Unlocking Bio-Instructive Polymers: A Novel Multi-Well Screening Platform Based on Secretome Sampling.

Biomaterials are designed to interact with biological systems to replace, support, enhance, or monitor their function. However, there are challenges associated with traditional biomaterials' development due to the lack of underlying theory governing cell response to materials' chemistry. This leads to the time-consuming process of testing different materials plus the adverse reactions in the body such as cytotoxicity and foreign body response. High-throughput screening (HTS) offers a solution to these challenges by enabling rapid and simultaneous testing of a large number of materials to determine their bio-interactions and biocompatibility. Secreted proteins regulate many physiological functions and determine the success of implanted biomaterials through directing cell behaviour. However, the majority of biomaterials' HTS platforms are suitable for microscopic analyses of cell behaviour and not for investigating non-adherent cells or measuring cell secretions. Here, we describe a multi-well platform adaptable to robotic printing of polymers and suitable for secretome profiling of both adherent and non-adherent cells. We detail the platform's development steps, encompassing the preparation of individual cell culture chambers, polymer printing, and the culture environment, as well as examples to demonstrate surface chemical characterisation and biological assessments of secreted mediators. Such platforms will no doubt facilitate the discovery of novel biomaterials and broaden their scope by adapting wider arrays of cell types and incorporating assessments of both secretome and cell-bound interactions. Key features • Detailed protocols for preparation of substrate for contact printing of acrylate-based polymers including O2 plasma etching, functionalisation process, and Poly(2-hydroxyethyl methacrylate) (pHEMA) dip coating. • Preparations of 7 mm × 7 mm polymers employing pin printing system. • Provision of confined area for each polymer using ProPlate® multi-well chambers. • Compatibility of this platform was validated using adherent cells [primary human monocyte-derived macrophages (MDMs)) and non-adherent cells (primary human monocyte-derived dendritic cells (moDCs)]. • Examples of the adaptability of the platform for secretome analysis including five different cytokines using enzyme-linked immunosorbent assay (ELISA, DuoSet®). Graphical overview.

Effects of 3-dimensional culture conditions (collagen-chitosan nano-scaffolds) on maturation of dendritic cells and their capacity to interact with T-lymphocytes.

In the body, there is a natural three-dimensional (3D) microenvironment in which immune cells, including dendritic cells (DC), play their functions. This study evaluated the impact of using collagen-chitosan 3D nano-scaffolds in comparisons to routine 2D culture plates on DC phenotype and functions. Bone marrow-derived DC were cultured on scaffolds and plates and then stimulated with lipopolysaccharide (LPS) or chitosan-based nanoparticles (NP) for 24 h. Thereafter, DC viability, expression of maturation markers and levels of cytokines secretion were evaluated. In another set of studies, the DC were co-cultured with allogenic T-lymphocytes in both the 2D and 3D systems and effects on DC-induction of T-lymphocyte proliferation and cytokine release were analyzed. The results indicated that CD40, CD86 and MHC II marker expression and interleukin (IL)-12, IL-6 and tumor necrosis factor (TNF)-α secretion by DC were enhanced in 3D cultures in comparison to by cells maintained in the 2D states. The data also showed that DNA/chitosan NP activated DC more than LPS in the 3D system. T-Lymphocyte proliferation was induced to a greater extent by DNA/NP-treated DC when both cell types were maintained on the scaffolds. Interestingly, while DC induction of T-lymphocyte interferon (IFN)-γ and IL-4 release was enhanced in the 3D system (relative to controls), there was a suppression of transforming growth factor (TGF)-ß production; effects on IL-10 secretion were variable. The results here suggested that collagen-chitosan scaffolds could provide a pro-inflammatory and activator environment to perform studies to analyze effects of exogenous agents on the induction of DC maturation, NP uptake and/or cytokines release, as well as for the ability of these cells to potentially interact with other immune system cells in vitro.

Barberry's (Berberis integerrima) ingredients suppress T-cell response and shift immune responses toward Th2: an in vitro study.

AIM: Food and medicinal applications of barberry date back to 2500 years ago. This study investigates Berberis integerrima impact on lymphocytic immune responses. MATERIALS & METHODS: Balb/c splenocytes were treated by 0.001-1000 µg/ml of B. integerrimaaqueous and alcoholic extracts in presence of phytohemagglutinin and lipopolysaccharide mitogens. Cell proliferation was assayed and cytokines were measured using ELISA. RESULTS: Both extracts suppressed proliferation of phytohemagglutinin stimulated splenocytes (as T cells), while alcoholic extract induced expansion of lipopolysaccharide activated cells (as B lymphocytes) and unstimulated cells (p < 0.05). Both barberry extracts suppressed IFN-γ production (p < 0.05) and enhanced IL-4, IL-10 and TGF-ß release from splenocytes (p < 0.05). CONCLUSION: Both extracts could suppress T-cell and enhance B-cell proliferation and shift immune responses toward Th2.

Immunomodulatory effects of clove (Syzygium aromaticum) constituents on macrophages: in vitro evaluations of aqueous and ethanolic components.

The present work sought to investigate potential suppressive effects on mouse macrophages by in vitro treatment with clove (Syzygium aromaticum) ethanolic extracted essential oil (containing eugenol) or its water-soluble extract. Using doses (ranging from 0.001-1000 µg/ml) of each material freshly prepared in the laboratory, cell survival and production of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12 by the treated cells (that in all cases also had received LPS stimulation) were measured. Results indicated that, except at doses ≥100 µg/ml, viability was unaffected in all groups. NO release by LPS-stimulated macrophages was generally significantly suppressed by either material; in contrast, low (i.e. 0.001-1 µg/ml) doses of either extract class appeared to enhance NO release by non-LPS (unstimulated)-treated macrophages. Among LPS-stimulated cells, TNFα release was also significantly affected by each extract; the ethanolic extract was suppressive at all doses tested, while the aqueous material was so up to 1 µg/ml and then became stimulatory. In contrast, nearly every dose of either extract appeared to stimulate IL-6 release from the LPS-treated cells. Effects on IL-12 production were overall inconsistent; in general, the ethanolic extract tended to be stimulatory of production by the LPS-treated cells. The data for the aqueous material showed no discernable pattern of effect. The results suggest that clove extracts do not have a distinct cytotoxic activity, but do impart potential anti- and pro-oxidant effects in cells, depending on their concentrations and on the activation state of the macrophages themselves at the time of exposure to the extracts. The impact of the extracts on macrophage cytokine release also displays a pattern of dose-relatedness.

Clove (Syzygium aromaticum) ingredients affect lymphocyte subtypes expansion and cytokine profile responses: An in vitro evaluation.

Clove (Syzygium aromaticum) has been used in folk medicine in many disorders. The present work aimed to investigate effects of clove essential oil as eugenol and water soluble ingredients on mouse splenocytes. Clove extracts were harvested and in different concentrations (0.001-1000 µg/mL) were affected to splenocytes and also phytohemagglutinin (PHA = 5 µg/mL) and lipopolysaccharide (LPS = 10 µg/mL) activated splenocytes; then splenocytes proliferation assayed using the MTT ([3-(4, 5-dimethylthiazole-2-yl) -2, 5-diphenyl tetrazolium bromide]) method were done. On the culture supernatant interferon (IFN)-γ, interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-ß cytokines were measured. Clove ingredients (100 µg/mL and 1000 µg/mL) reduced PHA stimulated splenocytes proliferation and enhanced LPS stimulated cells expansion. Treated splenocytes showed suppression of IFN-γ release and induction of IL-4, IL-10, and TGF-ß secretion (in the range of 0.1-1000 µg/mL). The results of this study suggest clove extracts could suppress the T cell cellular immunity and enhance humoral immune responses. In clove affection cytokine pattern shifted toward modulatory and Th2 responses and accelerator of humoral immunity cytokines.