Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34(+)lin(-) hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well receives a single hematopoietic stem cell candidate. During an experiment lasting several days to weeks, each cell-containing well is moved sequentially and serially to a microscopic imaging system. This movement is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Biobox built on the microscopic stage. New image analysis software facilitates tracking of cell movement, recording the time of cell division, and immunophenotyping of multiple, individual, or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and may be applicable to a wide range of other systems of interest in tissue engineering.