Advancing in vitro -in vivo toxicity correlations via high-throughput three-dimensional primary hepatocyte culture

AIChE Journal ◽  
2018 ◽  
Vol 64 (12) ◽  
pp. 4331-4340
Author(s):  
Dylan M. Bruckner ◽  
Jeannette J. Connerney ◽  
Jonathan S. Dordick
Keyword(s):  
High Throughput ◽  
Three Dimensional ◽  
Hepatocyte Culture ◽  
Primary Hepatocyte ◽  
Primary Hepatocyte Culture ◽  
In Vivo Toxicity

Patient-derived organoids for personalized drug screening in intrahepatic cholangiocarcinoma.

2020 ◽  
Vol 38 (4_suppl) ◽  
pp. 581-581
Author(s):  
Ricardo J. Antonia ◽  
Kan Toriguchi ◽  
Eveliina Karelehto ◽  
Dania Annuar ◽  
Luika Timmerman ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Intrahepatic Cholangiocarcinoma ◽  
Three Dimensional ◽  
Small Samples ◽  
Patient Specific ◽  
Mtor Inhibition ◽  
Drug Responses

581 Background: Despite standard treatment with gemcitabine and cisplatin, median survival for unresectable Intrahepatic Cholangiocarcinoma (ICC) is < 1 year. Clearly, novel therapeutic strategies are urgently needed. The paucity of targetable mutations in ICC and the as yet unproven benefit of genetically targeted drugs led us to ask whether a reliable clinical benefit may be revealed by patient-specific therapeutic testing in novel models of ICC. Here we describe our ability to establish patient-derived three-dimensional organoid cultures (PDO) that enable individualized identification of active single agents or drug combinations in surrogate models of ICC. Methods: To model patient-specific drug responses, we used the freshly resected ICCs from small samples of single patient tumors to generate PDXs and PDOs, small spheroidal clusters of tumor cells grown in vitro. We have employed a high-throughput drug screening platform using AI-enhanced robotics (Yamaha Motor Corporation) to identify and distribute single, uniformly sized PDOs into 384-well ultra-low adherent plates. This is coupled with a TECAN D300e drug dispenser that rapidly delivers nanoliter volumes of a 34-drug panel, thereby facilitating rapid, reliable drug response analyses. Results: Our data show that PDOs retain characteristic genomic and histological features of the patients’ tumors. Drug responses were specific to each patient tumor, but PDOs from all patients responded to a greater or lesser degree to mTOR inhibition, suggesting that this pathway is important in ICC. The responses of PDO to the mTOR inhibitor Sapanisertib (INK128), was recapitulated in the same patient’s PDX. Further, INK128 was synergistic with gemcitabine in patient 970 PDOs as well as in vivo in PDX also from patient 970. Conclusions: As it is believed that PDX can predict patient responses to drugs, our results suggest that PDO may also predict patient drug responses. The establishment of PDO may allow economical patient-specific, high throughput drug screens that could ultimately inform clinical practice. [Table: see text]

scholarly journals Three-Dimensional Primary Hepatocyte Culture in Synthetic Self-Assembling Peptide Hydrogel

2008 ◽  
Vol 14 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Sihong Wang ◽  
Deepak Nagrath ◽  
Pohun C. Chen ◽  
François Berthiaume ◽  
Martin L. Yarmush
Keyword(s):  
Three Dimensional ◽  
Hepatocyte Culture ◽  
Primary Hepatocyte ◽  
Primary Hepatocyte Culture ◽  
Self Assembling ◽  
Peptide Hydrogel

scholarly journals A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Terrence Cheng ◽  
Nelson S. Torres ◽  
Ping Chen ◽  
Anand Srinivasan ◽  
Sandra Cardona ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
High Throughput ◽  
Three Dimensional ◽  
Hanging Drop ◽  
Methicillin Resistant ◽  
Content Type ◽  
Throughput Model

ABSTRACT Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection. IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.

scholarly journals Three-Dimensional Primary Hepatocyte Culture in Synthetic Self-Assembling Peptide Hydrogel

2008 ◽  
pp. 110306233438005 ◽  
Author(s):  
Sihong Wang ◽  
Deepak Nagrath ◽  
Pohun C. Chen ◽  
François Berthiaume ◽  
Martin L. Yarmush
Keyword(s):  
Three Dimensional ◽  
Hepatocyte Culture ◽  
Primary Hepatocyte ◽  
Primary Hepatocyte Culture ◽  
Self Assembling ◽  
Peptide Hydrogel

scholarly journals Quantitative Live-Cell Confocal Imaging of 3D Spheroids in a High-Throughput Format

2018 ◽  
Vol 23 (3) ◽  
pp. 231-242 ◽  
Author(s):  
Elizabeth Leary ◽  
Claire Rhee ◽  
Benjamin T. Wilks ◽  
Jeffrey R. Morgan
Keyword(s):  
High Throughput ◽  
Fluorescent Dyes ◽  
Three Dimensional ◽  
3D Culture ◽  
Confocal Imaging ◽  
In Vivo Models ◽  
Ratio Imaging ◽  
3D Spheroids

Accurately predicting the human response to new compounds is critical to a wide variety of industries. Standard screening pipelines (including both in vitro and in vivo models) often lack predictive power. Three-dimensional (3D) culture systems of human cells, a more physiologically relevant platform, could provide a high-throughput, automated means to test the efficacy and/or toxicity of novel substances. However, the challenge of obtaining high-magnification, confocal z stacks of 3D spheroids and understanding their respective quantitative limitations must be overcome first. To address this challenge, we developed a method to form spheroids of reproducible size at precise spatial locations across a 96-well plate. Spheroids of variable radii were labeled with four different fluorescent dyes and imaged with a high-throughput confocal microscope. 3D renderings of the spheroid had a complex bowl-like appearance. We systematically analyzed these confocal z stacks to determine the depth of imaging and the effect of spheroid size and dyes on quantitation. Furthermore, we have shown that this loss of fluorescence can be addressed through the use of ratio imaging. Overall, understanding both the limitations of confocal imaging and the tools to correct for these limits is critical for developing accurate quantitative assays using 3D spheroids.

scholarly journals Induction of acyl-CoA oxidase and cytochrome P450IVA1 RNA in rat primary hepatocyte culture by peroxisome proliferators

1991 ◽  
Vol 280 (1) ◽  
pp. 249-253 ◽  
Author(s):  
D R Bell ◽  
C R Elcombe
Keyword(s):  
Time Course ◽  
Clofibric Acid ◽  
Hepatocyte Culture ◽  
Primary Hepatocyte ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferation ◽  
Rnase Protection ◽  
Primary Hepatocyte Culture ◽  
Acyl Coa Oxidase

We have characterized the induction of acyl-CoA oxidase and cytochrome P450IVA1 RNAs in a primary hepatocyte culture system in vitro, using a sensitive and specific RNAse protection assay. Hepatocytes were cultured with a maximal inducing dose of the peroxisome proliferator clofibric acid (1 mM), or vehicle control, for 4 days, and the level of RNAs compared with the level in rats which had been treated with corn oil or clofibric acid (300 mg/kg) for 4 days. The level of acyl-CoA oxidase and P450IVA1 RNAs in 4-day-old control hepatocytes was less than 2% of that in control liver. However, the level of these RNAs in RNA from treated hepatocytes was 61% of that in liver RNA from treated rats. Hepatocytes were treated with the potent peroxisome proliferator methylclofenapate (100 microM), and the induction of RNAs determined at various times after exposure. P450IVA1 RNA was significantly induced 1 h after dosing, rising to 34-fold above control after 8 h, whereas acyl-CoA oxidase RNA was not significantly induced until 4 h, increasing to 5.2-fold above control after 8 h. A similar time course of induction was seen after treatment of hepatocytes with 100 microM-nafenopin, 100 microM-methylclofenapate, 1 mM-clofibric acid or 1 mM-mono(ethylhexyl) phthalate, suggesting that the differential time course of induction of P450IVA1 and acyl-CoA oxidase RNAs is not related to the esterification, structure or potency of the peroxisome proliferator, but is intrinsic to the process of peroxisome proliferation. Hepatocytes were treated with methylclofenapate in the presence and absence of cycloheximide. P450IVA1 RNA was significantly induced by methylclofenapate in the presence of cycloheximide, rising to 17-fold above control after 8 h. However, no induction of acyl-CoA oxidase RNA was detected in the presence of cycloheximide. Therefore we characterize the induction of acyl-CoA oxidase and P450IVA1 RNAs in primary hepatocyte culture in vitro as a faithful model of the induction response in rat liver, and suggest that induction of P450IVA1 RNA is a primary event in the process of peroxisome proliferation.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

NT4X-167; Novel N-terminal Aβ specific antibody, prevents in vitro and in vivo toxicity of highly toxic Aβ4-42 species

2015 ◽  
Vol 48 (06) ◽  
Author(s):  
G Antonios ◽  
H Borgers ◽  
T Pilot ◽  
V Pena ◽  
T Bayer
Keyword(s):  
Specific Antibody ◽  
In Vivo Toxicity
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