Long-Term Dynamic in vitro System for Investigating Rat Pineal Melatonin Secretion

Maternal stress before or during the sensitive preimplantation phase is associated with reproduction failure. Upon real or perceived threat, glucocorticoids (classic stress hormones) as cortisol are synthesized. The earliest “microenvironment” of the embryo consists of the oviduct epithelium and the oviductal fluid generated via the epithelial barrier. However, to date, the direct effects of cortisol on the oviduct are largely unknown. In the present study, we used a compartmentalized in vitro system to test the hypothesis that a prolonged stimulation with cortisol modifies the physiology of the oviduct epithelium. Porcine oviduct epithelial cells were differentiated at the air–liquid interface and basolaterally stimulated with physiological levels of cortisol representing moderate and severe stress for 21 days. Epithelium structure, transepithelial bioelectric properties, and gene expression were assessed. Furthermore, the distribution and metabolism of cortisol was examined. The polarized oviduct epithelium converted basolateral cortisol to cortisone and thereby reduced the amount of bioactive cortisol reaching the apical compartment. However, extended cortisol stimulation affected its barrier function and the expression of genes involved in hormone signaling and immune response. We conclude that continuing maternal stress with long-term elevated cortisol levels may alter the early embryonic environment by modification of basic oviductal functions.

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Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

Nature Communications ◽

10.1038/s41467-020-18954-z ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Sandra Valle ◽

Sonia Alcalá ◽

Laura Martin-Hijano ◽

Pablo Cabezas-Sáinz ◽

Diego Navarro ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Ductal Adenocarcinoma ◽

In Vitro System ◽

Tumorigenic Potential ◽

Term Enrichment ◽

Pluripotency Gene ◽

Pancreatic Cancer Stem Cells

Abstract Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.

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Amplification of Sca-1+ Lin-WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential

Blood ◽

10.1182/blood.v83.1.128.128 ◽

1994 ◽

Vol 83 (1) ◽

pp. 128-136 ◽

Cited By ~ 106

Author(s):

VI Rebel ◽

W Dragowska ◽

CJ Eaves ◽

RK Humphries ◽

PM Lansdorp

Keyword(s):

Interleukin 6 ◽

Cell Number ◽

In Vitro System ◽

Serum Free ◽

Murine Bone Marrow ◽

Steel Factor ◽

Parallel Increase

Abstract Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/-0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in > or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum-and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype.

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The FRTL-5 thyroid cell strain as a model for studies on thyroid cell growth

Acta Endocrinologica ◽

10.1530/acta.0.114s242 ◽

1987 ◽

Vol 116 (1_Suppl) ◽

pp. S242-S245 ◽

Cited By ~ 15

Author(s):

Francesco Saverio Ambesi-Impiombato ◽

Giovanni Villone

Keyword(s):

Cell Proliferation ◽

Cell Growth ◽

Cell Strain ◽

Thyroid Cell ◽

In Vitro System ◽

Defined Media ◽

Thyroid Follicular Cells ◽

Rat Thyroid

Abstract. Thyroid cell proliferation has been studied using an in vitro system of rat thyroid follicular cell strain (FRTL-5). While growing in continuous culture, this strain is still differentiated and non-tumourigenic. Both advantages and limitations in the use of such system for studies of thyroid cell growth should be considered. Some obvious limitations should be considered, such as the species (rat) from which FRTL-5 cells were originated, their long-term growth outside the animals, the presence of a chronic TSH stimulation. On the other hand, several advantages as the growth in hormonally and chemically defined media, their dependence upon TSH in the medium, their genetic homogeneity and their widespread use in many laboratories render the FRTL-5 strain a useful experimental tool. Studies on cell proliferation and mechanism of action of hormones, growth factors and human autoimmune IgG have been and are being performed, with the assumption that FRTL-5 cells are the in vitro equivalent of thyroid follicular cells.

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Bioaccessibility of ochratoxin A from red wine in an in vitro dynamic gastrointestinal model

World Mycotoxin Journal ◽

10.3920/wmj2014.1744 ◽

2015 ◽

Vol 8 (1) ◽

pp. 107-112 ◽

Cited By ~ 3

Author(s):

C.A. González-Arias ◽

I. Piquer-Garcia ◽

S. Marin ◽

V. Sanchis ◽

A.J. Ramos

Keyword(s):

Ochratoxin A ◽

Red Wine ◽

Dialysis Membrane ◽

Fluorescence Detector ◽

Penicillium Species ◽

In Vitro System ◽

Small Doses ◽

Flow Dialysis

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium species with immunosuppressive, teratogenic, and carcinogenic properties. It has been determined that wine is the second largest source of OTA (10% of total OTA intake) in the European diet and that its presence, even in small doses, can be a problem in terms of long-term toxicity. In this paper, we evaluated the bioaccessibility of OTA in a spiked red wine sample under human fasting conditions using an in vitro dynamic digestion model that includes a continuous-flow dialysis system to simulate intestinal passage. To the best of our knowledge, this report is the first examining the bioaccessibility of OTA in wine. A liquid-liquid method was used to extract the OTA and ochratoxin alpha (OTα) from gastrointestinal juices, and the extracts were analysed by HPLC with a fluorescence detector. The bioaccessibility of OTA from the spiked red wine (1.0, 2.0 and 4 μg/l) was high in the gastric compartment (102.8, 128.3 and 122.3%, respectively), whereas in the simulated intestine, it did not exceed 26%, and the amount of OTA that crossed the dialysis membrane was very low (<3.3%). The amount of OTα in gastric chyme ranged from 5.1 to 19.1% of the spiked OTA, whereas in the intestinal compartment it did not exceed 5%. In conclusion, in the in vitro system assayed, OTA exhibited a high bioaccessibility in the simulated stomach, but it decreased after the intestinal digestion and passage through the dialysis membrane.

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Amplification of Sca-1+ Lin-WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential

Blood ◽

10.1182/blood.v83.1.128.bloodjournal831128 ◽

1994 ◽

Vol 83 (1) ◽

pp. 128-136 ◽

Cited By ~ 1

Author(s):

VI Rebel ◽

W Dragowska ◽

CJ Eaves ◽

RK Humphries ◽

PM Lansdorp

Keyword(s):

Interleukin 6 ◽

Cell Number ◽

In Vitro System ◽

Serum Free ◽

Murine Bone Marrow ◽

Steel Factor ◽

Parallel Increase

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/-0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in > or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum-and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype.

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69 EFFECTS OF ENDOTOXIN ON LIVER CELLS IN A LONG TERM IN VITRO SYSTEM

Shock ◽

10.1097/00024382-199505000-00070 ◽

1995 ◽

Vol 3 (5) ◽

pp. 21

Author(s):

H-C. Pape ◽

A. Baader ◽

D. Remmers ◽

O Crome ◽

N. Frühauf ◽

...

Keyword(s):

Liver Cells ◽

In Vitro System

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Identification of in vitro growth conditions for c-Kit–negative hematopoietic stem cells

Blood ◽

10.1182/blood-2003-04-1249 ◽

2003 ◽

Vol 102 (9) ◽

pp. 3120-3128 ◽

Cited By ~ 15

Author(s):

Kim Klarmann ◽

Mariaestela Ortiz ◽

Meghan Davies ◽

Jonathan R. Keller

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Growth Conditions ◽

Hematopoietic Stem ◽

In Vitro System ◽

Bone Marrow Cultures ◽

In Vitro Systems

AbstractOur laboratory recently identified a quiescent class of pluripotent hematopoietic stem cells (PHSCs) that are lineage negative (Linneg), lack c-Kit, and are able to give rise to c-Kit–positive (c-Kitpos) PHSCs in vivo. This population fails to proliferate in vitro but has delayed reconstituting activity in vivo. In this study, we purified these cells to enrich for the PHSCs and we identified in vitro conditions capable of supporting their maturation. The c-Kit–negative (c-Kitneg) cells exhibited differential expression of Sca-1, CD34, CD43, CD45, and Thy 1.2. We purified the cells based on Sca-1, as it is expressed on active PHSCs. We detected pre–colony-forming unit spleen (pre–CFU-s) activity in both the Sca-1neg and Sca-1pos populations, indicating the presence of primitive PHSCs in both populations. However, our in vitro studies suggest that the Sca-1pos population is enriched for PHSCs. The in vitro systems that support the growth of these dormant cells include a modified long-term marrow culture and various stromal cell lines. In modified long-term bone marrow cultures, c-Kitneg cells gave rise to c-Kitpos PHSCs, with long-term reconstitution activity in vivo. Thus we have established an in vitro system to examine PHSC maturation that will allow us to study the mediators of the c-Kitneg to c-Kitpos transition.

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