Growth factor contents of autologous human sera prepared by different production methods and their biological effects on chondrocytes

Abstract

To discuss the autologous serum production for cartilage tissue engineering, we compared three kinds of sera: whole blood-derived serum (WBS), platelet-containing plasma-derived serum (PCS), and plasma-derived serum (PDS), on the growth factor contents and their biological effects on human auricular chondrocytes. EGF, VEGF and PDGF levels were highest in WBS, while PCS and PDS followed WBS. The proliferation effects of WBS were the most pronounced, followed by that of PCS, both of which realized a 1000-fold-increase in chondrocyte numbers at the third passage, whereas PDS reached it after passage 4. No significant differences were observed in histology or cartilaginous matrix measurements of tissue-engineered cartilage produced from chondrocytes cultured under different serum conditions. WBS would be clinically useful because of its potent proliferation effects, while PCS, which possibly saves the red cell concentrate, may be an option in cases where there are elevated risks of blood loss.

Regenerative Cartilage made by Fusion of Cartilage Elements derived from Chondrocyte Sheets prepared in Temperature-Responsive Culture Dishes

“…The isolated chondrocytes were seeded in a 100 mm collagencoated plastic tissue culture dish (AGC Techno Glass, Chiba, Japan) at a density of 2,500 cells/cm 2 , and cultured in chondrocyte growth medium (DMEM/F12 containing 5% human serum, 100 ng/ml FGF-2, and 5 g/ml insulin) 13,14) in a 37 °C/5% CO 2 incubator. The medium was changed twice a week.…”

“…The fragments of cartilage were incubated for collagenase digestion in a 0.15 % collagenase solution at 37 o C in 24 h. The collected cells counted after dyeing with trypan blue were confirmed for cell viability. The isolated 2.0×10 5 chondrocytes were seeded in the 100 mm collagen-coated dish and cultured in DMEM/F12 containing 5 % autologous serum, 100 ng/ml FGF-2, and 5 g/ml insulin in a 37 o C /5% CO 2 incubator 13,14) . In the prepapration of autologous serum, whole blood was harvested from the cervical vein of each beagle, and was allowed to clot over 5 h at room temperature 13) .…”

“…The isolated 2.0×10 5 chondrocytes were seeded in the 100 mm collagen-coated dish and cultured in DMEM/F12 containing 5 % autologous serum, 100 ng/ml FGF-2, and 5 g/ml insulin in a 37 o C /5% CO 2 incubator 13,14) . In the prepapration of autologous serum, whole blood was harvested from the cervical vein of each beagle, and was allowed to clot over 5 h at room temperature 13) . The serum was separated by centrifugation of the clotted blood at 500 × g for 30 minutes.…”

Usefulness of Agarose Mold as a Storage Container for Three-Dimensional Tissue-Engineered Cartilage

“…The lysate was filtered through a cell strainer with a pore size of 100 μm to remove residues, and then centrifuged at 500 × g for 5 minutes to isolate human chondrocytes. The isolated chondrocytes were seeded in a collagen type I-coat plastic tissue culture dish at a density of 2500 cells/cm 2 , and cultured in chondrocyte growth medium (DMEM/F-12 containing 5% human serum, 100 ng/mL FGF-2, and 5 μg/mL insulin) [11,12] in a 37˚C /5% CO 2 incubator. The medium was changed twice a week.…”

Application of floating cells for improved harvest in human chondrocyte culture

“…However, we often observed cells that did not adhere to culture plates and continued to float over a certain time period after cell seeding. In general, we changed the culture medium every 2 or 3 days (23,24), at which time these floating cells were generally discarded. We reasoned that if adequate space is provided for the attachment of cells, the cells floating within the medium may effectively adhere to the plate and begin to proliferate.…”