scholarly journals Preventing Microbial Contamination during Long-Term In Vitro Culture of Human Granulosa-Lutein Cells: An Ultrastructural Analysis

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
C. O. Campos ◽  
M. P. Bernuci ◽  
A. A. Vireque ◽  
J. R. Campos ◽  
M. F. Silva-de-Sá ◽  
...  
Keyword(s):  
Bacterial Contamination ◽  
Microbial Contamination ◽  
High Dose ◽  
Transmission Electron ◽  
Cellular Debris ◽  
Reproductive Techniques ◽  
And Function ◽  
Cell Plating

Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.

Mesothelial Cells

2007 ◽  
Vol 27 (2_suppl) ◽  
pp. 110-115 ◽  
Author(s):  
Susan Yung ◽  
Chan Tak Mao
Keyword(s):  
Mesothelial Cell ◽  
In Vitro Models ◽  
Mesothelial Cells ◽  
Dynamic Membrane ◽  
Peritoneal Mesothelial Cells ◽  
Immunohistochemical Markers ◽  
Vitro Model ◽  
And Function

♦ Background The introduction of peritoneal dialysis (PD) as a modality of renal replacement therapy has provoked much interest in the biology of the peritoneal mesothelial cell. Mesothelial cells isolated from omental tissue have immunohistochemical markers that are identical to those of mesothelial stem cells, and omental mesothelial cells can be cultivated in vitro to study changes to their biologic functions in the setting of PD. ♦ Method The present article describes the structure and function of mesothelial cells in the normal peritoneum and details the morphologic changes that occur after the introduction of PD. Furthermore, this article reviews the literature of mesothelial cell culture and the limitations of in vitro studies. ♦ Results The mesothelium is now considered to be a dynamic membrane that plays a pivotal role in the homeostasis of the peritoneal cavity, contributing to the control of fluid and solute transport, inflammation, and wound healing. These functional properties of the mesothelium are compromised in the setting of PD. Cultures of peritoneal mesothelial cells from omental tissue provide a relevant in vitro model that allows researchers to assess specific molecular pathways of disease in a distinct population of cells. Structural and functional attributes of mesothelial cells are discussed in relation to long-term culture, proliferation potential, age of tissue donor, use of human or animal in vitro models, and how the foregoing factors may influence in vitro data. ♦ Conclusions The ability to propagate mesothelial cells in culture has resulted, over the past two decades, in an explosion of mesothelial cell research pertaining to PD and peritoneal disorders. Independent researchers have highlighted the potential use of mesothelial cells as targets for gene therapy or transplantation in the search to provide therapeutic strategies for the preservation of the mesothelium during chemical or bacterial injury.

Reproductive fluids, added to the culture media, contribute to minimizing phenotypical differences between in vitro-derived and artificial insemination-derived piglets

2022 ◽  
pp. 1-13
Author(s):  
Evelyne París-Oller ◽  
Cristina Soriano-Úbeda ◽  
Ramsés Belda-Pérez ◽  
Lucía Sarriás-Gil ◽  
Jordana S. Lopes ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Culture Media ◽  
Growth Parameters ◽  
Tolerance Test ◽  
Oral Glucose ◽  
Long Term Effects ◽  
Hematological Indices ◽  
Reproductive Techniques

Abstract The addition of reproductive fluids (RF) to the culture media has shown benefits in different embryonic traits but its long-term effects on the offspring phenotype are still unknown. We aimed to describe such effects in pigs. Blood samples and growth parameters were collected from piglets derived from in vitro-produced embryos (IVP) with or without RF added in the culture media versus those artificially inseminated (AI), from day 0 to month 6 of life. An oral glucose tolerance test was performed on day 45 of life. We show here the first comparative data of the growth of animals produced through different assisted reproductive techniques, demonstrating differences between groups. Overall, there was a tendency to have a larger size at birth and faster growth in animals derived from in vitro fertilization and embryo culture versus AI, although this trend was diminished by the addition of RFs to the culture media. Similarly, small differences in hematological indices and glucose tolerance between animals derived from AI and those derived from IVP, with a sex-dependent effect, tended to fade in the presence of RF. The addition of RF to the culture media could contribute to minimizing the phenotypical differences between the in vitro-derived and AI offspring, particularly in males.

Long-term administration of high-dose methylphenidate-induced cerebellar morphology and function damage in adult rats

2020 ◽  
Vol 103 ◽  
pp. 101712 ◽  
Author(s):  
Amir Raoofi ◽  
Abass Aliaghaei ◽  
Mohammad-Amin Abdollahifar ◽  
Mahdi Eskandarian Boroujeni ◽  
Sara Sadat Javadinia ◽  
...  
Keyword(s):  
High Dose ◽  
Adult Rats ◽  
Term Administration ◽  
And Function

Biocompatibility of Charcoal Hemoperfusion. Effects of Long-Term Treatment on Lymphocyte Characteristics and Function

1986 ◽  
Vol 9 (5) ◽  
pp. 301-304 ◽  
Author(s):  
S. Stefoni ◽  
A. Nanni Costa ◽  
G. Liviano D'Arcangelo ◽  
M. Biavati ◽  
S. lannelli ◽  
...  
Keyword(s):  
Antigen Expression ◽  
Term Treatment ◽  
T Cell Subsets ◽  
Long Term Treatment ◽  
Circulating Lymphocytes ◽  
And Function ◽  
Charcoal Hemoperfusion

Biocompatibility of charcoal hemoperfusion was studied in a group of 15 uremic patients, evaluating the effects of long-term treatment on some structural and functional parameters of circulating lymphocytes: in vivo distribution of T-cell subsets; surface T3, T4 and T8 antigen expression, in vivo and in vitro DNA synthesis. A comparative analysis was performed with patients on conventional dialysis using cuprophan membranes.

scholarly journals IL-2 administration increases CD4+CD25hi Foxp3+ regulatory T cells in cancer patients

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2409-2414 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Steven A. Rosenberg
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Interleukin 2 ◽  
Selective Inhibition ◽  
High Dose ◽  
Protein Levels ◽  
And Function ◽  
The Impact

Abstract Interleukin-2 (IL-2) is historically known as a T-cell growth factor. Accumulating evidence from knockout mice suggests that IL-2 is crucial for the homeostasis and function of CD4+CD25+ regulatory T cells in vivo. However, the impact of administered IL-2 in an immune intact host has not been studied in rodents or humans. Here, we studied the impact of IL-2 administration on the frequency and function of human CD4+CD25hi T cells in immune intact patients with melanoma or renal cancer. We found that the frequency of CD4+CD25hi T cells was significantly increased after IL-2 treatment, and these cells expressed phenotypic markers associated with regulatory T cells. In addition, both transcript and protein levels of Foxp3, a transcription factor exclusively expressed on regulatory T cells, were consistently increased in CD4 T cells following IL-2 treatment. Functional analysis of the increased number of CD4+CD25hi T cells revealed that this population exhibited potent suppressive activity in vitro. Collectively, our results demonstrate that administration of high-dose IL-2 increased the frequency of circulating CD4+CD25hi Foxp3+ regulatory T cells. Our findings suggest that selective inhibition of IL-2-mediated enhancement of regulatory T cells may improve the therapeutic effectiveness of IL-2 administration. (Blood. 2006;107:2409-2414)

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 283-293 ◽  
Author(s):  
M. Popelková ◽  
P. Chrenek ◽  
J. Pivko ◽  
A.V. Makarevich ◽  
E. Kubovičová ◽  
...  
Keyword(s):  
Ultrastructural Level ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Hatching Rate ◽  
Transmission Electron ◽  
Cellular Debris ◽  
Or Gene ◽  
Rabbit Gene

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p<0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not siginificantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.

scholarly journals The chemokine GROβ mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 860-869 ◽  
Author(s):  
Seiji Fukuda ◽  
Huimin Bian ◽  
Andrew G. King ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
And Function ◽  
Stimulating Factor ◽  
Cxcr4 Axis

Abstract Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GROβ (GROβΔ4/CXCL2Δ4) or the combination of GROβΔ4 plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF–mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GROβ-mobilized grafts. PBSCs mobilized by GROβΔ4 alone or with G-CSF contained significantly more Sca-1+-c-kit+-lineage− (SKL) cells and more primitive CD34−-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GROβΔ4-mobilized SKL cells adhered better to VCAM-1+ endothelial cells compared with G-CSF–mobilized cells. GROβΔ4-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1α in vitro that was associated with higher CD26 expression. However, GROβΔ4-mobilized SKL and c-Kit+ lineage− (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GROβΔ4-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GROβΔ4-mobilized cells is less dependent on the SDF-1α/CXCR4 axis.

Telomere Length of Hematopoietic Cells in Long-Term Post-Autograft Survivors: No Evidence of Accelerated Replicative Cell Senescence Despite the Persistent Reduction of Both Committed and Immature Progenitor Cell Compartments.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4123-4123
Author(s):  
Alberto Rocci ◽  
Irene Ricca ◽  
Chiara Della Casa ◽  
Paolo Longoni ◽  
Mara Compagno ◽  
...  
Keyword(s):  
Stem Cells ◽  
Telomere Length ◽  
Progenitor Cell ◽  
Hematopoietic Cells ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Replicative Stress

Abstract Telomere length is considered a valuable replicative capacity predictor of human hematopoietic stem cells. Indeed, a progressive telomere shortening affects hematopoietic cells upon in vitro expansion. However, less is known on the dynamics of telomere shortening in vivo following a non-physiological replicative stress. Aim of this study was to investigate markers for cellular senescence of hematopoietic cells exposed to replicative stress induced by bone marrow reconstitution following stem cell autograft. Thus, both telomere length and in vitro functional characteristics of bone marrow (BM) and peripheral blood (PB) were evaluated at long-term in subjects who had received intensive chemotherapy and autograft. Thirty-two adults with a previous diagnosis of lymphoma were examined, at a median time of 73 months (range 42–125) since autograft. They all had received a high-dose sequential chemotherapy treatment followed by peripheral blood progenitor cell (PBPC) autograft. There were 20 male and 12 female (median age at autograft: 40 yrs., range 21–60). A Southern blot procedure using a chemiluminescence-based assay was employed to determine telomere length on samples from grafted PBPC as well as on BM and PB samples obtained at long-term during follow-up. These latter samples were also studied for their in vitro growth characteristics, assessed by short and long-term culture assays. In all cases, autograft had been performed with large quantities of hematopoietic stem cells (median autografted CD34+ve cells/kg: 9.8 x 106, range 2–24), allowing a rapid and stable hematologic reconstitution. Telomere length was found slightly shorter in BM mononuclear cells from samples taken at follow-up compared to samples from grafted material (median telomere length: 6,895 bp vs 7,073 bp, respectively; p=ns). No marked differences were observed in telomere evaluation between BM and PB cells. No significant differences were observed as well when PB telomere length of follow-up samples was compared with telomere length of PB from age-related normal subjects. BM and PB samples were then assessed for their in vitro growth characteristics. Committed and stromal progenitors were grown from all samples in good though variable quantities. However, as compared to normal controls, a statistically significant reduction of marrow-derived hematopoietic progenitors (CFU-GM - BFU-E - CFU-Mix) as well as stromal progenitors (CFU-F) was observed. Additionally, the more immature LTC-IC progenitor cell compartment was dramatically reduced, both in BM and PB samples. The results indicate that: i. the proliferative stress induced by intensive chemotherapy and post-graft hematopoietic reconstitution does not imply marked telomere loss in BM and PB cells at long-term, provided that large quantities of PBPC are used for autograft; ii. stem cells present in the graft or surviving after high-dose therapy are capable of reconstituting a sufficiently adequate hematopoiesis although the committed progenitor cell compartment and even more the immature LTC-IC progenitors are persistently reduced even at up to 10 years since autograft.

Osteoblast Growth Inhibition by Unfractionated Heparin and by Low Molecular Weight Heparins: An in-vitro Investigation

2002 ◽  
Vol 8 (3) ◽  
pp. 251-255 ◽  
Author(s):  
H. J. Kock ◽  
A. E. Handschin
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Cell Cultures ◽  
Term Therapy ◽  
Low Molecular Weight ◽  
High Dose ◽  
Low Molecular Weight Heparins ◽  
In Vitro Investigation

Osteoporosis is a rare but potentially severe complication under high-dose, long-term unfractionated heparin therapy. Low-molecular-weight heparins (LMWHs) have gained increased importance in antithrombotic therapy over the past decade. Whether this heterogeneous group of drugs carries a comparable risk of osteoporosis in long-term application is unknown. In a standardized in vitro model, the effects of 4 different low-molecular-weight heparins (nadroparin, enoxaparin, dalteparin, certoparin) on osteoblast growth were studied at the same dose (50,μg/mL). As control, the effect of unfractionated heparin (Liquemin) was tested on human osteoblasts in vitro at an equal dose. Human osteoblast cell cultures were incubated with equal doses of the heparins, and cell concentrations were measured after 48 and 96 hours. In addition, a fluorescence assay was performed to detect potential cytotoxic effect of heparins on bone cells. In comparison to control groups of non-incubated cell cultures, LMWHs caused a significant inhibition of osteoblast growth (p<0.05). Therefore, the risk of osteoporosis under long-term therapy with high doses of LMWHs cannot be excluded and should be further evaluated in clinical trials.

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