Phase contrast image (100x) of chondrocyte and associated cells in day one (29 hours after innoculation) of culture as described (SC00A7 Protocol). Scale bar = 50 m. Chondrocyte is darker multicellular structure overlying attached cells, ~100 m in diameter.

Native MSC-Derived Human Chondrocytes Neuromics -Vitro Biopharma Quality Control Study Cell Culture and Viability of SC00A7 lot 006 January 2013

Labeled and unlabeled chondrocytes

Phase contrast image (100x) of chondrocyte and associated cells in day two (45 hours after innoculation) of culture as described (SC00A7 Protocol). Scale bar = 50

m. Chondrocyte is darker multicellular structure overlying attached cells, ~150 m in diameter.

Labeled and unlabeled chondrocytes

Phase contrast image (200x) of chondrocyte and associated cells in day five of culture as described (SC00A7 Protocol). Scale bar = 50 m. Chondrocyte is darker multicellular structure overlying attached cells, ~ 70 m in diameter.

Labeled and unlabeled chondrocytes

Cell viability of chondrocyte cultures determined by propidium iodide(PI) method in the second day of culture. Cells were cultured as above and 1 mg/ml PI was added to yield a final [PI] of 5 g/ml. PI positive (dead) cells are shown here as white. Viable was >/= 70%. Scale bar is 50 m. Direct measurement of viability by the PI method at day 5 yielded 83% viability.

Labeled and unlabeled chondrocytes

Conclusions: • Human MSC-derived chondrocytes were maintained viable in cell culture using Neuromics-Vitro Biopharma catalog number PC-004 Human Chondrocyte Maintenance Medium in Greiner Bio-One TC-coated 48-well cell culture plates innoculated with 20 to 50 l/well (2,000 to 5,000 cells per well) • Cell cultures consisted of multicellular chondrocytes together with attached cells, that are possibly MSCs derived from chondrocytes. • Cell viability was high throughout 5 full days of culture as determined by propidium Iodide permeation of cells. Cellular viability of cells detached from these cultures by Accutase treatment was 83% after five days in continuous culture.

Labeled and unlabeled chondrocytes Culturing Protocol