03541nas a2200277   4500008004100000022004200041245014500083210006900228250001000297260000800307300001500315490000700330520254200337653003902879653005002918653002802968653005402996653002003050653004203070653003703112653001103149100001603160700001403176700002503190856004803215       2006                        eng d  a1076-3279 (Print) 1076-3279 (Linking)00aEffect of stratified culture compared to confluent culture in monolayer on proliferation and differentiation of human articular chondrocytes0 aEffect of stratified culture compared to confluent culture in mo  a38986  cSep  a2397 - 4050 v123 aWith conventional tissue culture of cells, it is generally assumed that when the available 2D substrate is fully occupied, growth ceases or is greatly reduced.However, in nature wound repair mostly involves proliferation of cells that are attracted to the defect site in a 3D environment.Hence, proliferation continues in 3D until the defect site is filled with cells contributing to repair tissue. With this in mind,we examined the growth behavior of human articular chondrocytes during stratified culture as opposed to routine culture to confluency. Additionally, we studied the influence of growth factors on proliferation during stratified culture and differentiation thereafter. Chondrocytes were cultured in monolayer on tissue culture plastic to confluency or stratified for an additional 7 days. Culture medium was based on DMEM with 10% serum and either supplemented with high concentrations of nonessential amino acids (NEAA) and ascorbic acid (AsAP), or instead with basic fibroblastic growth factor (bFGF), platelet-derived growth factor (PDBF-BB), and/or transforming growth factor beta1 (TGF-beta). After expansion, cells were harvested, counted, and their differentiation capacity was examined in pellet culture assay. It was shown that chondrocytes, cultured stratified proliferate exponentially for up to an additional 4 days and that cell yield increased 5-fold. Furthermore, during stratified culture the number of cells increased further in the presence of bFGF, PDBF-BB, and TGFbeta1 or high concentrations of NEAA and AsAP. Depending on donor variation and factors supplemented the cell yield ranged from 0.06 up to 1.1 million cells/cm2 at the second passage. During stratified culture in the presence of either bFGF and PDGF or high concentrations of NEAA and AsAP, exponential growth continued for up to 7 days. Finally, cells maintained their differentiation capacity when cultured stratified with or without growth factors (bFGF, TGF-beta, and PDGF), but not when cultured with high levels of AsAP and NEAA. In contrast to other 3D culture techniques like microcarrier or suspension culture, nutrient consumption remained the same as with conventional expansion. Because this allows culturing of clinically relevant amounts of chondrocytes without increasing the amount of serum, chondrocytes can be fully expanded in the presence autologous serum, avoiding the risk of viral and/or prion disease transmission associated with the use of animal-derived serum or serum replacers with animal-derived constituents.10a*Cell Proliferation / drug effects10aCartilage, Articular / cytology / *physiology10aCell Culture Techniques10aCell Differentiation / drug effects / *physiology10aCells, Cultured10aChondrocytes / cytology / *physiology10aGrowth Substances / pharmacology10aHumans1 aHendriks, J1 aRiesle, J1 aVanblitterswijk, C A  uhttps://www.notifylibrary.org/reference/661