scholarly journals Digitonin-collagenase perfusion for efficient separation of periportal or perivenous hepatocytes

1985 ◽  
Vol 228 (3) ◽  
pp. 757-760 ◽  
Author(s):  
K O Lindros ◽  
K E Penttilä
Keyword(s):  
Lactate Dehydrogenase ◽  
Alanine Aminotransferase ◽  
High Yield ◽  
Cell Populations ◽  
Gamma Glutamyltransferase ◽  
Efficient Separation ◽  
Rat Liver Cells ◽  
Collagenase Perfusion ◽  
Portal Infusion

Intact rat liver cells from the perivenous region were isolated by collagenase perfusion after first destroying the periportal region by a brief portal infusion of digitonin. Periportal cells were isolated after retrograde digitonin infusion. Significantly higher alanine aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase activities and lower glutamate dehydrogenase and pyruvate kinase activities in periportal than in perivenous cells demonstrate marked separation. The high yield allows further characterization in vitro of the cell populations.

Stabilities of some constituents of marmoset (Callithrix jacchus) plasma under various conditions of storage.

1984 ◽  
Vol 30 (1) ◽  
pp. 101-104 ◽  
Author(s):  
C W Davy ◽  
M R Jackson ◽  
J M Walker
Keyword(s):  
Alkaline Phosphatase ◽  
Lactate Dehydrogenase ◽  
Alanine Aminotransferase ◽  
Room Temperature ◽  
Sequential Analysis ◽  
Callithrix Jacchus ◽  
Optimal Conditions ◽  
Gamma Glutamyltransferase ◽  
Aspartate Amino Transferase ◽  
The Stability

Abstract The stability of various marmoset (Callithrix jacchus) plasma constituents was investigated after storage at room temperature, 4 degrees C, and -20 degrees C. The method of sequential analysis ensured that the between-run bias of the methods of analysis used was drastically reduced, and the definitions of stability were linked to the imprecision of these methods. Optimal conditions for storage for as long as 48 h depended on the analyte being measured. Room temperature was optimal for cholinesterase and acetylcholinesterase; 4 degrees C for protein, albumin, alanine aminotransferase, isocitrate dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, and glutamate dehydrogenase; and -20 degrees C for glutathione reductase and alkaline phosphatase. For aspartate amino-transferase and gamma-glutamyltransferase, either 4 degrees C or -20 degrees C would be suitable. Reasons are advanced for some conflicting reports in the published work, and we emphasize the need to investigate each analyte and species separately.

scholarly journals Gluconeogenesis in periportal and perivenous hepatocytes of rat liver, isolated by a new high-yield digitonin/collagenase perfusion technique

1985 ◽  
Vol 229 (1) ◽  
pp. 221-226 ◽  
Author(s):  
B Quistorff
Keyword(s):  
Glucose Metabolism ◽  
Pyruvate Kinase ◽  
Rat Liver ◽  
Alanine Aminotransferase ◽  
Control Cell ◽  
High Yield ◽  
Perfusion Technique ◽  
Data Support ◽  
Collagenase Perfusion ◽  
Periportal Zone

A technique is described which allows preparations of hepatocytes, enriched in either periportal or perivenous hepatocytes (‘PP-cells’ and ‘PV-cells’ respectively), in a yield of about 30-50% compared with control cell preparations. The liver is first perfused for 40-60s with digitonin (4 mg/ml) to destroy selectively either the periportal or the perivenous part of the microcirculatory unit, and then the remaining hepatocytes are isolated by the ordinary collagenase perfusion technique. In periportal cells the activities of alanine aminotransferase and pyruvate kinase were 29.4 and 18.7 mumol/min per mg of DNA respectively. The rate of gluconeogenesis was 0.402 mumol/min per mg of DNA. In perivenous cells the corresponding values were 9.55, 22.1 and 0.244 mumol/min per mg of DNA respectively. These data support the concept of a zonation of glucose metabolism within the microcirculatory unit of the liver, with the afferent part (periportal zone) having a 2-fold, more active gluconeogenesis than the efferent part (perivenous zone).

Separation of two populations of fish hepatocytes by digitonin infusion: some metabolic patterns and hormonal responsiveness

1991 ◽  
Vol 69 (2) ◽  
pp. 427-435 ◽  
Author(s):  
C. Ottolenghi ◽  
D. Ricci ◽  
M. E. Gavioli ◽  
A. C. Puviani ◽  
E. Fabbri ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Portal Vein ◽  
Alanine Aminotransferase ◽  
Glycogen Phosphorylase ◽  
Liver Cells ◽  
Glycogen Depletion ◽  
Camp Content ◽  
Collagenase Perfusion ◽  
Ictalurus Melas ◽  
Two Populations

Two pools of liver cells, enriched either in periportal or in perivenous hepatocytes, were obtained from the catfish Ictalurus melas by the selective destruction of the liver by digitonin infusion. Following digitonin pulses directed either orthograde via the portal vein or retrograde via the hepatic vein, hepatocytes from undamaged perivenous and periportal zones, respectively, were isolated by the conventional collagenase perfusion procedure. Eluates from either periportal or perivenous zones were collected for a total of 9 min before, during, and immediately following the digitonin pulse. The eluates were assayed for protein, glycogen, glucose, and activities of lactate dehydrogenase and alanine aminotransferase, and their yield ("recovery") was estimated as a percentage of the total hepatic content. The aforementioned metabolic compounds and enzyme activities were measured also in periportal and perivenous liver cells. No significant differences were obtained between these cells. Both pools of hepatocytes were more than 90% viable and remained hormone responsive. Epinephrine (3 × 10−8 M) or salmon glucagon (3 × 10−8 M) elevated cAMP content, and stimulated glycogen phosphorylase a activity, glycogen depletion, and glucose release in periportal and perivenous liver cells. No differences were observed between periportal and perivenous cells in their metabolic responsiveness to either hormone.

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

2019 ◽  
Vol 14 (4) ◽  
pp. 305-319 ◽  
Author(s):  
Marietta Herrmann ◽  
Franz Jakob
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Adult Stem Cells ◽  
Current Knowledge ◽  
Cell Populations ◽  
Surface Markers ◽  
Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

Design, Synthesis, In vitro Cytotoxic Activity Evaluation, and Study of Apoptosis Inducing Effect of New Styrylimidazo[1,2-a]Pyridines as Potent Anti-Breast Cancer Agents

2019 ◽  
Vol 19 (2) ◽  
pp. 265-275 ◽  
Author(s):  
Faeze Khalili ◽  
Sara Akrami ◽  
Malihe Safavi ◽  
Maryam Mohammadi-Khanaposhtani ◽  
Mina Saeedi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cytotoxic Activity ◽  
High Yield ◽  
Breast Cancer Cell Lines ◽  
Pyridine Derivatives ◽  
One Pot ◽  
Standard Drug ◽  
Three Component Reaction ◽  
Anti Cancer

Background: This paper reports synthesis, cytotoxic activity, and apoptosis inducing effect of a novel series of styrylimidazo[1,2-a]pyridine derivatives. Objective: In this study, anti-cancer activity of novel styrylimidazo[1,2-a]pyridines was evaluated. Methods: Styrylimidazo[1,2-a]pyridine derivatives 4a-o were synthesized through a one-pot three-component reaction of 2-aminopyridines, cinnamaldehydes, and isocyanides in high yield. All synthesized compounds 4a-o were evaluated against breast cancer cell lines including MDA-MB-231, MCF-7, and T-47D using MTT assay. Apoptosis was evaluated by acridine orange/ethidium bromide staining, cell cycle analysis, and TUNEL assay as the mechanism of cell death. Results: Most of the synthesized compounds exhibited more potent cytotoxicity than standard drug, etoposide. Induction of apoptosis by the most cytotoxic compounds 4f, 4g, 4j, 4n, and 4m was confirmed through mentioned methods. Conclusion: In conclusion, these results confirmed the potency of styrylimidazo[1,2-a]pyridines for further drug discovery developments in the field of anti-cancer agents.

scholarly journals Optimized Supercritical CO2 Extraction Enhances the Recovery of Valuable Lipophilic Antioxidants and Other Constituents from Dual-Purpose Hop (Humulus lupulus L.) Variety Ella

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 918
Author(s):  
Nóra Emilia Nagybákay ◽  
Michail Syrpas ◽  
Vaiva Vilimaitė ◽  
Laura Tamkutė ◽  
Audrius Pukalskas ◽  
...  
Keyword(s):  
Supercritical Co2 ◽  
Antioxidant Properties ◽  
Extraction Yield ◽  
Biologically Active ◽  
High Yield ◽  
Acid Extraction ◽  
Supercritical Co2 Extraction ◽  
Bioactive Constituents ◽  
Total Extract

The article presents the optimization of supercritical CO2 extraction (SFE-CO2) parameters using response surface methodology (RSM) with central composite design (CCD) in order to produce single variety hop (cv. Ella) extracts with high yield and strong in vitro antioxidant properties. Optimized SFE-CO2 (37 MPa, 43 °C, 80 min) yielded 26.3 g/100 g pellets of lipophilic fraction. This extract was rich in biologically active α- and β-bitter acids (522.8 and 345.0 mg/g extract, respectively), and exerted 1481 mg TE/g extract in vitro oxygen radical absorbance capacity (ORAC). Up to ~3-fold higher extraction yield, antioxidant recovery (389.8 mg TE/g pellets) and exhaustive bitter acid extraction (228.4 mg/g pellets) were achieved under the significantly shorter time compared to the commercially used one-stage SFE-CO2 at 10–15 MPa and 40 °C. Total carotenoid and chlorophyll content was negligible, amounting to <0.04% of the total extract mass. Fruity, herbal, spicy and woody odor of extracts could be attributed to the major identified volatiles, namely β-pinene, β-myrcene, β-humulene, α-humulene, α-selinene and methyl-4-decenoate. Rich in valuable bioactive constituents and flavor compounds, cv. Ella hop SFE-CO2 extracts could find multipurpose applications in food, pharmaceutical, nutraceutical and cosmetics industries.

scholarly journals Generation of High-Yield, Functional Oligodendrocytes from a c-myc Immortalized Neural Cell Line, Endowed with Staminal Properties

2021 ◽  
Vol 22 (3) ◽  
pp. 1124
Author(s):  
Mafalda Giovanna Reccia ◽  
Floriana Volpicelli ◽  
Eirkiur Benedikz ◽  
Åsa Fex Svenningsen ◽  
Luca Colucci-D’Amato
Keyword(s):  
Cell Line ◽  
Low Cost ◽  
Neural Cell ◽  
New Drugs ◽  
High Yield ◽  
Time Saving ◽  
Interacting Protein ◽  
Neuronal Marker ◽  
Differentiation Status

Neural stem cells represent a powerful tool to study molecules involved in pathophysiology of Nervous System and to discover new drugs. Although they can be cultured and expanded in vitro as a primary culture, their use is hampered by their heterogeneity and by the cost and time needed for their preparation. Here we report that mes-c-myc A1 cells (A1), a neural cell line, is endowed with staminal properties. Undifferentiated/proliferating and differentiated/non-proliferating A1 cells are able to generate neurospheres (Ns) in which gene expression parallels the original differentiation status. In fact, Ns derived from undifferentiated A1 cells express higher levels of Nestin, Kruppel-like factor 4 (Klf4) and glial fibrillary protein (GFAP), markers of stemness, while those obtained from differentiated A1 cells show higher levels of the neuronal marker beta III tubulin. Interestingly, Ns differentiation, by Epidermal Growth Factors (EGF) and Fibroblast Growth Factor 2 (bFGF) withdrawal, generates oligodendrocytes at high-yield as shown by the expression of markers, Galactosylceramidase (Gal-C) Neuron-Glial antigen 2 (NG2), Receptor-Interacting Protein (RIP) and Myelin Basic Protein (MBP). Finally, upon co-culture, Ns-A1-derived oligodendrocytes cause a redistribution of contactin-associated protein (Caspr/paranodin) protein on neuronal cells, as primary oligodendrocytes cultures, suggesting that they are able to form compact myelin. Thus, Ns-A1-derived oligodendrocytes may represent a time-saving and low-cost tool to study the pathophysiology of oligodendrocytes and to test new drugs.

108 MCY-M11, a CAR-PBMC cell product transiently expressing a mesothelin targeted mRNA CAR, exhibits desirable functional and immune phenotype attributed to sustained antitumor immunity in vitro

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A120-A120
Author(s):  
Sashi Kasimsetty ◽  
Himavanth Gatla ◽  
Dhana Chinnasamy
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Antitumor Immunity ◽  
Memory T Cells ◽  
Cell Populations ◽  
Epitope Spreading ◽  
Antitumor Response ◽  
Cell Product

BackgroundMCY-M11, an anti-mesothelin CAR (Meso-CAR) mRNA transfected PBMC cell product manufactured through <1 day-process is under clinical evaluation for the treatment of advanced ovarian cancer and peritoneal mesothelioma. In this in-vitro study, we characterized the phenotypic and functional status of immune cell populations in MCY-M11 and their possible role in antitumor immunity.MethodsMCY-M11 cell product were generated using unmanipulated healthy donor PBMCs (n=5) by transfection of Meso-CAR mRNA using MaxCyte’s proprietary Flow Electroporation® system. Frozen MCY-M11 cell product was thawed and cultured for 18 hours, then co-cultured with hMSLNneg or hMSLNpos human mesothelioma cell line, MSTO-211H, or stimulated with anti-CD3/anti-CD28 antibodies in vitro for 8 days. Distinct cell populations in MCY-M11 were evaluated for kinetics and duration of CAR expression, differentiation, activation, exhaustion, and their ability to secrete various immunomodulatory molecules during in vitro stimulation. Antigen-specific proliferation and cytotoxicity of MCY-M11 against hMSLNpos tumor cells as well as their ability to mount long-term antitumor immunity through epitope spreading mechanisms were studied.ResultsIndividual cell populations in MCY-M11 exhibited a consistent but transient Meso-CAR expression persisting for about 7 days. Cell subsets in MCY-M11 acquired early signs of activation and differentiation within 18–24 hours post-culture, but only attained full activation and lineage-specific differentiation upon specific response to hMSLNpos tumor cells. hMSLN antigen experienced MCY-M11 retained significant fractions of Naïve and Central Memory T cells and increased percentage of Effector Memory T cells along with increased expression of CD62L, CD27, and chemokine receptors (CCR5, CCR7, and CXCR3). MCY-M11 exhibited strong antigen-specific cytotoxicity against hMSLNpos tumor cells with corresponding increase in activation and proliferation of CD4+ and CD8+ T cell subsets and displayed low or no acquisition of known exhaustion markers. NK cells also exhibited a functionally superior molecular signature exhibiting increased levels of NKG2D, NKp44, NKp46, FAS, and TRAIL. The Monocytes and B cells in MCY-M11 also acquired an activated, differentiated, and mature phenotype, expressing molecules required for antigen presentation (HLA-DR, HLA-ABC, and CD205) and T cell co-stimulation (CD80 and CD86) to mount a strong antitumor response. These phenotypic changes in cell subsets of MCY-M11 transpired with simultaneous secretion of potent immunostimulatory molecules and chemokines facilitating an extended antitumor response through epitope spreading.ConclusionsWe demonstrated that MCY-M11 is a unique cell product possessing a complete built-in immune cellular machinery with favorable phenotype and enhanced functions specialized in mediating an effective and long-term antitumor response.Trial RegistrationNCT03608618

Efficient trace-scale extraction method of reactor produced 199Au adequate for nuclear medicine applications

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mohamed F. Attallah ◽  
Ahmed M. Shahr El-Din ◽  
Mohamed A. Gizawy ◽  
Amal M. I. Ali
Keyword(s):  
Aqueous Media ◽  
Selective Extraction ◽  
Extraction Process ◽  
High Yield ◽  
High Recovery ◽  
Rapid Separation ◽  
Efficient Separation ◽  
Liquid Liquid Extraction ◽  
Cyanex 302 ◽  
Cyanex 923

Abstract Production of no carrier-added (NCA) 199Au through natPt(n, γ) reaction and subsequent purification using liquid-liquid extraction from other radioisotopes is studied in the context of theranostic application. Comparative separation of NCA 199Au after dissolution of activated Pt target using three Cyanex compounds (Cyanex-272, Cyanex-302 and Cyanex-923) is evaluated. The extraction process is optimized in terms of the type of extractant, the concentration of extractant, extraction time and aqueous media (HNO3, NH4OH). Among these extractants, the Cynaex-923 is efficient and promising for rapid separation and production of NCA 199Au from HNO3 by high extraction %. Selective extraction of 199Au from other Pt and Ir radioisotopes is observed. High recovery of 199Au was obtained in the case of Cyanex-923 using 0.05 M thiourea dissolved in HCl or 2 M NaOH. Our results find the Cyanex-923 as a promising extractant for efficient separation of 199Au from irradiated Pt target with high yield (99%).

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