Tissue Culture Polystyrene Surfaces Research Articles

Supramolecular Surface Coatings with Acetylated Polyrotaxane-Based Triblock Copolymers for Thermal Regulation of Cell Adhesion and Fabrication of Cell Sheets.

Polyrotaxanes (PRXs) containing acetylated α-cyclodextrins exhibit a temperature-dependent phase transition in aqueous solutions across their lower critical solution temperature (LCST) of approximately 26.6 °C. To gain insights into the interactions of acetylated PRXs (Ac-PRXs) with biological components, thermoresponsive supramolecular surfaces were prepared by coating tissue culture polystyrene (TCPS) surfaces with Ac-PRX triblock copolymers, and their surface properties across the LCST were evaluated. The wettability and protein adsorption of Ac-PRX-coated surfaces changed significantly between 10 and 37 °C, whereas the uncoated TCPS and unmodified PRX-coated surfaces did not alter the wettability and protein adsorption at 10 and 37 °C. The adhesion, proliferation, morphology, and adhesion strength of NIH/3T3 cells on Ac-PRX-coated surfaces were found to be similar to those of the uncoated and unmodified PRX-coated surfaces. However, the adhesion strength of NIH/3T3 cells on Ac-PRX-coated surfaces decreased drastically at 10 °C. Consequently, the cells spontaneously detached from the Ac-PRX-coated surfaces without enzymatic treatment. Additionally, when incubating confluent cells at 10 °C, the cells detached from Ac-PRX-coated surfaces as cell sheets while retaining extracellular matrix proteins. The findings of this study provide new directions for the design of thermoresponsive supramolecular biointerfaces for applications in bioseparation and cell manipulation.

Performance of the Polydopamine-Graphene Oxide Composite Substrate in the Osteogenic Differentiation of Mouse Embryonic Stem Cells.

Graphene oxide (GO) is a biocompatible material considered a favorable stem cell culture substrate. In this study, GO was modified with polydopamine (PDA) to facilitate depositing GO onto a tissue culture polystyrene (PT) surface, and the osteogenic performance of the PDA/GO composite in pluripotent embryonic stem cells (ESCs) was investigated. The surface chemistry of the PDA/GO-coated PT surface was analyzed by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). A high cell viability of ESCs cultured on the PDA/GO composite-coated surface was initially ensured. Then, the osteogenic differentiation of the ESCs in response to the PDA/GO substrate was assessed by alkaline phosphatase (ALP) activity, intracellular calcium levels, matrix mineralization assay, and evaluation of the mRNA and protein levels of osteogenic factors. The culture of ESCs on the PDA/GO substrate presented higher osteogenic potency than that on the uncoated control surface. ESCs cultured on the PDA/GO substrate expressed significantly higher levels of integrin α5 and β1, as well as bone morphogenetic protein receptor (BMPR) types I and II, compared with the control groups. The phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun-N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was observed in ESCs culture on the PDA/GO substrate. Moreover, BMP signal transduction by SMAD1/5/8 phosphorylation was increased more in cells on PDA/GO than in the control. The nuclear translocation of SMAD1/5/8 in cells was also processed in response to the PDA/GO substrate. Blocking activation of the integrin α5/β1, MAPK, or SMAD signaling pathways downregulated the PDA/GO-induced osteogenic differentiation of ESCs. These results suggest that the PDA/GO composite stimulates the osteogenic differentiation of ESCs via the integrin α5/β1, MAPK, and BMPR/SMAD signaling pathways.

A designer cell culture insert with a nanofibrous membrane toward engineering an epithelial tissue model validated by cellular nanomechanics.

Engineered platforms for culturing cells of the skin and other epithelial tissues are useful for the regeneration and development of in vitro tissue models used in drug screening. Recapitulating the biomechanical behavior of the cells is one of the important hallmarks of successful tissue generation on these platforms. The biomechanical behavior of cells profoundly affects the physiological functions of the generated tissue. In this work, a designer nanofibrous cell culture insert (NCCI) device was developed, consisting of a free-hanging polymeric nanofibrous membrane. The free-hanging nanofibrous membrane has a well-tailored architecture, stiffness, and topography to better mimic the extracellular matrix of any soft tissue than conventional, flat tissue culture polystyrene (TCPS) surfaces. Human keratinocytes (HaCaT cells) cultured on the designer NCCIs exhibited a 3D tissue-like phenotype compared to the cells cultured on TCPS. Furthermore, the biomechanical characterization by bio-atomic force microscopy (Bio-AFM) revealed a markedly altered cellular morphology and stiffness of the cellular cytoplasm, nucleus, and cell–cell junctions. The nuclear and cytoplasmic moduli were reduced, while the stiffness of the cellular junctions was enhanced on the NCCI compared to cells on TCPS, which are indicative of the fluidic state and migratory phenotype on the NCCI. These observations were corroborated by immunostaining, which revealed enhanced cell–cell contact along with a higher expression of junction proteins and enhanced migration in a wound-healing assay. Taken together, these results underscore the role of the novel designer NCCI device as an in vitro platform for epithelial cells with several potential applications, including drug testing, disease modeling, and tissue regeneration.

Laminin-511 and recombinant vitronectin supplementation enables human pluripotent stem cell culture and differentiation on conventional tissue culture polystyrene surfaces in xeno-free conditions.

Human pluripotent stem cells (hPSCs) are typically cultivated on extracellular matrix (ECM) protein-coated dishes in xeno-free culture conditions. We supplemented mixed ECM proteins (laminin-511 and recombinant vitronectin, rVT) in culture medium for hPSC culture on conventional polystyrene dishes. Three hPSC cell lines were successfully cultivated on uncoated polystyrene dishes in medium supplemented with optimal conditions of laminin-511 and rVT. Excellent colony shape and colony size as well as high expansion fold of hPSCs were found under these conditions, whereas the colony size was small and poor expansion fold was found solely on L-511-coated dishes. A small portion of L-511 in the culture medium supported hPSC adhesion and prevented the adhesion from being too strong on the uncoated dishes, and rVT in the culture medium further supported adhesion of hPSCs on the dishes by maintaining their pluripotency. Having the optimal composition of L-511 and rVT in the culture medium was important for generating good hPSC colony shapes and sizes as well as a high expansion fold. After long-term culture of hPSCs on uncoated dishes supplemented with the mixed proteins, the hPSCs successfully showed pluripotent markers and could differentiate into a specific lineage of cells, cardiomyocytes, with high efficiency.

Quantitative relation between thickness and grafting density of temperature‐responsive poly(N‐isopropylacrylamide‐co‐acrylamide) thin film using synchrotron‐source ATR‐FTIR and spectroscopic ellipsometry

Poly(N‐isopropylacrylamide‐co‐acrylamide) (PNIAM‐co‐AM) grafted onto tissue culture polystyrene surfaces (TCPSs) with different thicknesses and grafting densities were prepared by varying the spin‐coating speed and UV irradiation time for use in cell sheet engineering. The thickness of the PNIAM grafted surface, less than 30 nm, was believed to influence protein adhesion and cell attachment. However, methods for determining the thicknesses of a thin surface (nanometer scale) are still a challenge. In this study, a model was developed to relate the thickness of the PNIAM‐co‐AM film to its grafting density. Spectroscopic ellipsometry (SE) and attenuated total reflection‐Fourier transform infrared spectroscopy (ATR‐FTIR) with synchrotron radiation were used to quantify the thickness and the grafting density, respectively. The amount of the grafted copolymer was determined from quantitative ATR‐FTIR by relating the signals at wavenumber 1,654 to 1,600 cm−1 (A1,654/A1,600) to the known grafting density. A combination of SE and ATR‐FTIR was used to construct a linear model (h = 6.29σ + 2.24), to obtain the grafting density (σ) from the film thickness (h) with R2 = 0.93. To validate the model, random samples were prepared with different thicknesses and grafting densities. The linear model was used to predict the grafting density, which was nearly equal to the measured values.

Readily Adsorbable Thermoresponsive Polymers for the Preparation of Smart Cell-Culturing Surfaces on Site.

The efficacy of several cell therapy products is directly impacted by trypsinization, which can diminish the engrafting capacity of transplanted cells by cleaving cell surface receptors. Thermoresponsive surfaces can alleviate this drawback, enabling temperature-driven and enzyme-free cell harvesting. However, the production of thermoresponsive surfaces relies on dedicated and complex equipment, often involving protocols dependent on high surface activation energies that prevent the development of scalable and universal platforms. In this work, we developed thermoresponsive copolymers incorporating styrene units that enable the copolymer adsorption on tissue culture polystyrene surfaces from an alcoholic solution in a short time, regardless of the vessel size and geometry, and without any particular equipment. In this way, the procedure can be performed with minimal effort by the end user on any surface. The thermoresponsive copolymers were synthesized via reversible addition-fragmentation chain transfer polymerization, providing high control over the polymer microstructure, a key parameter for tuning its cloud point and architecture. Block copolymers comprising a thermoresponsive segment and a polystyrene block exhibited optimal adhesion on conventional cell culture surfaces and permitted a more efficient temperature-mediated harvesting of adipose-derived stromal cells and Chinese hamster ovary cells compared to their statistical counterparts. To expand the application of this polymer deposition protocol to serum-free cell culture, we also considered the polymer modification with the tripeptide arginine-glycine-aspartic acid, known to promote the cell adhesion to synthetic substrates. The incorporation of this peptide enabled the collection in serum-free conditions of intact cell sheets from surfaces prepared shortly before their usage.

Biological Effects of Polyrotaxane Surfaces on Cellular Responses of Fibroblast, Preosteoblast and Preadipocyte Cell Lines.

Biointerfaces based on polyrotaxane (PRX), consisting of α-cyclodextrins (α-CDs) threaded on a poly(ethylene glycol) (PEG) chain, are promising functionalized platforms for culturing cells. PRXs are characterized by the molecular mobility of constituent molecules where the threading α-CDs can move and rotate along the PEG chain. Taking advantage of this mobility, we have previously succeeded in demonstrating the regulation of cellular responses, such as cellular adhesion, proliferation, and differentiation. In the present study, we investigated differences in the cellular responses to PRX surfaces versus commercially available tissue culture polystyrene (TCPS) surfaces using fibroblasts, preosteoblasts, and preadipocytes. PRX surfaces were found to more significantly promote cellular proliferation than the TCPS surfaces, regardless of the cell type. To identify the signaling pathways involved in the activation of cellular proliferation, a DNA microarray analysis was performed. PRX surfaces showed a significant increase in the integrin-mediated cell adhesion and focal adhesion pathways. Furthermore, PRX surfaces also promoted osteoblast differentiation more than TCPS. These results suggest that structural features of PRX surfaces act as mechanical cues to dominate cellular proliferation and differentiation.

Synergistic Effect of Inducible Factors on the Differentiation of Human Adipose-Derived Stem Cells to Vascular Cells

The use of cells derived from the patient’s tissue is an attractive approach to develop autologous tissue-engineered vascular graft (TEVG) to bypass the diseased blood vessel. Multipotent human adipose-derived mesenchymal stem cells (hADMSCs) have the potential to differentiate into both smooth muscle cells (SMCs) and endothelial cells (ECs). However, the disease conditions affecting the growth and differentiation potential of cardiovascular disease (CVD) patient’s MSC (pADMSCs) is a hindrancee. This study aimed to derive both endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) from pADMSCs using a specific niche comprising fibrin and other inducers. pADMSCs were seeded on SMC-specific/EC-specific fibrin matrix coated on tissue culture polystyrene (TCPS) surface. In the third passage, pADMSCs were exposed to GFs for a specific period to induce pADMSCs to SMPCs. For inducing hADMSCs to EPCs, cells were exposed to GFs, followed by hypoxia. The EPCs were seeded in the lumen of a scaffold and applied shear stress to induce into ECs. The relative gene expressions at different stages were assessed by mRNA quantification and at the protein level by immunocytochemistry of final differentiated cells. A consistent matrix and GF dependent protocol facilitated derivation of SMPCs/SMCs with early, mid, and late markers. Sequential action by GF, hypoxia (HO), and shear stress (SS) differentiated pADMSCs to EPCs/ECS. The study established that patient-specific ECs and SMCs may be derived from ADMSCs for the construction of intimal and medial layers of TEVG aiming to fabricate autologous blood vessels.

110th Anniversary: Fast and Easy-to-Use Method for Coating Tissue Culture Polystyrene Surfaces with Nonfouling Copolymers To Prevent Cell Adhesion

Synthetic substrates able to prevent the adhesion of cells, commonly referred to as nonfouling surfaces, are useful in a wide range of applications, including the culture of multicellular spheroids and the engineering of complex cell sheets. Nonfouling properties are commonly conferred to surfaces through superhydrophilic polymer-based coatings. At the same time, this type of treatment requires difficult, expensive, and time-consuming procedures, leading to high prices of the final product. In this perspective, the development of quick single-step methods to coat surfaces with nonfouling polymers would dramatically reduce the costs and increase the flexibility of such products. Moreover, this strategy would enable the process to be carried out directly by the end user. In this work, we report a fast and easy-to-use coating method to prevent the nonspecific adhesion of cells on tissue culture polystyrene (TCPS) surfaces. Our approach is based on the adsorption of poly(styrene-co-3-sulfopropyl methacrylate) copolymers comprising nonfouling hydrophilic moieties as well as functionalities that enhance the polymer adhesion to the substrate. The polymer adsorption is obtained from an aqueous mixture with a procedure that can be conveniently performed in short time. The kinetic of polymer adsorption as well as the effect of the polymer concentration was studied in order to reduce the time and cost of the entire procedure. Additionally, the polymer composition and the polymer density were optimized to completely avoid the adhesion of three different adherent cell lines, that is, CHO, A375-P, and HFF-1 cells.

Investigating How Organic Solvents Affect Tissue Culture Polystyrene Surfaces through Responses of Mesenchymal Stem Cells.

Polymer coating of tissue culture polystyrene (TCPS) surfaces promotes their biofunctionality, which can aid manipulation of cellular functions. However, the effect of the solvent used for polymer coating is yet to be elucidated. In this study, solvent-treated TCPS surfaces using water, methanol, ethanol, 2-propanol, and dimethyl sulfoxide are fabricated. Solvent treatment of TCPS surfaces is performed by spreading solvents onto the surfaces and allowing them to dry. Solvent treatment changes the surface roughness and wettability, depending on the kind of solvents. In addition, these surface property changes affected the extension, proliferation, and differentiation of human bone marrow-derived mesenchymal stem cells. These results suggest that solvent selection for polymer coating is crucial in the regulation of cell responses. Further, treatment with an appropriate solvent can result in a more suitable culture environment for modulating cellular functions.

Influence of Cell Spreading Area on the Osteogenic Commitment and Phenotype Maintenance of Mesenchymal Stem Cells

Osteogenic differentiation and commitment of mesenchymal stem cells (MSCs) is a complex process that is induced and regulated by various biological factors and biophysical cues. Although cell spreading area, as a biophysical cue, has been demonstrated to play a critical role in the regulation of osteogenic differentiation of MSCs, it is unclear how it affects the maintenance of the committed phenotype after osteogenic differentiation of MSCs. In this study, poly (vinyl alcohol) was micropatterned on a tissue culture polystyrene surface, and the micropatterns were used to culture MSCs to control their cell spreading area. The influence of cell spreading area on osteogenic differentiation and maintenance of the differentiated phenotype of MSCs was investigated. MSCs with a larger spreading area showed a higher degree of osteogenic differentiation, slower loss of differentiated phenotype and slower re-expression of stem cell markers compared with MSCs with a smaller spreading area. A large cell spreading area was beneficial for osteogenic differentiation of MSCs and maintenance of their differentiated phenotype.

Construction of novel temperature-responsive hydrogel culture system based on the biomimetic method for stem cell sheet harvest

Temperature-responsive hydrogel culture system is considered as an ideal platform for cell sheet harvest, but its complex preparation methods and harsh reaction conditions limit its application. Inspired by the marine mussels, a biomimetic method presented here is to construct a novel temperature-responsive hydrogel culture system for stem cell sheet harvest. The tissue culture polystyrene is first modified with polydopamine coating, and then amine-terminated poly(N-isopropylacrylamide) is grafted onto the coating via the Schiff base or Michael addition reaction to construct the temperature-sensitive hydrogel culture system. Then, bone marrow stromal cells are cultured on the culture system to construct cell sheets. The prepared culture system shows significant temperature-sensitive property with the grafted concentrations of poly(N-isopropylacrylamide) ranging from 0.5 to 1 g/L. Meanwhile, the constructed culture system has low cytotoxicity and facilitates the stem cell adhesion, proliferation, and cell sheet formation at 37°C. When the culture system is placed in a 20°C environment, the cell sheet can be completely detached from the surface of tissue culture polystyrene without being treated with any enzymes. More importantly, the cell morphology, cell sheet thickness, and the fibril structure of the associated proteins are similar to the cells cultured on the tissue culture polystyrene without modification. The biomimetic, simple, inexpensive, and environmentally friendly preparation of the culture system enables it to be used for the harvest of cell sheet and even applied to tissue engineering for tissue regeneration.

Sulfonated Polyrotaxane Surfaces with Basic Fibroblast Growth Factor Alter the Osteogenic Potential of Human Mesenchymal Stem Cells in Short-Term Culture.

Human mesenchymal stem cells (hMSCs) are prone to senescence and lose their differentiation potential when expanded under nonfavorable conditions. This leads to the underutilization of hMSCs in clinical situations such as bone regeneration. The use of growth factors and small molecules as supplements and changing the physical properties of the cell culture surface have been explored to maintain the self-renewal and differentiation potential of hMSCs during the in vitro expansion phase. Here, we have explored the effect of polyrotaxanes (PRXs) with different molecular mobilities along with either soluble or immobilized fibroblast growth factor 2 (FGF2) in the maintenance of the osteogenic differentiation potential of hMSCs during in vitro expansion. We found that a less expanded shape of the hMSCs was associated with highly mobile PRX surfaces, and less mobile PRX surfaces led to flattened cell morphology. The presence of FGF2 induced further expansion of the cell shape and size. The immobilization of FGF2 helped to improve the yield of hMSCs on highly mobile surfaces by promoting cell attachment to the surfaces. hMSCs cultured on highly mobile PRX surfaces exhibited poor actin cytoskeletal organization and retention of the transcriptional regulator, yes-associated protein (YAP), in cytoplasm in contrast to the hMSCs on less mobile PRX surfaces. When the hMSCs that proliferated under these conditions were collected and subjected to osteogenic differentiation on tissue culture polystyrene (TCPS) surfaces, we found that only the hMSCs cultured on highly mobile PRXs with FGF2 in both soluble and immobilized forms showed mineralization indicative of osteogenic differentiation. Further, we found that hMSCs cultured on highly mobile PRX surfaces expressed higher levels of stemness marker genes, Nanog and Oct4. These results indicate that culturing hMSCs on PRX surfaces with different molecular mobilities even for a short period of time (4 days) was sufficient to cause a drastic change in the osteogenic potential. From these results, it is suggested that apart from the use of supplements such as FGF2 in its freely soluble or immobilized form, the consideration of proper molecular mobility of the substrates could enable us to design better culture conditions for the hMSCs with osteogenic potential.

Cell sheet tissue engineering: Cell sheet preparation, harvesting/manipulation, and transplantation.

Cell sheet tissue engineering is a concept for creating transplantable two-dimensional (2D) and three-dimensional (3D) tissues and organs. This review describes three elements of cell sheet tissue engineering in terms of the chemical and physical effects of material surfaces and the interfacial properties of cell sheets: preparation, harvesting/manipulation and transplantation of cell sheets. An essential technology for the preparation of cell sheets is the use of a temperature-responsive cell culture surface, where the surface of tissue culture polystyrene (TCPS) dish is modified with thin layer of temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). PIPAAm-immobilized TCPS allows cultured cells to be harvested as a contiguous cell sheet with extracellular matrices (ECMs) by reducing the temperature, while chemical and physical disruption impair ECMs, cell-cell junction, and membrane proteins. Ligand-immobilized and porous hydrophilic PIPAAm-grafted surfaces are able to accelerate cell sheet preparation and harvesting, respectively. In addition, the manipulation of harvested cell sheets with the aid of cell sheet manipulator facilitates the formation of 3D tissues. Cell sheet-based tissues and their transplantation are in seven clinical settings such as heart, cornea, esophagus, periodontal, middle chamber of ear, knee cartilage, and lung. In order to create thick and large 3D tissues and organs, large production of differentiated parenchymal cells from induced pluripotent stem (iPS) cells and vascularization within 3D tissues are key issues. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 955-967, 2019.

Milieu for Endothelial Differentiation of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (hASCs) have been shown to differentiate down many lineages including endothelial lineage. We hypothesized that hASCs would more efficiently differentiate toward the endothelial lineage when formed as three-dimensional (3D) spheroids and with the addition of vascular endothelial growth factor (VEGF). Three conditions were tested: uncoated tissue culture polystyrene (TCPS) surfaces that induced a 2D monolayer formation; elastin-like polypeptide (ELP)-collagen composite hydrogel scaffolds that induced encapsulated 3D spheroid culture; and ELP-polyethyleneimine-coated TCPS surfaces that induced 3D spheroid formation in scaffold-free condition. Cells were exposed to endothelial differentiation medium containing no additional VEGF or 20 and 50 ng/mL of VEGF for 7 days and assayed for viability and endothelial differentiation markers. While endothelial differentiation media supported endothelial differentiation of hASCs, our 3D spheroid cultures augmented this differentiation and produced more von Willebrand factor than 2D cultures. Likewise, 3D cultures were able to uptake LDL, whereas the 2D cultures were not. Higher concentrations of VEGF further enhanced differentiation. Establishing angiogenesis is a key factor in regenerative medicine. Future studies aim to elucidate how to produce physiological changes such as neoangiogenesis and sprouting of vessels which may enhance the survival of regenerated tissues.

Facile protein-resistant and anti-biofilm surface coating based on catechol-conjugated poly(N-vinylpyrrolidone)

A facile biofouling-resistant surface coating was developed based on catechol-conjugated poly(N-vinylpyrrolidone) (CA-PVP). CA-PVP can be securely and effectively coated on glass, gold, and tissue culture polystyrene (TCPS) surfaces under a mild condition as characterized by ATR-FTIR and PVP-complexation with iodine and hydrogen peroxide. PVP itself without catechol conjugation, on the other hand, cannot achieve well-coating on surfaces. The coated surfaces became more hydrophilic and protein-resistant due to the presence of PVP as measured by the reduced contact angles and surface plasmon resonance (SPR) responses. The significantly enhanced protein resistance created by CA-PVP coatings leads to its significant anti-biofilm and fibroblast cell adhesion resistance as observed by fluorescent microscope. CA-PVP coating also demonstrated its high biocompatibility that ≥ 95% cell viability of mouse fibroblast cells still can be maintained after 48 h cultivation.

Physical stimulation and scaffold composition efficiently support osteogenic differentiation of mesenchymal stem cells.

Despite significant achievements in the field of tissue engineering, simplification and improvement of the existing protocols are of great importance. The use of complex differentiation media, due to the presence of multiple factors, may have some undesired effects on cell health and functions. Thus, minimizing the number of involved factors, while maintaining the differentiation efficiency, provides less costly and controllable conditions. Adipose-derived Mesenchymal stem cells (ASCs), the adult stem cells present in adipose tissue, can be a suitable source of stem cells due to abundant and ease of access. The aim of this study is to optimize the osteogenic differentiation of ASCs by chemical composition of scaffold, in the first step, and then by electromagnetic treatments. ASCs were cultured on PVA/PES scaffold and tissue culture polystyrene surfaces (TCPS) and osteogenic differentiation was performed with either osteogenic medium, or electromagnetic field or both. The impact of each treatment on ASCs growth and proliferation was measured by MTT assay. Changes in gene expression levels of osteogenic-specific markers including ALP and RUNX2 were determined by Real Time PCR. Furthermore, alkaline phosphatase activity and calcium deposition were measured. The MTT assay showed the significant effects on cell growth and respiration in scaffold-seeded ASCs treated with electromagnetic field, compared to control TCPS plate. Also, the electromagnetic treatment, increased alkaline phosphatase activity and calcium deposition. Finally, Real Time PCR showed higher expression of ALP and RUNX2 genes in electromagnetic field groups compared to control groups. It can be concluded that PVA/PES scaffold used in this study improved the osteogenic capacity of ASCs. Moreover, the osteogenic potential of ASCs seeded on PVA/PES scaffold could be augmented by electromagnetic field without any chemical stimulation.

Extracellular matrix derived from periodontal ligament cells maintains their stemness and enhances redifferentiation via the wnt pathway.

Large numbers of viable cells cannot be obtained from periodontal ligament tissues of patients with periodontitis. Therefore, it is imperative to establish an ex vivo environment that can support cell proliferation and delay senescence. Here, we have successfully reconstructed a native extracellular matrix (ECM), derived from early-passage human periodontal ligament cells (PDLCs) using the NH4 OH/Triton X-100 protocol. The ECM was investigated by scanning electron microscopy and immunostaining for specific ECM proteins (collagen I and fibronectin). Late-passage ECM-expanded PDLCs exhibited a much higher proliferation index and lower levels of reactive oxygen species (ROS), confirmed by the increased expression of pluripotent markers and enhanced osteogenic capacity. Interestingly, the Wnt pathway was suppressed during the ECM expansion-mediated increase in pluripotency, but was activated in an osteogenic differentiation environment, as confirmed by treatment with the XAV-939 β-catenin inhibitor or the SP600125 c-Jun N-terminal kinase (JNK) inhibitor. Cell sheets formed by ECM-expanded PDLCs exhibited an enhanced periodontal tissue regeneration capacity compared to those formed on tissue culture polystyrene (TCP) surfaces in vivo. Taken together, the cell-free ECM provides a tissue-specific cell niche for the ex vivo expansion of PDLCs while retaining stemness and osteogenic potential, partially via the Wnt pathway. This represents a promising matrix for future applications in periodontal tissue regeneration therapy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 272-284, 2018.

Uniform neural tissue models produced on synthetic hydrogels using standard culture techniques.

The aim of the present study was to test sample reproducibility for model neural tissues formed on synthetic hydrogels. Human embryonic stem (ES) cell-derived precursor cells were cultured on synthetic poly(ethylene glycol) (PEG) hydrogels to promote differentiation and self-organization into model neural tissue constructs. Neural progenitor, vascular, and microglial precursor cells were combined on PEG hydrogels to mimic developmental timing, which produced multicomponent neural constructs with 3D neuronal and glial organization, organized vascular networks, and microglia with ramified morphologies. Spearman's rank correlation analysis of global gene expression profiles and a comparison of coefficient of variation for expressed genes demonstrated that replicate neural constructs were highly uniform to at least day 21 for samples from independent experiments. We also demonstrate that model neural tissues formed on PEG hydrogels using a simplified neural differentiation protocol correlated more strongly to invivo brain development than samples cultured on tissue culture polystyrene surfaces alone. These results provide a proof-of-concept demonstration that 3D cellular models that mimic aspects of human brain development can be produced from human pluripotent stem cells with high sample uniformity between experiments by using standard culture techniques, cryopreserved cell stocks, and a synthetic extracellular matrix. Impact statement Pluripotent stem (PS) cells have been characterized by an inherent ability to self-organize into 3D "organoids" resembling stomach, intestine, liver, kidney, and brain tissues, offering a potentially powerful tool for modeling human development and disease. However, organoid formation must be quantitatively reproducible for applications such as drug and toxicity screening. Here, we report a strategy to produce uniform neural tissue constructs with reproducible global gene expression profiles for replicate samples from multiple experiments.

Robust Generation of Quiescent Porcine Valvular Interstitial Cell Cultures.

BackgroundValvular interstitial cells (VICs) in the healthy aortic valve leaflet exhibit a quiescent phenotype, with <5% of VICs exhibiting an activated phenotype. Yet, in vitro culture of VICs on tissue culture polystyrene surfaces in standard growth medium results in rapid transformation to an activated phenotype in >90% of cells. The inability to preserve a healthy VIC phenotype during in vitro studies has hampered the elucidation of mechanisms involved in calcific aortic valve disease. This study describes the generation of quiescent populations of porcine VICs in 2‐dimensional in vitro culture and their utility in studying valve pathobiology.Methods and ResultsWithin 4 days of isolation from fresh porcine hearts, VICs cultured in standard growth conditions were predominantly myofibroblastic (activated VICs). This myofibroblastic phenotype was partially reversed within 4 days, and fully reversed within 9 days, following application of a combination of a fibroblast media formulation with culture on collagen coatings. Specifically, culture in this combination significantly reduced several markers of VIC activation, including proliferation, apoptosis, α‐smooth muscle actin expression, and matrix production, relative to standard growth conditions. Moreover, VICs raised in a fibroblast media formulation with culture on collagen coatings exhibited dramatically increased sensitivity to treatment with transforming growth factor β1, a known pathological stimulus, compared with VICs raised in either standard culture or medium with a fibroblast media formulation.ConclusionsThe approach using a fibroblast media formulation with culture on collagen coatings generates quiescent VICs that more accurately mimic a healthy VIC population and thus has the potential to transform the study of the mechanisms of VIC activation and dysfunction involved in the early stages of calcific aortic valve disease.