Characterization of CD326-positive human hepatic stem cells.

AIM OF THE STUDY: CD326 has been used as a single marker to enrich for hepatic stem cell populations in the liver. However, bile duct epithelium is also positive for CD326, which impedes the selection of pure hepatic stem cell populations. Some markers have been proposed to be co-expressed by hepatic stem cells but these have not been systematically compared. Therefore, we determined the percentages and compared the characteristics of human liver cells expressing potential stem cell surface markers. MATERIAL AND METHODS: We analyzed CD326 expression in human liver tissues from fetal, neonatal, pediatric, and adult stages using immunohistochemistry. In flow cytometry, we quantified fetal liver cells for their co-expression of CD326 with CD56, CD117, CD44, CD90, CD49f, LGR5 and SSEA4. We analyzed the various fractions for their quantitative expression of genes typically associated with progenitors and hepatic lineages. RESULTS: 12.5% of cells were positive for CD326; of these, 63.5% co-expressed CD44. The lowest co-expression percentages were for SSEA4 (2.1%) and LGR5 (0.7%). Fractions revealed distinct gene expression patterns. Of all combinations, cells that co-expressed surface CD326 and SSEA4 demonstrated the highest gene expression for the proliferation marker MKi67 and hepatic markers DLK1, AFP and ALB, and were the only fraction negative for the biliary epithelial marker KRT19. Histology of adult and fetal liver showed cells positive for CD326 and SSEA4 but negative for CK19. CONCLUSIONS: CD326-positive cells represent a heterogeneous population, which in combination with SSEA4 potentially distinguishes bile duct epithelium from hepatic stem cells. These findings can help to further classify human hepatic progenitor stages.

Conditioned Medium from Human Amnion-Derived Mesenchymal Stromal/Stem Cells Attenuating the Effects of Cold Ischemia-Reperfusion Injury in an In Vitro Model Using Human Alveolar Epithelial Cells.

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.

The degradation behavior of calcium-rich hydroxyapatite foams in vitro.

Hydroxyapatite (HA) is a well-known regenerative biomaterial. However, the slow degradation rate of HA is still an obstacle in clinical applications. In this study, we concentrated on investigating the degradation behavior of the calcium-rich HA foams, which had a demonstrated effect on blood differentiation in previous studies. The HA foams were processed by an emulsion method and were infiltrated with calcium nitrate to create a calcium carbonate second phase, heterogeneously distributed on and under the surface of the foam. During the 28-day solubility test, the calcium carbonate phase contributed to enhanced Ca2+ ion release into the saline compared to phase pure HA foams. Both types of foams were biocompatible as demonstrated by human endothelial cell culture on their surface. The release of calcium ions, the degradation behavior, and the endothelial cell differentiation behavior suggest this biphasic ceramic is a candidate for bone marrow in vitro culture and a possible bone substitute material.

Effects of Mesenchymal Stem Cell Coculture on Human Lung Small Airway Epithelial Cells.

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.

Tissue Engineering and Regenerative Medicine Therapies for Cell Senescence in Bone and Cartilage.

The bone repair and substitute have been developed for decades with the bone defect repair applied successfully in clinical. However, implant complications may occur in more challenging situations, bone cell senescence and osteoporosis (OP). Due to certain microenvironment conditions, including hormonal, nutritional, and aging factors, the bone cell responses are regulated to alteration and end into cell senescence and OP. Thus, the bone tissue regeneration is limited with site morbidity increased, leading to bone graft failure. In such pathological state, bone grafts and substitutes are companied with OP therapies to improve bone tissue engineering and to enhance bone graft healing. Those substitutes with OP therapeutic-based applications have been becoming a growing field of interest in bone repair. Bone grafts, such as scaffolds with antiosteoporotic drugs releasing and implants with surface therapeutic modified, are studied to significantly increase low bone density as well as improve impaired bone regeneration. In this review, we discuss a thorough understanding in bone remodeling process, bone cell senescence, and mechanisms of OA and OP. Based on the understanding, the review concentrates on the treatments to the OP and the bone grafts applied in the bone senescence model and provides a future direction in clinical trial. Impact Statement This review discusses the tissue engineering and regenerative medicine therapies for aging-related bone diseases, which have been highly concerned in aging society. These therapies can improve the efficacy of bone tissue regeneration, enhance the quality of bone repair, and provide new insights in the field of bone clinical treatments and aging regeneration.

Comparative study of the production of soluble factors in human placenta-derived mesenchymal stromal/stem cells grown in adherent conditions or as aggregates in a catheter-like device.

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.

Isolation and Characterization of a Human Fetal Mesenchymal Stem Cell Population: Exploring the Potential for Cell Banking in Wound Healing Therapies.

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.

Characterization of Human Mesenchymal Stem Cells from Different Tissues and Their Membrane Encasement for Prospective Transplantation Therapies.

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.

Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors.

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.

Hepatic progenitors of the fetal liver: Interactions with hematopoietic stem cells.

The aim of this review is to summarize and give an overview on the findings of signaling between hepatic and hematopoietic progenitors of the liver. To date, there are not many findings published in the field, and the aim of this review is to cover all current publications in this area. The liver is the main site of hematopoiesis during fetal development. However, little is known about how hepatic and other non-hematopoietic progenitors potentially influence hematopoiesis and vice versa. The concurrent peaks of hepatic and hematopoietic progenitor proliferation during development indicate interactions that could possibly be mediated through cell-cell contact, extracellular matrices, cytokines and growth factors, or other signaling molecules. For example, hepatic progenitors, such as hepatic stem cells and hepatoblasts, possess characteristic surface markers that can be cleaved, giving rise to fragments of various lengths. A surface molecule of hepatoblasts has been demonstrated to play an essential role in hematopoiesis. Particularly, these effects on hematopoiesis were distinct, depending on whether it was membrane-bound or cleaved. In this review, the various hepatic and hematopoietic progenitor cell types are concisely described, and the current findings of their potential interactions are summarized.

Calcium-Infiltrated Biphasic Hydroxyapatite Scaffolds for Human Hematopoietic Stem Cell Culture.

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.

Response of Human Fetal Liver Progenitor Cell Types to Temperature and pH Stresses In Vitro.

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.

Epithelial cell adhesion molecule fragments and signaling in primary human liver cells.

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.

Effect of Calcium-Infiltrated Hydroxyapatite Scaffolds on the Hematopoietic Fate of Human Umbilical Vein Endothelial Cells.

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.

A microstructural study of the degradation and calcium release from hydroxyapatite-calcium oxide ceramics made by infiltration.

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.

Transplantation of hepatocytes from genetically engineered pigs into baboons.

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.

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells.

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.

Response of Primary Human Bone Marrow Mesenchymal Stromal Cells and Dermal Keratinocytes to Thermal Printer Materials In Vitro.

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.

Multicompartmental Hollow-Fiber-Based Bioreactors for Dynamic Three-Dimensional Perfusion Culture.

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.