Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data.

INTRODUCTION: Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). METHODS: The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. RESULTS: The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties. CONCLUSIONS: We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.

Use of gene-modified keratinocytes and fibroblasts to enhance regeneration in a full skin defect.

BACKGROUND: With the development of cell-based gene transfer techniques, genetically modified human keratinocytes (Kc) and fibroblasts (Fb) have been proven to be a better choice in wound repair. METHODS: This study was designed to construct in one step a gene-modified artificial skin by a genetically engineered Kc expressing PDGF-BB and Fb expressing VEGF(165) and bFGF. The wound healing effect in a full-thickness wound model was then observed. Unmodified artificial skin served as control. On the post-operative days 7, 14, and 21, residual wound area was calculated and skin wound tissues were subjected to biopsy for further investigation. RESULTS: Compared with unmodified artificial skin, gene-modified artificial skin resulted in a reduced wound contraction and a well-organized human epidermis and better formed dermis. CONCLUSIONS: The results suggest that our two-layer, gene-modified artificial skin improved both vascularization and epidermalization for skin regeneration. This technique could bring about a new approach in the treatment of burns and chronic wounds.

Mesenchymal stem cell-dependent formation of heterotopic tendon-bone insertions (osteotendinous junctions).

Ligament-to-bone and tendon-to-bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)-signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon-inherent interference with bone formation and may thus allow the stem cell-dependent formation of tendon-bone interfaces. The tenogenic and osteo-/chondrogenic competence was mediated by the adeno- and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage-type OTJs, while, in contrast, the viral modification of primary human bone marrow-derived mesenchymal stromal/stem cells showed evidence of fibrous tendon-bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures. These findings may contribute to the establishment of stem cell-dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually.

Quality of Cell Products: Authenticity, Identity, Genomic Stability and Status of Differentiation.

Cellular therapies that either use modifications of a patient's own cells or allogeneic cell lines are becoming in vogue. Besides the technical issues of optimal isolation, cultivation and modification, quality control of the generated cellular products are increasingly being considered to be more important. This is not only relevant for the cell's therapeutic application but also for cell science in general. Recent changes in editorial policies of respected journals, which now require proof of authenticity when cell lines are used, demonstrate that the subject of the present paper is not a virtual problem at all. In this article we provide 2 examples of contaminated cell lines followed by a review of the recent developments used to verify cell lines, stem cells and modifications of autologous cells. With relative simple techniques one can now prove the authenticity and the quality of the cellular material of interest and therefore improve the scientific basis for the development of cells for therapeutic applications. The future of advanced cellular therapies will require production and characterization of cells under GMP and GLP conditions, which include proof of identity, safety and functionality and absence of contamination.

Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation.

BACKGROUND: Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (alphaT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. alphaT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser)3 amino acid sequence. RESULTS: Coexpression of alphaT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with alphaT2ib indicated interaction of alphaT2ib with its cognate antigen within cells. alphaT2ib inhibited NF-kappaB driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding alphaT2ib into HEK293 cells demonstrated high efficiency of the TLR2-alphaT2ib interaction. The alphaT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-alphaT2ib. Transduction with AdValphaT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNFalpha mRNA accumulation, as well as TNFalpha translation and release by macrophages were largely abrogated upon transduction of alphaT2ib. alphaT2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdValphaT2ib. Systemic transduction applying AdValphaT2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation. CONCLUSION: The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation in vitro and ex vivo. This indicates a therapeutic potential of alphaT2ib in microbial or viral infections.

Reduction of circulating soluble Flt-1 alleviates preeclampsia-like symptoms in a mouse model.

Preeclampsia (PE) is characterized by widespread endothelial damage with hypertension, proteinuria, glomeruloendotheliosis and elevated soluble Flt-1 (sFlt-1), a natural occurring antagonist of vascular endothelial growth factor (VEGF). Cancer patients receiving anti-VEGF therapy exhibit similar symptoms. We suggested that a decrease in circulating sFlt-1 would alleviate the symptoms associated with PE. Adenoviral (Adv) overexpression of sFlt-1 induced proteinuria, caused glomerular damage and increase in blood pressure in female Balb/c mice. Circulating level of sFlt-1 above 50 ng/ml plasma induced severe vascular damage and glomerular endotheliosis. Albumin concentration in urine was elevated up to 30-fold, compared to control AdvGFP-treated animals. The threshold of kidney damage was in the range of 20-30 ng/ml sFlt-1 in plasma (8-15 ng/ml in urine). Co-administration of AdvsFlt-1 with AdvVEGF to neutralize circulating sFlt-1 resulted in more than a 70% reduction in free sFlt-1 in plasma, more than 80% reduction in urine and rescued the damaging effect of sFlt-1 on the kidneys. This demonstrates that below a critical threshold sFlt-1 fails to elicit damage to the fenestrated endothelium and that co-expression of VEGF is able to rescue effects mediated by sFlt-1 overexpression.

Evaluating maturation and genetic modification of human dendritic cells in a new polyolefin cell culture bag system.

BACKGROUND: Dendritic cells (DCs) are applied worldwide in several clinical studies of immune therapy of malignancies, autoimmune diseases, and transplantations. Most legislative bodies are demanding high standards for cultivation and transduction of cells. Closed-cell cultivating systems like cell culture bags would simplify and greatly improve the ability to reach these cultivation standards. We investigated if a new polyolefin cell culture bag enables maturation and adenoviral modification of human DCs in a closed system and compare the results with standard polystyrene flasks. STUDY DESIGN AND METHODS: Mononuclear cells were isolated from HLA-A*0201-positive blood donors by leukapheresis. A commercially available separation system (CliniMACS, Miltenyi Biotec) was used to isolate monocytes by positive selection using CD14-specific immunomagnetic beads. The essentially homogenous starting cell population was cultivated in the presence of granulocyte-macrophage-colony-stimulating factor and interleukin-4 in a closed-bag system in parallel to the standard flask cultivation system. Genetic modification was performed on Day 4. After induction of maturation on Day 5, mature DCs could be harvested and cryopreserved on Day 7. During the cultivation period comparative quality control was performed using flow cytometry, gene expression profiling, and functional assays. RESULTS: Both flasks and bags generated mature genetically modified DCs in similar yields. Surface membrane markers, expression profiles, and functional testing results were comparable. The use of a closed-bag system facilitated clinical applicability of genetically modified DCs. CONCLUSIONS: The polyolefin bag-based culture system yields DCs qualitatively and quantitatively comparable to the standard flask preparation. All steps including cryopreservation can be performed in a closed system facilitating standardized, safe, and reproducible preparation of therapeutic cells.

Efficient generation of clinical-grade genetically modified dendritic cells for presentation of multiple tumor-associated proteins.

BACKGROUND: Dendritic cells (DCs) play a central role in the initiation and regulation of immune responses. DCs for clinical applications can be generated with high yield from leukapheresis products. Using adenoviral transduction we genetically modified human DCs to produce and present melanoma-associated antigens. Coexpression of green fluorescent protein and epitope tags were used to monitor genetic modification. Generation, genetic modification, and cryoconservation of gene modified human DCs on a clinical scale in a closed system is feasible. STUDY DESIGN AND METHODS: CD14-positive monomuclear cells were isolated from leukapheresis products of HLA-A* 0201 positive voluntary blood donors using immunomagnetic beads. Selected cells were cultivated for 7 days. Adenovirus transduction was optimal on Day 4. Maturation was induced on Day 5. Mature DC were aliquoted and cryoconserved on Day 7. Quality control was performed using flow cytometry, expression profiling, and functional assays (ELISPOT, CBA). RESULTS: We were able to generate sufficient genetically modified mature DCs in serum-free cultures that could be stored by cryopreservation. The use of a closed system facilitated development of methods for standardized production of clinically applicable genetically modified DCs. The adenoviral transduction system allowed simultaneous and flexible expression of tumor-associated antigens for prolonged presentation of multiple epitopes. CONCLUSION: The feasibility of a closed-bag system for the cultivation of genetically modified human DCs is shown. The immature DCs were genetically modified by recombinant replication-deficient adenoviruses to express multiple epitopes of tumor-associated proteins and then differentiated to mature antigen-presenting DCs.

Timing and targeting of cell-based VEGF165 gene expression in ischemic tissue.

BACKGROUND: Therapeutic angiogenesis has become a key technology in experimental and clinical medicine. Only few data are available on the effects of timing and targeting of therapeutic proteins after cell-based gene transfer. This work investigates such effects after temporary expression of vascular endothelial growth factor 165 (VEGF(165)), the most commonly used angiogenic protein for therapeutic purposes. METHODS: We established a cell-based gene-transfer model using fibroblasts to temporarily produce VEGF(165). Cells were implanted into 40 rats. Protein expression and angiogenic effects were measured by PCR, immunohistology, and microangiography. To determine an improvement for survival of ischemically challenged tissue, cells were implanted in an ischemic flap model at different locations and time points. RESULTS: After implantation of modified cells, a temporary increase was found in the target tissue for VEGF(165), endothelial cell counts, and capillary network formations. Four wk later, histological alterations in the target tissue area were not different from controls. Implantation of modified cells into flap plus wound margin 1 wk before surgery showed significant improvement of tissue survival demonstrated by planimetric measurements and blood vessels counting in the target tissue. CONCLUSION: In our model, temporary expression of VEGF(165) induces therapeutically relevant angiogenesis and improves blood supply only if applied 1 wk before ischemia. It is essential to include the surrounding area for induction of angiogenesis in this model. In contrast, the angiogenic effects are not effective in the target area and its surrounding tissue, if therapeutic gene expression is started during onset of ischemia or 2 wk before ischemia in this model.

Molecular Diagnostics in Transfusion Medicine: In Capillary, on a Chip, in Silico, or in Flight?

Serology, defined as antibody-based diagnostics, has been regarded as the diagnostic gold standard in transfusion medicine. Nowadays however the impact of molecular diagnostics in transfusion medicine is rapidly growing. Molecular diagnostics can improve tissue typing (HLA typing), increase safety of blood products (NAT testing of infectious diseases), and enable blood group typing in difficult situations (after transfusion of blood products or prenatal non-invasive RhD typing). Most of the molecular testing involves the determination of the presence of single nucleotide polymorphisms (SNPs). Antigens (e.g. blood group antigens) mostly result from single nucleotide differences in critical positions. However, most blood group systems cannot be determined by looking at a single SNP. To identify members of a blood group system a number of critical SNPs have to be taken into account. The platforms which are currently used to perform molecular diagnostics are mostly gel-based, requiring time-consuming multiple manual steps. To implement molecular methods in transfusion medicine in the future the development of higher-throughput SNP genotyping non-gel-based platforms which allow a rapid, cost-effective screening are essential. Because of its potential for automation, high throughput and cost effectiveness the special focus of this paper is a relative new technique: SNP genotyping by MALDI-TOF MS analysis.

High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells.

Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering. The ex vivo adenoviral vector mediated transduction into osteogenically differentiated MSCs revealed a highly efficient and long lasting expression of the transgenes. Biological activity of VEGF and Ang-1 secreted from transduced cells was confirmed by analyzing the sprouting, proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in response to conditioned medium obtained from transduced cells. The transduced osteogenically differentiated MSCs described in this report may be suitable for inducing neovascularization in bone tissue engineering applications.

Automatic registration of serial sections of mouse lymph node by using Image-Reg.

The first step towards the three-dimensional (3D) reconstruction of histological structures from serial sectioned tissue blocks is the proper alignment of microscope image sequences. We have accomplished an automatic rigid registration program, named Image-Reg, to align serial sections from mouse lymph node and Peyer's patch. Our approach is based on the calculation of the pixel-correlation of objects in adjacent images. The registration process is mainly divided into two steps. Once the foreground images have been segmented from the original images, the first step (primary alignment) is performed on the binary images of segmented objects; this process includes rotation by using the moments and translation through the X, Y axes by using the centroid. In the second step, the matching error of two binary images is calculated and the registration results are refined through multi-scale iterations. In order to test the registration performance, Image-Reg has been applied to an image and its transformed (rotated) version and subsequently to an image sequence of three serial sections of mouse lymph node. In addition, to compare our algorithm with other registration methods, three other approaches, viz. manual registration with Reconstruct, semi-automatic landmark registration with Image-Pro Plus and the automatic phase-correlation method with Image-Pro Plus, have also been applied to these three sections. The performance of our program has been also tested on other two-image data sets. These include: (a) two light microscopic images acquired by the automatic microscope (stitched with other software); (b) two images fluorescent images acquired by confocal microscopy (tiled with other software). Our proposed approach provides a fast and accurate linear alignment of serial image sequences for the 3D reconstruction of tissues and organs.

Serial sectioning and three-dimensional reconstruction of mouse Peyer's patch.

Peyer's patches (PPs) are typical gut-associated lymphoid tissues that are located along the wall of the small intestine and that serve as the major sites for generation of immunity to intestinal antigens. Their unique micro-organization is crucial for the generation of the immune response. Although many studies have been reported on the functional anatomy of PP, most investigations have relied on the random sampling of these organs, a procedure that is insufficient for the systemic scanning of the whole tissue or organ. By combining a variety of methods, we have accomplished 3D reconstructions of Peyer's patch. The complex reconstruction procedure includes several steps. First, the PP are serially sectioned at a thickness of 10 microm with a cryostat; (b) the serial sections are stained with haematoxylin-eosin; (c) multiple images from the PP are acquired with an automatic microscope and stitched together with Image Pro Plus to generate a composite image for the whole organ; (d) the serial images are reconstructed with Image J, Reconstruct and 3D Studio Max. The combinational approaches that we present here should be of value when extrapolated to the reconstruction of other tissues or organs. Moreover, the 3D model that we have created and our stereological analysis should be extremely helpful for further in vivo microscopic studies of PP with respect to the immune response.

Immunohistochemical study of the reticular and vascular network of mouse lymph node using vibratome sections.

The function of lymph nodes is greatly influenced by their unique microanatomy, in which distinct subpopulations of cells are compartmentalized by a meshwork of reticular cells and fibres, specialized blood and lymphatic vessels and nerves. Using antibodies against extracellular matrix (ECM) proteins (fibronectin, collagen IV and laminin), proteoglycan (perlecan), and a fibroblastic marker (ERTR-7), the distribution and molecular organization of the system of reticular fibres was investigated by three-dimensional (3D) reconstruction methods. Fibronectin, collagen IV and laminin are restricted to reticular fibres and have a similar distribution pattern, whereas perlecan is limited to the vascular system of the lymph node. Various compartments of the lymph node, such as the B-cell follicle, paracortex (including the high endothelial venules and paracortical cord), and medulla have been reconstructed to visualize their vasculature with respect to B and T cells. Since the morphology of lymph nodes may change significantly in pathological conditions, different compartments of reactive lymph node (after low-dose Listeria monocytogenes infection), especially germinal centres, were also investigated. The data presented here should facilitate our understanding of the 3D organization of non-immune cell components of lymph nodes, which is crucial for cell adhesion, migration, activation, and differentiation in normal and pathological conditions.

Use of Autostitch for automatic stitching of microscope images.

Image stitching is the process of combining multiple images to produce a panorama or larger image. In many biomedical studies, including those of cancer and infection, the use of this approach is highly desirable in order to acquire large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we describe the application of Autostitch, viz. software that is normally used for the generation of panoramas in photography, in the seamless stitching of microscope images. First, we tested this software on image sets manually acquired by normal light microscopy and compared the performance with a manual stitching approach performed with Paint Shop Pro. Secondly, this software was applied to an image stack acquired by an automatic microscope. The stitching results were then compared with that generated by a self-programmed rectangular tiling macro integrated in Image J. Thirdly, this program was applied in the image stitching of images from electron microscopy. Thus, the automatic stitching program described here may find applications in convenient image stitching and virtual microscopy in the biomedical research.

Six-color segmentation of multicolor images in the infection studies of Listeria monocytogenes.

Multiple immunofluorescent staining is a powerful strategy for visualizing the spatial and temporal relationship between antigens, cell populations, and tissue components in histological sections. To segment different cell populations from the multicolor image generated by immunostaining based on color addition theory, a systems approach is proposed for automatic segmentation of six colors. After image acquisition and processing, images are automatically segmented with the proposed approach and six-pseudo channels for individual or colocalized fluorescent dye are generated to distinguish different cell types. The principle of this approach is the classification of each pixel into one of six colors (red, green, blue, yellow, magenta, and cyan) by choosing the minimal angular deviation between the RGB vector of the given pixel and six classically defined edge vectors. In the present infection studies of Listeria monocytogenes, the new multicolor staining methods based on the color addition were applied and the proposed color segmentation was performed for multicolor analysis. Multicolor analysis was accomplished to study the migration and interaction of Listeria and different cell subpopulations such as CD4CD25 double positive T regulatory cells; we also visualized simultaneously the B cells, T cells, dendritic cells, macrophages, and Listeria in another experiment. After Listeria infection, ERTR9 macrophages and dendritic cells formed cluster with Listeria in the infection loci. The principle of color addition and the systems approach for segmentation may be widely applicable in infection and immunity studies requiring multicolor imaging and analysis. This approach can also be applied for image analysis in the multicolor in vivo imaging, multicolor FISH or karyotyping or other studies requiring multicolor analysis.

Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts.

The pathogenesis of fibrosis, especially involving post-translational modifications of collagen, is poorly understood. Lysyl hydroxylase 2 (long) (LH2 (long)) is thought to play a pivotal role in fibrosis by directing the collagen cross-link pattern. Here we show that LH2 (long) exerts a bimodal function on collagen synthesis in human dermal fibroblasts. Adenoviral-mediated overexpression of LH2 (long) resulted in a mRNA increase of collagen alpha1(I) but not of fibronectin and fibrillin-1. This was accompanied by a higher mRNA level of prolyl-4-hydroxylase but not of other ER proteins (Bip, Hsp47, LH1, LH3). The collagen mRNA increase led to an elevated collagen synthesis, which was higher in the fraction of extracellularly deposited, cell-associated collagen than in the medium. The cross-link pattern of cell-associated collagen showed an increase of the hydroxylysine-aldehyde-derived cross-link dihydroxylysinonorleucine and a decrease of the lysine-aldehyde-derived component hydroxylysinonorleucine. The helical lysyl hydroxylation of the procollagen molecule was unaltered. The increase of collagen synthesis in fibroblasts overexpressing LH2 (long) was independent from cross-linking as it was also observed in the presence of beta-aminopropionitril, a cross-linking inhibitor. Together our data identify LH2 (long) as a bifunctional protein and underscores its potential role in the pathogenesis of fibrosis.

Formation of three-dimensional fetal myocardial tissue cultures from rat for long-term cultivation.

Three-dimensional cardiomyocyte cultures offer new possibilities for the analysis of cardiac cell differentiation, spatial cellular arrangement, and time-specific gene expression in a tissue-like environment. We present a new method for generating homogenous and robust cardiomyocyte tissue cultures with good long-term viability. Ventricular heart cells prepared from fetal rats at embryonic day 13 were cultured in a scaffold-free two-step process. To optimize the cell culture model, several digestion protocols and culture conditions were tested. After digestion of fetal cardiac ventricles, the resultant cell suspension of isolated cardiocytes was shaken to initialize cell aggregate formation. In the second step, these three-dimensional cell aggregates were transferred onto a microporous membrane to allow further microstructure formation. Autonomously beating cultures possessed more than 25 cell layers and a homogenous distribution of cardiomyocytes without central necrosis after 8 weeks in vitro. The cardiomyocytes showed contractile elements, desmosomes, and gap junctions analyzed by immunohistochemistry and electron microscopy. The beat frequency could be modulated by adrenergic agonist and antagonist. Adenoviral green fluorescent protein transfer into cardiomyocytes was possible and highly effective. This three-dimensional tissue model proved to be useful for studying cell-cell interactions and cell differentiation processes in a three-dimensional cell arrangement.

Six-colour fluorescent imaging of lymphoid tissue based on colour addition theory.

Multi-colour imaging of immunofluorescently labelled tissue using confocal microscopy was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the co-localization of two or more markers on one cell for the identification of specific cell populations; (2) the co-localization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multi-step staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multi-pseudo-channels for individual or co-localized fluorescent dyes. With this new technique, we have been able to visualize six cell populations simultaneously in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient, and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.

[Influence of vascular endothelial growth factor gene modification on skin substitute grafted on nude mice].

OBJECTIVE: To investigate the influence of vascular endothelial growth factor (VEGF) gene modification on skin substitute grafted on nude mice. METHODS: Human fibroblasts were transfected with VEGF adenovirus vector. Then the genetic modified fibroblasts were seeded on patches of Integra artificial skin. Twenty-four hours later, the Integra patches were grafted onto full-thickness skin defects on nude mice. Seventy-two nude mice were divided into experiment (n = 18, E, with fibroblasts seeded on Integra which were transfected by adenovirus containing VEGF in advance), GFP control (n = 18, the fibroblasts were transfected with adenovirus containing labelled GFP segment as same as that in E group, but containing no VEGF gene), Fb control (n = 18, without gene transfection), and control (n = 18, no fibroblast was seeded on Integra) groups. The survival rate, the revascularization process and the histological changes in the grafts in gene modified group (experimental group) and control groups were observed and analyzed. RESULTS: The revascularization condition in the experimental group was much better than that in the control group. The grafts adhered firmly to the wound during early postoperation stage, and were more prone to bleed when separated from the wound. The survival rate was obviously higher, while the infection rate was much lower in experimental group (100.0%) compared with the control groups (83.3%, 75.0%, 77.8%, respectively) (P < 0.05). CONCLUSION: High expression of VEGF by gene modification can promote the vascularization process of skin substitute, hence improve the grafting result.