Formation and Differentiation of Three-Dimensional Rat Marrow Stromal Cell Culture on Microcarriers in a Rotating-Wall Vessel

Abstract

Using a high aspect ratio vessel (HARV), this study investigated the formation of 3-D rat marrow stromal cell culture on microcarriers and the expression of bone-related biochemical markers under conditions of simulated microgravity. In addition, it calculated the shear stresses imparted on the surface of microcarriers of different densities by the medium fluid in an HARV. Secondary rat marrow stromal cells were cultured on two types of microcarriers, Cytodex-3 beads and modified bioactive glass particles. Examination of cellular morphology by scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together. Mineralization was observed in the aggregates. Spherical cell-bead aggregates were observed in an HARV, while cell-bead assemblies were mostly loosely packed in a chain-like or branched structure in a cell bag. The expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown via the use of histochemical staining, immunolabeling, and confocal scanning electron microscopy. Using a numerical approach, it was found that at a given rotational speed and for a given culture medium, a larger density difference between the microcarrier and the culture medium (e.g., a modified bioactive glass particle) imparted a higher maximum shear stress on the microcarrier.

Selective differentiation of mammalian bone marrow stromal cells cultured on three-dimensional polymer foams

“…[7][8][9] We previously reported that 3D poly(glycolic acid) (PGA) meshes supported the formation of cartilaginous and bone-like tissues by avian BMSC expanded in the presence of fibroblast growth factor-2 (FGF-2). 10 However, mammalian (bo-vine calf) BMSC cultured on PGA meshes under the same conditions failed to differentiate over 4 weeks in vitro and cell-polymer constructs collapsed.…”

Bioreactor-based bone tissue engineering: The influence of dynamic flow on osteoblast phenotypic expression and matrix mineralization

“…To overcome the drawbacks associated with static culturing systems, considerable interest has been generated in tissue engineering by using the high-aspect-ratio vessel rotating bioreactor, which has the characteristic of low-shear, three dimensionality and highmass transfer and thus provides a dynamic flow culture condition to promote tissue synthesis (18)(19)(20)(21). Various cell types can form 3D assemblies in the rotating bioreactors (22)(23)(24)(25)(26). Rat stromal cells cultured on cytodex-3 beads have been shown to form aggregates and synthesize mineralized matrix, and cell aggregates formed by MC3T3 osteoblast-like cells produced collagen fibrils in the matrix between microspheres (17).…”

Materials in particulate form for tissue engineering. 2. Applications in bone

“…To overcome such limitations, Ducheyne and co-workers (Qiu et al, 1998(Qiu et al, , 2000(Qiu et al, , 2001) envisioned a strategy using the HARV bioreactor and microcarriers to engineer constructs that could be used for bone TE purposes. In a first approach, the authors used bioactive glass, Cytodex-3 beads and rat stromal cells for assessing the feasibility of culture using a HARV bioreactor (Qiu et al, 1998).…”

“…To overcome such limitations, Ducheyne and co-workers (Qiu et al, 1998(Qiu et al, , 2000(Qiu et al, , 2001) envisioned a strategy using the HARV bioreactor and microcarriers to engineer constructs that could be used for bone TE purposes. In a first approach, the authors used bioactive glass, Cytodex-3 beads and rat stromal cells for assessing the feasibility of culture using a HARV bioreactor (Qiu et al, 1998). It was observed that 3D multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together, as well as mineralization taking place, and the expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown (Qiu et al, 1998).…”

“…In a first approach, the authors used bioactive glass, Cytodex-3 beads and rat stromal cells for assessing the feasibility of culture using a HARV bioreactor (Qiu et al, 1998). It was observed that 3D multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together, as well as mineralization taking place, and the expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown (Qiu et al, 1998). The authors further developed bioactive ceramic hollow microspheres with an apparent density in the range 0.81.0 g/cm 3 as microcarriers for 3D bone tissue formation in rotating-wall vessels (RWV).…”