Chemically Crosslinked Methylcellulose Substrates for Cell Sheet Engineering.
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ABSTRACT:
SUBMITTER: Bonetti L
PROVIDER: S-EPMC8482237 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThere is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking.ResultsIn this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion.ConclusionsAll components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network.
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Project description:Postoperative adhesion and occlusion remain a serious issue associated with various surgeries, including endoscopic surgery, in which proliferated fibrous tissues stick to adjacent tissues and often cause severe complications. Cell sheet engineering has emerged as an effective approach not only for cell transplantation but also for the treatment of postoperative adhesion and occlusion. However, as the tissues in the body, such as middle ear and small intestine, and typical operative sites are non-flat and spatially complicated, tailored cell sheets with three-dimensional (3D) configurations may lead to widespread use of this approach. In the present study, we used microstereolithography, biocompatible gold plating, and electrochemical cell detachment to achieve this purpose. Various objects with dimensions ranging from millimeter- to micrometer-scale were fabricated with photocurable resin using lab-made equipment for microstereolithography. To coat the fabricated objects with a thin gold layer, conventional cyanide-based gold plating was unusable because it severely damaged almost all cells. Electroless non-cyanide gold plating we prepared was cytocompatible and suitable for electrochemical cell detachment. Cell sheets on the gold-plated substrate could be directly transplanted into a mouse intraperitoneally using electrochemical cell detachment. We further demonstrated that cell sheets grown on gold-coated 3D objects were rapidly detached along with the desorption of electroactive-oligopeptide monolayer and transferred to a surrounding hydrogel. This approach may provide a promising strategy to prepare and directly transplant tailor-made cell sheets with suitable configurations.
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Project description:Specific detection of circulating tumor cells and characterization of their aggressiveness could improve cancer diagnostics and treatment. Metastasis results from such tumor cells, and causes the majority of cancer deaths. Chemically modified viruses could provide an inexpensive and efficient approach to detect tumor cells and quantitate their cell surface biomarkers. However, non-specific adhesion between the cell surface receptors and the virus surface presents a challenge. This report describes wrapping the virus surface with different PEG architectures, including as fusions to oligolysine, linkers, spacers and scaffolded ligands. The reported PEG wrappers can reduce by >75% the non-specific adhesion of phage to cell surfaces. Dynamic light scattering verified the non-covalent attachment by the reported wrappers as increased sizes of the virus particles. Further modifications resulted in specific detection of prostate cancer cells expressing PSMA, a key prostate cancer biomarker. The approach allowed quantification of PSMA levels on the cell surface, and could distinguish more aggressive forms of the disease.
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Project description:Self-assembled monolayers (SAMs) of alkanethiolates on gold are chemically defined substrates that can be used to evaluate the effects of an immobilized biomolecule. However, the types of biomolecules that can influence stem cell behavior are numerous and inter-related, and efficient experimental formats are a critical need. Here we employed a SAM array technology to investigate the effects of multiple, distinct peptides and peptide combinations on human mesenchymal stem cell (hMSC) behavior. Specifically, we characterized the conjugation of peptide mixtures to SAM arrays and then investigated the combined effects of a bone morphogenic protein receptor-binding peptide (BR-BP), a heparin proteoglycan-binding peptide (HPG-BP), and varied densities of the integrin-binding ligand Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) on hMSC surface coverage and alkaline phosphatase activity. Results indicate that an amine reactive fluorescent probe can be used to characterize peptide composition after immobilization in SAM array spots. Furthermore, hMSC response to BR-BP and HPG-BP is dependent on GRGDSP density and at day 7, hMSC alkaline phosphatase expression is highly dependent on GRGDSP density. Taken together, we demonstrate how a SAM array approach can be used to probe the combinatorial effects of multiple peptides and motivate further investigations into potential synergies between cell adhesion and other bioactive peptides.
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Project description:Multicellular biology is dependent on the control of cell-cell interactions. These concepts have begun to be exploited for engineering of cell-based therapies. Herein, we detail the use of a multivalent lipidated scaffold for the rapid and reversible manipulation of cell-cell interactions. Chemically self-assembled nanorings (CSANs) are formed via the oligomerization of bivalent dihydrofolate reductase (DHFR2) fusion proteins using a chemical dimerizer, bis-methotrexate. With targeting proteins fused onto the DHFR2 monomers, the CSANs can target specific cellular antigens. Here, anti-EGFR or anti-EpCAM fibronectin-DHFR2 monomers incorporating a CAAX-box sequence were enzymatically prenylated, then assembled into the corresponding CSANs. Both farnesylated and geranylgeranylated CSANs efficiently modified the cell surface of lymphocytes and remained bound to the cell surface with a half-life of >3 days. Co-localization studies revealed a preference for the prenylated nanorings to associate with lipid rafts. The presence of antigen targeting elements in these bifunctional constructs enabled them to specifically interact with target cells while treatment with trimethoprim resulted in rapid CSAN disassembly and termination of the cell-cell interactions. Hence, we were able to determine that activated PBMCs modified with the prenylated CSANs caused irreversible selective cytotoxicity toward EGFR-expressing cells within 2 hours without direct engagement of CD3. The ability to disassemble these nanostructures in a temporally controlled manner provides a unique platform for studying cell-cell interactions and T cell-mediated cytotoxicity. Overall, antigen-targeted prenylated CSANs provide a general approach for the regulation of specific cell-cell interactions and will be valuable for a plethora of fundamental and therapeutic applications.
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Project description:Human mesenchymal stem cells (hMSCs) are a widely available and clinically relevant cell type with a host of applications in regenerative medicine. Current clinical expansion methods can lead to selective changes in hMSC phenotype potentially resulting from relatively undefined cell culture surfaces. Chemically defined synthetic surfaces can aid in understanding the influence of cell-material interactions on stem cell behavior. Here, a thin copolymer coating for hMSC culture on plastic substrates is developed. The random copolymer is synthesized by living free radical polymerization and characterized in solution before application to the substrate, ensuring a homogeneous coating and limiting the sample-to-sample variations. The ability to coat multiple substrate types and cover large surface areas is reported. Arg-Gly-Asp-containing peptides are incorporated into the coating under aqueous conditions via their lysine or cysteine side chains, resulting in amide and thioester linkages, respectively. Stability studies show amide linkages to be stable and thioester linkages to be labile under standard serum-containing culture conditions. In addition, chemically defined passaging of hMSCs using only ethylenediaminetetraacetic acid on polystyrene dishes is shown. After passage, the hMSCs can be seeded back onto the same plate, indicating potential reusability of the coating.
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Project description:Cell sheet technology is becoming increasingly popular in tissue engineering and regenerative medicine, due to integrity into versatile organ and manageable cell and tissue type from the bank, and no needs of large volume organ for transplantation. Cell sheets have still a room to resolve the mechanical resistance under load-bearing occasion, easy translocation into organ, and prompt shape modulation for regular application in vivo. Herein, a layer-by-layer (LbL) assembly of nanometer scaled film coating method was introduced to inter-planar cell sheet for multilayered cell sheet (M1) and a single cell before sheet formation (M2). Nano-films with collagen and alginate increased mechanical property of cell sheets without altering cell functions, viability, and proliferation. The moduli of triple layered cell sheet (M1) and (M2) were critically enhanced to 109% and 104%, compared to uncoated cell sheet (CON) with mono-layer, while modulus of CON with triple-layers were increased to 43%. LbL assembly to cell sheets offers increased modulus allowing cell sheet engineering to become a potential strategy under load-bearing environment.
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Project description:Analyte-responsive smart polymeric materials are of great interest and have been actively investigated in the field of regenerative medicine. Phenylboronate containing copolymers form gels with polyols under alkaline conditions. Monosaccharides, by virtue of their higher affinity towards boronate, can displace polyols and solubilize such gels. In the present study, we investigate the possibility of utilizing phenylboronate-polyol interactions at physiological pH in order to develop monosaccharide-responsive degradable scaffold materials for systems dealing with cells and tissues. Amine assisted phenylboronate-polyol interactions were employed to develop novel hydrogel and cryogel scaffolds at neutral pH. The scaffolds displayed monosaccharide inducible gel-sol phase transformability. In vitro cell culture studies demonstrated the ability of scaffolds to support cell adhesion, viability and proliferation. Fructose induced gel degradation is used to recover cells cultured on the hydrogels. The cryogels displayed open macroporous structure and superior mechanical properties. These novel phase transformable phenylboronate-polyol based scaffolds displayed a great potential for various cell sheet and tissue engineering applications. Their monosaccharide responsiveness at physiological pH is very useful and can be utilized in the fields of cell immobilization, spheroid culture, saccharide recognition and analyte-responsive drug delivery.
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