Tissue Culture Polystyrene Dishes Research Articles

AMPK activation enhances osteoblast differentiation on a titanium disc via autophagy

PurposeThe acquisition of osseointegration during implant therapy is slower and poorer in patients with diabetes compared with healthy persons. The serum concentration of adiponectin in patients with type II diabetes is lower than that of healthy persons via the suppression of AMP-activated protein kinase (AMPK). Therefore, we hypothesized that the AMPK activation enhances bone formation around implants, resulting in the improved acquisition of osseointegration. The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium disc (Ti).MethodsConfluent mouse pre-osteoblasts (MC3T3-E1) cells (1 × 105 cells/well) were cultured with BMP-2 for osteoblast differentiation, in the presence or absence AICAR, an AMPK activator. We examined the effects of AMPK activation on osteoblast differentiation and the underlying mechanism on a Ti using a CCK8 assay, a luciferase assay, quantitative RT-PCR, and western blotting.ResultsAlthough the proliferation rate of osteoblasts was not different between a Ti and a tissue culture polystyrene dish, the addition of AICAR, AMPK activator slightly enhanced osteoblast proliferation on the Ti. AICAR enhanced the BMP-2-dependent transcriptional activity on the Ti, leading to upregulation in the expression of osteogenesis-associated molecules. AICAR simultaneously upregulated the expression of autophagy-associated molecules on the Ti, especially LC3-II. AdipoRon, an adiponectin receptor type1/type2 activator activated AMPK, and upregulated osteogenesis-associated molecules on Ti.ConclusionsAMPK activation enhances osteoblast differentiation on a Ti via autophagy, suggesting that it promotes the acquisition of osseointegration during implant therapy.

Xeno-free culture and proliferation of hPSCs on 2D biomaterials.

Human pluripotent stem cells (human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)) have unlimited proliferative potential, whereas adult stem cells such as bone marrow-derived stem cells and adipose-derived stem cells have problems with aging. When hPSCs are intended to be cultured on feeder-free or xeno-free conditions without utilizing mouse embryonic fibroblasts or human fibroblasts, they cannot be cultured on conventional tissue culture polystyrene dishes, as adult stem cells can be cultured but should be cultivated on material surfaces grafted or coated with (a) natural or recombinant extracellular matrix (ECM) proteins, (b) ECM protein-derived peptides and specific synthetic polymer surfaces in xeno-free and/or chemically defined conditions. This review describes current developing cell culture biomaterials for the proliferation of hPSCs while maintaining the pluripotency and differentiation potential of the cells into 3 germ layers. Biomaterials for the cultivation of hPSCs without utilizing a feeder layer are essential to decrease the risk of xenogenic molecules, which contributes to the potential clinical usage of hPSCs. ECM proteins such as human recombinant vitronectin, laminin-511 and laminin-521 have been utilized instead of Matrigel for the feeder-free cultivation of hPSCs. The following biomaterials are also discussed for hPSC cultivation: (a) decellularized ECM, (b) peptide-grafted biomaterials derived from ECM proteins, (c) recombinant E-cadherin-coated surface, (d) polysaccharide-immobilized surface, (e) synthetic polymer surfaces with and without bioactive sites, (f) thermoresponsive polymer surfaces with and without bioactive sites, and (g) synthetic microfibrous scaffolds.

Hybrid coatings for orthopaedic implants formed by physical vapour deposition and microarc oxidation

This study is focused on the preparation of new hybrid layers intended for surface modification of Ti-6Al-4V alloys for potential orthopaedic and dental applications. Combination of the technology of physical vapour deposition (PVD) and subsequent micro-arc oxidation (MAO) was utilized for the deposition of Ti and ZrTi to form hybrid oxide layers. The oxide layers were prepared using an alkaline electrolyte with glycerol as an additive under micro-arc discharge conditions with different Si content on their surfaces. The hybrid ZrTi coatings with a Zr/Si structure achieved the best tribological properties described by a low friction coefficient of 0.3 and high abrasion resistance. There was also an increase in corrosion potential and polarization resistance of hybrid ZrTi coatings. Although the proliferation of human bone marrow mesenchymal stem cells was slower on these hydrophilic Ti and ZrTi coatings than both on uncoated Ti-6Al-4V and the reference tissue culture polystyrene dishes, both types of hybrid coating promoted greater osteogenic differentiation of these cells, indicated by approx. twice as high activity of alkaline phosphatase. The hybrid oxide layers newly developed in this study – especially the layers with Zr – are therefore promising for coating metallic bone implants.

Effect of extracellular matrix proteins on the differentiation of human pluripotent stem cells into mesenchymal stem cells.

The transplantation of human mesenchymal stem cells (hMSCs), such as bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs), has shown beneficial effects in protecting transplanted tissues and cells against graft-versus-host disease (GVHD). Human pluripotent stem cell (hPSC)-derived mesenchymal stem cells (MSCs) can also be used to generate hMSCs with stable characteristics without limitations. Therefore, we differentiated human induced pluripotent stem cells (hiPSCs, H-M5) and human embryonic stem cells (hESCs, H9) into hMSCs on dishes coated with different extracellular matrix (ECM) proteins to study the effect of cell culture biomaterials on hPSC differentiation into hMSCs. hPSC-derived MSCs cultured on Matrigel (MAT)-coated, collagen (COL)-coated and laminin-521 (LN-521)-coated tissue culture polystyrene (TCP) dishes showed excellent proliferation speed and reduced aging over 10 passages. High MSC surface marker (CD44, CD73, CD90 and CD105) expression was also observed on hPSC-derived MSCs cultured on MAT-coated, COL-coated and LN-521-coated TCP dishes as well as uncoated TCP dishes. Analysis of late osteogenic differentiation by evaluation of mineral deposition revealed that hPSC-derived MSCs cultured on fibronectin (FN)-coated and LN-521-coated TCP dishes showed high osteogenic differentiation. ECM proteins are effective as coating materials on cell culture biomaterials to regulate the proliferation and differentiation fate of hPSC-derived MSCs.

Enhanced culturing of adipose derived mesenchymal stem cells on surface modified polystyrene Petri dishes fabricated by plasma enhanced chemical vapor deposition system.

Mesenchymal stem cells (MSCs) have received considerable attention as therapeutic cells for regenerative medicine and tissue engineering, because of their ability to replace damaged cells or regenerate surrounding cells. There are many technical difficulties in the mass production of high-quality stem cells because the stem cells must maintain an efficient proliferative cell state during in vitro culture. The results of this study show that plasma surface-modification enhanced significantly the culture of adipose-derived mesenchymal stem cells (ASCs) on the polystyrene (PS) Petri dishes. Ar, O2 , pyrrole, and 4,7,10-trioxa-1,13-tridecanediamine (TTDDA) were used as the gas and/or precursors for plasma modification. Specifically, surfaces of PS Petri dishes, coated with plasma polymerized pyrrole (ppPy) and plasma polymerized TTDDA (ppTTDDA) were found to contain amine and carboxyl functional groups, respectively. Ar and O2 plasma-treated PS Petri dishes have similar culture abilities (±1.2 times) to commercially available tissue culture polystyrene (TCPS) dishes, and PS Petri dishes coated with ppPy and ppTTDDA have significantly enhanced culture abilities (2.4 times) at 96 hr compared with TCPS dishes. Western blotting was performed using antibodies against stem cell marker proteins to confirm the stemness properties of stem cells, in the sense that the expressions of the antibody proteins such as CD44, CD73, and CD105 in plasma modified samples were similar to or higher than those in TCPS dishes.

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.

Adhesion, proliferation, and detachment of cells on poly(N-isopropyl acrylamide) brushes tethered on polystyrene using surface-initiated atom transfer radical polymerization

The preparation of thermo-responsive cell culture dishes through grafting poly(N-isopropyl acrylamide) (PNIPAAm) brushes is described. The effect of different treatments, including ultraviolet-ozone (UVO) and ozone exposure on hydroxyl functionalization of the tissue culture polystyrene (TCPS) dishes, was investigated. The surface of TCPS dishes was activated by UVO irradiation, and the synthesized initiator was immobilized on the surface. PNIPAAm brushes were yielded by surface-initiated atom transfer radical polymerization (SI-ATRP). Surface composition and properties of grafted PNIPAAm on polystyrene surface were studied by X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), energy-dispersive X-ray (EDX) spectroscopy, and water contact angle (WCA). The results show that PNIPAAm brushes were successfully grafted on the TCPS, and the WCA of the surface was raised from 33 to 64° by increasing the temperature from 20 to 40 °C, which is suitable for cell sheet detachment. Different cell types, including MG, endothelial, and fibroblast cells, were cultured on the PNIPAAm-grafted TCPS. The cell culture results show that the attachment to the surface and proliferation were successfully carried out. The formed cell sheet was also detached by decreasing the temperature from 37 to 20 °C. Therefore, the grafted PNIPAAm brushes are useful for the preparation of cell sheets from different cell types.

A Chemically Defined Serum-Free Culture System for Spontaneous Human Mesenchymal Stem Cell Spheroid Formation.

Mesenchymal stem cells (MSCs) possess promising potential in tissue engineering and regenerative medicine. Previous studies demonstrated that spheroid formation of MSCs exhibited improved stemness maintenance and therapeutic potential compared with monolayer culture. To date, various spheroid culture systems have been developed but most of them required low adhesion conditions or special equipment. In this study, we demonstrated that inoculation of dissociated MSCs in TeSR-E8 medium could induce self-assemble spheroid formation in conventional tissue culture polystyrene dishes. Compared with monolayer culture, adipose-derived stem cell (ADSC) spheroids enhanced the proliferation and osteogenic capability of ADSCs compared with monolayer culture. When reseeded in normal serum-containing medium, the expression level of stemness biomarkers was even higher in spheroid-derived ADSCs than monolayer culture. Importantly, spheroid ADSCs could effectively promote the M2 polarization of macrophages both in vitro and in vivo. After transplantation into mouse, spheroid ADSCs improved the survival rate and significantly decreased serum levels of proinflammatory factors IL-1β and TNF-α following LPS challenge. In summary, we developed a 3D spheroid culture system through TeSR-E8 medium without the involvement of low adhesion conditions and special equipment, which provided a practical and convenient method for spheroid formation of MSCs with great potential for stem cell clinical therapy.

Design of injectable hydrogels of gelatin and alginate with ferric ions for cell transplantation

The objective of this study is to design bioabsorbable injectable hydrogels based on the physico-chemical interaction between biocompatible polymers and ferric ions, and evaluate the survival, proliferation, and osteogenic differentiation of cells encapsulated in the hydrogels. The injectable hydrogels were prepared by simply mixing mixed alginate/gelatin solution at various ratios and FeCl3 solution. The hydrogels prepared disappeared within a few days in the phosphate buffered-saline solution (PBS) with containing collagenase although the disappearance rate increased with an increase of the gelatin ratio in the hydrogel. For the hydrogel of alginate/gelatin low ratio, the survival and proliferation of cells in the hydrogel-encapsulated condition were significantly high compared with those of hydrogel at the higher ratios. The cells collected 3 days after cultured in the hydrogel also proliferated to a significantly higher extent than those collected from other hydrogels. The proliferation ability of cells was similar that of cells cultured on the standard tissue culture polystyrene (TCPS) dish. When evaluated to compare with cells cultured on the TCPS dish, the expression of runt-related transcription factor-2 (RUNX2) gene, the alkaline phosphatase (ALP) activity, and the calcium precipitation were significantly high. The cells were encapsulated by the mixed alginate/gelatin and FeCl3 hydrogel and injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the injectable hydrogel prepared by simple mixing mixed alginate/gelatin solution and FeCl3 solution is a promising material for the cell transplantation. Statement of SignificanceInjectable hydrogels prepared by simple mixing mixed alginate/gelatin solution at various ratios and FeCl3 solution. For the hydrogel of alginate/gelatin low ratio, the survival, the proliferation, and the differentiate properties of cells in the hydrogel-encapsulated condition were similar those of cells cultured on the TCPS dish. When the cells encapsulated hydrogels were injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the present injectable hydrogel is a promising material for the cell transplantation.

Neuropeptide Y increases differentiation of human olfactory receptor neurons through the Y1 receptor

Olfactory dysfunction significantly impedes the life quality of patients. Neuropeptide Y (NPY) is not only a neurotrophic factor in the rodent olfactory system but also an orexigenic peptide that regulates feeding behavior. NPY increases the olfactory receptor neurons (ORNs) responsivity during starvation; however, whether NPY can promote differentiation of human ORNs remains unexplored. This study investigates the effect of NPY on the differentiation of human olfactory neuroepithelial cells in vitro. Human olfactory neuroepithelium explants were cultured on tissue culture polystyrene dishes for 21 days. Then, cells were cultured with or without NPY at the concentration of 0.5 ng/mℓ for 7 days. The effects of treatment were assessed by phase contrast microscopy, immunocytochemistry and western blot analysis. The further mechanism was evaluated with NPY Y1 receptor-selected antagonist BIBP3226. NPY-treated olfactory neuroepithelial cells exhibited thin bipolar shape, low circularity, low spread area, and long processes. The expression levels of Ascl1, βIII tubulin, GAP43 and OMP were significantly higher in NPY-treated cells than in controls (p < 0.05). NPY-treated olfactory neuroepithelial cells expressed more components of signal transduction apparatuses, Golf and ADCY3, than those without NPY treatment. Western blot analysis also further confirmed these findings (p < 0.05). Additionally, the expression levels of Ascl1, βIII2 tubulin, GAP43, OMP, ADCY3, and Golf in BIBP3226 + NPY and controls were comparable (p > 0.05). NPY not only increases expressions of protein markers of human olfactory neuronal progenitor cells, but also promotes differentiation of ORN and enhances formation of components of olfactory-specific signal transduction pathway through Y1 receptors.

Design of Injectable Hydrogels of Gelatin and Alginate with Ferric Ions for Cell Transplantation

The objective of this study is to design bioabsorbable injectable hydrogels based on the physico-chemical interaction between biocompatible polymers and ferric ions, and evaluate the survival, proliferation, and osteogenic differentiation of cells encapsulated in the hydrogels. The injectable hydrogels were prepared by simply mixing mixed alginate/gelatin solution at various ratios and FeCl3 solution. The hydrogels prepared disappeared within a few days in the phosphate buffered-saline solution (PBS) with containing collagenase although the disappearance rate increased with an increase of the gelatin ratio in the hydrogel. For the hydrogel of alginate/gelatin low ratio, the survival and proliferation of cells in the hydrogel-encapsulated condition were significantly high compared with those of hydrogel at the higher ratios. The cells collected 3 days after cultured in the hydrogel also proliferated to a significantly higher extent than those collected from other hydrogels. The proliferation ability of cells was similar that of cells cultured on the standard tissue culture polystyrene (TCPS) dish. When evaluated to compare with cells cultured on the TCPS dish, the expression of runt-related transcription factor-2 (RUNX2) gene, the alkaline phosphatase (ALP) activity, and the calcium precipitation were significantly high. The cells were encapsulated by the mixed alginate/gelatin and FeCl3 hydrogel and injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the injectable hydrogel prepared by simple mixing mixed alginate/gelatin solution and FeCl3 solution is a promising material for the cell transplantation.

Characteristic differences of cell sheets composed of mesenchymal stem cells with different tissue origins.

IntroductionStem cell therapy with mesenchymal stem cells (MSCs) has been widely used in many clinical trials, and therapy with MSC sheets shows promise for patients. However, there are few reports characterizing MSC sheets. In the present study, the properties of MSC sheets derived from bone marrow, adipose tissue, and umbilical cord were evaluated.MethodsCell sheets were fabricated with MSCs from different tissue origins in temperature-responsive cell culture dishes with and without pre-coating of fetal bovine serum (FBS). MSC adhesion behavior in the culture dish was observed. Secretion of cytokines related to cell proliferation and immune regulation from MSC sheets was investigated by ELISA. The adhesion properties of the MSC sheets were investigated by time-lapse microscopy.ResultsDifferent cell adhesion and proliferation rates in temperature-responsive cell culture dishes were observed among the three types of MSCs. FBS pre-coating of the dishes enhanced cell attachment and proliferation in all cell types. Harvested cell sheets showed high attachment capacity to tissue culture polystyrene dish surfaces.ConclusionsMSC sheets can be fabricated from MSCs from different tissue origins using temperature-responsive cell culture dishes. The fabricated MSC sheets could be useful in cell transplantation therapies by choosing appropriate types of MSCs that secrete therapeutic cytokines for the targeted diseases.

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.

Laser-synthesized nanocrystalline, ferroelectric, bioactive BaTiO3/Pt/FS for bone implants.

The goal of our study is to design BaTiO3 ferroelectric layers that will cover metal implants and provide improved osseointegration. We synthesized ferroelectric BaTiO3 layers on Pt/fused silica substrates, and we studied their physical and bio-properties. BaTiO3 and Pt layers were prepared using KrF excimer laser ablation at substrate temperature Ts in the range from 200°C to 750°C in vacuum or under oxygen pressure of 10 Pa, 15 Pa, and 20 Pa. The BaTiO3/Pt and Pt layers adhered well to the substrates. BaTiO3 films of crystallite size 60-140 nm were fabricated. Ferroelectric loops were measured and ferroelectricity was also confirmed using Raman scattering measurements. Results of atomic force microscopy topology and the X-ray diffraction structure of the BaTiO3/Pt/fused silica multilayers are presented. The adhesion, viability, growth, and osteogenic differentiation of human osteoblast-like Saos-2 cells were also studied. On days 1, 3, and 7 after seeding, the lowest cell numbers were found on non-ferroelectric BaTiO3, while the values on ferroelectric BaTiO3, on non-annealed and annealed Pt interlayers, and on the control tissue culture polystyrene dishes and microscopic glass slides were similar, and were usually significantly higher than on non-ferroelectric BaTiO3. A similar trend was observed for the intensity of the fluorescence of alkaline phosphatase, a medium-term marker of osteogenic differentiation, and of osteocalcin, a late marker of osteogenic differentiation. At the same time, the cell viability, tested on day 1 after seeding, was very high on all tested samples, reaching 93-99%. Ferroelectric BaTiO3 films deposited on metallic bone implants through a Pt interlayer can therefore markedly improve the osseointegration of these implants in comparison with non-ferroelectric BaTiO3 films.

Methylcellulose Based Thermally Reversible Hydrogels.

The creation of single and multilayered adult stem cells (ASCs) sheets is presented. The stem cell sheets preserve the cell-cell and cell-extracellular matrices and are developed by utilizing a thermally reversible methylcellulose (MC) coated tissue culture polystyrene (TCPS) dish. This technique is an improvement and a simplification of earlier noninvasive cell retrieval methods based on the use of a temperature-responsive poly(N-isopropylacrylamide) (PIPAAm) coated TCPS dishes. The optimal combination of MC-water-salt was determined to be 12-14% of MC (mol. wt. of 15,000) in water with 0.5× PBS (~150 mOsm). This solution exhibited a gel formation temperature of ~32 °C. The addition (evenly spread) of 1 ml of 3 mg/ml rat tail type-I (pH adjusted to 7.5) over the MC coated surface at 37 °C improves ASC adhesion and proliferation on the methylcellulose system. Upon confluence, a continuous monolayer ASC sheet was formed on the surface of the MC hydrogel system. When the grown cell sheet was removed from the incubator and exposed to room temperature (~30 °C), it spontaneously and gradually detached from the surface of the thermoresponsive hydrogel, creating an ASC sheet.

Morphological adaptations in breast cancer cells as a function of prolonged passaging on compliant substrates.

Standard tissue culture practices involve propagating cells on tissue culture polystyrene (TCP) dishes, which are flat, 2-dimensional (2D) and orders of magnitude stiffer than most tissues in the body. Such simplified conditions lead to phenotypical cell changes and altered cell behaviors. Hence, much research has been focused on developing novel biomaterials and culture conditions that more closely emulate in vivo cell microenvironments. In particular, biomaterial stiffness has emerged as a key property that greatly affects cell behaviors such as adhesion, morphology, proliferation and motility among others. Here we ask whether cells that have been conditioned to TCP, would still show significant dependence on substrate stiffness if they are first pre-adapted to a more physiologically relevant environment. We used two commonly utilized breast cancer cell lines, namely MDA-MB-231 and MCF-7, and examined the effect of prolonged cell culturing on polyacrylamide substrates of varying compliance. We followed changes in cell adhesion, proliferation, shape factor, spreading area and spreading rate. After pre-adaptation, we noted diminished differences in cell behaviors when comparing between soft (1 kPa) and stiff (103 kPa) gels as well as rigid TCP control. Prolonged culturing of cells on complaint substrates further influenced responses of pre-adapted cells when transferred back to TCP. Our results have implications for the study of stiffness-dependent cell behaviors and indicate that cell pre-adaptation to the substrate needs consideration.

Effect of stem cell niche elasticity/ECM protein on the self-beating cardiomyocyte differentiation of induced pluripotent stem (iPS) cells at different stages

The transplantation of beating cardiomyocytes (BCMs) is expected to be made more effective for serious heart diseases. The identification of the appropriate engineering processes and suitable culture substrates for inducing stem cell differentiation into BCMs is thus indispensable. The differentiation can be divided into three major processes, the cardiac differentiation step, the beating-induction step and the beating-duration step. A protocol with the higher efficiency in all of the steps must be useful. In this study, we separately evaluated the effect of culture substrates at each three step. We clarified that the biological and the physical properties of the culture substrates required at these steps were different. We found useful criteria for effective cardiac cell niche systems design.

A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting

The 3D bioprinting and cell/tissue printing techniques open new possibilities for future applications. To facilitate the 3D bioprinting process, a large amount of living cells are required. Induced pluripotent stem cells (iPSCs) represent a promising cell source for bioprinting. However, the maintenance and expansion of undifferentiated iPSCs are expensive and time consuming. Therefore, in this study a culture method to obtain a sufficient amount of healthy and undifferentiated iPSCs in a short-term period was established. The iPSCs could be passaged for twice on tissue culture polystyrene (TCPS) dish with the conditional medium and could adapt to the feeder-free environment. Feeder-free dishes were further prepared from chitosan, chitosan-hyaluronan, silk fibroin, and polyurethane (PU1 and PU2) two-dimensional substrates. The iPSCs cultured on the chitosan substrates showed a higher proliferation rate without losing the stemness feature. Among the different materials, PU2 could be prepared as a thermoresponsive hydrogel, which was a potential ink for 3D bioprinting. The iPSCs cultured on PU2 substrates well survived when further embedded in PU2 hydrogel. Moreover, PU2 hydrogel printed with iPSCs remained structural integrity. The use of PU2 hydrogel to embed iPSCs reduced the injury to iPSCs by shear stress. These results indicate that iPSCs could be expanded on chitosan or PU2 membranes without the feeder layer and then printed in PU2 hydrogel. The combination of these steps could offer a new possibility for future applications of iPSC-based 3D bioprinting in tissue engineering.

Polychromatic light-induced osteogenic activity in 2D and 3D cultures.

Photobiomodulation (PBM) has been applied to manipulate cellular responses by using monochromatic light in different wavelengths from ultraviolet (UV) to infrared (IR) region. Until now, an effective wavelength has not been revealed to induce proliferation and/or differentiation of cells. Therefore, in the presented study, we decided to use a specially designed plasma arc light source providing wavelengths between 590 and 1500nm in order to investigate its biomodulatory effects on chitosan scaffold-supported three-dimensional (3D) cell cultures. For comparison, two-dimensional (2D) cell cultures were also carried out in tissue-culture polystyrene dishes (TCPS). The results showed that light-induced temperature rise did not affect cells when the distance between the light source and the cells was 10cm and the frequency of administration was daily. Moreover, light was applied for 5 and 10min to the cells in TCPS and in chitosan scaffold groups, respectively. Cell culture studies under static conditions indicated that polychromatic light significantly stimulated bone nodule formation via the prolonged cell survival and stimulated differentiation of MC3T3-E1 preosteoblastic cells in both TCPS and chitosan scaffold groups. In conclusion, specially designed plasma arc light source used in this study induces formation of bone tissue and so, this light source is proposed as an appropriate system for in vitro bone tissue engineering applications. Statistical analyses were performed with one-way ANOVA by using GraphPad Instat software and standard deviations were calculated by using data of three parallel samples for each group.

Switching of growth factor binding on heparin-functionalized thermoresponsive surface for hepatocyte sheet manipulation with maintenance of hepatic functions

Event Abstract Back to Event Switching of growth factor binding on heparin-functionalized thermoresponsive surface for hepatocyte sheet manipulation with maintenance of hepatic functions Jun Kobayashi1, Yoshikatsu Akiyama1*, Masayuki Yamato1* and Teruo Okano1* 1 Tokyo Women's Medical University, Institute of Advanced Biomedical Engineering and Science, Japan Introduction: Liver tissue engineering using primary hepatocytes is an attractive method for the treatment of liver diseases[1]. Engineered Hepatocyte sheets were effectively engrafted in pre-vascularized subcutaneous site and exhibited liver-specific functionalities. By contrast, cultured hepatocytes rapidly lose their viability and phenotypic functions on isolation from the native in vivo microenvironment of the liver. To overcome this problem, heparin-functionalized poly(N-isopropylacrylamide) (PIPAAm)-grafted cell culture surface[2],[3], which interacts with heparin-binding proteins such as heparin-binding EGF-like growth factor (HB-EGF), was been designed for maintaining hepatic functions during the cultivation (Figure 1, left). In addition, the detachment of the cultured hepatocytes as a sheet was examined when lowering temperature to 20°C (Figure 1, right). Figure 1. Schematic of affinity regulation between heparin-binding protein and immobilized heparin by conformational change of PIPAAm. Materials and Methods: Heparin-functionalized thermoresponsive surfaces were prepared as described previously[2]. Briefly, poly(IPAAm-co-2-carboxyisopropylacrylamide) (poly(IPAAm-co-CIPAAm))-grafted surfaces on tissue culture polystyrene (TCPS) dishes were prepared by electron beam irradiation. Heparin was covalently immobilized on the poly(IPAAm-co-CIPAAm)-grafted dishes by condensing reaction. Then, affinity binding of HB-EGF was performed on the heparinized thermoresponsive surfaces by incubation of HB-EGF solution at 37°C for 24 h. Amounts of bound HB-EGF on the heparinized surface were quantified using [125I]-labeled HB-EGF. Primary rat hepatocytes were seeded on the dishes in DMEM with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2. Results: The addition of soluble HB-EGF in the cell culture media was essential for the survival of hepatocytes. When the medium contained less than 10 ng/cm2 of soluble HB-EGF, the hepatocytes were not able to adhere and form their cell sheets. By contrast, hepatocytes adhered and formed their sheets on heparin-functionalized thermoresponsive surface with 10 ng/cm2 of bound HB-EGF. In addition, the secretion of albumin on bound HB-EGF was maintained and higher than that on PIPAAm-grafted surfaces with soluble HB-EGF. Therefore, bound HB-EGF gave a high activity of maintenance of hepatocyte adhesion and function compared with soluble HB-EGF. Finally, when lowering temperature to 20°C, the cultured hepatocyte sheets were detached from the surface through the reduction of affinity binding between HE-EGF and immobilized heparin with increasing the mobility of heparin and steric hindrance of the swollen PIPAAm chains (Figure 1, right). Conclusions: Heparin-functionalized thermoresponsive cell culture surfaces facilitated the manipulation of hepatic cell sheets with maintaining hepatic functions by changing temperature. Creation of transferable and functional liver tissues is considered to have a potential to treat liver disease. Part of this work was financially supported by JSPS KAKENHI Grant Numbers 23106009 and 15K01317