Three-Dimensional Culture Decreases the Angiogenic Ability of Mouse Macrophages

Macrophages play important roles in angiogenesis; however, previous studies on macrophage angiogenesis have focused on traditional 2D cultures. In this study, we established a 3D culture system for macrophages using collagen microcarriers and assessed the effect of 3D culture on their angiogenic capabilities. Macrophages grown in 3D culture displayed a significantly different morphology and arrangement under electron microscopy compared to those grown in 2D culture. Tube formation assays and chick embryo chorioallantoic membrane assays further revealed that 3D-cultured macrophages were less angiogenic than those in 2D culture. Whole-transcriptome sequencing showed that nearly 40% of genes were significantly differently expressed, including nine important angiogenic factors of which seven had been downregulated. In addition, the expression of almost all genes related to two important angiogenic pathways was decreased in 3D-cultured macrophages, including the two key angiogenic factors, VEGFA and ANG2. Together, the findings of our study improve our understanding of angiogenesis and 3D macrophage culture in tissues, and provide new avenues and methods for future research on macrophages.

Development of in vitro 3D culture system to mimic lung cancer tissue

A 3D culture system based on a photocurable matrix has been developed. The aim is to create a 3D printable platform mimicking lung cancer tissue, to study tumor microenvironment evolution, in terms of structural (architecture) and molecular (signalling) components.

Efficient Generation of Neural Stem Cells from Embryonic Stem Cells Using a Three-Dimensional Differentiation System

Mouse embryonic stem cells (ESCs) are useful tools for studying early embryonic development and tissue formation in mammals. Since neural lineage differentiation is a major subject of organogenesis, the development of efficient techniques to induce neural stem cells (NSCs) from pluripotent stem cells must be preceded. However, the currently available NSC differentiation methods are complicated and time consuming. This study aimed to propose an efficient method for the derivation of NSCs from mouse ESCs; early neural lineage commitment was achieved using a three-dimensional (3D) culture system, followed by a two-dimensional (2D) NSC derivation. To select early neural lineage cell types during differentiation, Sox1-GFP transgenic ESCs were used. They were differentiated into early neural lineage, forming spherical aggregates, which were subsequently picked for the establishment of 2D NSCs. The latter showed a morphology similar to that of brain-derived NSCs and expressed NSC markers, Musashi, Nestin, N-cadherin, and Sox2. Moreover, the NSCs could differentiate into three subtypes of neural lineages, neurons, astrocytes, and oligodendrocytes. The results together suggested that ESCs could efficiently differentiate into tripotent NSCs through specification in 3D culture (for approximately 10 days) followed by 2D culture (for seven days).

O-101 Neospermatogenesis benefits from a three-dimensional culture system

Study question Does a three-dimensional (3D) culture system increase the efficiency of male germline differentiation of mouse embryonic stem cells (mESC) over a bidimensional method? Summary answer Our 3D culture system based on direct spherification proves superior to the standard bidimensional plating in promoting neogametogenesis of mESC into post-meiotic male germ cells. What is known already Two-dimensional monolayer cell cultures are common in stem cell research. However, this method does not replicate a physiological 3D spatial relationship and may provide an inaccurate replication of in vivo environments. A 3D spherical structure allows us to mimic the seminiferous tubule, the site of in vivo spermatogenesis. By using spheroids as a scaffold to replicate cell culture systems, we can study spermatogenesis in a controlled setting. Direct spherification, a technique commonly used in molecular gastronomy, provides an opportunity to create spheroids that mimic in vivo events that materialize in the lab Study design, size, duration mESCs were initially cultured on a 6-well plate coated with fibroblasts and inserted into sodium alginate spheres. To coax differentiation, spheres (3 to 6 mm in diameter) were plunged directly into differentiation medium (DM) while the control mESC in 6-well dishes were layered with it. Cells obtained from both culture systems were tested by biomarkers for different germ cell stages Participants/materials, setting, methods Bidimensional mESC at 80% confluence were differentiated either on a plate or spherified for a 3D culture. Both systems underwent the same timeline of exposure to EpiLC medium with Activin A, bFGF and KSR for 3 days and PGCLC medium with BMP4, LIF, SCF and EGF for 7 days. Differentiated cells were retrieved from each method at day 3 and day 10 to assess for germ line differentiation markers, DAZL, VASA and BOULE Main results and the role of chance Under optic visualization through the sphere wall, cellular aggregation was seen on day 2 of culturing in EpiLC medium while this phenomenon was not observed on bidimensional plating. In the conventional method, cells expressed 7% DAZL (spermatogonium cell stage) and 1% VASA (pre-spermatid cell stage) whereas in direct spherification, cells expressed 20% DAZL (P < 0.001) and 15% VASA positivity (P < 0.0001). To further compare the different methods in later stages of germ-line differentiation, the remaining spheres were cultured in PGCLC medium for 7 days. At day 10, isolated cells were assessed for VASA and DAZL again. In the conventional method, 23% of cells expressed positivity for VASA and 29% DAZL whereas direct spherification achieved a positivity rate of 43% for VASA (P < 0.005) and 45% for DAZL (P < 0.005). This increased expression in both VASA and DAZL signify the increased number of cells undergoing germline differentiation. Additionally, BOULE was assessed for the presence of meiotic cells such as the spermatocyte. The conventional method yielded < 1% BOULE positivity whereas in direct spherification, there was 10% positivity (P < 0.005). Direct spherifcation result shows that differentiation almost doubled in comparison to the conventional method, yielding more post-meiotic cells in the same amount of time Limitations, reasons for caution Despite a higher differentiation rate in direct spherification, these cells would still need to be tested for their fertilization potential. The ability to achieve fertilization, blastocysts and live pups would provide final proof and reliability of this method of neogametogenesis Wider implications of the findings Differentiating ESCs through direct spherification provides an alternative to studying intercellular relationships. This provides an opportunity to study spermatogenesis in more detail by replicating the microenvironment of the seminiferous tubule. Once embryo developmental competence of the de novo gamete is confirmed, this may open a new chapter in human reproduction Trial registration number N/A

3D cell culture using a clinostat reproduces microgravity-induced skin changes

Exposure to microgravity affects human physiology in various ways, and astronauts frequently report skin-related problems. Skin rash and irritation are frequent complaints during space missions, and skin thinning has also been reported after returning to Earth. However, spaceflight missions for studying the physiological changes in microgravity are impractical. Thus, we used a previously developed 3D clinostat to simulate a microgravity environment and investigate whether physiological changes of the skin can be reproduced in a 3D in vitro setting. Our results showed that under time-averaged simulated microgravity (taSMG), the thickness of the endothelial cell arrangement increased by up to 59.75%, indicating skin irritation due to vasodilation, and that the diameter of keratinocytes and fibroblast co-cultured spheroids decreased by 6.66%, representing skin thinning. The α1 chain of type I collagen was upregulated, while the connective tissue growth factor was downregulated under taSMG. Cytokeratin-10 expression was significantly increased in the taSMG environment. The clinostat-based 3D culture system can reproduce physiological changes in the skin similar to those under microgravity, providing insight for understanding the effects of microgravity on human health before space exploration.

Lin28A Promotes the Amplification and Cancer Stemness of Lung Cancer Cells Via Activating MAPK Pathway Dependent on Let-7c Functions

Background: Metastasis and relapse of lung cancer are the main cause of disease-related deaths. It’s reported that tumor metastasis and relapse originated from cancer stem cells (CSCs) which possess more potential in proliferation and invasion. In our previous studies, we established a conditional BME-based three-dimensional culture (3D culture) system to mimic the growth environment in vivo and further amplified lung cancer stem cells (LCSCs) in our system. However, the molecular mechanisms of the amplification and development of LCSCs in our 3D culture system are still not very clear. Methods: We tested the expression of Lin28 and let7 by western blot and qPCR, and constructed A549 cells either knockdown of Lin28 or overexpression of let7, followed by investigating the expression of stemness markers by flow cytometry and qPCR, and stem cell like phenotypes including cell proliferation, colony formation, mammosphere culture, cell apoptosis, migration, invasion and drug resistance in vitro, as well as tumorigenicity in vivo. Results: Here we observed Lin28A/let-7c was dysregulated in LCSCs both from the 3D culture system and from lung cancer tissues. Further, the abnormal expression of Lin28A/let-7c was correlated with poor survival outcomes. We found over-expression let-7c inhibited the maintenance of LCSC properties, while the results for knockdown of Lin28A showed Lin28A was critical for the enrichment and amplification of LCSCs via MAPK signaling pathway. Importantly, we found that either knockdown of Lin28A or over-expression of let-7c inhibited carcinogenesis and disrupted LCSC expansion in vivo. Conclusions: Our study uncovered the functions and mechanisms of the "Lin28A/let-7c/MAPK" signaling pathway in promoting the amplification and cancer stemness of LCSCs, which might be a potential therapeutic target for lung cancer therapy by reducing and even eliminating LCSCs in the future.