scholarly journals The Immunoregulatory Effect of Mechanical Stress on Three-dimensional Cultured Mesenchymal Stem Cells After Extensive Expansion

2021 ◽  
Author(s):  
Fang-Ying Du ◽  
Na Zhao ◽  
Lei Bao ◽  
Jing Lei ◽  
An-Qi Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Mechanical Stress ◽  
Quantitative Polymerase Chain Reaction ◽  
Three Dimensional ◽  
3D Culture ◽  
Early Passage ◽  
Immunomodulatory Property ◽  
2D And 3D ◽  
Immunomodulatory Function

Abstract Background: Mesenchymal stem cells (MSCs) have been used to treat immunopathy, and three-dimensional (3D) cultured MSCs show enhanced immunomodulatory property compared with those in two-dimensional (2D) culture. However, both the regulatory mechanisms remain unclear. The aim of the study was to investigate the role of mechanical stress in maintaining the immunomodulatory function of 2D and 3D cultured MSCs.Methods: Umbilical cord mesenchymal stem cells (UC-MSCs) were plated on tissue culture plastic (TCP) as 2D culture and 3D cultured UC-MSCs were seeded in matrigel. Surface markers, clonogenicity, proliferation and immunoregulatory property of UC-MSCs were evaluated. Meanwhile, we established the mouse models of colitis and type 1 diabetes mellitus (T1DM) to reveal the pharmacotherapeutic effects of 3D cultured MSCs in vivo. The effect of changing mechanical stress by modulating Yes-associated protein (YAP) on immunomodulatory function of 2D and 3D cultured UC-MSCs was evaluated by immunofluorescent analysis, real-time quantitative polymerase chain reaction (qPCR) and western blot.Results: We verified early passage UC-MSCs in 2D and 3D cultures exhibited stemness, immunomodulatory property and therapeutic efficacy against immunopathy. However, these characteristics of 2D cultured UC-MSCs were impaired after extensive expansion, whereas 3D culture extended them for several passages by activating YAP. Moreover, prostaglandin E2 (PGE2) could up-regulate YAP to improve the immunomodulatory ability of 2D cultured UC-MSCs after extensive expansion. Conclusions: This work found for the first time that the significance of mechanical stress in maintaining immunoregulatory function of 2D and 3D cultured UC-MSCs, providing a new idea for improving the efficacy of MSCs-based immunotherapy.

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 Comparison of Immunosuppressive and Angiogenic Properties of Human Amnion-Derived Mesenchymal Stem Cells between 2D and 3D Culture Systems

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Vitale Miceli ◽  
Mariangela Pampalone ◽  
Serena Vella ◽  
Anna Paola Carreca ◽  
Giandomenico Amico ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Culture Method ◽  
Paracrine Factors ◽  
Human Amnion ◽  
Tissue Repair And Regeneration ◽  
3D Culture System

The secretion of potential therapeutic factors by mesenchymal stem cells (MSCs) has aroused much interest given the benefits that it can bring in the field of regenerative medicine. Indeed, the in vitro multipotency of these cells and the secretive capacity of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture, MSCs rapidly lose the expression of key transcription factors associated with multipotency and self-renewal, as well as the ability to produce functional paracrine factors. In our study, we show that a three-dimensional (3D) culture method is effective to induce MSC spheroid formation, to maintain the multipotency and to improve the paracrine activity of a specific population of human amnion-derived MSCs (hAMSCs). The regenerative potential of both 3D culture-derived conditioned medium (3D CM) and their exosomes (EXO) was assessed against 2D culture products. In particular, tubulogenesis assays revealed increased capillary maturation in the presence of 3D CM compared with both 2D CM and 2D EXO. Furthermore, 3D CM had a greater effect on inhibition of PBMC proliferation than both 2D CM and 2D EXO. To support this data, hAMSC spheroids kept in our 3D culture system remained viable and multipotent and secreted considerable amounts of both angiogenic and immunosuppressive factors, which were detected at lower levels in 2D cultures. This work reveals the placenta as an important source of MSCs that can be used for eventual clinical applications as cell-free therapies.

Mesenchymal stem cells from adipose tissue which have been differentiated into chondrocytes in three-dimensional culture express lubricin

2011 ◽  
Vol 236 (11) ◽  
pp. 1333-1341 ◽  
Author(s):  
Giuseppe Musumeci ◽  
Debora Lo Furno ◽  
Carla Loreto ◽  
Rosario Giuffrida ◽  
Silvia Caggia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Collagen Type ◽  
Three Dimensional ◽  
3D Culture ◽  
Collagen Type I ◽  
Type I ◽  
Human Mscs ◽  
Alternative Source

The present study focused on the isolation, cultivation and characterization of human mesenchymal stem cells (MSCs) from adipose tissue and on their differentiation into chondrocytes through the NH ChondroDiff medium. The main aim was to investigate some markers of biomechanical quality of cartilage, such as lubricin, and collagen type I and II. Little is known, in fact, about the ability of chondrocytes from human MSCs of adipose tissue to generate lubricin in three-dimensional (3D) culture. Lubricin, a 227.5-kDa mucinous glycoprotein, is known to play an important role in articular joint physiology, and the loss of accumulation of lubricin is thought to play a role in the pathology of osteoarthritis. Adipose tissue is an alternative source for the isolation of multipotent MSCs, which allows them to be obtained by a less invasive method and in larger quantities than from other sources. These cells can be isolated from cosmetic liposuctions in large numbers and easily grown under standard tissue culture conditions. 3D chondrocytes were assessed by histology (hematoxylin and eosin) and histochemistry (Alcian blue and Safranin-O/fast green staining). Collagen type I, II and lubricin expression was determined through immunohistochemistry and Western blot. The results showed that, compared with control cartilage and monolayer chondrocytes showing just collagen type I, chondrocytes from MSCs (CD44-, CD90- and CD105- positive; CD45-, CD14- and CD34-negative) of adipose tissue grown in nodules were able to express lubricin, and collagen type I and II, indicative of hyaline cartilage formation. Based on the function of lubricin in the joint cavity and disease and as a potential therapeutic agent, our results suggest that MSCs from adipose tissue are a promising cell source for tissue engineering of cartilage. Our results suggest that chondrocyte nodules producing lubricin could be a novel biotherapeutic approach for the treatment of cartilage abnormalities.

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 Mechanical and molecular parameters that influence the tendon differentiation potential of C3H10T1/2 cells in 2D- and 3D-culture systems

2019 ◽  
Author(s):  
Ludovic Gaut ◽  
Marie-Ange Bonnin ◽  
Isabelle Cacciapuoti ◽  
Monika Orpel ◽  
Mathias Mericskay ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
3D Culture ◽  
Culture Conditions ◽  
Negative Regulator ◽  
Plastic Substrate ◽  
Differentiation Potential ◽  
Molecular Parameters ◽  
2D And 3D ◽  
Cells Cultured

AbstractOne of the main challenges in tendon field relies in the understanding of regulators of the tendon differentiation program. The optimum culture conditions that favor tendon cell differentiation are not identified. Mesenchymal stem cells present the ability to differentiate into multiple lineages in cultures under different cues ranging from chemical treatment to physical constraints. We analyzed the tendon differentiation potential of C3H10T1/2 cells, a murine cell line of mesenchymal stem cells, upon different 2D- and 3D-culture conditions. We observed that C3H10T1/2 cells cultured in 2D conditions on silicone substrate were more prone to tendon differentiation assessed with the expression of the tendon markers Scx, Col1a1 and Tnmd as compared to cells cultured on plastic substrate. 3D fibrin environment was more favorable for Scx and Col1a1 expression compared to 2D-cultures. We also identified TGFβ2 as a negative regulator of Tnmd expression in C3H10T1/2 cells in 2D- and 3D-cultures. Altogether, our results provide us with a better understanding of the culture conditions that promote tendon gene expression and identify mechanical and molecular parameters on which we could play to define the optimum culture conditions that favor tenogenic differentiation in mesenchymal stem cells.

scholarly journals Rejuvenated ageing mesenchymal stem cells by stepwise preconditioning ameliorates surgery-induced osteoarthritis in rabbits

2021 ◽  
Vol 10 (1) ◽  
pp. 10-21
Author(s):  
Zhixian Zong ◽  
Xiaoting Zhang ◽  
Zhengmeng Yang ◽  
Weihao Yuan ◽  
Jianping Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Therapeutic Effect ◽  
Subchondral Bone ◽  
Adult Stem Cells ◽  
Cruciate Ligament ◽  
Quantitative Polymerase Chain Reaction ◽  
Cartilage Damage ◽  
Human Mesenchymal Stem Cells ◽  
Early Passage

Aims Ageing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model. Methods Mesenchymal stem cells (MSCs) were isolated from ageing patients and preconditioned with chondrogenic differentiation medium, followed by normal growth medium. Cellular assays including Bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrdU), quantitative polymerase chain reaction (q-PCR), β-Gal, Rosette forming, and histological staining were compared in the manipulated human mesenchymal stem cells (hM-MSCs) and their controls. The anterior cruciate ligament transection (ACLT) rabbit models were locally injected with two millions, four millions, or eight millions of hM-MSCs or phosphate-buffered saline (PBS). Osteoarthritis Research Society International (OARSI) scoring was performed to measure the pathological changes in the affected joints after staining. Micro-CT analysis was conducted to determine the microstructural changes in subchondral bone. Results Stepwise preconditioning approach significantly enhanced the proliferation and chondrogenic potential of ageing hMSCs at early passage. Interestingly, remarkably lower immunogenicity and senescence was also found in hM-MSCs. Data from animal studies showed cartilage damage was retarded and subchondral bone remodelling was prevented by the treatment of preconditioned MSCs. The therapeutic effect depended on the number of cells applied to animals, with the best effect observed when treated with eight millions of hM-MSCs. Conclusion This study demonstrated a reliable and feasible stepwise preconditioning strategy to improve the safety and efficacy of ageing MSCs for the prevention of OA development. Cite this article: Bone Joint Res 2021;10(1):10–21.

Tripeptide-based macroporous hydrogel improves the osteogenic microenvironment of stem cells

2021 ◽  
Author(s):  
Qian Li ◽  
He Zhang ◽  
Jijia Pan ◽  
Binhong Teng ◽  
Ziqian Zeng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Osteogenic Induction

Due to the ability to combine multiple osteogenic induction "cues" at the same time, hydrogels are widely used in the three-dimensional (3D) culture of human mesenchymal stem cells (hMSCs) and...

scholarly journals Efficacy of 3D Culture Priming is Maintained in Human Mesenchymal Stem Cells after Extensive Expansion of the Cells

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1031 ◽  
Author(s):  
Thomas J. Bartosh ◽  
Joni H. Ylostalo
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Early Passage ◽  
Animal Product ◽  
Large Numbers ◽  
Late Passage ◽  
Fetal Bovine ◽  
Free Media

The use of non-optimal preparations of mesenchymal stem cells (MSCs), such as extensively expanded cells, might be necessary to obtain the large numbers of cells needed for many clinical applications. We previously demonstrated that minimally expanded (early passage) MSCs can be pre-activated as spheroids to produce potentially therapeutic factors in 3D cultures. Here, we used extensively expanded (late passage) MSCs and studied their 3D-culture activation potential. MSCs were culture-expanded as 2D monolayers, and cells from various passages were activated by 3D culture in hanging drops with either fetal bovine serum (FBS)-containing media or a more clinically-applicable animal product-free (xeno-free) media. Gene expression analyses demonstrated that MSC spheroids prepared from passage 3, 5, and 7 cells were similar to each other but different from 2D MSCs. Furthermore, the expression of notable anti-inflammatory/immune-modulatory factors cyclooxygenase-2 (PTGS2), TNF alpha induced protein 6 (TNFAIP6), and stanniocalcin 1 (STC-1) were up-regulated in all spheroid preparations. This was confirmed by the detection of secreted prostaglandin E2 (PGE-2), tumor necrosis factor-stimulated gene 6 (TSG-6, and STC-1. This study demonstrated that extensively expanded MSCs can be activated in 3D culture through spheroid formation in both FBS-containing and xeno-free media. This work highlights the possibility of activating otherwise less useable MSC preparations through 3D culture generating large numbers of potentially therapeutic MSCs.

scholarly journals Three-dimensional Differentiated Human Mesenchymal Stem Cells Exhibit Robust Antifibrotic Potential and Ameliorates Mouse Liver Fibrosis

2021 ◽  
Vol 30 ◽  
pp. 096368972098752
Author(s):  
Ja Sung Choi ◽  
Young-Jin Park ◽  
Sung-Whan Kim
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Growth Factor ◽  
Liver Fibrosis ◽  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
Enzyme Linked Immunosorbent Assay ◽  
Therapeutic Mechanism ◽  
3D Culture System

Recently, three-dimensional (3D)-cultured adipose mesenchymal stem cells (ASCs) have provided an effective therapy for liver fibrosis. This study aimed to enhance the potential of human ASCs for antifibrosis or hepatocyte regeneration using a 3D culture system and investigate their therapeutic mechanism in experimental liver fibrosis. ASC-3Dc were generated in a 3D culture system and stimulated with four growth factors, namely epidermal growth factor, insulin-like growth factor (IGF)-1, fibroblast growth factor-2, and vascular endothelial growth factor-A. The expression levels of antifibrotic or hepatic regeneration factors were then measured using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The therapeutic effects of ASC-3Dc were determined using a liver fibrosis model induced by thioacetamide. Histological analysis was performed to elucidate the therapeutic mechanism. ASC-3Dc exhibited high levels of hepatocyte growth factor (HGF), IGF-1, stromal cell-derived factor (SDF)-1 genes, and protein expression. In addition, injecting ASC-3Dc significantly prevented hepatic fibrosis and improved liver function in vivo. Moreover, high numbers of ki-67-expressing hepatocytes were detected in the ASC-3Dc-injected livers. Albumin-expressing ASC-3Dc engrafted in fibrotic livers augmented HGF expression. Thus, short-term 3D-cultured ASCs may be a novel alternative to the conventional treatment for liver damage in clinical settings.

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