Culture and Growth Characteristics of Chondrocytes Encapsulated in Alginate Beads

Abstract

Methodology is described for the culture of avian and mammalian chondrocytes in ionotrophically gelled "semi-solid" and "hollow" alginate beads. Chondrocytes grown in "semi-solid" gels exhibited a spherical shape as opposed to a fibroblastic morphology observed in monolayer culture. In the "semi-solid" beads, the cells grew as small clumps and as large aggregates. The aggregates were round or elliptical in appearance and surrounded by a dense Alcian Blue positive halo. Preliminary studies with collagen and chitosan matrixes encapsulated in "hollow" beads suggest that cell growth and morphology are profoundly influenced by the composition of the cellular environment. Chondrocyte structure and function in the "semi-solid" and "hollow" beads were partially characterized by light microscopy, histochemical and biochemical means. The encapsulation methodology is readily applicable for the culture of chondrocytes in single beads, in multiwell dishes, or mass culture.

Viability of equine articular chondrocytes in alginate beads exposed to different oxygen tensions

“…In our model, the chondrocyte concentration in alginate beads was about 1 × 10 6 chondrocytes for 18 alginate beads. This cell density was higher than that observed in histological preparations of articular cartilage, but corresponded to the cell density used by other authors in similar models (Guo et al, 1989;Sanchez et al, 2002).…”

Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes

“…The released chondrocytes were thoroughly washed, counted, and then suspended in a solution of alginate (Keltone LV, 1.2% in 150 mM NaCl; Kelco, Chicago, IL). Beads of alginate gel, each containing approximately 40,000 entrapped chondrocytes, were prepared as described previously, by dropwise release of the cell suspension into a sterile solution of isotonic CaCI, (102 mM) (36,38). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO, in air and fed for the first 96 hours with complete medium, consisting of 45% Ham's F-12, 45% Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS; Hyclone, UT), and 25 pg of ascorbate per ml.…”

“…To obtain a measure of 3sS-sulfate incorporation per mg of DNA, additional beads from each donor were treated similarly, but were not labeled. After release from the alginate gel with chelating agents as reported previously (38), the cells were solubilized by treatment with papain at 60"C, and the content of DNA was evaluated by fluorescence using Hoechst dye 33258 (41). For each donor and each culture condition (i.e., with and without FBS), synthesis of 35S-PG was expressed as the ratio of the counts per minute per mg of DNA in treated and untreated cultures.…”

Mechanical confinement regulates cartilage matrix formation by chondrocytes

“…Alginate is a polysaccharide that does not present any cell-adhesion ligands for cells to bind to, and is not degradable by mammalian enzymes 23 . Alginate can be crosslinked into a 3D hydrogel with divalent cations such as calcium, and these hydrogels have been widely used for 3D culture of cells, including chondrocytes 24–26 . Ionic crosslinking results in viscoelastic hydrogels 27,28 , as the weak ionic crosslinks can unbind under stress or strain, allowing the hydrogel polymer matrix to flow 28 .…”