Materials and Methods for Cryopreserved Bone Contstructs
The technology developed includes materials and methods for cryo-preservation of HCG-cell constructs. In one embodiment, porous HCG scaffolds are provided in a perfusion bioreactor having perfusion chambers that can contain the HCG scaffolds, cells are then seeded in the HCG scaffolds in the perfusion bioreactor, cell culture media is perfused through and the bioreactor operated so as to allow for cell seeding and growth in the HCG scaffold. After a suitable period of time, the cell culture media is removed and the HCG containing cells (HCG-cell constructs) can be washed with a suitable buffer, such as phosphate-buffered saline (PBS). The HCG-cell constructs are then perfused with a suitable cryo-preservation fluid transversely across the HCG-cell constructs in the bioreactor. The cryo-preservant can comprise one or more of the following: DMSO, trehalose, glycerol, ethylene glycol, and serum for cell culture (e. g., fetal bovine serum (FBS)). In one embodiment, the HCG-cell constructs are perfused for a suitable period of time with cryo-preservant fluid using transverse flow of the fluid in the bioreactor at a suitable flow rate. The HCG-cell constructs (or the perfusion chambers containing them) are then removed from the bioreactor and placed in a cryo-preservant media and maintained at increasingly colder temperatures until temperatures reach about -80° C. The frozen HCG-cell constructs (or the chambers containing them) can then be stored at a suitable cryogenic temperature (e.g., in liquid nitrogen) until needed. When needed, frozen HCG-cell constructs can be removed from cold storage and thawed using suitable means (e. g., 37°C. water bath). Cells contemplated for use in the presentinvention include stem cells, such mesenchymal stem cells.Cells can be animal cells, such as mammalian cells or human cells.