In Vitro Follicle Growth, Maturation, and Gene Expression: Three-Dimensional (3D) Matrigel Culture versus Two-Dimensional Culture (2D)

This research aimed to compare the In Vitro growth, maturation, and gene expression in ovarian follicles collected from adult mice (6–8-week-old) between two-dimensional and three-dimensional cultures. First, we confirmed In Vitro follicle growth and maturation using adult mice with outbred characteristics and analyzed the expression of genes related to follicular development. We found that the three-dimensional culture system utilizing a Matrigel drop to create an in vivo-like ovarian microenvironment was more efficient in terms of In Vitro follicle growth, maturation, and gene expression than the two-dimensional system (non-physical environment). The in vivo-like three-dimensional culture of ovarian follicles provides new insights into the physiology and development of ovarian follicle in vivo, thereby contributing to new strategies to improve female fertility.

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Recapitulating tumour microenvironment in chitosan–gelatin three-dimensional scaffolds: an improved in vitro tumour model

Journal of The Royal Society Interface ◽

10.1098/rsif.2012.0564 ◽

2012 ◽

Vol 9 (77) ◽

pp. 3288-3302 ◽

Cited By ~ 29

Author(s):

Neha Arya ◽

Viren Sardana ◽

Meera Saxena ◽

Annapoorni Rangarajan ◽

Dhirendra S. Katti

Keyword(s):

Cancer Progression ◽

Expression Profiles ◽

Three Dimensional ◽

Gene Expression Profiles ◽

Petri Dish ◽

Tumour Microenvironment ◽

Two Dimensional ◽

Tumour Model

Owing to the reduced co-relationship between conventional flat Petri dish culture (two-dimensional) and the tumour microenvironment, there has been a shift towards three-dimensional culture systems that show an improved analogy to the same. In this work, an extracellular matrix (ECM)-mimicking three-dimensional scaffold based on chitosan and gelatin was fabricated and explored for its potential as a tumour model for lung cancer. It was demonstrated that the chitosan–gelatin (CG) scaffolds supported the formation of tumoroids that were similar to tumours grown in vivo for factors involved in tumour-cell–ECM interaction, invasion and metastasis, and response to anti-cancer drugs. On the other hand, the two-dimensional Petri dish surfaces did not demonstrate gene-expression profiles similar to tumours grown in vivo . Further, the three-dimensional CG scaffolds supported the formation of tumoroids, using other types of cancer cells such as breast, cervix and bone, indicating a possible wider potential for in vitro tumoroid generation. Overall, the results demonstrated that CG scaffolds can be an improved in vitro tool to study cancer progression and drug screening for solid tumours.

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Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab136 ◽

2021 ◽

Author(s):

Yu Takahashi ◽

Yu Inoue ◽

Keitaro Kuze ◽

Shintaro Sato ◽

Makoto Shimizu ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Gene Expression Regulation ◽

Intestinal Epithelial Cells ◽

Three Dimensional ◽

Culture Conditions ◽

Dependent Manner ◽

Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

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Three-dimensional imaging of nucleosomes from transcriptionally active genes using electron spectroscopic imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162739 ◽

1996 ◽

Vol 54 ◽

pp. 54-55

Author(s):

G. J. Czarnota ◽

D. P. Bazett-Jones ◽

F. P. Ottensmeyer

Keyword(s):

Gene Expression ◽

Three Dimensional ◽

Spectroscopic Imaging ◽

Dimensional Structure ◽

Crystallographic Structure ◽

Electron Spectroscopic Imaging ◽

Nucleosome Structure ◽

Active Genes

The three-dimensional structure of the nucleosome was determined using particles purified from transcriptionally active genes in conjunction with electron spectroscopic imaging, and quaternion-assisted angular reconstitution procedures. The results reveal a configuration which is very different from the canonical compact crystallographic structure for this fundamental chromosome subunit, implying a structural disruption of the nucleosome with the activation of gene expression in accord with numerous physico-chemical observations.Previous analyses of nucleosomes purified from transcriptionally quiescent genes have indicated numerous structural states dependent on factors in vitro which modify charge based interactions in nucleoprotein complexes. Nucleosomes from transcriptionally active genes undergo chemical alterations in vivo which similarly modify charge based interactions. In order to investigate the effects of the gene expression associated chemical alterations on nucleosome structure, particles were purified from transcriptionally active genes using mercury affinity chromatography. These nucleosome particles are hyperacetylated with respect to particles from transcriptionally quiescent genes. Here additionally, sulphydryls normally buried within the protein core of the transcriptionally inactive particle are exposed to chemical modifying agents thus facilitating purification as described.

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Stability and lability of circadian period of gene expression in the cyanobacterium Synechococcus elongatus

Microbiology ◽

10.1099/mic.0.022343-0 ◽

2009 ◽

Vol 155 (2) ◽

pp. 635-641 ◽

Cited By ~ 6

Author(s):

Eugenia M. Clerico ◽

Vincent M. Cassone ◽

Susan S. Golden

Keyword(s):

Gene Expression ◽

Physiological State ◽

Three Dimensional ◽

Synechococcus Elongatus ◽

Circadian Period ◽

Normal Period ◽

Promoter Recognition ◽

Molecular Aspects

Molecular aspects of the circadian clock in the cyanobacterium Synechococcus elongatus have been described in great detail. Three-dimensional structures have been determined for the three proteins, KaiA, KaiB and KaiC, that constitute a central oscillator of the clock. Moreover, a temperature-compensated circadian rhythm of KaiC phosphorylation can be reconstituted in vitro with the addition of KaiA, KaiB and ATP. These data suggest a relatively simple circadian system in which a single oscillator provides temporal information for all downstream processes. However, in vivo the situation is more complex, and additional components contribute to the maintenance of a normal period, the resetting of relative phases of circadian oscillations, and the control of rhythms of gene expression. We show here that two well-studied promoters in the S. elongatus genome report different circadian periods of expression under a given set of conditions in wild-type as well as mutant genetic backgrounds. Moreover, the period differs between these promoters with respect to modulation by light intensity, growth phase, and the presence or absence of a promoter-recognition subunit of RNA polymerase. These data contrast sharply with the current clock model in which a single Kai-based oscillator governs circadian period. Overall, these findings suggest that complex interactions among the circadian oscillator, perhaps other oscillators, and other cellular machinery result in a clock that is plastic and sensitive to the environment and to the physiological state of the cell.

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Linear Polyp Measurement at CT Colonography: In Vitro and in Vivo Comparison of Two-dimensional and Three-dimensional Displays

Radiology ◽

10.1148/radiol.2363041534 ◽

2005 ◽

Vol 236 (3) ◽

pp. 872-878 ◽

Cited By ~ 88

Author(s):

Perry J. Pickhardt ◽

Andrew D. Lee ◽

Elizabeth G. McFarland ◽

Andrew J. Taylor

Keyword(s):

Ct Colonography ◽

Three Dimensional ◽

Two Dimensional

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Recent Advances in Three-Dimensional Stem Cell Culture Systems and Applications

Stem Cells International ◽

10.1155/2021/9477332 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Xiaowen Wu ◽

Junxiang Su ◽

Jizhen Wei ◽

Nan Jiang ◽

Xuejun Ge

Keyword(s):

Cell Culture ◽

Culture System ◽

Three Dimensional ◽

Culture Method ◽

Future Research ◽

Two Dimensional ◽

Culture Methods ◽

Three Dimensional Culture ◽

Dimensional Cell

Cell culture is one of the most core and fundamental techniques employed in the fields of biology and medicine. At present, although the two-dimensional cell culture method is commonly used in vitro, it is quite different from the cell growth microenvironment in vivo. In recent years, the limitations of two-dimensional culture and the advantages of three-dimensional culture have increasingly attracted more and more attentions. Compared to two-dimensional culture, three-dimensional culture system is better to realistically simulate the local microenvironment of cells, promote the exchange of information among cells and the extracellular matrix (ECM), and retain the original biological characteristics of stem cells. In this review, we first present three-dimensional cell culture methods from two aspects: a scaffold-free culture system and a scaffold-based culture system. The culture method and cell characterizations will be summarized. Then the application of three-dimensional cell culture system is further explored, such as in the fields of drug screening, organoids and assembloids. Finally, the directions for future research of three-dimensional cell culture are stated briefly.

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428. Normal follicle growth and maturation in a three-dimensional in vitro culture system: follicular environment manipulation and assessment of oocyte outcomes

Reproduction Fertility and Development ◽

10.1071/srb08abs428 ◽

2008 ◽

Vol 20 (9) ◽

pp. 108

Author(s):

K. R. Dunning ◽

L. K. Akison ◽

D. L. Russell ◽

R. J. Norman ◽

R. L. Robker

Keyword(s):

Culture System ◽

Cumulus Cell ◽

Three Dimensional ◽

Oocyte Quality ◽

Metabolic Effects ◽

Follicle Growth ◽

Cumulus Expansion ◽

Follicle Diameter

In vivo, the oocyte matures in a niche environment surrounded by somatic cells, and later in ovarian follicular development, by follicular fluid. Maternal diet influences the environment in which an oocyte matures but the mechanisms by which an altered metabolic profile, such as hyperinsulinemia, affects oocyte quality are not known. We investigated the use of a three dimensional follicle culture system allowing direct manipulation of the follicular environment thus circumventing systemic hormonal and metabolic effects. Secondary follicles (113.4 ± 1.02µm, n = 54) were isolated from mice at d12, encapsulated individually in 2µl of alginate matrix, and cultured in aMEM/5%FCS/10 mIU/mL LH/100 mIU FSH at 37°C/5%CO2, with media sampling and replacement every second day. Following 12 days of culture there was a significant 3-fold increase in follicle diameter (320 ± 10.1µm, n = 51). Histological analysis showed normal follicular morphology and antrum formation. Analysis of oestradiol (15.0ng/mL), androstenedione (7.8ng/mL) and progesterone (23.7ng/mL) in the media at d12 confirmed normal steroidogenesis and differentiation. Treatment of follicles with an ovulatory stimulus (1.5IU/mL hCG/5ng/mL Egf), resulted in cumulus expansion and hyaluronan localising to the cumulus oocyte complex (COC) and follicular basement membrane. These analyses were consistent with follicle growth and induction of ovulation in vivo. Further, COCs isolated from follicles and matured in vitro (IVM) in the presence of Egf and FSH, underwent cumulus expansion (CEI 2.8 ± 0.2) and were capable of fertilisation and blastocyst development. LH did not induce IVM COC expansion (CEI 1.36 ± 0.2), reflecting the normal in vivo differentiation process. However, culturing follicles in high insulin (5ug/mL) led to a significant increase in the degree of IVM cumulus expansion in response to LH (CEI 2.1 ± 0.3) indicating inappropriate cumulus cell differentiation, which may lead to poorer oocyte quality. These results demonstrate that this technique recapitulates normal in vivo folliculogenesis and is useful for manipulation of the follicular environment and assessment of oocyte outcomes.

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Bi-functional oxidized dextran–based hydrogel inducing microtumors: An in vitro three-dimensional lung tumor model for drug toxicity assays

Journal of Tissue Engineering ◽

10.1177/2041731417718391 ◽

2017 ◽

Vol 8 ◽

pp. 204173141771839 ◽

Cited By ~ 2

Author(s):

Dhaval Kedaria ◽

Rajesh Vasita

Keyword(s):

Drug Screening ◽

Drug Toxicity ◽

Disease Modeling ◽

Lung Tumor ◽

Three Dimensional ◽

Two Dimensional ◽

Chitosan Hydrogel ◽

Oxidized Dextran

Cancer is a serious death causing disease having 8.2 million deaths in 2012. In the last decade, only about 10% of chemotherapeutic compounds showed productivity in drug screening. Two-dimensional culture assays are the most common in vitro drug screening models, which do not precisely model the in vivo condition for reliable preclinical drug screening. Three-dimensional scaffold–based cell cultures perhaps mimic tumor microenvironment and recapitulate physiologically more relevant tumor. This study was carried out to develop bi-functional oxidized dextran–based cell instructive hydrogel that provides three-dimensional environment to cancer cells for inducing microtumor. Oxidized dextran was blended with thiolated chitosan to fabricate an in situ self-gelable hydrogel (modified dextran–chitosan) in a one-step process. The hydrogels characterization revealed cross-linked network structure with highly porous structure and water absorption. The modified dextran–chitosan hydrogel showed reduced hydrophobicity and has reduced protein absorption, which resulted in changing the A549 cell adhesiveness, and encouraged them to form microtumor. The cells were proliferated in clusters having spherical morphology with randomly oriented stress fiber and large nucleus. Further microtumors were studied for hypoxia where reactive oxygen species generation demonstrated 15-fold increase as compared to monolayer culture. Drug-sensitivity results showed that microtumors generated on modified dextran–chitosan hydrogel showed resistance to doxorubicin with having 33%–58% increased growth than two-dimensional monolayer model at concentrations of 25–100 µM. In summary, the modified dextran–chitosan scaffold can provide surface chemistry that induces three-dimensional microtumors with physiologically relevant properties to in vivo tumor including growth, morphology, extracellular matrix production, hypoxic phenotype, and drug response. This model can be potentially utilized for drug toxicity studies and cancer disease modeling to understand tumor phenotype and progression.

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Gene expression in Acetabularia. II. Analysis of in vitro translation products

Journal of Cell Science ◽

10.1242/jcs.58.1.35 ◽

1982 ◽

Vol 58 (1) ◽

pp. 35-48

Author(s):

R.L. Shoeman ◽

H.G. Schweiger

Keyword(s):

Gene Expression ◽

Gel Electrophoresis ◽

Wheat Germ ◽

In Vitro Translation ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis ◽

In Vitro System ◽

Acetabularia Mediterranea

The translation products induced by poly(A)+ RNA from Acetabularia mediterranea, A. cliftonii and A. ryukyuensis in a modified, highly efficient wheat germ cell-free in vitro system were separated by two-dimensional gel electrophoresis. A comparison of the translation products on the basis of their molecular weight and their isoelectric point revealed only a limited similarity between the patterns of the three species. The pronounced species specificity will permit the study of the in vivo translation of heterologous poly(A)+ RNA in Acetabularia cytoplasm.

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