scholarly journals Serum-Free Culture System for Spontaneous Human Mesenchymal Stem Cell Spheroid Formation

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Guoyi Dong ◽  
Shengpeng Wang ◽  
Yuping Ge ◽  
Qiuting Deng ◽  
Qi Cao ◽  
...  
Keyword(s):  
Clinical Research ◽  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cell ◽  
Spheroid Formation ◽  
Culture Methods ◽  
Serum Replacement ◽  
Knockout Serum Replacement

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

scholarly journals Serum-free culture system for spontaneous human mesenchymal stem cell spheroids formation

2019 ◽  
Author(s):  
Guoyi Dong ◽  
Shengpeng Wang ◽  
Yuping Ge ◽  
Qiuting Deng ◽  
Qi Cao ◽  
...  
Keyword(s):  
Clinical Research ◽  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cell ◽  
Culture Methods ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement

AbstractHuman mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids, but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stems medium (MiPS), adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were up-regulated in spheroids at day 6, and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were re-plated in normal FBS medium, cells formed a typical spindle-shaped morphology, and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

scholarly journals Mesenchymal stem cell spheroids: potential cell materials for cell therapy

STEMedicine ◽  
2020 ◽  
Vol 2 (5) ◽  
pp. e67
Author(s):  
Zhongjuan Xu ◽  
Xingzhi Liu ◽  
Yu Wei ◽  
Zhe Zhao ◽  
Junjun Cao ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Tissue Injury ◽  
Three Dimensional ◽  
3D Culture ◽  
Research Field ◽  
Three Dimensions ◽  
Spheroid Formation ◽  
Culture Methods ◽  
Differentiation Potential

Mesenchymal stromal/stem cells (MSCs) have been applied in clinical trials with an increasing number in recent years. MSCs showed their great potentials in regenerative medicine for their extensive sources, multilineage differentiation potential, low immunogenicity and self-renewal ability. However, the clinical application of MSCs still confronts many challenges including the requirement of large quantity of cells, low survival ability in vivo and the loss of main original characteristics due to two-dimensional (2D) culture although it is beneficial to cells fast expansion. Three-dimensional (3D) culture artificially creates an environment that permits cells to grow or interact with their surroundings in all three dimensions. Therefore, 3D culture was widely regarded as a more preferable and closer physiological microenvironment for cells growth. Recently, many different 3D spheroid culture methods have been developed to optimize MSCs biological characteristics to meet the demand of regenerative medicine. In this review, we comprehensively discussed the merits and demerits of different spheroid formation methods, expounded the mechanisms of spheroid formation and its microenvironment, and illustrated their optimized biological functions and the pre-clinical applications in various tissue injury and regeneration. In the end, we prospected the trends of this research field and proposed the key problems needed to be solved in the future.

Extracellular vesicles from three dimensional culture of human placental mesenchymal stem cells ameliorated renal ischemia/reperfusion injury

2021 ◽  
pp. 039139882098680
Author(s):  
Xuefeng Zhang ◽  
Nan Wang ◽  
Yuhua Huang ◽  
Yan Li ◽  
Gang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Extracellular Vesicles ◽  
Therapeutic Potential ◽  
Expression Profiles ◽  
Ischemia Reperfusion ◽  
Three Dimensional ◽  
3D Culture ◽  
Placental Mesenchymal Stem Cells ◽  
2D And 3D

Background: Three-dimensional (3D) culture has been reported to increase the therapeutic potential of mesenchymal stem cells (MSCs). The present study assessed the therapeutic efficacy of extracellular vesicles (EVs) from 3D cultures of human placental MSCs (hPMSCs) for acute kidney injury (AKI). Methods: The supernatants from monolayer culture (2D) and 3D culture of hPMSCs were ultra-centrifuged for EVs isolation. C57BL/6 male mice were submitted to 45 min bilateral ischemia of kidney, followed by renal intra-capsular administration of EVs within a 72 h reperfusion period. Histological, immunohistochemical, and ELISA analyses of kidney samples were performed to evaluate cell death and inflammation. Kidney function was evaluated by measuring serum creatinine and urea nitrogen. The miRNA expression profiles of EVs from 2D and 3D culture of hPMSCs were evaluated using miRNA microarray analysis. Results: The 3D culture of hPMSCs formed spheroids with different diameters depending on the cell density seeded. The hPMSCs produced significantly more EVs in 3D culture than in 2D culture. More importantly, injection of EVs from 3D culture of hPMSCs into mouse kidney with ischemia-reperfusion (I/R)-AKI was more beneficial in protecting from progression of I/R than those from 2D culture. The EVs from 3D culture of hPMSCs were more efficient against apoptosis and inflammation than those from 2D culture, which resulted in a reduction in tissue damage and amelioration of renal function. MicroRNA profiling analysis revealed that a set of microRNAs were significantly changed in EVs from 3D culture of hPMSCs, especially miR-93-5p. Conclusion: The EVs from 3D culture of hPMSCs have therapeutic potential for I/R-AKI.

scholarly journals Modified parylene-N films as chemical microenvironments for differentiation and spheroid formation of osteoblast cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tae-Hun Kim ◽  
Jong-Sook Lee ◽  
Hanhee Jo ◽  
Yusun Park ◽  
Mijin Yun ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Human Mesenchymal Stem Cells ◽  
Gene Expression Profiles ◽  
Global Gene Expression ◽  
Spheroid Formation ◽  
Hydrophobic Property ◽  
Osteoblast Cells ◽  
Bone Differentiation ◽  
Physical Treatments ◽  
Differentiation And Proliferation

Abstract In this work, the influence of parylene N film on the spheroid formation of osteoblast-like cells (MG-63) was determined and compared with that of high-hydrophilicity microenvironments, such as hydrophilic culture matrix and ultraviolet-treated parylene N film. To elucidate the change in cell properties due to the microenvironment of parylene N film, global gene expression profiles of MG-63 cells on parylene N film were analyzed. We confirmed the upregulated expression of osteoblast differentiation- and proliferation-related genes, such as Runx2, ALPL, and BGLAP and MKi67 and PCNA, respectively, using the real-time polymerase chain reaction. In addition, the differentiation and proliferation of osteoblast cells cultured on parylene N film were validated using immunostaining. Finally, the formation of spheroids and regulation of differentiation in human mesenchymal stem cells (MSCs) on parylene N film was demonstrated. The results of this study confirm that the microenvironment with the controlled hydrophobic property of parylene N film could effectively trigger the bone differentiation and maintains the proliferation of MSCs, similar to MG-63 cells without any scaffold structures or physical treatments.

scholarly journals Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Shuling Zhou ◽  
Karolina Szczesna ◽  
Anna Ochalek ◽  
Julianna Kobolák ◽  
Eszter Varga ◽  
...  
Keyword(s):  
Neural Progenitor Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Neural Progenitor Cell ◽  
Neural Progenitor ◽  
Culture Methods ◽  
Differentiation Potential ◽  
Differentiated Cells ◽  
Astrocyte Differentiation ◽  
Induced Pluripotent

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers,PAX6andNESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAPandaquaporin 4(AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.

scholarly journals Microfluidic-Based Droplets for Advanced Regenerative Medicine: Current Challenges and Future Trends

Biosensors ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 20
Author(s):  
Hojjatollah Nazari ◽  
Asieh Heirani-Tabasi ◽  
Sadegh Ghorbani ◽  
Hossein Eyni ◽  
Sajad Razavi Bazaz ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Regenerative Medicine ◽  
Cell Therapy ◽  
Large Scale ◽  
Three Dimensional ◽  
3D Culture ◽  
Cell Encapsulation ◽  
Cost Effective ◽  
Culture Methods

Microfluidics is a promising approach for the facile and large-scale fabrication of monodispersed droplets for various applications in biomedicine. This technology has demonstrated great potential to address the limitations of regenerative medicine. Microfluidics provides safe, accurate, reliable, and cost-effective methods for encapsulating different stem cells, gametes, biomaterials, biomolecules, reagents, genes, and nanoparticles inside picoliter-sized droplets or droplet-derived microgels for different applications. Moreover, microenvironments made using such droplets can mimic niches of stem cells for cell therapy purposes, simulate native extracellular matrix (ECM) for tissue engineering applications, and remove challenges in cell encapsulation and three-dimensional (3D) culture methods. The fabrication of droplets using microfluidics also provides controllable microenvironments for manipulating gametes, fertilization, and embryo cultures for reproductive medicine. This review focuses on the relevant studies, and the latest progress in applying droplets in stem cell therapy, tissue engineering, reproductive biology, and gene therapy are separately evaluated. In the end, we discuss the challenges ahead in the field of microfluidics-based droplets for advanced regenerative medicine.

scholarly journals Development of a 3D human osteoblast cell culture model for studying mechanobiology in orthodontics

2020 ◽  
Vol 42 (4) ◽  
pp. 387-395
Author(s):  
Damien Brezulier ◽  
Pascal Pellen-Mussi ◽  
Sylvie Tricot-Doleux ◽  
Agnès Novella ◽  
Olivier Sorel ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Gene Expression Profiles ◽  
Tissue Response ◽  
Cell Culture Model ◽  
Glucose Levels ◽  
Culture Model ◽  
Polymerase Chain ◽  
Adjustable Weight

Summary Objectives Mechanobiology phenomena constitute a major element of the cellular and tissue response during orthodontic treatment and the implantation of a biomaterial. Better understanding these phenomena will improve the effectiveness of our treatments. The objective of this work is to validate a model of three-dimensional (3D) culture of osteoblasts to study mechanobiology. Materials and methods The hFOB 1.19 cell line was cultured either traditionally on a flat surface or in aggregates called spheroids. They were embedded in 0.8% low-melting agarose type VII and placed in a polyethylene terephthalate transwell insert. Compressive forces of 1 and 4 g/cm2 were applied with an adjustable weight. Proliferation was evaluated by measuring diameters, monitoring glucose levels, and conducting Hoechst/propidium iodide staining. Enzyme-linked immunosorbent assays focusing on the pro-inflammatory mediators interleukin (IL)-6 and IL-8 and bone remodelling factor osteoprotegerin were performed to evaluate soluble factor synthesis. quantitative reverse transcription-polymerase chain reaction was performed to evaluate bone marker transcription. Results The 3D model shows good cell viability and permits IL dosing. Additionally, three gene expression profiles are analysable. Limitations The model allows analysis of conventional markers; larger exploration is needed for better understanding osteoblast mechanobiology. However, it only allows an analysis over 3 days. Conclusion The results obtained by applying constant compressive forces to 3D osteoblastic cultures validate this model system for exploring biomolecule release and analysing gene transcription. In particular, it highlights a disturbance in the expression of markers of osteogenesis.

scholarly journals Comparison of Pluripotency, Differentiation, and Mitochondrial Metabolism Capacity in Three-Dimensional Spheroid Formation of Dental Pulp-Derived Mesenchymal Stem Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Young-Bum Son ◽  
Dinesh Bharti ◽  
Saet-Byul Kim ◽  
Chan-Hee Jo ◽  
Eun-Yeong Bok ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Dental Pulp ◽  
Time Course ◽  
Three Dimensional ◽  
3D Culture ◽  
Mitochondrial Metabolism ◽  
Osteogenic Potential ◽  
Spheroid Formation ◽  
Differentiation Potential

Mesenchymal stem cells (MSCs) are valuable candidates in tissue engineering and stem cell-based therapy. Traditionally, MSCs derived from various tissues have been successfully expanded in vitro using adherent culture plates commonly called as monolayer two-dimensional (2D) cultures. Recently, many studies demonstrated that stemness and multilineage differentiation potential could be enhanced to greater extent when MSCs are cultured as suspended aggregates by means of three-dimensional (3D) culturing techniques. However, there are limited reports on changed mitochondrial metabolism on 3D spheroid formation of MSCs. Therefore, the present study was aimed at investigating the stemness, differentiation potential, and mitochondrial metabolism capacity of 3D dental pulp-derived MSC (DPSC) spheroids in comparison to monolayer cultured DPSCs. We isolated dental pulp-derived MSCs (DPSCs) and successfully developed a 3D culture system which facilitated the formation of MSC spheroids. The cell aggregation was observed after 2 hours, and spheroids were formed after 24 hours and remained in shape for 72 hours. After spheroid formation, the levels of pluripotent markers increased along with enhancement in adipogenic and osteogenic potential compared to 2D cultured control cells. However, decreased proliferative capacity, cell cycle arrest, and elevated apoptosis rate were observed with the time course of the 3D culture except for the initial 24-hour aggregation. Furthermore, oxygen consumption rates of living cells decreased with the time course of the aggregation except for the initial 24 hours. Overall, our study indicated that the short-term 3D culture of MSCs could be a suitable alternative to culture the cells.

scholarly journals Zebrafish skeletal muscle cell cultures: Monolayer to three-dimensional tissue engineered collagen constructs

2020 ◽  
Author(s):  
K.K Vishnolia ◽  
N.R.W Martin ◽  
D.J Player ◽  
E Spikings ◽  
M.P Lewis
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Morphological Characteristics ◽  
Hypertrophic Growth ◽  
3D Cultures

AbstractZebrafish (Danio rerio) are a commonly used model organism to study human muscular myopathies and dystrophies. To date, much of the work has been conducted in vivo due to limitations surrounding the consistent isolation and culture of zebrafish muscle progenitor cells (MPCs) in vitro and the lack of physiologically relevant models.Here we report a robust, repeatable, and cost-effective protocol for the isolation and culture of zebrafish MPCs in conventional monolayer (2D) and have successfully transferred these cells to 3D culture in collagen based three-dimensional (3D) tissue-engineered constructs. Zebrafish MPC’s cultured in 2D were consistently reported to be Desmin positive reflecting their muscle specificity, with those demonstrating Desmin positivity in the 3D cultures. In addition, mRNA expression of muscle markers specific for proliferation, differentiation and maturation measured from both monolayer and 3D cultures at appropriate developmental stages were found consistent with previously published from other species in vitro and in vivo muscle data.Collagen constructs seeded with zebrafish MPC’s were initially characterised for optimal seeding density, followed by macroscopic characterisation (three-fold contraction) of the matrix. Direct comparison between the morphological characteristics (proportion of cells) and gene expression profiles of cells cultured in collagen constructs revealed higher maturation and differentiation compared to monolayer cultures. In this regard, cells embedded in 3D collagen constructs revealed higher fusion index, Desmin positivity, hypertrophic growth, myotube maturity and myogenic mRNA expression when compared to in monolayer.In conclusion, these methods and models developed herein will facilitate in vitro experiments, which would complement in vivo zebrafish studies used to investigate the basic developmental, myopathies and dystrophies in skeletal muscle cells.

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