Characterization of the in vitro hepatocyte model for toxicological evaluation: repeated growth stimulation and glutathione response

1993 ◽  
Vol 71 (1-2) ◽  
pp. 7-13 ◽  
Author(s):  
Josée Guillemette ◽  
Michel Marion ◽  
Francine Denizeau ◽  
Michel Fournier ◽  
Pauline Brousseau
Keyword(s):  
Growth Factors ◽  
Dna Synthesis ◽  
Hepatocyte Proliferation ◽  
Initial Increase ◽  
Albumin Secretion ◽  
Toxic Response ◽  
Toxic Agents ◽  
Synthesis Stimulation

Hepatocytes in culture represent a useful model for investigating the effects of toxic agents on liver cells. However, further development of this model is hampered by the difficulty in promoting cell proliferation over prolonged periods and the lack of knowledge about the biochemical status of the cells relevant to the toxic response under proliferation conditions. In an effort to overcome these limitations, this work focused on the establishment of conditions to ameliorate the promotion of hepatocyte proliferation in vitro. It also examined the effects of growth stimuli on the levels of glutathione (GSH), a highly significant parameter influencing the resistance against toxic agents. In addition, albumin secretion was monitored as an indicator of liver-specific functions. Two modified L-15 media were developed: medium A for supporting cell differentiation, and medium B for promotion of proliferation. Collagen and Matrigel were used as substrata. In medium A, the time course of GSH levels was comparable for both substrata, with an initial increase followed by a plateau and then by a progressive decrease from the second to the fourth week. Hepatocytes cultured on collagen and sequentially exposed to medium B (containing epidermal growth factor ± norepinephrine) and medium A, showed repeated responsiveness to stimulation of DNA synthesis. Moreover, for cultures on collagen, a higher GSH content was observed in parallel with DNA synthesis stimulation, while albumin secretion was diminished. Although cells on Matrigel were refractory to DNA synthesis stimulation, GSH levels were still increased upon exposure to the growth factors, while under these conditions, albumin synthesis remained unaltered. These results show the possibility of expanding growth stimulus applications in hepatocyte cultures when it is of interest in cell pathology studies, such as those involving the effects of long-term exposure to xenobiotics, especially carcinogens. The results also suggest that hepatocytes subjected to a growth stimulus may have better protection against toxic injury.Key words: hepatocyte, glutathione, growth factors, long-term culture, collagen.

Stroma-free long-term growth of primary acute myeloid leukemia (AML) cells.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e18535-e18535
Author(s):  
Jing Chen ◽  
Glorymar Ibanez Sanchez ◽  
Paul Calder ◽  
Mark G. Frattini
Keyword(s):  
Growth Factors ◽  
Conditioned Medium ◽  
Stromal Cells ◽  
Drug Sensitivity ◽  
Growth Conditions ◽  
Leukemic Cells ◽  
Sensitivity Testing ◽  
Drug Sensitivity Testing

e18535 Background: To individualize therapy for relapsed/refractory AML patients, optimal in-vitro culture conditions to support primary leukemic cells are essential for drug sensitivity testing. Our lab has validated a high throughput chemosensitivity assay with primary AML cells maintained by growth factors (cytokines); however, growth factors have not been shown to support long-term assays of primary AML cells. Stromal cells of the tumor microenvironment are crucial to maintain normal hematopoiesis and leukemic cells and have been shown to support long term in-vitro expansion of primary AML cells. However, there is little information characterizing these growth conditions. The aim of this study was to compare long-term proliferation and phenotypes of primary AML cells with growth factors or stromal support to best determine their utility as a platform for drug sensitivity testing in functional assays. Methods: Patient-derived AML cells were cultured in 96-well plates in: 1) cell culture medium only 2) Human HS5 or HS27 stromal cells 3) HS5 or HS27 stroma-conditioned media or 4) cytokine cocktail. Viability readout by Guava ViaCount and leukemic cell surface phenotypes by fluorescently-conjugated antibodies were performed weekly over 3 weeks. Results: Primary AML cells cultured with only cytokines maintained proliferation at 3 weeks. In comparison, AML cells cultured in HS5 stroma-conditioned medium also maintained proliferation at a similar rate at 3 weeks, while co-culture with HS5 stromal cells demonstrated significantly higher proliferation. Leukemic immunophenotypes were maintained for all growth conditions over 3 weeks. Conclusions: Contrary to known data, primary AML cells with cytokines continued to expand at 3 weeks, at a similar rate to HS5 stroma-conditioned medium, a finding that has not been reported. Consistent with previous studies, we confirmed that stromal cells such as HS5 can provide long-term in-vitro expansion of primary AML cells, which cannot be substituted by stroma-conditioned medium. The ability to maintain long-term expansion of primary AML cells by both cytokines and stromal cells sets up a platform for a direct comparison of high throughput drug sensitivity testing under these growth conditions.

scholarly journals Organotypic Cultures of Intervertebral Disc Cells: Responses to Growth Factors and Signaling Pathways Involved

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Harris Pratsinis ◽  
Dimitris Kletsas
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Intervertebral Disc ◽  
Dna Synthesis ◽  
Signaling Pathways ◽  
Collagen Type I ◽  
Cell Physiology ◽  
Type I ◽  
Collagen Gels

Intervertebral disc (IVD) degeneration is strongly associated with low back pain, a major cause of disability worldwide. An in-depth understanding of IVD cell physiology is required for the design of novel regenerative therapies. Accordingly, aim of this work was the study of IVD cell responses to mitogenic growth factors in a three-dimensional (3D) organotypic milieu, comprising characteristic molecules of IVD’s extracellular matrix. In particular, annulus fibrosus (AF) cells were cultured inside collagen type-I gels, while nucleus pulposus (NP) cells in chondroitin sulfate A (CSA) supplemented collagen gels, and the effects of Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-Like Growth Factor-I (IGF-I) were assessed. All three growth factors stimulated DNA synthesis in both AF and NP 3D cell cultures, with potencies similar to those observed previously in monolayers. CSA supplementation inhibited basal DNA synthesis rates, without affecting the response to growth factors. ERK and Akt were found to be phosphorylated following growth factor stimulation. Blockade of these two signaling pathways using pharmacologic inhibitors significantly, though not completely, inhibited growth factor-induced DNA synthesis. The proposed culture systems may prove useful for further in vitro studies aiming at future interventions for IVD regeneration.

scholarly journals The Cultivation of Human Granulosa Cells

2008 ◽  
Vol 51 (3) ◽  
pp. 165-172 ◽  
Author(s):  
Lenka Brůčková ◽  
Tomáš Soukup ◽  
Jiří Moos ◽  
Martina Moosová ◽  
Jana Pavelková ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Doubling Time ◽  
Ovarian Follicle ◽  
Vitro Fertilization ◽  
Thecal Cells ◽  
Cell Doubling Time

The major functions of granulosa cells (GCs) include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells) to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome). GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 °C. GCs expansion medium consisted of DMEM/F12, 2 % FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF). GCs transported in follicular fluid and cultivated in 2 % FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92 % and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.

scholarly journals Activation and proliferation signals in murine macrophages: synergistic interactions between the hematopoietic growth factors and with phorbol ester for DNA synthesis

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1574-1580 ◽  
Author(s):  
JA Hamilton ◽  
G Vairo ◽  
NA Nicola ◽  
A Burgess ◽  
D Metcalf ◽  
...  
Keyword(s):  
Growth Factors ◽  
Dna Synthesis ◽  
Phorbol Ester ◽  
Synergistic Effects ◽  
Hematopoietic Growth Factors ◽  
Murine Bone Marrow ◽  
Gm Csf ◽  
Synergistic Interactions ◽  
Kinase C

Abstract There has been recent interest in the synergistic interactions between the growth factors involved in the in vitro control of hematopoiesis and other cell lineages. As a convenient model system, such interactions governing the DNA synthesis in murine bone marrow-derived macrophages (BMMs) were studied. By themselves, murine colony- stimulating factor-1 (CSF-1) and recombinant murine granulocyte- macrophage CSF (GM-CSF) were stimulators of DNA synthesis in quiescent or noncycling BMMs, whereas recombinant murine interleukin-3 (IL-3) and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), were weak mitogens. On the other hand, murine granulocyte CSF (G-CSF), concanavalin A (Con A), and lipopolysaccharide (LPS) were inactive on their own. When the quiescent BMMs were exposed to combinations of the CSFs, there were striking synergistic effects for both GM-CSF and IL-3 with suboptimal doses of CSF-1, with a smaller effect for GM-CSF with IL-3 and little or no effect for CSF-1 with G-CSF. CSF-1, GM-CSF, and IL-3 could also synergize with TPA; CSF-1 cooperated with 1-oleoyl-2- acetylglycerol (OAG), both sets of results pointing to an interaction with protein kinase C. LPS completely abolished the CSF-1-mediated stimulation of DNA synthesis. We propose that BMMs are suitable normal cells in which to examine in depth the various mechanistic possibilities for these interactions.

scholarly journals Response patterns of hairy cell leukemia to B-cell mitogens and growth factors

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2091-2097
Author(s):  
BA Barut ◽  
MK Cochran ◽  
C O'Hara ◽  
KC Anderson
Keyword(s):  
Growth Factors ◽  
Dna Synthesis ◽  
B Cell ◽  
Tumor Cells ◽  
Hairy Cell Leukemia ◽  
Proliferative Response ◽  
Tartrate Resistant Acid Phosphatase ◽  
Hairy Cell ◽  
Cell Leukemia

The effect of mitogens and/or recombinant B-cell growth factors (M/GFs) on the in vitro growth of hairy cells was examined. Tumor cells were isolated from the spleens of four patients with hairy cell leukemia (HCL) by Ficoll-Hypaque sedimentation and E-rosetting. Enrichment for tumor cells was confirmed with intracytoplasmic immunoglobulin (Ig) staining, tartrate resistant acid phosphatase (TRAP) staining, and staining using monoclonal antibodies (MoAbs) directed at B, T, myeloid, and monocytoid antigens (Ags) in indirect immunofluorescence assays. Tumor cells were B1(CD20)+ B2(CD21)- B4(CD19)+ IL-2R(CD25)+ PCA-1 +/- TRAP+. HCLs neither synthesized DNA nor secreted Ig in response to culture with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-3, IL-4, IL-5, or IL-6. However, a proliferative response (stimulation index greater than or equal to 3.0) without Ig secretion was triggered in HCLs by mitogens or combinations of GFs. Specifically, DNA synthesis was induced at 3 days in three of four HCL samples cultured with Staphylococcus aureus Cowan A (SAC) or the combination of phorbol ester (TPA) and the calcium ionophore A 23187 (Ca2+); DNA synthesis was triggered later (day 7) by tumor necrosis factor (TNF) or by IL-4 and IL-5. In contrast, the fourth patient, a nonresponder to SAC or TPA/Ca2+, demonstrated increased DNA synthesis at day 3 when cocultured with IL-4 and IL-5. Both autoradiography and staining with antibromodeoxyuridine (BrdU) MoAb conjugated to fluorescein confirmed DNA synthesis by only a minority (5% to 23%) of tumor cells within each patient. Dual staining confirmed that responsive cells were both BrdU+ and TRAP+. DNA synthesis induced by TPA/Ca2+ was blocked specifically by anti-IL-6 Ab; in contrast, the HCL proliferative response to SAC, TNF, or IL-4 and IL- 5 was not inhibited by anti-IL-6 Ab. alpha-Interferon inhibited the response to TPA/Ca2+, TNF, or IL-4 and IL-5 without any effect on response to SAC. Finally, peroxidase-antiperoxidase staining demonstrated that HCLs are induced by TPA/Ca2+, but not by SAC, to produce intracytoplasmic IL-6. These data demonstrate IL-4, IL-5, and IL-6 mediated DNA synthesis by HCLs in vitro and suggest a possible in vivo role for these growth factors in the pathophysiology of HCL.

scholarly journals Autocrine stimulation of human hepatocytes triggers late DNA synthesis and stabilizes long-term differentiation in vitro

2008 ◽  
Author(s):  
Kerstin Leckel ◽  
Christoph Strey ◽  
Wolf Bechstein ◽  
Kim Boost ◽  
Marcus Auth ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Human Hepatocytes ◽  
Autocrine Stimulation ◽  
Stimulation Of

Bombesin and bradykinin increase inositol phosphates and cytosolic free Ca2+, and stimulate DNA synthesis in human endometrial stromal cells

1991 ◽  
Vol 131 (2) ◽  
pp. 313-318 ◽  
Author(s):  
T. Endo ◽  
H. Fukue ◽  
M. Kanaya ◽  
M. Mizunuma ◽  
M. Fujii ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Phospholipase C ◽  
Stromal Cells ◽  
Inositol Phosphates ◽  
Initial Increase ◽  
Second Phase ◽  
Extracellular Medium ◽  
Endometrial Stromal Cells

ABSTRACT The present studies were carried out to investigate the effect of several growth factors on human endometrial stromal cells. In human endometrial stromal cells, bombesin and bradykinin provoked an increase in intracellular free Ca2+ and in labelled inositol phosphates when pre-incubated with [3H]myoinositol. Some or possibly all of the initial increase in intracellular free Ca2+ represented a mobilization of Ca2+ from intracellular stores and the second phase of the response depended on Ca2+ influx from the extracellular medium. [3H]Thymidine was added to human cultured endometrial stromal cells with bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F2α, vasopressin and platelet-derived growth factor. Bombesin, bradykinin and EGF stimulated the incorporation of [3H]thymidine into DNA in quiescent cells. In conclusion, bombesin and bradykinin are growth factors which activate phospholipase C in human endometrial stromal cells, while EGF stimulates DNA synthesis without the activation of phospholipase C. Journal of Endocrinology (1991) 131, 313–318

scholarly journals Transforming growth factor-α-induced DNA synthesis and c-myc expression in primary rat hepatocyte cultures is modulated by indomethacin

1992 ◽  
Vol 281 (3) ◽  
pp. 729-733 ◽  
Author(s):  
G G Skouteris ◽  
M McMenamin
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Phospholipase A2 ◽  
Culture Medium ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Receptor Gene ◽  
Hepatocyte Proliferation ◽  
Transforming Growth Factor Α

Primary hepatocytes stimulated with epidermal growth factor (EGF) secrete prostaglandins into the culture medium as soon as 1 h after the addition of the EGF. Transforming growth factor-alpha (TGF alpha), a potent hepatocyte mitogen, shares the same receptor with EGF, and its expression is increased after partial hepatectomy. TGF alpha is also secreted in culture. We have observed that TGF alpha induced hepatocyte DNA synthesis (30 h after addition) and at the same time stimulated the production of prostaglandins E2 and F2 alpha by the cultured hepatocytes. Indomethacin at 20-100 microM inhibited the TGF alpha-induced hepatocyte DNA synthesis, and this effect was specifically due to the inhibition of prostaglandin formation. Indomethacin also inhibited a TGF-alpha-induced increase in hepatocyte c-myc expression, indicating that prostaglandins mediate this increase, as previously shown for EGF. TGF alpha increased the expression of the EGF receptor gene, and this was prevented by the presence of an antibody against TGF alpha in the culture medium. We therefore suggest that TGF alpha induces hepatocyte proliferation either through coupling with its receptor (i.e. the EGF receptor) or by subsequent phosphorylation of lipocortin I. This leads to activation of phospholipase. A2, which seems to regulate the metabolism of arachidonic acid and the formation of prostaglandins. Thus hepatocyte proliferation in vitro appears to be controlled by a self-regulatory autocrine pathway involving activation of phospholipase A2 and secretion of prostaglandins and TGF alpha.

Role of p53 in Hematopoietic Recovery After Cytotoxic Treatment

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2998-3006 ◽  
Author(s):  
Pawel Wlodarski ◽  
Mariusz Wasik ◽  
Mariusz Z. Ratajczak ◽  
Cinzia Sevignani ◽  
Grazyna Hoser ◽  
...  
Keyword(s):  
Growth Factors ◽  
Bone Marrow Cells ◽  
Positive Impact ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Cytotoxic Treatment

Prompt reconstitution of hematopoiesis after cytoreductive therapy is essential for patient recovery and may have a positive impact on long-term prognosis. We examined the role of the p53 tumor suppressor gene in hematopoietic recovery in vivo after treatment with the cytotoxic drug 5-fluorouracil (5-FU). We used p53 knock-out (p53−/−) and wild-type (p53+/+) mice injected with 5-FU as the experimental model. Analysis of the repopulation ability and clonogenic activity of hematopoietic stem cells (HSCs) and their lineage-committed descendants showed a greater number of HSCs responsible for reconstitution of lethally irradiated recipients in p53−/− bone marrow cells (BMCs) recovering after 5-FU treatment than in the corresponding p53+/+ BMCs. In post–5-FU recovering BMCs, the percentage of HSC-enriched Lin− Sca-1+c-Kit+ cells was about threefold higher in p53−/− than in p53+/+ cells. Although the percentage of the most primitive HSCs (Lin− Sca-1+ c-Kit+CD34low/−) did not depend on p53, the percentage of multipotential HSCs and committed progenitors (Lin−Sca-1+ c-Kit+ CD34high/+) was almost fourfold higher in post–5-FU recovering p53−/− BMCs than in their p53+/+ counterparts. The pool of HSCs from 5-FU–treated p53−/− BMCs was exhausted more slowly than that from the p53+/+ population as shown in vivo using pre–spleen colony-forming unit (CFU-S) assay and in vitro using long-term culture-initiating cells (LTC-ICs) and methylcellulose replating assays. Clonogenic activity of various lineage-specific descendants was significantly higher in post–5-FU regenerating p53−/− BMCs than in p53+/+ BMCs, probably because of their increased sensitivity to growth factors. Despite all these changes and the dramatic difference in sensitivity of p53−/− and p53+/+ BMCs to 5-FU–induced apoptosis, lineage commitment and differentiation of hematopoietic progenitors appeared to be independent of p53 status. These studies suggest that suppression of p53 function facilitates hematopoietic reconstitution after cytoreductive therapy by: (1) delaying the exhaustion of the most primitive HSC pool, (2) stimulating the production of multipotential HSCs, (3) increasing the sensitivity of hematopoietic cells to growth factors, and (4) decreasing the sensitivity to apoptosis.

scholarly journals Both Stroma and Stem Cell Factor Maintain Long-Term Growth of ELM Erythroleukemia Cells, but Only Stroma Prevents Erythroid Differentiation in Response to Erythropoietin and Interleukin-3

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1548-1555 ◽  
Author(s):  
Jim O'Prey ◽  
Nick Leslie ◽  
Katsukiko Itoh ◽  
Wolfram Ostertag ◽  
Chris Bartholomew ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Stem Cell Factor ◽  
Neutralizing Antibodies ◽  
High Efficiency ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Interleukin 3

Abstract Defining how the stromal requirements of hematopoietic progenitors change during leukemia progression is an important topic that is not well understood at present. The murine ELM erythroleukemia is an interesting model because the erythroid progenitors retain dependence on bone marrow-derived stromal cells for long-term growth in vitro, and they also undergo erythroid differentiation in the presence of erythropoietin (EPO) and interleukin-3 (IL-3). In this report, we have shown using neutralizing antibodies that stem cell factor (SCF), insulin-like growth factor (IGF)-1, and integrin signaling pathways are all involved. We then determined whether ELM cells can be maintained long-term without stroma in various combinations of growth factors produced by stroma cells or growth factors for which ELM cells have receptors. This showed that ELM cells could be maintained with high efficiency in SCF alone; furthermore, the cells remained absolutely SCF-dependent and did not become more tumorigenic than cells maintained on stroma. In contrast, ELM cells underwent clonal extinction when serially cloned in IGF1; any cells that survived long-term growth in IGF-1 were found to be IGF1-independent. One important difference between maintaining ELM cells on stroma and growth in SCF is that stroma reversibly inhibits their differentiation in response to EPO and IL-3, whereas SCF does not.

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