An injectable hydrogel having proteoglycan-like hierarchical structure supports chondrocytes delivery and chondrogenesis.

The ECM of cartilage is composed of proteoglycans (PG) that contain glycosaminoglycan (GAG), aggrecan, hyaluronic acid (HA) and other molecular components which play an important role in regulating chondrocyte functions via cell-matrix interactions, integrin-mediated signalling etc. Implantation of chondrocytes encapsulated in scaffolds that mimic the micro-architecture of proteoglycan, is expected to enhance cartilage repair. With an aim to create a hydrogel having macromolecular structure that resembles the cartilage-specific ECM, we constructed a hierarchal structure that mimic the PG. The bottle brush structure of the aggrecan was obtained using chondroitin sulphate and carboxymethyl cellulose which served as GAG and core protein mimic respectively. A proteoglycan-like structure was obtained by cross-linking it with modified chitosan that served as a HA substitute. The physico-chemical characteristics of the above cross-linked injectable hydrogel supported long term human articular chondrocyte subsistence and excellent post-injection viability. The chondrocytes encapsulated in the PMH expressed significant levels of articular cartilage specific markers like collagen II, aggrecan, GAGs etc., indicating the ability of the hydrogel to support chondrocyte differentiation. The biocompatibility and biodegradability of the hydrogels was confirmed using suitable in vivo studies. The results revealed that the PG-mimetic hydrogel could serve as a promising scaffold for chondrocyte implantation.

Preparation and evaluation of gellan gum hydrogel reinforced with silk fibers with enhanced mechanical and biological properties for cartilage tissue engineering.

Various research about cartilage regeneration using biomaterials has been done recently. Particularly, gellan gum hydrogel (GG) is reported to be suitable as a biomaterial for cartilage tissue engineering (TE) for its water uptaking ability, producibility, and environmental resemblance of native cartilage. Despite these advantages, mechanical and cell adhesion properties are still difficult to modulate. Reinforcement is essential to overcome these problems. Herein, GG was modified by physically blending with different lengths of silk fiber (SF). As SF is expected to improve such disadvantages of GG, mechanical and biological properties were characterized to confirm its reinforcement ability. Mechanical properties such as degradation rate, swelling rate, compression strength, and viscosity were studied and it was confirmed that SF significantly reinforces the mechanical properties of GG. Furthermore, in vitro study was carried out to confirm morphology, biocompatibility, proliferation, and chondrogenesis of chondrocytes encapsulated in the hydrogels. Overall, chondrocytes in the GG blended with SF (SF/GG) showed enhanced cell viability and growth. According to this study, SF/GG can be a promising biomaterial for cartilage TE biomaterial.

Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells.

Human periimplantation development requires the transformation of the naive pluripotent epiblast into a polarized epithelium. Lumenogenesis plays a critical role in this process, as the epiblast undergoes rosette formation and lumen expansion to form the amniotic cavity. Here, we present a high-throughput in vitro model for epiblast morphogenesis. We established a microfluidic workflow to encapsulate human pluripotent stem cells (hPSCs) into monodisperse agarose microgels. Strikingly, hPSCs self-organized into polarized epiblast spheroids that could be maintained in self-renewing and differentiating conditions. Encapsulated primed hPSCs required Rho-associated kinase inhibition, in contrast to naive hPSCs. We applied microgel suspension culture to examine the lumen-forming capacity of hPSCs and reveal an increase in lumenogenesis during the naive-to-primed transition. Finally, we demonstrate the feasibility of co-encapsulating cell types across different lineages and species. Our work provides a foundation for stem cell-based embryo models to interrogate the critical components of human epiblast self-organization and morphogenesis.

Growth factors regional patterned and photoimmobilized scaffold applied to bone tissue regeneration.

Bone diseases such as osteomalacia, osteoporosis, and osteomyelitis are major illnesses that threaten the health of human. This study aimed to provide an idea at the molecular level of material properties determined with UV specific surface approaches. The tert-butyl hydroperoxide (t-BHP) exposure aging model bone mesenchymal stem cells (BMSCs) were reverted by using a poly-hybrid scaffold (PS), which is a carbon nanotube (CNT) coated polycaprolactone (PCL) and polylactic acid (PLA) scaffold, combined with insulin-like growth factor-1 (IGF). Then, the region-specific PS photo-immobilized with different growth factors (GFs) was obtained by interference and diffraction of ultraviolet (UV) light. Additionally, the reverted BMSCs were regionally pattern differentiated into three kinds of cells on the GF immobilized PS (GFs/PS). In vivo, the GFs/PS accelerate bone healing in injured Sprague-Dawley (SD) rats. The data showed that GFs/PS effectively promoted the differentiation of reverted BMSCs in the designated area on 21st day. These results suggest region-specific interface immobilization of GFs concurrently differentiating reverted BMSCs into three different cells in the same scaffold. This method might be considered as a short-time, low cost, and simple operational approach to scaffold modification for tissue regeneration in the future.

Application of double network of gellan gum and pullulan for bone marrow stem cells differentiation towards chondrogenesis by controlling viscous substrates.

Hydrogels have a large amount of water that provides a cartilage-like environment and is used in tissue engineering with biocompatibility and adequate degradation rates. In order to differentiate stem cells, it is necessary to adjust the characteristics of the matrix such as stiffness, stress-relaxing time, and microenvironment. Double network (DN) hydrogels provide differences in cellular biological behavior and have interpenetrating networks that combine the advantages of the components. In this study, by varying the viscous substrate of pullulan (PL), the DN hydrogels of gellan gum (GG) and PL were prepared to determine the cartilage differentiation of bone marrow stem cell (BMSC). The characteristics of GG/PL hydrogel were investigated by examining the swelling ratio, weight loss, sol fraction, compressive modulus, and gelation temperature. The viability, proliferation, and toxicity of BMSCs encapsulated in hydrogels were evaluated. Cartilage phenotype and cartilage differentiation were confirmed by morphology, GAG content, and cartilage-specific gene expression. Overall results demonstrate that GG/PL hydrogels can form cartilage differentiation of BMSCs and can be applied for tissue engineering purposes.

Enhanced osteogenesis of human mesenchymal stem cells by self-assembled peptide hydrogel functionalized with glutamic acid templated peptides.

Self-assembling peptide (SAP) hydrogel has been shown to be an excellent biological material for three-dimensional cell culture and stimulatie cell migration and differentiation into the scaffold, as well as for repairing bone tissue defects. Herein, we designed one of the SAP scaffolds KLD (KLDLKLDLKLDL) through direct coupling to short bioactive motif O1 (EEGGC) and O2 (EEEEE) of which bioactivity on osteogenic differentiation was previously demonstrated and self-assembled in different concentrations (0.5%, 1%, and 2%). Our aim was to enhance osteogenesis and biomineralization of injectable SAP hydrogels with controlled mechanical properties so that the peptide hydrogel also becomes capable of being injected to bone defects. The molecular integration of the nanofibrous peptide scaffolds was observed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The rheological properties and degradation profile of SAP hydrogels were evaluated to ensure stability of SAPs. Compared with pure KLD scaffold, we found that these designed bioactive peptide scaffolds significantly promoted hMSCs proliferation depicted by biochemical analysis of alkaline phosphatase (ALP) activity, total calcium deposition. Moreover, key osteogenic markers of ALP activity, collagen type I (COL-1), osteopontin (OP), and osteocalcin (OCN) expression levels determined by real-time polymerase chain reaction (PCR) and immunofluorescence analysis were also significantly increased with the addition of glutamic acid residues to KLD. We demonstrated that the designed SAP scaffolds promoted the proliferation and osteogenic differentiation of hMSCs. Our results suggest that these designed bioactive peptide scaffolds may be useful for promoting bone tissue regeneration.

Improved human islets' viability and functionality with mesenchymal stem cells and arg-gly-asp tripeptides supplementation of alginate micro-encapsulated islets in vitro.

INTRODUCTION: The extension of islet transplantation to a wider number of type 1 diabetes patients is compromised by severe adverse events related to the immunosuppressant therapy required for allogenic islet transplantation. In this context, microencapsulation offers the prospects of immunosuppressive-free therapy by physically isolating islets from the immune system. However, current biomaterials need to be optimized to: improve biocompatibility, guaranty the maintenance of graft viability and functionality, and prevent fibrosis overgrowth around the capsule in vivo. Accumulating evidence suggest that mesenchymal stem cells (MSCs) and anchor points consisting of tripeptides arg-gly-asp (RGD) have cytoprotective effects on pancreatic islets. Here, we investigated the effect of supplementing reference M-rich alginate microcapsules with MSCs and RGD-G rich alginate on bioprocessing as well as on human pancreatic islets viability and functionality. METHODS: We characterized the microcapsules components, and then for the new microcapsule composite product: we analyzed the empty capsules biocompatibility and then investigated the benefits of MSCs and RGD-G rich alginate on viability and functionality on the encapsulated human pancreatic islets in vitro. We performed viability tests by confocal microscopy and glucose stimulated insulin secretion (GSIS) test in vitro to assess the functionality of naked and encapsulated islets. RESULTS: Encapsulation in reference M-rich alginate capsules induced a reduction in viability and functionality compared to naked islets. This side-effect of encapsulation was in part counteracted by the presence of MSCs but the restoration was complete with the combination of both MSCs and the RGD-G rich alginate. CONCLUSIONS: The present findings show that bioprocessing a favorable composite environment inside the M-rich alginate capsule with both MSCs and RGD-G rich alginate improves human islets survival and functionality in vitro.

A High Cell-Bearing Capacity Multibore Hollow Fiber Device for Macroencapsulation of Islets of Langerhans.

Macroencapsulation of islets of Langerhans is a promising strategy for transplantation of insulin-producing cells in the absence of immunosuppression to treat type 1 diabetes. Hollow fiber membranes are of interest there because they offer a large surface-to-volume ratio and can potentially be retrieved or refilled. However, current available fibers have limitations in exchange of nutrients, oxygen, and delivery of insulin potentially impacting graft survival. Here, multibore hollow fibers for islets encapsulation are designed and tested. They consist of seven bores and are prepared using nondegradable polymers with high mechanical stability and low cell adhesion properties. Human islets encapsulated there have a glucose induced insulin response (GIIS) similar to nonencapsulated islets. During 7 d of cell culture in vitro, the GIIS increases with graded doses of islets demonstrating the suitability of the microenvironment for islet survival. Moreover, first implantation studies in mice demonstrate device material biocompatibility with minimal tissue responses. Besides, formation of new blood vessels close to the implanted device is observed, an important requirement for maintaining islet viability and fast exchange of glucose and insulin. The results indicate that the developed fibers have high islet bearing capacity and can potentially be applied for a clinically applicable bioartificial pancreas.

Cell subtype-dependent formation of breast tumor spheroids and their variable responses to chemotherapeutics within microfluidics-generated 3D microgels with tunable mechanics.

Tumor spheroids have been considered valuable miniaturized three dimensional (3D) tissue models for fundamental biological investigation as well as drug screening applications. Most tumor spheroids are generated utilizing the inherent aggregate behavior of tumor cells, and the effect of microenvironmental factors such as extracellular matrix (ECM) on tumor spheroid formation has not been extensively elucidated to date. Herein, uniform-sized spherical microgels encapsulated with different subtypes of breast tumor cells, based on tumor aggressiveness, are developed by flow-focusing microfluidics technology. Mechanical properties of microgels are controlled in a wide range via polymer concentration, and their influence on tumor physiology and spheroid formation is shown to be highly dependent on cell subtype. Specifically, the formation of polyploid/multinucleated giant cancer cells is a key early step in determining initial proliferation and eventual tumor spheroid generation within microgels with varying mechanics. In addition, chemotherapeutic screening performed on these tumor spheroids in microgels also display significantly variable cytotoxic effects based on microgel mechanics for each cell subtype, further highlighting the importance of microenvironmental factors on tumor spheroid physiology.

Viability and distribution of bacteria immobilized on Sawdust@silica: The removal mechanism of phenanthrene in soil.

Immobilized cells (ICs) have been widely used to enhance the remediation of organic-contaminated soil (e.g., polycyclic aromatic hydrocarbons, PAHs). Once ICs are added to the heterogeneous soil, degradation hotspots are immediately formed near the carrier, leaving the remaining soil lack of degrading bacteria. Therefore, it remains unclear how ICs efficiently utilize PAHs in soil. In this study, the viability of Silica-IC (Cells@Sawdust@Silica) and the distribution of inoculated ICs and phenanthrene (Phe) in a slurry system (soil to water ratio 1:2) were investigated to explore the removal mechanism of PAHs by the ICs. Results showed that the Silica-IC maintained (i) good reproductive ability (displayed by the growth curve in soil and water phase), (ii) excellent stability, which was identified by the ratio of colony forming units in the soil phase to the water phase, the difference between the colony number and the DNA copies, and characteristics of the biomaterial observed by the FESEM, and (iii) high metabolic activity (the removal percentages of Phe in soil by the ICs were more than 95% after 48 h). Finally, the possible pathways for the ICs to efficiently utilize Phe in soil are proposed based on the distribution and correlation of Phe and ICs between the soil and water phase. The adsorption-degradation process was dominant, i.e., the enhanced degradation occurred between the ICs and carrier-adsorbed Phe. This study provided new insights on developing a bio-material for efficient bio-remediation of PAHs-contaminated soil.

Fast fabrication of reusable polyethersulfone microbial biosensors through biocompatible phase separation.

In biosensors fabrication, entrapment in polymeric matrices allows efficient immobilization of the biorecognition elements without compromising their structure and activity. When considering living cells, the biocompatibility of both the matrix and the polymerization procedure are additional critical factors. Bio-polymeric gels (e.g. alginate) are biocompatible and polymerize under mild conditions, but they have poor stability. Most synthetic polymers (e.g. PVA), on the other hand, present improved stability at the expense of complex protocols involving chemical/physical treatments that decrease their biological compatibility. In an attempt to explore new solutions to this problem we have developed a procedure for the immobilization of bacterial cells in polyethersulfone (PES) using phase separation. The technology has been tested successfully in the construction of a bacterial biosensor for toxicity assessment. Biosensors were coated with a 300  µm bacteria-containing PES membrane, using non-solvent induced phase separation (membrane thickness ≈ 300 µm). With this method, up to 2.3 × 106 cells were immobilized in the electrode surface with an entrapment efficiency of 8.2%, without compromising cell integrity or viability. Biosensing was performed electrochemically through ferricyanide respirometry, with metabolically-active entrapped bacteria reducing ferricyanide in the presence of glucose. PES biosensors showed good stability and reusability during dry frozen storage for up to 1 month. The analytical performance of the sensors was assessed carrying out a toxicity assay in which 3,5-dichlorophenol (DCP) was used as a model toxic compound. The biosensor provided a concentration-dependent response to DCP with half-maximal effective concentration (EC50) of 9.2 ppm, well in agreement with reported values. This entrapment methodology is susceptible of mass production and allows easy and repetitive production of robust and sensitive bacterial biosensors.

Plasmonic Colloidosome-Based Multifunctional Platform for Bacterial Identification and Antimicrobial Resistance Detection.

Antimicrobial resistance (AMR) is an urgent threat to public health. Rapid bacterial identification and AMR tests are important to promote personalized treatment of patients and to limit the spread of AMR. Herein, we explore the utility of plasmonic colloidosomes in bacterial analysis based on mass spectrometry (MS) and Raman scattering. It is found that colloidosomes can provide a rigid micrometer-size platform for bacterial culture and analysis. Coupled with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, this platform enables bacterial identification at the species level with cell counts as low as 50, >100 times more sensitive than the standard method of MALDI-TOF MS based bacterial identification. Coupled with Raman scattering, it can distinguish single bacterial cells at the strain level and recognize AMR at the single-cell level. These reveal the broad potential of the platform for flexible and versatile bacterial detection and typing.

YestroSens, a field-portable S. cerevisiae biosensor device for the detection of endocrine-disrupting chemicals: Reliability and stability.

Farming, industry and urbanization lead to increases in the concentrations of potentially harmful compounds in waste, surface and drinking waters. One example of such pollution are estrogens, the steroidal female reproductive hormones. Already at a few nanograms per litre, these hormones can trigger endocrine disruption and cause acute and chronic health problems in humans and wildlife. Here, we present a Saccharomyces cerevisiae estrogen biosensor capable of detecting estradiol, as well as ethinylestradiol, at concentrations of 1 nM. After an initial characterization of the sensor strain performance in an optimal laboratory setting, we focused on developing a biosensor device. We addressed current limitations of biosensors, such as the requirement of the cells for a liquid growth matrix, controlled storage conditions required to preserve cell viability, and the usually required bulky, as well as expensive, laboratory equipment. Our study provides significant new insights into the field of applied biosensors. The system presented in this work takes microorganism-based analytics one step closer to field application in decentralized locations.

Anti-oxidant activity reinforced reduced graphene oxide/alginate microgels: Mesenchymal stem cell encapsulation and regeneration of infarcted hearts.

Mesenchymal stem cell (MSC) transplantation is promising for repairing heart tissues post myocardial infarction (MI). In particular, paracrine effects of the transplanted MSCs have been highlighted to play major roles in heart regeneration by secreting multiple growth factors and immune-modulatory cytokines. Nevertheless, its therapeutic efficacy still remains low, which is strongly associated with low viability and activity of the transplanted stem cells, because the transplanted MSCs are exposed to high shear stress during injection and harsh environments (e.g., high oxidative stress and host immune reactions) post injection. In this study, we aimed to develop novel injectable MSC-delivery microgel systems possessing high anti-oxidant activities. Specifically, we encapsulated MSCs in graphene oxide (GO)/alginate composite microgels by electrospraying. To further enhance the anti-oxidizing activities of the gels, we developed reduced MSC-embedded GO/alginate microgels (i.e., r(GO/alginate)), which have the potential to protect MSCs from the abovementioned harsh environments within MI tissues. Our in vitro studies demonstrated that the MSCs encapsulated in the r(GO/alginate) microgels showed increased viability under oxidative stress conditions with H2O2. Furthermore, cardiomyocytes (CMs), co-cultured with the encapsulated MSCs in transwells with H2O2 treatment, showed higher cell viability and cardiac maturation compared to monolayer cultured CMs, likely due to ROS scavenging by the gels and positive paracrine signals from the encapsulated MSCs. In vivo experiments with acute MI models demonstrated improved therapeutic efficacy of MSC delivery in r(GO/alginate) microgels, exhibiting significant decreases in the infarction area and the improvement of cardiac function. We believe that our novel MSC encapsulation system with GO, alginate, and mild reduction, which exhibits high cell protection capacity (e.g., anti-oxidant activity), will serve as an effective platform for the delivery of stem cells and other therapeutic cell types to treat various injuries and diseases, including MI.

Dual delivery of encapsulated BM-MSCs and BMP-2 improves osteogenic differentiation and new bone formation.

Stem cell-based therapies provide a promising approach for bone repair. In the present work, we developed a novel 3D vehicle system for dual-delivery of encapsulated bone marrow mesenchymal stem cells (BM-MSCs) and bone morphogenetic protein-2 (BMP-2) for treatment of large bone defects. The vehicle system consists of sodium alginate microcapsules and polylactic acid (PLLA) microspheres. BM-MSCs are encapsulated in the microcapsules, and BMP-2 proteins are encapsulated in the PLLA microspheres. This vehicle system acted as a multicore structure for sustained release of BMP-2, which enabled pulsed dosing induction of osteogenic differentiation of the co-embedded BM-MSCs. in vitro experiments showed that the loaded BMP-2 was constitutively released up to 30 days. Bioactivity of the incorporated BMP-2 in the microspheres was preserved and osteogenic differentiation of the BM-MSCs in the microcapsules was improved. In vivo, osteogenesis studies demonstrated that satisfactory degree of repair of a rat calvarial defect was achieved with the delivery of either encapsulated BM-MSCs alone or encapsulated BMP-2 alone. Transplantation of encapsulated both BM-MSCs and BMP-2 exhibited the greatest repair potential following 4- or 8-weeks treatment. In conclusion, microencapsulation of BM-MSCs and BMP-2 promoted the maturity of newly generated bone and improved new bone formation. Transplantation of BM-MSCs and BMP-2 in our novel 3-D vehicle system is a promising strategy for regenerative therapies of large bone defects.

Injectable stem cell-laden supramolecular hydrogels enhance in situ osteochondral regeneration via the sustained co-delivery of hydrophilic and hydrophobic chondrogenic molecules.

Hydrogels have been widely used as the carrier material of therapeutic cell and drugs for articular cartilage repair. We previously demonstrated a unique host-guest macromer (HGM) approach to prepare mechanically resilient, self-healing and injectable supramolecular gelatin hydrogels free of chemical crosslinking. In this work, we show that compared with conventional hydrogels our supramolecular gelatin hydrogels mediate more sustained release of small molecular (kartogenin) and proteinaceous (TGF-ß1) chondrogenic agents, leading to enhanced chondrogenesis of the encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro and in vivo. More importantly, the supramolecular nature of our hydrogels allows injection of the pre-fabricated hydrogels containing the encapsulated hBMSCs and chondrogenic agents, and our data show that the injection process has little negative impact on the viability and chondrogenesis of the encapsulated cells and subsequent neocartilage development. Furthermore, the stem cell-laden supramolecular hydrogels administered via injection through a needle effectively promote the regeneration of both hyaline cartilage and subchondral bone in the rat osteochondral defect model. These results demonstrate that our supramolecular HGM hydrogels are promising delivery biomaterials of therapeutic agents and cells for cartilage repair via minimally invasive procedures. This unique capability of injecting cell-laden hydrogels to target sites will greatly facilitate stem cell therapies.

Hybridization of a phospholipid polymer hydrogel with a natural extracellular matrix using active cell immobilization.

Three-dimensional tissue organization is still an obstacle in the field of tissue engineering, which generally involves cell immobilization, proliferation, and organization. As an artificial extracellular matrix (ECM) for providing a suitable environment of cells to construct tissues, combination of cytocompatible polymer hydrogels and natural ECM produced by the immobilized cells was considered. In this research, we designed a spontaneously forming hydrogel system between two water-soluble polymers for the immobilization of cells. These polymers were poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid-co-N-succinimidyloxycarbonyl tetra(ethylene glycol)methacrylate) (PMBVS) and poly(vinyl alcohol) (PVA) to form a PMBVS/PVA hydrogel in a cell culture medium under mild conditions. Basic fibroblast growth factor (bFGF) was conjugated with PMBVS (PMBV-bFGF). To enhance the growth of the immobilized cells, mouse fibroblast L929 cells were immobilized in the PMBVS/PVA hydrogel and the PMBV-bFGF/PVA hydrogel, and their proliferation and secretion of the ECM under stimulation with bFGF was observed. The ECM infiltrated and replaced the hydrogel, resulting in the formation of a hybrid hydrogel with the ECM and laden cells.

Oxygen generating biomaterial improves the function and efficacy of beta cells within a macroencapsulation device.

Tissue-engineered devices have the potential to significantly improve human health. A major impediment to the success of clinically scaled transplants, however, is insufficient oxygen transport, which leads to extensive cell death and dysfunction. To provide in situ supplementation of oxygen within a cellular implant, we developed a hydrolytically reactive oxygen generating material in the form of polydimethylsiloxane (PDMS) encapsulated solid calcium peroxide, termed OxySite. Herein, we demonstrate, for the first time, the successful implementation of this in situ oxygen-generating biomaterial to support elevated cellular function and efficacy of macroencapsulation devices for the treatment of type 1 diabetes. Under extreme hypoxic conditions, devices supplemented with OxySite exhibited substantially elevated beta cell and islet viability and function. Furthermore, the inclusion of OxySite within implanted macrodevices resulted in the significant improvement of graft efficacy and insulin production in a diabetic rodent model. Translating to human islets at elevated loading densities further validated the advantages of this material. This simple biomaterial-based approach for delivering a localized and controllable oxygen supply provides a broad and impactful platform for improving the therapeutic efficacy of cell-based approaches.

Development of 3D printable conductive hydrogel with crystallized PEDOT:PSS for neural tissue engineering.

Bioelectronic devices enable efficient and effective communication between medical devices and human tissue in order to directly treat patients with various neurological disorders. Due to the mechanical similarity to human tissue, hydrogel-based electronic devices are considered to be promising for biological signal recording and stimulation of living tissues. Here, we report the first three-dimensionally (3D) printable conductive hydrogel that can be photocrosslinked while retaining high electrical conductivity. In addition, we prepared dorsal root ganglion (DRG) cell-encapsulated gelatin methacryloyl (GelMA) hydrogels which were integrated with the 3D printed conductive structure and evaluated for efficiency neural differentiation under electrical stimulation (ES). For enhanced electrical conductivity, a poly(3,4-ethylenedioxythiophene) (PEDOT): polystyrene sulfonate (PSS) aqueous solution was freeze-dried and mixed with polyethylene glycol diacrylate (PEGDA) as the photocurable polymer base. Next, the conductive hydrogel was patterned on the substrate by using a table-top stereolithography (SLA) 3D printer. The fabricated hydrogel was characterized for electrochemical conductivity. After printing with the PEDOT:PSS conductive solution, the patterned hydrogel exhibited decreased printing diameters with increasing of PEDOT:PSS concentration. Also, the resultant conductive hydrogel had significantly increased electrochemical properties with increasing PEDOT:PSS concentration. The 3D printed conductive hydrogel provides excellent structural support to systematically transfer the ES toward encapsulated DRG cells for enhanced neuronal differentiation. The results from this study indicate that the conductive hydrogel can be useful as a 3D printing material for electrical applications.

3D-3 Tumor Models in Drug Discovery for Analysis of Immune Cell Infiltration.

The cross talk between tumor cells and other cells present in the tumor microenvironment such as stromal and immune cells highly influences the behavior and progression of disease. Understanding the underlying mechanisms of interaction is a prerequisite to develop new treatment strategies and to prevent or at least reduce therapy failure in the future. Specific reactivation of the patient's immune system is one of the major goals today. However, standard two-dimensional (2D) cell culture techniques lack the necessary complexity to address related questions. Novel three-dimensional (3D) in vitro models-embedded in a matrix or encapsulated in alginate-recapitulate the in vivo situation much better. Cross talk between different cell types can be studied starting from co-cultures. As cancer immune modulation is becoming a major research topic, 3D in vitro models represent an important tool to address immune regulatory/modulatory questions for T, NK, and other cells of the immune system. The 3D systems consisting of tumor cells, fibroblasts, and immune cells (3D-3) already proved as a reliable tool for us. For instance, we made use of those models to study the molecular mechanisms of the cross talk of non-small cell lung cancer (NSCLC) and fibroblasts, to unveil macrophage plasticity in the tumor microenvironment and to mirror drug responses in vivo. Generation of those 3D models and how to use them to study immune cell infiltration and activation will be described in the present book chapter.