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Substrate-independent immunomodulatory characteristics of mesenchymal stem cells in three-dimensional culture.

ABSTRACT: Mesenchymal stem cells (MSCs) play important roles in tissue regeneration, and multi-lineage differentiation and immunomodulation are two major characteristics of MSCs that are utilized in stem cell therapy. MSCs in vivo have a markedly different three-dimensional (3D) niche compared to the traditional two-dimensional (2D) culture in vitro. A 3D scaffold is predicted to provide an artificial 3D environment similar to the in vivo environment. Significant changes in MSC differentiation are shown to be occurred when under 3D culture. However, the immunomodulatory characteristics of MSCs under 3D culture remain unknown. In this study, 3D culture systems were constructed using different substrates to evaluate the common immunomodulatory characteristics of MSCs. Compared to the MSCs under 2D culture, the MSCs under 3D culture, which had higher stemness and maintained cell phenotype, showed altered immunophenotypic pattern. Gene expression profile analysis at mRNA and protein level detected by gene chip and protein chip, respectively, further revealed the difference between 3D cultured MSCs and 2D cultured MSCs, which was mainly concentrated in the immunoregulation related aspects. Moreover, the immunoregulatory role of 3D culture was confirmed by our immunosuppressive experiments. These findings demonstrated that the immunomodulatory capacities of MSCs were enhanced by the 3D geometry of substrates.

SUBMITTER: Li J

PROVIDER: S-EPMC6224081 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Substrate-independent immunomodulatory characteristics of mesenchymal stem cells in three-dimensional culture.

Li Jing J Chen Tong T Huang Xiahe X Zhao Yunshan Y Wang Bin B Yin Yanyun Y Cui Yi Y Zhao Yannan Y Zhang Ruiping R Wang Xiujie X Wang Yingchun Y Dai Jianwu J

PloS one 20181108 11

Mesenchymal stem cells (MSCs) play important roles in tissue regeneration, and multi-lineage differentiation and immunomodulation are two major characteristics of MSCs that are utilized in stem cell therapy. MSCs in vivo have a markedly different three-dimensional (3D) niche compared to the traditional two-dimensional (2D) culture in vitro. A 3D scaffold is predicted to provide an artificial 3D environment similar to the in vivo environment. Significant changes in MSC differentiation are shown t ...[more]

PMID: 30408051

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Project description:Background:Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved. Methods:We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production. Results:The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture. Conclusion:These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.

Project description:BackgroundIntestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine.ObjectivesTo describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities.Study designDescriptive study.MethodsIntestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed.ResultsIntestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle.Main limitationsTissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time.ConclusionsThe successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease (colic).

Dataset Information

Substrate-independent immunomodulatory characteristics of mesenchymal stem cells in three-dimensional culture.

Publications

Substrate-independent immunomodulatory characteristics of mesenchymal stem cells in three-dimensional culture.

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