Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.     

Mesenchymal stem cell cultivation in electrospun scaffolds: mechanistic modeling for tissue engineering

Tissue engineering is a multidisciplinary field of research in which the cells, biomaterials, and processes can be optimized to develop a tissue substitute. Three-dimensional (3D) architectural features from electrospun scaffolds, such as porosity, tortuosity, fiber diameter, pore size, and interconnectivity have a great impact on cell behavior. Regarding tissue development in vitro, culture conditions such as pH, osmolality, temperature, nutrient, and metabolite concentrations dictate cell viability inside the constructs. The effect of different electrospun scaffold properties, bioreactor designs, mesenchymal stem cell culture parameters, and seeding techniques on cell behavior can be studied individually or combined with phenomenological modeling techniques. This work reviews the main culture and scaffold factors that affect tissue development in vitro regarding the culture of cells inside 3D matrices. The mathematical modeling of the relationship between these factors and cell behavior inside 3D constructs has also been critically reviewed, focusing on mesenchymal stem cell culture in electrospun scaffolds.     

Microscale methods to assemble mammalian cells into tissue-like structures

Different cell types make up tissues and organs hierarchically and communicate within a complex, three-dimensional (3D) environment. The in vitro recapitulation of tissue-like structures is meaningful, not only for fundamental cell biology research, but also for tissue engineering (TE). Currently, TE research adopts either the top-down or bottom-up approach. The top-down approach involves defining the macroscopic tissue features using biomaterial scaffolds and seeding cells into these scaffolds. Conversely, the bottom-up approach aims at crafting small tissue building blocks with precision-engineered structural and functional microscale features, using physical and/or chemical approaches. The bottom-up strategy takes advantage of the repeating structural and functional units that facilitate cell-cell interactions and cultures multiple cells together as a functional unit of tissue. In this review, we focus on currently available microscale methods that can control mammalian cells to assemble into 3D tissue-like structures.     

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.     

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.^{1, 2, 3} Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.⁴ An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.     

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(L-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions. gene expression; osteogenesis; angiogenesis     

Development of an Ibuprofen‐releasing biodegradable PLA/PGA electrospun scaffold for tissue regeneration

Our aim was to develop a biodegradable fibrous dressing to act as a tissue guide for in situ wound repair while releasing Ibuprofen to reduce inflammation in wounds and reduce pain for patients on dressing changes. Dissolving the acid form of Ibuprofen (from 1% to 10% by weight) in the same solvent as 75% polylactide, 25% polyglycolide (PLGA) polymers gave uniformly loaded electrospun fibers which gave rapid release of drug within the first 8 h and then slower release over several days. Scaffolds with 10% Ibuprofen degraded within 6 days. The Ibuprofen released from these scaffolds significantly reduced the response of fibroblasts to major pro‐inflammatory stimulators. Fibroblast attachment and proliferation on scaffolds was unaffected by the addition of 1–5% Ibuprofen. Scaffolds loaded with 10% Ibuprofen initially showed reduced cell attachment but this was restored by soaking scaffolds in media for 24 h. In summary, addition of Ibuprofen to electrospun biodegradable scaffolds can give acute protection of adjacent cells to inflammation while the scaffolds provide an open 3D fibrous network to which cells can attach and migrate. By 6 days, such scaffolds will have completely dissolved into the wound bed obviating any need for dressing removal. Biotechnol. Bioeng. 2010; 105: 396–408. © 2009 Wiley Periodicals, Inc.     

Three-dimensional gastric cancer cell culture using nanofiber scaffold for chemosensitivity test

A three-dimensional (3D) culture of cancer cells has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. To investigate the sensitivity of cancer cells to anticancer drugs, nanofiber scaffolds composed of PHBV and collagen peptide were fabricated by electrospinning. A 3D culture of cancer cells was successfully achieved by the use of nanofiber scaffolds. From the result of a chemosensitivity test, it was found that higher concentrations of anticancer drugs were required to achieve a comparable cytotoxic effect in 3D culture due to their structural architecture. These data demonstrate that the electrospun nanofiber scaffolds can provide a 3D model particularly appropriate for investigating mechanisms involved in cancer cell sensitivity to anticancer drugs.     

Cellular infiltration on nanofibrous scaffolds using a modified electrospinning technique

Electrospinning is currently used to fabricate nanofibrous scaffolds for tissue engineering applications. The major problem of these scaffolds is their intrinsically two-dimensional nature which inhibits cellular migration and in-growth. In this study, we have introduced a modified setup of electrospinning to produce three-dimensional nanofibrous scaffolds which allows improved infiltration of cells. An array of focused halogen light bulbs was used to localize the heat in the path of electrospun jet near the collector. The fabricated mats were then seeded with cells in order to evaluate migration and infiltration. After 14 days of culture, a homogenous distribution of cells was observed throughout the scaffolds and showed the three-dimensional architecture of nanofibrous mats. By this novel and simple setup, the prepared electrospun mats will allow the seeded cells to obtain a three-dimensional arrangement which is ideal for tissue engineering applications.     

Influence of biological matrix and artificial electrospun scaffolds on proliferation,differentiation and trophic factor synthesis of rat embryonic stem cells

Two-dimensional vs three-dimensional culture conditions, such as the presence of extracellular matrix components, could deeply influence the cell fate and properties. In this paper we investigated proliferation, differentiation, survival, apoptosis, growth and neurotrophic factor synthesis of rat embryonic stem cells (RESCs) cultured in 2D and 3D conditions generated using Cultrex® Basement Membrane Extract (BME) and in poly-(l-lactic acid) (PLLA) electrospun sub-micrometric fibres. It is demonstrated that, in the absence of other instructive stimuli, growth, differentiation and paracrine activity of RESCs are directly affected by the different microenvironment provided by the scaffold. In particular, RESCs grown on an electrospun PLLA scaffolds coated or not with BME have a higher proliferation rate, higher production of bioactive nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) compared to standard 2D conditions, lasting for at least 2 weeks. Due to the high mechanical flexibility of PLLA electrospun scaffolds, the PLLA/stem cell culture system offers an interesting potential for implantable neural repair devices.     

A 3D Fibrous Scaffold Inducing Tumoroids: A Platform for Anticancer Drug Development

The development of a suitable three dimensional (3D) culture system for anticancer drug development remains an unmet need. Despite progress, a simple, rapid, scalable and inexpensive 3D-tumor model that recapitulates in vivo tumorigenesis is lacking. Herein, we report on the development and characterization of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and mono-methoxypolyethylene glycol (mPEG) designated as 3P. Cancer cells cultured on the 3P scaffold formed tight irregular aggregates similar to in vivo tumors, referred to as tumoroids that depended on the topography and net charge of the scaffold. 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression. 3P tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT and reduced tumoroid formation, diameter and number. Fine needle aspirates, collected from tumor cells implanted in mice when cultured on 3P scaffolds formed tumoroids, but showed decreased sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding. These results show that 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds and tailor a personalized cancer treatment.     

BMSCs在PLGA-[ASP-PEG]基质材料表面粘附及增殖的研究

目的：探讨大鼠骨髓间充质干细胞BMSCs在聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段多元共聚物 PLGA-[ASP-PEG]表面粘附、增殖的情况,为组织工程学体外诱导种子细胞生长提供新的生物材料。方法：在PLGA支架材料中引入聚乙二醇（PEG）和含有多个功能位点的天冬氨酸（ASP）,制成PLGA-[ASP-PEG]高分子支架材料。 将PLGA-[ASP-PEG]支架材料与BMSCs复合培养,以未改性的PLGA支架材料作对照,通过沉淀法、MTT法和考马斯亮蓝法分别检测BMSCs的粘附和增殖变化;扫描电镜观察黏附细胞的形态。结果 BMSCs在PLGA-[ASP-PEG]材料表面帖壁生长,细胞数目明显多于单纯PLGA组。细胞粘附率检测显示：改性后的PLGA-[ASP-PEG]表面BMSCs的粘附性能和增殖能力明显高于对照组,P＜0.05。MTT比色试验,BMSCs在三嵌段材料上培养20d后,吸光值A=1.336,约为对照组0.780的两倍。细胞内蛋白总量间接反映细胞黏附及增殖情况。培养12d时,在PLGA-[ASP-PEG]材料组细胞的蛋白含量为66.44μg/孔,单纯PLGA组为41.23μg/孔,间接说明了三嵌段材料生物相容性好,细胞黏附力强的特点。结论PLGA-[ASP-PEG]能促进组织工程种子细胞在骨基质材料表面的黏附、增殖并能较好地保持细胞的形态。     

Development of a 3D cell culture system for investigating cell interactions with electrospun fibers

There are many variables to be considered in studying how cells interact with 3D scaffolds used in tissue engineering. In this study we investigated the influence of the fiber diameter and interfiber spaces of 3D electrospun fiber scaffolds on the behavior of human dermal fibroblasts. Fibers of two dissimilar model materials, polystyrene and poly-L-lactic acid, with a broad range of diameters were constructed in a specifically developed 3D cell culture system. When fibroblasts were introduced to freestanding fibers, and encouraged to "walk the plank," a minimum fiber diameter of 10 microm was observed for cell adhesion and migration, irrespective of fiber material chemistry. A distance between fibers of up to 200 microm was also observed to be the maximum gap that could be bridged by cell aggregates--a behavior not seen in conventional 2D culture. This approach has identified some basic micro-architectural parameters for electrospun scaffold design and some key differences in fibroblast growth in 3D. We suggest the findings will be of value for optimizing the integration of cells in these scaffolds for skin tissue engineering.     

In vitro 3D human small intestinal villous model for drug permeability determination

We present a novel method for testing drug permeability that features human cells cultured on hydrogel scaffolds made to accurately replicate the shape and size of human small intestinal villi. We compared villous scaffolds to more conventional 2D cultures in paracellular drug absorption and cell growth experiments. Our results suggest that 3D villous platforms facilitate cellular differentiation and absorption more similar to mammalian intestines than can be achieved using conventional culture. To the best of our knowledge, this is the first accurate 3D villus model offering a well-controlled microenvironment that has strong physiological relevance to the in vivo system.     

Effect of spatial architecture on cellular colonization

The spatial cell-material interaction remains vital issue in forming biodegradable scaffolds in Tissue Engineering. In this study, to understand the influence of spatial architecture on cellular behavior, 2D and 3D chitosan scaffolds of 50-190 kD and >310 kD MW were synthesized through air drying and controlled rate freezing/lypohilization technique, respectively. In addition, chitosan was emulsified with 19, 76, and 160 kD 50:50 poly lactide-co-glycolide (PLGA) using 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) as stabilizer. 2D and 3D scaffolds were formed by air drying and lyophilization as before. Tensile and compressive properties of films and scaffolds were analyzed in wet conditions at 37 degrees C. Alterations in the cell spreading, proliferation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) were studied. These results showed that the formed 3D chitosan scaffolds had interconnected open pore architecture (50-200 microm size). HUVECs and MEFs had reduced spreading areas and circular morphology on 2D chitosan membranes compared with 3D chitosan scaffolds. The fluorescence photomicrographs for actin (using Alexa Fluor 488 phalloidin) and cytoplasm staining (using carboxyfluorescein diacetate-succinimidyl ester) demonstrated that the cells spread within 3D chitosan matrix. 2D and 3D emulsified chitosan and chitosan/PLGA scaffolds reduced the spreading of HUVECs and MEFs even further. Proliferation results, analyzed via MTT-Formazan assay and BrdU uptake assay, correlated with the spreading characteristics. The reductions in cell spreading area on emulsified surfaces were not detrimental to the viability and endocytic activity but to proliferation. The observed alterations in cellular colonization are in part due to the substrate stiffness and surface topography. In summary, these results suggest a significant influence of spatial architecture on cellular colonization.     

A scaffold-free in vitro model for osteogenesis of human mesenchymal stem cells

For studying cellular processes three-dimensional (3D) in vitro models are of a high importance. For tissue engineering approaches osseous differentiation is performed on 3D scaffolds, but material depending influences promote cellular processes like adhesion, proliferation and differentiation. To investigate developmental processes of mesenchymal stem cells without cell-substrate interactions, self-contained in vitro models mimicking physiological condition are required. However, with respect to scientific investigations and pharmaceutical tests, it is essential that these tissue models are well characterised and are of a high reproducibility. In order to establish an appropriate in vitro model for bone formation, different protocols are compared and optimised regarding their aggregate formation efficiency, homogeneity of the aggregates, the viability and their ability to induce differentiation into the osteogenic lineage. The protocols for the generation of 3D cell models are based on rotation culture, hanging drop technique, and the cultivation in non adhesive culture vessels (single vessels as well as 96 well plates). To conclude, the cultivation of hMSCs in 96 well non adhesive plates facilitates an easy way to cultivate homogenous cellular aggregates with high performance efficiency in parallel. The size can be controlled by the initial cell density per well and within this spheroids, bone formation has been induced.     

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.     

Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds

The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair.