Assessment of viability and osteogenic ability of human mesenchymal stem cells after being stored in suspension for clinical transplantation.

Human mesenchymal stem cells (MSCs) were suspended in phosphate-buffered saline (PBS) and stored up to 24 h at 4 degrees C, 24 degrees C, and 37 degrees C. More than 80% viability was maintained at any temperature for at least 1 h, then gradually decreased over time. After 24 h, the viabilities at 4 degrees C, 24 degrees C, and 37 degrees C were about 81%, 70%, and 62%, respectively. The MSCs suspended/stored in PBS at 4 degrees C for 24 h also exhibited in vitro osteogenic differentiation capability as evidenced by mineralized matrix formation as well as high alkaline phosphatase activity when cultured in an osteogenic medium. Furthermore, in vivo implantation experiments using the MSCs also demonstrated new bone formation. Because MSCs are known to possess multipotential stem cell characteristics, these data indicate that human MSCs stored in PBS at 4 degrees C could be delivered to distant medical facilities for the purpose of hard tissue and other types of tissue regeneration therapy.

Observation and quantitative analysis of rat bone marrow stromal cells cultured in vitro on newly formed transparent beta-tricalcium phosphate.

To observe living cell morphology on ceramics by light microscopy, we fabricated a new material-transparent beta - tricalcium phosphate (t-beta TCP) ceramic-for the purpose of serving as a tissue culture substrate. Bone marrow stromal cells (BMSCs) were obtained from rat femora and cultured on both t-beta TCP ceramic disks and culture grade polystyrene (PS) dishes in an osteogenic medium. After 1 day of culture, cell attachment and spreading on both the t-beta TCP and PS substrata were equally and clearly detected by ordinary light microscopy. After 14 days of culture, extensive cell growth, alkaline phosphatase (ALP) staining, and bone mineral deposition could be detected on both substrata. In addition, quantitative biochemical analyses revealed high DNA content, ALP activity, and osteocalcin content of these cultures. This experiment is significant in that all of the results were similarly observed on both the t-beta TCP and PS substrata, indicating the excellent properties of beta TCP ceramics for BMSCs culture towards osteogenic differentiation.

Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells.

Human bone marrow-derived mesenchymal cells contain mesenchymal stem cells (MSCs), which are well known for their osteo/chondrogenic potential and can be used for bone reconstruction. This article reports the viability of cryopreserved human mesenchymal cells and a comparison of the osteogenic potential between noncryopreserved and cryopreserved human mesenchymal cells with MSC-like characteristics, derived from the bone marrow of 28 subjects. The viability of cryopreserved mesenchymal cells was approximately 90% regardless of the storage term (0.3 to 37 months). It is clear by fluorescence-activated cell sorter analysis that the cell surface antigens of both noncryopreserved and cryopreserved mesenchymal cells were negative for hematopoietic cell markers such as CD14, CD34, CD45, and HLA-DR but positive for mesenchymal characteristics such as CD29 and CD105. To monitor the osteogenic potential of the cells, such as alkaline phosphatase (ALP) activity and in vitro mineralization, a subculture was conducted in the presence of dexamethasone, ascorbic acid, and glycerophosphate. No difference in osteogenic potential was found between cells with or without cryopreservation treatment. In addition, cells undergoing long-term cryopreservation (about 3 years) maintained high osteogenic potential. In conclusion, cryopreserved as well as noncryopreserved human mesenchymal cells could be applied for bone regeneration in orthopedics.

Clinical application of marrow mesenchymal stem cells for hard tissue repair.

Human marrow mesenchymal stem cells were cultured in a medium containing glycerophosphate, ascorbic acid, and dexamethasone (Dex) on alumina ceramic discs and on tissue culture polystyrene (TCPS) dishes. Cell proliferation followed by osteogenic differentiation was observed to be equal on both culture substrata. The differentiation resulted in the appearance of bone-forming osteoblasts, which fabricated mineralized matrices on these substrata. Stem cells kept at 4 degrees C for 24 h outside a CO2 incubator maintained a viability level of more than 90%. The regenerative cultured bone outside the incubator also maintained high alkaline phosphatase activity for several hours. These results verified that cultured bone fabricated at a cell processing center can be transported to distant hospitals for use in hard tissue repair. To date, the tissue engineered cultured bone formed on alumina ceramics in this environment have already been used in clinical situations, such as total ceramic ankle replacements.