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PubMedDate not supplied

Spatial composition of prostate cancer spheroids in mixed and static cultures.

Aggregation of neoplastic cells produces multicellular spheroids resembling micrometastases. The objective of this study was to investigate the effects of mixing culture medium on the spatial composition of spheroids prepared from well (LNCaP) and poorly (DU 145) differentiated human prostate cancer cells. Spheroids were cultured in a mixed suspension within a high-aspect rotating wall vessel and static liquid-overlay plate. Results from this study demonstrate that mixed cultures consistently manifested differences in morphology and composition between DU 145 and LNCaP spheroids. For example, 40 +/- 12% of DU 145 cells were Ki-67 positive 100 microm from the surface within mixed spheroids versus 0% for LNCaP cells; there was no significant difference in this spatial profile for static cultures. The results suggest that poorly differentiated spheroids may be more likely to experience a change in composition from mixing culture medium than well-differentiated spheroids, due to low tissue density. Immunostaining for P-glycoprotein is representative of this trend; average staining intensity increased 50% for DU 145 spheroids on mixing but was unchanged for LNCaP spheroids. The effects of mixing on spheroid composition were attributed to faster interstitial mass transport. Applications include drug development and delivery, as well as basic research on drug action and resistance.

Cell Line, Tumor↗
PubMedDate not supplied

Ultrastructural and functional investigations of adult hepatocyte spheroids during in vitro cultivation.

Cultivation of primary adult hepatocytes creates a challenge because of their loss of hepatocellular functions if prevented from attaining polarized cell-cell and cell-matrix interactions. Thus, when hepatocytes are seeded in nonadhesive porous alginate scaffolds, they form 100-microm-diameter spheroids with enhanced cell-cell interactions. Using transmission electron microcopy (TEM), histology, and functional studies, we investigated the state of hepatocyte spheroids during in vitro cultivation. TEM of day 3 spheroids revealed multiple cell layers, with tight junctions between adjacent cells and microvillus-lined channels that resembled bile canaliculi, both structurally and functionally. When copper ions were added to the external medium, the spheroidal hepatocytes performed endocytosis and eventually secreted the heavy metal ions into the bile lumens. From day 8 on, however, there was a rapid decline in cell viability. Histology and TEM analysis of day 13 spheroids revealed a necrotic center, with one viable cell layer on the outskirts. The absence of DNA laddering and negative results in TUNEL assay indicated that apoptosis is not the main process leading to cell death. Cell necrosis may be a result of accumulated bile secretions in the compacted spheroids. Collectively, our results suggest that spheroids derived from adult hepatocytes may have limited utility in long-term applications.

Animals↗
PubMedDate not supplied

Influence of pancreatic islets on spheroid formation and functions of hepatocytes in hepatocyte-pancreatic islet spheroid culture.

Hepatotrophic stimulation of hepatocytes is necessary to preserve long-term function of hepatocytes in hepatocyte transplantation and bioartificial liver system. The main source of hepatotrophic factors in portal venous blood seems to be the pancreatic islets. It was also reported that hepatocyte spheroids, tightly packed multicellular aggregates, showed enhanced liver-specific activities and a prolonged differentiated state compared with cells that were maintained as a monolayer. On the basis of these two facts, the authors tried to form hepatocyte-pancreatic islet spheroids and to evaluate the influence of pancreatic islets on spheroid formation and functions of hepatocytes in spheroid culture. Hepatocytes and pancreatic islet cells were harvested from adult male Sprague-Dawley rats weighing 200-250 g. Hepatocytes were cultured in spinner flasks with either basic nonstimulated medium (hepatocytes only [group BH] and cocultures with islet cells [group BI]) or hormone-stimulated medium (hepatocytes only [group HH] and cocultures with islet cells [group HI]). The size and morphology of spheroids, as determined by phase-contrast microscopy, and liver-specific functions, such as albumin secretion, urea synthesis, and ammonia removal, were compared among groups. The results were as follows: the size of spheroids, 66 +/- 53.4 microm, in group BH on day 2 was smaller than in group BI (179 +/- 66.2 microm on day 2, p < 0.05). In group BI, group HH, and group HI, smooth spheroids were observed on culture day 2. However, in group BH rugged incomplete aggregates were observed on the same day. In groups with basal medium, group BI showed better results in terms of hepatocyte-specific function such as albumin secretion, urea synthesis, and ammonia removal compared with group BH on days 2 and 3 (p < 0.05). In groups with hormone-defined medium, cocultures had no impact on albumin secretion rate, urea synthetic rate, and ammonia removal rate. In conclusion, we made a new type of hepatocyte-pancreatic islet spheroid, using a rotational culture method. Pancreatic islets in a spheroid culture system stimulated hepatocyte spheroid formation and some hepatocyte-specific functions in vitro.

Animals↗
PubMedDate not supplied

Dexamethasone effects on rat hepatocyte spheroid formation and function.

Hepatocytes cultured on moderately adhesive surfaces or in spinner flasks spontaneously self-assemble into spherical tissue-like aggregates (spheroids). These spheroids have smooth surfaces and tissue-like polarized cell morphology, including bile canalicular-like channels, and maintain high viability and liver-specific functions for extended culture periods. Dexamethasone (DEX), a synthetic glucocorticoid, is known to elicit various responses in gene expression, and is often added to hepatocyte culture medium. The morphology and liver-specific protein production of hepatocyte spheroids were assessed under DEX concentrations ranging from 50 nM to 10 microM. DEX altered the kinetics of spheroid formation in a concentration-dependent fashion, with increasing concentrations inhibiting aggregation and promoting aggregate disassembly on culture dishes. DEX addition to spinner cultures resulted in smaller, more irregularly shaped spheroids and a higher incidence of aggregate clumping. Albumin and urea production were also higher in DEX cultures, but this effect was not as sensitive to concentration and occurred irrespective of the state of aggregation. RTPCR was utilized to assess the mRNA levels of extracellular matrix proteins, E-cadherin, and cytochrome P-450 enzymes. Results indicated a slight increase in fibronectin and collagen III mRNA early in the cultures, possibly contributing to the changes in morphology observed.

Animals↗
PubMed2006

Antagonizing inactivated tumor suppressor genes and activated oncogenes by a versatile transgenesis system: application in mantle cell lymphoma.

A broad range of malignant diseases, such as mantle cell lymphoma (MCL), is associated with complex genomic alterations, demanding multimodal functional testing of candidate genes. To assess such candidate disease genes, we have developed a bidirectional targeted transgenesis tool, which allows well-controlled modulation of individual gene activities within a cellular MCL system. The engineered versatile transgenesis system permits functional analysis of virtually any candidate gene: for tumor suppressor genes by complementation via integration of respective genomic DNA or for oncogenes by inactivation via integrated shRNA coding plasmids. Complementation by genomic DNA ensures wild-type (WT) regulated gene expression, whereas genomic integration of shRNA coding inserts by an advanced RNAi-strategy mediates specific knock-down of gene expression. Site-specific genomic integration of an unmodified BAC, which contains the CDKN2A/B genes absent in the MCL model system, restored CDKN2A/B expression resulting in the inhibition of cell proliferation. CCND1, strongly overexpressed in the model system, was down-regulated via shRNA expression, again inhibiting proliferation. Notably, the presented site-specific shRNA-strategy circumvents interference by IFN-response induced when using other RNAi gene knock-down methods. In conclusion, we here demonstrate that adequate restoration of a range of different gene activities yields in a desired antiproliferative effect in MCL-derived cells. By antagonizing inactivated tumor suppressor genes or activated oncogenes, the presented approach can be readily used for the functional analysis of a broad range of disease-related genetic defects.

Cell Line, Tumor↗
PubMed2001

Aligned microcontact printing of biomolecules on microelectronic device surfaces.

Microcontact printing (muCP) of extracellular matrix proteins is a fascinating approach to control cell positioning and outgrowth, which is essential in the development of applications ranging from cellular biosensors to tissue engineering. Microelectronic devices can be used to detect the activity from a large number of recording sites over the long term. However, signals from cells can only be recorded at small sensitive spots. In this paper, we present an innovative setup to perform aligned muCP of extracellular matrix proteins on microelectronic devices in order to guide the growth of electrogenic cells specifically to these sensitive spots. Our system is based on the combination of a fine-placer with redesigned micro stamps having a rigid glass cylinder as backbone for attachment in the alignment tool. Alignment is performed moving the device with an optical table under microscopic control of the superimposed images from stamp and device surface. After successful alignment, the stamp is brought into contact with the device surface by means of a high-precision lever. With our setup, we were able to pattern up to 40 devices per hour. A lateral alignment accuracy of < 2microm has been achieved. Aligned neuronal growth on patterned devices was demonstrated with dissociated hippocampal neurons.

Animals↗
PubMed2005

Artificial skin as a valuable adjunct to surgical treatment of a large squamous cell carcinoma in a patient with epidermolysis bullosa.

BACKGROUND: Among tissue-engineered skins, two bilayered cellular constructs and one cryopreserved dermal substitute have been approved for the treatment of epidermolysis bullosa. Nevertheless, the application of artificial skin technology to surgical treatment of squamous cell carcinomas in a patient with epidermolysis bullosa has never been reported. OBJECTIVE: To reconstruct the large defect remaining after squamous cell carcinoma excision in a patient with dominantly inherited dystrophic epidermolysis bullosa. METHODS: To apply a 10 x 15 cm Integra sheet (Integral Life-sciences Corporation, Plainsboro, NJ, USA) (an acellular collagen matrix coated with a thin polysiloxane elastomer) to the excised area and 3 weeks later to cover the Integra sheet with an ultrathin meshed skin graft. RESULTS: The graft take was complete, and the donor site totally regenerated, except for three small bullae at 7 weeks postoperatively. CONCLUSION: Integra offers the advantage of filling huge defects with its dermal layer of collagen fibers and provides an optimal graft bed. This first step makes it possible to use very thin grafts 3 weeks later.

Adult↗
PubMed1993

New developments in the virus therapy of cancer: a historical review.

Since the 1920s, viruses had been used for oncolysis. Natural human viral infections can rarely induce remissions of leukemias or lymphomas. Inoculation of tumor-bearing patients with live viruses very seldom resulted in durable complete remissions. Genetically engineered or tumor-adapted virus strains may perform better. Virally modified tumor cell membrane vaccines can induce in the host rejection strength antitumor immunity. Modern technology and much more work is needed before the optimal procedures for viral oncolysis or active antitumor immunization with virally modified tumor cell vaccines are learned and can be implemented in the clinical practice. Laboratory monitoring of the host's immunological reactions accompanying failure and success of tumor rejection is essential for the recognition and duplication of the successful and for the avoidance of the unsuccessful interventions.

Genetic Engineering↗
PubMed2004

Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice.

Recent evidence indicates that vascular progenitor cells may be the source of smooth muscle cells (SMCs) that accumulate in atherosclerotic lesions, but the origin of these progenitor cells is unknown. To explore the possibility of vascular progenitor cells existing in adults, a variety of tissues from ApoE-deficient mice were extensively examined. Immunohistochemical staining revealed that the adventitia in aortic roots harbored large numbers of cells having stem cell markers, e.g., Sca-1(+) (21%), c-kit(+) (9%), CD34(+) (15%), and Flk1(+) cells (4%), but not SSEA-1(+) embryonic stem cells. Explanted cultures of adventitial tissues using stem cell medium displayed a heterogeneous outgrowth, for example, islands of round-shaped cells surrounded by fibroblast-like cell monolayers. Isolated Sca-1(+) cells were able to differentiate into SMCs in response to PDGF-BB stimulation in vitro. When Sca-1(+) cells carrying the LacZ gene were transferred to the adventitial side of vein grafts in ApoE-deficient mice, beta-gal(+) cells were found in atherosclerotic lesions of the intima, and these cells enhanced the development of the lesions. Thus, a large population of vascular progenitor cells existing in the adventitia can differentiate into SMCs that contribute to atherosclerosis. Our findings indicate that ex vivo expansion of these progenitor cells may have implications for cellular, genetic, and tissue engineering approaches to vascular disease.

Animals↗
PubMed2006

Self-management protocol for a moving-actuator type artificial heart.

In this paper, we describe the development of a self-management protocol for a controller of a moving-actuator type artificial heart, AnyHeart. The developed protocol analyzes motor current signals and detects abnormal pumping statuses. If preset abnormal pumping statuses are detected by an implemented algorithm, a controller triggers an emergency management procedure and transmits an alarm message to predetermined medical and engineering staffs via a cellular phone network to notify them of an abnormal pumping status occurrence and its likely cause. Results of in vitro performance experiments showed that the developed protocol can detect simulated abnormalities in motor current, manage the operating status of the blood pump during an emergency, and transmit an alarm message as desired.

Algorithms↗
PubMed2005

Fibrin sealant tissue adhesive--review and update.

The field of surgical tissue adhesives is rapidly developing, with multiple products now available. Fibrin sealant, the first widely available tissue adhesive, continues to be used in traditional as well as in new and creative ways. The product is now fully mature as a hemostatic agent in cardiac, liver, and spleen surgery. It is also useful as a sealant in a variety of clinical applications, including procedures such as colonic anastomosis, as well as in seroma prevention following soft tissue dissection. Newer uses such as reduced suture vascular and intestinal anastomosis, fistula closure, surgery in patients with hemophilia, and laparoscopic/endoscopic procedures are being studied in both laboratory and clinical settings. The most recent additions to the capabilities of fibrin sealant include the use of this tissue adhesive as a slow-release drug delivery system and as a substrate for cellular growth and tissue engineering. This article will review the basis of fibrin sealant effectiveness and will also cover the recent developments in the rapidly evolving employment of this material.

Algorithms↗
PubMed2003

Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion.

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte-colony stimulating factor. By week 4-5, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule-1, human intracellular adhesion molecule-1, human CD31, E-selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34(+) cells, and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.

Antibodies↗
PubMed1999

Gene therapy for the treatment of AIDS: animal models and human clinical experience.

Although antiretroviral drug therapy has had a significant impact on the natural history of HIV infection, complete virus eradication still remains an unattainable goal. Drug-mediated virological control only occurs transiently, in part as a result of the development of drug resistance. Gene therapy for the treatment of AIDS is a promising area of research that has as its goal the replacement of the HIV-infected cellular pool with cells engineered to resist virus replication. A variety of anti-HIV genes have been designed and tested in laboratory systems, and available results from pilot clinical trials demonstrate the safety and feasibility of this approach. Obstacles to effective application of this technology include partial protection of HIV resistance genes, lack of effective vectoring systems, and unregulated gene expression. Herein, we review recent advances in transduction methods, data from in vivo preclinical studies in relevant animal models, and emerging results derived from pilot clinical gene therapy studies.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Expression of MMPs and TIMPs in primary epithelial cell cultures of the upper aerodigestive tract seeded on the surface of a novel polymeric biomaterial.

INTRODUCTION: Using standard cell biological and biochemical experimental approaches we were able to test the ability of a particular polymer construct to support the adhesion, proliferation, and the cellular acitivity of pharyngeal cells. The delicate balance between Matrix Metalloproteinases (MMPs) and their endogenous inhibitors (Tissue Inhibitor of MMPs, TIMPs) have a decisive function in the remodeling of the extracellular matrix during cellular ingrowth. Novel polymeric biomaterials may be useful to develop new therapeutic options in head and neck surgery. METHODS: Primary cell cultures of the pharynx of Sprague-Dawley rats were seeded on the surface of a thermoplastic multi-block copolymer and on a polystyrene surface as control. Conditioned media of the primary cells was analyzed for MMPs and TIMPs. The MMP and TIMP expression was analysed by zymography and a radiometric enzyme assay. RESULTS: No statistically significant differences in the levels of MMP-1, MMP-2, MMP-9 and TIMPs were detected between cells grown on the novel polymer surface versus control. CONCLUSION: An appropriate understanding of the molecular machinery that regulates gene expression and cellular growth in tissue engineered constructs is the requirement for an optimal adaptation of biodegradable biomaterials to develop new therapeutic options in otolaryngology and head and neck surgery.

Animals↗
PubMed2005

The role of stem cells in atherosclerosis.

Accumulating evidence indicates the involvement of stem cells and/or progenitors in the development of arteriosclerosis, including transplant arteriosclerosis, angioplasty-induced restenosis, vein graft atherosclerosis and spontaneous atherosclerosis. Recently, it was demonstrated that stem/progenitor cells existing in the circulation and adventitia contribute to endothelial repair and smooth muscle cell (SMC) accumulation. Atherosclerosis can be initiated by endothelial death in specific areas, e.g. bifurcation regions, and subsequent replacement by stem/progenitor cells. Meanwhile, progenitor cells from blood and the adventitia migrate into the intima where they proliferate and differentiate into neo-SMC. Stem/progenitor cells are responsible for the formation of atherosclerotic lesions, which appear as an inflammatory disease. Thus, these cells may be a source of endothelial cells and SMC, and might have implications for cellular, genetic, and tissue engineering approaches to vascular disease.

Arteriosclerosis↗
PubMed1990

Double staining to increase the sensitivity of protein detection in polyacrylamide gels.

As more and more researchers are examining proteins that are available only in extremely limited quantities, i.e., cellular extracts or genetic engineering products, it is critical to utilize staining methods that maximize sensitivity. The protocol we describe here--double staining of polyacrylamide electrophoresis gels with Pro-Blue (colloidal blue stain) followed by silver staining--yields an extremely sensitive, nonspecific protein stain. On average, this double-staining technique resulted in a 40-fold increase in sensitivity and intensity vs. silver stain alone. This is a tremendous return for a small investment in additional time and materials.

Electrophoresis, Polyacrylamide Gel↗