmentosa, and age-related macular degeneration. A variety of transplantable products, delivered as cell suspensions or as preformed 3D structures combining cells and natural or artificial substrates, are in the pipeline. Here we review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing. While recognizing the challenges, we look forward to a deep portfolio of sight-restoring, stem cell-based medicine. VIDEO ABSTRACT.

Polarized, Cobblestone, Human Retinal Pigment Epithelial Cell Maturation on a Synthetic PEG Matrix.

Cell attachment is essential for the growth and polarization of retinal pigment epithelial (RPE) cells. Currently, surface coatings derived from biological proteins are used as the gold standard for cell culture. However, downstream processing and purification of these biological products can be cumbersome and expensive. In this study, we constructed a library of chemically modified nanofibers to mimic the Bruch's membrane of the retinal pigment epithelium. Using atmospheric-pressure plasma-induced graft polymerization with a high-throughput screening platform to modify the nanofibers, we identified three polyethylene glycol (PEG)-grafted nanofiber surfaces (PEG methyl ether methacrylate, n = 4, 8, and 45) from a library of 62 different surfaces as favorable for RPE cell attachment, proliferation, and maturation in vitro with cobblestone morphology. Compared with the biologically derived culture matrices such as vitronectin-based peptide Synthemax, our newly discovered synthetic PEG surfaces exhibit similar growth and polarization of retinal pigment epithelial (RPE) cells. However, they are chemically defined, are easy to synthesize on a large scale, are cost-effective, are stable with long-term storage capability, and provide a more physiologically accurate environment for RPE cell culture. To our knowledge, no one has reported that PEG derivatives directly support attachment and growth of RPE cells with cobblestone morphology. This study offers a unique PEG-modified 3D cell culture system that supports RPE proliferation, differentiation, and maturation with cobblestone morphology, providing a new avenue for RPE cell culture, disease modeling, and cell replacement therapy.

Restriction of Cancer Metastatic Potential Using Embryonic Stem Cells Encapsulated in Alginate Hydrogel Microstrands.

Current treatments focused on eradicating metastatic tumors have proven unsuccessful due to cancer's ability to quickly undergo epithelial-to-mesenchymal transition (EMT) and metastasize to secondary sites. Using human triple negative breast cancer cells (BCCs) as a model system, this work establishes a platform for the study of aggressive cancer phenotypes by demonstrating the inhibition of human metastatic cancer cells with 3D cultured embryonic stem cells (ESCs) encapsulated in alginate microstrands (ESC-microstrands), which mimic the embryonic microenvironment and recapitulate pluripotent signaling. Coculture with ESC-microstrands significantly decreases triple negative BCC proliferation and survival and reverses abnormal cancer metabolism. In particular, coculture with ESC-microstrands markedly restricts the metastatic potential of highly aggressive cancer cells, demonstrated as decreased migration and invasion, and reversed EMT marker expression. This indicates that pluripotent signaling from 3D ESC-microstrands could restrict cancer metastasis through restriction and reversion of EMT. Furthermore, two soluble factors associated with dysregulated oncogenic signaling were identified which display altered relative mRNA expression following coculture with ESC-microstrands. Future application of this model to mechanistic studies will enable a better understanding of cancer metastasis and the discovery of therapeutic targets for metastatic diseases.