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The magnetic labeling of living cells has become of major interest in the areas of cell therapy and tissue engineering. Magnetically labeled cells have been reported to allow increased and controlled seeding, tracking, and targeting of cells. In this work, we comprehensively characterize magnetic nanoparticles (MNPs) possessing a magnetite core of about 11 nm, and which are coated with the fluorinated surfactant F(CF2)nCH2CH2SCH2CH2C(O)OLi and 1,9-nonandithiol (NDT) for the nonspecific labeling of human pulmonary epithelial (H441) cells. We achieved a non-specific cell loading of 38 pg Fe/cell. In this work we combine magnetic cell labeling with subsequent genetic modification of the cells with non-viral transfection complexes associated with PEI-Mag2 magnetic nanoparticles upon gradient magnetic field application called magnetofection. The magnetic responsiveness and magnetic moment of the MNP-labeled cells and the magnetic transfection complexes were evaluated by measuring changes in the turbidity of prepared cells suspensions and complexes in a defined magnetic gradient field. The magnetic responsiveness of cells that were loaded with NDT-Mag1 MNPs (20-38 pg Fe/cell) was sufficient to engraft these labeled cells magnetically onto the luminal surface of a culture tube. This was achieved using a solenoid electromagnet that produced a radial magnetic field of 20-30 mT at the seeding area and an axial gradient field of approx. 4 T/m. The MNP-labeled cells were magnetofected in 2D arrays (well plates) and at the luminal surface of cell culture tube. The optimized magnetic pre-labeling of cells did not interfere with, or even increased, the efficiency of magnetofection in both culture systems without causing cell toxicity. Cell loading of 38 pg Fe/cell of NDT-Mag1 MNPs resulted in high transverse relaxivities r2*, thus allowing the MRI detection of cell concentrations that were equivalent to (or higher than) 1.2 microg Fe/ml. Multi-echo gradient echo imaging and R2* mapping detected as few as 1533 MNP-labeled H441 cells localized within a 50 microl fibrin clot and MNP-labeled cell monolayers that were engrafted on the luminal surface of a cell culture tube. Further loading of cells with MNPs did not increase either the magnetic responsiveness of the cells or the sensitivity of MR imaging. In summary, the NDT-Mag1 magnetic nanoparticles provided a high cell-loading efficiency, resulting in strong cell magnetic moments and a high sensitivity to MRI detection. The transfection ability of the labeled cells was also maintained, thereby increasing the magnetofection efficiency.

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surgery of the nose or auricle.