Influence of Medium Exchange Schedules on Metabolic, Growth, and GM‐CSF Secretion Rates of Genetically Engineered NIH‐3T3 Cells

Abstract

The metabolic and secretory characteristics of NIH-3T3 fibroblasts transfected with a cDNA encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were examined as a function of the culture medium exchange schedule. The rates of glucose and glutamine consumption and of lactate and ammonia production were measured over exchange schedules ranging from complete medium replacement weekly (1/week) to complete medium replacement daily (7/week). All measured metabolic rates increased with increased medium exchange rates and accelerated sharply between exchange rates of 3.5/week and 7/week. The lactate/glucose and ammonia/glutamine yield coefficients, however, remained invariant at about 1.9 and 1.0 mol/mol, respectively, under all medium perfusion conditions. A shift-up in medium perfusion rates from 3.5/week to 7/week resulted in increased metabolic rates that resembled those observed in the cultures that were exchanged at the 7/week rate throughout, showing that the metabolic rates could be directly controlled by the perfusion rate. Differential regulation of medium versus serum perfusion demonstrated that increased NIH-3T3 cell metabolism was directly proportional to the serum flux to which the cells were exposed. Thus a limiting serum component is responsible for the altered metabolic and growth rates. The GM-CSF production by the transfected 3T3 cells was stable but exhibited substantial transient increases during periods of cell proliferation, demonstrating that the secretion of transfected gene products can be highly modulated even when the cDNA is driven from a constitutive promoter. These studies show that the metabolic and secretory behavior of genetically engineered cells is influenced by the medium exchange schedule.

Rapid medium perfusion rate significantly increases the productivity and longevity of human bone marrow cultures.

“…We have previously found that the metabolism of normal human bone marrow cells is greatly enhanced by medium exchange schedules that more closely approximate those seen in vivo. Moreover, changes in medium and serum perfusion appear to directly stimulate the production of granulocyte/macrophage-colony-stimulating factor by stromal cells (9) and transfected NIH 3T3 cells (8 Similarly, studies on the addition of recombinant hematopoietic growth factors to long-term bone marrow cultures have shown improved production of CFU-GM and nonadherent cells in Dexter cultures over untreated controls. However, even with addition of these growth factors, CFU-GM and nonadherent cell production decreased by 1.5-2 logarithmic units by 5 weeks in culture (15 …”

“…Long-term in vitro human hematopoietic (Dexter) cultures are limited both in their longevity (8)(9)(10)(11)(12) weeks) and in their cell production over time. Hematopoiesis in vivo continues unabated throughout life, suggesting that failure to obtain continuous hematopoiesis in vitro is probably due to the failure of culture conditions to accurately reproduce the in vivo state.…”

“…This perfusion rate is much more rapid than that used in typical Dexter cultures, which vary from 0.5 to 2 medium volume exchanges per week (1,6). We have observed (8,9) that cell growth and hematopoietic growth factor production by bone marrow stroma and transfected NIH 3T3 cells is significantly influenced by the medium and serum perfusion rate with increased activity when the medium/serum perfusion rate approaches levels that prevail in vivo.…”

Haematopoietic Culture Systems

“…Hence, various feeding strategies have been developed in the cultivation of haematopoietic cells to try and recapitulate the needs of the HIM (Caldwell et al 1991a;Martinson 1998;McAdams 1995;Schwartz et al 1991). Medium exchange is required in order to overcome growth restrictions imposed by mass transfer limitations of oxygen and nutrients (Koller 1996;Oh 1994;Zandstra et al 1994) and the depletion of substrates and the accumulation of the metabolic by-products such as lactate that result in a drop in culture pH (Caldwell et al 1991a). Frequent medium exchange enhanced the cell output and CFU-GM in static cultures, especially at high cell densities of BM MNCs in the presence of preformed stroma (Koller 1996).…”

The influence of extra-cellular matrix and stroma remodeling on the productivity of long-term human bone marrow cultures

“…The function of the supporting stromal cell layer (mostly fibroblast, with some adipocytes and endothelial cells) has been shown to be significantly influenced by the medium perfusion rate, or the medium exchange schedule. Metabolic function, growth, and perhaps most importantly growth factor secretion have all been shown to be influenced by the medium exchange rate for normal human bone marrow fibroblasts (Caldwell et al, 1991), and even for transfected NIH-3T3 murine cells (Caldwell et al, 1991a). The ability of stroma to support human hematopoiesis ex vivo has been demonstrated to be enhanced by rapid medium exchange (Schwartz et al, 1991).…”