scholarly journals An Optimized Protocol for Histochemical Detection of Senescence-associated Beta-galactosidase Activity in Cryopreserved Liver Tissue

2020 ◽  
Vol 68 (4) ◽  
pp. 269-278
Author(s):  
Giulia Jannone ◽  
Milena Rozzi ◽  
Mustapha Najimi ◽  
Anabelle Decottignies ◽  
Etienne M. Sokal
Keyword(s):  
Liver Tissue ◽  
Hepatic Tissue ◽  
Histochemical Reaction ◽  
Bile Duct Resection ◽  
Fresh Tissue ◽  
Histochemical Detection ◽  
Optimized Protocol ◽  
Beta Galactosidase ◽  
Tissue Material

Senescence-associated beta-galactosidase (SA-β-gal) activity assay is commonly used to evaluate the increased beta-galactosidase (β-gal) activity in senescent cells related to enhanced lysosomal activity. Although the optimal pH for β-gal is 4.0, this enzymatic activity has been most commonly investigated at a suboptimal pH by using histochemical reaction on fresh tissue material. In the current study, we optimized a SA-β-gal activity histochemistry protocol that can also be applied on cryopreserved hepatic tissue. This protocol was developed on livers obtained from control rats and after bile duct resection (BDR). A significant increase in β-gal liver activity was observed in BDR rats vs controls after 2 hr of staining at physiological pH 4.0 (6.98 ± 1.19% of stained/total area vs 0.38 ± 0.22; p<0.01) and after overnight staining at pH 5.8 (24.09 ± 6.88 vs 0.12 ± 0.08; p<0.01). Although we noticed that β-gal activity staining decreased with cryopreservation time (from 4 to 12 months of storage at −80C; p<0.05), the enhanced staining observed in BDR compared with controls remained detectable up to 12 months after cryopreservation ( p<0.01). In conclusion, we provide an optimized protocol for SA-β-gal activity histochemical detection at physiological pH 4.0 on long-term cryopreserved liver tissue:

scholarly journals 3′ MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage

2020 ◽  
Vol 100 (10) ◽  
pp. 1345-1355 ◽  
Author(s):  
Stefaniya Boneva ◽  
Anja Schlecht ◽  
Daniel Böhringer ◽  
Hans Mittelviefhaus ◽  
Thomas Reinhard ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Storage Time ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Fresh Tissue ◽  
Long Term Storage ◽  
Ffpe Samples ◽  
The Impact ◽  
Term Storage

Abstract This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

scholarly journals Poly(ε-Caprolactone) Hollow Fiber Membranes for the Biofabrication of a Vascularized Human Liver Tissue

Membranes ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 112
Author(s):  
Simona Salerno ◽  
Franco Tasselli ◽  
Enrico Drioli ◽  
Loredana De Bartolo
Keyword(s):  
Tissue Engineering ◽  
Endothelial Cells ◽  
Hollow Fiber ◽  
Liver Tissue ◽  
External Surface ◽  
Internal Surface ◽  
Hepatic Tissue ◽  
Operational Parameters ◽  
Human Organ ◽  
Human Liver Tissue

The creation of a liver tissue that recapitulates the micro-architecture and functional complexity of a human organ is still one of the main challenges of liver tissue engineering. Here we report on the development of a 3D vascularized hepatic tissue based on biodegradable hollow fiber (HF) membranes of poly(ε-caprolactone) (PCL) that compartmentalize human hepatocytes on the external surface and between the fibers, and endothelial cells into the fiber lumen. To this purpose, PCL HF membranes were prepared by a dry-jet wet phase inversion spinning technique tailoring the operational parameters in order to obtain fibers with suitable properties. After characterization, the fibers were applied to generate a human vascularized hepatic unit by loading endothelial cells in their inner surface and hepatocytes on the external surface. The unit was connected to a perfusion system, and the morpho-functional behavior was evaluated. The results demonstrated the large integration of endothelial cells with the internal surface of individual PCL fibers forming vascular-like structures, and hepatocytes covered completely the external surface and the space between fibers. The perfused 3D hepatic unit retained its functional activity at high levels up to 18 days. This bottom-up tissue engineering approach represents a rational strategy to create relatively 3D vascularized tissues and organs.

Effect of spaceflight on rat hepatocytes: a morphometric study

1992 ◽  
Vol 73 (2) ◽  
pp. S136-S141 ◽  
Author(s):  
R. N. Racine ◽  
S. M. Cormier
Keyword(s):  
Image Analysis ◽  
Light Microscopy ◽  
Cell Population ◽  
Kupffer Cells ◽  
Rat Hepatocytes ◽  
Morphometric Study ◽  
Hepatic Tissue ◽  
Computer Assisted ◽  
Computer Assisted Image

Hepatic tissue from flight, synchronous, vivarium, and tail-suspended rats was examined by light microscopy and computer-assisted image analysis. Glycogen levels in flight rats were found to be significantly elevated over those in controls. Lipid was also higher but not significantly different. Hepatocytes appeared larger in flight animals because of area attributed to increased glycogen. Sinusoids were less prominent in flight animals than in controls. The total Kupffer cell population appeared to be reduced in flight animals and may represent changes in defensive capacity of the liver. Alterations in the storage of glycogen and number of Kupffer cells suggest an important effect of spaceflight on the function of the liver that may have important implications for long-term spaceflight.

scholarly journals Generation of functional liver tissue by establishing hepatobiliary connections ex vivo

2020 ◽  
Author(s):  
Naoki Tanimizu ◽  
Norihisa Ichinohe ◽  
Yasushi Sasaki ◽  
Tohru Itoh ◽  
Ryo Sudo ◽  
...  
Keyword(s):  
Liver Tissue ◽  
Ex Vivo ◽  
Drainage System ◽  
Bile Canaliculi ◽  
Mouse Hepatocyte ◽  
Functional Connections ◽  
Intrahepatic Bile Ducts ◽  
Metabolic Functions ◽  
Bile Drainage

Abstract In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. We have developed a hepatobiliary organoid using mouse hepatocyte progenitors and cholangiocytes. Hepatocyte metabolites were secreted into the bile canaliculi, and then transported into the biliary structure. Hepatocytes in the organoid acquired and maintained metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we established functional liver tissue incorporating a bile drainage system ex vivo. This hepatobiliary organoid enabled us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

Short-term Resistance Training Increases APPL1 Content in the Liver and the Insulin Sensitivity of Mice Fed a Long-term High-fat Diet

2019 ◽  
Vol 128 (01) ◽  
pp. 30-37
Author(s):  
Luciele Guerra Minuzzi ◽  
Gabriel Keine Kuga ◽  
Leonardo Breda ◽  
Rafael Calais Gaspar ◽  
Vitor Rosetto Muñoz ◽  
...  
Keyword(s):  
Insulin Sensitivity ◽  
Resistance Training ◽  
High Fat Diet ◽  
Calorie Intake ◽  
Hepatic Tissue ◽  
Chow Diet ◽  
Adapter Protein ◽  
High Fat ◽  
Short Term

Abstract Background APPL1, an adapter protein, interact directly with adiponectin receptors mediating adiponectin signaling and acting as a critical regulator of the crosstalk between adiponectin and insulin signaling pathway. The inadequate level of physical activity, high-calorie intake, or both lead to adverse consequences on health, like insulin resistance. On the order hand, physical exercise acts positively in the insulin action. Purpose Here, we investigated the effects of short-term resistance training (RT) on APPL1 content and adiponectin pathway in the liver of mice fed a long-term high-fat diet. Methods Swiss mice were distributed into 3 groups: Mice that fed a chow diet (CTR); Mice fed a high-fat diet for 16 months (HFD); and mice fed a high-fat diet for 16 months and submitted to a climbing ladder exercise (RT) for 7 days (HFD-EXE). Results The results show that short-term RT increases the APPL1 content but wasn’t able to alter AdipoR1 and AdipoR2 content in the liver of HFD-EXE mice. However, this increase in the APPL1 content in response to RT was accompanied by improvement in the insulin sensitivity. Conclusion In summary, our data suggested that short-term RT improves glycemic homeostasis and increases APPL1 in the hepatic tissue of mice treated with long-term high-fat diet.

scholarly journals Reversible Growth-Arrest of a Spontaneously-Derived Human MSC-Like Cell Line

2020 ◽  
Vol 21 (13) ◽  
pp. 4752 ◽  
Author(s):  
Catharina Melzer ◽  
Roland Jacobs ◽  
Thomas Dittmar ◽  
Andreas Pich ◽  
Juliane von der Ohe ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Layer ◽  
Growth Arrest ◽  
Surface Proteins ◽  
Therapeutic Approaches ◽  
Cell Source ◽  
One Year ◽  
Beta Galactosidase

Life cycle limitation hampers the production of high amounts of primary human mesenchymal stroma-/stem-like cells (MSC) and limits cell source reproducibility for clinical applications. The characterization of permanently growing MSC544 revealed some differentiation capacity and the simultaneous presence of known MSC markers CD73, CD90, and CD105 even after continuous long-term culture for more than one year and 32 passages. The expression of CD13, CD29, CD44, and CD166 were identified as further surface proteins, all of which were also simultaneously detectable in various other types of primary MSC populations derived from the umbilical cord, bone marrow, and placenta suggesting MSC-like properties in the cell line. Proliferating steady state MSC544 exhibited immune-modulatory activity similar to a subpopulation of long-term growth-inhibited MSC544 after 189d of continuous culture in confluency. This confluent connective cell layer with fibroblast-like morphology can spontaneously contract and the generated space is subsequently occupied by new cells with regained proliferative capacity. Accordingly, the confluent and senescence-associated beta-galactosidase-positive MSC544 culture with about 95% G0/G1 growth-arrest resumed re-entry into the proliferative cell cycle within 3d after sub-confluent culture. The MSC544 cells remained viable during confluency and throughout this transition which was accompanied by marked changes in the release of proteins. Thus, expression of proliferation-associated genes was down-modulated in confluent MSC544 and re-expressed following sub-confluent conditions whilst telomerase (hTERT) transcripts remained detectable at similar levels in both, confluent growth-arrested and proliferating MSC544. Together with the capability of connective cell layer formation for potential therapeutic approaches, MSC544 provide a long term reproducible human cell source with constant properties.

Sign in / Sign up

Export Citation Format

Share Document