This protocol describes an effective method for the production of spherical microtissues (microspheres), which can be used for a variety of tissue-engineering purposes. The obtained microtissues are well suited for the study of osteogenesis in vitro when multipotent stem cells are used. The dimensions of the microspheres can easily be adjusted according to the cell numbers applied in an individual experiment. Thus, microspheres allow for the precise administration of defined cell numbers at well-defined sites. Here we describe a detailed workflow for the production of microspheres using unrestricted somatic stem cells from human umbilical cord blood and adapted protocols for the use of these microspheres in histological analysis. RNA extraction methods for mineralized microtissues are specifically modified for optimum yields. The duration of running the complete protocol without preparatory cell culture but including 2 weeks of microsphere incubation, histological staining and RNA isolation is about 3 weeks.

This study was performed to systematically assess the prevalence, topography and prognostic impact of disseminated tumor cells (DTCs) in lymph nodes (LN) of patients with primary, regional and distant metastasis-free head and neck squamous cell carcinoma (HNSCC) who underwent resection with elective neck dissection. From the routinely processed resection specimen, we could prospectively analyze a total of 1.137 exactly mapped LNs of 50 pN0-HNSCC patients, classified as tumor free by routine histopathology. Three immunohistochemistry (IHC) assays using antibodies directed against CK5/14, a broad spectrum of CKs (1-8, 10, 14-16 and 19), and CD44v6, respectively, were applied on 4.190 LN sections to detect DTCs. The IHC results were correlated with clinicopathologic parameters and clinical follow-up data. We detected seven micrometastases (MM) in five patients and 31 DTCs in 12 patients. Overall, 15 (30%) patients were positive for DTCs or MMs. Strikingly, the anatomical distribution of LN affected with DTCs was not random, but was dependent on the lateralization of the primary tumor and clustered significantly most proximal to the primary tumor. None of the investigated patients developed loco-regional lymphatic or distant metastasis during the mean follow-up period of 71 months. Our results reveal clinically occult tumor cell dissemination as an early and frequent event in HNSCC. Considering that higher rates of recurrences in therapeutic LN dissection concepts have been reported than in elective neck dissection strategies, our DTC-data support to perform elective neck dissections, since they appear to be effective in preventing loco-regional lymphatic recurrence from LN DTCs or MMs.