In vitro encapsulation and expansion of T and CAR-T cells using 3D synthetic thermo-responsive matrices.

Suspension cell culture and rigid commercial substrates are the most common methods to clinically manufacture therapeutic CAR-T cells ex vivo. However, suspension culture and nano/micro-scale commercial substrates poorly mimic the microenvironment where T cells naturally develop, leading to profound impacts on cell proliferation and phenotype. To overcome this major challenge, macro-scale substrates can be used to emulate that environment with higher precision. This work employed a biocompatible thermo-responsive material with tailored mechanical properties as a potential synthetic macro-scale scaffold to support T cell encapsulation and culture. Cell viability, expansion, and phenotype changes were assessed to study the effect of two thermo-responsive hydrogel materials with stiffnesses of 0.5 and 17 kPa. Encapsulated Pan-T and CAR-T cells were able to grow and physically behave similar to the suspension control. Furthermore, matrix stiffness influenced T cell behavior. In the softer polymer, T cells had higher activation, differentiation, and maturation after encapsulation obtaining significant cell numbers. Even when terpolymer encapsulation affected the CAR-T cell viability and expansion, CAR T cells expressed favorable phenotypical profiles, which was supported with cytokines and lactate production. These results confirmed the biocompatibility of the thermo-responsive hydrogels and their feasibility as a promising 3D macro-scale scaffold for in vitro T cell expansion that could potentially be used for cell manufacturing process.

In vitro assessment of inflammatory skin potential of poly(methyl methacrylate) at non-cytotoxic concentrations.

Poly(methyl methacrylate) (PMMA) is considered an attractive substrate material for fabricating wearable skin sensors such as fitness bands and microfluidic devices. Despite its widespread use, inflammatory and allergic responses have been attributed to the use of this material. Therefore, the main objective of this study was to obtain a comprehensive understanding of potential biological effects triggered by PMMA at non-cytotoxic concentrations using in vitro models of NIH3T3 fibroblasts and reconstructed human epidermis (RhE). It was hypothesized that concentrations that do not reduce cell viability are sufficient to activate pathways of inflammatory processes in the skin. The study included cytotoxicity, cell metabolism, cytokine quantification, histopathological, and gene expression analyses. The NIH3T3 cell line was used as a testbed for screening cell toxicity levels associated with the concentration of PMMA with different molecular weights (MWs) (i.e., MW ~5,000 and ~15,000 g/mol). The lower MW of PMMA had a half-maximal inhibitory concentration (IC50 ) value of 5.7 mg/cm2 , indicating greater detrimental effects than the higher MW (IC50 = 14.0 mg/cm2 ). Non-cytotoxic concentrations of 3.0 mg/cm2 for MW ~15,000 g/mol and 0.9 mg/cm2 for MW ~5,000 g/mol) induced negative metabolic changes in NIH3T3 cells. Cell viability was severely reduced to 7% after the exposure to degradation by-products generated after thermal and photodegradation degradation of PMMA. PMMA at non-cytotoxic concentrations still induced overexpression of pro-inflammatory cytokines, chemokines, and growth factors (IL1B, CXCL10, CCL5, IL1R1, IL7, IL17A, VEGFA, FGF2, IFNG, IL15) on the RhE model. The inflammatory response was also supported by histopathological and gene expression analyses of PMMA-treated RhE, indicating tissue damage and gene overexpression. Results suggested that non-cytotoxic concentrations of PMMA (3.0 to 5.6 mg/cm2 for MW ~15,000 g/mol and 0.9 to 2.1 mg/cm2 for MW ~5,000 g/mol) were sufficient to negatively alter NIH3T3 cells metabolism and activate inflammatory events in the RhE skin.

Synthetic Thermo-Responsive Terpolymers as Tunable Scaffolds for Cell Culture Applications.

The use of tailored synthetic hydrogels for in vitro tissue culture and biomanufacturing provides the advantage of mimicking the cell microenvironment without issues of batch-to-batch variability. To that end, this work focused on the design, characterization, and preliminary evaluation of thermo-responsive, transparent synthetic terpolymers based on N-isopropylacrylamide, vinylphenylboronic acid, and polyethylene glycol for cell manufacturing and in vitro culture applications. Polymer physical properties were characterized by FT-IR, 1H-NMR, DLS, rheology, and thermal-gravimetric analysis. Tested combinations provided polymers with a lower critical solution temperature (LCST) between 30 and 45 °C. Terpolymer elastic/shear modulus varied between 0.3 and 19.1 kPa at 37 °C. Cellular characterization indicated low cell cytotoxicity on NIH-3T3. Experiments with the ovarian cancer model SKOV-3 and Jurkat T cells showed the terpolymers' capacity for cell encapsulation without interfering with staining or imaging protocols. In addition, cell growth and high levels of pluripotency demonstrated the capability of terpolymer to culture iPSCs. Characterization results confirmed a promising use of terpolymers as a tunable scaffold for cell culture applications.

Real-time monitoring of human Schwann cells on heparin-collagen coatings reveals enhanced adhesion and growth factor response.

In this work, we evaluate the enhancing effect of six bilayers of heparin/collagen (HEP/COL)6 layer-by-layer coatings on human Schwann cell (hSCs) adhesion and proliferation in the presence or absence of nerve growth factor (NGF). hSCs behavior and in vitro bioactivity were studied during six days of culture using end-point viability and proliferation assays as well as an impedance-based real-time monitoring system. An end-point viability assay revealed that hSCs cultured on the (HEP/COL)6 coatings increased their growth by more than 230% compared to controls. However, an EdU proliferation assay revealed that the proliferation rate of hSCs in all conditions were similar, with 45% of cells proliferating after 18 hours of incubation. Fluorescence microscopy revealed that hSCs spreading was similar between the tissue culture plastic control and the (HEP/COL)6. The presence of NGF in solution resulted in cells with a larger spread area. Real-time monitoring of hSCs seeded on (HEP/COL)6 with and without NGF reveals that initial cell adhesion is improved by the presence of the (HEP/COL)6 coatings, and it is further improved by the presence of NGF. Our results suggest that (HEP/COL)6 coatings enhance Schwann cell behavior and response to NGF. This simple modification could be applied to current nerve regeneration strategies to improve the repair of damaged nerve.

Correction: Hydrophobicity of Antifungal ß-Peptides Is Associated with Their Cytotoxic Effect on In Vitro Human Colon Caco-2 and Liver HepG2 Cells.

[This corrects the article DOI: 10.1371/journal.pone.0149271.].

Hydrophobicity of Antifungal ß-Peptides Is Associated with Their Cytotoxic Effect on In Vitro Human Colon Caco-2 and Liver HepG2 Cells.

The widespread distribution of fungal infections, with their high morbidity and mortality rate, is a global public health problem. The increase in the population of immunocompromised patients combined with the selectivity of currents treatments and the emergence of drug-resistant fungal strains are among the most imperative reasons to develop novel antifungal formulations. Antimicrobial ß-peptides are peptidomimetics of natural antimicrobial peptides (AMPs), which have been proposed as developmental platforms to enhance the AMPs selectivity and biostability. Their tunability allows the design of sequences with remarkable activity against a wide spectrum of microorganisms such as the human pathogenic Candida spp., both in planktonic and biofilm morphology. However, the ß-peptide's effect on surrounding host cells remains greatly understudied. Assessments have mainly relied on the extent of hemolysis that a candidate peptide is able to cause. This work investigated the in vitro cytotoxicity of various ß-peptides in the Caco-2 and HepG2 mammalian cell lines. Results indicated that the cytotoxic effect of the ß-peptides was influenced by cell type and was also correlated to structural features of the peptide such as hydrophobicity. We found that the selectivity of the most hydrophobic ß-peptide was 2-3 times higher than that of the least hydrophobic one, for both cell types according to the selectivity index parameter (IC50/MIC). The IC50 of Caco-2 and HepG2 increased with hydrophobicity, which indicates the importance of testing putative therapeutics on different cell types. We report evidence of peptide-cell membrane interactions in Caco-2 and HepG2 using a widely studied ß-peptide against C. albicans.

Plasma biomarkers discriminate clinical forms of multiple sclerosis.

Multiple sclerosis, the most common cause of neurological disability in young population after trauma, represents a significant public health burden. Current challenges associated with management of multiple sclerosis (MS) patients stem from the lack of biomarkers that might enable stratification of the different clinical forms of MS and thus prompt treatment for those patients with progressive MS, for whom there is currently no therapy available. In the present work we analyzed a set of thirty different plasma cytokines, chemokines and growth factors present in circulation of 129 MS patients with different clinical forms (relapsing remitting, secondary progressive and primary progressive MS) and 53 healthy controls, across two independent cohorts. The set of plasma analytes was quantified with Luminex xMAP technology and their predictive power regarding clinical outcome was evaluated both individually using ROC curves and in combination using logistic regression analysis. Our results from two independent cohorts of MS patients demonstrate that the divergent clinical and histology-based MS forms are associated with distinct profiles of circulating plasma protein biomarkers, with distinct signatures being composed of chemokines and growth/angiogenic factors. With this work, we propose that an evaluation of a set of 4 circulating biomarkers (HGF, Eotaxin/CCL11, EGF and MIP-1ß/CCL4) in MS patients might serve as an effective tool in the diagnosis and more personalized therapeutic targeting of MS patients.

Low DPP4 expression and activity in multiple sclerosis.

Multiple sclerosis (MS) is a prototypic Th1/Th17 chronic autoimmune disease of the central nervous system. Dipeptidyl peptidase 4 (DPP4 or CD26) is a multifunctional molecule involved in autoimmune diseases' pathophysiology. We sought to integrate disparate pieces of data and analyze the plasma levels of sDPP4, DPP activity and DPP4 surface expression on T-cells in 129 MS patients with different clinical forms and 53 healthy controls, across two independent cohorts. Herein, we provide new evidence that sDPP4 concentration and DPP activity are significantly lower in MS patients than controls (p < 0.0001 and p < 0.01, respectively). In contrast, the frequency of circulating CD8(+)DPP4(hi) T-cells (p = 0.02) was increased in MS patients. This is the first study that simultaneously analyzes DPP4 expression and function in a large cohort of MS patients. Our data indicate a putative role for DPP4 in MS pathophysiology and suggest that a deeper understanding of surface versus shed DPP4 biology is warranted.

Synthesis, Stability, Cellular Uptake, and Blood Circulation Time of Carboxymethyl-Inulin Coated Magnetic Nanoparticles.

Iron oxide nanoparticles were coated with the biocompatible, biodegradable, non-immunogenic polysaccharide inulin by introduction of carboxyl groups into the inulin structure and conjugation with amine groups on the surface of iron oxide nanoparticles grafted with 3-aminopropyltriethoxysilane. The resulting nanoparticles were characterized by FT-IR spectroscopy, transmission electron microscopy, dynamic light scattering, zeta potential, SQUID magnetometry, and with respect to their energy dissipation rate in applied alternating magnetic fields. The nanoparticles had a hydrodynamic diameter in the range of 70 ± 10 nm and were superparamagnetic, with energy dissipation rates in the range of 58-175 W/g for an applied field frequency of 233 kHz and an applied field amplitude in the range of 20-48 kA/m. The nanoparticles were stable in a range of pH, at temperatures between 23°C and 53°C, and in short term storage in water, PBS, and culture media. The particles were non-cytotoxic to the immortalized human cancer cell lines Hey A8 FDR, A2780, MDA 468, MCF-7 and Caco-2. The nanoparticles were readily taken up by Caco-2 cells in a time and concentration dependent fashion, and were found to have a pharmacokinetic time constant of 47 ± 3 min. The small size, non-cytotoxicity, and efficient energy dissipation of the particles could make them useful for biomedical applications such as magnetic fluid hyperthermia.

Enhanced reduction in cell viability by hyperthermia induced by magnetic nanoparticles.

Colloidal suspensions of iron oxide magnetic nanoparticles are known to dissipate energy when exposed to an oscillating magnetic field. Such energy dissipation can be employed to locally raise temperature inside a tumor between 41°C and 45°C (hyperthermia) to promote cell death, a treatment known as magnetic fluid hyperthermia (MFH). This work seeks to quantify differences between MFH and hot-water hyperthermia (HWH) in terms of reduction in cell viability using two cancer cell culture models, Caco-2 (human epithelial colorectal adenocarcinoma) and MCF-7 (human breast cancer). Magnetite nanoparticles were synthesized via the co-precipitation method and functionalized with adsorbed carboxymethyl dextran. Cytotoxicity studies indicated that in the absence of an oscillating magnetic field, cell viability was not affected at concentrations of up to 0.6 mg iron oxide/mL. MFH resulted in a significant decrease in cell viability when exposed to a magnetic field for 120 minutes and allowed to rest for 48 hours, compared with similar field applications, but with shorter resting time. The results presented here suggest that MFH most likely induces apoptosis in both cell types. When compared with HWH, MFH produced a significant reduction in cell viability, and these effects appear to be cell-type related.