XTT Test Research Articles

CRYOPRESERVED HUMAN HEPATOCYTES FROM CELL BANK: IN VITRO FUNCTION AND CLINICAL APPLICATION

P658 Aims: the development of a National Bank of Human Hepatocytes, isolated from livers unwanted for transplantation at harvesting, allows to obtain live hepatic tissue usable for in vitro and clinical evaluation of cell therapy and transplantation and bioartificial organs. The aim of this study is to analyze in vitro function of isolated and cryopreserved human hepatocytes from cell Bank and define their potential clinical application in a bioartificial liver (BAL) device. Methods: 103 not transplantable livers were utilized for human hepatocytes isolation and cryopreservation from July 2001 to December 2003 in the Nord Italia Transplant area (NITp and AIRT). Hepatocytes were isolated by collagenase-enzymatic digestion. Isolated hepatic cells were analyzed in terms of yield, viability (tripan blue and XTT test), diazepam metabolism (cyt P450 activity) and production of human albumin (immunonephelometry and mRNA by RT-PCR) after isolation (fresh hepatocytes) and after cryopreservation (10% DMSO) in liquid nitrogen. Results: macrosteatosis >60%, ischemic damage due to prolonged donor hypotension or cardiac arrest and non-viral cirrhosis were the causes of refusal of the liver for transplantation respectively in 60%, 11% and 8% of cases. Cell yield averaged 7 million of hepatocytes per gram of liver tissue digested; mean viability was 80%±13%. The best results in terms of cell yield and viability were obtained from steatotic organs, while cirrhotic livers yielded less cell/gr of liver (3x106 vs 7x105 p<0.043 and 81% vs 66% p=0.27, respectively). Viability of cryopreserved hepatocytes after thawing averaged 50%. However thawed hepatocytes had a normal diazepam metabolism (fig 1) and human albumin synthesis was comparable to that of fresh cells (fig 2).FigureFigureCryopreserved human hepatocytes (CHH) were utilized as the biological component of a BAL device for temporaneous support of two patients affected by FHF awaiting for urgent transplant. A total of 3 BAL applications were performed. Ten to 13 billion viable CHH have been loaded in each BAL. No adverse events were recorded and vital functions remained table during the procedures. Biochemical liver functions showed bilirubin and ammonia reduction at the end of each treatment with CHH based BAL. One patient was successfully bridged to emergency OLTx performed after a single BAL; in the second case there was a spontaneous recovery of liver function after two BAL treatment and surgical arterialization of the portal vein. Both patients are still alive and well at home. Conclusions: the recovery of donor human livers unwanted for transplantation at harvesting, allowed isolation and cryopreservation of viable and functionally active human hepatocytes which have been banked and successfully used for clinical application of a BAL device.

Polimery wykorzystywane do rekonstrukcji kosci - ocena wybranych podlozy polimerowych w hodowli in vitro osteoblastow

In a literature review the theoretical fundamentals and achievements in osseous implants' investigations have been presented. Especially polymeric materials used in implants were discussed as well as the criteria of the proper polymers choice. These criteria cover the structure of a polymer, porosity, mechanical and surface properties and biodegradation process course. Modification of osseous implant surface with proteins has been also discussed. The part concerning the own research contains the results of evaluation of bone cells' (osteoblasts) adhesion to various types of biomaterials, used as porous osseous implants. A new way of evaluation, presented in Scheme A, was used. Contact angle values (CA, Fig. 1) of the surfaces obtained were measured. Also viability of cells after one-week incubation was investigated basing on the measurements of activity of enzymes of succinate dehydrogenase (XTT Test, Fig. 2) or alkaline phosphatase (ALP Test, Fig. 3). 11 series of measurements differing in the substrate type were done. The best results have been obtained for the surfaces with dextran as well as with the systems: dextran + poly(acrylic acid) (PAA), dextran + Ca(NO3)2 or dextran + PAA + Ca(NO3)2. We did not find an unambiguous correlation between biological tests results and hydrophylic or hydrophobic character of polymeric substrate.

Synthetic inhibitors of CDKs induce different responses in androgen sensitive and androgen insensitive prostatic cancer cell lines

Aims: Because of the high prevalence of prostatic cancer and the limitations of its treatment, enormous effort has been put into the development of new therapeutic modalities. One potential tool...

In vitro toxicity of surfactants in U937 cells: Cell membrane integrity and mitochondrial function

Surface-active agents are components of many drugs and cosmetics. In order to examine their cytotoxic and membrane-toxic potential, various surfactants were examined in U937 cells using the tetrazolium reduction assay EZ4U, a modified XTT test, and the arachidonic acid release test (AART). EZ4U measures the ability of living cells to reduce a colorless tetrazolium salt to an orange water-soluble formazan derivative by mitochondrial dehydrogenases. [3H]arachidonic acid ([3H]AA) is rapidly incorporated into cell membrane phospholipids. Due to membrane disintegration or enzymatic catalysis, it is released into the cell culture medium and can be measured by scintillation technique. The results after 24-hour-exposure are as follow (CC50 microg/ml; RC50 microg/ml): benzalkonium chloride (0.6), Cremophor A25 (1.4; 5.9), sodium cetearyl sulfate (1.6; 19.9), Brij78 (1.6; 7.7), TEGO betaine E (3.0; 19.3), TEGO betaine CKD (4.1; 20.2), TEGO betaine L7 (7.5; 20.0), sodium dodecyl sulfate (7.5; 39.0), Triton X-100 (8.9; 110), polysorbate 80 (48.1; 491), soybean lecithin (7920; 19940).

Toxicity and Cell Density Monitoring in Monolayer and Three-dimensional Cultures with the XTT Assay

The application of viability criteria (MTT and XTT tests) to monolayer cultures and immobilised cells in three-dimensional systems was investigated in order to assess cell viability and cell proliferation. The suitability and accuracy of these tests were compared with the conventional criteria (cellular protein and DNA content) used in monolayer cultures for the same purpose. The colorimetric assay based on the metabolic reduction of the tetrazolium salt XTT to a water-soluble formazan proved to be very useful, rapid and sensitive. This automated spectrophotometric enzymatic method, due to its lack of toxicity, also permits repeated nondestructive assays on a single cellular culture for the long-term monitoring of cytotoxicity, cell survival and cell proliferation, and can be performed in 96-well plates with minimal handling. This method could offer a solution for cellular density evaluation in complex cell cultures that do not permit visual examination; it is also the best choice for protein-based, three-dimensional systems such as collagen gels.

Induction of rat liver parenchymal cell apoptosis by hepatic myofibroblasts via transforming growth factor beta

The induction of apoptosis of rat liver parenchymal cells (PC) by transforming growth factor beta (TGF-beta)-expressing transforming fat-storing cells (FSC), i.e., myofibroblasts (MFB), was studied under culture conditions and compared with the apoptotic effect of human recombinant TGF-beta1. MFB were obtained by subculture of FSC. The TGF-beta concentration in the conditioned medium of myofibroblast (MFBcM) determined with the Mink cell proliferation inhibition assay was <0.25 ng/mL/24 in the native medium, but 1.9 ng/mL24 h after transient acidification. MFBcM added in various dilutions and for different times to PC monolayers induced progressive cell detachment from the plastic support and increase of lactate dehydrogenase (LDH) activity in the medium. The reduction of mitochondrial dehydrogenase activity in PC (XTT or WST-1 test) was an early sign of MFBcM-induced functional impairment of PC. Short term exposure of PC with MFBcM for 3 hours was sufficient to induce the deleterious effects on PC, but neither native (nonactivated) MFBcM nor conditioned medium of untransformed FSC (FSCcM), in which TGF-beta was not detectable, were able to impair function and viability of PC. Activated MFBcM increased strongly (up to 21-fold) the concentration of oligonucleosomal DNA fragments both in the adherent and detached fraction of PC. Internucleosomal DNA fragments (DNA ladder) were demonstrated by electrophoresis of extracted DNA on agarose gels and by in situ end-labeling of DNA breaks (TUNEL reaction) only in MFBcM-exposed PC. MFBcM-treated PC exhibited intense fluorescence after staining with DNA-binding dye Hoechst 33342 and an increased number of cells with fragmented nuclei. All these criteria point to MFBcM-generated apoptosis of cultured PC, which were found to be very similar to those induced by human recombinant TGF-beta1. The exclusive role of active TGF-beta in MFBcM as mediator of the apoptotic effects of MFB was proven by preincubation of the conditioned medium with human recombinant latency-associated peptide, which reversed completely MFBcM induced reduction of the XTT-test and the MFBcM-generated increase of oligonucleosomal DNA fragments. Partial reversibility was reached by preincubation of the medium with recombinant soluble type II TGF-beta receptor. The data let us conclude that transformed FSC, i.e., MFB in damaged liver, could participate in the mechanisms of PC apoptosis by paracrine loops involving TGF-beta. (Hepatology 1996 Mar;23(3):571-81)

Induction of rat liver parenchymal cell apoptosis by hepatic myofibroblasts via transforming growth factor beta.

The induction of apoptosis of rat liver parenchymal cells (PC) by transforming growth factor beta (TGF-beta)-expressing transforming fat-storing cells (FSC), i.e., myofibroblasts (MFB), was studied under culture conditions and compared with the apoptotic effect of human recombinant TGF-beta1. MFB were obtained by subculture of FSC. The TGF-beta concentration in the conditioned medium of myofibroblast (MFBcM) determined with the Mink cell proliferation inhibition assay was <0.25 ng/mL/24 in the native medium, but 1.9 ng/mL24 h after transient acidification. MFBcM added in various dilutions and for different times to PC monolayers induced progressive cell detachment from the plastic support and increase of lactate dehydrogenase (LDH) activity in the medium. The reduction of mitochondrial dehydrogenase activity in PC (XTT or WST-1 test) was an early sign of MFBcM-induced functional impairment of PC. Short term exposure of PC with MFBcM for 3 hours was sufficient to induce the deleterious effects on PC, but neither native (nonactivated) MFBcM nor conditioned medium of untransformed FSC (FSCcM), in which TGF-beta was not detectable, were able to impair function and viability of PC. Activated MFBcM increased strongly (up to 21-fold) the concentration of oligonucleosomal DNA fragments both in the adherent and detached fraction of PC. Internucleosomal DNA fragments (DNA ladder) were demonstrated by electrophoresis of extracted DNA on agarose gels and by in situ end-labeling of DNA breaks (TUNEL reaction) only in MFBcM-exposed PC. MFBcM-treated PC exhibited intense fluorescence after staining with DNA-binding dye Hoechst 33342 and an increased number of cells with fragmented nuclei. All these criteria point to MFBcM-generated apoptosis of cultured PC, which were found to be very similar to those induced by human recombinant TGF-beta1. The exclusive role of active TGF-beta in MFBcM as mediator of the apoptotic effects of MFB was proven by preincubation of the conditioned medium with human recombinant latency-associated peptide, which reversed completely MFBcM induced reduction of the XTT-test and the MFBcM-generated increase of oligonucleosomal DNA fragments. Partial reversibility was reached by preincubation of the medium with recombinant soluble type II TGF-beta receptor. The data let us conclude that transformed FSC, i.e., MFB in damaged liver, could participate in the mechanisms of PC apoptosis by paracrine loops involving TGF-beta.