3D Printing as a Strategy to Scale-Up Biohybrid Hydrogels for T Cell Manufacture.

The emergence of cellular immunotherapy treatments is introducing more efficient strategies to combat cancer as well as autoimmune and infectious diseases. However, the cellular manufacturing procedures associated with these therapies remain costly and time-consuming, thus limiting their applicability. Recently, lymph-node-inspired PEG-heparin hydrogels have been demonstrated to improve primary human T cell culture at the laboratory scale.

Migration of human T cells can be differentially directed by electric fields depending on the extracellular microenvironment.

T cell migration plays an essential role in the immune response and T cell-based therapies. It can be modulated by chemical and physical cues such as electric fields (EFs). The mechanisms underlying electrotaxis (cell migration manipulated by EFs) are not fully understood and systematic studies with immune cells are rare.

pH-Sensitive Acrylic Terpolymers for the Coating of Orally Administered Drugs Used for Colonic Release.

Polymeric coatings are a promising option for the development of delivery systems for orally administered drugs. However, the gastrointestinal conditions to which they are subjected, which include low pH and solubility as well as peristaltic movements, can limit their applications. In this work, different formulations of polymeric coatings were produced using pH-sensitive materials consisting of copolymers of methyl acrylate, methyl methacrylate, and methacrylic acid.

Three-dimensional cell culture of chimeric antigen receptor T cells originated from peripheral blood mononuclear cells towards cellular therapies.

Background Aims: With the objective of improving the ex vivo production of therapeutic chimeric antigen receptor (CAR) T cells, we explored the addition of three-dimensional (3D) polystyrene scaffolds to standard suspension cell cultures.

Methods: We aimed to mimic the structural support given by the lymph nodes during in vivo lymphocyte expansion.

Results: We observed an increase in cell proliferation compared with standard suspension systems as well as an enhanced cytotoxicity toward cancer cells.

Biohybrid Hydrogels for Tumoroid Culture.

Tumoroids are 3D in vitro models that recapitulate key features of in vivo tumors, such as their architecture - hypoxic center and oxygenated outer layer - in contrast with traditional 2D cell cultures. Moreover, they may be able to preserve the patient-specific signature in terms of cell heterogeneity and mutations. Tumoroids are, therefore, interesting tools for improving the understanding of cancer biology, developing new drugs, and potentially designing personalized therapeutic plans.

A Novel Generation of Tailored Antimicrobial Drugs Based on Recombinant Multidomain Proteins.

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions.

Ratiometric Nanothermometer Based on a Radical Excimer for In Vivo Sensing.

Ratiometric fluorescent nanothermometers with near-infrared emission play an important role in in vivo sensing since they can be used as intracellular thermal sensing probes with high spatial resolution and high sensitivity, to investigate cellular functions of interest in diagnosis and therapy, where current approaches are not effective. Herein, the temperature-dependent fluorescence of organic nanoparticles is designed, synthesized, and studied based on the dual emission, generated by monomer and excimer species, of the tris(2,4,6-trichlorophenyl)methyl radical (TTM) doping organic nanoparticles (TTMd-ONPs), made of optically neutral tris(2,4,6-trichlorophenyl)methane (TTM-αH), acting as a matrix. The excimer emission intensity of TTMd-ONPs decreases with increasing temperatures whereas the monomer emission is almost independent and can be used as an internal reference.

Hierarchical Quatsome-RGD Nanoarchitectonic Surfaces for Enhanced Integrin-Mediated Cell Adhesion.

The synthesis and study of the tripeptide Arg-Gly-Asp (RGD), the binding site of different extracellular matrix proteins, e.g., fibronectin and vitronectin, has allowed the production of a wide range of cell adhesive surfaces.

Enhanced human T cell expansion with inverse opal hydrogels.

Advanced personalized immunotherapies still have to overcome several biomedical and technical limitations before they become a routine cancer treatment in spite of recent achievements. In adoptive cell therapy (ACT), the capacity to obtain adequate numbers of therapeutic T cells in the patients following treatment should be improved. Moreover, the time and costs to produce these T cells should be reduced.

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain.

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics.

Methods for Processing Protein Aggregates into Surfaces.

The processing of inclusion bodies (IBs) into surfaces is of great interest for cell culture applications due to the combined physical and biological cues these particles provide. The arrangement of these IBs into defined and tunable micropatterns can be useful for basic research purposes regarding the mechanical properties needed for cell adhesion and migration, among other responses. There are several approaches that can be used when functionalizing a substrate with IBs, regarding both the strategy used and also the kind of surface-particle interaction.

Methods for the Characterization of Protein Aggregates.

The physicochemical characterization of protein aggregates yields an important contribution to further our understanding on many diseases for which the formation of protein aggregates is one of the pathological hallmarks. On the other hand, bacterial inclusion bodies (IBs) have recently been shown to be highly pure proteinaceous aggregates of a few hundred nanometers, produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. Despite the wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering, very few is known about their physicochemical properties.

Polylactide, Processed by a Foaming Method Using Compressed Freon R134a, for Tissue Engineering.

Fabricating polymeric scaffolds using cost-effective manufacturing processes is still challenging. Gas foaming techniques using supercritical carbon dioxide (scCO) have attracted attention for producing synthetic polymer matrices; however, the high-pressure requirements are often a technological barrier for its widespread use. Compressed 1,1,1,2-tetrafluoroethane, known as Freon R134a, offers advantages over CO in manufacturing processes in terms of lower pressure and temperature conditions and the use of low-cost equipment.

A Spatiotemporal Microenvironment Model to Improve Design of a Three-Dimensional Bioreactor for Red Cell Production.

Cellular microenvironments provide stimuli, including paracrine and autocrine growth factors and physicochemical cues, which support efficient cell production unmatched by current biomanufacturing platforms. While three-dimensional (3D) culture systems aim to recapitulate niche architecture and function of the target tissue/organ, they are limited in accessing spatiotemporal information to evaluate and optimize cell/tissue process development. Herein, a mathematical modeling framework is parameterized by single-cell phenotypic imaging and multiplexed biochemical assays to simulate the nonuniform tissue distribution of nutrients/metabolites and growth factors in cell niche environments.

CCL21-loaded 3D hydrogels for T cell expansion and differentiation.

Recent achievements in the field of immunotherapy, such as the development of engineered T cells used in adoptive cell therapy, are introducing more efficient strategies to combat cancer. Nevertheless, there are still many limitations. For example, these T cells are challenging to manufacture, manipulate, and control.

Stable anchoring of bacteria-based protein nanoparticles for surface enhanced cell guidance.

In tissue engineering, biological, physical, and chemical inputs have to be combined to properly mimic cellular environments and successfully build artificial tissues which can be designed to fulfill different biomedical needs such as the shortage of organ donors or the development of in vitro disease models for drug testing. Inclusion body-like protein nanoparticles (pNPs) can simultaneously provide such physical and biochemical stimuli to cells when attached to surfaces. However, this attachment has only been made by physisorption.

High-Throughput Cell Motility Studies on Surface-Bound Protein Nanoparticles with Diverse Structural and Compositional Characteristics.

Eighty areas with different structural and compositional characteristics made of bacterial inclusion bodies formed by the fibroblast growth factor (FGF-IBs) were simultaneously patterned on a glass surface with an evaporation-assisted method that relies on the coffee-drop effect. The resulting surface patterned with these protein nanoparticles enabled to perform a high-throughput study of the motility of NIH-3T3 fibroblasts under different conditions including the gradient steepness, particle concentrations, and area widths of patterned FGF-IBs, using for the data analysis a methodology that includes "heat maps". From this analysis, we observed that gradients of concentrations of surface-bound FGF-IBs stimulate the total cell movement but do not affect the total net distances traveled by cells.

Enantioselective Synthesis of 3-Heterosubstituted-2-amino-1-ols by Sequential Metal-Free Diene Aziridination/Kinetic Resolution.

A general protocol for the enantioselective synthesis of 3-heterosubstituted-2-amino-1-ols was developed based on metal- free intramolecular regio- and stereoselective diene aziridination and regioselective opening. Kinetic resolution of the resulting (1'-NR R and 1'-SR)-4-oxazolidinones was performed using ABCs organocatalysts, expanding the application of this methodology.

Functionalization of polyacrylamide for nanotrapping positively charged biomolecules.

Engineering new materials which are capable of trapping biomolecules in nanoscale quantities, is crucial in order to achieve earlier diagnostics in different diseases. This article demonstrates that using free radical copolymerization, polyacrylamide can be successfully functionalized with specific synthons for nanotrapping positively charged molecules, such as numerous proteins, through electrostatic interactions due to their negative charge. Specifically, two functional random copolymers, acrylamide/acrylic acid (1) and acrylamide/acrylic acid/-(pyridin-4-yl-methyl)acrylamide (2), whose negative net charges differ in their water solutions, were synthetized and their ability to trap positively charged proteins was studied using myoglobin as a proof-of-concept example.

Combining Adhesive Nanostructured Surfaces and Costimulatory Signals to Increase T Cell Activation.

Adoptive cell therapies are showing very promising results in the fight against cancer. However, these therapies are expensive and technically challenging in part due to the need of a large number of specific T cells, which must be activated and expanded in vitro. Here we describe a method to activate primary human T cells using a combination of nanostructured surfaces functionalized with the stimulating anti-CD3 antibody and the peptidic sequence arginine-glycine-aspartic acid, as well as costimulatory agents (anti-CD28 antibody and a cocktail of phorbol 12-myristate 13-acetate, ionomycin, and protein transport inhibitors).

Artificial 3D Culture Systems for T Cell Expansion.

Adoptive cell therapy, i.e., the extraction, manipulation, and administration of ex vivo generated autologous T cells to patients, is an emerging alternative to regular procedures in cancer treatment.

Surface-Bound Gradient Deposition of Protein Nanoparticles for Cell Motility Studies.

A versatile evaporation-assisted methodology based on the coffee-drop effect is described to deposit nanoparticles on surfaces, obtaining for the first time patterned gradients of protein nanoparticles (pNPs) by using a simple custom-made device. Fully controllable patterns with specific periodicities consisting of stripes with different widths and distinct nanoparticle concentration as well as gradients can be produced over large areas (∼10 cm) in a fast (up to 10 mm/min), reproducible, and cost-effective manner using an operational protocol optimized by an evolutionary algorithm. The developed method opens the possibility to decorate surfaces "a-la-carte" with pNPs enabling different categories of high-throughput studies on cell motility.

Stimuli-Responsive Functionalization Strategies to Spatially and Temporally Control Surface Properties: Michael vs Diels-Alder Type Additions.

Stimuli-responsive self-assembled monolayers (SAMs) are used to confer switchable physical, chemical, or biological properties to surfaces through the application of external stimuli. To obtain spatially and temporally tunable surfaces, we present microcontact printed SAMs of a hydroquinone molecule that are used as a dynamic interface to immobilize different functional molecules either via Diels-Alder or Michael thiol addition reactions upon the application of a low potential. In spite of the use of such reactions and the potential applicability of the resulting surfaces in different fields ranging from sensing to biomedicine through data storage or cleanup, a direct comparison of the two functionalization strategies on a surface has not yet been performed.

Enantioselective Synthesis of Aminodiols by Sequential Rhodium-Catalysed Oxyamination/Kinetic Resolution: Expanding the Substrate Scope of Amidine-Based Catalysis.

Regio- and stereoselective oxyamination of dienes through a tandem rhodium-catalysed aziridination-nucleophilic opening affords racemic oxazolidinone derivatives, which undergo a kinetic resolution acylation process with amidine-based catalysts (ABCs) to achieve s values of up to 117. This protocol was applied to the enantioselective synthesis of sphingosine.

Integrin-Assisted T-Cell Activation on Nanostructured Hydrogels.

Adoptive cell therapy (ACT) has shown very promising results as treatment for cancer in a few clinical trials, such as the complete remissions of otherwise terminal leukemia patients. Nevertheless, the introduction of ACT into clinics requires overcoming not only medical but also technical challenges, such as the ex vivo expansion of large amounts of specific T-cells. Nanostructured surfaces represent a novel T-cell stimulation technique that enables us to fine-tune the density and orientation of activating molecules presented to the cells.