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Depending on the experimental protocol, the Caco-2 cells are seeded in multi-well plates (6, 12, 24, or 96-well) and cultured for 21–25 days .
[ { "end": 50, "label": "CellLine", "start": 44, "text": "Caco-2" } ]
ChemBL_V1
Long-term culture of Caco-2 cells leads to their spontaneous differentiation into mature enterocyte-like cells resembling enterocytes of the small intestine in vivo .
[ { "end": 27, "label": "CellLine", "start": 21, "text": "Caco-2" } ]
ChemBL_V1
Differentiated Caco-2 cells exhibit functional tight junctions between the neighboring cells and well-developed microvilli on the apical surface .
[ { "end": 21, "label": "CellLine", "start": 15, "text": "Caco-2" } ]
ChemBL_V1
The Caco-2 cell monolayer’s integrity is assessed with transepithelial electrical resistance (TEER) measurements, performed in a non-invasive manner using ohmic resistance .
[ { "end": 10, "label": "CellLine", "start": 4, "text": "Caco-2" } ]
ChemBL_V1
The TERR values in the Caco-2 monolayers from several permeability tests are shown in Table 5.
[ { "end": 29, "label": "CellLine", "start": 23, "text": "Caco-2" } ]
ChemBL_V1
The TEER vales in the range of 500–1100 ohms are considered acceptable for fully differentiated cultures .
[]
ChemBL_V1
However, the data presented (Table 5) show that Caco-2 monolayers with a TERR of at least 300 ohms are commonly used for permeability studies.
[ { "end": 54, "label": "CellLine", "start": 48, "text": "Caco-2" } ]
ChemBL_V1
According to the biowaiver guidelines, the integrity of the Caco-2 monolayer should be confirmed every time before and after permeability tests .
[ { "end": 66, "label": "CellLine", "start": 60, "text": "Caco-2" } ]
ChemBL_V1
The permeability values of the tested compounds should be reported only if the TEER after the transport experiment is at least 75% of the initial value (before the experiment) .
[]
ChemBL_V1
Additionally, selecting monolayers with similar TEER values (measured before the permeability test started) is useful in preventing significant variations in the determination of transport rates due to unintended overgrowth .
[]
ChemBL_V1
Moreover, the literature’s data indicate the possibility of increasing the performance of the Caco-2 in vitro model by using the same monolayer for further permeation tests.
[ { "end": 100, "label": "CellLine", "start": 94, "text": "Caco-2" } ]
ChemBL_V1
To restore the integrity of the Caco-2 monolayer after permeability tests, two days of incubation in a culture medium are necessary.
[ { "end": 38, "label": "CellLine", "start": 32, "text": "Caco-2" } ]
ChemBL_V1
The presented protocol allows for two additional permeability determinations to be performed using the same Caco-2 monolayer .
[ { "end": 114, "label": "CellLine", "start": 108, "text": "Caco-2" } ]
ChemBL_V1
The permeability of compounds across the Caco-2 monolayer is carried out in two transport directions: apical-to-basolateral (A–B) and basolateral-to-apical (B–A).
[]
ChemBL_V1
The compounds are dissolved in the transport buffer and applied to the appropriate compartment in accordance with the tested transport direction .
[]
ChemBL_V1
Permeability studies are commonly performed in HBSS and HEPES , and MES or PBS are used less frequently.
[]
ChemBL_V1
The transport experimental conditions should mimic the actual physiological environment that drug molecules may encounter in the gastrointestinal tract.
[]
ChemBL_V1
Therefore, transport buffers with a pH of 6.5 (duodenum and jejunum) and 7.4. (
[]
ChemBL_V1
ileum and colon) are commonly used for permeability tests .
[]
ChemBL_V1
However, Lee and al. showed that a transport buffer with a pH of 7.4 is a good qualitative predictor for physiological intestinal permeability from the duodenum to the colon .
[]
ChemBL_V1
Transport experimental conditions should be established on the basis of individual preliminary experiments for each tested drug.
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ChemBL_V1
However, the transport time should end before the drug concentration in the receiver compartment has reached the equilibrium concentration of the system or before the total amount of drug has been removed by the sampling.
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ChemBL_V1
The time of transport testing should be adjusted to the physicochemical properties of substances, including their lipophilicity and the number of hydrogen bond atoms.
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ChemBL_V1
Commonly, the sampling time ranges from 5 min (more lipophilic substance) to 1–2 h (more hydrophilic substance).
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ChemBL_V1
Sampling intervals should be selected such that the transport experiment ends before the concentration in the receiver exceeds 10% of the donor concentration per time interval .
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ChemBL_V1
Permeability tests are generally performed in at least three repetitions .
[]
ChemBL_V1
Based on the standardization of the Caco-2 cell line, a positive control (a drug from the high-permeability group) and a negative control (drugs with low and moderate permeability) should be selected.
[]
ChemBL_V1
Then, in order to demonstrate the suitability of the Caco-2 model, permeability studies of the tested compound should be performed in the presence of selected control drugs.
[]
ChemBL_V1
For this purpose, it is recommended to use multi-well plates or to perform subsequent tests on the same Caco-2 monolayer.
[ { "end": 110, "label": "CellLine", "start": 104, "text": "Caco-2" } ]
ChemBL_V1
The positive and negative controls selected for permeability testing should not exhibit any significant physical, chemical, or permeation interactions with the tested drug .
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ChemBL_V1
Moreover, for regulatory validation purposes (e.g., BCS classification), an additional high-permeability internal standard (HP-IS) and low-permeability internal standard (LP-IS) should be established.
[]
ChemBL_V1
The HP-IS is the substance from the high-permeability group which in the validation tests has the lowest Papp value (from among at least five tested compounds in this permeability group).
[]
ChemBL_V1
Similarly, the LP-IS is the substance from the low-permeability group that has the highest Papp value in the validation tests.
[]
ChemBL_V1
These additional standards define the lower limit of high permeability (HP-IS) and the upper limit of low permeability (LP-IS), from which the permeability of the tested compound is assessed.
[]
ChemBL_V1
Thus, the test drug is classified as a highly permeable substance when its Papp value is equal to or greater than Papp of the selected HP-IS .
[]
ChemBL_V1
The selected transport conditions across the Caco-2 monolayer from scientific and regulatory studies are shown in Table 6.
[ { "end": 51, "label": "CellLine", "start": 45, "text": "Caco-2" } ]
ChemBL_V1
The in vitro transport studies based on the protocols described by Tavelin and Hubatsch are widely used in permeability research.
[]
ChemBL_V1
However, the transport protocols may require modification due to the properties of the tested compounds and the heterogeneity of the Caco-2 cell line .
[]
ChemBL_V1
As previously mentioned, the use of the Caco-2 cell line in the pharmaceutical industry requires the development of an experimental protocol that will ensure the efficiency, consistency, and reliability of the analytical methods used.
[ { "end": 46, "label": "CellLine", "start": 40, "text": "Caco-2" } ]
ChemBL_V1
Therefore, the conditions for permeability tests should be determined based on preliminary transport experiments.
[]
ChemBL_V1
However, the presented data (Table 6) indicate that the transport time across the Caco-2 monolayer does not exceed three hours.
[ { "end": 88, "label": "CellLine", "start": 82, "text": "Caco-2" } ]
ChemBL_V1
Therefore, for preliminary tests, the transport time can be set at two hours, with samples taken at intervals of 15 to 30 min.
[]
ChemBL_V1
Furthermore, to establish transport conditions through the Caco-2 monolayer, preliminary experiments can be performed only in one direction (B–A) in a transport buffer at a pH of 7.4 .
[ { "end": 65, "label": "CellLine", "start": 59, "text": "Caco-2" } ]
ChemBL_V1
Due to the significant increase in permeability studies using the Caco-2 cell line in recent years, the need to standardize this biological model seems justified.
[ { "end": 72, "label": "CellLine", "start": 66, "text": "Caco-2" } ]
ChemBL_V1
In summary, in this review, we presented the main aspects to consider for the standardization and validation of the Caco-2 cell line in relation to the pharmaceutical guidelines.
[ { "end": 122, "label": "CellLine", "start": 116, "text": "Caco-2" } ]
ChemBL_V1
The combination of the culture conditions, he number of passages, cell differentiation, and monolayer transport conditions has a decisive influence on the development of the monolayer for further suitable permeability studies.
[]
ChemBL_V1
Therefore, the development of intra-laboratory protocols for the cultivation and differentiation of Caco-2 cells is necessary to provide consistently cultivated functional cell monolayers and reduce in-house variability.
[ { "end": 106, "label": "CellLine", "start": 100, "text": "Caco-2" } ]
ChemBL_V1
The main conclusions concerning Caco-2 cell line cultivation and permeability test conditions are described in Table 7.
[ { "end": 38, "label": "CellLine", "start": 32, "text": "Caco-2" } ]
ChemBL_V1
The aspects described in Table 7 may help develop protocols for preparing the cell line for validation purposes (regulatory studies), as well as for permeability studies across biological membranes (scientific studies).
[]
ChemBL_V1
Although the Caco-2 model, like any biological model, has several limitations, mainly concerning cell line heterogeneity and external variability, its appropriate standardization may provide the most controlled conditions for establishing compound permeability.
[ { "end": 19, "label": "CellLine", "start": 13, "text": "Caco-2" } ]
ChemBL_V1
Human embryonic stem cells (hESCs) and neural progenitor (NP) cells are excellent models for recapitulating early neuronal development in vitro, and are key to establishing strategies for the treatment of degenerative disorders.
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CellFinder
While much effort had been undertaken to analyze transcriptional and epigenetic differences during the transition of hESC to NP, very little work has been performed to understand post-transcriptional changes during neuronal differentiation.
[]
CellFinder
Alternative RNA splicing (AS), a major form of post-transcriptional gene regulation, is important in mammalian development and neuronal function.
[ { "end": 135, "label": "Tissue", "start": 127, "text": null }, { "end": 110, "label": "Tissue", "start": 101, "text": null } ]
CellFinder
Human ESC, hESC-derived NP, and human central nervous system stem cells were compared using Affymetrix exon arrays.
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CellFinder
We introduced an outlier detection approach, REAP (Regression-based Exon Array Protocol), to identify 1,737 internal exons that are predicted to undergo AS in NP compared to hESC.
[]
CellFinder
Experimental validation of REAP-predicted AS events indicated a threshold-dependent sensitivity ranging from 56% to 69%, at a specificity of 77% to 96%.
[]
CellFinder
REAP predictions significantly overlapped sets of alternative events identified using expressed sequence tags and evolutionarily conserved AS events.
[]
CellFinder
Our results also reveal that focusing on differentially expressed genes between hESC and NP will overlook 14% of potential AS genes.
[]
CellFinder
In addition, we found that REAP predictions are enriched in genes encoding serine/threonine kinase and helicase activities.
[]
CellFinder
An example is a REAP-predicted alternative exon in the SLK (serine/threonine kinase 2) gene that is differentially included in hESC, but skipped in NP as well as in other differentiated tissues.
[]
CellFinder
Lastly, comparative sequence analysis revealed conserved intronic cis-regulatory elements such as the FOX1/2 binding site GCAUG as being proximal to candidate AS exons, suggesting that FOX1/2 may participate in the regulation of AS in NP and hESC.
[]
CellFinder
In summary, a new methodology for exon array analysis was introduced, leading to new insights into the complexity of AS in human embryonic stem cells and their transition to neural stem cells.
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CellFinder
Author SummaryDeriving neural progenitors (NP) from human embryonic stem cells (hESC) is the first step in creating homogeneous populations of cells that will differentiate into myriad neuronal subtypes necessary to form a human brain.
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CellFinder
During alternative RNA splicing (AS), noncoding sequences (introns) in a pre-mRNA are differentially removed in different cell types and tissues, and the remaining sequences (exons) are joined to form multiple forms of mature RNA, playing an important role in cellular diversity.
[]
CellFinder
The authors utilized Affymetrix exon arrays with probes targeting hundreds of thousands of exons to study AS comparing human ES to NP.
[ { "end": 127, "label": "CellType", "start": 119, "text": null }, { "end": 127, "label": "CellType", "start": 125, "text": null } ]
CellFinder
To accomplish this, a novel computational method, REAP (Regression-based Exon Array Protocol), is introduced to analyze the exon array data.
[]
CellFinder
The authors showed that REAP candidates are consistent with other types of methods for discovering alternative exons.
[]
CellFinder
In addition, REAP candidate alternative exons are enriched in genes encoding serine/theronine kinases and helicase activities.
[]
CellFinder
An example is the alternative exon in the SLK (serine/threonine kinase 2) gene that is included in hESC, but excluded in NP as well as in other differentiated tissues.
[]
CellFinder
Finally, by comparing genomic sequences across multiple mammals, the authors identified dozens of conserved candidate binding sites that were enriched proximal to REAP candidate exons.
[ { "end": 63, "label": "Tissue", "start": 56, "text": null } ]
CellFinder
The human central nervous system is composed of thousands of neuronal subtypes originating from neural stem cells (NSCs) that migrate from the developing neural tube.
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CellFinder
Such neuronal complexity is generated by a vast repertoire of molecular, genetic, and epigenetic mechanisms, such as the active retrotransposition of transposable elements [1], alternative promoter usage, alternative RNA splicing (AS), alternative polyadenylation, RNA editing, post-translational modifications, and epigenetic modulation [2].
[ { "end": 13, "label": "Tissue", "start": 5, "text": null } ]
CellFinder
Understanding the processes that generate neuronal diversity is key to gaining insights into neuronal development and paving new avenues for biomedical research.
[ { "end": 101, "label": "Tissue", "start": 93, "text": null }, { "end": 50, "label": "Tissue", "start": 42, "text": null } ]
CellFinder
Human embryonic stem cells (hESCs) are pluripotent cells that propagate perpetually in culture as undifferentiated cells and can be induced to differentiate into a multitude of cell types both in vitro and in vivo [3].
[ { "end": 26, "label": "CellType", "start": 0, "text": null }, { "end": 55, "label": "CellType", "start": 39, "text": null }, { "end": 15, "label": "Tissue", "start": 6, "text": null }, { "end": 33, "label": "CellType", "start": 28, "text": null } ]
CellFinder
As hESCs can theoretically generate all cell types that make up an organism, they serve as an important model for understanding early human embryonic development.
[ { "end": 149, "label": "Tissue", "start": 140, "text": null }, { "end": 8, "label": "CellType", "start": 3, "text": null } ]
CellFinder
In addition, the hESCs are a nearly infinite source for generating specialized cells such as neurons and glia for potential therapeutic purposes [4,5].
[ { "end": 100, "label": "CellType", "start": 93, "text": null }, { "end": 22, "label": "CellType", "start": 17, "text": null }, { "end": 109, "label": "CellType", "start": 105, "text": null } ]
CellFinder
In recent years, methods have been introduced to induce hESCs to differentiate into neural progenitors (NPs) [6,7] and neuronal and glial subtypes [8–12].
[ { "end": 146, "label": "CellType", "start": 132, "text": null }, { "end": 136, "label": "CellType", "start": 132, "text": null }, { "end": 61, "label": "CellType", "start": 56, "text": null }, { "end": 102, "label": "CellType", "start": 84, "text": null }, { "end": 107, "label": "CellType", "start": 104, "text": null }, { "end": 127, "label": "Tissue", "start": 119, "text": null } ]
CellFinder
The therapeutic interest in understanding the molecular basis of pluripotency and differentiation has led to many studies comparing transcriptional profiles in different hESC lines and the study of expression changes during the differentiation of hESCs to various lineages [13–17].
[ { "end": 252, "label": "CellType", "start": 247, "text": null } ]
CellFinder
NSCs and progenitor cells (NPs) are present throughout development and persist into adulthood [18–20].
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CellFinder
They are critical for both basic research and developing approaches to treat neurological disorders, such as Parkinson disease and amyotrophic lateral sclerosis (ALS), and stroke or head injuries [21,22].
[ { "end": 186, "label": "Tissue", "start": 182, "text": null } ]
CellFinder
NSCs and NPCs can be isolated from human fetal brain tissue [23–26], as well as from several regions of the adult human brain, such as the cortex, hippocampus, and the subventricular zone (SVZ) of the lateral ventricles [26–35].
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CellFinder
Several studies have explored expression patterns of NPCs.
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CellFinder
For example, Wright et al. identified “expressed” and “not expressed” genes in NPCs isolated from the human embryonic cortex [24]; Cai et al. used the massively parallel signature sequencing profiling (MPSS) technique to analyze expression of fetal NPCs in comparison to hESCs and astrocyte precursors [27]; Maisel et al. used Affymetrix Gene Chip arrays to compare adult and fetal NPCs propagated in neurospheres [35].
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CellFinder
However, as with hESCs, the focus thus far has been primarily on transcriptional differences, which ignores differential RNA processing such as AS, polyadenylation, degradation, or promoter usage.
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CellFinder
AS is frequently used to regulate gene expression and to generate tissue-specific mRNA and protein isoforms [36–39].
[]
CellFinder
Recent studies using splicing-sensitive microarrays suggested that up to 75% of human genes undergo AS, where multiple isoforms are derived from the same genetic loci [40].
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CellFinder
This functional complexity underscores the challenge and importance of elucidating AS regulation.
[]
CellFinder
AS appears to play a dominant role in regulating neuronal gene expression and function [41,42].
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CellFinder
Examples of splicing regulators that are enriched and function specifically in neuronal cells include the brain-specific splicing factor Nova [43,44] and neural-specific polypyrimidine tract binding protein (nPTB), which antagonizes its paralogous PTB to regulate exon exclusion in neuronal cells [45–47].
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CellFinder
Finally, an early report estimating that 15% of point mutations disrupt splicing underscores the importance of splicing in human disease [48].
[]
CellFinder
Indeed, the disruption of specific AS events has been implicated in several human genetic diseases, such as frontotemporal dementia and parkinsonism, Frasier syndrome, and atypical cystic fibrosis [49].
[]
CellFinder
While insights into the regulation of AS have come predominantly from the molecular dissection of individual genes [36,49], it is becoming clear that molecular rules can be identified from large-scale studies of both constitutive splicing and AS [40].
[]
CellFinder
Most systematic global analyses on AS have focused on comparisons across differentiated human tissues [50–52].
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CellFinder
Only one study, utilizing expressed sequence tag (EST) collections from stem cells, has attempted to find AS differences between embryonic and hematopoietic stem cells [53].
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CellFinder
However, utilizing ESTs to identify AS has intrinsic problems, as ESTs tend to be biased for the 3′ ends of genes, and full coverage of the genome by ESTs is severely limited by sequencing costs.
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CellFinder
The commercial availability of Affymetrix exon arrays provides an alternative approach to interrogate the expression of every known and predicted exon in the human genome.
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CellFinder
The Affymetrix GeneChip Human Exon 1.0 ST array contains ∼5.4 million features used to interrogate ∼1 million exon clusters (collections of overlapping) of known and predicted exons with more than 1.4 million probesets, with an average of four probes per exon.
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CellFinder
Our goal was to identify and characterize AS events that distinguish pluripotent hESCs from multipotent NPs, paving the way for future candidate gene approaches to study the impact of AS in hESCs and NPs.
[ { "end": 203, "label": "CellType", "start": 200, "text": null }, { "end": 195, "label": "CellType", "start": 190, "text": null } ]
CellFinder
However, as different hESC lines were established under different culture conditions from embryos with unique genetic backgrounds, we expected that hESCs and their derived NPs might have distinct epigenetic and molecular signatures [54].
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CellFinder
As both common and cell-line specific alternatively spliced exons are likely to be important in regenerative research, in our study two separate hESC lines were used, with independent protocols for differentiating the hESCs into NPs positive for Sox1, an early neuroectodermal marker.
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CellFinder