Long-term culture system for deep-sea mussels Gigantidas childressi

The simulation of deep-sea conditions in laboratories is technically challenging but necessary for experiments that aim at a deeper understanding of physiological mechanisms or host-symbiont interactions of deep-sea organisms. In a proof-of-concept study, we designed a recirculating system for long-term culture (>2 years) of deep-sea mussels Gigantidas childressi (previously Bathymodiolus childressi). Mussels were automatically (and safely) supplied with a maximum stable level of ~60 μM methane in seawater using a novel methane-air mixing system. Experimental animals also received daily doses of live microalgae. Condition indices of cultured G. childressi remained high over years, and low shell thickness growth could be detected, which is indicative of positive energy budgets. Using stable isotope data, we demonstrate that G. childressi in our culture system gained energy, both, from digestion of methane oxidizing endosymbionts and from digesting particulate food (microalgae). Limitations of the system, as well as opportunities for future experimental approaches involving deep-sea mussels are discussed.

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The human hepatoma Hep3B cell line as an experimental model in the study of the long-term regulation of acute-phase proteins by cytokines

Biochemical Journal ◽

10.1042/bj2870255 ◽

1992 ◽

Vol 287 (1) ◽

pp. 255-259 ◽

Cited By ~ 8

Author(s):

M Hiron ◽

M Daveau ◽

P Arnaud ◽

J Bauer ◽

J P Lebreton

Keyword(s):

Cell Line ◽

Acute Phase ◽

Culture System ◽

Acute Phase Protein ◽

Hepatoma Cell Line ◽

Mrna Levels ◽

Human Hepatoma ◽

Long Term Culture ◽

Phase Protein

The regulation of the synthesis by the cytokines interleukin-1 (IL-1) and IL-6 of the positive acute-phase protein alpha 1-acid glycoprotein (AGP) and of the negative acute-phase protein alpha 2-HS glycoprotein (AHSG) has been studied in a long-term culture system of the human hepatoma cell line Hep3B. The culture system contained 30 nM-sodium selenite as the only supplement. This allowed maintenance of the synthesis of the proteins under study at a near steady state for over 3 months. An increase in AGP mRNA and a decrease in AHSG mRNA were observed when cells were treated for two successive 48 h-periods with monocyte-conditioned medium. A return to basal levels was obtained after cessation of the cytokine addition. Two further additions of cytokines led to alterations in mRNA levels similar to those observed following the first cytokine treatment. The amounts of AGP and AHSG secreted were altered in accordance with the mRNA modifications. These results suggest that new cytokine receptors were being constantly synthesized during cell culture. When cytokines were present in the culture medium for 10 days, maximum alterations in AGP and AHSG synthesis were obtained following 2 and 4 days of treatment respectively, but further alterations in protein levels could not be observed afterwards. Expression of IL-6 receptor mRNA was not up-regulated by cytokines, but only by 1 microM-dexamethasone. Our results show that, in this long-term culture system, cytokines induce a response in hepatoma cells similar to that observed in vivo during human inflammatory states. This model could be used to evaluate the effects of agonists or antagonists of cytokines responsible for the hepatic acute-phase protein response.

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Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+7+ NK progenitor

Blood ◽

10.1182/blood.v83.9.2594.2594 ◽

1994 ◽

Vol 83 (9) ◽

pp. 2594-2601 ◽

Cited By ~ 146

Author(s):

JS Miller ◽

KA Alley ◽

P McGlave

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Culture System ◽

Interleukin 2 ◽

Early Event ◽

Hematopoietic Progenitors ◽

Long Term Culture ◽

Lineage Differentiation ◽

Marrow Stroma

Abstract We have recently described a marrow stroma-dependent long-term culture system that supports differentiation of CD34+ human marrow primitive progenitors into natural killer (NK) cells. We postulate that CD7 expression may be an early event in commitment of hematopoietic progenitors to the NK lineage. Here we compare the characteristics of CD34+7- and CD34+7+ marrow cells cultivated in the stroma-based NK culture system. These CD34+ populations were further compared with a marrow derived, more committed, CD34–7+ progenitor to emphasize the continuum of NK development and to highlight differences between progenitors in our assays. No progenitor proliferated when plated in media without stroma, underscoring the importance of stroma in NK differentiation. Plating progenitor populations in interleukin-2 containing media directly on preestablished, allogeneic, irradiated marrow stroma for 5 weeks resulted in CD56+CD3- NK cells; however, characteristics of the cultured populations differed. Fold expansion and cloning efficiency of the CD34+7+ population, determined by a functional limiting dilution assay was significantly higher than of the CD34+7- or CD34+7+ populations. This suggests that the CD34+7+ population is highly enriched for an NK progenitor and a possible intermediate in NK lineage differentiation. Further dividing the CD34+7+ population by the relative fluorescence of CD7 into CD34+7+dim and CD34+7+bright populations showed that the CD34+7+bright population exhibited a significantly higher cloning frequency than parallel experiments with CD34+7+dim cells (11.8% +/- 2.4% v 2.4% +/- 0.7%, n = 6; P = .005). Plating of the more primitive CD34+7- population in a transwell system (which separates progenitors from stroma by a microporous membrane) prevents differentiation into NK cells. In contrast, plating of CD34+7+ progenitors in transwells resulted in generation of NK cells. These data suggest that primitive, but not more mature NK progenitors may require direct contact with stroma for the initial differentiation steps. Finally, differentiation of the NK progenitors in this stroma-dependent model results in expression of CD2 not present on any of the starting populations. This observation suggests that marrow stroma can stimulate CD2 expression on NK progenitors in a previously undescribed fashion that may be analogous to the thymic effect on CD2 expression in immature T lymphocytes. These observations identify early steps in the commitment of primitive marrow CD34+ hematopoietic progenitors to a lymphoid lineage and underscore the importance of coexpression of CD7 with CD34 as an early lymphoid commitment characteristic and direct progenitor-stroma interactions in this process.

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Response of myelodysplastic syndrome marrow progenitor cells to stimulation with cytokine combinations in a stroma‐free long‐term culture system

British Journal of Haematology ◽

10.1046/j.1365-2141.1996.374910.x ◽

1996 ◽

Vol 92 (3) ◽

pp. 548-558 ◽

Cited By ~ 15

Author(s):

D. A. Soligo ◽

S. Campiglio ◽

F. Servida ◽

P. Bossolasco ◽

L. Romitti ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Progenitor Cells ◽

Culture System ◽

Long Term Culture

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Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains

mBio ◽

10.1128/mbio.03536-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Diane G. Edmondson ◽

Bridget D. DeLay ◽

Lindsay E. Kowis ◽

Steven J. Norris

Keyword(s):

Culture System ◽

Treponema Pallidum ◽

Basal Medium ◽

Human Pathogen ◽

Low Oxygen ◽

Term Survival ◽

Content Type ◽

Long Term Culture

ABSTRACT The bacterium that causes syphilis, Treponema pallidum subsp. pallidum, has now been cultured in vitro continuously for periods exceeding 3 years using a system consisting of coculture with Sf1Ep rabbit epithelial cells in TpCM-2 medium and a low-oxygen environment. In addition, long-term culture of several other syphilis isolates (SS14, Mexico A, UW231B, and UW249B) and the T. pallidum subsp. endemicum Bosnia A strain has been achieved. During in vitro passage, T. pallidum subsp. pallidum exhibited a typical bacterial growth curve with logarithmic and stationary phases. Sf1Ep cells are required for sustained growth and motility; however, high initial Sf1Ep cell numbers resulted in reduced multiplication and survival. Use of Eagle’s minimal essential medium as the basal medium was not effective in sustaining growth of T. pallidum subsp. pallidum beyond the first passage, whereas CMRL 1066 or M199 supported long-term culture, confirming that additional nutrients present in these more complex basal media are required for long-term culture. T. pallidum subsp. pallidum growth was dependent upon the presence of fetal bovine serum, with 20% (vol/vol) being the optimal concentration. Omission of reactive oxygen species scavengers dithiothreitol, d-mannitol, or l-histidine did not dramatically affect survival or growth. Additionally, T. pallidum subsp. pallidum can be successfully cultured in a Brewer jar instead of a specialized low-oxygen incubator. Phosphomycin or amphotericin B can be added to the medium to aid in the prevention of bacterial or fungal contamination, respectively. These results help define the parameters of the T. pallidum subsp. pallidum culture system that are required for sustained, long-term survival and multiplication. IMPORTANCE Syphilis is caused by the bacterium Treponema pallidum subsp. pallidum. Until recently, this pathogen could only be maintained through infection of rabbits or other animals, making study of this important human pathogen challenging and costly. T. pallidum subsp. pallidum has now been successfully cultured for over 3 years in a tissue culture system using a medium called TpCM-2. Here, we further define the growth requirements of this important human pathogen, promoting a better understanding of the biology of this fastidious organism.

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Electrospun nanofibers facilitate better alignment, differentiation, and long-term culture in an in vitro model of the neuromuscular junction (NMJ)

Biomaterials Science ◽

10.1039/c8bm00720a ◽

2018 ◽

Vol 6 (12) ◽

pp. 3262-3272 ◽

Cited By ~ 15

Author(s):

Baiwen Luo ◽

Lingling Tian ◽

Nuan Chen ◽

Seeram Ramakrishna ◽

Nitish Thakor ◽

...

Keyword(s):

Neuromuscular Junction ◽

Culture System ◽

In Vitro Model ◽

Electrospun Nanofibers ◽

Nanofibrous Scaffold ◽

Long Term Culture ◽

Vitro Model

An electrospun nanofibrous scaffold is used as a novel in vitro culture system to provide long-term support for NMJ formation.

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Analysis of peripheral blood CD34+ cells mobilized with granulocyte colony-stimulating factor (G-CSF) using a long-term culture system

Bone Marrow Transplantation ◽

10.1038/sj.bmt.1701162 ◽

1998 ◽

Vol 21 (8) ◽

pp. 751-757 ◽

Cited By ~ 6

Author(s):

T Suzuki ◽

K Muroi ◽

Y Amemiya ◽

Y Miura

Keyword(s):

Peripheral Blood ◽

Culture System ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Long Term Culture ◽

Peripheral Blood Cd34 ◽

Cd34 Cells ◽

Stimulating Factor ◽

Factor G

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Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+7+ NK progenitor

Blood ◽

10.1182/blood.v83.9.2594.bloodjournal8392594 ◽

1994 ◽

Vol 83 (9) ◽

pp. 2594-2601 ◽

Cited By ~ 8

Author(s):

JS Miller ◽

KA Alley ◽

P McGlave

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Culture System ◽

Interleukin 2 ◽

Early Event ◽

Hematopoietic Progenitors ◽

Long Term Culture ◽

Lineage Differentiation ◽

Marrow Stroma

We have recently described a marrow stroma-dependent long-term culture system that supports differentiation of CD34+ human marrow primitive progenitors into natural killer (NK) cells. We postulate that CD7 expression may be an early event in commitment of hematopoietic progenitors to the NK lineage. Here we compare the characteristics of CD34+7- and CD34+7+ marrow cells cultivated in the stroma-based NK culture system. These CD34+ populations were further compared with a marrow derived, more committed, CD34–7+ progenitor to emphasize the continuum of NK development and to highlight differences between progenitors in our assays. No progenitor proliferated when plated in media without stroma, underscoring the importance of stroma in NK differentiation. Plating progenitor populations in interleukin-2 containing media directly on preestablished, allogeneic, irradiated marrow stroma for 5 weeks resulted in CD56+CD3- NK cells; however, characteristics of the cultured populations differed. Fold expansion and cloning efficiency of the CD34+7+ population, determined by a functional limiting dilution assay was significantly higher than of the CD34+7- or CD34+7+ populations. This suggests that the CD34+7+ population is highly enriched for an NK progenitor and a possible intermediate in NK lineage differentiation. Further dividing the CD34+7+ population by the relative fluorescence of CD7 into CD34+7+dim and CD34+7+bright populations showed that the CD34+7+bright population exhibited a significantly higher cloning frequency than parallel experiments with CD34+7+dim cells (11.8% +/- 2.4% v 2.4% +/- 0.7%, n = 6; P = .005). Plating of the more primitive CD34+7- population in a transwell system (which separates progenitors from stroma by a microporous membrane) prevents differentiation into NK cells. In contrast, plating of CD34+7+ progenitors in transwells resulted in generation of NK cells. These data suggest that primitive, but not more mature NK progenitors may require direct contact with stroma for the initial differentiation steps. Finally, differentiation of the NK progenitors in this stroma-dependent model results in expression of CD2 not present on any of the starting populations. This observation suggests that marrow stroma can stimulate CD2 expression on NK progenitors in a previously undescribed fashion that may be analogous to the thymic effect on CD2 expression in immature T lymphocytes. These observations identify early steps in the commitment of primitive marrow CD34+ hematopoietic progenitors to a lymphoid lineage and underscore the importance of coexpression of CD7 with CD34 as an early lymphoid commitment characteristic and direct progenitor-stroma interactions in this process.

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