Search tips
Search criteria

Results 1-6 (6)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
Document Types
1.  Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes 
PLoS ONE  2013;8(7):e69334.
Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.
PMCID: PMC3713052  PMID: 23874947
2.  Engineered Three-Dimensional Liver Mimics Recapitulate Critical Rat-Specific Bile Acid Pathways 
Tissue Engineering. Part A  2010;17(5-6):677-689.
A critical hepatic function is the maintenance of optimal bile acid (BA) compositions to achieve cholesterol homeostasis. BAs are rarely quantified to assess hepatic phenotype in vitro since existing analytical techniques have inadequate resolution. We report a detailed investigation into the biosynthesis and homeostasis of eight primary rat BAs in conventional in vitro hepatocyte cultures and in an engineered liver mimic. The three-dimensional (3D) liver mimic was assembled with layers of primary rat hepatocytes and liver sinusoidal endothelial cells. A high-pressure liquid chromatography and mass spectrometry technique was developed with a detection limit of 1 ng/mL for each BA, which is significantly lower than previous approaches. Over a 2-week culture, only 3D liver mimics exhibited the ratio of conjugated cholic acid to chenodeoxycholic acid that has been observed in vivo. This ratio, an important marker of BA homeostasis, was significantly higher in stable collagen sandwich cultures indicating significant deviation from physiological behavior. The biosynthesis of tauro-β-muricholic acid, a key primary rat BA, doubled only in the engineered liver mimics while decreasing in the other systems. These trends demonstrate that the 3D liver mimics provide a unique platform to study hepatic metabolism.
PMCID: PMC3043955  PMID: 20929286
3.  A Comparative Study of Genome-Wide Transcriptional Profiles of Primary Hepatocytes in Collagen Sandwich and Monolayer Cultures 
Tissue Engineering. Part C, Methods  2010;16(6):1449-1460.
Two commonly used culture systems in hepatic tissue engineering are the collagen sandwich (CS) and monolayers of cells. In this study, genome-wide gene expression profiles of primary hepatocytes were measured over an 8-day period for each cell culture system using Affymetrix GeneChips and compared via gene set enrichment analysis to elicit biologically meaningful information at the level of gene sets. Our results demonstrate that gene expression in hepatocytes in CS cultures steadily and comprehensively diverges from that in monolayer cultures. Gene sets up-regulated in CS cultures include several associated with liver metabolic and synthesis functions, such as metabolism of lipids, amino acids, carbohydrates, and alcohol, and synthesis of bile acids. Monooxygenases such as Cytochrome-P450 enzymes do not show any change between the culture systems after 1 day, but exhibit significant up-regulation in CS cultures after 3 days in comparison to hepatocyte monolayers. These data provide insights into the up- and down-regulation of several liver-critical gene sets and their subsequent effects on liver-specific functions. These results provide a baseline for further explorations into the systems biology of engineered liver mimics.
PMCID: PMC2988646  PMID: 20412007
4.  The Design of In Vitro Liver Sinusoid Mimics Using Chitosan–Hyaluronic Acid Polyelectrolyte Multilayers 
Tissue Engineering. Part A  2010;16(9):2731-2741.
Interactions between hepatocytes and liver sinusoidal endothelial cells (LSECs) are essential for the development and maintenance of hepatic phenotypic functions. We report the assembly of three-dimensional liver sinusoidal mimics comprised of primary rat hepatocytes, LSECs, and an intermediate chitosan–hyaluronic acid polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30 to 55 nm and exhibited a shear modulus of ∼100 kPa. Hepatocyte–PEM cellular constructs exhibited stable urea and albumin production over a 7-day period, and these values were either higher or similar to cells cultured in a collagen sandwich. This is of significance because the thickness of a collagen gel is ∼1000-fold higher than the height of the chitosan–hyaluronic acid PEM. In the hepatocyte–PEM–LSEC liver-mimetic cellular constructs, LSEC phenotype was maintained, and these cultures exhibited stable urea and albumin production. CYP1A1/2 activity measured over a 7-day period was significantly higher in the hepatocyte–PEM–LSEC constructs than in collagen sandwich cultures. A 16-fold increase in CYP1A1/2 activity was observed for hepatocyte–PEM–10,000 LSEC samples, thereby suggesting that interactions between hepatocytes and LSECs are critical in enhancing the detoxification capability in hepatic cultures in vitro.
PMCID: PMC2928042  PMID: 20491586
5.  IDIOPATHIC ACUTE TRANSVERSE MYELITIS IN CHILDREN: An analysis and discussion of MRI findings 
There is lack of reported MRI studies of idiopathic acute transverse myelitis in children.
To describe the imaging features of idiopathic acute transverse myelitis in children..
We retrospectively analyzed the spinal MRI findings of children diagnosed with acute transverse myelitis . The anatomic regions, vertebral segmental length, gray or white matter involvement, cord expansion and gadolinium enhancement were examined.
27 children were diagnosed with isolated monophasic acute transverse myelitis with a mean follow-up of 5.2 years. Two children later diagnosed with neuromyelitis optica were excluded from the pediatric ATM cohort. None of the patients had a subsequent diagnosis of multiple sclerosis. The mean age of onset was 9.5 years (0.5-16.9). Spinal MRIs were abnormal in 21 (78%). The mean interval between symptom onset and the MRI was 1.7 days (0-19 days). Central cord hyperintensity involving gray matter was seen in all patients. A majority (67%) of the patients demonstrated long segment lesions with a mean segment length of 6.4.
Central cord inflammation extending over three or more segments is the most common finding of idiopathic monophasic transverse myelitis in children. The risk of multiple sclerosis in children who experience isolated transverse myelitis as a first demyelinating event is low.
PMCID: PMC3022414  PMID: 20858691
Acute transverse myelitis; MRI; demyelinating disease; pediatric onset multiple sclerosis; spinal cord inflammation
6.  3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes 
PLoS ONE  2010;5(11):e15456.
Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.
PMCID: PMC2980491  PMID: 21103392

Results 1-6 (6)