Ammonium-Chloride-Potassium Lysing Buffer Treatment of Fully Differentiated Cells Increases Cell Purity and Resulting Neotissue Functional Properties.

Juvenile and fetal, primary, fully differentiated cells are widely considered to be ideal cell types for tissue engineering applications. However, their use in tissue engineering may be hindered through contamination by undesirable cell types. These include blood-associated cells as well as unwanted resident cell types found both in healthy and pathologic donor tissues. Ammonium-chloride-potassium (ACK) lysing buffer is used to lyse red blood cells (RBCs) during the isolation of stem cell populations, but has not been explored for the purification of fully differentiated cells. This study sought to investigate the effect of ACK buffer treatment of freshly isolated, fully differentiated cells to increase cell purity and enhance the formation of biofunctional engineered neotissues; this was tested in the well-established cartilage tissue engineering model of the self-assembling process using fetal ovine articular chondrocytes (foACs) and juvenile bovine articular chondrocytes (jbACs). ACK buffer treatment of foACs and jbACs decreased the number of contaminating RBCs by over 60% and additionally reduced the number of apoptotic chondrocytes in the cell isolates. Reducing the number of contaminating RBCs removed cellular detractors to the self-assembling process and eliminated an apoptotic stimulus, thus improving neocartilage homogeneity, chondrocyte distribution, and extracellular matrix deposition within the neotissues. For example, in foAC neocartilage, ACK buffer treatment ultimately led to a 170% increase in compressive aggregate modulus, a 130% increase in shear modulus, an 80% increase in tensile modulus, and a 130% increase in ultimate tensile strength of the neocartilage. This work represents the first time that ACK buffer has been used to purify fully differentiated cells and subsequently increase the functional properties of neotissue.