Biomimetic brain tumor niche regulates glioblastoma cells towards a cancer stem cell phenotype.

Glioblastoma (GBM) is the most malignant primary brain tumor and contains tumorigenic cancer stem cells (CSCs), which support the progression of tumor growth. The selection of CSCs and facilitation of the brain tumor niches may assist the development of novel therapeutics for GBM. Herein, hydrogel materials composed of agarose and hydroxypropyl methyl cellulose (HMC) in different concentrations were established and compared to emulate brain tumor niches and CSC microenvironments within a label-free system. Human GBM cell line, U-87 MG, was cultured on a series of HMC-agarose based culture system. Cell aggregation and spheroids formation were investigated after 4 days of culture, and 2.5% HMC-agarose based culture system demonstrated the largest spheroids number and size. Moreover, CD133 marker expression of GBM cells after 6 days of culture in 2.5% HMC-agarose based culture system was 60%, relatively higher than the control group at only 15%. Additionally, cells on 2.5% HMC-agarose based culture system show the highest chemoresistance, even at the high dose of 500 µM temozolomide for 72 h, the live cell ratio was still > 80%. Furthermore, the results also indicate that the expression of ABCG2 gene was up-regulated after culture in 2.5% HMC-agarose based culture system. Therefore, our results demonstrated that biomimetic brain tumor microenvironment may regulate GBM cells towards the CSC phenotype and expression of CSC characteristics. The microenvironment selection and spheroids formation in HMC-agarose based culture system may provide a label-free CSC selection strategy and drug testing model for future biomedical applications.

Rapid fabrication method of a microneedle mold with controllable needle height and width.

The main issue of transdermal drug delivery is that macromolecular drugs cannot diffuse through the stratum corneum of skin. Many studies have pursued micro-sized needles encapsulated with drugs to overcome this problem, as these needles can pierce the stratum corneum and allow drugs to enter the circulatory system of the human body. However, most microneedle fabrication processes are time-consuming and require expensive equipment. In this study, we demonstrate a rapid method for fabricating a microneedle mold using drawing lithography and a UV-cured resin. The mold was filled with a water-soluble material, polyvinylpyrrolidone (PVP), which was then demolded to produce a water-soluble microneedle array. The results of an in vitro skin insertion test using PVP microneedles and pig ear skin demonstrated the feasibility of the microneedle mold. In addition, by controlling the viscosity of the UV-cured resin through various heat treatments, microneedles with different heights and aspect ratios were produced. Compared with other methods, this technology significantly simplifies and accelerates the mold fabrication process. In addition, the required equipment is relatively simple and inexpensive. Through this technology, we can rapidly fabricate microneedle molds with controllable dimensions for various applications.

Successful differentiation of neural stem/progenitor cells cultured on electrically adjustable indium tin oxide (ITO) surface.

In order to control differentiation of neural cells and guide the developed neurites to targets, polyelectrolyte multilayer (PEM) films were used because of their capability of modulation of electrical charged characteristics, thickness, and stiffness. In this work, we suggested that indium tin oxide (ITO) is an alternative surface to achieve the above-mentioned objectives. A microfluidic system with four culture chambers was developed and each chamber consisted of parallel ITO surfaces for the application of adjustable electrical field. Neural stem/progenitor cells (NSPCs) were respectively cultured on the ITO surfaces with and without PEM film, constructed by alternate adsorption of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PLGA). Analyses of cell morphology, cytotoxicity, process outgrowth, differentiated cell types, and neuron functionality were compared between both surfaces. In this study, NSPCs successfully differentiated on ITO surface with electrical stimulation. The optimal electrical potential was found to be 80 mV that could stimulate the longest process, i.e., >300 µm, after 3 days culture. Cell differentiation, process development, and functionality of differentiated neuron on ITO surface were shown to be strongly controlled by the electrical stimulation that can be simply adjusted by external equipment. The electrically adjustable cell differentiation reported here could potentially be applied to neurochip for the study of neural signal transmission in a well-constructed network.

Dual crosslinking silk fibroin/pectin-based bioink development and the application on neural stem/progenitor cells spheroid laden 3D bioprinting.

The human nervous system is an incredibly intricate physiological network, and neural cells lack the ability to repair and regenerate after a brain injury. 3-dimensional (3D) bioprinting technology offers a promising strategy for constructing biomimetic organ constructs and in vitro brain/disease models. The bioink serves as a pivotal component that emulates the microenvironment of biomimetic construct and exerts a profound influence on cellular behaviors. In this study, a series of mechanically adjustable and dual crosslinking bioinks were developed using photocrosslinkable methacrylated silk fibroin (SilMA) in combination with the ionic crosslinking material, pectin, or pectin methacryloyl (PecMA) with silk fibroin (SF) supplementation. SilMA/pectin exhibited superior properties, with SilMA providing biocompatibility and adjustable mechanical properties, while the addition of pectin enhanced printability. The porous structure supported neural cell growth, and 15 % SilMA/0.5 % pectin bioinks displayed excellent printability and shape fidelity. Neural stem/progenitor cells (NSPCs)-loaded bioinks were used to construct a 3D brain model, demonstrating sustained vitality and high neuronal differentiation without the need for growth factors. The SilMA/pectin bioinks demonstrated adjustable mechanical properties, favorable biocompatibility, and an environment highly conducive to neural induction, offering an alternative approach for neural tissue engineering applications or in vitro brain models.

Selection, enrichment, and maintenance of self-renewal liver stem/progenitor cells utilizing polypeptide polyelectrolyte multilayer films.

Recent progress has led to the identification of liver stem/progenitor cells as suitable sources for generating transplantable liver cells. However, the great variability in methods utilized to isolate liver stem/progenitor cells is a considerable challenge for clinical applications. The polyelectrolyte-multilayer technique can constitute a useful method for selective cell adhesion. Whether enrichment of liver stem/progenitor cells can be achieved utilizing polypeptide polyelectrolyte-multilayer films was investigated in current work. Fetal liver cells isolated from E13.5 mouse embryos were seeded on the poly-l-glutamic acid/poly-l-lysine alternating films, and we revealed that fetal liver stem/progenitor cells were selected and formed colonies. These undifferentiated colonies were maintained on the films composed of four alternating layers, with the topmost poly-l-glutamic acid layer judged by the constitutive expression of stem-cell markers such as Dlk-1, CD49f, and CD133 and self-renew marker-beta-catenin. Our work has demonstrated that highly tunable polyelectrolyte-multilayer films were suitable for selective enrichment of liver stem/progenitor cells in vitro.

Designing a three-dimensional expanded polytetrafluoroethylene-poly(lactic-co-glycolic acid) scaffold for tissue engineering.

The purpose of this study was to design a three-dimensional expanded polytetrafluoroethylene (ePTFE)-poly(lactic-co-glycolic acid) (PLGA) scaffold for tissue engineering. To test the feasibility of this composite scaffold, a series of two-dimensional culture experiments were performed to investigate the behavior of anterior cruciate ligament (ACL) cells on the ePTFE and PLGA membranes. It was found PLGA provided a cell-favorable substrate for cell adhesion, migration, and growth, indicating PLGA is an ACL cell-conductive material. Conversely, poor adhesion and proliferation of ACL cells were observed on the ePTFE, even on the collagen-coated ePTFE. Therefore, the scaffold was not fabricated by coating PLGA on the ePTFE surface because it is difficult to coat anything on the extremely hydrophobic ePTFE surface. Instead, the ePTFE embedded in the PLGA matrix was prepared by immersing ePTFE scrim yarns into the PLGA solution, and then precipitating PLGA to form a three-dimensional construction with porous morphology. The role of ePTFE is regarded as a reinforcing constituent to improve the mechanical strength of porous PLGA matrix to provide early repair strength for tissue healing. However, porous PLGA matrix acts as a supportive environment for allowing cell adhesion, migration, and growth to guide the repair and regeneration of ligament tissue. To test this assumption, a preliminary animal experiment of rabbit ACL wound healing with this three-dimensional ePTFE-PLGA scaffold was performed. These results are very encouraging because such a new scaffold made of ePTFE scrim yarns embedded in PLGA may serve as ACL prostheses in the ligament tissue engineering.

Role of phase diagram of membrane formation system in controlling the crystallinity and degradation rate of PLLA membranes.

In this work, the theoretical phase diagram of membrane formation system of ethanol, methylene chloride, and poly-L-lactide (PLLA) was studied. On the basis of the phase diagram, particulate and porous membranes, dominated by crystallization and liquid-liquid demixing, respectively, were prepared. Furthermore, degradation of PLLA membranes with particulate, porous, and dense morphologies was performed in phosphate buffered solution (PBS) at 37 degrees C for 168 days and was investigated by mass loss, scanning electron microscopy (SEM), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). Besides the membrane morphology, a close relationship between the phase behavior of the membrane formation system and the membrane crystallinity was found, which in turn influenced the degradation rate of these membranes significantly. In the case of dense membranes, it showed the lowest initial crystallinity and the greatest rate of mass loss and molecular weight decrease compared with particulate and porous membranes. In contrast, the particulate membranes had the highest crystallinity and the slowest degradation rate in this study. Therefore, the phase diagram of membrane formation system could not only anticipate membrane morphology, but could also control the membrane crystallinity and degradation rate simultaneously.

Label-free selection and enrichment of liver cancer stem cells by surface niches build up with polyelectrolyte multilayer films.

Recent studies indicate that a small population of cancer cells exhibits stem cell properties and are referred to as cancer-initiating or cancer stem cells (CSCs). The selection and identification of cancer stem cells through methods require well-defined biomarkers and immunolabeling procedures are complicated and often unreliable. Herein, we fabricated a series of microenviroment by using polyelectrolyte multilayers (PEM) nanofilms to program and mimic hepatocellular carcinoma CSCs niches for CSCs selection with a label-free method. When cultured on PEM substrates, human cancer cell lines-Huh7 cells grew into individual round colonies and these cells displayed high marker expression of CSCs. Especially, these selected cells demonstrated significant chemo-resistant property in comparison with normal population. Therefore, we believed that niches selection and colony formation method may provide a new strategy on CSCs selection and drug evaluation for cancer therapy.

Biomimetic niche for neural stem cell differentiation using poly-L-lysine/hyaluronic acid multilayer films.

Polyelectrolyte multilayer films have been suggested as tunable substrates with flexible surface properties that can modulate cell behavior. However, these films' biological effects on neural stem/progenitor cells have rarely been studied. Herein, biomimetic multilayer films composed of hyaluronic acid and poly-L-lysine were chosen to mimic the native extracellular matrix niche of brain tissue and were evaluated for their inductive effects, without the addition of chemical factors. Because neural stem/progenitor cells are sensitive to substrate properties, it is important that this system provides control over the surface charge, and slight stiffness variations are also possible. Both of these factors affect neural stem/progenitor cell differentiation. The results showed that neural stem/progenitor cells were induced to differentiate on the poly-L-lysine/hyaluronic acid multilayer films with 0.5-4 alternating layers. In addition, the neurite outgrowth length was regulated by the surface charge of the terminal layer but did not increase with the layer number. In contrast, the quantity of differentiated neurons was enhanced slightly as the number of layers increased but was not affected by the surface charge of the terminal layer. In sum, material pairs in the form of native poly-L-lysine/hyaluronic acid films achieved important targets for neural regenerative medicine, including enhancement of the neurite outgrowth length, regulation of neuron differentiation, and the formation of a network. These extracellular matrix-mimetic poly-L-lysine/hyaluronic acid multilayer films may provide a versatile platform that could be useful for surface modification for applications in neural engineering.

Preparation of PLLA membranes with different morphologies for culture of MG-63 Cells.

In this work, poly L-lactide (PLLA) membranes with different morphologies were prepared and the equilibrium phase diagram of membrane formation system of ethanol, methylene chloride, and PLLA was studied. Based on the phase diagram, particulate and porous membranes, dominated by crystallization and liquid-liquid demixing, respectively, could be prepared by changing the PLLA concentration of casting solution. In addition, in vitro interaction of MG-63 osteosarcoma cells and PLLA membranes with dense, porous and particulate morphologies was investigated. It was found that the particulate membrane not only could improve cell adhesion and growth, but also could upregulate the osteoblastic phenotype. Therefore, the PLLA membrane with particulate morphology satisfies the biomaterial requirement necessary for temporary scaffold to transplanted osteoblasts and provides a means for the architectural design of more complex tissue-engineered systems.

Facilitating neural stem/progenitor cell niche calibration for neural lineage differentiation by polyelectrolyte multilayer films.

Neural stem/progenitor cells (NSPCs) are a possible candidate for advancing development and lineage control in neural engineering. Differentiated protocols have been developed in this field to generate neural progeny and to establish neural networks. However, continued refinement is required to enhance differentiation specificity and prevent the generation of unwanted cell types. In this study, we fabricated a niche-modulated system to investigate surface effects on NSPC differentiation by the formation of polyelectrolyte multilayer (PEM) films governed by electrostatic interactions of poly-l-glutamine acid as a polyanion and poly-l-lysine as a polycation. The serum- and chemical agent-free system provided a clean and clear platform to observe in isolation the interaction between surface niche and stem cell differentiation. We found that NSPCs were inducible on PEM films of up to eight alternating layers. In addition, neurite outgrowth, neuron percentage, and synaptic function were regulated by layer number and the surface charge of the terminal layer. The average process outgrowth length was over 500µm on PLL/PLGA(n=7.5) only after 3 days of culture. Moreover, the quantity and quality of the differentiated neurons were enhanced as the number of layers increased, especially when the terminal layer was poly-l-lysine. Our results achieve important targets of neural engineering, including long processes, large neural network size, and large amounts of functional neurons. Our methodology for nanoscale control of material deposition can be successfully applied for surface modification, neural niche modulation, and neural engineering applications.

Assembly of polyelectrolyte multilayer films on supported lipid bilayers to induce neural stem/progenitor cell differentiation into functional neurons.

The key factors affecting the success of neural engineering using neural stem/progenitor cells (NSPCs) are the neuron quantity, the guidance of neurite outgrowth, and the induction of neurons to form functional synapses at synaptic junctions. Herein, a biomimetic material comprising a supported lipid bilayer (SLB) with adsorbed sequential polyelectrolyte multilayer (PEM) films was fabricated to induce NSPCs to form functional neurons without the need for serum and growth factors in a short-term culture. SLBs are suitable artificial substrates for neural engineering due to their structural similarity to synaptic membranes. In addition, PEM film adsorption provides protection for the SLB as well as the ability to vary the surface properties to evaluate the effects of physical and mechanical signals on NSPC differentiation. Our results revealed that NSPCs were inducible on SLB-PEM films consisting of up to eight alternating layers. In addition, the process outgrowth length, the percentage of differentiated neurons, and the synaptic function were regulated by the number of layers and the surface charge of the outermost layer. The average process outgrowth length was greater than 500 µm on SLB-PLL/PLGA (n = 7.5) after only 3 days of culture. Moreover, the quantity and quality of the differentiated neurons were obviously enhanced on the SLB-PEM system compared with those on the PEM-only substrates. These results suggest that the PEM films can induce NSPC adhesion and differentiation and that an SLB base may enhance neuron differentiation and trigger the formation of functional synapses.

The behavior of mesenchymal stem cells on micropatterned PLLA membranes.

The aim of this study was to evaluate the behaviors of mesenchymal stem cells (MSCs) on Poly(L-Lactic acid) (PLLA) membranes with different surface topographies. The double-sided micropatterns, island-patterned, and sunken-patterned PLLA membranes with diameters of 60 and 100 microm, were fabricated by the soft lithography method. The cell viability of MSCs on the island- and sunken-patterned PLLA membranes were characterized by scanning electron microscopy (SEM), MTT assay, and flow cytometric analysis. Cell adhesion and proliferation capability were superior for the MSCs seeding on the island-patterned PLLA membranes than those on the sunken-patterned PLLA membranes. Especially, we observed the best biocompatibility for MSCs on the island-patterned surface with diameter of 100 microm. In addition, the improvement of cell attachment and augmenting subsequent cellular response are investigated after the island-patterned membranes precoating with collagen and fibronectin. Furthermore, the flow cytometric analysis reveals the MSCs can expand and maintain the phenotype on these PLLA membranes without losing its potential for differentiation. Since scale-up of cell production and optimization of culture conditions are important for stem cell engineering, to control the stem cell proliferation and differentiation is necessary. Therefore, besides topographical properties play a crucial role on the stem cells attachment and proliferative activity, it is suggested that the "relative scale" between cell and pattern also affects the cell adhesion morphologies and cell behaviors. Based on the overall cellular response, this study provides a valuable guidance to prepare appropriate topographic surface for tissue engineering application.