RELATIONSHIP BETWEEN CELL FUNCTION AND INITIAL CELL SEEDING DENSITY OF PRIMARY PORCINE CHONDROCYTES IN VITRO

Abstract

Maintenance of differentiated functional phenotype within in vitro chondrocyte culture requires seeding at high densities with large numbers of cells. However, optimal cell seeding numbers and densities remain elusive due to multiple varying parameters and different methodologies utilized in previous studies. In the current study, we tried to investigate the relationship between cell seeding number and differentiated functional phenotype of in vitro cultured chondrocytes. Varying numbers of primary porcine chondrocytes (0.25, 2.5, 25 and 250 K) were seeded in 96 well-plates and cultured for 4 weeks. Cell proliferation, glycosaminoglycan (GAG) production and gene expression levels of Sox9, aggrecan, COL II and COL I were evaluated. The results showed that GAG content was high in the 0.25 and 25 K groups, gene expression of Sox9 was high in the 2.5, 25 and 250 K groups and expression of COL II was high in the 25 K group, whereas expression of COL I was low in the 0.25, 25 and 250 K groups. It is concluded that the seeding number and density of the 25 K (78 K cells/cm2) group achieved the optimal balance between functional phenotype of individual cells and the total ECM production for in vitro cultured chondrocytes.

3D Culture of Chondrocytes in Gelatin Hydrogels with Different Stiffness

“…Another reason may be that the GelMA hydrogels with high stiffness could promote the chondrocytes cluster formation. The chondrocytes aggregation has been reported to facilitate the outcome of cartilage repair [ 50 , 51 ], because cell-cell interaction plays a critical role in chondrogenesis [ 52 , 53 , 54 ]. The staining results indicated that GelMA hydrogels with high stiffness could support the chondrogenic phenotype.…”

Scaffold channel size influences stem cell differentiation pathway in 3-D printed silica hybrid scaffolds for cartilage regeneration

“…* indicates P < 0.05. tures, but extremely high cell density could result in the formation of tight cell sheets or agglomerates, limiting oxygen, nutrient and waste product exchange, leading to apoptosis and reduced ECM formation. 43,44 It has also been reported that in high cell density cultures, Sox9 could inhibit the expression of hyaline cartilage marker collagen Type II. 45,46 In addition to cell-cell interaction, cell-material (cellprotein-material) interaction also plays an important role in maintaining phenotypic chondrocyte characteristics.…”

Influence of Cellular Microenvironment on Human Articular Chondrocyte Cell Signaling

“…17,18 Spherical cell morphology, decreased actin cytoskeletal organization, and cell clusters that facilitate the outcome of cartilage repair 26,45 improved chondrogenesis due to the increase in intercellular contacts. 46,47 Suboptimal chondrogenic expression levels were found when cells were subjected to conditions that deviate from the native-like cellular microenvironment (i.e., the Soft and Stiff Col-Tgels, 0.9 ± 0.1 and 40 ± 10 kPa, respectively).…”