Safety and efficacy of cardiopoietic stem cells in the treatment of post-infarction left-ventricular dysfunction - From cardioprotection to functional repair in a translational pig infarction model.

To date, clinical success of cardiac cell-therapies remains limited. To enhance the cardioreparative properties of stem cells, the concept of lineage-specification through cardiopoietic-guidance has been recently suggested. However, so far, only results from murine studies and from a clinical pilot-trial in chronic heart-failure (CHF) are available, while systematic evidence of its therapeutic-efficacy is still lacking. Importantly, also no data from large animals or for other indications are available. Therefore, we here investigate the therapeutic-efficacy of human cardiopoietic stem cells in the treatment of post-infarction LV-dysfunction using a translational pig-model. Using growth-factor priming, lineage-specification of human bone-marrow derived MSCs was achieved to generate cardiopoietic stem cells according to GMP-compliant protocols. Thereafter, pigs with post-infarction LV-dysfunction (sub-acute phase;1-month) were randomized to either receive transcatheter NOGA 3D electromechanical-mapping guided intramyocardial transplantation of cardiopoietic cells or saline (control). After 30days, cardiac MRI (cMRI) was performed for functional evaluation and in-vivo cell-tracking. This approach was coupled with a comprehensive post-mortem cell-fate and mode-of-repair analysis. Cardiopoietic cell therapy was safe and ejection-fraction was significantly higher when compared to controls (p = 0.012). It further prevented maladaptive LV-remodeling and revealed a significantly lower relative and total infarct-size (p = 0.043 and p = 0.012). As in-vivo tracking and post-mortem analysis displayed only limited intramyocardial cardiopoietic cell-integration, the significant induction of neo-angiogenesis (∼40% higher; p = 0.003) and recruitment of endogenous progenitors (∼2.5x higher; p = 0.008) to the infarct border-zone appeared to be the major modes-of-repair. This is the first report using a pre-clinical large animal-model to demonstrate the safety and efficacy of cardiopoietic stem cells for the treatment of post-infarction LV-dysfunction to prevent negative LV-remodeling and subsequent CHF. It further provides insight into post-delivery cardiopoietic cell-fate and suggests the mechanisms of cardiopoietic cell-induced cardiac-repair. The adoption of GMP-/GLP-compliant methodologies may accelerate the translation into a phase-I clinical-trial in patients with post-ischemic LV-dysfunction broadening the current indication of this interesting cell-type.

Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension.

Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

The ultrastructure of fibronectin fibers pulled from a protein monolayer at the air-liquid interface and the mechanism of the sheet-to-fiber transition.

Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ultrastructural characterization is then contrasted with previous FRET studies that characterized the molecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio- or biomimetic polymers.

Effect of matrine on primary human hepatocytes in vitro.

Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

Intramyocardial transplantation and tracking of human mesenchymal stem cells in a novel intra-uterine pre-immune fetal sheep myocardial infarction model: a proof of concept study.

Although stem-cell therapies have been suggested for cardiac-regeneration after myocardial-infarction (MI), key-questions regarding the in-vivo cell-fate remain unknown. While most available animal-models require immunosuppressive-therapy when applying human cells, the fetal-sheep being pre-immune until day 75 of gestation has been proposed for the in-vivo tracking of human cells after intra-peritoneal transplantation. We introduce a novel intra-uterine myocardial-infarction model to track human mesenchymal stem cells after direct intra-myocardial transplantation into the pre-immune fetal-sheep. Thirteen fetal-sheep (gestation age: 70-75 days) were included. Ten animals either received an intra-uterine induction of MI only (n = 4) or MI+intra-myocardial injection (IMI;n = 6) using micron-sized, iron-oxide (MPIO) labeled human mesenchymal stem cells either derived from the adipose-tissue (ATMSCs;n = 3) or the bone-marrow (BMMSCs;n = 3). Three animals received an intra-peritoneal injection (IPI;n = 3; ATMSCs;n = 2/BMMSCs;n = 1). All procedures were performed successfully and follow-up was 7-9 days. To assess human cell-fate, multimodal cell-tracking was performed via MRI and/or Micro-CT, Flow-Cytometry, PCR and immunohistochemistry. After IMI, MRI displayed an estimated amount of 1×10(5)-5×10(5) human cells within ventricular-wall corresponding to the injection-sites which was further confirmed on Micro-CT. PCR and IHC verified intra-myocardial presence via detection of human-specific ß-2-microglobulin, MHC-1, ALU-Sequence and anti-FITC targeting the fluorochrome-labeled part of the MPIOs. The cells appeared viable, integrated and were found in clusters or in the interstitial-spaces. Flow-Cytometry confirmed intra-myocardial presence, and showed further distribution within the spleen, lungs, kidneys and brain. Following IPI, MRI indicated the cells within the intra-peritoneal-cavity involving the liver and kidneys. Flow-Cytometry detected the cells within spleen, lungs, kidneys, thymus, bone-marrow and intra-peritoneal lavage, but not within the heart. For the first time we demonstrate the feasibility of intra-uterine, intra-myocardial stem-cell transplantation into the pre-immune fetal-sheep after MI. Utilizing cell-tracking strategies comprising advanced imaging-technologies and in-vitro tracking-tools, this novel model may serve as a unique platform to assess human cell-fate after intra-myocardial transplantation without the necessity of immunosuppressive-therapy.

Transcatheter based electromechanical mapping guided intramyocardial transplantation and in vivo tracking of human stem cell based three dimensional microtissues in the porcine heart.

Stem cells have been repeatedly suggested for cardiac regeneration after myocardial infarction (MI). However, the low retention rate of single cell suspensions limits the efficacy of current therapy concepts so far. Taking advantage of three dimensional (3D) cellular self-assembly prior to transplantation may be beneficial to overcome these limitations. In this pilot study we investigate the principal feasibility of intramyocardial delivery of in-vitro generated stem cell-based 3D microtissues (3D-MTs) in a porcine model. 3D-MTs were generated from iron-oxide (MPIO) labeled human adipose-tissue derived mesenchymal stem cells (ATMSCs) using a modified hanging-drop method. Nine pigs (33 ± 2 kg) comprising seven healthy ones and two with chronic MI in the left ventricle (LV) anterior wall were included. The pigs underwent intramyocardial transplantation of 16 × 10(3) 3D-MTs (1250 cells/MT; accounting for 2 × 10(7) single ATMSCs) into the anterior wall of the healthy pigs (n = 7)/the MI border zone of the infarcted (n = 2) of the LV using a 3D NOGA electromechanical mapping guided, transcatheter based approach. Clinical follow-up (FU) was performed for up to five weeks and in-vivo cell-tracking was performed using serial magnet resonance imaging (MRI). Thereafter, the hearts were harvested and assessed by PCR and immunohistochemistry. Intramyocardial transplantation of human ATMSC based 3D-MTs was successful in eight animals (88.8%) while one pig (without MI) died during the electromechanical mapping due to sudden cardiac-arrest. During FU, no arrhythmogenic, embolic or neurological events occurred in the treated pigs. Serial MRI confirmed the intramyocardial presence of the 3D-MTs by detection of the intracellular iron-oxide MPIOs during FU. Intramyocardial retention of 3D-MTs was confirmed by PCR analysis and was further verified on histology and immunohistochemical analysis. The 3D-MTs appeared to be viable, integrated and showed an intact micro architecture. We demonstrate the principal feasibility and safety of intramyocardial transplantation of in-vitro generated stem cell-based 3D-MTs. Multimodal cell-tracking strategies comprising advanced imaging and in-vitro tools allow for in-vivo monitoring and post-mortem analysis of transplanted 3D-MTs. The concept of 3D cellular self-assembly represents a promising application format as a next generation technology for cell-based myocardial regeneration.

An operational concept for long-term cinemicrography of cells in mono- and co-culture under highly controlled conditions--the SlideObserver.

Cell morphology, proliferation and motility, as well as mono- and heterotypic cell-to-cell interactions, are of increasing interest for in vitro experiments. However, tightly controlling culture conditions whilst simultaneously monitoring the same set of cells is complicated. Moreover, video-microscopy of distinct cells or areas of cells over a prolonged period of time represents a technical challenge. The SlideObserver was designed for cinemicrography of cells in co-and monoculture. The core elements of the system are the SlideReactors, miniaturised hollow fibre-based bioreactors operated in closed perfusion loops. Within the SlideReactors, cells can be cultured under adaptable conditions as well as in direct- and indirect co-culture. The independent perfusion loops enable controlled variation of parameters such as medium, pH, and oxygenation. A combined automated microscope stage and camera set-up allows for micrograph acquisition of multiple user-defined regions of interest within the bioreactor units. For proof of concept, primary cells (HUVEC, human hepatocytes) and cell lines (HuH7, THP-1) were cultured under stable and varying culture conditions, as well as in mono- and co-culture. The operational system enabled non-stop imaging and automated control of process parameters as well as elective manipulation of either reactor. As opposed to non-perfused culture systems or comparable devices for cinemicrographic analysis, the SlideObserver allows simultaneous morphological monitoring of an entire culture of cells in multiple bioreactors.

Modification of aminosilanized superparamagnetic nanoparticles: feasibility of multimodal detection using 3T MRI, small animal PET, and fluorescence imaging.

PURPOSE: The aim of our study was to modify an aminosilane-coated superparamagnetic nanoparticle for cell labeling and subsequent multimodal imaging using magnetic resonance imaging (MRI), positron emission tomography (PET), and fluorescent imaging in vivo. PROCEDURES: We covalently bound the transfection agent HIV-1 tat, the fluorescent dye fluorescein isothiocyanate, and the positron-emitting radionuclide gallium-68 to the particle and injected them intravenously into Wistar rats, followed by animal PET and MRI at 3.0 T. As a proof of principle hepatogenic HuH7 cells were labeled with the particles and observed for cell toxicity as well as detectability by MRI and biodistribution in vivo. RESULTS: PET imaging and MRI revealed increasing hepatic and splenic accumulation of the particles over 24 h. Adjacent in vitro studies in hepatogenic HuH7 cells showed a rapid intracellular accumulation of the particles with high labeling efficiency and without any signs of toxicity. In vivo dissemination of the labeled cells could be followed by dynamic biodistribution studies. CONCLUSIONS: We conclude that our modified superparamagnetic nanoparticles are stable under in vitro and in vivo conditions and are therefore applicable for efficient cell labeling and subsequent multimodal molecular imaging. Moreover, their multiple free amino groups suggest the possibility for further modifications and might provide interesting opportunities for various research fields.

Monitoring of Liver Cell Transplantation in a Preclinical Swine Model Using Magnetic Resonance Imaging.

Liver cell transplantation (LCT) is a promising treatment approach for certain liver diseases, but clinical implementation requires methods for noninvasive follow-up. Labeling with superparamagnetic iron oxide particles can enable the detection of cells with magnetic resonance imaging (MRI). We investigated the feasibility of monitoring transplanted liver cells by MRI in a preclinical swine model and used this approach to evaluate different routes for cell application. Liver cells were isolated from landrace piglets and labeled with micron-sized iron oxide particles (MPIO) in adhesion. Labeled cells (n = 10), native cells (n = 3), or pure particles (n = 4) were transplanted to minipigs via intraportal infusion into the liver, direct injection into the splenic parenchyma, or intra-arterial infusion to the spleen. Recipients were investigated by repeated 3.0 Tesla MRI and computed tomography angiography up to 8 weeks after transplantation. Labeling with MPIO, which are known to have a strong effect on the magnetic field, enabled noninvasive detection of cell aggregates by MRI. Following intraportal application, which is commonly applied for clinical LCT, MRI was able to visualize the microembolization of transplanted cells in the liver that were not detected by conventional imaging modalities. Cells directly injected into the spleen were retained, whereas cell infusions intra-arterially into the spleen led to translocation and engraftment of transplanted cells in the liver, with significantly fewer microembolisms compared to intraportal application. These findings demonstrate that MRI can be a valuable tool for noninvasive elucidation of cellular processes of LCT and-if clinically applicable MPIO are available-for monitoring of LCT under clinical conditions. Moreover, the results clarify mechanisms relevant for clinical practice of LCT, suggesting that the intra-arterial route to the spleen deserves further evaluation.

Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.

BACKGROUND: A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications. RESULTS: Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion. CONCLUSION: This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications.

Stretched extracellular matrix proteins turn fouling and are functionally rescued by the chaperones albumin and casein.

While evidence is mounting that cells exploit protein unfolding for mechanochemical signal conversion (mechanotransduction), what mechanisms are in place to deal with the unwanted consequences of exposing hydrophobic residues upon force-induced protein unfolding? Here, we show that mechanical chaperones exist that can transiently bind to hydrophobic residues that are freshly exposed by mechanical force. The stretch-upregulated binding of albumin or casein to fibronectin fibers is reversible and does not inhibit fiber contraction once the tension is released.

Methylprednisolone and tacrolimus prevent hypothermia-induced endothelial dysfunction.

BACKGROUND: Hypothermia is used to preserve organs for transplantation and is the oldest method to protect organs during complex pediatric cardiac surgery. Loss of tissue function and tissue edema are common complications in children undergoing corrective cardiac surgery and heart transplantation. The present study was designed to examine the effects of methylprednisolone and tacrolimus on endothelial cell function and morphology after deep hypothermia and rewarming. METHODS: Human umbilical vein endothelial cells were pre-treated with methylprednisolone or tacrolimus, or both, incubated within a specially designed bioreactor or in monolayers, and then exposed to a dynamic cooling and rewarming protocol. Immunocytochemistry, time-lapse video microscopy, cell permeability and adherence assays, and Western blot analysis were performed. RESULTS: Confluent endothelial cells exposed to hypothermia displayed elongated cell shapes with intercellular gap formation, increased endothelial cell-layer permeability, and loss in adherence. Upon rewarming, however, endothelial cell integrity was restored. Opening and closing of intercellular gaps was dependent on extracellular signal-regulated kinase 1 and 2 (ERK 1/2) activation and connexin 43 expression. The combined treatment with methylprednisolone and tacrolimus inhibited these hypothermia-induced changes. CONCLUSIONS: These results suggest that methylprednisolone and tacrolimus inhibit hypothermia-induced endothelial gap formation by phosphorylated ERK 1/2 inhibition and connexin 43 stabilization. Application of combined drugs that affect multiple targets may therefore be considered as a possible new therapeutic strategy to prevent endothelial dysfunction after hypothermia and rewarming.

Imaging of primary human hepatocytes performed with micron-sized iron oxide particles and clinical magnetic resonance tomography.

Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For the visualization of the hepatocytes during and following cell application and the ability of a timely response to potential complications, a non-invasive modality for imaging the transplanted cells has to be established. The aim of this study was to label primary human hepatocytes with micron-sized iron oxide particles (MPIOs), enabling the detection of cells by clinical magnetic resonance imaging (MRI). Primary human hepatocytes isolated from 13 different donors were used for the labelling experiments. Following the dose-finding studies, hepatocytes were incubated with 30 particles/cell for 4 hrs in an adhesion culture. Particle incorporation was investigated via light, fluorescence and electron microscopy, and labelled cells were fixed and analysed in an agarose suspension by a 3.0 Tesla MR scanner. The hepatocytes were enzymatically resuspended and analysed during a 5-day reculture period for viability, total protein, enzyme leakage (aspartate aminotransferase [AST], lactate dehydrogenase [LDH]) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and the primary human hepatocytes were clearly detectable by MR instrumentation. The particle load was not affected by resuspension and showed no alternations during the culture period. Compared to control groups, labelling and resuspension had no adverse effects on the viability, enzyme leakage and metabolic activity of the human hepatocytes. The feasibility of preparing MPIO-labelled primary human hepatocytes detectable by clinical MR equipment was shown in vitro. MPIO-labelled cells could serve for basic research and quality control in the clinical setting of human hepatocyte transplantation.

Isolation of primary human hepatocytes after partial hepatectomy: criteria for identification of the most promising liver specimen.

Demands for primary human hepatocytes are continuously increasing, while supply is insufficient due to limited cell sources. To improve cell availability, the present study investigates the influence of donor liver characteristics on the outcome of hepatocyte isolation from surgically removed liver tissue (n = 50). Hepatocytes were isolated from liver specimens using a standardized two-step collagenase perfusion technique. The patient's sex, previous chemotherapy, or histopathology have shown no influence. Donor age significantly affected the isolation outcome, but was not found suitable for predicting cell yields. Preoperative blood parameters did not correlate with cell yield, although cell function was affected: total protein, albumin synthesis, and cell viability were significantly decreased for serum gamma-glutamyl-transferase (GGT) levels >60 U/L. Specimens from patients with benign diseases gave significantly higher cell yields than tissue removed due to secondary and primary tumors, respectively. The indication for surgery is a valuable basis for identifying the most yielding specimens. Hepatocytes from donors with high GGT levels appear to show reduced functional properties.

Isolation and culture of different epidermal and dermal cell types from human scalp suitable for the development of a therapeutical cell spray.

BACKGROUND: Previous studies demonstrated, that cultured epithelial autografts (CEA) can be isolated and skin cell sprays can be produced for application on different types of wounds. The purpose of the present study was to determine which cell types can be isolated from the human scalp and whether these cells can be used for spray transplantation. METHODS: Outer root sheath cells (ORS), keratinocytes, melanocytes, dermal papilla cells (DP), and dermal sheath cells (DSC) were isolated from human scalp tissue. Isolated cells were characterized, expanded and sprayed in an in vitro model. Growth behaviour, morphology and cell counts were compared with non-sprayed cells. RESULTS: With acceptable time, equipment and laboratory personnel a sufficient amount of keratinocytes, ORS, melanocytes, DP cells and DSC cells could be achieved. The cells are sufficient for application as a cell spray. Cells, positive for Integrin alpha6, Cytokeratin 19, CD73 and CD105 were identified within the cultures. CONCLUSIONS: Human scalp is suitable to gain epidermal and dermal cells for the development of therapeutic cell spray transplantation. Further studies have to determine, whether these cells can be combined to produce wound specific skin substitutes.

Hypothermia suppresses inflammation via ERK signaling pathway in stimulated microglial cells.

Hypothermic perfusion is a standard method for neuroprotection during cardiac surgery in children. However, the cellular responses underlying these mechanisms have not been clearly elucidated. In the present study we demonstrated that the inflammatory response of stimulated microglial cells is significantly reduced after moderate hypothermia. Continuous hypothermia caused a diminished NO release. Moderate hypothermia and rewarming caused a downregulation of phosphorylated MEK, ERK and iNOS-expression, diminished cytokine release and reduced CD-11a and ICAM-1 expression. Thus, neuroprotection offered by hypothermia could be attributed to reduced cytotoxic products released from stimulated microglial cells mediated by the MEK/ERK signal transduction pathway.

Continuously microscopically observed and process-controlled cell culture within the SlideReactor: proof of a new concept for cell characterization.

Certain cell types, especially primary human cells, favor a well-defined culture environment offering continuous supply of nutrients and oxygen and waste product removal. Several bioreactors based on special matrices or hollow fibers have been developed that provide such conditions. However, characterization of matrix re-organization or growth of tissue within these systems is possible only after culture termination. Evaluation of the influence of certain medium additives or culture conditions (e.g., temperature, oxygenation) on cell viability, expansion, and differentiation within these systems remains a challenging task. The SlideReactor, a miniaturized hollow fiber-based bioreactor, was developed to enable the observation of cells during culture. An operation concept offering predefined conditions for various cell types has been designed. For proof of concept, primary human cells (hepatocytes, fibroblasts, keratinocytes) and cell lines (HepG2, HuH7, C3A, WiDr, SkHep1) were cultured and observed. A series of experiments (n=40) showed the feasibility of the set-up; determination of process parameters and continuous observation is possible. The SlideReactor may serve as a simple and cost-efficient tool for cell characterization and optimization of cell-culture conditions.

Cryopreservation of primary human hepatocytes: the benefit of trehalose as an additional cryoprotective agent.

Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 +/- 13 vs. 46.9 +/- 11%, P < 0.01) and plating efficiency (41.5 +/- 18 vs. 17.6 +/- 13%, P < 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation.

Evaluation of primary human liver cells in bioreactor cultures for extracorporeal liver support on the basis of urea production.

Primary human liver cells from donor organs unsuitable for transplantation were cultivated in bioreactors developed for extracorporeal liver support. Because each system contains cells originating from an individual organ, each bioreactor culture must be individually characterized. The objective of this study was to identify suitable decisive parameters for the evaluation of cell culture performance. We analyzed the data from 47 bioreactor cultures containing 437 +/- 110 g of cells. Choosing urea production as the decisive parameter, the bioreactor cultures were divided into high-performance (daily urea production > or = 110 mg per bioreactor between culture days 3 and 14) and low-performance cultures. Comparing the mean courses of the groups revealed a significant distinction in most other investigated biochemical parameters. In conclusion, urea production seems to be an appropriate parameter for evaluating the performance of liver cell cultures in bioreactors because it corresponds to all other evaluated parameters of cell function.

A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle.

Protein transduction domains (PTDs) have been used to deliver a variety of biologically active cargo across cellular membranes. However the potential of PTDs to mediate transport of nanoparticular structures into the cytoplasm bypassing the endosomal compartment remains unclear. Cell-permeable virus-like particles (VLPs) harboring a marker gene based on hepatitis B virus nucleocaspids were established. Cell permeability was achieved by fusion with translocation motif (TLM)-PTD. Electron and confocal microscopy revealed that these VLPs translocate as complete particles across the plasma membrane and transverse the cytoplasm toward the nucleus. Inhibition of endocytosis did not affect translocation of these VLPs into the cytoplasm. Based on these particles, a gene transfer system was developed. To this end the particles were loaded with DNA-encoding small hepatitis B virus surface antigen (SHBs) or green fluorescence protein (eGFP) that served as marker genes. Although the DNA-packaging efficiency was very low, applying the appropriate number of VLPs to primary human hepatocytes a gene transfer efficiency of approximately 95% was observed. In conclusion, the TLM-PTD has the potential to mediate efficient transfer of assembled particles and its cargo, nucleic acids, into primary human hepatocytes. This provides the basis for development of novel transducible therapeutic or diagnostic particles.