- [Show abstract] [Hide abstract] ABSTRACT: Non-cultured, autologous cell-spray grafting is an alternative to mesh grafting for larger partial- and deep partial-thickness burn wounds. The treatment uses a suspension of isolated cells, from a patient's donor site skin tissue, and cell-spray deposition onto the wound that facilitates re-epithelialization. Existing protocols for therapeutic autologous skin cell isolation and cell-spray grafting have defined the donor site area to treatment area ratio of 1:80, substantially exceeding the coverage of conventional mesh grafting. However, ratios of 1:100 are possible by maximizing the wound treatment area with harvested cells from a given donor site skin tissue according to a given burn area. Although cell isolation methods are very well described in the literature, a rational approach addressing critical aspects of these techniques are of interest in planning clinical study protocols. We considered in an experimental study the cell yield as a function of the donor site skin tissue, the cell density for spray grafting, the liquid spray volume, the sprayed distribution area, and the percentage of surface coverage. The experimental data was then used for the development of constants and mathematical equations to give a rationale for the cell isolation and cell-spray grafting processes and in planning for clinical studies.
- [Show abstract] [Hide abstract] ABSTRACT: Partial and deep partial-thickness burn wounds present a difficult diagnosis and prognosis that makes the planning for a conservative treatment versus mesh grafting problematic. A non-invasive treatment strategy avoiding mesh grafting is often chosen by practitioners based on their clinical and empirical evidence. However, a delayed re-epithelialization after conservative treatment may extend the patient's hospitalization period, increase the risk of infection, and lead to poor functional and aesthetic outcome. Early spray grafting, using non-cultured autologous cells, is under discussion for partial and deep partial-thickness wounds to accelerate the re-epithelialization process, reducing the healing time in the hospital, and minimizing complications. To address planning for future clinical studies on this technology, suitable indications will be interesting. We present case information on severe second-degree injuries after gas, chemical, electrical, gasoline, hot water, and tar scalding burns showing one patient per indication. The treatment results with autologous non-cultured cells, support rapid, uncomplicated re-epithelialization with aesthetically and functionally satisfying outcomes. Hospital stays averaged 7.6±1.6 days. Early autologous cell-spray grafting does not preclude or prevent simultaneous or subsequent traditional mesh autografting when indicated on defined areas of full-thickness injury.
- [Show abstract] [Hide abstract] ABSTRACT: The creation of larger-scale three-dimensional tissue constructs depends on proper medium mass and gas exchange, as well as removal of metabolites, which cannot be achieved in conventional static two-dimensional petri dish culture. In cultures of tissue-density this problem can be addressed by decentral perfusion through artificial micro-capillaries. While the static medium exchange in petri dishes leads to metabolite peaks, perfusion culture provides a dynamic medium supply, thereby preventing non-physiological peaks. To overcome the limitations of conventional static two-dimensional culture, a three-dimensional perfusion bioreactor technology has been developed, providing decentral and high-performance mass exchange as well as integral oxygenation. Similar to organ systems in vivo, the perfusion with medium provides nutrition and removes waste metabolites, and the perfusion with gas delivers oxygen and carbon dioxide for pH regulation. Such bioreactors are available at various dimensions ranging from 0.2 to 800 mL cell compartment volumes (manufactured by StemCell Systems, Berlin, Germany). Here, we describe in detail the setup and maintenance of a small-scale 4-chamber bioreactor with its tubing circuit and perfusion system.
- [Show abstract] [Hide abstract] ABSTRACT: Advancement in thermal three-dimensional printing techniques has greatly increased the possible applications of various materials in medical applications and tissue engineering. Yet, potential toxic effects on primary human cells have been rarely investigated. Therefore, we compared four materials commonly used in thermal printing for bioengineering, namely thermally printed acrylonitrile butadiene styrene, MED610, polycarbonate, and polylactic acid, and investigated their effects on primary human adult skin epidermal keratinocytes and bone marrow mesenchymal stromal cells (BM-MSCs) in vitro. We investigated indirect effects on both cell types caused by potential liberation of soluble substances from the materials, and also analyzed BM-MSCs in direct contact with the materials. We found that even in culture without direct contact with the materials, the culture with MED610 (and to a lesser extent acrylonitrile butadiene styrene) significantly affected keratinocytes, reducing cell numbers and proliferation marker Ki67 expression, and increasing glucose consumption, lactate secretion, and expression of differentiation-associated genes. BM-MSCs had decreased metabolic activity, and exhibited increased cell death in direct culture on the materials. MED610 and acrylonitrile butadiene styrene induced the strongest expression of genes associated to differentiation and estrogen receptor activation. In conclusion, we found strong cell-type-specific effects of the materials, suggesting that materials for applications in regenerative medicine should be carefully selected not only based on their mechanical properties but also based on their cell-type-specific biological effects. Electronic supplementary material The online version of this article (doi:10.1007/s40846-016-0118-z) contains supplementary material, which is available to authorized users.
- [Show abstract] [Hide abstract] ABSTRACT: Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.
Dataset: Supplementary Material
- [Show abstract] [Hide abstract] ABSTRACT: Autologous cell-spray grafting of non-cultured epidermal cells is an innovative approach for the treatment of severe second-degree burns. After treatment, wounds are covered with dressings that are widely used in wound care management; however, little is known about the effects of wound dressings on individually isolated cells. The sprayed cells have to actively attach, spread, proliferate, and migrate in the wound for successful re-epithelialization, during the healing process. It is expected that exposure to wound dressing material might interfere with cell survival, attachment, and expansion. Two experiments were performed to determine whether some dressing materials have a negative impact during the early phases of wound healing. In one experiment, freshly isolated cells were seeded and cultured for one week in combination with eight different wound dressings used during burn care. Cells, which were seeded and cultured with samples of Adaptic®, Xeroform®, EZ Derm®, and Mepilex® did not attach, nor did they survive during the first week. Mepitel®, N-Terface®, Polyskin®, and Biobrane® dressing samples had no negative effect on cell attachment and cell growth when compared to the controls. In a second experiment, the same dressings were exposed to pre-cultured cells in order to exclude the effects of attachment and spreading. The results confirm the above findings. This study could be of interest for establishing skin cell grafting therapies in burn medicine and also for wound care in general.
- [Show abstract] [Hide abstract] ABSTRACT: Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale-up of stem cell culture is necessary. Bioreactors for dynamic three-dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow-fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 x 10(6) mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 x 10(6) mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four-compartment bioreactor including further cultivation of remaining cells. This article is protected by copyright. All rights reserved.
- [Show abstract] [Hide abstract] ABSTRACT: An alternative approach for traditional clinical mesh grafting in burn wound treatment is the use of expanded autologous keratinocytes in suspension or sheets that are cultured over 2-4 weeks in a remote service facility. While a wound reepithelialization has been described, the functional and aesthetic outcome is under debate. Cell isolation from split-skin donor tissue aims to preserve the valuable stem cell progenitors from the basal epidermal layer and to provide patients with a rapid wound reepithelialization and a satisfying outcome. While the presence of epidermal progenitors in the cell graft is thought to enable an improved epidermal surface post reepithelialization, we investigated a feasible clinical approach involving cultured versus noncultured epidermal cells comparing the α6int(high)/K15(high)/FSC(low)/SSC(low) and α6int(high)/K5(high)/FSC(low)/SSC(low) keratinocyte progenitor subpopulations before and after in vitro culture process. Our results show a significant increase of cell size during in vitro passaging and a decrease of progenitor markers linked to a gradual differentiation. A provision of the regenerative epidermal progenitors, isolated from the split-skin biopsy and applied directly onto the wound in an on-site setting of isolation and cell spray grafting in the operation room, could be of interest when choosing options for skin wound care with autologous cells. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
- [Show abstract] [Hide abstract] ABSTRACT: Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
- [Show abstract] [Hide abstract] ABSTRACT: The development of functional engineered tissue constructs depends on high cell densities and appropriate vascularization. In this study we implemented a four-compartment three-dimensional (3D) perfusion bioreactor culture model for studying the effects of medium perfusion on endothelial, hepatic and hematopoietic cell populations of primary human fetal liver in an in-vivo like environment. Human fetal liver cells were cultured in bioreactors configured to provide either perfusion or diffusion conditions. Metabolic activity of the cultures was monitored daily by measuring glucose consumption and lactate production. Cell viability during culture was analyzed by lactate dehydrogenase activity. Hepatic functionality was determined by the release of albumin and alpha-fetoprotein (AFP) in culture medium samples. After 4 days of culture cells were analyzed for the expression of a variety of endothelial, hepatic and hematopoietic genes, as well as the surface marker expression of CD31 and CD34 in flow cytometry. We found that medium perfusion increased expression of endothelial genes such as CD31, vWF, CD140b, CD309 and CD144 while decreasing expression of the erythrocyte specific hematopoietic gene CD235a. Hepatic differentiation was promoted under perfusion conditions as demonstrated by lower AFP and higher albumin secretion compared to cultures not exposed to medium perfusion. Additionally, cultures exposed to medium perfusion gave higher rates of glucose consumption and lactate production, indicating increased metabolic activity. In conclusion, high-density bioreactors configured to provide constant medium perfusion significantly induced hepatic and endothelial cell differentiation and provided improved conditions for the culture of human fetal liver cells compared to cultures without perfusion.
- [Show abstract] [Hide abstract] ABSTRACT: Investigation A novel active wound dressing (AWD) concept based on a microporous hollow fiber membrane network was investigated in an animal model. It provides a local solution-perfused environment for regenerative cell nutrition, wound irrigation, debris removal, electrolyte balancing, pH regulation, and topical antibiosis. The device is capable of supplying soluble factors, as tested experimentally for the recombinant human growth and differentiation factor-5 (rhGDF-5). Methods Following in vitro studies for rhGDF-5 using primary human keratinocytes and dermal fibroblasts, we employed a porcine partial thickness wound model with five distinct wounds on each back of n = 8 pigs. Four wound groups were perfused differently over 9 days and compared with a negative control wound without perfusion: (1) 1% trehalose solution, pH 5.5; (2) rhGDF-5 (150 ng/ml) in 1% trehalose solution, pH 5.5; (3) nutrition solution; and (4) rhGDF-5 (150 ng/ml) in nutrition solution with 1% trehalose, pH 5.5. Results Promoted wound healing was observed within group 1 and more pronounced within group 2. Groups 3 and 4, with nutrition solution, showed significant adverse effects on wound healing (p < 0.05). Conclusions The investigated AWD concept appears to be an interesting therapeutic tool to study further wound healing support. Additionally, topical application of rhGDF-5 could be promising.
- [Show abstract] [Hide abstract] ABSTRACT: The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to six weeks and compared to Petri-dish controls. Cells were analysed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of six weeks. Compared to Petri-dish culture controls, bioreactor perfusion culture improved in both the short and long term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture. Biotechnol. Bioeng. © 2014 Wiley Periodicals, Inc.
- [Show abstract] [Hide abstract] ABSTRACT: AimThe transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) has been shown to play an important role in liver development, cell proliferation and differentiation. It is, however, largely unknown, if C/EBPα regulates cell differentiation and proliferation differently in the diverse cell types of the human liver. We investigated the role of C/EBPα in primary human fetal liver cells and liver cell sub-populations in vitro using a three-dimensional perfusion bioreactor as an advanced in vivo-like human organ culture model.Methods Human fetal liver cells were investigated in vitro. C/EBPα gene expression was knocked down using siRNA or over-expressed by plasmid transfection. Cell type specific gene expression was studied, cell populations and their proliferation were investigated, and metabolic parameters were analyzed.ResultsWhen C/EBPα gene expression was knocked down, we observed a significantly reduced expression of typical endothelial, hematopoietic and mesenchymal genes such as CD31, vWF, CD90, CD45 and α-smooth muscle actin in fetal cells. Theintracellular expression of hepatic proteins and genes for liver specific serum proteins α–fetoprotein and albumin were reduced, their protein secretion was increased. Fetal endothelial cell numbers were reduced and hepatoblast numbers were increased. C/EBPα over-expression in fetal cells resulted in increased endothelial numbers, but did not affect mesenchymal cell types or hepatoblasts.Conclusions We demonstrated that the effects of C/EBPα are specific for the different human fetal liver cell types, using an advanced three-dimensional perfusion bioreactor as a humanin vivo-like model.
- [Show abstract] [Hide abstract] ABSTRACT: Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
- [Show abstract] [Hide abstract] ABSTRACT: Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions. Copyright © 2012 John Wiley & Sons, Ltd.
- [Show abstract] [Hide abstract] ABSTRACT: The presence of mesenchymal stem cells (MSCs) has been described in various organs. Pericytes possess a multilineage differentiation potential and have been suggested to be one of the developmental sources for MSCs. In human liver, pericytes have not been defined. Here, we describe the identification, purification, and characterization of pericytes in human adult and fetal liver. Flow cytometry sorting revealed that human adult and fetal liver contains 0.56%±0.81% and 0.45%±0.39% of CD146(+)CD45(-)CD56(-)CD34(-) pericytes, respectively. Of these, 41% (adult) and 30% (fetal) were alkaline phosphatase-positive (ALP(+)). In situ, pericytes were localized around periportal blood vessels and were positive for NG2 and vimentin. Purified pericytes could be cultured extensively and had low population doubling times. Immunofluorescence of cultures demonstrated that cells were positive for pericyte and mesenchymal cell markers CD146, NG2, CD90, CD140b, and vimentin, and negative for endothelial, hematopoietic, stellate, muscle, or liver epithelial cell markers von Willebrand factor, CD31, CD34, CD45, CD144, CD326, CK19, albumin, α-fetoprotein, CYP3A7, glial fibrillary acid protein, MYF5, and Pax7 by gene expression; myogenin and alpha-smooth muscle actin expression were variable. Fluorescence-activated cell sorting analysis of cultures confirmed surface expression of CD146, CD73, CD90, CD10, CD13, CD44, CD105, and ALP and absence of human leukocyte antigen-DR. In vitro differentiation assays demonstrated that cells possessed robust osteogenic and myogenic, but low adipogenic and low chondrogenic differentiation potentials. In functional in vitro assays, cells had typical mesenchymal strong migratory and invasive activity. In conclusion, human adult and fetal livers harbor pericytes that are similar to those found in other organs and are distinct from hepatic stellate cells.
- [Show abstract] [Hide abstract] ABSTRACT: An emulsion casting method was chosen for the processing of highly porous hydroxyapatite (HAp) foams to be used for the in vitro culturing of primary human liver cells. The volume fraction of heptane added to the aqueous dispersion of HAp particles could be varied to achieve a wide range of porosities (10-90 vol.-%) and pore morphologies. The emulsion derived open foam has a distinctive morphology, including a continuous large pore phase and solid struts containing smaller isolated pores. The effect of the heptane content of the slip on this sintered structure was quantified using linear intercept measurement parameters. Additionally, macropores necessary for perfusion were created using polymer tubes placed in the casting mould. When the tube spacing approached the pore size in the foam, the pore morphology was strongly affected.
- [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Cell banked epidermal skin progenitor cells have the potential to provide an "off-the-freezer" product. Such cells may provide a skin donor area-independent cell-spray grafting therapy for the treatment of burns. We first characterized fetal skin samples of gestational ages ranging from 6 to 21 weeks. As the results suggest that the phenotypic differentiation occurs after 10 weeks, which may complicate follow-up in vitro studies, we developed and compared different cell isolation techniques for human fetal skin-derived epithelial cells from tissue ages 6 to 9 weeks. We initially screened seven methods of characterization, concluding that two methods warranted further investigation: incubating the epidermal tissue in Petri-dishes with culture medium for spontaneous cell outgrowth, and wiping the epidermal tissue onto a dry Petri-dish culture surface followed by adding culture medium. Non-controllable culture contamination with dermal cells was the reason for excluding the other five methods. The results suggest that epidermal cells can be isolated from tissue exhibiting a single homogeneous layer of CK15(+) basal keratinocytes up to week 9. At later gestational ages, the ongoing skin differentiation results in a multi-layer basal structure and progenitors associated with the hair bulb would have to be considered. Spraying the resulting cells with a clinical spray device was successfully demonstrated in an in vitro model. Conclusion: Gestational age 6-9 weeks epidermal human fetal skin cells from the basal layer can be reproducibly isolated and transferred into culture for studies on the development of skin cell transplantation therapies.
- [Show abstract] [Hide abstract] ABSTRACT: Properly regulated inflammation facilitates recognition and reaction to injury or infection, but inadequate or overly robust inflammation can lead to disease. Sepsis is an inflammatory disease that accounts for nearly 10% of total U.S. deaths, costing more than $17 billion. Acute inflammation in sepsis may evolve too rapidly to be modulated appropriately, and we suggest that therapies should focus not on abolishing inflammation, but rather on attenuating the positive feedback cycle of inflammation/damage/inflammation. In Gram-negative sepsis, bacterial endotoxin causes inflammation and is driven and regulated by the cytokine tumor necrosis factor-α (TNF-α), which is, in turn, negatively regulated via its endogenous inhibitor, soluble TNF-α receptor (sTNFR). We generated stably gene-modified variants of human HepG2 hepatocytes, using lentiviral constructs coding for mouse sTNFR driven by the constitutive cytomegalovirus promoter, and seeded them in a scaled-down, experimental liver bioreactor. When connected to anesthetized, cannulated rats subjected to endotoxin infusion and maintained solely by the animals' circulation, this biohybrid device elevated circulating sTNFR, reduced the levels of TNF-α and other key inflammatory mediators, alleviated hypotension, and reduced circulating markers of organ damage. This novel class of biohybrid devices may bemodified for patient- and disease-specific application, and, thus, may represent a disruptive strategy that offers the potential for rational inflammation reprogramming.
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
- • Department of Surgery
- • McGowan Institute for Regenerative Medicine
Università della Calabria
Rende, Calabria, Italy
- Department of Chemical Engineering and Materials
San Diego Regenerative Medicine InstituteSan Diego, California, United States
Humboldt-Universität zu Berlin
Berlín, Berlin, Germany
- Department of Biology
Freie Universität Berlin
Berlín, Berlin, Germany
- Institute of Chemistry and Biochemistry