3D Printed Nanocellulose Scaffolds as a Cancer Cell Culture Model System.
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ABSTRACT:
SUBMITTER: Rosendahl J
PROVIDER: S-EPMC8301137 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nanocellulose has been demonstrated as a suitable material for cell culturing, given its similarity to extracellular matrices. Taking advantage of the shear thinning behavior, nanocellulose suits three-dimensional (3D) printing into scaffolds that support cell attachment and proliferation. Here, we propose aqueous suspensions of acetylated nanocellulose of a low degree of substitution for direct ink writing (DIW). This benefits from the heterogeneous acetylation of precursor cellulosic fibers, which eases their deconstruction and confers the characteristics required for extrusion in DIW. Accordingly, the morphology of related 3D-printed architectures and their performance during drying and rewetting as well as interactions with living cells are compared with those produced from typical unmodified and TEMPO-oxidized nanocelluloses. We find that a significantly lower concentration of acetylated nanofibrils is needed to obtain bioinks of similar performance, affording more porous structures. Together with their high surface charge and axial aspect, acetylated nanocellulose produces dimensionally stable monolithic scaffolds that support drying and rewetting, required for packaging and sterilization. Considering their potential uses in cardiac devices, we discuss the interactions of the scaffolds with cardiac myoblast cells. Attachment, proliferation, and viability for 21 days are demonstrated. Overall, the performance of acetylated nanocellulose bioinks opens the possibility for reliable and scale-up fabrication of scaffolds appropriate for studies on cellular processes and for tissue engineering.
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Project description:Biocatalysis is increasingly becoming an alternative method for the synthesis of industrially relevant complex molecules. This can be realized by using enzyme immobilized polysaccharide-based 3D scaffolds as compatible carriers, with defined properties. Especially, immobilization of either single or multiple enzymes on a 3D printed polysaccharide scaffold, exhibiting well-organized interconnected porous structure and morphology, is a versatile approach to access the performance of industrially important enzymes. Here, we demonstrated the use of nanocellulose-based 3D porous scaffolds for the immobilization of glycosyltransferases, responsible for glycosylation in natural biosynthesis. The scaffolds were produced using an ink containing nanofibrillated cellulose (NFC), carboxymethyl cellulose (CMC), and citric acid. Direct-ink-writing 3D printing followed by freeze-drying and dehydrothermal treatment at elevated temperature resulted in chemically cross-linked scaffolds, featuring tunable negative charges (2.2-5.0 mmol/g), pore sizes (10-800 μm), fluid uptake capacity, and exceptional dimensional and mechanical stability in the wet state. The negatively charged scaffolds were applied to immobilize two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Zbasic2-CGT; sucrose synthase, Zbasic2-SuSy), each harboring a cationic binding module (Zbasic2) to promote charge-based enzyme adsorption. Both enzymes were immobilized at ∼30 mg of protein/g of dry carrier (∼20% yield), independent of the scaffold used. Their specific activities were 0.50 U/mg (Zbasic2-CGT) and 0.19 U/mg (Zbasic2-SuSy), corresponding to an efficacy of 37 and 18%, respectively, compared to the soluble enzymes. The glycosyltransferases were coimmobilized and shown to be active in a cascade reaction to give the natural C-glycoside nothofagin from phloretin (1.0 mM; ∼95% conversion). All enzyme bound scaffolds showed reusability of a maximum of 5 consecutive reactions. These results suggest that the 3D printed and cross-linked NFC/CMC-based scaffolds could present a class of solid carriers for enzyme (co)-immobilization, with promising applications in glycosyltransferase-catalyzed synthesis and other fields of biocatalysis.
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Project description:Vascularization is a fundamental prerequisite for large bone construct development and remains one of the main challenges of bone tissue engineering. Our current study presents the combination of 3D printing technique with a hydrogel-based prevascularization strategy to generate prevascularized bone constructs. Human adipose derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) were encapsulated within our bioactive hydrogels, and the effects of culture conditions on in vitro vascularization were determined. We further generated composite constructs by forming 3D printed polycaprolactone/hydroxyapatite scaffolds coated with cell-laden hydrogels and determined how the co-culture affected vascularization and osteogenesis. It was demonstrated that 3D co-cultured ADMSC-HUVEC generated capillary-like networks within the porous 3D printed scaffold. The co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis. The prevascularized constructs were subcutaneously implanted into nude mice to evaluate the in vivo vascularization capacity and the functionality of engineered vessels. The hydrogel systems facilitated microvessel and lumen formation and promoted anastomosis of vascular networks of human origin with host murine vasculature. These findings demonstrate the potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1788-1798, 2018.
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Project description:In skin research, widely used in vitro 2D monolayer models do not sufficiently mimic physiological properties. To replace, reduce, and refine animal experimentation in the spirit of '3Rs', new approaches such as 3D skin equivalents (SE) are needed to close the in vitro/in vivo gap. Cell culture inserts to culture SE are commercially available, however, these inserts are expensive and of limited versatility regarding experimental settings. This study aimed to design novel cell culture inserts fabricated on commercially available 3D printers for the generation of full-thickness SE. A computer-aided design model was realized by extrusion-based 3D printing of polylactic acid filaments (PLA). Improvements in the design of the inserts for easier and more efficient handling were confirmed in cell culture experiments. Cytotoxic effects of the final product were excluded by testing the inserts in accordance with ISO-norm procedures. The final versions of the inserts were tested to generate skin-like 3D scaffolds cultured at an air-liquid interface. Stratification of the epidermal component was demonstrated by histological analyses. In conclusion, here we demonstrate a fast and cost-effective method for 3D-printed inserts suitable for the generation of 3D cell cultures. The system can be set-up with common 3D printers and allows high flexibility for generating customer-tailored cell culture plastics.
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Project description:Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.
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Project description:In this paper, we describe the use of 3D printed devices for both static and flow studies that contain electrospun collagen scaffolds and can accommodate transepithelial/transendothelial electrical resistance (TEER) measurements. Electrospinning was used to create the collagen scaffold, followed by an optimized 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS) cross-linking procedure to produce stable collagen fibers that are similar in size to fibers in vivo. LC/MS was used to study the leaching of solvent and NHS from the scaffold, with several rinsing steps being shown to eliminate the leaching and promote the culture of Madin-Darby Canine Kidney (MDCK) epithelial cells on the scaffold. Both static and flow 2-part devices were successfully fabricated by 3D printing using either VeroClear or MED610 material (PolyJet printing) and assembling the scaffold between laser cut Teflon gaskets. The devices were designed to easily accommodate commonly used STX2 chopstick electrodes for TEER measurements. A detailed comparison was made between the use of collagen scaffolds vs other electrospun materials for cell culture. The collagen extracellular matrix model displayed a high barrier functionality for up to 7 days. In addition, a different 3D printed device with a collagen scaffold is described to incorporate continuous flow and replenishment of media under the cell layer in a manner that also enables periodic recording of TEER measurements. Overall, this work shows that the combination of biological ECM materials such as collagen into microfluidic devices that incorporate flow have great potential to form more realistic cell culture models in areas such as blood brain barrier research.
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Project description:The aim of the present study was to investigate the additive manufacturing process for high consistency nanocellulose. Unlike thermoformable plastics, wood derived nanocelluloses are typically processed as aqueous dispersions because they are not melt-processable on their own. The ability to use nanocellulose directly in additive manufacturing broadens the possibilities regarding usable raw materials and achievable properties thereof. Modern additive manufacturing systems are capable of depositing nanocellulose with micrometer precision, which enables the printing of accurate three-dimensional wet structures. Typically, these wet structures are produced from dilute aqueous fibrillar dispersions. As a consequence of the high water content, the structures deform and shrink during drying unless the constructs are freeze-dried. While freeze-drying preserves the geometry, it results in high porosity which manifests as poor mechanical and barrier properties. Herein, we study an additive manufacturing process for high consistency enzymatically fibrillated cellulose nanofibers in terms of printability, shape retention, structure, and mechanical properties. Particular emphasis is placed on quantitative shape analysis based on 3D scanning, point cloud analysis, and x-ray microtomography. Despite substantial volumetric as well as anisotropic deformation, we demonstrate repeatability of the printed construct and its properties.
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Project description:Three-dimensional (3D) printing technology allowed fast and cheap prototype fabrication in numerous segments of industry and it also became an increasingly versatile experimental platform in life sciences. Yet, general purpose software tools to control printer hardware are often suboptimal for bioprinting applications. Here we report a package of open source software tools that we developed specifically to meet bioprinting requirements: Machine movements can be (i) precisely specified using high level programming languages, and (ii) easily distributed across a batch of tissue culture dishes. To demonstrate the utility of the reported technique, we present custom fabricated, biocompatible 3D-printed plastic structures that can control cell spreading area or medium volume, and exhibit excellent optical properties even at 50 ul sample volumes. We expect our software tools to be helpful not only to manufacture customized in vitro experimental chambers, but for applications involving printing cells and extracellular matrices as well.
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Project description:For tissue engineering applications, a porous scaffold with an interconnected network is essential to facilitate the cell attachment and proliferation in a three dimensional (3D) structure. This study aimed to fabricate the scaffolds by an extrusion-based 3D printer using a blend of polycaprolactone (PCL), and graphene oxide (GO) as a favorable platform for bone tissue engineering. The mechanical properties, morphology, biocompatibility, and biological activities such as cell proliferation and differentiation were studied concerning the two different pore sizes; 400??m, and 800??m, and also with two different GO content; 0.1% (w/w) and 0.5% (w/w). The compressive strength of the scaffolds was not significantly changed due to the small amount of GO, but, as expected scaffolds with 400??m pores showed a higher compressive modulus in comparison to the scaffolds with 800??m pores. The data indicated that the cell attachment and proliferation were increased by adding a small amount of GO. According to the results, pore size did not play a significant role in cell proliferation and differentiation. Alkaline Phosphate (ALP) activity assay further confirmed that the GO increase the ALP activity and further Elemental analysis of Calcium and Phosphorous showed that the GO increased the mineralization compared to PCL only scaffolds. Western blot analysis showed the porous structure facilitate the secretion of bone morphogenic protein-2 (BMP-2) and osteopontin at both day 7 and 14 which galvanizes the osteogenic capability of PCL and PCL?+?GO scaffolds.
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Project description:A bioengineered spinal cord is fabricated via extrusion-based multi-material 3D bioprinting, in which clusters of induced pluripotent stem cell (iPSC)-derived spinal neuronal progenitor cells (sNPCs) and oligodendrocyte progenitor cells (OPCs) are placed in precise positions within 3D printed biocompatible scaffolds during assembly. The location of a cluster of cells, of a single type or multiple types, is controlled using a point-dispensing printing method with a 200 μm center-to-center spacing within 150 μm wide channels. The bioprinted sNPCs differentiate and extend axons throughout microscale scaffold channels, and the activity of these neuronal networks is confirmed by physiological spontaneous calcium flux studies. Successful bioprinting of OPCs in combination with sNPCs demonstrates a multicellular neural tissue engineering approach, where the ability to direct the patterning and combination of transplanted neuronal and glial cells can be beneficial in rebuilding functional axonal connections across areas of central nervous system (CNS) tissue damage. This platform can be used to prepare novel biomimetic, hydrogel-based scaffolds modeling complex CNS tissue architecture in vitro and harnessed to develop new clinical approaches to treat neurological diseases, including spinal cord injury.
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