Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR.

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.

Fluorescence-activated cell sorting of PCK-26 antigen-positive cells enables selection of ovine esophageal epithelial cells with improved viability on scaffolds for esophagus tissue engineering.

OBJECTIVE: For esophagus tissue engineering, isolation and proliferation of esophageal epithelial cells (EEC) is a pre-requisite for scaffold seeding to create constructs. The aim of this study was to sort EEC expressing cytokeratin markers and their proliferative subpopulations; also, to investigate the viability of differentiated EEC subpopulations on collagen scaffolds. METHODS: Ovine esophageal epithelial cells (OEECs) from sheep esophagus were analyzed using flow cytometry for pan cytokeratin (PCK-26) and proliferation cell nuclear antigen (PCNA). Using fluorescent-activated cell sorting, OEEC were separated and analyzed for PCNA expression. The OEEC subpopulations were seeded on collagen scaffolds for a week in vitro culture. RESULTS: Proliferation cell nuclear antigen was expressed in >45% of OEEC isolated. In flow cytometry, 30% OEEC were PCK-26 positive which exhibited a high-proliferative capacity of 80%. PCK-26-negative OECC exhibited a low-proliferative capability of 13%. Scanning electron microscopy demonstrated organized attachment and uniform scaffold coverage in PCK-26-positive cells. CONCLUSION: Ovine esophageal epithelial cells can be divided into PCK-26-positive and negative subpopulations. PCK-26-positive OEEC constitute one-third of the isolated cells with high-proliferative capability. Seeding of PCK-26-positive OEEC on collagen scaffolds leads to uniform distribution of cells in vitro. In esophagus, tissue engineering PCK-26-positive OEEC subpopulation is important for optimal construct generation.

A novel sandwich ELISA for alpha1 domain based detection of soluble HLA-G heavy chains.

The detection of soluble human leukocyte antigen G (HLA-G) has been a technically demanding task for several years now and various enzyme linked immunosorbent assay (ELISA) formats have been designed. However, no ELISA test has been described so far which is able to detect all possible kinds of soluble HLA-G (sHLA-G) molecules that might occur in bio fluids. Here we describe a new ELISA approach able to recognize soluble alpha1 domain containing heavy chains of all HLA-G isoforms. The detection limit is shown to be at about 150 pg soluble recombinant HLA-G1 heavy chain per milliliters. Detectable HLA-G fragments are shown to occur in the supernatants of different HLA-G transfected cell lines and appear to be particularly abundant in supernatant of trophoblast derived choriocarcinoma cell lines. The novel ELISA employs the well characterized HLA-G mAbs 4H84 and MEM-G1 which ensure high HLA-G specificity. A negative control ELISA format, designed against non-existing analytes, has been established to reveal non-specific signal interference.

Esophagus tissue engineering: in situ generation of rudimentary tubular vascularized esophageal conduit using the ovine model.

PURPOSE: Esophagus replacement using the present surgical techniques is associated with significant morbidity. Tissue engineering of the esophagus may provide the solution for esophageal loss. In our attempts to engineer the esophagus, this study aimed to investigate the feasibility of generating vascularized in situ esophageal conduits using the ovine model. METHODS: Esophageal biopsies were obtained from lambs, and ovine esophageal epithelial cells (OEEC) were proliferated. The OEEC were seeded on to bovine collagen sheets preseeded with fibroblasts. After 2 weeks of maintaining the constructs in vitro, the constructs were tubularized on stents to create a tube resembling the esophagus and implanted into the omentum for in situ tissue engineering. The edges of the omentum were sutured using nonabsorbable suture material. The implanted constructs were retrieved after 8 and 12 weeks. RESULTS: The omental wrap provided vascular growth within and around the constructs as they were integrated along the outer surface area of the scaffold. After removal of the stents, the engineered conduit revealed a structure similar to the esophagus. Histologic investigations demonstrated esophageal epithelium organization into patches on the luminal side and vascular ingrowths on the conduit's outer perimeter. CONCLUSION: Our study demonstrated the seeding of OEEC on collagen scaffolds and formation of a rudimentary conduit resembling esophageal morphology after in situ omental implantation. Vascular coverage and ingrowth in the periphery of the construct could also be demonstrated. These findings hold future promise for the engineering of the esophagus with improved microarchitecture.

Esophagus tissue engineering: hybrid approach with esophageal epithelium and unidirectional smooth muscle tissue component generation in vitro.

PURPOSE: The aim of this study was to engineer the two main components of the esophagus in vitro: (a) esophageal epithelium and (b) smooth muscle tissue. Furthermore, (a) survivability of esophageal epithelial cells (EEC) on basement membrane matrix (BMM)-coated scaffolds and (b) oriented smooth muscle tissue formation on unidirectional BMM-coated collagen scaffolds was investigated. METHODS: Both EEC and smooth muscle cells (SMC) were sourced from Sprague-Dawley rats. The EEC were maintained in vitro and seeded onto BMM-coated 2-D collagen scaffolds. Similarly, smooth muscle cells were obtained using an explants technique and seeded on unidirectional 3-D BMM-coated collagen scaffolds. Cell-polymer constructs for EEC and SMC were maintained in vitro for 8 weeks. RESULTS: Protocols to obtain higher yield of EEC were established. EEC formed a layer of differentiated epithelium after 14 days. EEC survivability on polymers was observed up to 8 weeks. Unidirectional smooth muscle tissue strands were successfully engineered. CONCLUSION: Esophageal epithelium generation, survivability of EEC on BMM-coated scaffolds, and engineering of unidirectional smooth muscle strands were successful in vitro. The hybrid approach of assembling individual tissue components in vitro using BMM-coated scaffolds and later amalgamating them to form composite tissue holds promises in the tissue engineering of complex organ systems.

Smooth muscle tissue engineering for hybrid tubular organs: scanning electron microscopic investigations of cell interactions with collagen scaffolds.

Tissue engineering of transplantable tubular tissue necessitates a composite approach to assembling the individual components. In the engineering of tubular structures (gastrointestinal, urogenital and vascular tissues) there is a demand for smooth muscle tissue in addition to the primary organ-specific tissue. This study aims to investigate the interaction of smooth muscle cells on bovine collagen scaffolds cross-linked with glutaraldehyde under in vitro conditions, using scanning electron microscopy. The density of tissue generated over the 8 week period was demonstrated in terms of: (a) scaffold coverage and attachment; (b) cell density; (c) cell viability deep inside the scaffolds; and (d) cell differentiation.