Smart Sensor Control and Monitoring of an Automated Cell Expansion Process.

Immune therapy for cancer patients is a new and promising area that in the future may complement traditional chemotherapy. The cell expansion phase is a critical part of the process chain to produce a large number of high-quality, genetically modified immune cells from an initial sample from the patient. Smart sensors augment the ability of the control and monitoring system of the process to react in real-time to key control parameter variations, adapt to different patient profiles, and optimize the process. The aim of the current work is to develop and calibrate smart sensors for their deployment in a real bioreactor platform, with adaptive control and monitoring for diverse patient/donor cell profiles. A set of contrasting smart sensors has been implemented and tested on automated cell expansion batch runs, which incorporate advanced data-driven machine learning and statistical techniques to detect variations and disturbances of the key system features. Furthermore, a 'consensus' approach is applied to the six smart sensor alerts as a confidence factor which helps the human operator identify significant events that require attention. Initial results show that the smart sensors can effectively model and track the data generated by the Aglaris FACER bioreactor, anticipate events within a 30 min time window, and mitigate perturbations in order to optimize the key performance indicators of cell quantity and quality. In quantitative terms for event detection, the consensus for sensors across batch runs demonstrated good stability: the AI-based smart sensors (Fuzzy and Weighted Aggregation) gave 88% and 86% consensus, respectively, whereas the statistically based (Stability Detector and Bollinger) gave 25% and 42% consensus, respectively, the average consensus for all six being 65%. The different results reflect the different theoretical approaches. Finally, the consensus of batch runs across sensors gave even higher stability, ranging from 57% to 98% with an average consensus of 80%.

Evaluation of Neurosecretome from Mesenchymal Stem Cells Encapsulated in Silk Fibroin Hydrogels.

Physical and cognitive disabilities are hallmarks of a variety of neurological diseases. Stem cell-based therapies are promising solutions to neuroprotect and repair the injured brain and overcome the limited capacity of the central nervous system to recover from damage. It is widely accepted that most benefits of different exogenously transplanted stem cells rely on the secretion of different factors and biomolecules that modulate inflammation, cell death and repair processes in the damaged host tissue. However, few cells survive in cerebral tissue after transplantation, diminishing the therapeutic efficacy. As general rule, cell encapsulation in natural and artificial polymers increases the in vivo engraftment of the transplanted cells. However, we have ignored the consequences of such encapsulation on the secretory activity of these cells. In this study, we investigated the biological compatibility between silk fibroin hydrogels and stem cells of mesenchymal origin, a cell population that has gained increasing attention and popularity in regenerative medicine. Although the survival of mesenchymal stem cells was not affected inside hydrogels, this biomaterial format caused adhesion and proliferation deficits and impaired secretion of several angiogenic, chemoattractant and neurogenic factors while concurrently potentiating the anti-inflammatory capacity of this cell population through a massive release of TGF-Beta-1. Our results set a milestone for the exploration of engineering polymers to modulate the secretory activity of stem cell-based therapies for neurological disorders.

Safety and tolerability of silk fibroin hydrogels implanted into the mouse brain.

At present, effective therapies to repair the central nervous system do not exist. Biomaterials might represent a new frontier for the development of neurorestorative therapies after brain injury and degeneration. In this study, an in situ gelling silk fibroin hydrogel was developed via the sonication-induced gelation of regenerated silk fibroin solutions. An adequate timeframe for the integration of the biomaterial into the brain tissue was obtained by controlling the intensity and time of sonication. After the intrastriatal injection of silk fibroin the inflammation and cell death in the implantation area were transient. We did not detect considerable cognitive or sensorimotor deficits, either as examined by different behavioral tests or an electrophysiological analysis. The sleep and wakefulness states studied by chronic electroencephalogram recordings and the fitness of thalamocortical projections and the somatosensory cortex explored by evoked potentials were in the range of normality. The methodology used in this study might serve to assess the biological safety of other biomaterials implanted into the rodent brain. Our study highlights the biocompatibility of native silk with brain tissue and extends the current dogma of the innocuousness of this biomaterial for therapeutic applications, which has repercussion in regenerative neuroscience. STATEMENT OF SIGNIFICANCE: The increasingly use of sophisticated biomaterials to encapsulate stem cells has changed the comprehensive overview of potential strategies for repairing the nervous system. Silk fibroin (SF) meets with most of the standards of a biomaterial suitable to enhance stem cell survival and function. However, a proof-of-principle of the in vivo safety and tolerability of SF implanted into the brain tissue is needed. In this study we have examined the tissue bioresponse and brain function after implantation of SF hydrogels. We have demonstrated the benign coexistence of silk with the complex neuronal circuitry that governs sensorimotor coordination and mechanisms such as learning and memory. Our results have repercussion in the development of advances strategies using this biomaterial in regenerative neuroscience.

Chondrogenic potential of human dermal fibroblasts in a contractile, soft, self-assembling, peptide hydrogel.

The present paper describes a simple approach to obtain three-dimensional (3D) cartilage constructs using human normal dermal fibroblasts (hNDFs) cultured in a self-assembling peptide nanofibre scaffold. During the first days of culture, the 3D constructs underwent morphological changes consisting of a substantial contraction process that ended in a small compact structure. During this process the system became sensitive to induction with standard chondrogenic medium, evidenced by the expression of specific markers of mature cartilage. First, it was detected that the samples become highly stained with toluidine blue dye over time (40-50 days), indicating that the system produced significantly high amounts of glycosaminoglycans. By quantitative PCR, it was confirmed that the system significantly upregulated the expression of the proteoglycan aggrecan, a good indicator of cartilage commitment. Moreover, collagen type II was upregulated at protein level, confirming that the system differentiated to a chondrocyte-like construct. Additionally, during the first days of culture in control medium analysed hNDFs proliferation capacity in this 3D system was analysed. This platform could be used in the future to obtain an autologous source of cells from a simple patient skin biopsy, which could be easily translated into a low-cost and effective regenerative therapy.

Mechanical behaviour and formation process of silkworm silk gut.

High performance silk fibers were produced directly from the silk glands of silkworms (Bombyx mori) following an alternative route to natural spinning. This route is based on a traditional procedure that consists of soaking the silk glands in a vinegar solution and stretching them by hand leading to the so called silkworm guts. Here we present, to the authors' best knowledge, the first comprehensive study on the formation, properties and microstructure of silkworm gut fibers. Comparison of the tensile properties and microstructural organization of the silkworm guts with those of naturally spun fibers allows gain of a deeper insight into the mechanisms that lead to the formation of the fiber, as well as the relationship between the microstructure and properties of these materials. In this regard, it is proved that an acidic environment and subsequent application of tensile stress in the range of 1000 kPa are sufficient conditions for the formation of a silk fiber.

Unexpected behavior of irradiated spider silk links conformational freedom to mechanical performance.

Silk fibers from Argiope trifasciata and Nephila inaurata orb-web weaving spiders were UV irradiated to modify the molecular weight of the constituent proteins. Fibers were characterized either as forcibly silked or after being subjected to maximum supercontraction. The effect of irradiation on supercontraction was also studied, both in terms of the percentage of supercontraction and the tensile properties exhibited by irradiated and subsequently supercontracted fibers. The effects of UV exposure at the molecular level were assessed by polyacrylamide gel electrophoresis and mass spectrometry. It is shown that UV-irradiated fibers show a steady decrease in their main tensile parameters, most notably, tensile strength and strain. The combination of the mechanical and biochemical data suggests that the restricted conformational freedom of the proteins after UV irradiation is critical in the reduction of these properties. Consequently, an adequate topological organization of the protein chains emerges as a critical design principle in the performance of spider silk.

Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes.

The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz's model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens.

Spider silk gut: development and characterization of a novel strong spider silk fiber.

Spider silk fibers were produced through an alternative processing route that differs widely from natural spinning. The process follows a procedure traditionally used to obtain fibers directly from the glands of silkworms and requires exposure to an acid environment and subsequent stretching. The microstructure and mechanical behavior of the so-called spider silk gut fibers can be tailored to concur with those observed in naturally spun spider silk, except for effects related with the much larger cross-sectional area of the former. In particular spider silk gut has a proper ground state to which the material can revert independently from its previous loading history by supercontraction. A larger cross-sectional area implies that spider silk gut outperforms the natural material in terms of the loads that the fiber can sustain. This property suggests that it could substitute conventional spider silk fibers in some intended uses, such as sutures and scaffolds in tissue engineering.

Development of a three-dimensional bone-like construct in a soft self-assembling peptide matrix.

This work describes the development of a three-dimensional (3D) model of osteogenesis using mouse preosteoblastic MC3T3-E1 cells and a soft synthetic matrix made out of self-assembling peptide nanofibers. By adjusting the matrix stiffness to very low values (around 120 Pa), cells were found to migrate within the matrix, interact forming a cell-cell network, and create a contracted and stiffer structure. Interestingly, during this process, cells spontaneously upregulate the expression of bone-related proteins such as collagen type I, bone sialoprotein, and osteocalcin, indicating that the 3D environment enhances their osteogenic potential. However, unlike MC3T3-E1 cultures in 2D, the addition of dexamethasone is required to acquire a final mature phenotype characterized by features such as matrix mineralization. Moreover, a slight increase in the hydrogel stiffness (threefold) or the addition of a cell contractility inhibitor (Rho kinase inhibitor) abrogates cell elongation, migration, and 3D culture contraction. However, this mechanical inhibition does not seem to noticeably affect the osteogenic process, at least at early culture times. This 3D bone model intends to emphasize cell-cell interactions, which have a critical role during tissue formation, by using a compliant unrestricted synthetic matrix.

Differentiation of mouse embryonic stem cells in self-assembling peptide scaffolds.

Here, we describe the capacity of mouse embryonic stem cells (mESCs) to differentiate into osteoblast-like cells in a three-dimensional (3D) self-assembling peptide scaffold, a synthetic nanofiber biomaterial with future applications in regenerative medicine. We have previously demonstrated that classical tissue cultures (two-dimensional) as well as 3D-systems promoted differentiation of mESCs into cells with an osteoblast-like phenotype expressing osteopontin (OPN) and collagen type I (Col I), as well as high alkaline phosphatase (Alk Phos) activity and calcium phosphate mineralization. Interestingly, in 3D self-assembling peptide scaffold cultures, the frequency of appearance of embryonic stem-cell-like colonies was substantially enhanced, suggesting that this particular 3D microenvironment promoted the generation of a stem-cell-like niche that allows the maintenance of a small pool of undifferentiated cells. We propose that the 3D system provides a unique microenvironment permissive to promote differentiation of mESCs into osteoblast-like cells while maintaining its regenerative capacity.

Nanometric self-assembling peptide layers maintain adult hepatocyte phenotype in sandwich cultures.

BACKGROUND: Isolated hepatocytes removed from their microenvironment soon lose their hepatospecific functions when cultured. Normally hepatocytes are commonly maintained under limited culture medium supply as well as scaffold thickness. Thus, the cells are forced into metabolic stress that degenerate liver specific functions. This study aims to improve hepatospecific activity by creating a platform based on classical collagen sandwich cultures. RESULTS: The modified sandwich cultures replace collagen with self-assembling peptide, RAD16-I, combined with functional peptide motifs such as the integrin-binding sequence RGD and the laminin receptor binding sequence YIG to create a cell-instructive scaffold. In this work, we show that a plasma-deposited coating can be used to obtain a peptide layer thickness in the nanometric range, which in combination with the incorporation of functional peptide motifs have a positive effect on the expression of adult hepatocyte markers including albumin, CYP3A2 and HNF4-alpha. CONCLUSIONS: This study demonstrates the capacity of sandwich cultures with modified instructive self-assembling peptides to promote cell-matrix interaction and the importance of thinner scaffold layers to overcome mass transfer problems. We believe that this bioengineered platform improves the existing hepatocyte culture methods to be used for predictive toxicology and eventually for hepatic assist technologies and future artificial organs.

FUNCTIONALIZED, SWELLABLE HYDROGEL LAYERS AS A PLATFORM FOR CELL STUDIES.

This paper reports the design, synthesis and characterization of thin films as a platform for studying the separate influences of physical and chemical cues of a matrix on the adhesion, growth and final phenotype of cells. Independent control of the physical and chemical properties of functionalized, swellable hydrogel thin films was achieved using initiated Chemical Vapor Deposition (iCVD). The systematic variation in crosslink density is demonstrated to control the swelling ability of the iCVD hydrogel films based on 2-hydroxyethyl methacrylate (HEMA). At the same time, the incorporation of controllable concentrations of the active ester pentafluorophenyl methacrylate (PFM) allows easy immobilization of aminated bioactive motifs, such as bioactive peptides. Initial cell culture results with Human Umbilical Vein Endothelial Cells (HUVEC) indicated that the strategy of using PFM to immobilize a cell-adhesion peptide motif onto the hydrogel layers promotes proper HUVEC growth and enhances their phenotype.