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Liver cells cultured in 3D bioreactors is an interesting option for temporary extracorporeal liver support in the treatment of acute liver failure and for animal models for preclinical drug screening. Bioreactor capacity to eliminate drugs is generally used for assessing cell metabolic competence in different bioreactors or to scale-up bioreactor design and performance for clinical or preclinical applications. However, drug adsorption and physical transport often disguise the intrinsic drug biotransformation kinetics and cell metabolic state. In this study, we characterized the intrinsic kinetics of lidocaine elimination and adsorption by porcine liver cells cultured in 3D four-compartment hollow fiber membrane network perfusion bioreactors. Models of lidocaine transport and biotransformation were used to extract intrinsic kinetic information from response to lidocaine bolus of bioreactor versus adhesion cultures. Different from 2D adhesion cultures, cells in the bioreactors are organized in liver-like aggregates. Adsorption on bioreactor constituents significantly affected lidocaine elimination and was effectively accounted for in kinetic analysis. Lidocaine elimination and cellular monoethylglicinexylidide biotransformation featured first-order kinetics with near-to-in vivo cell-specific capacity that was retained for times suitable for clinical assist and drug screening. Different from 2D cultures, cells in the 3D bioreactors challenged with lidocaine were exposed to close-to-physiological lidocaine and monoethylglicinexylidide concentration profiles. Kinetic analysis suggests bioreactor technology feasibility for preclinical drug screening and patient assist and that drug adsorption should be accounted for to assess cell state in different cultures and when laboratory bioreactor design and performance is scaled-up to clinical use or toxicological drug screening.
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The clinical results of lung transplantation (LTx) are still less favorable than other solid organ transplants in both the early and long term. The fragility of the lungs limits the procurement rate and can favor the occurrence of ischemia-reperfusion injury (IRI). Ex vivo lung perfusion (EVLP) with Steen SolutionTM (SS) aims to address problems, and the implementation of EVLP to alleviate the activation of IRI-mediated processes has been achieved using mesenchymal stromal/stem cell (MSC)-based treatments. In this study, we investigated the paracrine effects of human amnion-derived MSCs (hAMSCs) in an in vitro model of lung IRI that includes cold ischemia and normothermic EVLP. We found that SS enriched by a hAMSC-conditioned medium (hAMSC-CM) preserved the viability and delayed the apoptosis of alveolar epithelial cells (A549) through the downregulation of inflammatory factors and the upregulation of antiapoptotic factors. These effects were more evident using the CM of 3D hAMSC cultures, which contained an increased amount of immunosuppressive and growth factors compared to both 2D cultures and encapsulated-hAMSCs. To conclude, we demonstrated an in vitro model of lung IRI and provided evidence that a hAMSC-CM attenuated IRI effects by improving the efficacy of EVLP, leading to strategies for a potential implementation of this technique.
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Células Epiteliales Alveolares/efectos de los fármacos , Isquemia Fría/métodos , Medios de Cultivo Condicionados/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Daño por Reperfusión/tratamiento farmacológico , Células A549 , Células Epiteliales Alveolares/metabolismo , Amnios/citología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Citocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , FN-kappa B/genética , Daño por Reperfusión/fisiopatologíaRESUMEN
Mesenchymal stem cells (MSCs) and their secreted extracellular vesicles have been used effectively in different lung disease animal models and clinical trials. Their specific beneficial effects, the potential differences between MSCs derived from different organs, and interactions between MSC products and target cells still need to be studied further. Therefore, we investigated the effects of secreted products of human MSCs derived from the bone marrow and adipose tissue on human lung small airway epithelial (AE) cells in vitro. AE cells were cocultured with MSCs in inserts that allowed the free exchange of medium but did not allow direct cell-to-cell contact. We examined the effects on AE cell viability, proliferation, cell numbers, expression of AE cell-specific genes, and CD54 (intercellular adhesion molecule 1 (ICAM1)) surface positivity, as well as the secretion/uptake of growth factors relevant for AE cell. We found that coculture increased the viability of AE cells. The majority of AE cells expressed CD54 on their surface, but the percentage of cells being positive for CD54 did not increase in coculture. However, ICAM1 gene expression was increased in coculture. Also, we observed increased gene expression of mucin (MUC1), a lung-enriched cell surface glycoprotein. These observed effects were the same between bone marrow and adipose tissue MSCs. However, MSCs derived from adipose tissue reduced angiopoietin concentrations in coculture, whereas those from the bone marrow did not. Conclusively, MSCs influenced AE cells positively by increasing their viability and affecting gene expression, with some effects being specific for the tissue origin of MSCs.
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Técnicas de Cocultivo/métodos , Células Epiteliales/citología , Pulmón , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Animales , Acuaporina 5 , Células de la Médula Ósea/citología , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , HumanosRESUMEN
Different approaches have been studied in both preclinical and clinical settings to develop cell-based therapies and/or engineered cell-based therapies to better integrate grafts with the host. In these techniques, much attention is addressed to the use of adult stem cells such as mesenchymal stem cells (MSCs), but identifying and obtaining sufficient numbers of therapeutic cells, and the right route of administration, is often a challenge. In this study, we tested the feasibility of encapsulating human amnion-derived MSCs (hAMSCs) in a semipermeable and biocompatible fiber as a new approach for regenerative medicine. Our data showed that hAMSCs aggregated in the device constitutes an effective system for enhancing, or at least for maintaining, the paracrine activity of these cells in order to better promote tissue regeneration in an immune isolated state. In our new experimental approach, the hAMSCs retained their therapeutic potential, as shown by both the production of specific immunomodulatory/angiogenic factors and immunomodulatory and angiogenic ability observed in vitro. Unlike cell infusion methods, the use of encapsulated-cells leads to minimally invasive approaches, avoiding a direct interaction with the host. Therefore, the potentiality of an allograft or xenograft without the need for immunosuppression, and the lack of tumorigenesis is very intriguing.
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Amnios/citología , Técnicas de Cultivo de Célula/instrumentación , Células Madre Mesenquimatosas/citología , Placenta/citología , Inductores de la Angiogénesis/metabolismo , Materiales Biocompatibles/química , Adhesión Celular , Agregación Celular , Técnicas de Cultivo de Célula/métodos , Movimiento Celular , Supervivencia Celular , Células Inmovilizadas/citología , Células Inmovilizadas/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Factores Inmunológicos/metabolismo , Células Madre Mesenquimatosas/metabolismo , EmbarazoRESUMEN
Various cell-based therapies are in development to address chronic and acute skin wound healing, for example for burns and trauma patients. An off-the-shelf source of allogeneic dermal cells could be beneficial for innovative therapies accelerating the healing in extensive wounds where the availability of a patient's own cells is limited. Human fetal-derived dermal fibroblasts (hFDFs) show high in vitro division rates, exhibit low immunological rejection properties, and present scarless wound healing in the fetus, and previous studies on human fetal tissue-derived cell therapies have shown promising results on tissue repair. However, little is known about cell lineage stability and cell differentiation during the cell expansion process, required for any potential therapeutic use. We describe an isolation method, characterize a population, and investigate its potential for cell banking and thus suitability as a potential product for cell grafting therapies. Our results show hFDFs and a bone marrow-derived mesenchymal stem cell (BM-MSC) line shared identification markers and in vitro multilineage differentiation potential into osteogenic, chondrogenic, and adipogenic lineages. The hFDF population exhibited similar cell characteristics as BM-MSCs while producing lower pro-inflammatory cytokine IL-6 levels and higher levels of the wound healing factor hepatocyte growth factor. We demonstrate in vitro differentiation of hFDFs, which may be a problem in maintaining long-term lineage stability, potentially limiting their use for cell banking and therapy development.
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Bancos de Muestras Biológicas , Técnicas de Cultivo de Célula/métodos , Feto/citología , Fibroblastos/citología , Células Madre Mesenquimatosas/citología , Piel/citología , Cicatrización de Heridas , Adipogénesis/genética , Linaje de la Célula/genética , Movimiento Celular/genética , Proliferación Celular/genética , Condrogénesis/genética , Citocinas/metabolismo , Feto/metabolismo , Fibroblastos/metabolismo , Fibroblastos/trasplante , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/genética , Piel/metabolismo , Trasplantes , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Human mesenchymal stem cells can be isolated from various organs and are in studies on therapeutic cell transplantation. Positive clinical outcomes of transplantations have been attributed to both the secretion of cytokines and growth factors as well as the fusion of donor cells with that of the host. We compared human mesenchymal stem cells from six different tissues for their transplantation-relevant potential. Furthermore, for prospective allogenic transplantation we developed a semipermeable hollow-fiber membrane enclosure, which would prevent cell fusion, would provide an immune barrier, and would allow for easy removal of donor cells from patients after recovery. We investigated human mesenchymal stem cells from adipose tissue, amniotic tissue, bone marrow, chorionic tissue, liver, and umbilical cord. We compared their multilineage differentiation potential, secretion of growth factors, and the expression of genes and surface markers. We found that although the expression of typical mesenchymal stem cell-associated gene THY1 and surface markers CD90 and CD73 were mostly similar between mesenchymal stem cells from different donor sites, their expression of lineage-specific genes, secretion of growth factors, multilineage differentiation potential, and other surface markers were considerably different. The encasement of mesenchymal stem cells in fibers affected the various mesenchymal stem cells differently depending on their donor site. Conclusively, mesenchymal stem cells isolated from different tissues were not equal, which should be taken into consideration when deciding for optimal sourcing for therapeutic transplantation. The encasement of mesenchymal stem cells into semipermeable membranes could provide a physical immune barrier, preventing cell fusion.
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Membrana Celular/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Biomarcadores/metabolismo , Adhesión Celular , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Forma de la Célula , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , L-Lactato Deshidrogenasa/metabolismoRESUMEN
Although the hepatic and hematopoietic progenitors of the liver are well characterized, the interactions between these two lineages remain mostly elusive. Hepatoblasts express delta-like noncanonical Notch ligand 1 (Dlk1), whose cleaved extracellular domain can become a soluble protein. We assessed the effects of DLK1 gene expression knockdown in cultures of total fetal liver cells. Furthermore, we separated Dlk1+ hepatoblasts from the total liver cell fraction and investigated effects of direct cell contact. Dlk1- cells were cultured either without Dlk1+ hepatoblasts, in direct contact with hepatoblasts, or separated from hepatoblasts by a porous membrane in inserts to inhibit cell contact but allow free exchange of molecules. Expression of the hepatic and hematopoietic genes, colony forming unit potential of various hematopoietic progenitors, and cell numbers and types were investigated. We found that DLK1 knockdown in total fetal liver cell cultures decreased total cell numbers. The expression of hepatic progenitor genes and mature hematopoietic genes was affected. Hematopoietic BFU-E and CFU-GM colony numbers were reduced significantly. The depletion of Dlk1+ hepatoblasts in culture decreased the potential of all hematopoietic progenitors to form colonies of all types and reduced the percentage of mature hematopoietic cells. The addition of hepatoblasts in inserts to Dlk1- cells further decreased the potential to form the CFU-GM and CFU-GEMM colonies and the percentage of mature hematopoietic cells but increased total cell numbers. Conclusively, direct contact of Dlk1 supports hematopoietic progenitor expansion and functionality that cannot be reconstituted in coculture without direct cell contact.
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Long-term in vitro expansion of hematopoietic stem cells (HSCs), while maintaining their functionality and multilineage differentiation potential, is still challenging. In this study, three-dimensional (3D) high-porosity hydroxyapatite (HA) foams have been designed to closely mimic the chemistry and physical structure of cancellous bone. Furthermore, calcium oxide was distributed in the HA ceramics to provide surface calcium ion release, hypothesizing that a local surface calcium gradient supports HSC localization and maintenance. Primary human HSCs and osteoblasts were cocultured for 6 weeks. Controls were cultured in two-dimensional dishes, while scaffold cultures were performed with calcium nitrate-infiltrated HA scaffolds and untreated HA scaffolds. Cells were analyzed for surface markers by flow cytometry, metabolic activity, and hematopoietic multilineage differentiation potential. The release of calcium into culture medium was also determined. The implementation of HA scaffolds had a positive effect on erythrocyte colony formation capacity of HSCs, with an increased osteoblast fraction observed when compared to control cultures without scaffolds. The presentation of scaffolds did not affect metabolic turnover when compared to control cultures. In conclusion, 3D open-porous HA scaffolds provide a bone-like structure and enable the long-term maintenance of primary HSCs.
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Calcio/farmacología , Durapatita/farmacología , Células Madre Hematopoyéticas/metabolismo , Andamios del Tejido/química , Calcio/química , Técnicas de Cultivo de Célula , Células Cultivadas , Durapatita/química , Células Madre Hematopoyéticas/citología , Humanos , PorosidadRESUMEN
Prolonged physiological stresses, including abnormal pH and temperature, are deleterious. However, human hepatic progenitors have been shown to be quite tolerant of temporary temperature stress such as in cold ischemia. We aimed at identifying how various stresses affect liver progenitors, and at determining whether distinct effects exist on different progenitor cells of the human liver. Total fetal liver cells were exposed to low (25°C), normal (37°C), or high (40°C) temperatures, or low (6.76), normal (7.35), or high (7.88) pH in vitro. Culture at 25°C increased cell numbers and percentages of proliferation marker Ki67+ total cells. In total cell cultures, percentages of CD326+ hepatic progenitors co-expressing DLK1 (delta-like 1 homolog), SSEA4, or CD90 increased, as well as proliferation of SSEA4+ and CD235a+ progenitors. Analyses of presorted hepatic progenitors revealed that culture at 25°C increased cell numbers of CD326+ hepatic stem/progenitor cells but not DLK+ hepatoblasts. The expression of several mesenchymal genes was reduced, and distinct hepatic stem/progenitor cell colonies emerged. At 40°C, numbers of adherent hepatic cells decreased but those of hematopoietic nonadherent cells increased. High pH did not cause major effects. Acidic pH resulted in decreased total cell numbers and affected hematopoietic cells. Percentages of DLK1+ hepatoblasts were increased, but those of hematopoietic mature CD45+ cells were decreased. In particular, proliferation of adherent hepatic CD326+, SSEA4+ progenitors, and hematopoietic CD45+ cells and CD235a+ erythroblasts was reduced. Conclusively, our data indicate that low-temperature stress stimulates hepatic progenitor and erythroblast proliferation, whereas acidic pH promotes hepatic maturation and reduces hematopoietic cells.
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Eritroblastos/citología , Hepatocitos/citología , Hígado/citología , Hígado/embriología , Células Madre/citología , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Separación Celular , Supervivencia Celular , Células Cultivadas , Isquemia Fría , Citometría de Flujo , Perfilación de la Expresión Génica , Edad Gestacional , Células Madre Hematopoyéticas/citología , Humanos , Concentración de Iones de Hidrógeno , Magnetismo , TemperaturaRESUMEN
Cell-spray autografting is an innovative early treatment option for deep partial-thickness burn wounds. As an alternative to non-operative management, cell-spray autografting can achieve rapid wound re-epithelialization, particularly in large wounds. When compared to traditional mesh autografting for deep partial-thickness burn wounds, cell-spray autografting can accomplish re-epithelialization with a much smaller donor site. In this review, we describe the development of a biomedical engineering method for isolation and immediate distribution of autologous, non-cultured, adult epidermis-, and adult dermis-derived stem cells. We present data on cell isolation procedures in 44 patients with deep partial-thickness burns performed over five years under an innovative practice IRB. Treated patients presented with a variety of burn wound etiologies and a wide range of TBSA. Overall clinical results were very satisfying. The average hospital length of stay following treatment was seven days. Over the time period, the donor-site to burn-wound surface area ratio was enhanced from 1:80 to 1:100. A detailed analysis of all process-related biotechnology and operative problems, pitfalls, and solutions was performed and is reported herein. Strategies for future clinical studies are discussed.
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Quemaduras/terapia , Separación Celular/métodos , Trasplante de Células/métodos , Repitelización , Ingeniería Biomédica , Superficie Corporal , Quemaduras/epidemiología , Desbridamiento , Humanos , Tiempo de Internación , Obesidad/epidemiología , Trasplante de Piel , Fumar/epidemiología , Trasplante Autólogo/métodos , Resultado del TratamientoRESUMEN
Epithelial Cell Adhesion Molecule (EpCAM), or CD326, is a trans-membrane glycoprotein expressed by multiple normal epithelia as well as carcinoma. Human hepatic stem cells and bile duct epithelium of the liver are EpCAM positive. In tumor cell lines, its intracellular domain can be released after cleavage of the extracellular domain. Within the cell nucleus, it induces cell proliferation, but cleavage depends on cell contact. Fragments of various lengths have been described in tumor cells. Despite its described important role in proliferation in tumor cells, there is not much known about the expression and role of EpCAM fragments in primary human liver cells. Here, we demonstrate that EpCAM protein fragments and function are considerable different between tumor cells, normal fetal and adult liver cells. Contrary to previously reported findings in tumor cells, gene knockdown or treatment with an inhibitor of the cleavage enzyme ADAM17 (TACE) rather increased cell numbers in primary human fetal liver-derived EpCAM-positive cells. EpCAM fragment sizes were not affected by treatment with inhibitor. Knockdown of EPCAM gene expression by siRNA in sorted cells did not significantly affect proliferation-associated genes or cell numbers. The intracellular domain could not be detected within cell nuclei of fetal and adult liver cells. In conclusion, signaling through the intracellular domain of EpCAM appears to be a mechanism that induces proliferation specifically in tumorigenic cells but not in normal primary EpCAM-positive liver cells.
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Células Madre Adultas/metabolismo , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Células Madre Fetales/metabolismo , Hígado/metabolismo , Células Madre Neoplásicas/metabolismo , Fragmentos de Péptidos/metabolismo , Transducción de Señal , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Células Madre Adultas/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Molécula de Adhesión Celular Epitelial/genética , Células Madre Fetales/efectos de los fármacos , Regulación de la Expresión Génica , Glicosilación , Células HT29 , Humanos , Ácidos Hidroxámicos/farmacología , Hígado/citología , Hígado/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Cultivo Primario de Células , Dominios Proteicos , Transducción de Señal/efectos de los fármacosRESUMEN
Foamed hydroxyapatite offers a three-dimensional scaffold for the development of bone constructs, mimicking perfectly the in vivo bone structure. In vivo, calcium release at the surface is assumed to provide a locally increased gradient supporting the maintenance of the hematopoietic stem cells niche. We fabricated hydroxyapatite scaffolds with high surface calcium concentration by infiltration, and used human umbilical vein endothelial cells (HUVECs) as a model to study the effects on hematopoietic lineage direction. HUVECs are umbilical vein-derived and thus possess progenitor characteristics, with a prospective potential to give rise to hematopoietic lineages. HUVECs were cultured for long term on three-dimensional porous hydroxyapatite scaffolds, which were either infiltrated biphasic foams or untreated. Controls were cultured in two-dimensional dishes. The release of calcium into culture medium was determined, and cells were analyzed for typical hematopoietic and endothelial gene expressions, surface markers by flow cytometry, and hematopoietic potential using colony-forming unit assays. Our results indicate that the biphasic foams promoted a hematopoietic lineage direction of HUVECs, suggesting an improved in vivo-like scaffold for hematopoietic bone tissue engineering.
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Diferenciación Celular , Linaje de la Célula , Durapatita/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ingeniería de Tejidos/métodos , Andamios del Tejido , Biomarcadores/metabolismo , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Fenotipo , Porosidad , Propiedades de Superficie , Factores de TiempoRESUMEN
Hydroxyapatite pellets, partially densified in a low-temperature heat treatment, were infiltrated with calcium nitrate solution followed by in-situ precipitation of Ca(OH)2 and CaCO3. The infiltrated bodies were then densified to high relative density and the calcium carbonate transformed to calcium oxide during sintering and resulted in biphasic hydroxyapatite-CaO ceramics. This work investigated the influence of the infiltration on surface morphology, weight change, and microstructural-level degradation caused by exposure to saline at pH=7.4 and a temperature of 20°C. The CaO rendered the materials more susceptible to degradation, and released calcium into the saline faster than single phase, calcium deficient hydroxyapatite (HA) that were used as a control. In consequence, these ceramics could be used to release calcium into the culture microenvironments of bone tissue or bone marrow cells next to a scaffold surface.
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Compuestos de Calcio/química , Calcio/química , Cerámica/química , Durapatita/química , Ensayo de Materiales/métodos , Óxidos/química , Microscopía , Solubilidad , Soluciones , Propiedades de Superficie , Factores de Tiempo , Agua/química , Difracción de Rayos XRESUMEN
BACKGROUND: Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes. METHODS: Baboons (n=3) were recipients of GTKO/hCD46 pig hepatocytes. All three baboons received anti-thymocyte globulin (ATG) induction and tapering methylprednisolone. Baboon 1 received maintenance immunosuppressive therapy with tacrolimus and rapamycin. Baboons 2 and 3 received an anti-CD40mAb/rapamycin-based regimen that prevents sensitization to pig solid organ grafts. The baboons were euthanized 4 or 5 weeks after hepatocyte transplantation. The baboon immune response was monitored by the measurement of anti-non-Gal IgM and IgG antibodies (by flow cytometry) and CFSE-mixed lymphocyte reaction. Monitoring for hepatocyte survival and function was by (i) real-time PCR detection of porcine DNA, (ii) real-time PCR for porcine gene expression, and (iii) pig serum albumin levels (by ELISA). The sites of hepatocyte injection were examined microscopically. RESULTS: Detection of porcine DNA and porcine gene expression was minimal at all sites of hepatocyte injection. Serum levels of porcine albumen were very low-500-1000-fold lower than in baboons with orthotopic pig liver grafts, and approximately 5000-fold lower than in healthy pigs. No hepatocytes or infiltrating immune cells were seen at any of the injection sites. Two baboons (Baboons 1 and 3) demonstrated a significant increase in anti-pig IgM and an even greater increase in IgG, indicating sensitization to pig antigens. DISCUSSION AND CONCLUSIONS: As a result of this disappointing experience, the following points need to be considered. (i) Were the isolated pig hepatocytes functionally viable? (ii) Are pig hepatocytes more immunogenic than pig hearts, kidneys, artery patch grafts, or islets? (iii) Does injection of pig cells (antigens) into the spleen and/or lymph nodes stimulate a greater immune response than when pig tissues are grafted at other sites? (iv) Did the presence of the recipient's intact liver prevent survival and proliferation of pig hepatocytes? (v) Is pig CD47-primate SIRP-α compatibility essential? In conclusion, the transplantation of genetically engineered pig hepatocytes into multiple sites in immunosuppressed baboons was associated with very early graft failure. Considerable further study is required before clinical trials should be undertaken.
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Supervivencia de Injerto/inmunología , Hepatocitos/inmunología , Trasplante Heterólogo , Animales , Animales Modificados Genéticamente , Anticuerpos/inmunología , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Antígenos/inmunología , Supervivencia de Injerto/efectos de los fármacos , Hepatocitos/trasplante , Terapia de Inmunosupresión/métodos , Inmunosupresores/farmacología , Papio hamadryas/inmunología , Porcinos , Trasplante Heterólogo/métodosRESUMEN
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.
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Quemaduras/terapia , Separación Celular/métodos , Queratinocitos/trasplante , Trasplante de Piel/métodos , Superficie Corporal , Recuento de Células , Centrifugación , Colagenasas , Endopeptidasas , Humanos , Técnicas In Vitro , Sitio Donante de Trasplante , Trasplante Autólogo , Tripsina , Cicatrización de HeridasRESUMEN
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.
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Traumatismos del Brazo/terapia , Quemaduras/terapia , Separación Celular/métodos , Traumatismos de la Mano/terapia , Queratinocitos/trasplante , Repitelización , Trasplante de Piel/métodos , Traumatismos Torácicos/terapia , Adolescente , Adulto , Quemaduras Químicas/terapia , Quemaduras por Electricidad/terapia , Endopeptidasas , Humanos , Trasplante Autólogo , Tripsina , Cicatrización de HeridasRESUMEN
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.
RESUMEN
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.
Asunto(s)
Reactores Biológicos , Técnicas de Cultivo de Célula/instrumentación , Perfusión/instrumentación , Aire/análisis , Animales , Dióxido de Carbono/análisis , Células Cultivadas , Diseño de Equipo , Gases/análisis , Humanos , Ingeniería de Tejidos/instrumentaciónRESUMEN
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.
Asunto(s)
Quemaduras/terapia , Queratinocitos/trasplante , Apósitos Oclusivos , ARN Mensajero/metabolismo , Antígenos CD34/genética , Apoptosis/genética , Caspasa 3/genética , Técnicas de Cultivo de Célula , Supervivencia Celular , Trasplante de Células , Células Cultivadas , Materiales Biocompatibles Revestidos , Expresión Génica , Humanos , Técnicas In Vitro , Integrina alfa6/genética , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinas/genética , Antígeno Ki-67/genética , Proteínas de la Membrana/genética , Microscopía de Contraste de Fase , Fenoles , Repitelización , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Siliconas , Cicatrización de Heridas , Receptor fas/genéticaRESUMEN
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 × 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 × 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.