Stem Cell Bioprocessing of Skin-Derived Precursor Cells (SKPs) in Stirred Suspension Bioreactors

Skin-derived precursors (SKPs) have recently emerged as a promising candidate for dermal regeneration following split-thickness skin grafts (STSGs) for burn patients. SKPs are stem cells that reside in the dermal papilla and dermal sheath of the hair follicle and can be isolated and cultured in vitro.  Transplanted rodent SKPs can generate dermal cells and fill in a wound following injury.  When grown in either static (T-flask) or dynamic (stirred suspension bioreactor) culture, they form spherical aggregates as cells proliferate.  When examining the design of a bioprocess for the controlled expansion of these cells to treat burn patients, there are a number of important issues that need to be considered.  First, there is a limited time frame between the injury and the time when a population of cells will be needed.  This necessitates a fast expansion under controlled conditions from a limited starting population.  Second, as this is an autologous treatment, there will be donor-to-donor variations that will mean any bioprocess will need to be rigorously tested on a wide variety of donors (age, sex).  In addition, bioprocessing parameters will need to be examined for cells from a wide diversity of donor sites (trunk, scalp, etc), as the frequency and proliferation capacity of SKPs from each site may vary.  Third, it is also important to maintain a phenotype that is associated with strong regeneration capability.

We have expanded human SKPs from a number of different sources in stirred suspension bioreactors and have achieved expansion that is superior to standard static culture in T-flasks.  This has been observed for donors of different ages and sex, as well as for different donor sites. SKPs proliferate faster in the stirred suspension bioreactors than in static conditions.  Evaluations of important process variables such as agitation rate and feeding conditions have been performed.  Initial phenotype characterization by immunostaining also looks promising and suggests that SKPs grown in stirred suspension bioreactors maintain attributes consistent with endogenous dermal stem cells following bioreactor expansion.  We will present data showing that a stirred suspension bioreactor system is a viable method for expanding populations of human SKPs for use in clinical cell therapy settings.