Search Results
Biocompatibility assessment of silk nanoparticles: hemocompatibility and internalization by human blood cells
Many nanoparticles are designed for use as potential nanomedicines for parenteral administration. However, emerging evidence suggests that hemocompatibility is important, but is highly particle- and test-bed dependent. Thus, knowledge of bulk material properties does not predict the hemocompatibility of uncharacterized nanoparticles, including silk nanoparticles. This study compares the hemocompatibility of silk versus silica nanoparticles, using whole human blood under quasi-static and flow conditions. Substantial hemocompatibility differences are noted for some nanoparticles in quasi-static versus dynamic studies; i.e., the inflammatory response to silk nanoparticles is significantly lower under flow versus quasi-static conditions. Silk nanoparticles also have very low coagulant properties - an observation that scales from the macro- to the nano-level. These nanoparticle hemocompatibility studies are complemented by preliminary live cell measurements to evaluate the endocytosis and trafficking of nanoparticles in human blood cells. Overall, this study demonstrates that nanoparticle hemocompatibility is affected by several factors, including the test bed design.
Oxygen producing microscale spheres affect cell survival in conditions of oxygen-glucose deprivation in a cell specific manner: Implications for cell transplantation
This study outlines the synthesis of microscale oxygen producing spheres, which, when used in conjunction with catalase, can raise the dissolved oxygen content of cell culture media for 16-20 hours. In conditions of oxygen and glucose deprivation, designed to mimic the graft environment in vivo, the spheres rescue SH-SY5Y cells and meschymal stem cells, showing that oxygen producing biomaterials may hold potential to improve the survival of cells post-transplantation.
Limbal stromal cells derived from porcine tissue demonstrate mesenchymal characteristics in vitro
Limbal stromal cells (LSCs) from the human ocular surface display mesenchymal stromal cell characteristics in vitro. In this study, we isolated cells from the porcine limbal stroma (pLSCs), characterised them, and evaluated their ability to support angiogenesis and the culture of porcine limbal epithelial stem cells (pLESCs). The isolated cells adhered to plastic and grew in monolayers in vitro using serum-supplemented or serum-free medium. The pLSCs demonstrated expression of CD29, and cross-reactivity with anti-human CD45, CD90, CD105, CD146, and HLA-ABC. However, expression of CD105, CD146 and HLA-ABC reduced when cultured in serum-free medium. PLSCs did not undergo adipogenic or osteogenic differentiation, but differentiated towards the chondrogenic lineage. Isolated cells were also co-cultured with human umbilical vein endothelial cells (HUVECs) in star-shaped Poly(ethylene glycol) (starPEG)-heparin hydrogels to assess their pericyte capacity which supported angiogenesis networks of HUVECs. PLSCs supported the three dimensional HUVEC network for 7 days. The isolated cells were further growth-arrested and evaluated as feeder cells for pLESC expansion on silk fibroin membranes, as a potential carrier material for transplantation. PLSCs supported the growth of pLESCs comparably to murine 3T3 cells. In conclusion, although pLSCs were not completely comparable to their human counterpart, they display several mesenchymal-like characteristics in vitro.
Bone marrow niche-mimetics modulate HSPC function via integrin signaling
The bone marrow (BM) microenvironment provides critical physical cues for hematopoietic stem and progenitor cell (HSPC) maintenance and fate decision mediated by cell-matrix interactions. However, the mechanisms underlying matrix communication and signal transduction are less well understood. Contrary, stem cell culture is mainly facilitated in suspension cultures. Here, we used bone marrow-mimetic decellularized extracellular matrix (ECM) scaffolds derived from mesenchymal stromal cells (MSCs) to study HSPC-ECM interaction. Seeding freshly isolated HSPCs adherent (AT) and non-adherent (SN) cells were found. We detected enhanced expansion and active migration of AT-cells mediated by ECM incorporated stromal derived factor one. Probing cell mechanics, AT-cells displayed naïve cell deformation compared to SN-cells indicating physical recognition of ECM material properties by focal adhesion. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be induced. Signaling focal contacts via ITGβ3 were identified to facilitate cell adhesion, migration and mediate ECM-physical cues to modulate HSPC function.
Exploring Structure–Property Relationships of GAGs to Tailor ECM-Mimicking Hydrogels
Glycosaminoglycans (GAGs) are a class of linear polysaccharides that are ubiquitous in the extracellular matrix (ECM) and on cell surfaces. Due to their key role in development, homeostasis, pathogenesis, and regeneration, GAGs are increasingly used in the design of ECM-mimicking hydrogels to stimulate tissue formation and regenerative processes via specifically orchestrated cell-instructive signals. These applications first and foremost build on the ability of GAGs to effectively bind, protect, and release morphogens. The specificity and strength of morphogen-GAG interactions are largely governed by the number and spatial distribution of negatively charged sulfate groups carried by GAGs. Herein, we summarize a mean-field approach to quantify the density of ionizable groups, GAG concentration, and cross-linking degree of GAG-containing hydrogels on the basis of microslit electrokinetic experiments. We further present and discuss a continuum model of mucosa that accounts for charge regulation by glycan-ion pairing in biological contexts and under conditions of macromolecular crowding. Finally, we discuss the modulation of the morphogen binding and transport in GAG hydrogels by selective desulfation of the GAG component.
Three-Dimensional In Vitro Hydro- and Cryogel-Based Cell-Culture Models for the Study of Breast-Cancer Metastasis to Bone
Bone is the most common site for breast-cancer invasion and metastasis, and it causes severe morbidity and mortality. A greater understanding of the mechanisms leading to bone-specific metastasis could improve therapeutic strategies and thus improve patient survival. While three-dimensional in vitro culture models provide valuable tools to investigate distinct heterocellular and environmental interactions, sophisticated organ-specific metastasis models are lacking. Previous models used to investigate breast-to-bone metastasis have relied on 2.5D or singular-scaffold methods, constraining the in situ mimicry of in vitro models. Glycosaminoglycan-based gels have demonstrated outstanding potential for tumor-engineering applications. Here, we developed advanced biphasic in vitro microenvironments that mimic breast-tumor tissue (MCF-7 and MDA-MB-231 in a hydrogel) spatially separated with a mineralized bone construct (human primary osteoblasts in a cryogel). These models allow distinct advantages over former models due to the ability to observe and manipulate cellular migration towards a bone construct. The gels allow for the binding of adhesion-mediating peptides and controlled release of signaling molecules. Moreover, mechanical and architectural properties can be tuned to manipulate cell function. These results demonstrate the utility of these biomimetic microenvironment models to investigate heterotypic cell–cell and cell–matrix communications in cancer migration to bone.
Defined Geldrop Cultures Maintain Neural Precursor Cells
Distinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.
A three-dimensional ex vivo tri-culture model mimics cell-cell interactions between acute myeloid leukemia and the vascular niche
Ex vivo studies of human disease, such as acute myeloid leukemia, are generally limited to the analysis of two-dimensional cultures which often misinterpret the effectiveness of chemotherapeutics and other treatments. Here we show that matrix metalloproteinase-sensitive hydrogels prepared from poly(ethylene glycol) and heparin functionalized with adhesion ligands and pro-angiogenic factors can be instrumental to produce robust three-dimensional culture models, allowing for the analysis of acute myeloid leukemia development and response to treatment. We evaluated the growth of four leukemia cell lines, KG1a, MOLM13, MV4-11 and OCI-AML3, as well as samples from patients with acute myeloid leukemia. Furthermore, endothelial cells and mesenchymal stromal cells were co-seeded to mimic the vascular niche for acute myeloid leukemia cells. Greater drug resistance to daunorubicin and cytarabine was demonstrated in three-dimensional cultures and in vascular co-cultures when compared with two-dimensional suspension cultures, opening the way for drug combination studies. Application of the C-X-C chemokine receptor type 4 (CXCR4) inhibitor, AMD3100, induced mobilization of the acute myeloid leukemia cells from the vascular networks. These findings indicate that the three-dimensional tri-culture model provides a specialized platform for the investigation of cell-cell interactions, addressing a key challenge of current testing models. This ex vivo system allows for personalized analysis of the responses of patients’ cells, providing new insights into the development of acute myeloid leukemia and therapies for this disease.
Retargeting of UniCAR T cells with an in vivo synthesized target module directed against CD19 positive tumor cells
Recent treatments of leukemias with T cells expressing chimeric antigen receptors (CARs) underline their impressive therapeutic potential but also their risk of severe side effects including cytokine release storms and tumor lysis syndrome. In case of cross-reactivities, CAR T cells may also attack healthy tissues. To overcome these limitations, we previously established a switchable CAR platform technology termed UniCAR. UniCARs are not directed against typical tumor-associated antigens (TAAs) but instead against a unique peptide epitope: Fusion of this peptide epitope to a recombinant antibody domain results in a target module (TM). TMs can cross-link UniCAR T cells with tumor cells and thereby lead to their destruction. So far, we constructed TMs with a short half-life. The fast turnover of such a TM allows to rapidly interrupt the treatment in case severe side effects occur. After elimination of most of the tumor cells, however, longer lasting TMs which have not to be applied via continous infusion would be more convenient for the patient. Here we describe and characterize a TM for retargeting UniCAR T cells to CD19 positive tumor cells. Moreover, we show that the TM can efficiently be produced in vivo from producer cells housed in a sponge-like biomimetic cryogel and, thereby, serving as an in vivo TM factory for an extended retargeting of UniCAR T cells to CD19 positive leukemic cells.
Impact of the springtail’s cuticle nanotopography on bioadhesion and biofilm formation in vitro and in the oral cavity
Springtails (Collembola) have a nanostructured cuticle. To evaluate and to understand anti-biofouling properties of springtail cuticles’ morphology under different conditions, springtails, shed cuticles and cuticle replicates were studied after incubation with protein solutions and bacterial cultures using common in vitro models. In a second step, they were exposed to human oral environment in situ in order to explore potential application in dentistry. In vitro, the cuticular structures were found to resist wetting by albumin solutions for up to 3 h and colonization by Staphylococcus epidermidis was inhibited. When exposed in the oral cavity, initial pellicle formation was of high heterogeneity: parts of the surface were coated by adsorbed proteins, others remained uncoated but exhibited locally attached, ‘bridging’, proteinaceous membranes spanning across cavities of the cuticle surface; this unique phenomenon was observed for the first time. Also the degree of bacterial colonization varied considerably. In conclusion, the springtail cuticle partially modulates bioadhesion in the oral cavity in a unique and specific manner, but it has no universal effect. Especially after longer exposure, the nanotextured surface of springtails is masked by the pellicle, resulting in subsequent bacterial colonization, and, thus, cannot effectively avoid bioadhesion in the oral cavity comprehensively. Nevertheless, the observed phenomena offer valuable information and new perspectives for the development of antifouling surfaces applicable in the oral cavity.