3D Mesenchymal Stem Cells Research Articles

Cellular uptake of FITC-labeled Ce0.8Gd0.2O2-x nanoparticles in 2D and 3D mesenchymal stem cell systems

Cellular uptake of FITC-labeled Ce0.8Gd0.2O2-x nanoparticles in 2D and 3D mesenchymal stem cell systems

Distinctive Biological Properties between Mesenchymal Stem Cell Spheroids and Clumps of Mesenchymal Stem Cells/Extracellular Matrix Complexes in 3D Culture Systems

Background: Cells typically function and behave within a three-dimensional (3D) environment. Mesenchymal stem cells (MSCs), known for their self-renewal, multi-lineage differentiation capabilities, and paracrine effects, have garnered significant medical interest. MSC spheroid culture is widely adopted to study the biological properties of MSCs in a 3D context. In contrast, we previously developed 3D clumps of MSC/ECM complexes termed C-MSCs. C-MSCs consisted of cells and self-produced ECM proteins, allowing grafting into tissue defects without any artificial scaffolds. This present study aimed to elucidate the fundamental biological distinctions between 3D MSC spheroids and C-MSCs. Methods: MSC spheroids and C-MSCs are generated from human bone-marrow-derived MSCs. The physical properties, histological structures, and gene expression patterns were compared in vitro. Results: Macroscopic and histological examinations revealed that, whereas MSC spheroids are dense cell clusters primarily formed through Cadherin-mediated cell–cell interactions, C-MSCs are cell aggregates anchored by the ECM component COL1, enabling them to form larger structures. Furthermore, transcriptome analysis showed that C-MSCs possess enhanced capacities to produce immunomodulatory and cytoprotective factors, a prominent biological characteristic of MSCs. Conclusion: Recognizing the distinct attributes of each cell aggregate offers insights into the potential evolution of 3D cell culture techniques and possible therapeutic implications.

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) alleviate excessive autophagy of ovarian granular cells through VEGFA/PI3K/AKT/mTOR pathway in premature ovarian failure rat model

BackgroundPremature ovarian failure (POF) is one of the leading causes of female infertility and is accompanied by abnormal endocrine, seriously affecting female quality of life. Previous studies have demonstrated that mesenchymal stem cells (MSCs) transplantation is a promising therapeutic strategy for POF. However, the mechanism remains obscure. This study aims to investigate the therapeutic effect of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on ovarian function in the POF rat model and explore the underlying mechanisms.MethodsThe ovarian function was evaluated by ovarian morphology, histology, estrous cycle, hormone levels (AMH, E2, FSH, and LH), and fertility ability to investigate the effect of hUC-MSCs on the POF rats model. The cytokines levels were assayed in serum using protein array to explore the mechanisms of hUC-MSCs therapy for POF. The excessive autophagy levels were evaluated using a co-culture system of 3D MSCs spheroids with human ovarian granulosa cell line (KGN) or primary ovarian granulosa cells (GCs) to understand the paracrine effect of hUC-MSCs on GCs. The related proteins expression of autophagy and PI3K/AKT/mTOR pathway was detected using Western Blotting and/or in various inhibitors supplement to further demonstrate that vascular endothelial growth factor A (VEGFA) secreted by hUC-MSCs can alleviate excessive autophagy of ovarian GCs via PI3K/AKT/mTOR signaling pathway. The ovarian culture model in vitro was applied to confirm the mechanism.ResultsThe ovarian function of POF and the excessive autophagy of ovarian GCs were restored after hUC-MSCs transplantation. The protein array result demonstrated that VEGF and PI3K/AKT might improve ovarian function. in vitro experiments demonstrated that VEGFA secreted by hUC-MSCs could decrease oxidative stress and inhibit excessive autophagy of ovarian GCs via PI3K/AKT/mTOR pathway. The ovarian culture model results confirmed this mechanism in vitro.ConclusionThe hUC-MSCs can alleviate excessive autophagy of ovarian GCs via paracrine VEGFA and regulate the PI3K/AKT/mTOR signaling pathway, thereby improving the ovarian function of POF.

Synergistic Therapeutic Potential of Dual 3D Mesenchymal Stem Cell Therapy in an Ischemic Hind Limb Mouse Model.

Three-dimensional (3D) culture systems have been widely used to promote the viability and metabolic activity of mesenchymal stem cells (MSCs). The aim of this study was to explore the synergistic benefits of using dual 3D MSC culture systems to promote vascular regeneration and enhance therapeutic potential. We used various experimental assays, including dual 3D cultures of human adipose MSCs (hASCs), quantitative reverse transcription polymerase chain reaction (qRT-PCR), in vitro cell migration, Matrigel tube network formation, Matrigel plug assay, therapeutic assays using an ischemic hind limb mouse model, and immunohistochemical analysis. Our qRT-PCR results revealed that fibroblast growth factor 2 (FGF-2), granulocyte chemotactic protein-2 (GCP-2), and vascular endothelial growth factor-A (VEGF-A) were highly upregulated in conventional 3D-cultured hASCs (ASC-3D) than in two-dimensional (2D)-cultured hASCs. Hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and stromal-cell-derived factor-1 (SDF-1) showed higher expression levels in cytokine-cocktail-based, 3D-cultured hASCs (ASC-3Dc). A conditioned medium (CM) mixture of dual 3D ASCs (D-3D; ASC-3D + ASC-3Dc) resulted in higher migration and Matrigel tube formation than the CM of single 3D ASCs (S-3D; ASC-3D). Matrigel plugs containing D-3D contained more red blood cells than those containing S-3D. D-3D transplantation into ischemic mouse hind limbs prevented limb loss and augmented blood perfusion when compared to S-3D transplantation. Transplanted D-3D also revealed a high capillary density and angiogenic cytokine levels and transdifferentiated into endothelial-like cells in the hind limb muscle. These findings highlight the benefits of using the dual 3D culture system to optimize stem-cell-based therapeutic strategies, thereby advancing the therapeutic strategy for ischemic vascular disease and tissue regeneration.

ROS filter coating scaffold protects 3D mesenchymal stem cell spheroids for dual-phase treatment of spinal cord injury

Implanting mesenchymal stem cells (MSCs) has offered a great promise in spinal cord injury (SCI) repair. However, the harsh environment accompanied with excessive ROS following SCI seriously threatens MSC survival and compromises its efficacy. Additionally, the intricate pathology distinguished as the acute phase and chronic phase makes MSC therapeutics alone difficult to alleviate severe inflammation and promote functional recovery simultaneously. Despite scaffold systems for local stem cell delivery being extensively explored, dual-phase management for SCI using one protective scaffold system has rarely been reported. Herein, a composite scaffold system (RF-pGel-MS), which consisted of an outer ROS filter (RF) and an inner porous GelMA hydrogel (pGel) harbored with MSC spheroids (MSs), was designed for SCI repair. Specifically, RF rapidly scavenged ROS, protecting the MS from peroxidation damage in the acute phase. MS constructed via 3D culture of MSCs presented enhanced paracrine effects, which remarkably facilitated neuron repair and functional recovery in the chronic phase. The dual-phase therapeutic efficacy of RF-pGel-MS led to significant locomotor and electrophysiology recovery in the SCI rats. Hence, the currently proposed strategy using RF-pGel-MS is efficient for ROS scavenging and boosting the stem cell therapeutics, which is insightful for the SCI treatment.

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

BACKGROUNDThe 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.AIMTo 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.METHODSWe 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.RESULTSWe 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. CONCLUSIONChanges in cell membrane fluidity may be a novel path to elucidate the complicated physiological alterations in 3D spheroid-forming cells.

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies.

Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

Immunomodulatory Properties of Cytokine-preconditioned Compact-bone Derived Mesenchymal Stem Cells Cultured in 2D and 3D Culture Conditions

Abstract. Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties and therefore represent a promising therapeutic tool for the treatment of immune-related diseases. Recently, preconditioning has been proposed as a strategy to improve the immunomodulatory activity of MSCs. In this study, we focused to investigate the effects of preconditioning with pro-inflammatory cytokines (TNF-α and IFN-γ) on immunomodulatory activity of mouse compact bone-derived MSCs (CB MSCs) in 2D and 3D culture conditions. Mouse CB MSCs for the 2D condition were cultured in a standard tissue culture plate. 3D spheroid MSCs were formed by enforced composing in Nunclon Sphera 96-well U-bottom plate. MSCs in 2D and 3D spheroid cultures were preconditioned with TNF-α and IFN-γ alone and in combination for 24 hours. The levels of immunomodulatory factors (PGE2, IL-6, IL-10, TNF-α, IFN-γ) were measured by ELISA. The immunomodulatory properties of MSCs were examined by macrophage inflammation and lymphocyte co-culture assays. Our results showed that TNF-α and IFN-γ more effectively increased the secretion of PGE2 and IL-6 by 3D spheroid MSCs compared with 2D MSCs culture.Сytokine-preconditioned 3D spheroid MSCs significantly decreased the production of TNF-α, IFN-γ, IL-10 in splenic lymphocytes and TNF-α and IL-6 in macrophages. Сytokine-preconditioned 3D spheroid MSCs increased IL-6 and IL-10 levels in lymphocytes and macrophages respectively. It was found that 3D spheroid MSCs more effectively suppressed the proliferation of T cells compared with 2D MSCs culture. Moreover, preconditioning of 3D spheroid MSCs using TNF-α suppressed more effectively in vitro immune response than preconditioning with IFN-γ or with the combination of two cytokines. Thus, our data suggest that preconditioning with pro-inflammatory cytokines more effectively increases the immunomodulatory activity of 3D MSC spheroids compared to 2D MSC culture.

Immunosuppressive mesenchymal stem cells aggregates incorporating hydrogel microspheres promote an invitro invasion of cancer cells.

IntroductionThe objective of this study is to design a co-culture system of cancer cells and three-dimensional (3D) mesenchymal stem cells (MSC) aggregates for the in vitro evaluation of cancer invasion.MethodsFirst, the MSC of an immunosuppressive phenotype (MSC2) were prepared by the MSC stimulation of polyriboinosinic polyribocytidylic acid. By simple mixing MSC2 and gelatin hydrogel microspheres (GM) in a U-bottomed well of 96 well plates which had been pre-coated with poly (vinyl alcohol), 3D MSC2 aggregates incorporating GM were obtained. The amount of chemokine (C–C motif) ligand 5 (CCL5) secreted from the MSC2 aggregates incorporating GM. Finally, an invasion assay was performed to evaluate the cancer invasion rate by co-cultured cancer cells and the 3D MSC2 incorporating GM.ResultsThe amount of CCL5 secreted for the 3D MSC2 aggregates incorporating GM was significantly higher than that of two-dimensional (2D) MSC, 2D MSC2, and 3D MSC aggregates incorporating GM. When MDA-MB-231 human breast cancer cells were co-cultured with the 3D MSC2 aggregates incorporating GM, the invasion rate of cancer cells was significantly high compared with that of 2D MSC or 2D MSC2 and 3D MSC aggregates incorporating GM. In addition, high secretion of matrix metalloproteinase-2 was observed for the 3D MSC2 aggregates/cancer cells system.ConclusionsIt is concluded that the co-culture system of 3D MSC2 aggregates incorporating GM and cancer cells is promising to evaluate the invasion of cancer cells in vitro.

Enhancing chondrogenic potential via mesenchymal stem cell sheet multilayering.

Advanced tissue engineering approaches for direct articular cartilage replacement in vivo employ mesenchymal stem cell (MSC) sources, exploiting innate chondrogenic potential to fabricate hyaline-like constructs in vitro within three-dimensional (3D) culture conditions. Cell sheet technology represents one such advanced 3D scaffold-free cell culture platform, and previous work has shown that 3D MSC sheets are capable of in vitro hyaline-like chondrogenic differentiation. The present study aims to build upon this understanding and elucidate the effects of an established cell sheet manipulation technique, cell sheet multilayering, on fabrication of MSC-derived hyaline-like cartilage 3D layered constructs in vitro. To achieve this goal, multilayered MSC sheets are prepared and assessed for structural and biochemical transitions throughout chondrogenesis. Results support MSC multilayering as a means of increasing construct thickness and 3D cellular interactions related to in vitro chondrogenesis, including N-cadherin, connexin 43, and integrin β-1. Data indicate that increasing construct thickness from 14 μm (1-layer construct) to 25 μm (2-layer construct) increases these cellular interactions and subsequent in vitro MSC chondrogenesis. However, a clear initial thickness threshold (33 μm - 3-layer construct) is evident that decreases the rate and extent of in vitro chondrogenesis, specifically chondrogenic gene expressions (Sox9, aggrecan, type II collagen) and sulfated proteoglycan accumulation in deposited extracellular matrix (ECM). Together, these data support the utility of cell sheet multilayering as a platform for tailoring construct thickness and subsequent MSC chondrogenesis for future articular cartilage regeneration applications.

3D spheroids of human placenta-derived mesenchymal stem cells attenuate spinal cord injury in mice

Mesenchymal stem cell (MSC) is an absorbing candidate for cell therapy in treating spinal cord injury (SCI) due to its great potential for multiple cell differentiation, mighty paracrine secretion as well as vigorous immunomodulatory effect, of which are beneficial to the improvement of functional recovery post SCI. However, the therapeutic effects of MSC on SCI have been limited because of the gradual loss of MSC stemness in the process of expanding culture. Therefore, in this study, we aimed to maintain those beneficial properties of MSC via three-dimensional spheroid cell culture and then compared them with conventionally-cultured MSCs in the treatment of SCI both in vitro and in vivo with the aid of two-photon microscope. We found that 3D human placenta-derived MSCs (3D-HPMSCs) demonstrated a significant increase in secretion of anti-inflammatory factors and trophic factors like VEGF, PDGF, FGF via QPCR and Bio-Plex assays, and showed great potentials on angiogenesis and neurite morphogenesis when co-cultured with HUVECs or DRGs in vitro. After transplantation into the injured spinal cord, 3D-HPMSCs managed to survive for the entire experiment and retained their advantageous properties in secretion, and exhibited remarkable effects on neuroprotection by minimizing the lesion cavity, inhibiting the inflammation and astrogliosis, and promoting angiogenesis. Further investigation of axonal dieback via two-photon microscope indicated that 3D-HPMSCs could effectively alleviate axonal dieback post injury. Further, mice only treated with 3D-HPMSCs obtained substantial improvement of functional recovery on electrophysiology, BMS score, and Catwalk analysis. RNA sequencing suggested that the 3D-HPMSCs structure organization-related gene was significantly changed, which was likely to potentiate the angiogenesis and inflammation regulation after SCI. These results suggest that 3D-HPMSCs may hold great potential for the treatment of SCI.

Modulation of Inherent Niches in 3D Multicellular MSC Spheroids Reconfigures Metabolism and Enhances Therapeutic Potential.

Multicellular spheroids show three-dimensional (3D) organization with extensive cell–cell and cell–extracellular matrix interactions. Owing to their native tissue-mimicking characteristics, mesenchymal stem cell (MSC) spheroids are considered promising as implantable therapeutics for stem cell therapy. Herein, we aim to further enhance their therapeutic potential by tuning the cultivation parameters and thus the inherent niche of 3D MSC spheroids. Significantly increased expression of multiple pro-regenerative paracrine signaling molecules and immunomodulatory factors by MSCs was observed after optimizing the conditions for spheroid culture. Moreover, these alterations in cellular behaviors may be associated with not only the hypoxic niche developed in the spheroid core but also with the metabolic reconfiguration of MSCs. The present study provides efficient methods for manipulating the therapeutic capacity of 3D MSC spheroids, thus laying solid foundations for future development and clinical application of spheroid-based MSC therapy for regenerative medicine.

Tailoring the proliferation of fibroblast cells by multiresponsive and thermosensitive stem cells composite F127 hydrogel containing folic acid.MgO:ZnO/chitosan hybrid microparticles for skin regeneration

In this study, biodegradable and thermosensitive F127 hydrogel containing folic acid.MgO:ZnO/chitosan hybrid particles (FMZC) was fabricated as a 3D mesenchymal stem cells (MSCs) delivery vehicle for regenerative medicine and wound healing purposes, in such a way to be responsive to lysozyme and UVA irradiation. The results showed that F127 hydrogel containing FMZC is a suitable and nontoxic construct for encapsulation of MSCs in the presence of lysozyme and UVA irradiation, bearing high stem cell viability and proliferation. The final hydrogel, MSC&FMZC, in response to lysozyme induced a higher proliferation rate and migration in human foreskin fibroblast cells (HFF). These phenomena were attributed to the released F.MgO:ZnO nanocomposites from chitosan microparticles and paracrine factors from MSCs within the hydrogel, resulting in synergistic biological effects. Moreover, lysozyme-treated MSC&FMZC hydrogel showed higher antibacterial and anti-biofilm activity against both Gram-positive and Gram-negative bacteria than bare hydrogel. However, a significant increase in the antibacterial activity of MSC&FMZC was observed as the treated bacteria were subjected to UVA irradiation owing to the photocatalytic activity of F.MgO:ZnO nanocomposites. Regarding the antibacterial activity and stimulating skin cell behavior of MSC&FMZC hydrogel that can promote the regenerative activities of skin, it could be considered as a promising scaffold for bacteria-accompanied wound healing.

Intravascular Application of Labelled Cell Spheroids: An Approach for Ischemic Peripheral Artery Disease.

Mesenchymal stem cells (MSC) are known for their vascular regeneration capacity by neoangiogenesis. Even though, several delivery approaches exist, particularly in the case of intravascular delivery, only limited number of cells reach the targeted tissue and are not able to remain on site. Applicated cells exhibit poor survival accompanied with a loss of functionality. Moreover, cell application techniques lead to cell death and impede the overall MSC function and survival. 3D cell spheroids mimic the physiological microenvironment, thus, overcoming these limitations. Therefore, in this study we aimed to evaluate and assess the feasibility of 3D MSCs spheroids for endovascular application, for treatment of ischemic peripheral vascular pathologies. Multicellular 3D MSC spheroids were generated at different cell seeding densities, labelled with ultra-small particles of iron oxide (USPIO) and investigated in vitro in terms of morphology, size distribution, mechanical stability as well as ex vivo with magnetic resonance imaging (MRI) to assess their trackability and distribution. Generated 3D spheroids were stable, viable, maintained stem cell phenotype and were easily trackable and visualized via MRI. MSC 3D spheroids are suitable candidates for endovascular delivery approaches in the context of ischemic peripheral vascular pathologies.

Trends in Articular Cartilage Tissue Engineering: 3D Mesenchymal Stem Cell Sheets as Candidates for Engineered Hyaline-Like Cartilage.

Articular cartilage defects represent an inciting factor for future osteoarthritis (OA) and degenerative joint disease progression. Despite multiple clinically available therapies that succeed in providing short term pain reduction and restoration of limited mobility, current treatments do not reliably regenerate native hyaline cartilage or halt cartilage degeneration at these defect sites. Novel therapeutics aimed at addressing limitations of current clinical cartilage regeneration therapies increasingly focus on allogeneic cells, specifically mesenchymal stem cells (MSCs), as potent, banked, and available cell sources that express chondrogenic lineage commitment capabilities. Innovative tissue engineering approaches employing allogeneic MSCs aim to develop three-dimensional (3D), chondrogenically differentiated constructs for direct and immediate replacement of hyaline cartilage, improve local site tissue integration, and optimize treatment outcomes. Among emerging tissue engineering technologies, advancements in cell sheet tissue engineering offer promising capabilities for achieving both in vitro hyaline-like differentiation and effective transplantation, based on controlled 3D cellular interactions and retained cellular adhesion molecules. This review focuses on 3D MSC-based tissue engineering approaches for fabricating “ready-to-use” hyaline-like cartilage constructs for future rapid in vivo regenerative cartilage therapies. We highlight current approaches and future directions regarding development of MSC-derived cartilage therapies, emphasizing cell sheet tissue engineering, with specific focus on regulating 3D cellular interactions for controlled chondrogenic differentiation and post-differentiation transplantation capabilities.

Enzymatically Cross-linked Hydrogels Based on Synthetic Poly(α-amino acid)s Functionalized with RGD Peptide for 3D Mesenchymal Stem Cell Culture.

Injectable hydrogel scaffolds combined with stem cell therapy represent a promising approach for minimally invasive surgical tissue repair. In this study, we developed and characterized a fully synthetic, biodegradable poly(N5-(2-hydroxyethyl)-l-glutamine)-based injectable hydrogel modified with integrin-binding arginine-glycine-aspartic acid (RGD) peptide (PHEG-Tyr-RGD). The biodegradable hydroxyphenyl polymer precursor derivative of PHEG-Tyr was enzymatically cross-linked to obtain injectable hydrogels with different physicochemical properties. The gelation time, gel yield, swelling behavior, and storage modulus of the PHEG-Tyr hydrogels were tuned by varying the concentrations of the PHEG-Tyr precursors and horseradish peroxidase as well as the nH2O2/nTyr ratio. The mechanical properties and gelation time of the PHEG-Tyr hydrogel were optimized for the encapsulation of rat mesenchymal stem cells (rMSCs). We focused on the 2D and 3D spreading and viability of rMSCs within the PHEG-Tyr-RGD hydrogels with different physicochemical microenvironments in vitro. Encapsulation of rMSCs shows long-term survival and exhibits cell-matrix and cell-cell interactions reflective of both the RGD concentration and hydrogel stiffness. The presented biomaterial represents a suitable biological microenvironment to guide 3D spreading and may act as a promising 3D artificial extracellular matrix for stem cell therapy.

Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets

Cell and tissue engineering approaches for articular cartilage regeneration increasingly focus on mesenchymal stem cells (MSCs) as allogeneic cell sources, based on availability and innate chondrogenic potential. Many MSCs exhibit chondrogenic potential as three-dimensional (3D) cultures (i.e. pellets and seeded biomaterial scaffolds) in vitro; however, these constructs present engraftment, biocompatibility, and cell functionality limitations in vivo. Cell sheet technology maintains cell functionality as scaffold-free constructs while enabling direct cell transplantation from in vitro culture to targeted sites in vivo. The present study aims to develop transplantable hyaline-like cartilage constructs by stimulating MSC chondrogenic differentiation as cell sheets. To achieve this goal, 3D MSC sheets are prepared, exploiting spontaneous post-detachment cell sheet contraction, and chondrogenically induced. Results support 3D MSC sheets’ chondrogenic differentiation to hyaline cartilage in vitro via post-contraction cytoskeletal reorganization and structural transformations. These 3D cell sheets’ initial thickness and cellular densities may also modulate MSC-derived chondrocyte hypertrophy in vitro. Furthermore, chondrogenically differentiated cell sheets adhere directly to cartilage surfaces via retention of adhesion molecules while maintaining the cell sheets’ characteristics. Together, these data support the utility of cell sheet technology for fabricating scaffold-free, hyaline-like cartilage constructs from MSCs for future transplantable articular cartilage regeneration therapies.

Mesenchymal Stem Cell Spheroids Embedded in an Injectable Thermosensitive Hydrogel: An In Situ Drug Formation Platform for Accelerated Wound Healing.

The ability of mesenchymal stem cells (MSCs) to enhance cutaneous wound healing has been well established. Extensive expansion of cells to reach sufficient cell numbers for regenerating tissues has always limited cell-based therapies. An ingenious solution to address this challenge is to develop a strategy to increase the immunomodulatory effects of MSCs without expanding them. In this study, we employed a simple characteristic of cells. It was observed that an optimized three-dimensional (3D) MSC culture in spheroid forms significantly improved their paracrine effects. An electrospray (ES) encapsulation apparatus was used to encapsulate individual or 3D spheroid MSCs into microscale alginate beads (microbeads). Furthermore, alginate microbeads were embedded in an injectable thermosensitive hydrogel matrix, which gels at skin temperature. The hydrogel fills and seals the wounds cavities, maintains high humidity at the wound area, absorbs exudate, and fixes microbeads, protecting them from direct contact with the harsh wound environment. In vitro investigations revealed that secretion of interleukin 10 (IL-0) and transforming growth factor β1 (TGF-β1) gene was gradually enhanced, providing a delivery platform for prolonged release of bioactive molecules. In vivo study on full-thickness wounds showed granulation and re-epithelialization, only after 7 days. Moreover, increased expression of α-smooth muscle actin (α-SMA) in the first 14 days after treatment ensured wound contraction. Besides, a gradual decrease in α-SMA secretion resulted in reduced scar formation. Well-organized collagen fibrils and high expression of the angiogenesis biomarker CD31 confirmed the promoting effect of the hydrogel on the wound-healing process. The proposed wound-dressing system would potentially be used in scalable and effective cell-based wound therapies.

Design and characterization of microspheres for a 3D mesenchymal stem cell culture

Recent studies have shown the relevance of growing mesenchymal stem cells (MSCs) in three-dimensional environments with respect to the monolayer cell culture on an adherent substrate. In this sense, macroporous scaffolds and hydrogels have been used as three-dimensional (3D) supports. In this work, we explored the culture of MSCs in a 3D environment created by microspheres, prepared with a fumarate-vinyl acetate copolymer and chitosan. In this system, the environment that the cells feel has similarities to that found by the cells encapsulated in a hydrogel, but the cells have the ability to reorganize their environment since the microspheres are mobile. We evaluated their biocompatibility in vitro using RAW 264.7 macrophages and bone marrow mesenchymal stem cells (BMSCs). The results with RAW 264.7 cells showed good cell viability, without evident signs of cytotoxicity. BMSCs not only proliferate, but also rearrange to grow in clusters, thus highlighting the advantages of microspheres as 3D environments.

Specificity of 3D MSC Spheroids Microenvironment: Impact on MSC Behavior and Properties.

Mesenchymal stem cells (MSC) have been considered the promising candidates for the regenerative and personalized medicine due to their self-renewal potential, multilineage differentiation and immunomodulatory capacity. Although these properties have encouraged profound MSC studies in recent years, the majority of research has been based on standard 2D culture utilization. The opportunity to resemble in vivo characteristics of cells native niche has been provided by implementation of 3D culturing models such as MSC spheroid formation assesed through cells self-assembling. In this review, we address the current literature on physical and biochemical features of 3D MSC spheroid microenvironment and their impact on MSC properties and behaviors. Starting with the reduction in the cells' dimensions and volume due to the changes in adhesion molecules expression and cytoskeletal proteins rearrangement resembling native conditions, through the microenvironment shifts in oxygen, nutrients and metabolites gradients and demands, we focus on distinctive and beneficial features of MSC in spheroids compared to cells cultured in 2D conditions. By summarizing the data for 3D MSC spheroids regarding cell survival, pluripotency, differentiation, immunomodulatory activities and potential to affect tumor cells growth we highlighted advantages and perspectives of MSC spheroids use in regenerative medicine. Further detailed analyses are needed to deepen our understanding of mechanisms responsible for modified MSC behavior in spheroids and to set future directions for MSC clinical application.