The authors have taken a new approach to finding optimal conditions for sti
mulating conservative division of single isolated CD34+lin(-) hematopoietic
stem cell candidates from human umbilical cord blood. The approach require
d the design and development of a novel multi-well single cell combinatoria
l culture system. This system incorporates the use of a multi-well tissue c
ulture plate in which each well, can receive a single hematopoietic stem ce
ll candidate. Sequential movement of each cell-containing well to a microsc
opic imaging system, serially over a several-day to several-week experiment
, is facilitated by computer control of a motorized stage and stabilization
of the experiment in an environmentally controlled Bio-box built on the mi
croscope stage. New image analysis software facilitates in the tracking of
cell movement, recording of the time of fell division, and immunophenotypin
g of each of multiple individual or recently doubled cells in real time by
a robotically controlled pipetting station. The principles of single cell c
ulture should help solve many problems in human hematopoietic stem cell exp
ansion and also may be applicable to a wide range of other systems of inter
est in tissue engineering.