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Hematopoietic stem and progenitor cells
In its natural environment, hematopoiesis resides in a microenvi- ronment characterized by local geometry (structure and vasculature), by accessory cells of mixed origin (stromal cells) and the extracellular matrix produced by them (Nielsen, 1999). Since the first in vitro reconstruction of the in vivo murine hematopoietic microenvironment to culture Hematopoietic Stem and Progenitor Cells (HSPCs) by Dexter et al.(1973), which was later adapted for human cells (Gartner and Kaplan, 1980), hematopoietic cell cultures have been typically performed in static conditions (Haylock et al., 1992; Lemoli et al., 1992).
The potential of stirred suspension cultures to support hemato- poiesis ex-vivo has been investigated since the 1990s. HSPCs are relatively shear-sensitive cells, and agitation is thought to affect surface marker expression (McDowell and Papoutsakis, 1998), thus low agitation rates (30–60 rpm) are necessary in these systems in order to avoid cell damage (Collins et al., 1998a; Sardonini and Wu, 1993; Zandstra et al., 1994).
The short-term maintenance of both colony-forming cell (CFC) numbers and their precursors, detected as long-term culture initiating cells (LTC-IC), was initially demonstrated to be possible in stirred suspension (Zandstra et al., 1994). After 4 weeks the number of LTC-ICs and CFCs present in stirred cultures initiated with 1 million cells increased an average of 7- and 22-fold, respectively. Later on, the same authors studied the parameters that possibly limit the cytokine- mediated expansion of primitive hematopoietic cells in stirred suspension cultures (Zandstra et al., 1997). More primitive cells (LTC- ICs) were shown to deplete cytokines from the medium much more rapidly than their more mature progeny according to a mechanism that is strongly dependent on the concentration of cytokines to which the cells are exposed.
Cultures of umbilical cord blood (UCB) mononuclear cells (MNCs), peripheral blood (PB) MNCs, and PB CD34+ cells were also performed in spinner flasks and in T-flasks, both in serum-containing and serum-free media (Collins et al., 1997). Glucose and lactate metabolic rates were determined and correlated with the percentage of CFC present in the culture for a broad range of culture conditions. The proliferation and differentiation characteristics of these popula- tions in spinner flask cultures were also examined by the same authors (Collins et al., 1998a). Cell proliferation in spinner flasks was dependent on both agitator design and agitation rate, as well as on the establishment of critical inoculum densities. The expansion of UCB and PB MNCs in a stirred-tank bioreactor system with pH and dissolved oxygen control was also described, as well as oxygen uptake and lactate production in these cultures (Collins et al., 1998b). Expansion of total cells and CFCs was greatly enhanced by the use of a cell-dilution feeding protocol (as compared to a cell-retention feeding protocol). The different metabolic profile of CFCs and more mature cells may allow the prediction of the content of several cell types in culture by monitoring the uptake or production of oxygen, lactate and other metabolites.
A number of perfusion reactors have also been developed for HSPCs culture. The greatest success has been achieved with two flat- bed reactor systems: a multipass perfusion system (Koller et al., 1993a), and one single-pass perfusion reactor (Koller et al., 1993b; Palsson et al., 1993). Both systems support 10- to 20-fold total cell expansion and ~ 10-fold progenitor expansion, whereas expansion of primitive cells has only been reported for the second system. The multipass reactor was further extended for use with or without stroma by the introduction of multiple microgrooves at the chamber bottom, allowing rapid medium exchange with low shear stress (Horner et al., 1998; Sandstrom et al., 1995, 1996).
The single-pass system has been employed in several clinical trials. The cell production system consists in a disposable cassette where cells are injected on top of a layer of stromal cells grown on a tissue culture plastic surface. Nutrients are continuously perfused to the cassette,