3D spheroids
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Standardní kultivace nádorových buněčných linií ve 2D uspořádání je dobře zavedeným a finančně dostupným experimentálním mode-lem pro in vitro testování biologických účinků potenciálních protinádorových léčiv. 2D kultury však postrádají metabolické a proliferační gradienty, důležité buněčné interakce a signalizace, které jsou přítomné in vivo. 3D buněčné sféroidy zohledňují gradienty živin, kyslíku či odpadních metabolitů, důležitost interakcí mezi buňkami a extracelulární matrix a navozují tak situaci bližší reálným podmínkám. Bio-logické vlastnosti 3D sféroidů a jejich odpovědi na účinky léčiv se značně liší ve srovnání s 2D kulturami. Hodnocením protinádorových účinků potenciálních léčiv na 3D kulturách se zásadně zvyšuje šance na výběr farmakologicky relevantních struktur a snížit tak riziko neúspěchu v průběhu klinického testování.
Traditional cultivation of cancer cell lines in 2D arrangement is well established and affordable experimental model for in vitro testing of biological effects of potential anticancer drugs. However, 2D cultures lack metabolic and proliferative gradients, important cell interac-tions and signaling that are present in vivo. Within 3D spheroids the gradients of nutrients, oxygen or waste metabolites, the importance of interactions between the cells and the extracellular matrix are included, and thus 3D can better simulate in vivo tumor microenviro-ment. The biological properties of 3D spheroids and their responses to drug effects vary greatly compared to 2D cultures. The evaluation of anticancer drug effects on 3D spheroids increases the chances of selection of pharmacologically relevant structures and thus reduce clinical trial failure risk.
- Klíčová slova
- solidní nádory,
- MeSH
- biologické modely MeSH
- buněčné sféroidy * fyziologie klasifikace účinky léků MeSH
- lidé MeSH
- nádorové buňky kultivované cytologie mikrobiologie MeSH
- nádory diagnostické zobrazování MeSH
- preklinické hodnocení léčiv MeSH
- protinádorové látky farmakokinetika MeSH
- techniky 3D buněčné kultury metody MeSH
- techniky in vitro metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.
Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.
- MeSH
- buněčná diferenciace * fyziologie MeSH
- buněčné kultury metody MeSH
- buněčné linie MeSH
- buněčné sféroidy * metabolismus MeSH
- myši MeSH
- neurony metabolismus MeSH
- prionové nemoci * metabolismus patologie MeSH
- priony metabolismus MeSH
- techniky 3D buněčné kultury metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mesenchymal stem cells (MSC) have been considered the promising candidates for the regenerative and personalized medicine due to their self-renewal potential, multilineage differentiation and immunomodulatory capacity. Although these properties have encouraged profound MSC studies in recent years, the majority of research has been based on standard 2D culture utilization. The opportunity to resemble in vivo characteristics of cells native niche has been provided by implementation of 3D culturing models such as MSC spheroid formation assesed through cells self-assembling. In this review, we address the current literature on physical and biochemical features of 3D MSC spheroid microenvironment and their impact on MSC properties and behaviors. Starting with the reduction in the cells' dimensions and volume due to the changes in adhesion molecules expression and cytoskeletal proteins rearrangement resembling native conditions, through the microenvironment shifts in oxygen, nutrients and metabolites gradients and demands, we focus on distinctive and beneficial features of MSC in spheroids compared to cells cultured in 2D conditions. By summarizing the data for 3D MSC spheroids regarding cell survival, pluripotency, differentiation, immunomodulatory activities and potential to affect tumor cells growth we highlighted advantages and perspectives of MSC spheroids use in regenerative medicine. Further detailed analyses are needed to deepen our understanding of mechanisms responsible for modified MSC behavior in spheroids and to set future directions for MSC clinical application.
- MeSH
- buněčná diferenciace MeSH
- buněčné mikroprostředí * MeSH
- buněčné sféroidy cytologie MeSH
- epigeneze genetická MeSH
- lidé MeSH
- mezenchymální kmenové buňky cytologie metabolismus MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- MeSH
- buněčné sféroidy * MeSH
- hepatocyty * MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- komentáře MeSH
- práce podpořená grantem MeSH
The ability to simulate the 3D structure of a human body is essential to increase the efficiency of drug development. In vivo conditions are significantly different in comparison to in vitro conditions. A standardly used cell monolayer on tissue culture plastic (2D cell culture) is not sufficient to simulate the transfer phenomena occurring in living organisms, therefore, cell growth in a 3D space is desired. Drug absorption, distribution, metabolism, excretion and toxicity could be tested on 3D cell aggregates called spheroids, decrease the use of animal models and accelerate the drug development. In this work, the formation of spheroids from HT-29 human colorectal adenocarcinoma cells was successfully achieved by means of the so-called liquid marbles, which are liquid droplets encapsulated by a hydrophobic powder. During the cultivation in the medium inside the liquid marbles, cells spontaneously formed spherical agglomerates (spheroids) without the need of any supporting scaffold. The study focused on the influence of different parameters—namely liquid marble volume, seeding cell density and time of cultivation—on the final yield and quality of spheroids. This work has shown that using liquid marbles as microbioreactors is a suitable method for the cultivation of HT-29 cells in the form of spheroids.
- Klíčová slova
- liquid marble,
- MeSH
- bioreaktory MeSH
- buněčné sféroidy MeSH
- buňky HT-29 MeSH
- lidé MeSH
- techniky 3D buněčné kultury * metody MeSH
- uhličitan vápenatý MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
The efficiency of clinical trials involving transplantation of multipotent mesenchymal stromal cells (MSCs) is often insufficient due to harsh conditions present within the target tissue including hypoxia, low nutrient supply as well as inflammatory reactions. This indicates the necessity for optimization of cell-based therapy approaches which might include either modification of the cell manufacturing process or specific cell pretreatment procedures prior to transplantation. Recent reports confirm evidence that the aggregation of MSCs into three-dimensional (3D) multicellular spheroids results in enhancement of the overall therapeutic potential of cells, by improving the anti-inflammatory and angiogenic properties, stemness and survival of MSCs after transplantation. Such an MSCs spheroid generation approach may open new opportunities for the enlargement of MSCs applications in clinical research and therapy. However, the unification and optimization of 3D spheroid generation techniques, including the selection of appropriate clinical-grade culture conditions and methods for their large-scale production, are still of great importance. The current review addresses questions regarding therapeutic-associated properties of 3D multicellular MSCs spheroids in vitro and during preclinical animal studies, with special attention to the possibilities of translating these research achievements toward further clinical manufacturing and applications.
- MeSH
- buněčné kultury MeSH
- buněčné sféroidy cytologie metabolismus transplantace MeSH
- cytokiny metabolismus MeSH
- kardiovaskulární nemoci patologie terapie MeSH
- lidé MeSH
- mezibuněčné signální peptidy a proteiny metabolismus MeSH
- multipotentní kmenové buňky cytologie metabolismus MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The pregnane X receptor (PXR) is a ligand-activated nuclear receptor controlling hepatocyte expression of numerous genes. Although expression changes in xenobiotic-metabolizing, lipogenic, gluconeogenic and bile acid synthetic genes have been described after PXR activation, the temporal dynamics of their expression is largely unknown. Recently, 3D spheroids of primary human hepatocytes (PHHs) have been characterized as the most phenotypically relevant hepatocyte model. We used 3D PHHs to assess time-dependent expression profiles of 12 prototypic PXR-controlled genes in the time course of 168 h of rifampicin treatment (1 or 10 μM). We observed a similar bell-shaped time-induction pattern for xenobiotic-handling genes (CYP3A4, CYP2C9, CYP2B6, and MDR1). However, we observed either biphasic profiles for genes involved in endogenous metabolism (FASN, GLUT2, G6PC, PCK1, and CYP7A1), a decrease for SHP or oscillation for PDK4 and PXR. The rifampicin concentration determined the expression profiles for some genes. Moreover, we calculated half-lives of CYP3A4 and CYP2C9 mRNA under induced or basal conditions and we used a mathematical model to describe PXR-mediated regulation of CYP3A4 expression employing 3D PHHs. The study shows the importance of long-term time-expression profiling of PXR target genes in phenotypically stable 3D PHHs and provides insight into PXR function in liver beyond our knowledge from conventional 2D in vitro models.
Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.
- MeSH
- biologické markery MeSH
- buněčná diferenciace * MeSH
- buněčné kultury MeSH
- buněčné sféroidy MeSH
- exprese genu MeSH
- kardiomyocyty cytologie metabolismus MeSH
- kyslík metabolismus MeSH
- lidé MeSH
- pluripotentní kmenové buňky cytologie metabolismus MeSH
- sarkoplazmatické retikulum metabolismus MeSH
- srdeční mitochondrie metabolismus MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Three-dimensional cell culture systems are increasingly used for biological and anticancer drug screening as they mimic the structure and microenvironment of tumors more closely than conventional two-dimensional cell models. In this study, the growth kinetics of colon adenocarcinoma-derived spheroids (HT-29 cell line) cultivated in liquid marble micro-bioreactors and nonadherent PDMS-coated well plates was investigated in detail and enabled precise control of the spheroid size by the seed cell density and cultivation time. The therapeutic effect of 5-fluorouracil and irinotecan hydrochloride in 2D monolayer cell culture and 3D tumor spheroids revealed an unexpected twist in their efficacy due to different ability to penetrate through 3D microtissue. For 5-fluorouracil, the inhibitory concentration IC50 after 48 h exposure increased from 11.3 μM for a 2D cell culture to 707.7 μM for a 3D spheroid. In the case of irinotecan, IC50 increased from 24.9 μM to 77.8 μM. Despite its higher molar weight, irinotecan appeared to penetrate the 3D spheroid structure more efficiently than 5-fluorouracil. While 5-fluorouracil mainly caused a suppression of spheroid growth from the outside, irinotecan affected the entire spheroid and caused its originally compact structure to disintegrate. The acquired results highlight the need to screen cancer chemotherapeutics on 3D tumor models, as contrasting results can be obtained compared to standard 2D cell cultures.
- MeSH
- adenokarcinom * MeSH
- buněčné sféroidy MeSH
- cytostatické látky * farmakologie MeSH
- fluoruracil farmakologie MeSH
- irinotekan farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové mikroprostředí MeSH
- nádory tračníku * farmakoterapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
