Co-culture technology of mesenchymal stem cells and macrophages / 中国组织工程研究

BACKGROUND:In the co-culture environment of mesenchymal stem cells and macrophages,mesenchymal stem cells can promote the polarization of macrophages into anti-inflammatory macrophages to reduce inflammation,and macrophages can promote the osteogenic differentiation of mesenchymal stem cells.The co-culture of both plays an important role in regulating the immune system and promoting tissue regeneration. OBJECTIVE:To summarize the methods,influencing factors and possible mechanisms of co-culture between mesenchymal stem cells and macrophages,and to provide theoretical basis and experimental methods for the application of co-culture of mesenchymal stem cells and macrophages in tissue engineering. METHODS:The first author searched the relevant articles published from January 1970 to September 2023 in PubMed and CNK by computer from January to September 2023.The Chinese and English key words were"mesenchymal stem cells,macrophages,co-culture".Finally,63 articles were included and analyzed. RESULTS AND CONCLUSION:(1)In vitro co-culture of mesenchymal stem cells and macrophages can be divided into direct contact co-culture and indirect contact co-culture according to the model,and two-dimensional cell co-culture and three-dimensional cell co-culture according to the dimension.(2)The co-culture of mesenchymal stem cells and macrophages can promote the polarization of macrophages towards M2 type and enhance the osteogenic effect of mesenchymal stem cells.(3)In the co-culture model,the methods of co-culture,the proportion and time of co-culture,the phenotype of macrophages,and the cell source and conditions all affected the immune regulation of macrophages and the osteogenesis of mesenchymal stem cells.(4)Cell interaction in co-culture may regulate the immune function of macrophages,proliferation,migration and osteogenesis of mesenchymal stem cells through cell-secreted soluble factors,extracellular vesicles,cell-cell contact,and metabolic pathways.(5)Mesenchymal stem cells and macrophages can enhance cardiac function after acute myocardial infarction,promote epithelial wound healing,reduce lung inflammation,improve renal function,and accelerate bone repair.(6)There are still some problems in co-culture of mesenchymal stem cells and macrophages,such as the selection of co-culture conditions,the maintenance of good cell state and interaction of co-cultured cells.(7)The co-culture of mesenchymal stem cells and macrophages can improve the local inflammatory microenvironment and promote tissue regeneration and repair,so it will have a broad application prospect in tissue engineering.

Ideas and methods of in vitro model establishment for cell microenvironment in COPD / 中国药理学通报

Chronic obstructive pulmonary disease ( COPD ) major chronic disease threatening public health with complex pathological mechanisms. The change of the cell microenvironment of the lung is an important part of the pathophysiology of COPD. Cell culture technology is an important method to investigate the pathological mechanism of COPD and evaluate the pharmacological effect of medicine. Here we introduce the composition of the cell microenvironment of the lung, the change of the cell microenvironment in the pathological process of COPD, and summarize the application of in vitro model mimics cell microenvironment of COPD in the study of mechanism. In addition, we aim to put forward the ideas of the in vitro model establishment of cell microenvironment of COPD.

Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

BACKGROUND: Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection. RESULTS: Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-ß, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium. CONCLUSIONS: Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.

Progress in antitumor effect of tumor-associated macrophages in co-culture system / 中华微生物学和免疫学杂志

Macrophages are important cells of the immune system. Tumor-associated macrophages are enriched macrophages near tumor cells or tissues. Their role is mainly to promote the construction of tumor inflammatory microenvironment and inhibit tumor immune response. Cell co-culture system is a symbiotic culture system formed by mimicking the internal environment of the body in vitro. The co-culture condition is relatively consistent with the environment in vivo, enabling better information exchange and material exchange between cells, which is a supplement to the monolayer cell culture and animal experiments. Tumor-associated macrophages and tumor cells co-exist in the tumor microenvironment. Thus, constructing a co-culture system for tumor-associated macrophages and tumor cells would be conducive to studying the antitumor effect of tumor-associated macrophages and developing new immunotherapy drugs. The co-culture system would provide a new direction for treating malignant tumors. This article mainly reviewed the co-culture patterns of macrophages and the antitumor effects of different phenotypes of macrophages, and highlighted the importance of using immunotherapy to treat malignant tumors in the tumor microenvironment.

Advances in tumor-endothelial cells co-culture and interaction on microfluidics / 药物分析学报

The metastasis in which the cancer cells degrade the extracellular matrix (ECM) and invade to the sur-rounding and far tissues of the body is the leading cause of mortality in cancer patients. With a lot of advancement in the field, yet the biological cause of metastasis are poorly understood. The microfluidic system provides advanced technology to reconstruct a variety of in vivo-like environment for studying the interactions between tumor cells (TCs) and endothelial cells (ECs). This review gives a brief account of both two-dimensional models and three-dimensional microfluidic systems for the analysis of TCs-ECs co-culture as well as their applications to anti-cancer drug screening. Furthermore, the advanced methods for analyzing cell-to-cell interactions at single-cell level were also discussed.

Research progress of cell co-culture method / 中华危重病急救医学

Cell culture technology is the most commonly used method in the in vitro experiments at present. However, monolayer cell culture technology has been unable to meet the demand of the researchers. This is because that monolayer cell culture cannot mimic the cellular environment in which multiple cells interact with each other in the body. We cannot discuss the relationship of many cells, because we do not know the relationship between cells through a single kind of cell. So cell co-culture medicine arises at the historic moment for the demand. With the development of research method in recent years, cell co-culture method also has been improved in practice: from direct contact co-cultures to indirect contact co-cultures, from two-dimensional co-cultures to three-dimensional co-cultures. Cell co-culture method is closer to the human body. It is also more advantageous to study the interaction among cells. Nowadays, there are more researchers tend to select this method to study the physiological and pathological in vitro model, tissue engineering, and cell differentiation research. At the same time, it has become the focus of drug research and development, drug analysis, mechanism of drug action, and drug targets. This article will review the studies of cell co-culture method, summarize advantages and disadvantages of various methods, so as to promote improvement of cell culture methods, to build cells co-culture system that more close to human body, and build the in vitro model that simulate internal circulation of human body further.

Development of a neuromuscular junction model on surfaces modified by plasma polymerization / Desarrollo de un modelo de unión neuromuscular sobre superficies modificadas por polimerización por plasma

The aim of this work is to implement a biological model of neuromuscular junctions to study the mechanisms involved in intra and inter cellular processes using cell co-cultures. To optimize growth and development of the neuromuscular junction, cells were seeded on plasma polymerized pyrrole which has proven suitable for other types of cell cultures. The cell lines used were motor neuron NG108-15 and skeletal muscle C2C12. Cells were evaluated according to their morphology and electrophysiological characteristics. To observe the expression of specific proteins of the nerve synapse, immunocytochemical techniques were applied using dying antibodies. Proteins localized in nerve terminals were dyed and imaged by fluorescence microscopy. Images of cell co-cultures showed the formation of neuromuscular junctions. The preparation of neuromuscular junctions described in this work will allow the study of the mechanisms involved in their functions.

El objetivo de este trabajo es implementar un modelo biológico de unión neuromuscular para el estudio de los mecanismos involucrados en los procesos intra e intercelulares empleando co-cultivos celulares. Con el fin de optimizar el crecimiento y desarrollo de las uniones neuromusculares, las células se cultivaron sobre superficies de polipirrol obtenidas mediante polimerización por plasma que han mostrado ser adecuadas en otros tipos de cultivos celulares. Las líneas celulares que se emplearon fueron los modelos de motoneurona NG108-15 y muscular C2C12. Las células se evaluaron de acuerdo a su morfología y características electrofisiológicas. Para observar la expresión de proteínas clave de la sinapsis, se aplicaron técnicas inmunocitoquímicas utilizando anticuerpos específicos para la marcación de proteínas localizadas en las terminales nerviosas adquiriendo imágenes con microscopía de fluorescencia. Las imágenes de los co-cultivos celulares mostraron la formación de uniones neuromusculares. El método de preparación de uniones neuromusculares que se describe en este trabajo permitirá estudiar los mecanismos involucrados en sus funciones.

Vero Cell Co-culture Counteracts the Detrimental Effects of Hydrosalpinx Fluid on the Development of Mouse Embryos in vitro

Recent studies have suggested that the hydrosalpinx has a negative effect on pregnancy outcome, with markedly diminished implantation and increased early pregnancy loss. Fluid from the hydrosalpinx may leak into and accumulate in the uterine cavity. It is not clear, however if this creates a hostile local environment in the uterus for embryo implantation or exerts a direct embryotoxic effect. This study was conducted to investigate the detrimental effects of hydrosalpinx fluid (HSF) on the development of mouse embryos in vitro and to demonstrate whether Vero cells overcome these adverse effects. HSF was collected from three women with bilateral hydrosalpinx at the time of laparoscopic surgery. Collected fluid was centrifuged and the supernatant was frozen at -20degrees C. For co-culture, Vero cells were commercially obtained in a frozen state and cultured using Ham's F10 medium. Single-cell mouse embryos (B6CBAF1) were cultured for 5 days in 0, 0.4, 0.8, and 1.2% of HSF in media with and without Vero cells and examined daily to record the number of embryos reaching expanded blastocyst and hatching stage. Co-culture of mouse embryos with Vero cells at 0.8% HSF concentration significantly enhanced embryo development, but not at 1.2% hydrosalpinx fluid concentration. These results suggest that HSF is highly embryotoxic and Vero cells are likely to overcome these detrimental effects to some degree.

The cytotoxic activity against tumor cells induced rapidly in vitro the cell co-culture system by rhIL-18 / 中国免疫学杂志

Objective:To investigate the availability for rhIL-18 in the in vitro culture system to stimulate PBMHCs,with inducing cytotoxicities against tumor cells and to analyze the influencing factors for the effects.Methods:The NK cells,T cells and dendritic cells were separated from fresh PBMNCs by using the Stem Sep TM immunomagnetic beads.Cell phenotypes of the purified cell populations were identified with FCM technique.The cell co-culture system was established as follows.The PBMNCs or the cell preparations deleting the definite cell subset with immunoscreening were co-cultivated with mitotic-inactivated tumor cells in the presence of rhIL-18.Cytotoxicities of the various effector cell preparations to a series of tumor cell lines were examined by the isotope releasing assay.Results:In the in vitro cell co-culture system,rhIL-18 rapidly induces activation of the cytolytic responses against various tumor cell lines mediated by PBMNCs.The rapid induction within 24 h of culture was dependent on the dosage of rhIL-18,with the optimal dose of 100 ng/ml of the cytokine.The activated cytotoxicities were abrogated by deleting NK cells prior to the cell co-culture but did not vary when either T cells or DCs were removed.The cytotoxic responses were shown as a pattern of broad-spectrum to the target cells used and were not blocked by anti-MHC moAbs.Conclusion:NK cells were responsible for the rapid induction of cytotoxicities against tumor cells by rhIL-18.