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Human Liver Stem Cells:
Published in Gianfranco Alpini, Domenico Alvaro, Marco Marzioni, Gene LeSage, Nicholas LaRusso, The Pathophysiology of Biliary Epithelia, 2020
These MAPC have remarkable multi-potential characteristics. When cultured under carefully defined conditions, they can be induced to differentiate into functional, mature hepatocytes.68 Cells require multiple passages before studies can be undertaken and the differentiation response requires a complex set of conditions and growth factors, principally the presence of HGF and FGF4. Significantly, also, it was dependent on matrix derived signals, since the investigators used Matrigel to support the clonal expansion and subsequent differentiation.68 Although the chief constituents of Matrigel are known and include the three main basement membrane components, laminin, heparan sulphate proteoglycan and collagen IV,69 it is also know to contain significant levels of contaminating growth factors cytokines and other macro-molecules. Thus the differentiation signals are more complex than just two factors.
Collection and Expansion of Stem Cells
Published in Richard K. Burt, Alberto M. Marmont, Stem Cell Therapy for Autoimmune Disease, 2019
Ideally, human ES cell lines should be established in the absence of MEF layers, perhaps on human embryonic fibroblasts and in defined synthetic media. Xu et al demonstrated that human ES cells do not require physical contact with MEFs and can be maintained on a substrate of Matrigel (Becton Dickinson, Franklin Lakes, and NJ).143 Matrigel is a basement membrane matrix prepared from a mouse sarcoma cell line using medium previously conditioned by MEFs. Matrigel consists of collagen V and laminin, although other components are not fully defined. A suitable fully defined synthetic medium for the culture of human ES cells has not been described. Leukemia inhibitory factor (LIF) is sufficient to support the growth of mouse ES cells in the absence of feeders but does not appear to play a similar role in human ES cells.137
Methods to Study the Vasculature in ADPKD
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Patricia Outeda, Terry Watnick
The capillary-like or tube-like tubulogenesis assay is another well known “in vitro” method to study angiogenesis (Figure 7.7a). This is a versatile method since it allows the use of different endothelial cells (e.g., HUVECs, HMEC-1, or primary cells isolated from genetically modified animals) and different extracellular matrix components, and it is ideal for a quick screening of factors modulating angiogenesis.151–155 When plated on a matrix (commonly Matrigel, fibrin, or collagen), endothelial cells quickly reorganize, migrate, and differentiate to interconnect and form a complex network with quantifiable tube-like structures that mimics “in vivo” angiogenesis.156,157 These tube-like structures can be visualized using a phase-contrast or fluorescent microscope (as illustrated in Figure 7.7a). The most common matrix for 3D culture is Matrigel, which is a mixture of proteins secreted by murine Engelbreth-Holm-Swarm sarcoma cells and which resembles the extracellular environment of many tissues.158,159 Collagen gels are extensively used in angiogenesis assays and fibrin gels have also become more popular.160,161 It is important to keep variables such as passage number (especially for primary cells), cell density, and time allowed for the capillary network to form consistent across experimental groups, and therefore optimization may be required.
Culturing human pluripotent stem cells for regenerative medicine
Published in Expert Opinion on Biological Therapy, 2023
Hiroki Ozawa, Takuya Matsumoto, Masato Nakagawa
An obvious solution to this problem was feeder-free culture. Developing feeder-free culture methods requires a substrate (coating material) to replace feeder cells. Matrigel is a commonly used cell culture matrix primarily composed of basement membrane ECM proteins such as laminin, collagen IV, entactin, and heparan sulfate proteoglycan perlecan [52]. Furthermore, defined ECM molecules have been developed to support the pluripotency and proliferation of hPSCs. These include laminin [52], vitronectin [53], and SyntheMax, which is a synthetic peptide-acrylate surface [54]. Of the various laminin isoforms, laminin 511 [55,56] and laminin 521 [57] are particularly well suited for culturing hPSCs. Since full-length laminins are large heterotrimeric proteins, their large-scale production as recombinant proteins can be challenging. Consequently, an active fragment of the laminin 511 molecule called LN511-E8 was developed. This fragment has been shown to outperform the original full-length protein [58].
3D high resolution clonogenic survival measurement of xrs-5 cells in low-dose region of carbon ion plans
Published in International Journal of Radiation Biology, 2023
Dea Kartini, Olga Sokol, Chutima Talabnin, Chinorat Kobdaj, Marco Durante, Michael Krämer, Martina Fuss
First, matrigel matrix (Corning) was diluted with ice-cold culture medium to a final ECM protein concentration of 5 mg/mL. In order to compensate the V-shape of the well plate and prevent cells from attaching to the bottom of the well forming monolayers, a pre-coating with 30 µL of the diluted matrigel was applied and the plates were incubated for an hour or until the matrigel layer was hardened. Next, 2.5 µL of cell suspension containing 10,000 cells was mixed with 22.5 µL of matrigel. The mixture was added on top of the first gel layer and incubated for 1 h. After that, a third matrigel layer (10 µL) was added and incubated for an hour to provide additional space for cell growth and protecting the embedded cell layer from dissolving or mechanical damage. Finally, culture medium (29 µL) was added as a final layer to keep the gel hydrated and provide nutrients, and then cells were incubated for 48 h. The layered matrigel structure inside the well is illustrated in Figure 1.
Upregulation of microRNA-155 Enhanced Migration and Function of Dendritic Cells in Three-dimensional Breast Cancer Microenvironment
Published in Immunological Investigations, 2021
Pengxiang Yang, Xingjian Cao, Huilong Cai, Xiang Chen, Yihua Zhu, Yue Yang, Weiwei An, Jing Jie
DCs often display an immature status due to weak immunogenicity and the immune-suppressive tumor microenvironment, resulting in tumor escape (Kohnepoushi et al. 2019; Sabado and Bhardwaj 2015; Son et al. 2013). To increase confidence in DCs immunotherapy and to demonstrate its great clinical value, we explored whether cell function could be enhanced by a strategy that involves manipulating the intrinsic regulatory function of miR-155 (Gardner and Ruffell 2016; Palucka and Banchereau 2013). Three-dimensional cell culture is now of increasing interest in research that attempts to predict the pharmacodynamics and toxicity of drugs and the effects of vaccine candidates (Markey et al. 2017). Because 3D cell culture is superior to standard 2D cell culture conditions, 3D cell culture may better mimic complex physiological phenomena in vivo (Sprague et al. 2014; Yang et al. 2018). Recently, hydrogel matrices were used as the mechanical support of immune cells for further research (Groell et al. 2018). In this study, we developed a 3D cell culture model that encapsulates DCs in a simple Matrigel scaffold that mimics the mechanical properties of tissues. Matrigel is a basement membrane-derived preparation extracted from mouse sarcoma tumors that closely mimics the immunosuppressive tumor microenvironment in vivo (Caliari and Burdick 2016). The viability and function of DCs was studied in this model, which was shown to be applicable and effective.