Table 5.

Experimental studies evaluating post-thaw BM-MSCs proliferation potential

Study	Species	Results post-thaw	Method of assessment
Human
Bruder et al. [91]	Human	No effect on proliferation	Cell re-plated for one passage post-thaw; crystal violet dye-binding method
Haack-Sorensen et al. [19]	Human	No effect on proliferation	PKH26-GL cell linker kit
Xiang et al. [93]	Human	No effect on proliferation	Growth curves
Zhao et al. [94]	Human (with chronic myeloid leukaemia)	No effect on proliferation	Cell count and cell-doubling time
Doan et al. [95]	Human	No effect on proliferation	NA
Ginis et al. [50]	Human	Proliferation of cryopreserved cells after 1 or 5 months of storage was higher than non-cryopreserved cells	Calcein-AM staining (at day 1, 4, 7 and 14 after post-thaw plating)
Mamidi et al. [33]	Human	No effect on proliferation	Population doublings, cumulative population doublings and population doubling time
Matsumura et al. [26]	Human	No effect on proliferation	Cell count; population doubling time (24 h, 48 h, 72 h and 96 h post-thaw)
Holubova et al. [69]	Human	No effect on proliferation	Cell count
Al-Saqi et al. [66]	Human	No significant difference in population doubling time but cells cryopreserved in DMSO had longer population doubling time compared to fresh	Population doubling (first and second passage post-thaw)
Luetzkendorf et al. [40]	Human	No effect on proliferation	Population doublings; Population doubling time
Pollock et al. [67]	Human	Population doublings decreased with increasing pre-freeze passage number	Population doublings
Lechanteur et al. [34]	Human	Very low recovery until day 4 then a slight increase indicating re-proliferation	Cell count (0–5 days after thawing)
Yuan et al. [52]	Human (BM-MSC engineered to express TRAIL)	No effect on proliferation	XTT assay
Other species
Edamura et al. [36]	Dog	DMSO and FBS-free freezing resulted in similar proliferative capacity as non-cryopreserved; DMSO and FBS containing freezing media gave lower proliferative capacity	Cell count (2, 4, 6,8, 10 and 12 days post-thaw)
Tokumoto et al. [48]	Monkey	No effect on proliferation	DNA quantification at 4, 8 and 12 days
Lauterboeck et al. [49]	Monkey	No effect on proliferation	Population doubling time
Heino et al. [39]	Minipig	Two to sixfolds decrease in the proliferative capacity of cells	Population doublings
Romanek et al. [98]	Pig (BM-MSC treated with a high hydrostatic pressure (HHP) before freezing)	Cells treated with HHP showed better proliferation rate	Cell count
Mitchell et al. [32]	Horse	No effect on proliferation	Cell staining with CellTrace label
Colony-forming unit ability
Human
Verdanova et al. [25]	Human	Best number of colonies obtained when cells were frozen with 5% DMSO with 5% sericin in culture medium	Cells seeded 60 cm Petri dishes for 2 weeks, Crystal Violet stained and colonies counted (light microscope)
Other species
Ock and Rho, [51]	Pig	All cryopreserved cells showed significantly lower numbers of colonies compared to fresh; Lower DMSO produced higher number of colonies	Cells seeded in 6-well plates for 2 weeks, 4% Giemsa stained and colonies counted (light microscope)
Mitchell et al. [32]	Horse	No effect on colony-forming unit ability	Cells seeded in 10 cm plates for 1 week, Crystal Violet stained and colonies counted (light microscope)

The key results on bone-marrow derived mesenchymal stem cell proliferation 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