The Influence of Tacrolimus on Cellular Morphology, Cellular Viability, Osteogenic Differentiation, and mRNA Expression within Stem Cell Spheroids.

Background and Objectives: Tacrolimus is a macrolide lactone compound derived from the bacterium Streptomyces tsukubensis, widely known as an immunosuppressant. In basic research, the effects of tacrolimus on osteogenic differentiation have been tested using mesenchymal stem cells. In this study, tacrolimus's effects on the cellular survival and osteogenic differentiation of stem cell spheroids were investigated. Materials and Methods: Concave microwells were used to form stem cell spheroids in the presence of tacrolimus at final concentrations of 0 µg/mL, 0.1 µg/mL, 1 µg/mL, 10 µg/mL, and 100 µg/mL. A microscope was used to test cellular vitality qualitatively, and an assay kit based on water-soluble tetrazolium salt was used to measure cellular viability quantitatively. Alkaline phosphatase activity and an anthraquinone dye test for measuring calcium deposits were used to assess osteogenic differentiation. To assess the expression of osteogenic differentiation, a quantitative polymerase chain reaction, Western blot, and RNA sequencing were performed. Results: Spheroids across all concentrations maintained a relatively uniform and spherical shape. Cell viability assay indicated that tacrolimus, up to a concentration of 100 µg/mL, did not significantly impair cell viability within spheroids cultured in osteogenic media. The increase in calcium deposition, particularly at lower concentrations of tacrolimus, points toward an enhancement in osteogenic differentiation. There was an increase in COL1A1 expression across all tacrolimus concentrations, as evidenced by the elevated mean and median values, which may indicate enhanced osteogenic activity. Conclusions: This study showed that tacrolimus does not significantly impact the viability of stem cell spheroids in osteogenic media, even at high concentrations. It also suggests that tacrolimus may enhance osteogenic differentiation, as indicated by increased calcium deposition and COL1A1 expression. These findings advance our understanding of tacrolimus's potential roles in tissue repair, regeneration, and stem cell-based therapeutic applications.

Endochondral Repair of Jawbone Defects Using Periosteal Cell Spheroids.

Recapitulation of the natural healing process is receiving increasing recognition as a strategy to induce robust tissue regeneration. Endochondral ossification has been recognized as an essential reparative approach in natural jawbone defect healing. However, such an approach has been overlooked in the recent development of cell-based therapeutics for jawbone repair. Therefore, this study aimed to explore a bioinspired stem cell-based strategy for jawbone repair by mimicking the mesenchymal condensation of progenitor cells during the early endochondral ossification process. For this purpose, passage 3 of jawbone periosteum-derived cells (jb-PDCs) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 96.4 ± 5.8 µm after 48 h. Compared to monolayer culture, the jb-PDC spheroids showed a significant reduction of stemness marker expression evidenced by flow cytometry. Furthermore, a significant upregulation of chondrogenic transcription factor SOX9 in both gene and protein levels was observed in the jb-PDC spheroids after 48 h of chondrogenic induction. RNA sequencing and Western blotting analysis further suggested that the enhanced SOX9-mediated chondrogenic differentiation in jb-PDC spheroids was attributed to the activation of the p38 MAPK pathway. Impressively, inhibition of p38 kinase activity significantly attenuated chondrogenic differentiation jb-PDC spheroids, evidenced by a significant decline of SOX9 in both gene and protein levels. Strikingly, the jb-PDC spheroids implanted in 6- to 8-wk-old male C57BL/6 mice with critical-size jawbone defects (1.8 mm in diameter) showed an evident contribution to cartilaginous callus formation after 1 wk, evidenced by histological analysis. Furthermore, micro-computed tomography analysis showed that the jb-PDC spheroids significantly accelerated bone healing after 2 wk in the absence of exogenous growth factors. In sum, the presented findings represent the successful development of cell-based therapeutics to reengineer the endochondral bone repair process and illustrate the potential application to improve bone repair and regeneration in the craniofacial skeleton.

Simulated Microgravity Exposure Induces Antioxidant Barrier Deregulation and Mitochondria Enlargement in TCam-2 Cell Spheroids.

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell-cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force.

Coacervation-Mediated Cytocompatible Formation of Supramolecular Hydrogels with Self-Evolving Macropores for 3D Multicellular Spheroid Culture.

Coacervation driven liquid-liquid phase separation of biopolymers has aroused considerable attention for diverse applications, especially for the construction of microstructured polymeric materials. Herein, a coacervate-to-hydrogel transition strategy is developed to create macroporous hydrogels (MPH), which are formed via the coacervation process of supramolecular assemblies (SA) built by the host-guest complexation between γ-cyclodextrin and anthracene dimer. The weak and reversible supramolecular crosslinks endow the SA with liquid-like rheological properties, which facilitate the formation of SA-derived macroporous coacervates and the subsequent transition to MPH (pore size ≈ 100 µm). The excellent structural dynamics (derived from SA) and the cytocompatible void-forming process of MPH can better accommodate the dramatic volumetric expansion associated with colony growth of encapsulated multicellular spheroids compared with the non-porous static hydrogel with similar initial mechanical properties. The findings of this work not only provide valuable guidance to the design of biomaterials with self-evolving structures but also present a promising strategy for 3D multicellular spheroid culture and other diverse biomedical applications.

Changes of cell membrane fluidity for mesenchymal stem cell spheroids on biomaterial surfaces.

BACKGROUND: The therapeutic potential of mesenchymal stem cells (MSCs) in the form of three-dimensional spheroids has been extensively demonstrated. The underlying mechanisms for the altered cellular behavior of spheroids have also been investigated. Cell membrane fluidity is a critically important physical property for the regulation of cell behavior, but it has not been studied for the spheroid-forming cells to date. AIM: To explore the association between cell membrane fluidity and the morphological changes of MSC spheroids on the surface of biomaterials to elucidate the role of membrane fluidity during the spheroid-forming process of MSCs. METHODS: We generated three-dimensional (3D) MSC spheroids on the surface of various culture substrates including chitosan (CS), CS-hyaluronan (CS-HA), and polyvinyl alcohol (PVA) substrates. The cell membrane fluidity and cell morphological change were examined by a time-lapse recording system as well as a high-resolution 3D cellular image explorer. MSCs and normal/cancer cells were pre-stained with fluorescent dyes and co-cultured on the biomaterials to investigate the exchange of cell membrane during the formation of heterogeneous cellular spheroids. RESULTS: We discovered that vesicle-like bubbles randomly appeared on the outer layer of MSC spheroids cultured on different biomaterial surfaces. The average diameter of the vesicle-like bubbles of MSC spheroids on CS-HA at 37 °C was approximately 10 µm, smaller than that on PVA substrates (approximately 27 µm). Based on time-lapse images, these unique bubbles originated from the dynamic movement of the cell membrane during spheroid formation, which indicated an increment of membrane fluidity for MSCs cultured on these substrates. Moreover, the membrane interaction in two different types of cells with similar membrane fluidity may further induce a higher level of membrane translocation during the formation of heterogeneous spheroids. CONCLUSION: Changes in cell membrane fluidity may be a novel path to elucidate the complicated physiological alterations in 3D spheroid-forming cells.

3D cell cultures toward quantitative high-throughput drug screening.

3D cell cultures are being utilized for drug discovery and development. However, there are still challenges to implementing them generally in quantitative high-throughput screening (HTS) due to the complexity of the 3D architecture, the time- and labor-consuming process, and the lack of compatibility with traditional screening protocols. Therefore, there is a great need for the integration of microfabrication techniques, automation systems, and high-throughput analytical tools that reveal the pharmacological and toxicological effects of therapeutics using 3D cultures. We first review the current advances in 3D culture models and discuss their key challenges in HTS. Last, we review recent progress and breakthroughs in the automation and high-throughput imaging of 3D culture models, which can be integrated with machine-learning (ML) tools to aid quantitative HTS for drug discovery and development.

A Spheroid Model of Early and Late-Stage Osteosarcoma Mimicking the Divergent Relationship between Tumor Elimination and Bone Regeneration.

Osteosarcoma is the most diagnosed bone tumor in children. The use of tissue engineering strategies after malignant tumor resection remains a subject of scientific controversy. As a result, there is limited research that focuses on bone regeneration postresection, which is further compromised following chemotherapy. This study aims to develop the first co-culture spheroid model for osteosarcoma, to understand the divergent relationship between tumor elimination and bone regeneration. By manipulating the ratio of stromal to osteosarcoma cells the modelled cancer state (early/late) is modified, as is evident by the increased tumor growth rates and an upregulation of a panel of well-established osteosarcoma prognostic genes. Validation of the authors' model is conducted by analyzing its ability to mimic the cytotoxic effects of the FDA-approved chemotherapeutic Doxorubicin. Next, the model is used to investigate what effect osteogenic supplements have, if any, on tumor growth. When their model is treated with osteogenic supplements, there is a stimulatory effect on the surrounding stromal cells. However, when treated with chemotherapeutics this stimulatory effect is significantly diminished. Together, the results of this study present a novel multicellular model of osteosarcoma and provide a unique platform for screening potential therapeutic options for osteosarcoma before conducting in vivo experiments.

Assessment of the Impacts of Centipeda minima (L.) on Cell Viability, and Osteogenic Differentiation of Mesenchymal Stem Cell Spheroids.

Background and Objectives: Centipeda minima (L.) is a well-known and traditional pharmaceutical that has been utilized to treat different conditions controlling rhinitis, soothe pain, and decrease swelling. We assessed the impacts of Centipeda minima (L.) extricates (CMTs) on the osteogenic differentiation of cell spheroids made of human-bone-marrow-derived mesenchymal stem cells. Materials and Methods: Mesenchymal stem cells (MSCs) in spheroid 3D culture were generated and propagated in the presence of CMTs ranging from 0 to 1 µg/mL. Cell morphology was measured on Days 1, 3, 5, and 7. The quantitative cellular viability was evaluated on Days 1, 3, 5, and 7. Alkaline phosphatase activity assays were designed to measure the osteogenic differentiation of mesenchymal stem cell spheroids on Day 7. Alizarin Red S staining was performed to investigate the mineralization of cell spheroids on Days 7 and 14. Real-time polymerase chain reactions were used to measure the expression levels of RUNX2 and COL1A1 on Day 14. Western blot techniques were performed to identify the protein expression of Runt-related transcription factor 2 and type I collagen. Results: The control group's mesenchymal stem cells displayed a spheroid shape. There was no noticeable change in morphology with the addition of CMTs at final concentrations of 0.001, 0.01, 0.1, and 1 µg/mL compared with the untreated (control) group. The application of CMTs did not induce a significant change in cell viability. The relative alkaline phosphatase activity values in the 0.001, 0.01, 0.1, and 1 µg/mL CMT groups were 114.4% ± 8.2%, 130.6% ± 25.3%, 87.8% ± 3.4%, and 92.1% ± 6.8%, respectively, considering a control of 100% (100.0% ± 17.9%). On Day 14, calcium deposits were clearly observed in each group. The relative values of Alizarin Red S staining in the 0.001, 0.01, 0.1, and 1 µg/mL CMT groups were 100.1% ± 8.9%, 105.9% ± 0.0%, 109.7% ± 19.1%, and 87.0% ± 40.9%, respectively, considering a control of 100% (100.0% ± 28.7%). The addition of CMT significantly increased RUNX2 expression in the 0.01 µg/mL group and COL1A1 in the 0.001 and 0.01 µg/mL groups. Normalization of protein expression showed that the addition of CMTs significantly increased type I collagen expression in the 0.001, 0.01, and 1 µg/mL groups. Conclusions: In conclusion, CMTs influence the osteogenic differentiation of bone-marrow-derived mesenchymal stem cells and the use of CMTs may positively influence the osteogenic differentiation of cell spheroids.

Magnetic levitation assisted biofabrication, culture, and manipulation of 3D cellular structures using a ring magnet based setup.

Diamagnetic levitation is an emerging technology for remote manipulation of cells in cell and tissue level applications. Low-cost magnetic levitation configurations using permanent magnets are commonly composed of a culture chamber physically sandwiched between two block magnets that limit working volume and applicability. This work describes a single ring magnet-based magnetic levitation system to eliminate physical limitations for biofabrication. Developed configuration utilizes sample culture volume for construct size manipulation and long-term maintenance. Furthermore, our configuration enables convenient transfer of liquid or solid phases during the levitation. Before biofabrication, we first calibrated/ the platform for levitation with polymeric beads, considering the single cell density range of viable cells. By taking advantage of magnetic focusing and cellular self-assembly, millimeter-sized 3D structures were formed and maintained in the system allowing easy and on-site intervention in cell culture with an open operational space. We demonstrated that the levitation protocol could be adapted for levitation of various cell types (i.e., stem cell, adipocyte and cancer cell) representing cells of different densities by modifying the paramagnetic ion concentration that could be also reduced by manipulating the density of the medium. This technique allowed the manipulation and merging of separately formed 3D biological units, as well as the hybrid biofabrication with biopolymers. In conclusion, we believe that this platform will serve as an important tool in broad fields such as bottom-up tissue engineering, drug discovery and developmental biology.

Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering.

Skeletal reconstruction is necessary in cases of bone defects created by tumors, trauma, and abnormalities. Regeneration of bone defects remains a critical problem, and current approaches are based on biocompatible scaffolds. Spheroids represent a simple 3D system since no supporting material is required for cell growth. Different techniques are used to generate spheroids, such as hanging drop, low-attachment plates, and magnetic nanoparticles. The idea of using magnetic nanoparticles is to cross-link through cell membrane overnight to create complex 3D cellular spheroid by using magnets to guide the cellular response. Herein, the current study aimed to achieve 3D human fetal osteoblast (hFOB) spheroid under magnetic levitation. Formation of 3D spheroid culture under magnetic levitation was evaluated by cell viability at 3, 7, and 14 days. Morphology of the 3D hFOB spheroid was analyzed by SEM and fluorescence microscopy and the differentiation towards mineralized lineage by ALP assay, qPCR, and alizarin red staining. The cell viability indicated that the 3D hFOB spheroid still viable after 14 days of culture. ALP assay, qPCR analysis expression of Col1, ALP, and Itg-ß1 molecules, and calcium deposition with alizarin red showed a high level of bioactivity of the 3D hFOB spheroid. SEM images allowed the morphological analysis of the 3D microtissue-like spheroid with the presence of matrix deposition. These results indicate that magnetic levitation culture enables 3D stable osteoblast spheroids and could be a promising strategy for engineering application in the 3D construct in surgery regeneration of mineralized tissue.

Multi-drug-resistance efflux in cisplatin-naive and cisplatin-exposed A2780 ovarian cancer cells responds differently to cell culture dimensionality.

A primary reason for chemotherapy failure is chemoresistance, which is driven by various mechanisms. Multi-drug resistance (MDR) is one such mechanism that is responsible for drug extrusion from the intracellular space. MDR can be intrinsic and thus, may pre-exist the first application of chemotherapy. However, MDR may also be acquired during tumor exposure to chemotherapeutic agents. To understand whether cell clustering can influence intrinsic and acquired MDR, the present study assessed cultured monolayers (representing individual cells) and spheroids (representing clusters) formed by cisplatin-naïve (intrinsic MDR) and cisplatin-exposed (acquired MDR) lines of ovarian cancer A2780 cells by determining the cytometry of reaction rate constant (CRRC). MDR efflux was characterized using accurate and robust cell number vs. MDR efflux rate constant (k MDR) histograms. Both cisplatin-naïve and cisplatin-exposed monolayer cells presented unimodal histograms; the histogram of cisplatin-exposed cells was shifted towards a higher k MDR value suggesting greater MDR activity. Spheroids of cisplatin-naïve cells presented a bimodal histogram indicating the presence of two subpopulations with different MDR activity. In contrast, spheroids of cisplatin-exposed cells presented a unimodal histogram qualitatively similar to that of the monolayers of cisplatin-exposed cells but with a moderate shift towards greater MDR activity. A flow-cytometry assessment of multidrug resistance-associated protein 1 transporter levels in monolayers and dissociated spheroids revealed distributions similar to those of k MDR, thus, suggesting a plausible molecular mechanism for the observed differences in MDR activity. The observed greater effect of cell clustering on intrinsic rather than in acquired MDR can help guide the development of new therapeutic strategies targeting clusters of circulating tumor cells.

Insulin-like growth factor 2-enhanced osteogenic differentiation of stem cell spheroids by regulation of Runx2 and Col1 expression.

Insulin-like growth factor 2 (IGF-2) is a growth factor that is involved in various functions of cells, including stem cells. The effects of IGF-2 on the cellular viability and osteogenic differentiation of stem cell spheroids were investigated in the present study. Stem cell spheroids were formed using concave microwells in the presence of IGF-2 at final concentrations of 0, 10 and 100 ng/ml. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. The level of alkaline phosphatase activity, and an anthraquinone dye assay for calcium deposit evaluation, were used to assess osteogenic differentiation. A quantitative PCR analysis was conducted to evaluate the expression of Runx2 and Col1. Spheroid formation was noticed on day 1 in the microwells, and the spheroidal shape was maintained up to day 7. The cell viability assay values for IGF-2 at 0, 10 and 100 ng/ml at day 1 were 0.193±0.002, 0.191±0.002 and 0.201±0.006, respectively (P>0.05). The absorbance values at 405 nm for the alkaline phosphatase activity assays on day 21 were 0.221±0.006, 0.375±0.010 and 0.280±0.015 for IGF-2 at 0, 10 and 100 ng/ml, respectively. There were significantly higher values for IGF-2 in the 10 and 100 ng/ml groups when compared with the control (P<0.05). Significantly higher Alizarin red staining was noted for IGF-2 in the 10 ng/ml group when compared with the unloaded control at day 21 (P<0.05). Quantitative PCR revealed that mRNA levels of Runx2 and Col1 were significantly higher at 100 ng/ml on day 7. Conclusively, the present study demonstrated that the application of IGF-2 increased alkaline phosphatase activity, Alizarin red staining, and Runx2 and Col1 expression of stem cell spheroids.

Morphological stability, cellular viability and stem cell marker expression of three-dimensional cultures of stem cells from bone marrow and periodontium.

The aim of the present study was to evaluate the morphology, cellular viability and stem cell marker expression of three-dimensional cultures of bone marrow and gingiva-derived stem cells in different ratios. Stem cell spheroids were made with bone marrow and gingiva-derived stem cells using ratios of 6:0 (Group 1), 4:2 (Group 2), 3:3 (Group 3), 2:4 (Group 4) and 0:6 (Group 5), respectively. The viability of cell spheroids was analyzed using a Live/Dead kit assay and a Cell Counting Kit-8 assay. Total RNA extraction and reverse transcription-quantitative PCR were performed to detect the mRNA expression levels of Nanog and ß-actin in each group. Stem cell spheroids were well formed in silicone elastomer-based concave microwells with different ratios of bone marrow and gingiva-derived stem cells. The shape of the spheroids and their viability were maintained throughout the entirety of the experimental procedure. Statistically significant increases in spheroid diameters were noted in Groups 4 and 5 on day 1 when compared with Group 1 on day 1. There was a significant increase in the cell viability values seen in Group 3 on day 1 when compared with Group 1 on day 1. Highest levels of Nanog expression was seen in Group 3 on day 10, but the increase was not significant when compared with Group 1 on day 1. Co-culturing with higher ratios of gingiva-derived stem cells produced stem cell spheroids with larger diameters and increased cellular viability. This co-culture technique may be used in stem cell therapy with allogenic stem cell transplantation.

Effects of platelet-derived growth factor-BB on cellular morphology and cellular viability of stem cell spheroids composed of bone-marrow-derived stem cells.

Platelet-derived growth factor-BB (PDGF-BB) is a potent mitogenic, angiogenic and chemoattractant, and is one of the most abundant growth factors in platelet-derived products. The goal of the present study was to examine the effects of PDGF-BB on cellular morphology and cellular viability using 3D stem cell cultures. On day 1, spheroids formed well in silicon-elastomer-based concave microwells. The addition of 10 or 100 ng/ml PDGF-BB did not affect the morphology of the cell spheroids. During longer periods of incubation, the cell spheroids maintained their shape without noticeable alterations. The majority of cells in the spheroids exhibited green fluorescence when analyzed using a live/dead assay, indicative of live cells. On day 1, the Cell Counting Kit-8 (CCK-8) assay values for PDGF-BB at 0, 10 and 100 ng/ml were 0.241±0.003, 0.227±0.001 and 0.241±0.004, respectively; on day 3, the CCK-8 assay values for PDGF-BB were 0.233±0.005, 0.278±0.001 and 0.194±0.003, respectively; and on day 7, they were 0.248±0.014, 0.293±0.031 and 0.346±0.034, respectively. The 100 ng/ml group showed significantly higher values compared with the control group on day 7. Together, the results of the present study showed that the addition of 10 and 100 ng/ml PDGF-BB increased cellular viability, suggesting that PDGF-BB may be usable in cell therapy.

Bone morphogenetic protein 2-enhanced osteogenic differentiation of stem cell spheres by regulation of Runx2 expression.

Bone morphogenetic protein 2 (BMP-2) is a growth factor that is used to induce osteogenic differentiation in stem cells. The present study assessed the effects of BMP-2 on stem cell spheroid morphology, viability and osteogenic differentiation. Stem cell spheres were constructed and treated with BMP-2 at predetermined concentrations (0-100 ng/ml) using concave microwells. Cell viability was qualitatively and quantitatively analyzed via microscopy and a water-soluble tetrazolium salt assay kit, respectively. Alkaline phosphatase activity was assessed and an anthraquinone dye for calcium deposit evaluation was performed to determine osteogenic differentiation. The expressions of (runt-related transcription factor 2) and collagen 1 were also determined via quantitative PCR. Spherical shapes were formed using concave microwells on day 1, which were maintained up to day 21. On day 1, the relative cell viability of 0, 10 and 100 ng/ml BMP-2 treated cells was 100.0±1.9, 97.3±4.4 and 101.3±2.6%, respectively. Significantly higher values for alkaline phosphatase activity were determined in the 100 ng/ml treated group when compared with the control group. Additionally, Runx2 mRNA levels were significantly higher in the 100 ng/ml BMP-2 group compared with the control group, as determined via quantitative PCR. The results of the present study indicated that BMP-2 enhanced the differentiation of stem cell spheres, which was demonstrated by increased alkaline phosphatase activity and Runx2 expression.

Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids in part by regulating RUNX2 and BGLAP expression.

Fibroblast growth factors (FGFs) are growth factors that were initially identified as proteins that stimulate fibroblast proliferation. The aim of the present study was to examine the effects of FGF-4 on the morphology, cellular viability and osteogenic differentiation of stem cell spheroids. Stem cell spheroids were generated using concave microwells in the presence of FGF-4 at concentrations of 0, 50, 100 and 200 ng/ml. Cellular viability was qualitatively assessed by a fluorometric live/dead assay using a microscope and quantitatively determined by using Cell Counting Kit-8. Furthermore, alkaline phosphatase activity and calcium deposition were determined to assess osteogenic differentiation. Reverse transcription-quantitative PCR (RT-qPCR) was performed to evaluate the mRNA expression levels of Runt-related transcription factor 2 (RUNX2) and bone γ-carboxyglutamate protein (BGLAP). Spheroidal shapes were achieved in the microwells on day 1 and a significant increase in the spheroid diameter was observed in the 200 ng/ml FGF-4 group compared with the control group on day 1 (P<0.05). The results regarding viability using Cell Counting Kit-8 in the presence of FGF-4 at 50, 100 and 200 ng/ml at day 1 were 98.0±2.5, 106.2±17.6 and 99.5±6.0%, respectively, when normalized to the control group (P>0.05). Furthermore, the alkaline phosphatase activity was significantly elevated in the 200 ng/ml group, when compared with the control group. The RT-qPCR results demonstrated that the mRNA expression levels of RUNX2 and BGLAP were significantly increased at 200 ng/ml. Therefore, the present results suggested that the application of FGF-4 maintained cellular viability while enhancing the osteogenic differentiation of stem cell spheroids, at least partially by regulating RUNX2 and BGLAP expression levels.

Multi-Spheroid-Loaded Human Acellular Dermal Matrix Carrier Preserves Its Spheroid Shape and Improves In Vivo Adipose-Derived Stem Cell Delivery and Engraftment.

BACKGROUND: Current in vivo adult stem cell delivery presents limited clinical effects due to poor engraftment and survival. To overcome current challenges in cell delivery and promote surgical cell delivery for soft tissue repair, a multi-spheroid-loaded thin sectioned acellular dermal matrix (tsADM) carrier which preserves loaded spheroids' three-dimensional (3D) structure, was developed. METHODS: Adipose-derived stem cells (ASCs) were used for spheroid delivery. After generating spheroids in 3D cell culture dishes, spheroid plasticity and survival in-between coverslips were evaluated. Spheroids were loaded onto tsADM, their shape changes were followed up for 14 days, and then imaged. Spheroid adhesion stability to tsADM against shear stress was also evaluated. Finally, cell delivery efficacy was compared with cell-seeded tsADM by in vivo implantation and histological evaluation. RESULTS: Spheroids withstood cyclic compression stress and maintained their 3D shape without fusion after 48 h of culture in-between coverslips. Cell survival improved when spheroids were cultured on tsADM in-between the coverslips. Spheroid-loaded tsADM with coverslips maintained their spheroid outline for 14 days of culture whereas without coverslips, the group lost their outline due to spreading after 4 days in culture. Spheroids loaded onto tsADMs were more stable after six rather than 3 days in culture. Spheroid-loaded tsADMs showed about a 2.96-fold higher ASCs transplantation efficacy than cell-seeded tsADMs after 2 weeks of in vivo transplantation. CONCLUSION: These results indicate that transplantation of spheroid-loaded tsADMs significantly improved cell delivery. These findings suggest that a combined approach with other cells, drugs, and nanoparticles may improve cell delivery and therapeutic efficacy.

Lovastatin increases the proliferation and osteoblastic differentiation of human gingiva-derived stem cells in three-dimensional cultures.

Lovastatin is a cholesterol-lowering agent that also has effects of cell proliferation and apoptosis. The present study was performed to evaluate the effects of lovastatin on the proliferation and osteogenic differentiation of three-dimensional cell spheroids formed from human gingiva-derived stem cells (GDSCs) using concave microwells. GDSCs were plated on polydimethylsiloxane-based concave micromolds and grown in the presence of lovastatin at concentrations of 0, 2 and 6 µM. The morphology of the cells was viewed under an inverted microscope, and cell viability was determined with Cell Counting kit-8 on days 2, 7 and 14. Alkaline phosphatase activity assays were performed to evaluate the osteogenic differentiation on days 2 and 8. Alizarin red-S staining was also used to assess the mineralization of the stem cell spheroids at day 14. The results confirmed that GDSCs formed spheroids in concave microwells. No significant changes were noted with longer incubation time, and no significant differences in cell viability were noted between the three lovastatin groups at each time point. Higher osteogenic differentiation was observed in the 2 µM group when compared with the control. Mineralized extracellular deposits were visible after Alizarin red-S staining, and higher mineralization was noted in the 2 and 6 µM lovastatin groups when compared with the 0 µM control. The relative mineralization values of the 0, 2 and 6 µM groups on day 14 were 39.0±9.6, 69.3±6.0 and 60.9±7.5, respectively. This study demonstrated that the application of lovastatin enhanced the osteogenic differentiation of cell spheroids formed from GDSCs. This suggests that combinations of lovastatin and stem cell spheroids may have the potential for use in tissue engineering.

A Study of the Effects of Doxorubicin-Containing Liposomes on Osteogenesis of 3D Stem Cell Spheroids Derived from Gingiva.

The objective of the present investigation is to determine the effects of neutral, anionic, and cationic liposomes loaded with doxorubicin with thin-lipid-film-hydration method on the cellular viability and osteogenesis of stem cell spheroids. Spheroid formation and morphology of the three-dimensional spheroid were noted with an inverted microscope. Quantitative cellular viability was assessed using a commercially available kit. Osteogenic potential was evaluated by applying alkaline phosphatase activity and anthraquinone dye of Alizarin Red S. Western blot analysis was performed using collagen I expression. Spheroids were formed in each silicon elastomer-based concave microwell on Day 1. Noticeable changes of the spheroid were seen with a higher concentration of doxorubicin, especially in the cationic liposome group at Days 5 and 7. We found that the application of doxorubicin for 5 days significantly reduced the cellular viability. A higher concentration of doxorubicin produced a significant decrease in alkaline phosphatase activity. Alizarin Red S staining showed that extracellular calcium deposits were evenly noted in each group. An increase of calcium deposits was noted on Day 14 when compared to Day 7. The morphology of the groups with higher concentrations of doxorubicin showed to be more dispersed. We noticed that doxorubicin-loaded cationic liposomes resulted in the highest uptake of the examined cell spheroids and that doxorubicin-loaded liposomes affected the osteogenic differentiation. The implication of this study is that the type of liposome should be selected based on the purpose of the application.

Uptake and efflux of FL118 and two FL118 derivatives in 3D cell model.

Drug uptake and efflux are two of the critical factors required in order to be able to define drug efficacy. This study aims to investigate cytotoxicity and uptake mechanisms of two FL118 analogues (7-Q20 and val-FL118) in parallel with FL118 in three-dimensional multi-cellular spheroids model. The influence of compound concentration, time, temperature, cell lines, and the inhibitors of P-gp, BCRP and LAT1 on drug uptake and efflux were investigated. In vitro cytotoxicity studies revealed that FL118, 7-Q20 and val-FL118 exhibited sensitive cytotoxicity to the HCT-116 cell line and the water-soluble compound 7-Q20 showed the lowest IC50. Cellular uptake and efflux of FL118 was independent of efflux pump proteins. Uptake and efflux of 7-Q20 were affected by P-gp, which was one of reasons that caused a lower uptake at 37 °C than at 4 °C. The carrier protein LAT1 played a role in the cellular intakes of val-FL118. These findings provided basic information for FL118 and the two novel FL118 derivatives for further development.