Hydrogels For 3D Culture Research Articles

Determining Which Hydrostatic Pressure Regimes Promote Osteogenesis in Human Mesenchymal Stem Cells.

Compressive loading of bone causes hydrostatic pressure changes which have been proposed as an osteogenic differentiation stimulus for mesenchymal stem cells (hMSCs). We hypothesised that hMSCs are adapted to differentiate only in response to cyclic hydrostatic pressures above critical thresholds of magnitude and frequency which correspond to physiological levels of anabolic bone loading. Using a pneumatic-hydrostatic bioreactor, we applied hydrostatic pressure regimes to human hMSCs in 3D collagen hydrogel cultures for 1h/day over 28days to determine which levels of pressure and frequency stimulated osteogenesis in vitro. Stimulation of the 3D cultures with 0-280kPa cyclic hydrostatic pressure at 1Hz resulted in up to 75% mineralisation in the hydrogel (without exogenous growth factors), whilst static culture or variations of the regime with either constant high pressure (280kPa, 0Hz), low-frequency (0.05Hz, 280kPa) or low-magnitude (70kPa, 1Hz) stimulation had no osteogenic effects (< 2% mineralisation). Nuclear translocation of YAP was observed following cyclic hydrostatic pressure in mature MLO-A5 osteoblasts but not in hMSCs, suggesting that cyclic hydrostatic pressure activates different mechanotransduction pathways in undifferentiated stem cells and committed osteoblasts. Hydrostatic pressure is a potent stimulus for differentiating MSC into highly active osteoblasts and may therefore be a versatile tool for translational cell engineering. We have demonstrated that there are minimum levels of force and frequency needed to trigger osteogenesis, i.e. a pressure 'switch', which corresponds to the physiological forces experienced by cells in their native mesenchymal niche. The mechanotransduction mechanisms underpinning these effects are the subject of further study.

Utilizing quantitative phase microscopy to localize fluorescence in three dimensions via the transport of intensity equation.

We demonstrate the use of a novel, to the best of our knowledge, localization algorithm for digitally refocusing fluorescence images from a three-dimensional cell culture. Simultaneous phase and fluorescence intensity images are collected through a multimodal system that combines digital holography via quantitative phase microscopy (QPM) and fluorescence microscopy. Defocused fluorescence images are localized to a specific z-plane within the three-dimensional (3D) matrix using the transport of intensity equation (TIE) and depth-resolved information derived from the QPM measurements. This technique is applied to cells stained with different fluorescent tags suspended in 3D collagen hydrogel cultures. Experimental findings demonstrate the localization of defocused images, facilitating the analysis and comparison of cells within the hydrogel matrix. This method holds promise for comprehensive cellular imaging of fluorescence labeling in three-dimensional environments, enabling detailed investigations into cellular behavior and interactions.

Epigenetic Priming Enhances Chondrogenic Potential of Expanded Chondrocytes.

Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure, such as matrix-induced autologous chondrocyte implantation. Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to (1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists after the transfer to a three-dimensional (3D) culture; and (2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded bovine chondrocytes, used as a model system. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0 population doublings, 8 population doublings, and 16 population doublings (PD16) in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a 3D hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for PD16 and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded bovine chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.

248 Divergent Satellite Cell Profiles of Bovine Trapezius, Longissimus, and Bicep Femoris Muscles in Utero

Abstract As we look to better understand whole body growth and development, there are still many mechanisms of individual muscle growth that we have yet to comprehend. Therefore, the objective of this study was to compare proliferative capacity and satellite cell number within the trapezius (TP), longissimus (LM), and bicep femoris (BF) muscles of fetal bovine throughout mid and late gestation. We hypothesized a difference in satellite cell number across these three muscles may further explain the differential growth pattern of each of these muscles in utero. Muscle samples were harvested from bovine fetuses at a commercial abattoir and aged using crown rump length (~150-220d in utero). These samples were then dissected into single fibers and seeded in a 3D hydrogel culture with 20% FBS media. Cells were fixed using 4% paraformaldehyde and stained for DAPI, Pax-7, Myf-5 (Invitrogen, Bedford, MA) at 72, 96, and 120h. Wells were imaged and analyzed for proliferation rate using the Cytation 5 (Agilent; La Jolla, CA) Prime Gen 5 software. Data were analyzed as a two-way ANOVA with repeated measures using GraphPad Prism (Version 9.5). For Myf-5, Pax-7, and dual positive cells (P ≤ 0.05) there was a difference across muscle type with BF having an increased number of positive cells compared with the TP muscle and LM being intermediate (Myf-5 mean = 51, 23, and 40 respectively; Pax-7 mean = 45, 30, and 20 respectively; Dual mean = 39, 20, 16 respectively). This variation was further supported by a similar divergence in proliferation rate across muscle type (P = 0.04), with BF muscle having increased proliferative capacity over TP muscle and LM muscle being intermediary. These findings suggest contrasting growth patterns across muscle types in utero that may influence postnatal growth patterns. Further research is needed to understand how the variability in satellite cell number contributes to differences in individual muscle growth.

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

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

235.3: Large-scale long-term 3D-hydrogel culture yields high-quality islets suitable for transplantation

235.3: Large-scale long-term 3D-hydrogel culture yields high-quality islets suitable for transplantation

Single-cell analysis reveals TLR-induced macrophage heterogeneity and quorum sensing dictate population wide anti-inflammatory feedback in response to LPS.

The role of macrophages in controlling tissue inflammation is indispensable to ensure a context-appropriate response to pathogens whilst preventing excessive tissue damage. Their initial response is largely characterized by high production of tumor necrosis factor alpha (TNFα) which primes and attracts other immune cells, thereafter, followed by production of interleukin 10 (IL-10) which inhibits cell activation and steers towards resolving of inflammation. This delicate balance is understood at a population level but how it is initiated at a single-cell level remains elusive. Here, we utilize our previously developed droplet approach to probe single-cell macrophage activation in response to toll-like receptor 4 (TLR4) stimulation, and how single-cell heterogeneity and cellular communication affect macrophage-mediated inflammatory homeostasis. We show that only a fraction of macrophages can produce IL-10 in addition to TNFα upon LPS-induced activation, and that these cells are not phenotypically different from IL-10 non-producers nor exhibit a distinct transcriptional pathway. Finally, we demonstrate that the dynamics of TNFα and IL-10 are heavily controlled by macrophage density as evidenced by 3D hydrogel cultures suggesting a potential role for quorum sensing. These exploratory results emphasize the relevance of understanding the complex communication between macrophages and other immune cells and how these amount to population-wide responses.

Pressure does not affect fibrotic function of dermal fibroblast through an in vitro 3D hydrogel culture model.

Pressure therapy has been used for the prevention and treatment of hypertrophic scars for decades. However, the cellular and molecular mechanisms of this treatment modality have not been fully elaborated, leading to long-lasting controversies regarding its clinical effectiveness. In this current study, we adopted an in vitro 3D culture and compression model to explore the effect of pressure force on fibroblasts, in order to further explain the working mechanism of compression force during pressure treatment. Human dermal fibroblasts were cultured in the 3D culture hydrogel and treated with 1.5atm of external compression force through a syringe tube device, for 4, 8, and 20 h respectively. RNA-seq identified 437 differentially regulated genes after an 8-h compression intervention compared with control cells, among which 256 genes were up-regulated and 181 genes were down-regulated. Further q-PCR analysis confirmed that early growth response 1(EGR1)and c-fos were down-regulated after an 8-h compression intervention. However, the down-regulation of EGR1 and c-fos at the mRNA level does not lead to altered protein synthesis through western blot, for both 8 and 20-h time points after pressure intervention. Genes closely related to the fibrotic function of fibroblasts including type I collagen (COL1), type III collagen (COL3), transforming growth factor β1(TGF-β1), matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 1 (TIMP1), connective tissue growth factor (CTGF), α smooth muscle actin (α-SMA), and fibronectin 1 (FN1), were also unaffected after pressure treatment for 8h. The current study indicated that in our 3D hydrogel culture model, pressure does not directly affect the fibrotic function of dermal fibroblast in vitro. Indirect regulation including reducing oedema, blood perfusion, and tension could be a more possible mechanism of pressure therapy.

3D Hydrogel Cultures for High-Throughput Drug Discovery.

Our increased understanding of how a cell's microenvironment influences its behavior has fueled an interest in three-dimensional (3D) cell cultures for drug discovery. Particularly, scaffold-based 3D cultures are expected to recapitulate in vivo tissue stiffness and extracellular matrix composition more accurately than standard two-dimensional (2D) monolayer cultures. Here we present a 3D hydrogel cell culture setup suitable for automated screening with standard high-throughput screening (HTS) liquid handling equipment commonly found in a drug discovery laboratory. Further, we describe the steps required to validate the assay system for compound screening.

Integrin binding peptides facilitate growth and interconnected vascular-like network formation of rat primary cortical vascular endothelial cells in vitro.

Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2'-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional (3D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.

Studies on oxygen availability and the creation of natural and artificial oxygen gradients in gelatin-methacryloyl hydrogel 3D cell culture.

Three-dimensional (3D) cultivation platforms allow the creation of cell models, which more closely resemble in vivo-like cell behavior. Therefore, 3D cell culture platforms have started to replace conventional two-dimensional (2D) cultivation techniques in many fields. Besides the advantages of 3D culture, there are also some challenges: cultivation in 3D often results in an inhomogeneous microenvironment and therefore unique cultivation conditions for each cell inside the construct. As a result, the analysis and precise control over the singular cell state is limited in 3D. In this work, we address these challenges by exploring ways to monitor oxygen concentrations in gelatin methacryloyl (GelMA) 3D hydrogel culture at the cellular level using hypoxia reporter cells and deep within the construct using a non-invasive optical oxygen sensing spot. We could show that the appearance of oxygen limitations is more prominent in softer GelMA-hydrogels, which enable better cell spreading. Beyond demonstrating novel or space-resolved techniques of visualizing oxygen availability in hydrogel constructs, we also describe a method to create a stable and controlled oxygen gradient throughout the construct using a 3D printed flow-through chamber.

Abstract 199: A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments

Abstract Mantle cell lymphomas (MCL) are rare B-cell neoplasms belonging to the non-Hodgkin’s lymphomas and often show an aggressive clinical course. MCL respond relatively briefly to conventional chemotherapy and are characterized by frequent relapses (Dreyling et al., Ann Oncol, 2017). Therapy for MCL remains a challenge also due to limited cultivation time of primary cells. In vitro suspension culture of primary human MCL cells is not easily feasible (Bryant et al., Lab Invest, 2000) and cells undergo spontaneous apoptosis ex vivo (Zhang et al., Blood, 2012; Medina et al., Heamatologica, 2012). Useful in vitro MCL models that allow long-term culture of primary MCL cells could be helpful in the biological and therapeutic study of MCL. For this purpose, we established a 3D hydrogel culture model for cultivation of primary MCL cells that were first tested with cell lines. After 3 days of culture, the cells cultured in the hydrogel can successfully be analysed for cell viability (live-dead confocal laser scanning microscope-based assay, flow cytometry), metabolism (pH, glucose, lactate), and biomarker expression by immunohistochemistry. The 3D hydrogel cultivation model allows a variety of analytical methods, also because it is reversible and the cells can easily be separated as single cell suspension from hydrogel for further analytical purposes, such as flow cytometric analyses. Next, we cultured primary MCL cells in the alginate hydrogel 3D culture system. MCL cells were isolated from blood of different patients and separated by Ficoll centrifugation. The isolated cell population was identified as MCL cells by double-staining of anti-CD20 and anti-CD5 followed by flow cytometric analysis. Cells were further cultured in medium as conventional suspension culture or in alginate hydrogel as 3D culture. Cell death was assessed with AnnexinV by staining phospholipids of apoptotic cells, allowing flow cytometric quantification. All samples had less than 6% AnnexinV positive (+) cells at day 0 before the experiment started. After 2 days cultivation, primary MCL cells showed a strong induction of cell death in suspension culture with 65.5% (±4.2%) AnnexinV+ cells versus 21.9% (±0.6%) AnnexinV+ cells in 3D hydrogel culture. Subsequently after 3 days cultivation, 74.2% (±12.2%) AnnexinV+ cells in suspension versus 33.5% (±1.3%) AnnexinV+ cells in the hydrogel 3D culture were determined. Therefore, we conclude that our novel 3D culture system might be a more suitable cultivation method for primary MCL cells than conventional suspension culture in cell culture medium. We have developed a 3D cultivation system with alginate hydrogel that allows nearly tripling the in vitro cultivation time of primary MCL. This could pave the way for new long-term treatments in research in the future and serve a better understanding of the cells. Citation Format: Kathrin Böpple, Annette M. Staiger, Heike Horn, German Ott, Walter E. Aulitzky, Meng Dong. A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 199.

Multiple roles for Bcl-3 in mammary gland branching, stromal collagen invasion, involution and tumor pathology

BackgroundThe Bcl-3 protein is an atypical member of the inhibitor of -κB family that has dual roles as a transcriptional repressor and a coactivator for dimers of NF-κB p50 and p52. Bcl-3 is expressed in mammary adenocarcinomas and can promote tumorigenesis and survival signaling and has a key role in tumor metastasis. In this study, we have investigated the role of Bcl-3 in the normal mammary gland and impact on tumor pathology.MethodsWe utilized bcl-3−/− mice to study mammary gland structure in virgins and during gestation, lactation and early involution. Expression of involution-associated genes and proteins and putative Bcl-3 target genes was examined by qRT-PCR and immunoblot analysis. Cell autonomous branching morphogenesis and collagen I invasion properties of bcl-3−/− organoids were tested in 3D hydrogel cultures. The role of Bcl-3 in tumorigenesis and tumor pathology was also assessed using a stochastic carcinogen-induced mammary tumor model.ResultsBcl-3−/− mammary glands demonstrated reduced branching complexity in virgin and pregnant mice. This defect was recapitulated in vitro where significant defects in bud formation were observed in bcl-3−/− mammary organoid cultures. Bcl-3−/− organoids showed a striking defect in protrusive collective fibrillary collagen I invasion associated with reduced expression of Fzd1 and Twist2. Virgin and pregnant bcl-3−/− glands showed increased apoptosis and rapid increases in lysosomal cell death and apoptosis after forced weaning compared to WT mice. Bcl-2 and Id3 are strongly induced in WT but not bcl-3−/− glands in early involution. Tumors in WT mice were predominately adenocarcinomas with NF-κB activation, while bcl-3−/− lesions were largely squamous lacking NF-κB and with low Bcl-2 expression.ConclusionsCollectively, our results demonstrate that Bcl-3 has a key function in mammary gland branching morphogenesis, in part by regulation of genes involved in extracellular matrix invasion. Markedly reduced levels of pro-survival proteins expression in bcl-3 null compared to WT glands 24 h post-weaning indicate that Bcl-3 has a role in moderating the rate of early phase involution. Lastly, a reduced incidence of bcl-3−/− mammary adenocarcinomas versus squamous lesions indicates that Bcl-3 supports the progression of epithelial but not metaplastic cancers.

Alginate/gelatin-based hybrid hydrogels with function of injecting and encapsulating cells in situ

Multi-network hydrogels with high strength and toughness have attracted increasing attention. Herein, a hybrid hydrogel consisting of alginate, gelatin, and polyacrylamide was constructed with the combination of advantages of natural and synthetic polymers. Alginate grafted with host-guest complex of βCD/Ad-AAm was first prepared, namely Alg-βCD/Ad-AAm, then further crosslink with gelatin methacryloyl (GelMA) to form hydrogel via one-step UV light initiation. The hydrogel produced by this method has more uniform and well-crosslinked networks. The hydrogels demonstrated uniform porosity, adjustable hydrophilicity (water contact angle within 32.7–91.5°), and desired mechanical properties (maximum tensile strain of 242.8%, tensile strength of 75.9 kPa, and Young's modulus of 28.5 kPa). The hydrogel also possessed self-healing ability and pH sensitivity, showing higher mechanical tensile strength at lower pH. The temperature-adjustable viscosity of pre-gel solution (sol-gel transition point of 20.4 °C) endowed it to be 3D printed as a bioink, and the printed scaffold exhibited good resilience and toughness. Moreover, HUVEC, L929, and 3T3 cells were cultured on hydrogel surfaces for 28 days and were enveloped within the hydrogels for 3D culture, indicating excellent cytocompatibility of the hydrogels. Therefore, this hybrid hydrogel system can be used potentially in cell culture scaffold and tissue engineering.

Tissue-Specific Hydrogels for Three-Dimensional Printing and Potential Application in Peripheral Nerve Regeneration.

Decellularized extracellular matrix hydrogel (dECM-G) has demonstrated its significant tissue-specificity, high biocompatibility, and versatile utilities in tissue engineering. However, the low mechanical stability and fast degradation are major drawbacks for its application in three-dimensional (3D) printing. Herein, we report a hybrid hydrogel system consisting of dECM-Gs and photocrosslinkable gelatin methacrylate (GelMA), which resulted in significantly improved printability and structural fidelity. These premixed hydrogels retained high bioactivity and tissue-specificity due to their containing dECM-Gs. More specifically, it was realized that the hydrogel containing dECM-G derived from porcine peripheral nerves (GelMA/pDNM-G) effectively facilitated neurite growth and Schwann cell migration from two-dimensional cultured dorsal root ganglion explants. The nerve cells were also encapsulated in the GelMA/pDNM-G hydrogel for 3D culture or underwent cell-laden bioprinting with high cell viability. The preparation of such GelMA/dECM-G hydrogels enabled the recapitulation of functional tissues through extrusion-based bioprinting, which holds great potential for applications in regenerative medicine. Impact statement Tissue-derived decellularized matrices have drawn broad interests for their versatile applications in tissue engineering and regenerative medicine, especially the decellularized peripheral nerve matrix, which can effectively facilitate axonal extension, remyelination, and neural functional restoration after peripheral nerve injury. However, neither decellularized porcine nerve matrix (pDNM) nor pDNM hydrogel (pDNM-G) can be directly used in three-dimensional printing for personalized nerve constructs or cell transplantation. This work developed a hybrid hydrogel consisting of decellularized extracellular matrix hydrogel (dECM-G) and photocrosslinkable gelatin methacrylate (GelMA), which resulted in significantly improved printability and structural fidelity. The GelMA/pDNM-G hydrogel retained high bioactivity and tissue-specificity due to its dECM-G content. Such hybrid hydrogel systems built up a springboard in advanced biomaterials for neural tissue engineering, as well as a promising strategy for dECM containing bioprinting.

Recent advances in 3D hydrogel culture systems for mesenchymal stem cell-based therapy and cell behavior regulation.

Mesenchymal stem cells (MSCs) have been increasingly recognized as a resource for disease treatment and regenerative medicine. Meanwhile, the unique chemical and physical properties of hydrogels provide innate advantages to achieve high quality MSCs on a large scale. Tremendous kinds of biomaterials have been employed to form hydrogels providing a controllable microenvironment for culturing MSCs. The development of materials science makes it possible to mimic the natural extracellular matrix (ECM), providing an effective means to understand mechanisms such as sensing and remodeling of the different microenvironments by MSCs. The mechanical cues, the formation mechanisms, material types and combination hydrogels are all discussed in this review for three-dimensional (3D) hydrogel culture systems. This article also focuses on the latest development of hydrogel culture systems applied both in vivo and in vitro. Besides the innovation of materials, the culture methods and spatiotemporal cues during the culture stage are other directions of exploration for 3D culture systems. The ultimate goal of hydrogel 3D culture systems is to perfectly mimic the native microenvironment for the study of MSC behavior or the applications of MSC-based therapies.

Multicomponent Peptide Hydrogels as an Innovative Platform for Cell-Based Tissue Engineering in the Dental Pulp.

In light of the increasing levels of antibiotic resistance, nanomaterials and novel biologics are urgently required to manage bacterial infections. To date, commercially available self-assembling peptide hydrogels have not been studied extensively for their ability to inhibit micro-organisms relevant to tissue engineering sites such as dental root canals. In this work, we assess the biocompatibility of dental pulp stem/stromal cells with commercially available multicomponent peptide hydrogels. We also determine the effects of dental pulp stem/stromal cell (DPSC) culture in hydrogels on growth factor/cytokine expression. Furthermore, to investigate novel aspects of self-assembling peptide hydrogels, we determine their antimicrobial activity against the oral pathogens Staphylococcus aureus, Enterococcus faecalis, and Fusobacterium nucleatum. We show that self-assembling peptide hydrogels and hydrogels functionalized with the adhesion motif Arg-Gly-Asp (RGD) are biocompatible with DPSCs, and that cells grown in 3D hydrogel cultures produce a discrete secretome compared with 2D-cultured cells. Furthermore, we show that soluble peptides and assembled hydrogels have antimicrobial effects against oral pathogens. Given their antibacterial activity against oral pathogens, biocompatibility with dental pulp stem/stromal cells and enhancement of an angiogenic secretome, multicomponent peptide hydrogels hold promise for translational use.

Abstract 2638: Developing 3d hydrogel model for patient-derived organoids of metastatic colorectal cancer

Abstract The advances in the development of preclinical models for various cancers have tremendously helped study of the biology and genetics of human cancers as well as for development of therapeutics. Recently, patient-derived organoids (PDOs) have emerged as a robust preclinical platform that can provide insight into the patient specific genetic mechanisms, cancer progression and drug susceptibility. We have developed a novel PDO platform by embedding patient-derived organoids in a 3D hydrogel suspension culture. Our previous studies have shown that, when cultured in hydrogel, spheroids derived from cancer cell lines demonstrated upregulated gene expression signatures in relation to migration and angiogenesis compared to cultures in media, suggesting a more hypoxic, nutrient deficient environment. Given that hypoxia and nutrient deficiency are important feathers of solid tumors, the hydrogel environment could provide a good platform for in vitro study of tumor development and resistance to treatment. In the current study, we embedded PDOs derived from liver metastasis of colorectal cancer into the 3D suspension hydrogel culture. The PDOs adapted well in the hydrogel gel environment. We assessed the response of PDOs in hydrogel to anticancer agents, and compared that to those cultured in media. The PDOs cultured in hydrogel showed significantly elevated resistance to the treatments. The IC50s of chemotherapeutic drugs 5-fluorouracil and oxaliplatin are increased by ~5-10 fold when cultured in hydrogel compared to those in media. Further studies through RNAseq analysis revealed differentially activated signaling pathways. In particular, the levels of the CXC ligand family chemokines are significantly up-regulated in the PDOs cultured in the hydrogel. The CXC ligand family chemokines are important immune stimulants that are known to play key role in the chemotaxis and tumor recruitment of immune cells including neutrophil, T-cell and B-cells. Interestingly, the elevated levels of CXC-ligands have been shown to relate to resistance of colorectal cancer against drug treatment such as 5-fluorouracil. Our hydrogel model could help elucidate the mechanisms behind such resistance. Future study will focus on evaluating how well the hydrogel PDO platform can recapitulate tumor microenvironment and its potential in screening for personalized medicine. Citation Format: He Wei, Eveliina Karelehto, Karen Dubbin, Aimy Sebastian, Robert Warren, Monica Moya, Gaby Loots, Elizabeth Wheeler, Matthew Coleman. Developing 3d hydrogel model for patient-derived organoids of metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2638.

Engineering Fiber-Based Nervous Tissue Constructs for Axon Regeneration

Biomaterial-based scaffolds used in nerve conduits including channels for confining regenerating axons and 3-dimensional (3D) gels as substrates for growth have made improvements in models of nerve repair. Many biomaterial strategies, however, continue to fall short of autologous nerve grafts, which remain the current gold standard in repairing severe nerve lesions (<20 mm). Intraluminal nerve conduit fibers have also shown considerable promise in directing regenerating axons in vitro and in vivo and have gained increasing interest for nerve repair. It is unknown, however, how growing axons respond to a fiber when encountered in a 3D environment. In this study, we considered a construct consisting of a compliant collagen hydrogel matrix and a fiber component to assess contact-guided axon growth. We investigated preferential axon outgrowth on synthetic and natural polymer fibers by utilizing small-diameter microfibers of poly-L-lactic acid and type I collagen representing 2 different fiber stiffnesses. We found that axons growing freely in a 3D hydrogel culture preferentially attach, turn and follow fibers with outgrowth rates and distances that far exceed outgrowth in a hydrogel alone. Wet-spun type I collagen from rat tail tendon performed the best, associated with highly aligned and accelerated outgrowth. This study also evaluated the response of dorsal root ganglion neurons from adult rats to provide data more relevant to axon regenerative potential in nerve repair. We found that ECM treatments on fibers enhanced the regeneration of adult axons indicating that both the physical and biochemical presentation of the fibers are essential for enhancing axon guidance and growth.

Engineered Hydrogels as Valve Tissue Mimetics That Recapitulate Sexual Dimorphisms Observed in Calcification and Osteopontin Activity

Objective: Recent clinical reports indicate that patients with calcific aortic valve stenosis (AVS) have sexually dimorphic disease phenotypes, where female patients have reduced valve calcification compared to males. Yet, the cellular mechanisms contributing to this dimorphism remain unknown. Our results reveal that diseased valve leaflets from a female tend to have reduced and smaller-scale microcalcifications compared to males. Additionally, osteopontin (OPN) expression, a pro-fibrotic but anti-calcific bone sialoprotein, is elevated in human female valve tissue sections. We hypothesize that OPN may play a role in reducing calcification in female aortic valves. Methods: To this end, we use a hydrogel system with cellularly-degradable crosslinkers and adhesive peptides to culture VICs isolated from male and female pigs in 3D. We further introduced a secondary interpenetrating network of collagen type I and studied differences in the cellular response to osteogenic stimuli. Results: The hydrogel matrix supported VIC survival, matrix remodeling, and recapitulated the mineralization differences that are observed in human diseased valve tissue. Specifically, female valve tissue and female VICs cultured in pro-calcifying hydrogels have more punctate calcification (< 3000 μm2) relative to male samples (> 3000 μm2) (Figure 1). Further, OPN localization shifted from the hydrogel extracellular matrix to the nucleus in female cells in response to exogenously delivered calcification stimuli. Conclusions: OPN localization corresponded to sites of VIC-mediated matrix calcification, suggesting OPN may have a role in suppressing mineralization growth in AVS progression. This 3D hydrogel culture system should prove useful to test additional hypotheses related to sex-specific AVS calcification and potentially guide the development of sex-specific treatments. Figure 1. Von Kossa stained histological sections display reduced calcification of female samples (left) relative to male (right), in both tissue sections of human diseased aortic valve (top) and within in vitro 3D hydrogel valve mimetics (bottom). Scale bar = 100 μm