Challenges to Adopting 3D Cultures in HTS Programs

Fucoxanthin (Fx) is a carotenoid derived from marine organisms that exhibits anticancer activities. However, its role as a potential drug adjuvant in breast cancer (BC) treatment is still poorly explored. Firstly, this study investigated the cytotoxic effects of Fx alone and combined with doxorubicin (Dox) and cisplatin (Cis) on a panel of 2D-cultured BC cell lines (MCF7, SKBR3 and MDA-MB-231) and one non-tumoral cell line (MCF12A). Fucoxanthin induced cytotoxicity against all the cell lines and potentiated Dox cytotoxic effects towards the SKBR3 and MDA-MB-231 cells. The combination triggering the highest cytotoxicity (Fx 10 µM + Dox 1 µM in MDA-MB-231) additionally showed significant induction of cell death and genotoxic effects, relative to control. In sequence, the same combination was tested on 3D cultures using a multi-endpoint approach involving bioactivity assays and microscopy techniques. Similar to 2D cultures, the combination of Fx and Dox showed higher cytotoxic effects on 3D cultures compared to the isolated compounds. Furthermore, this combination increased the number of apoptotic cells, decreased cell proliferation, and caused structural and ultrastructural damages on the 3D models. Overall, our findings suggest Fx has potential to become an adjuvant for Dox chemotherapy regimens in BC treatment.

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The Effects of Tgfb1 and Csf3 on Chondrogenic Differentiation of iPS Cells in 2D and 3D Culture Environment

International Journal of Molecular Sciences ◽

10.3390/ijms22062978 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2978

Author(s):

Chie-Hong Wang ◽

Chun-Hao Tsai ◽

Tsung-Li Lin ◽

Shih-Ping Liu

Keyword(s):

Cell Proliferation ◽

Chondrogenic Differentiation ◽

Expression Profiles ◽

Es Cells ◽

3D Culture ◽

Ips Cells ◽

3D Cultures ◽

Positive Effects ◽

Cartilaginous Matrix ◽

Culture Environment

Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor β1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.

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GENETIC SUSCEPTIBILITY TO IBD OPERATES VIA CONTROL OF APICAL JUNCTIONAL COMPLEXES

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.070 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S30-S30

Author(s):

Isabelle Hébert-Milette ◽

Chloé Lévesque ◽

Guy Charron ◽

John Rioux

Keyword(s):

Tight Junction ◽

Tight Junctions ◽

Intestinal Inflammation ◽

Protein Localization ◽

Junctional Complex ◽

Epithelial Permeability ◽

3D Cultures ◽

Apical Junctional Complex ◽

Junction Protein ◽

The Impact

Abstract Introduction Intestinal permeability is increased in unaffected 1st degree relatives of patients with inflammatory bowel disease (IBD), and is considered a risk factor for the development of IBD, likely increasing the interactions between intestinal microorganisms and the immune system. We recently reported that C1orf106, a gene located within a genomic region associated with IBD, regulates epithelial permeability. We further demonstrated that a rare coding variant within C1orf106 (p.Y333F) decreases protein stability and that lower levels of C1orf106 protein leads altered stability of adherens junctions (AJ) and to an increase in epithelial permeability. Hypothesis In addition to altering AJ, we believe that C1orf106 is also involved in the regulation of tight junction (TJ) formation, which also impacts epithelial permeability. Objectives The objectives of the project are to (a) validate the impact of C1orf106 on tight junctions and (b) verify the impact of C1orf106 IBD-associated variants on intestinal barrier integrity. Results We observed that knocking down the expression of C1orf106 in Caco-2 cells leads to a number of phenotypes in human epithelial monolayer (2D) and spheroid (3D) cultures that are associated with alterations in TJs. Specifically, when studying the dynamic reformation of TJ in 2D cultures after transient withdrawal of calcium, which is required for TJ stability, we observed that lower levels of C1orf106 resulted in (1) decreased recovery of barrier function as measured by transepithelial electrical resistance (TEER); (2) an alteration of tight junction protein localization; and (3) thickening of the circumferential actin belt. Moreover, in 3D cultures, we observed an altered spheroid formation associated with impaired epithelial polarization. In addition, our preliminary studies of human induced pluripotent stem cell (hiPSC)-derived epithelial cultures support that Y333F heterozygotes also have altered structure and function of their tight junctions. Conclusion Our observations indicate an important role of C1orf106 in apical junctional complex (AJC) formation likely mediated by a regulation of the circumferential actin belt. This can affect other functions of AJC, like the establishment of cell polarity. AJC formation is important for epithelial repair after an injury and its dysregulation impairs the formation of an impermeable epithelial barrier, which likely facilitates the passage of microorganisms and the induction and maintenance of intestinal inflammation.

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Erratum: Stiffness of the microenvironment upregulates ERBB2 expression in 3D cultures of MCF10A within the range of mammographic density

Scientific Reports ◽

10.1038/srep31680 ◽

2016 ◽

Vol 6 (1) ◽

Author(s):

Qingsu Cheng ◽

Cemal Cagatay Bilgin ◽

Gerald Fontenay ◽

Hang Chang ◽

Matthew Henderson ◽

...

Keyword(s):

Mammographic Density ◽

3D Cultures ◽

Erbb2 Expression

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Investigation of Adaptive Responses in Bystander Cells in 3D Cultures Containing Tritium-Labeled and Unlabeled Normal Human Fibroblasts

Radiation Research ◽

10.1667/rr1866.1 ◽

2010 ◽

Vol 174 (2) ◽

pp. 216-227 ◽

Cited By ~ 16

Author(s):

Massimo Pinto ◽

Edouard I. Azzam ◽

Roger W. Howell

Keyword(s):

Human Fibroblasts ◽

Adaptive Responses ◽

3D Cultures ◽

Normal Human ◽

Bystander Cells

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Challenges to Adopting 3D Cultures in HTS Programs

3D Cell-Based Biosensors in Drug Discovery Programs ◽

10.1201/ebk1420073492-18 ◽

2010 ◽

pp. 287-306

Keyword(s):

3D Cultures

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FTIR imaging of MCF-7 colonies and their vicinity in Matrigel-embedded 3D cultures

Biomedical Spectroscopy and Imaging ◽

10.3233/bsi-160139 ◽

2016 ◽

Vol 5 (2) ◽

pp. 155-166 ◽

Cited By ~ 1

Author(s):

Margarita Smolina ◽

Erik Goormaghtigh

Keyword(s):

Ftir Imaging ◽

3D Cultures ◽

Mcf 7

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Microfluidic Live-Imaging Technology to Perform Research Activities in 3D Models

10.20944/preprints202103.0264.v2 ◽

2021 ◽

Author(s):

Arnaud Martino Capuzzo ◽

Daniele Vigo

Keyword(s):

Fluorescence Intensity ◽

Regulatory Networks ◽

3D Structure ◽

Live Imaging ◽

Two Dimensions ◽

Live Cells ◽

Straight Line Passing ◽

3D Cultures ◽

Research Activities

Morphological dissimilarity and its evolution over time are one of the most unexpected variations found when comparing cell cultures in 2D and 3D. Monolayer cells appear to flatten in the lower part of the plate, adhering to and spreading in the horizontal plane while not extending vertically. Consequently, cells developed in two dimensions have a forced apex-basal polarity. Co-cultivation and crosstalking between multiple cell types, which control development and formation in the in vivo counterpart, are possible in 3D cultures. With or without a scaffold matrix, 3D model culture may exhibit more in vivo-like morphology and physiology. 3D cultures mimic relevant physiological cellular processes, transforming them into one-of-a-kind drug screening platforms. The structures and dynamics of regulatory networks, which are increasingly studied with live-imaging microscopy, must be considered to help and guarantee the functional maintenance of a 3D structure. However, commercially available technologies that can be used for current laboratory needs are minimal, despite the need to make it easier to acquire cellular kinetics with high spatial and temporal resolution, in order to improve visual efficiency and, as a result, experimentation performance. The CELLviewer is a newly developed multi-technology instrument that integrates and synchronizes the work of various scientific disciplines. The aim of this study is to test the device using two different models: a single Jurkat cell and an MCF-7 spheroid. The two models are loaded into the microfluidic cartridge for each experiment after they have been grown and captured in time-lapse for a total of 4 hours. The samples used are tracked under the operation of the optics after adaptive autofocus, while slipping inside the cartridge chamber, and the 3D rotation was successfully obtained experimentally. The MitoGreen dye, a fluorescence marker selectively permeable to live cells, was then used to determine cell viability. To measure the model diameter, construct fluorescence intensity graphs along a straight line passing through the cell, and visualize the spatial fluorescence intensity distribution in 3D, ImageJ software was used.

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