Development of a new technique for muscle tissues-stem cells co-colture

INTRODUCTION: Adult stem cells reside in all tissues, where they maintain homeostatic conditions and respond to injuries. These cells are regulated and supported by the surrounding microenvironment, called stem cell “niche”, composed by cellular and molecular factors, that interact with and regulate stem cell fate. Key niche components are represented by growth factors, cell-cell interactions and cell-matrix adhesion. Also in muscle tissue niches are present, especially in myofiber basal lamina, where a network of extracellular matrix components and secreted growth factors stimulate muscle stem cell survival, activation and/or proliferation. In order to better understand the influence of muscle tissue secreted factors on stem cells, we developed a new technical approach to perform a muscle tissue-stem cells co-culture; we focused on peripheral blood derived CD133 cells, a population known to possess a myogenic potential. METHODS: muscle tissue sections were cut with a tissue chopper from fresh quadriceps of C57BL mice and inserted in a culture well upon a porous membrane, above cells suspension; cells were isolated through immunomagnetic separation column; immunophenotypic characterization was performed with Cytomics FC500 RESULTS: the culture system developed creates a physical separation between tissue section and stem cells, allowing soluble factors exchange and preventing tissue-cell contamination; the absence of cell mix was evaluated through cytogenetic analysis of cell karyotype; the reproducibility of the technique has been demonstrated through muscle slice weight monitoring. Preliminary proliferation experiments show an increase in CD133+ stem cells rate of proliferation in presence of muscle tissue; cell immunophenotype monitoring confirmed stemness maintenance. CONCLUSIONS: we have developed a reproducible and standardized technique and designed a culture system that guarantees, spatial division of culture environment, no cellular contaminations between culture compartments, in vitro tissue survival, stem cell viability and proliferation, communication between tissue and cells through chemical signals (factors release).