A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification.

Monoclonal antibodies (mAbs) are commonly used biologic drugs for the treatment of diseases such as rheumatoid arthritis, multiple sclerosis, COVID-19 and various cancers. They are produced in Chinese hamster ovary cell lines and are purified via a number of complex and expensive chromatography-based steps, operated in batch mode, that rely heavily on protein A resin. The major drawback of conventional procedures is the high cost of the adsorption media and the extensive use of chemicals for the regeneration of the chromatographic columns, with an environmental cost. We have shown that conventional protein A chromatography can be replaced with a single crystallization step and gram-scale production can be achieved in continuous flow using the template-assisted membrane crystallization process. The templates are embedded in a membrane (e.g., porous polyvinylidene fluoride with a layer of polymerized polyvinyl alcohol) and serve as nucleants for crystallization. mAbs are flexible proteins that are difficult to crystallize, so it can be challenging to determine the optimal conditions for crystallization. The objective of this protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAb purification platform to replace at least the protein A affinity stage in traditional chromatography-based purification platforms. The procedure provides details on how to establish the optimal parameters for separation (crystallization conditions, choice of templates, choice of membrane) and advice on analytical and characterization methods.

NiSe and CoSe Topological Nodal-Line Semimetals: A Sustainable Platform for Efficient Thermoplasmonics and Solar-Driven Photothermal Membrane Distillation.

The control of heat at the nanoscale via the excitation of localized surface plasmons in nanoparticles (NPs) irradiated with light holds great potential in several fields (cancer therapy, catalysis, desalination). To date, most thermoplasmonic applications are based on Ag and Au NPs, whose cost of raw materials inevitably limits the scalability for industrial applications requiring large amounts of photothermal NPs, as in the case of desalination plants. On the other hand, alternative nanomaterials proposed so far exhibit severe restrictions associated with the insufficient photothermal efficacy in the visible, the poor chemical stability, and the challenging scalability. Here, it is demonstrated the outstanding potential of NiSe and CoSe topological nodal-line semimetals for thermoplasmonics. The anisotropic dielectric properties of NiSe and CoSe activate additional plasmonic resonances. Specifically, NiSe and CoSe NPs support multiple localized surface plasmons in the optical range, resulting in a broadband matching with sunlight radiation spectrum. Finally, it is validated the proposed NiSe and CoSe-based thermoplasmonic platform by implementing solar-driven membrane distillation by adopting NiSe and CoSe nanofillers embedded in a polymeric membrane for seawater desalination. Remarkably, replacing Ag with NiSe and CoSe for solar membrane distillation increases the transmembrane flux by 330% and 690%, respectively. Correspondingly, costs of raw materials are also reduced by 24 and 11 times, respectively. The results pave the way for the advent of NiSe and CoSe for efficient and sustainable thermoplasmonics and related applications exploiting sunlight within the paradigm of the circular blue economy.

Membrane bioreactor for investigation of neurodegeneration.

To gain a better understanding of neurodegeneration mechanisms and for preclinical evaluation of new therapeutics more accurate models of neuronal tissue are required. Our strategy was based on the implementation of advanced engineered system, like membrane bioreactor, in which neurons were cultured in the extracapillary space of poly(l-lactic acid) (PLLA) microtube array (MTA) membranes within a dynamic device designed to recapitulate specific microenvironment of living neuronal tissue. The high membrane permeability and the optimized fluid dynamic conditions created by PLLA-MTA membrane bioreactor provide a 3D low-shear stress environment fully controlled at molecular level with enhanced diffusion of nutrients and waste removal that successfully develops neuronal-like tissue. This neuronal membrane bioreactor was employed as in vitro model of ß-amyloid -induced toxicity associated to Alzheimer's disease, to test for the first time the potential neuroprotective effect of the isoflavone glycitein. Glycitein protected neurons from the events induced by ß-amyloid aggregation, such as the production of ROS, the activation of apoptotic markers and ensuring the viability and maintenance of cellular metabolic activity. PLLA-MTA membrane bioreactor has great potential as investigational tool in preclinical research, contributing to expand the available in vitro devices for drug screening.

Kinetics of oxygen uptake by cells potentially used in a tissue engineered trachea.

Synthetic polymer scaffold seeded with autologous cells have a clinical translational potential. A rational design oriented to clinical applications must ensure an efficient mass transfer of nutrients as a function of specific metabolic rates, especially for precariously vascularized tissues grown in vitro or integrated in vivo. In this work, luminescence lifetime-based sensors were used to provide accurate, extensive and non-invasive measurements of the oxygen uptake rate for human mesenchymal stem cells (hMSCs), tracheal epithelial cells (hTEpiCs) and human chondrocytes (hCCs) within a range of 2-40% O2 partial pressure. Estimated Michaelis-Menten parameters were: V(max) = 0.099 pmol/cell⋅h and K(M) = 2.12 × 10(-7) mol/cm(3) for hMSCs, V(max) = 1.23 pmol/cell⋅h and K(M) = 2.14 × 10(-7) mol/cm(3) for hTEpiCs, V(max) = 0.515 pmol/cell⋅h and K(M) = 1.65 × 10(-7) mol/cm(3) for hCCs. Kinetics data served as an input to a preliminary computational simulation of cell culture on a poly-ethylene terephthalate (PET) tracheal scaffold obtaining an efficient mass transfer at cell density of 10(6) cell/cm(3). Oxygen concentration affected the glucose uptake and lactate production rates of cells that adapted their metabolism according to energy demand in hypoxic and normoxic conditions.

Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system.

Isolated hepatocytes in spheroid configuration exhibit a high degree of cell-cell contacts, which are important in the maintenance of viability and liver specific functions. In the absence of a vascular network, the cells in a large spheroid size experience mass transfer limitations of metabolites and oxygen in the core of aggregates. In this paper transport phenomena related to the diffusion and reaction of oxygen, glucose and lactate are mathematically described and experimentally verified for hepatocyte spheroids cultured in a rotating-wall polystyrene system (RWPS) not permeable for gases and in a rotating-wall membrane system (RWMS) with oxygen-permeable membrane. The concentration profiles of glucose, oxygen and lactate in the hepatocyte spheroids were estimated for different diameters of aggregates by solving the mass transfer equations for simultaneous diffusion and reaction, by finite element method. Simulation results evidenced that, for aggregates with size lower than 300 microm cultured in both RWPS and RWMS systems, the concentration profiles of glucose and lactate towards the core of spheroids (effective diffusion coefficients in the order of 10(-11)m(2)/s) are not significantly affected by the metabolic rate (c.a 10(-6)microg/mm(3)/s for glucose and about one order of magnitude less for lactate). On the contrary, the transport of oxygen (diffusion coefficient: 3.4 x 10(-10)m(2)/s, reaction rate: 1.5 x 10(-5)microg/mm(3)/s) is critically affected by the size of the multicellular spheroids and significant gradients in oxygen concentration may develop in spheroids. Aggregates with a size greater than 200 microm suffer severe oxygen limitation in the most part of its size attaining the lowest partial pressure in the centre. The improved viability predicted by the model culturing hepatocyte spheroids in the RWMS, characterized by a higher O(2) permeability with respect to RWPS, was experimentally confirmed. The results demonstrated that the mathematical model used in this study represents a useful support to experimental procedures in order to obtain hepatocyte spheroids with optimal size.

Fetuin-A gene expression, synthesis and release in primary human hepatocytes cultured in a galactosylated membrane bioreactor.

This paper reports on human hepatocytes cultured in a galactosylated membrane bioreactor in order to explore the modulation of the effects of a pro-inflammatory cytokine, Interleukin-6 (IL-6) on the liver cells at molecular level. In particular the role of IL-6 on gene expression and production of a glycoprotein, fetuin-A produced by hepatocytes, was investigated by culturing hepatocytes in the membrane bioreactor, both in the absence and presence of IL-6 (300 pg/ml). IL-6 modulated the fetuin-A gene expression, synthesis and release by primary human hepatocytes cultured in the bioreactor. A 75% IL-6-induced reduction of fetuin-A concentration in the medium was associated with a 60% increase of C-reactive protein in the same samples. Real-time-PCR demonstrated an 8-fold IL-6-induced reduction of fetuin-A gene expression. These results demonstrate that the hepatocyte galactosylated membrane bioreactor is a valuable tool to study IL-6 effects and gave evidence, for the first time, that IL-6 down-regulates the gene expression and synthesis of fetuin-A by primary human hepatocytes. The human hepatocyte bioreactor behaves like the in vivo liver, reproducing the same hepatic acute-phase response that occurs during the inflammatory process.