USE OF ADIPOSE TISSUE-DERIVED STROMAL CELLS FOR CHONDROCYTE DIFFERENTIATION AND CARTILAGE REPAIR
Methods and compositions for directing adipose-derived stromal cells cultivated in vitro to differentiate into cells of the chondrocyte lineage are disclosed. The invention further provides a variety of chondroinductive agents which can be used singly or in combination with other nutrient components to induce chondrogenesis in adipose-derived stromal cells either in cultivating monolayers or in a biocompatible lattice or matrix in a three-dimensional configuration. Use of the differentiated chondrocytes for the therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo is disclosed.
1 . A method for differentiating adipose tissue derived stromal cells into chondrocytic cells, comprising:
a) pelleting said stromal cells by centrifuging between 50,000 to 5 million cells at 500×g for 2 to 20 minutes in sterile tubes containing a medium such as Dulbecco's Modified Eagle's Medium (DMEM) or alpha modified Minimal Essential Medium (αMEM) or Roswell Park Memorial Institute media 1640 (RPMI Media 1640);
b) plating isolated stromal cells at a density of 500 to 20,000 cells/cm 2 in a differentiating medium;
c) supplementing said medium with:
(i) a chondroinductive agent capable of activating any cellular signal transduction pathway leading to the mature chondrocyte phenotype;
(ii) an antibiotic;
(iii) a nutrient supplemented with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins;
(iv) ascorbate or related vitamin C analog;
(v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and
d) incubating said cells at about 31° C. to 37° C. for about 3-4 weeks in with 5% CO 2 and between 1% and 20% oxygen.
2 . A method for differentiating adipose tissue derived stromal cells into chondrocytic cells, comprising:
a) suspending stromal cells at a concentration of 0.5 to 10 million cells per ml in calcium alginate or any other biocompatible lattice or matrix of supporting chondrogenesis in a three-dimensional configuration;
b) transferring cells to 35 mm culture dishes and plating cells at a density of 500 to 20,000 cells/cm 2 in a differentiating medium comprising a chemically defined culture medium having or supplemented with:
(i) a chondroinductive agent capable of activating any cellular transduction pathway leading to the mature chondrocyte phenotype;
(ii) an antibiotic;
(iii) a nutrient supplemented with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins;
(iv) ascorbate or related vitamin C analog;
(v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and
(c) incubating said cells at about 31 to 37° C. for about 3-4 weeks in an incubator with 5% CO 2 and between 1% and 20% oxygen.