Expansion and Differentiation of Human Hematopoietic Cells from Static Cultures through Small‐Scale Bioreactors

Abstract

Maintenance of the progenitor cells responsible for hematopoiesis has generally been accomplished using a feeder layer of stromal cells in stationary culture. Here, we compared the expansion of the total cell and progenitor cell populations using low-density mononuclear cells (LDMCs) obtained from human bone marrow in static culture (T-flasks) and in different cell culture bioreactors designed for the scale-up of mammalian cells. Static cultures were performed without the presence of a previously established stromal cell layer. Expansion of marrow in all cases was accomplished through the use of added cytokines such as IL-3, GM-CSF, and c-kit ligand. The results for the total cell expansion in static culture ranged from 4.4- to 32-fold. The cell number increase was affected by such factors as patient to patient variability, freeze-thawing, and the combination of cytokines used. Due to widespread use and the small amount of marrow needed, static cultures were used as a basis for comparison with other expansion systems. The cell culture systems used to evaluate the scale-up of marrow cultures included suspension, microcarrier, airlift, and hollow fiber bioreactors. Using identical media, cytokines, and feed schedules, LDMCs in the suspension bioreactor expanded to a value of 1.6 compared to a normalized value of 1.0 for static cultures for the two runs investigated. Expansion results for microcarrier cultures averaged 0.75 when compared to static cultures. A cell number increase in the airlift bioreactor resulted in an expansion which was 0.70 of the control static culture. Granulocyte-macrophage and erythroid progenitor assay data were also evaluated for the suspension, microcarrier, and airlift bioreactors.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: Shear, proliferation, and the IL-2 receptor

“…Myeloid lineage hematopoietic cells have been grown in spinner flasks at scales of 30-250 mL (Collins et al, 1998;Sardonini and Wu, 1993;Zandstra et al, 1994). Collins et al (1998) noted that hematopoietic cells (derived from mobilized peripheral blood) were very susceptible to damage by even low levels of agitation, such that cultures grown at 30 rpm in spinner flasks showed greater total cell expansion than those grown at 60 rpm.…”

“…Additionally, Collins et al (1998) showed that factors such as impeller diameter and seeding density were critically important for successful cell expansion in stirred culture vessels. Bone marrow-derived mononuclear cells were successfully expanded in spinner flask cultures employing agitation rates of 40 and 45 rpm (Sardonini and Wu, 1993;Zandstra et al, 1994). Natural killer cells have been grown successfully in both 250-mL spinner flasks and in a 750-mL computer-controlled stirred tank bioreactor at 60 rpm (Pierson et al, 1996).…”

Cellular determinants affecting the rate of cytokine in cultures of human hematopoietic cells

“…Stirred suspension cultures also have the advantage of exposing cells to a relatively homogeneous environment that should be possible to control automatically. Moreover, preliminary experiments indicated the feasibility of their use (Sardonini and Wu, 1993;Zandstra et al, 1994). The goal of the present studies was, therefore, to determine whether manipulating extracellular cytokine levels might increase primitive progenitor outputs in this system.…”

Functional Cells Cultured on Microcarriers for Use in Regenerative Medicine Research

“…Hematopoietic stem/progenitor cells have been expanded or differentiated with or without coculture with It is possible to establish an economical and c-GMPcompliant microcarrier system to produce human fibrobone marrow cells grown in microcarrier cultures (23,74,101 …”