The multiparametric effects of hydrodynamic environments on stem cell culture

Abstract

Stem cells possess the unique capacity to differentiate into many clinically relevant somatic cell types, making them a promising cell source for tissue engineering applications and regenerative medicine therapies. However, in order for the therapeutic promise of stem cells to be fully realized, scalable approaches to efficiently direct differentiation must be developed. Traditionally, suspension culture systems are employed for the scale-up manufacturing of biologics via bioprocessing systems that heavily rely upon various types of bioreactors. However, in contrast to conventional bench-scale static cultures, large-scale suspension cultures impart complex hydrodynamic forces on cells and aggregates due to fluid mixing conditions. Stem cells are exquisitely sensitive to environmental perturbations, thus motivating the need for a more systematic understanding of the effects of hydrodynamic environments on stem cell expansion and differentiation. This article discusses the interdependent relationships between stem cell aggregation, metabolism, and phenotype in the context of hydrodynamic culture environments. Ultimately, an improved understanding of the multifactorial response of stem cells to mixed culture conditions will enable the design of bioreactors and bioprocessing systems for scalable directed differentiation approaches.

FIG. 1.

Stem cell differentiation formats. Stem cells can be cultured in monolayer or in suspension, either adherent to spherical microcarriers or as aggregates of cells. Suspension cultures generally increase the density of cells, and thus increase the overall cell yield per volume or media. Although suspension cultures are more scalable, the three-dimensional aggregate structure increases the diffusive distance between the media and cells at the center, which may result in decreased transport and the development of gradients of nutrients and metabolites throughout the spheroid. Color images available online at www.liebertonline.com/teb

FIG. 2.

Methods of embryoid body formation and propagation. (A) Aggregates of stem cells can be formed and maintained statically by physically separating cells in small volume drops or by spontaneous aggregation of cells within bulk suspension cultures in liquid or semi-solid media. (B) Dynamic cultures are amenable to supporting increased culture volumes (10^1–3 L) for the production of large cell yields in various formats, such as rotating wall vessels, or stirred bioreactors, which employ external or internal agitation, respectively. Color images available online at www.liebertonline.com/teb

FIG. 3.

Stem cell aggregate formation. (A) Stem cells may form small aggregates or large agglomerates in suspension, with resulting spheroid size being influenced by hydrodynamic properties. Spheroid size is mediated by the number of collisions between cells in solution, which is impacted by (B) the transport parameters of the system (mass transfer coefficient, k, which increases as a power law of impeller speed, with the exponent (a), being a function of geometrical properties of the bioreactor system), as well as the (C) collision frequency, which is modulated by the mass (m) and number (N) of cells and aggregates in suspension. Additionally, final spheroid size is altered by (D) the binding kinetics (koff) of the adhesion molecules, such as integrins and cadherins, which changes as a function of forces exerted on the bonds.

FIG. 4.

Macro- and microscale changes in transport and stem cell metabolism. (A) Macro-scale transport in various stem cell culture formats. Blue intensity represents relative concentrations of soluble morphogens and nutrients (such as O₂) within populations of single cells and (B, C) different-sized aggregates of stem cells. Concentration gradients arise in spheroid culture, with diffusive limitations in large aggregates. (D) Alteration of metabolic pathways in dynamic hESC culture leads to decreases (green) and increases (red) in local concentrations of nutrients and metabolites, respectively; the increased uptake of glucose and glutamine and excretion of lactate and ammonia lead to a highly proliferative state favoring expansion and decreases the pH of the extracellular environment. TCA, tricarboxylic acid cycle; LDH, lactate dehydrogenase. Color images available online at www.liebertonline.com/teb