Culture Of Mesenchymal Stem Cells Research Articles

In Vitro Culture and Multipotency Evaluation of Broiler Umbilical Cord Mesenchymal Stem Cells

HIGHLIGHTS Broiler-derived UMSCs line was successfully isolated for the first time. UMSCs possessed stemness characteristics, such as hereditary stability, self-renewal ability, and expression of specific markers. UMSCs possessed multilineage potential and could be differentiated into cells of three layers.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance Angiogenesis Through Upregulation of the VWF and Flk1 Genes in Endothelial Cells.

Exosomes derived from mesenchymal stem cells (MSCs) are crucial mediators of the paracrine effects as well as tissue repair and have promising clinical applications. They enhance tissue regeneration by reducing inflammatory responses, enhancing proliferation, inhibiting apoptosis, and stimulating angiogenesis. This study aimed to evaluate the mechanism of angiogenesis supported by exosomes derived from MSCs. Exosomes were isolated via ultracentrifugation of a conditioned medium collected from human umbilical cord MSC (hUCMSC) cultures. These exosomes were characterized using transmission electron microscopy, and the expression of specific markers (CD9, CD81, and CD63) was evaluated. To understand the mechanism of angiogenesis, we evaluated the effects of exosomes in endothelial cells (HUVECs). The obtained exosomes were supplemented at a dose of 20μg/mL into two kinds of culture media for HUVECs (M200 medium and endothelial cell growth medium), while phosphate-buffered saline was added to these media as a control. The effects of the exosomes were evaluated based on the formation of a tubular structure in the culture and the expression of angiogenic genes (MMP-2, Ephrin B2, Ephrin B4, Flk1, Flt1, VWF, VE-cadherin, CD31, ANG1, ANG2, and HGF) via RT-PCR. The exosomes were obtained from the hUCMSCs at a concentration of 0.7±0.029μg/mL. They accelerated the formation of new blood vessels by upregulating HGF, VWF, CD31, Flt1, and Flk1 (especially VWF and Flt1). Exosomes derived from hUCMSCs can promote angiogenesis through upregulation of VWF and Flt1 in endothelial cells.

Protocol for 3D Bioprinting Mesenchymal Stem Cell-derived Neural Tissues Using a Fibrin-based Bioink.

Three-dimensional bioprinting utilizes additive manufacturing processes that combine cells and a bioink to create living tissue models that mimic tissues found in vivo. Stem cells can regenerate and differentiate into specialized cell types, making them valuable for research concerning degenerative diseases and their potential treatments. 3D bioprinting stem cell-derived tissues have an advantage over other cell types because they can be expanded in large quantities and then differentiated to multiple cell types. Using patient-derived stem cells also enables a personalized medicine approach to the study of disease progression. In particular, mesenchymal stem cells (MSC) are an attractive cell type for bioprinting because they are easier to obtain from patients in comparison to pluripotent stem cells, and their robust characteristics make them desirable for bioprinting. Currently, both MSC bioprinting protocols and cell culturing protocols exist separately, but there is a lack of literature that combines the culturing of the cells with the bioprinting process. This protocol aims to bridge that gap by describing the bioprinting process in detail, starting with how to culture cells pre-printing, to 3D bioprinting the cells, and finally to the culturing process post-printing. Here, we outline the process of culturing MSCs to produce cells for 3D bioprinting. We also describe the process of preparing Axolotl Biosciences TissuePrint - High Viscosity (HV) and Low Viscosity (LV) bioink, the incorporation of MSCs to the bioink, setting up the BIO X and the Aspect RX1 bioprinters, and necessary computer-aided design (CAD) files. We also detail the differentiation of 2D and 3D cell cultures of MSC to dopaminergic neurons, including media preparation. We have also included the protocols for viability, immunocytochemistry, electrophysiology, and performing a dopamine enzyme-linked immunosorbent assay (ELISA), along with the statistical analysis. Graphical overview.

Guideline for design of substrate stiffness for mesenchymal stem cell culture based on heterogeneity of YAP and RUNX2 responses.

Mesenchymal stem cells (MSCs) have the potential for self-renewal and multipotency to differentiate into various lineages. Thus, they are of great interest in regenerative medicine as a cell source for tissue engineering. Substrate stiffness is one of the most extensively studied exogenous physical factors; however, consistent results have not always been reported for controlling MSCs. Conventionally used stiff culture substrates, such as tissue-culture polystyrene and glass, enhance nuclear localization of a mechanotransducer YAP and a pre-osteogenic transcription factor RUNX2, and bias MSCs towards the osteogenic lineage, even without osteogenic-inducing soluble factors. The mechanosensitive nature and intrinsic heterogeneity present challenges for obtaining reproducible results. This review summarizes the heterogeneity in human MSC response, specifically, nuclear/cytoplasmic localization changes in the mechanotransducer yes-associated protein (YAP) and the osteogenic transcription factor RUNX2, in response to substrate stiffness. In addition, a perspective on the intracellular factors attributed to response heterogeneity is discussed. The optimal range of stiffness parameters, Young's modulus, for MSC expansion culture to suppress osteogenic differentiation bias through the suppression of YAP and RUNX2 nuclear localization, and cell cycle progression is likely to be surprisingly narrow for a cell population from an identical donor and vary among cell populations from different donors. We believe that characterization of the heterogeneity of MSCs and understanding their biological meaning is an exciting research direction to establish guidelines for the design of culture substrates for the sophisticated control of MSC properties.

Chitosan: a green adhesive for surface functionalization and fabrication of thermoplastic biomedical microdevices.

Chitosan (CS) is a natural polymer that exhibits many biological properties and is used as a biomaterial for antibacterial coatings, tissue engineering, cell research, drug delivery, and negatively charged molecule capture. In our previous study, we used a CS-polydopamine mixture to realize UV-assisted bonding between poly(methyl methacrylate) (PMMA) substrates to fabricate microdevices for self-assembled stem cell spheroid cultures. Herein, we attained reliable adhesive bonding between PMMAs using CS at room temperature assisted by oxygen plasma. The bond strength of adhesion was as high as 2.1 MPa, which could be stable for over two months according to the leak test. The adhesive bonding and surface functionalization of the microchannels were simultaneously completed such that the microdevices could be directly used for mesenchymal stem cell culture for spheroid generation and DNA purification for point-of-care testing (POCT) devices. Surface characterization was performed by contact angle measurements, Fourier-transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. The POCT device allows sequential on-chip DNA purification, amplification, and colorimetric detection of pathogenic bacteria. This method provides a convenient and reliable strategy for the fabrication of PMMA microdevices that can be directly implemented in biological studies and POCT applications without involving prior surface modification steps.

Overcoming the resistance of hepatocellular carcinoma to PD-1/PD-L1 inhibitor and the resultant immunosuppression by CD38 siRNA-loaded extracellular vesicles

ABSTRACT Extracellular vesicles (EVs) are promising tools for drug delivery across different biological barriers. Here, we evaluated the potential of EVs-mediated delivery of CD38 siRNA on the immunosuppression of hepatocellular carcinoma (HCC). EVs were isolated from bone marrow mesenchymal stem cell culture medium and loaded with CD38 siRNA to prepare EVs/siCD38. Loss-of-function assays were conducted to investigate the biological functions of EVs/siCD38 in HCC cells. Xenograft mouse models were performed for further validation. High CD38 expression was found in HCC. EVs/siCD38 inhibited CD38 enzyme activity, decreased adenosine production, and promoted macrophage repolarization to M1 type, thus inhibiting HCC cell growth and metastasis in vitro as well as tumor growth in mice. Mechanistically, CD38 was upregulated in mice resistant to PD-1/PD-L1 inhibitor and EVs/siCD38 reversed the resistance of tumor to PD-1/PD-L1 inhibitor in vivo. Our results provide functional evidence for the use of EV-mediated delivery of CD38 siRNA to prevent immunosuppression feature of HCC.

Modulation of NLRP3 inflammasomes activation contributes to improved survival and function of mesenchymal stromal cell spheroids

Mesenchymal stem cells (MSCs) are ubiquitous multipotent cells that exhibit significant therapeutic potentials in a variety of disorders. Nevertheless, their clinical efficacy is limited owing to poor survival, low rate of engraftment, and impaired potency upon transplantation. Spheroidal three-dimensional (3D) culture of MSCs (MSC3D) has been proven to better preserve their invivo functional properties. However, the molecular mechanisms underlying the improvement in MSC function by spheroid formation are not clearly understood. NLRP3 inflammasomes, a key component of the innate immune system, have recently been shown to play a role in cell fate decision of MSCs. The present study examined the role of NLRP3 inflammasomes in the survival and potency of MSC spheroids. We found that MSC3D led to decreased activation of NLRP3 inflammasomes through alleviation of ER stress in an autophagy-dependent manner. Importantly, downregulation of NLRP3 inflammasomes signaling critically contributes to the enhanced survival rate in MSC3D through modulation of pyroptosis and apoptosis. The critical role of NLRP3 inflammasome suppression in the enhanced therapeutic efficacy of MSC spheroids was further confirmed in an invivo mouse model of DSS-induced colitis. These findings suggest that 3D culture confers survival and functional advantages to MSCs by suppressing NLRP3 inflammasome activation.

Current problems and persprectives of the treatment of cartilage injuries using mesenchimal stem cells

Annotation. Since standard methods of treatment of cartilage trauma and inflammatory diseases lack effectiveness, at present one of the most promising strategies is mesenchymal stem cell transplantation. Despite the successes in this field, many issues, such as the selection of the tissue-source of mesenchymal stem cells (MSCs) and their optimization or modification in culture in order to achieve the maximum therapeutic effect, remains an actual task for both global and domestic scientists. The purpose of the study is to prepare a review of literature sources for the period 2004-2022 (in the vast majority on publications of the last five years) and to systematize new data on the biological characteristics of MSCs from various tissue sources, which are used in strategies for the treatment of cartilage lesions tissues, to analyze existing problems and further prospects, as well as potential ways of optimizing MSC cultivation methods to increase their regenerative potential. A study of 49 sources that met the inclusion criteria was carried out. A significant variety of strategies for the use of MSCs in animal models and in clinical practice was revealed. The main mechanisms ensuring the therapeutic effect of MSCs have been determined and can be used as criteria for optimizing cellular material. However, it was found that the question of the effectiveness of the use of differentiated MSCs, the selection of the tissue-source of MSCs, and the specifics of the use of MSCs from neonatal sources remains open. Thus, the analysis of literary sources revealed further promising ways to improve the therapeutic efficiency of MSC grafting in a wide range of cartilaginous lesions. There is a need to research the effects of MSCs from neonatal tissues, and especially the umbilical cord. This will become possible due to experimental research that will be carried out in the future.

EMT and Tumor Turning Point Analysis in 3D Spheroid Culture of HNSCC and Mesenchymal Stem Cells.

The prognosis, metastasis, and behavior of head and neck squamous cancer cells are influenced by numerous factors concerning the tumor microenvironment, intercellular communication, and epithelial-to-mesenchymal transition (EMT). The aim of this study was to examine the codependent interaction of the mesenchymal stroma with head and neck squamous cell carcinoma (HNSCC) in a 3D spheroid structure. To simulate stroma-rich and -poor 3D tumor microenvironments, cells of the established cell SCC-040 were cultured with human mesenchymal stromal cells (MSCs), forming 3D stroma-tumor spheroids (STSs). STSs were compared to uniform spheroids of SCC-040 and MSC, respectively. The expressions of CD24, β-catenin, SNAI2, and ZEB2 were analyzed via RT-qPCR. The immunohistochemical expressions of E-cadherin, connexin 43, vimentin, and emmprin were analyzed, and protein expression pathways as well as Akt signaling were assessed via protein analysis. A promotive effect on the expressions of EMT markers ZEB2 (p = 0.0099), SNAI2 (p = 0.0352), and β-catenin (p = 0.0031) was demonstrated in STSs, as was the expression of Akt pathway proteins mTOR (p = 0.007), Erk1/2 (p = 0.0045), and p70 S6 Kinase (p = 0.0016). Our study demonstrated a change in genetic expression patterns early on in tumor development, indicating a tumor turning point.

Processing by Laser Stereolithography and <i>In Vitro</i> Biological Evaluation of Hydroxyapatite Scaffolds Mimicking Human Trabecular Bone Architecture

Hydroxyapatite (HA) ceramic scaffolds are commonly used as bone graft substitutes. Design of such scaffolds is a challenge to improve biological properties and extend the applications of HA ceramics in the field of bone tissue engineering. In this work, we investigated the processing and the in vitro properties of HA ceramic scaffolds mimicking human trabecular bone architecture. Samples of human tibial trabecular bone were collected (University Hospital Center of Limoges) and scanned by X-Ray μ-computed tomography (μ-CT) to generate 3D model database. From this computer-aided design, HA ceramic scaffolds were shaped layer-by-layer by additive manufacturing using laser stereolithography (SLA). Then, green parts were sintered to obtain dense ceramic scaffolds. The shaped parts were compared to the model (wall thickness, size, and geometry of the porous network) using image analysis. A good agreement was found. Only small differences were detected due to a light overpolymerization or to some unprinted very small details that were not linked to a polymerized area of the previous layer. Due to part shrinkage during sintering a magnifying factor has to be applied to the scanned CAO model to match the real dimensions of the trabecular bone sample. Human mesenchymal stem cell (hMSC) cultures were performed to investigate the biological properties of these scaffolds (cell attachment and proliferation of hMSC). These preliminary biological evaluations show the good biocompatibility and cell adhesion of the HA substitute. This work evidences the efficiency of SLA to produce ceramic scaffold architectures mimicking that of the natural trabecular bone with promising biological behavior.

Improving extracellular vesicles production through a Bayesian optimization-based experimental design

With the growing demand and diversity of biological drugs, developing optimal processes for their accelerated production with minimal resource utilization is a pressing challenge. Typically, such optimization involves multiple target properties, such as production yield, biological activity, and product purity. Therefore, strategic experimental design techniques that can characterize the parameter space while simultaneously arriving at the optimal process satisfying multiple target properties are required. To achieve this, we propose the use of a multi-objective batch Bayesian optimization (MOBBO) algorithm and illustrate its successful application for the production of extracellular vesicles (EVs) from a 3D culture of mesenchymal stem cells (MSCs) considering three objectives, namely to maximize the vesicle-to-protein ratio, maximize the enzymatic activity of the MSC-EV protein CD73, and minimize the amount of calregulin impurities. We show that the optimal combination of the process parameters to address the intended objectives could be achieved with only 32 experiments. For the four parameters considered (i.e., microcarrier concentration, seeding density, centrifugation time, and impeller speed), this number of experiments is comparable to or lower than the classical design of experiments (DoE) and the traditional one-factor-at-a-time (OFAT) approach. We illustrate how the algorithm adaptively samples in the process parameter space, selectively excluding unfavorable regions, thus minimizing the number of experiments required to reach optimal conditions. Finally, we compare the obtained solutions to the literature data and present possible applications of the collected data for other modeling activities such as Quality by Design, process monitoring, control, and scale-up.

Development of a biocompatible allogenic extra cellular matrix (MEC) for gradual dispensation of active substances

AbstractPurpose: TBF aims to design an allogeneic extra cellular matrix that can be loaded with a large range of active substances: antibiotic, anti‐inflammatory or biological components and able to provide a gradual dispensation of these substances.Methods: TBF developed an amniotic membrane of umbilical cord (AM‐UC). The product is a moulded foam or fibres to be reconstituted in dry eyes or gel. The product is virally inactivated, freeze‐dried and sterilized. Products are safe and biocompatible. The testing product consists in a moulded small strip that can be inserted behind the lower eyelid. These products contain hyaluronic acid, growth factors and proteoglycans. They have been impregnated after gamma irradiation with a known quantity of gentamycin, dexamethasone or extracellular micro‐vesicles extracted from mesenchymal stem cells cultures. Impregnated products have been placed in an extraction medium, which has been removed and replaced at defined times. Actives substances have then been quantified in the different extracts according to appropriate methods. Gentamycin has been quantified according to the Method A from the Eur. Ph., dexamethasone by an ELISA detection kit then supported by HPLC‐MS/MS method (after a bioreactor study) and exosomes using an ELISA detection kit based on CD63 protein binding.Results: For gentamycin and dexamethasone, quantifications have shown that the products enable a progressive release during the first 6 h of extraction of 40%–60% of the active substance with still extraction after 24 h. Then a threshold is reached, and the release is stabilized over the remaining time of extraction until reaching 55%–70% of extraction. Exosomes are constantly released in extraction medium; all observation points are similar.Conclusions: The results obtained tend to show that the product developed can be impregnated with different type of active substances and allow a gradual dispensation. It could be used in ophthalmological pathologies in order to increase contact duration between the eye and the active substance.

A physiological adipose-on-chip disease model to mimic adipocyte hypertrophy and inflammation in obesity

The adipose tissue is a metabolically active endocrine organ with a dynamic secretome that is known to be implicated in metabolic disorders. Various studies have demonstrated detrimental downstream endocrinal effects of dysfunctional adipose tissue on other metabolic tissues, such as skeletal muscle and liver. In vitro ‘Adipose-on-Chip’ (AOC) models have been developed as an animal-alternative experimental platform to mimic adipose dysfunction in metabolic diseases. However, existing AOCs have not modeled both overtime lipid accumulation and inflammation of adipocytes in the presence of excess circulating free fatty acids (FFA), which are hallmarks of dysfunctional adipose tissue in obesity. This study reports for the first time, the establishment of a physiologically-relevant AOC disease model, which mimics adipose tissue pathophysiology in obesity via excessive FFA loading. The AOC model supports 3D perfusion culture of human bone marrow mesenchymal stem cell (BMMSC) differentiated adipocytes with improved adipogenic phenotypes as compared to conventional 2D well-plate cultures. Adipocytes in the AOC can be induced into a diseased phenotype on-chip, where they become both hypertrophic and inflamed when treated with an FFA mixture. This AOC disease model provides a more physiological experimental system to study the effects of adipose tissue dysfunction on downstream tissues for mechanistic investigations into obesity-related metabolic diseases. • Our adipose-on-chip (AOC) platform supports dynamic 3D culture of adipocytes with enhanced adipogenic phenotype. • Treating our AOC platform with free fatty acids drove adipocyte inflammation and formation of hypertrophic lipid droplets. • Our AOC may be coupled to liver/muscle-on-chip platforms to study effects of adipose tissue on other metabolic organs.

Improvement in Skin Elasticity Using Red Deer Umbilical Cord Lining Mesenchymal Stem Cell Conditioned Media.

Significant improvement in skin tone was reported after topical application of a facial cream (CALECIM® Professional Multi-Action Cream, CALECIM Cosmeceuticals, Singapore) containing conditioned media (CM) derived from Red Deer Umbilical Cord Lining Mesenchymal Stem Cell (RD-CLMSC) culture. This study investigates the paracrine effects of RD-CLMSC-CM on human dermal fibroblasts (HDF) to understand how it may increase skin turgor and elasticity. Skin aging is associated with lower levels of extracellular matrix components such as hyaluronic acid (HA) and elastin, resulting in poor skin turgor and elasticity. Histochemical staining followed by photocolorimetry demonstrated that RD-CLMSC-CM upregulated HDF expression of elastin by 56% and HA by 83% compared with DMEM/10% Fetal Calf Serum (FCS).To further quantify the effects of CM, a proliferation assay was used to assess HDF response to RD-CLMSC-CM exposure. Exposure to RD-CLMSC-CM resulted in the highest increase in HDF proliferation over DMEM/10% FCS (113%) followed by Human (H)-CLMSC-CM (112%), then Human Foreskin Fibroblast (FSF)-CM (16%).These experimental results demonstrate both the cross-species efficacy and lack of toxicity of RD-CLMSC-CM on HDF. These pre-clinical studies also suggest the clinical effects of RD-CLMSC-CM on skin turgor may be related to increased HA and elastin production by HDF, as well as enhanced proliferation. J Drugs Dermatol. 2023;21(1):82-89. doi:10.36849/JDD.6906.

Lipoplex-Functionalized Thin-Film Surface Coating Based on Extracellular Matrix Components as Local Gene Delivery System to Control Osteogenic Stem Cell Differentiation.

A gene-activated surface coating is presented as a strategy to design smart biomaterials for bone tissue engineering. The thin-film coating is based on polyelectrolyte multilayerscomposed of collagen I and chondroitin sulfate, two main biopolymers of the bone extracellular matrix, which are fabricated by layer-by-layer assembly. For further functionalization, DNA/lipid-nanoparticles (lipoplexes) are incorporated into the multilayers. The polyelectrolyte multilayer fabrication and lipoplex deposition are analyzed by surface sensitive analytical methods that demonstrate successful thin-film formation, fibrillar structuring of collagen, and homogenous embedding of lipoplexes. Culture of mesenchymal stem cells on the lipoplex functionalized multilayer results in excellent attachment and growth of them, and also, their ability to take up cargo like fluorescence-labelled DNA from lipoplexes. The functionalization of the multilayer with lipoplexes encapsulating DNA encoding for transient expression of bone morphogenetic protein 2 induces osteogenic differentiation of mesenchymal stem cells, which is shown by mRNA quantification for osteogenic genes and histochemical staining. In summary, the novel gene-functionalized and extracellular matrix mimicking multilayer composed of collagen I, chondroitin sulfate, and lipoplexes, represents a smart surface functionalization that holds great promise for tissue engineering constructs and implant coatings to promote regeneration of bone and other tissues.

Investigation of the Apoptotic Effects of Mesenchymal Stem Cells on KG-1 Leukemic Cell Line

Background: Mesenchymal stem cells (MSCs) are undifferentiated cells with the ability of multi-potency, pluripotency, and self-renewal. MSCs show great promise in cancer therapy due to their unique features. MSC secrete various cytokines with multifunctional properties, although their roles are unclear. Methods: We have investigated the influence of secreted cytokines from MSCs on KG-1 cells as a cell model of acute myeloid leukemia (AML). For this purpose, following the culture and characterization of MSCs, a trans-well system was used for co-culturing MSCs and KG-1. To determine apoptosis induction Ki/Caspase-3 assay was conducted for cultured KG-1 alone and in co-culture with MSCs (10:1) on day 7. In the following step, the protein was isolated from both groups (control and experimental) and western blotting was done for investigating the BAX and BCL-2 proteins expression. Results: It was found that MSCs significantly enhanced caspase-3 activity in KG-1 cells (P<0.05). Besides, A significant increase in protein expression of BAX was detected, while BCL-2 displayed a dramatic reduction (P<0.01). Conclusion: As a concluding remark, MSCs have a contributory role in the apoptosis of KG-1 cells that is mediated by Caspase-3, BAX, and BCL2 expression.

Regulatory T-Cell Enhancement, Expression of Adhesion Molecules, and Production of Anti-Inflammatory Factors Are Differentially Modulated by Spheroid-Cultured Mesenchymal Stem Cells.

The culture of mesenchymal stem cells (MSCs) as spheroids promotes a more physiological cellular behavior, as it more accurately reflects the biological microenvironment. Nevertheless, mixed results have been found regarding the immunosuppressive properties of spheroid-cultured MSCs (3D-MSCs), the mechanisms of immunoregulation of 3D-MSCs being scarcely described at this point. In the present study, we constructed spheroids from MSCs and compared their immunosuppressive potential with that of MSCs cultured in monolayer (2D-MSCs). First, we evaluated the ability of 2D-MSCs and 3D-MSCs to control the activation and proliferation of T-cells. Next, we evaluated the percentage of regulatory T-cells (Tregs) after the co-culturing of peripheral blood mononuclear cells (PBMCs) with 2D-MSCs and 3D-MSCs. Finally, we investigated the expression of adhesion molecules, as well as the expressions of several anti-inflammatory transcripts in 2D-MSCs and 3D-MSCs maintained in both inflammatory and non-inflammatory conditions. Interestingly, our data show that several anti-inflammatory genes are up-regulated in 3D-MSCs, and that these cells can control T-cell proliferation. Nevertheless, 2D-MSCs are more efficient in suppressing the immune cell proliferation. Importantly, contrary to what was observed in 3D-MSCs, the expressions of ICAM-1 and VCAM-1 are significantly upregulated in 2D-MSCs exposed to an inflammatory environment. Furthermore, only 2D-MSCs are able to promote the enhancement of Tregs. Taken together, our data clearly show that the immunosuppressive potential of MSCs is significantly impacted by their shape, and highlights the important role of cell-cell adhesion molecules for optimal MSC immunomodulatory function.

Osteogenic differentiation and proliferation potentials of human bone marrow and umbilical cord-derived mesenchymal stem cells on the 3D-printed hydroxyapatite scaffolds

Mesenchymal stem cells (MSCs) are a promising candidate for bone repair. However, the maintenance of MSCs injected into the bone injury site remains inefficient. A potential approach is to develop a bone-liked platform that incorporates MSCs into a biocompatible 3D scaffold to facilitate bone grafting into the desired location. Bone tissue engineering is a multistep process that requires optimizing several variables, including the source of cells, osteogenic stimulation factors, and scaffold properties. This study aims to evaluate the proliferation and osteogenic differentiation potentials of MSCs cultured on 2 types of 3D-printed hydroxyapatite, including a 3D-printed HA and biomimetic calcium phosphate-coated 3D-printed HA. MSCs from bone marrow (BM-MSCs) and umbilical cord (UC-MSCs) were cultured on the 3D-printed HA and coated 3D-printed HA. Scanning electron microscopy and immunofluorescence staining were used to examine the characteristics and the attachment of MSCs to the scaffolds. Additionally, the cell proliferation was monitored, and the ability of cells to differentiate into osteoblast was assessed using alkaline phosphatase (ALP) activity and osteogenic gene expression. The BM-MSCs and UC-MSCs attached to a plastic culture plate with a spindle-shaped morphology exhibited an immunophenotype consistent with the characteristics of MSCs. Both MSC types could attach and survive on the 3D-printed HA and coated 3D-printed HA scaffolds. The MSCs cultured on these scaffolds displayed sufficient osteoblastic differentiation capacity, as evidenced by increased ALP activity and the expression of osteogenic genes and proteins compared to the control. Interestingly, MSCs grown on coated 3D-printed HA exhibited a higher ALP activity and osteogenic gene expression than those cultured on the 3D-printed HA. The finding indicated that BM-MSCs and UC-MSCs cultured on the 3D-printed HA and coated 3D-printed HA scaffolds could proliferate and differentiate into osteoblasts. Thus, the HA scaffolds could provide a suitable and favorable environment for the 3D culture of MSCs in bone tissue engineering. Additionally, biomimetic coating with octacalcium phosphate may improve the biocompatibility of the bone regeneration scaffold.

Three-Dimensional Culture of Equine Bone Marrow-Derived Mesenchymal Stem Cells Enhances Anti-Inflammatory Properties in a Donor-Dependent Manner.

Three-dimensional (3D) culture of human mesenchymal stem cells (MSCs) as spheroids enhances the production of important regulators of inflammation: prostaglandin E2 (PGE2), interleukin (IL)-6, and tumor necrosis factor-inducible gene 6 (TSG-6). The horse is a model species and suffers from musculoskeletal, ocular, and systemic inflammatory disease. It is unknown if 3D culture promotes enhanced production of immunomodulatory cytokines and regulators in equine MSCs and if there is variation between individual cell donors. We evaluated the feasibility, cell viability, and stem cell marker stability of 3D-cultured equine bone marrow-derived MSCs (eBMSCs) and determined the effect of inflammatory stimulation upon gene expression and secretion of key regulators of inflammation [PGE2, TSG-6, IL-10, IL-6, stromal cell-derived factor 1 (SDF-1)]. Variations in anti-inflammatory phenotype between six donors were investigated, with and without IL-1β stimulation, in either monolayer [two-dimensional (2D)] or 3D culture. Our results showed that eBMSCs self-aggregate in 3D culture while maintaining cell viability and markers of stemness CD90, CD44, CD104, and Oct4. In addition, 3D culture enhances the anti-inflammatory phenotype regardless of inflammatory stimulation by increasing PGE2, IL-6, TSG-6, SDF-1, and IL-10. Finally, anti-inflammatory phenotype was enhanced by IL-1β exposure but showed significant variation between cell lines in the degree of gene upregulation, and what genes were expressed. We conclude that 3D culture of eBMSCs as spheroids alters their anti-inflammatory phenotype, but this effect is influenced by cytokine exposure and cell donor.

Extracellular matrix protein gelatin provides higher expansion, reduces size heterogeneity, and maintains cell stiffness in a long-term culture of mesenchymal stem cells

Extracellular matrices (ECM) present in our tissues play a significant role in maintaining tissue homeostasis through various physical and chemical cues such as topology, stiffness, and secretion of biochemicals. They are known to influence the behavior of resident stem cells. It is also known that ECM type and coating density on cell culture plates strongly influence in vitro cellular behavior. However, the influence of ECM protein coating on long-term mesenchymal stem cell expansion has not been studied yet. To address this gap, we cultured bone-marrow derived hMSCs for multiple passages on the tissue culture plastic plates coated with 25 μg/ml of various ECM proteins. We found that cells on plates coated with ECM proteins had much higher proliferation compared to the regular tissue culture plates. Further, gelatin-coated plates helped the cells to grow faster compared to collagen, fibronectin, and laminin coated plates. Additionally, the use of gelatin showed less size heterogeneity among the cells when expanded from passages 3 to 9 (P3 to P9). Gelatin also helped in maintaining cellular stiffness which was not observed across other ECM proteins. In summary, in this research, we have shown that gelatin which is the least expensive compared to other ECM proteins, provides a better platform for mesenchymal stem cell expansion.