Development of tissue culture system with automated pulsation and Kalman filter control for an artificial artery model.

Tissue-engineered arterial vessels have been used as substitutes for unnecessary animal experiments to evaluate the pharmacokinetics of drugs targeting various arteriopathies caused by structural or physiological arterial defects. An arterial tissue culture system was established to simulate the mechanical characteristics of a heart-beating pump and to do online feedback control of lactate and glucose concentrations. The mechanically controlled flow pump mimicked the heart pumping inside a tissue-engineered artery composed of muscle and endothelial cells within a nanofibrous scaffold. After monitoring the pH of the culture medium online, lactate and glucose were estimated using the Kalman filter algorithm, and the set-point online control was operated to maintain glucose for artery tissue engineering. The composition of the artificial artery was confirmed by immunofluorescence staining, and its mechanical characteristics were examined. The online automated system successfully demonstrated its applicability as a standardized process for arterial tissue culture to replace animal arterial experiments.

In vitro Models of the Small Intestine for Studying Intestinal Diseases.

The small intestine is a digestive organ that has a complex and dynamic ecosystem, which is vulnerable to the risk of pathogen infections and disorders or imbalances. Many studies have focused attention on intestinal mechanisms, such as host-microbiome interactions and pathways, which are associated with its healthy and diseased conditions. This review highlights the intestine models currently used for simulating such normal and diseased states. We introduce the typical models used to simulate the intestine along with its cell composition, structure, cellular functions, and external environment and review the current state of the art for in vitro cell-based models of the small intestine system to replace animal models, including ex vivo, 2D culture, organoid, lab-on-a-chip, and 3D culture models. These models are described in terms of their structure, composition, and co-culture availability with microbiomes. Furthermore, we discuss the potential application for the aforementioned techniques to these in vitro models. The review concludes with a summary of intestine models from the viewpoint of current techniques as well as their main features, highlighting potential future developments and applications.

A systematic correlation analysis of carotenoids, chlorophyll, non-pigmented cell mass, and cell number for the blueprint of Dunaliella salina culture in a photobioreactor.

Microalgal carotenoids are attractive health ingredients, but their production should be optimized to improve cost-effectiveness. Understanding cellular physiology centered on carotenoid synthesis is the prerequisite for this work. Therefore, systematic correlation analyses were conducted among chlorophyll, carotenoids, non-pigmented cell mass, and cell number of Dunaliella salina in a specified condition over a relatively long culture time. First, an integrated correlation was performed: a temporal profile of the carotenoids was correlated with those of other factors, including chlorophyll, non-pigmented cell mass, and cell number. Pearson and Spearman correlation analyses were performed to identify linearity and monotonicity of the correlation, respectively, and then cross-correlation was executed to determine if the correlation had a time lag. Second, to understand the cellular potential of metabolism, the procedure was repeated to provide a data set composed of the specific synthesis rates of the factors or growth rate, which additionally provided kinetic correlations among the constituting components of the cell, excluding the effect of cell number. This systematic approach could generate a blueprint model that is composed of only what it needs, which could make it possible to efficiently control and optimize the process.

Author Correction: Artificial controlled model of blood circulation system for adhesive evaluation.

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Artificial controlled model of blood circulation system for adhesive evaluation.

Since there are several casualties due to uncontrolled bleeding resulting from simple injury to surgery, effective styptic or vessel adhesives are important; however, their development is limited by the lack of standardized systems to evaluate potential compounds. The current study outlines the development of an aorta styptic evaluation system, comprising of decellularized swine aorta tissue and a heart pump-mimicking system. Although the cells in the swine aorta were removed, the structural stability of the aorta was sustained due to the maintenance of the extracellular matrix. Using a control adhesive, Cyanoacrylate, the developed model was found to have similar adhesive efficacy to intact aorta. The circulatory-mimicking system was designed to mimic the beat rate and strength of blood-flow from the heart, which was necessary to evaluate the adherent efficacy. The decellularized aorta improves instabilities of intact tissues, which occurs on account of storage and origin, thereby allowing for a more standardized system. The system was able to simulate several symptoms of circulation, according to patient age and health, by adjusting pumping frequency and intensity. Therefore, this system can be used as a standardized evaluation system for screening adhesives. Further, it would also evaluate other medical devices, such as stent or medications.

Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device.

Artificial skin or skin equivalents have been used for clinical purpose to skin graft and as substitutes for animal experiments. The culture of cell lines such as HaCaT has the potential to produce large amounts of artificial skin at a low cost. However, there is a limit to keratinization due to the restriction of differentiation in HaCaT. In this study, a culture device that mimics the in vivo keratinization mechanism, co-stimulated by air-exposure and mechanical stimulation, was developed to construct skin equivalents. The device can reconstruct the epidermal morphology, including the cornified layer, similar to its formation in vivo. Under the condition, epidermis was differentiated in the spinous and granular layers. Formation of the stratum corneum is consistent with the mRNA and protein expressions of differentiation markers. The device is the first of its kind to combine air-exposure with mechanical stress to co-stimulate keratinization, which can facilitate the economically viable production of HaCaT-based artificial skin substitutes.

Thermodynamic Insights and Conceptual Design of Skin-Sensitive Chitosan Coated Ceramide/PLGA Nanodrug for Regeneration of Stratum Corneum on Atopic Dermatitis.

Atopic dermatitis (AD) is a complex skin disease primarily characterized by psoriasis of the stratum corneum. AD drugs have usually been used in acidic and hydrophilic solvents to supply moisture and prevent lipid defects. Ceramide is a typical treatment agent to regenerate the stratum corneum and relieve symptoms of AD. However, ceramide has limitation on direct use for skin because of its low dispersion properties in hydrophilic phase and side effects at excessive treatment. In this study, ceramide imbedded PLGA nanoparticles were developed with chitosan coating (Chi-PLGA/Cer) to overcome this problem. The chitosan coating enhanced initial adherence to the skin and prevented the initial burst of ceramide, but was degraded by the weakly acidic nature of skin, resulting in controlled release of ceramide with additional driving force of the squeezed PLGA nanoparticles. Additionally, the coating kinetics of chitosan were controlled by manipulating the reaction conditions and then mathematically modeled. The Chi-PLGA/Cer was not found to be cytotoxic and ceramide release was controlled by pH, temperature, and chitosan coating. Finally, Chi-PLGA/Cer was demonstrated to be effective at stratum corneum regeneration in a rat AD model. Overall, the results presented herein indicated that Chi-PLGA/Cer is a novel nanodrug for treatment of AD.

Regulation of astrocyte activity via control over stiffness of cellulose acetate electrospun nanofiber.

Astrocytes are involved in neuron protection following central nervous system (CNS) injury; accordingly, engineered astrocytes have been investigated for their usefulness in cell therapy for CNS injury. Nanofibers have attracted a great deal of attention in neural tissue engineering, but their mechanical properties greatly influence physiology. Cellulose acetate (CA) has been studied for use in scaffolds owing to its biocompatibility, biodegradability, and good thermal stability. In this study, stiffness of CA nanofibers controlled by heat treatment was shown to regulate astrocyte activity. Adhesion and viability increased in culture as substrate became stiffer but showed saturation at greater than 2 MPa of tensile strength. Astrocytes became more active in terms of increasing intermediate filament glial fibrillary acidic protein (GFAP). The results of this study demonstrate the effects of stiffness alone on cellular behaviors in a three-dimensional culture and highlight the efficacy of heat-treated CA for astrocyte culture in that the simple treatment enables control of astrocyte activity.

Chitosan nanoparticle/PCL nanofiber composite for wound dressing and drug delivery.

Many investigations of wound dressings equipped with drug delivery systems have recently been conducted. Chitosan is widely used not only as a material for wound dressing by the efficacy of its own, but also as a nanoparticle for drug delivery. In this study, an electrospun polycaprolactone nanofiber composite with chitosan nanoparticles (ChiNP-PCLNF) was fabricated and then evaluated for its drug release and biocompatibility to skin fibroblasts. ChiNP-PCLNF complexes showed no cytotoxicity and nanoparticles adsorbed by van der Waals force were released into aquatic environments and then penetrated into rat primary fibroblasts. Our studies demonstrate the potential for application of ChiNP-PCLNF as a wound dressing system with drug delivery for skin wound healing without side effects.

Development of a complex bone tissue culture system based on cellulose nanowhisker mechanical strain.

In bone tissue engineering, scaffolds have been investigated for their ability to support osteoblast growth and differentiation for recovery of damaged bones. Tunicate cellulose nanowhisker (CNW) film and mechanical strain were assessed for their suitability for osteoblasts. In this study, sulfuric acid hydrolysis extraction of tunicates integuments was conducted to obtain CNWs, which were found to be acceptable for adhering, growing, and differentiating osteoblasts without cytotoxicity. Mechanical stress enhanced osteoblast differentiation, and cell survival rate was recovered at around day 3, although there was a slight increase in cell death at day 1 after stimulation. We also found that intracellular flux of calcium ion was related to increased differentiation of CNWs under mechanical stress. Overall, we demonstrated the suitability of tunicate CNWs as a scaffold for bone tissue engineering and developed a complex system based on CNW for osteoblast growth and differentiation that will be useful for bone substitute fabrication.

Implications of the oxygenated electrospun poly(ɛ-caprolactone) nanofiber for the astrocytes activities.

Astrocytes support structure of central nervous system (CNS) and provide nutrients to neurons. When CNS is injured, astrocytes are activated and produce glia scar. There are debates if the reactive astrocytes give beneficial or harmful effects on neuronal regeneration. In vitro tissue culture systems successfully have been used to investigate how the astrocytes activity is regulated in response to environmental conditions. Physicochemical characteristics of supporting materials for tissue culture are one of the most important environmental conditions. Electrospun nanofiber has physical uniqueness such as high surface area to volume ratio and high porosity, which is favorable to tissue culture. However, cellular activities can also be regulated in response to surface chemistry, which can be modified easily and diversely. Poly(ε-caprolactone) (PCL) is widely used for a scaffold for tissue culture. In this research, oxygen plasma-treated PCL nanofiber was assessed to ascertain whether it can have such potentials to regulate astrocytes activity. As a result, oxygen plasma treatment increased the hydrophilicity of the PCL nanofiber which made adhesion and viability of astrocytes enhanced without cytotoxicity Activation of astrocytes in the plasma treated scaffolds was confirmed by the fact of upregulation of glial fibrillary acidic protein. Above all, oxygenated nanofiber provides an initial culture environment which makes astrocytes activated.

Astrocyte behavior and GFAP expression on Spirulina extract-incorporated PCL nanofiber.

Nanomaterials are attractive for use in biological systems due to their ability to control the microenvironment of cells. Additionally, nanofibers can mimic fibrous characteristics of natural tissues. This study was conducted to assess astrocyte activity and infiltration behavior on Spirulina extract-embedded polycaprolactone (SP-PCL) nanofiber. Astrocytes moved along with the nanofiber, and developed an elongated and stellate shape, which is similar to those in the natural neural tissue. In addition, the expression of GFAP, a biomarker representing the activation of astrocytes, was gradually up-regulated with the increase of the concentration of Spirulina extract, indicating that Spirulina extract can control astrocyte activation. Overall, the results presented herein indicate that SP-PCL nanofiber could be used in astrocyte tissue engineering for neuronal regeneration.

Effect of topography of an electrospun nanofiber on modulation of activity of primary rat astrocytes.

Several biomaterials for neural tissue engineering have recently been proposed for regeneration of damaged tissue and promotion of axonal guidance following CNS injury. When implanted into damaged nerve tissue, biomaterials should favorably induce cell infiltration and axonal guiding while suppressing inflammation. Nanofiber scaffolds are regarded as adequate materials to meet the above requirements; however, most studies of these materials conducted to date have targeted neuronal cells, not glial cells, despite their important function in the injured CNS. In this study, an electrospun nanofibrous scaffold of polycaprolactone (PCL) was investigated with respect to its topographic effects on astrocyte behavior and expression of GFAP. The results revealed that the PCL nanofiber topograghy promoted adhesion, but GFAP expression was down-regulated, leading to reduced astrocytes activity. Taken together, these results indicate that the topographic structure of electrospun nanofibers provides a scaffold that is favorable to neural regeneration via alleviation of astrogliosis.

Assessment of Spirulina-PCL nanofiber for the regeneration of dermal fibroblast layers.

Skin is a barrier which protects injured tissues, and thus, skin regeneration is one of many important medical issues. Tissue engineering is an attractive approach to make artificial tissue or regenerate lost tissues. While constituting artificial tissues, cells must infiltrate through scaffolds, maintaining viability and proliferation. However, a three-dimensional tissue culture involves stressful environments due to several reasons such as mass or gas transport and high cell density. Once stressed, cells produce reactive oxygen species, resulting in alleviating cellular viability and activity. Spirulina is well known to have antioxidant molecules, which have been known to modulate oxidative stress to cells. Electrospun nanofiber has widely been used as a scaffold to mimic natural extracellular matrix. In this research, we assessed Spirulina extract-imbedded nanofiber as a scaffold for an artificial skin tissue. Spirulina extract was proven to positively affect viability and proliferation of mouse fibroblasts. In addition, fibroblasts infiltrated through Spirulina extract-imbedded electrospun nanofiber without cytotoxicity.

Controlled activity of mouse astrocytes on electrospun PCL nanofiber containing polysaccharides from brown seaweed.

The central nervous system (CNS), once injured, rarely recovers original function mainly due to its limited regeneration ability. Astrocytes are cells that play critical roles in neural regeneration. Several biomaterials have been studied to replace and regenerate lost tissues within injured CNS. Seaweeds have extracellular polymeric substances (EPS) with bioactive properties such as antiviral and antioxidant properties. In this study, astrocyte activity was assessed, after being cultured on an electrospun polycaprolactone (PCL) nanofibrous mat containing a brown seaweed EPS. Laminarin and fucoidan, two main components of EPS extract from the brown seaweed, were concluded to increase or decrease astrocyte activity with respect to their concentration. When the concentration was under 10 µg/ml, the astrocytes tended to increase their viability. In contrast, over 10 µg/ml EPS in media suppressed the viability of astrocytes. In addition, when contained in PCL nanofiber, the EPS extract was also proven to influence astrocyte activity in the same way as the case when astrocytes were exposed to EPS in solution. This implies that the brown seaweed EPS-PCL nanofiber mat can be used for temporal control of astrocyte activity by EPS concentration. Through this research, we propose that the electrospun EPS-PCL nanofiber could be used as a nanomedicine or scaffold to treat CNS injuries.

In vitro evaluation of the effects of electrospun PCL nanofiber mats containing the microalgae Spirulina (Arthrospira) extract on primary astrocytes.

The blue-green microalgae, Spirulina, a harmless food and pharmaceutical additive, has several bioactive compounds that have therapeutic functions. Polycaprolactone (PCL) is a biocompatible and biodegradable polymer that has widely been used for tissue engineering. The electrospun PCL nanofiber containing Spirulina (PCL-Spirulina) was fabricated and tested as a potential extracellular matrix material for a culture of primary astrocytes, which play important roles in CNS injured systems. Spirulina extract was observed to increase growth and metabolic activity of rat primary astrocytes without any harm once added to the culture media. However, PCL-Spirulina nanofiber was proven to alleviate astrocyte activity. Through this research and to the best of our knowledge, we first suggest a novel composite nanomaterial, an electrospun PCL-Spirulina nanofiber that could be used to treat CNS injured systems.