Table 4.
Published experimental studies detailing BM-MSCs post-thaw differentiation potential
| Study | Species | Results post-thaw | Method of assessment |
|---|---|---|---|
| Human | |||
| Bruder et al. [91] | Human | No effect on osteogenic differentiation ability | Cell re-plated for one passage post-thaw then re-plated and incubated with osteogenic supplements; quantification of alkaline phosphatase activity |
| Hirose et al. [41] | Human | No effect on osteogenic differentiation ability | Incubation with osteogenic media for 25 days; quantitative fluorescence analysis of calcein uptake |
| Kotobuki et al. [35] | Human | No effect on osteogenic differentiation ability | Incubation with osteogenic medium for 2 weeks; calcium and alkaline phosphatase activity staining |
| Kotobuki et al. [92] | Human | No effect on osteogenic differentiation ability | Incubation in osteogenic media for 2 weeks; quantification of alkaline phosphatase activity and calcein uptake |
| Xiang et al. [93] | Human | No effect on adipogenic or neuro genic differentiation ability | Cells at P15 post-thaw incubated in adipogenesis medium for 12 days; Oil Red O staining |
| Cells at P15 post-thaw incubated in neurogenesis induction medium for 1 or 6 days; fluorescent staining and RT-qPCR | |||
| Zhao et al. [94] | BM-MSC (human with chronic myeloid leukaemia) | No effect on differentiation ability | Incubation in osteogenic medium for 21 days; Von Kossa staining and RT-qPCR |
| Incubation in adipogenic media for 14 days; Oil Red staining and RT-qPCR | |||
| Incubation in neurogenic medium; Immunocytochemistry and western blotting for NF, GFAP and GalC | |||
| Endothelial differentiation for 2 weeks; immunohistochemical and western blotting for CD31 and vWF | |||
| Liu et al. [28] | Human | Serum-free reduced-DMSO freezing solution gives comparable differentiation to 10% DMSO | Incubation in osteogenic or adipogenic media for 2 weeks, and chondrogenic media for 3 weeks |
| Doan et al. [95] | Human | No effect on adipogenic differentiation ability | Incubation in adipogenic medium for 2–3 weeks; Oil Red staining |
| Ginis et al. [50] | Human | No effect on osteogenic differentiation ability | Incubation with osteogenic media; quantification of alkaline phosphatase activity on days 7 and 14 after incubation as well as flow cytometry analysis at day 14 after incubation of alkaline phosphatase surface expression |
| Quantification of calcium deposition at day 21 after incubation | |||
| Mamidi et al. [33] | Human | No effect on tri-lineage differentiation ability | Incubation in osteogenic differentiation media for 3 weeks; Alizarin red staining |
| Incubation in adipogenic differentiation media for 3 weeks; Oil Red O staining | |||
| Incubation in chondrogenic differentiation media for 3 weeks; Alcian blue staining | |||
| Matsumura et al. [26] | Human | No effect on tri-lineage differentiation ability | Incubation in osteogenic differentiation media for 14 days; Alizarin red staining and alkaline phosphatase activity |
| Incubation in adipogenic differentiation media for 14 days; Oil Red O staining and GPDN activity | |||
| Incubation in chondrogenic differentiation media for 14 days; Alcian blue staining | |||
| Kumazawa et al. [37] | Human | No effect on adipogenic or osteogenic differentiation ability | Incubation in osteogenic medium for 1, 2, and 3 weeks then alkaline phosphatase activity, calcium levels, alizarin red staining and RT-qPCR |
| Incubation in adipogenic medium for 1, 2, and 3 weeks; Oil Red O staining | |||
| Luetzkendorf et al. [40] | Human | No effect on adipogenic and osteogenic differentiation ability | Incubation in diff media until morphological signs of differentiation were visible (10–15 days) |
| Lechanteur et al. [34] | Human | No effect on differentiation ability | NA |
| Yuan et al. [52] | Human (BM-MSC engineered to express TRAIL) | No effect on tri-lineage differentiation ability | Differentiation procedures performed using StemPro differentiation kits according to manufacturer’s instructions |
| Other species | |||
| Liu et al. [29] | Rat, mouse and calf | No effect on adipogenic or osteogenic differentiation ability | Incubation with adipogenic media for 2 weeks; Oil Red O staining and alkaline phosphatase activity expression staining with BCIP/NBT |
| Incubation with adipogenic or osteogenic media for 2 weeks then Oil Red O staining and alkaline phosphatase activity expression staining with BCIP/NBT | |||
| Naaldijk et al. [27] | Rat | In general, no difference in differentiation was observed (qualitative observation); Quantification of ALP: ALP activity is lower at ‘high’ (> 5%) levels of DMSO compared to solutions with a higher HES 450 content | Incubation with osteogenesis, adipogenesis and chondrogenic media for 2 weeks |
| Quantification using alkaline phosphatase assay | |||
| Li et al. [96] | Dog | No effect on osteogenic differentiation ability | Incubation in osteogenic media for 5, 10 and 15 days; alkaline phosphatase activity measurement |
| Incubation in osteogenic media for 21 days; number of mineralized nodules determined | |||
| Zhu et al. [46] | Dog | No effect on osteogenic differentiation ability | Incubation with osteogenic media (21 days); measurement of alkaline phosphatase activity at 5, 10 and 15 days and Von Kossa staining and nodules counting at day 21 |
| Edamura et al. [36] | Dog | No effect on neurogenic differentiation ability | Incubation with neurogenic media for 6 h then RT-qPCR |
| Tokumoto et al. [48] | Monkey | Cryopreserved cells had a slightly higher ALP enzyme activity than non-cryopreserved cells | Incubation with osteogenic media for 4, 8 and 12 days; quantification of alkaline phosphatase enzyme activity |
| Nitsch et al. [97] | Monkey | No effect on adipogenic or osteogenic differentiation ability | Incubation with adipogenic media for 5 weeks; oil-red O staining |
| Incubation with osteogenic medium for 21 days; Von Kossa staining | |||
| Lauterboeck et al. [49] | Monkey | Significant decrease in oil droplet formation | Incubation with adipogenic medium for 20 days; Oil Red O staining |
| No difference in osteogenic differentiation ability | Incubation with osteogenic induction medium for 3 weeks; Alizarin red staining | ||
| Heino et al. [39] | Minipig | Cells lost their osteogenic differentiation potential | Cells incubated in osteogenic medium; stained for alkaline phosphatase activity (tested 9 days post-thaw) |
Results on bone-marrow derived mesenchymal stem cell tri-linage are presented in this table. For further details on the cryopreservation experimental details refer to either Table 1 or Additional file 2 which provide the individual freezing protocols outlined in the extracted papers alongside the concentration and passage of cells at the point of cryopreservation and the process of thawing