Hi Swarnapriya,

They should not take more than a few days to grow robustly.  I suspect that whoever froze them did a poor job.  Not much you can do if they were frozen as too crowded in the tube, used bad DMSO, did not cool them at a slow rate, etc...

Sorry!

Steve

When you say spherical, do you mean they are not attaching? That could possibly be dead cells due to the reasons Steve mentions. I assume they were kept in liquid nitrogen? Viability decreases over time at minus 80.

One thing we have noticed is the function of frozen cells is always reduced compared to fresh cell of the same passage that were never frozen. So when doing experiements, either always use fresh cells or always use frozen cells. Never mix or the standard deviations on your collated data at the end become huge.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088734/

https://www.atcc.org/~/media/PDFs/Cryopreservation_Technical_Manual.ashx

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MSCs from bone marrow and cord blood are cryopreservable by the methods used to cryopreserve their haematopoietic analogues, and slow freezing protocols utilising DMSO as cryoprotectant are in use by many groups . Recent studies by Liu et al.  have confirmed that 10% DMSO and slow cooling/rapid warming does not affect the viability or differentiation potential of adipose-derived MSCs. Adult stem cells derived from human dental pulp also showed high post-thaw recovery and trilineage differentiation potential after slow cooling in 1–1.5 mol/1 DMSO (∼7.5–10%). Furthermore, DMSO was shown to be superior to both ethylene glycol and propylene glycol under these conditions .

DMSO is known to have an effect on the epigenetic profile of, and induce differentiation in, murine stem cells . This, together with its the known adverse reactions when employed as a cryoprotectant for haematopoietic cell therapy, plus its cellular toxicity, have led to attempts to cryopreserve MSCs with other cryoprotectants; either on their own or in combination with DMSO at lowered concentrations. Many of the alternative cryoprotectant formulations have also attempted to remove animal serum from the cryoprotectant solution; both to reduce cost and to improve clinical utility through reducing the likelihood of zoonotic infections in the absence of terminal sterilisation procedures. This has led to substitution with human serum or human serum albumin. However, this too is costly and introduces at least the potential for transmission of human pathogens.

The use of polyvinylpyrrolidone (PVP), an extracellular cryoprotectant, has been investigated as an alternative to cryopreservation with DMSO and foetal calf serum (FCS). Cellular recovery and differentiation capacity were studied after equilibration in a number of different cryopreservation media followed by ‘dump’ freezing to −80 °C and storage in liquid nitrogen. Recovery of cells cryopreserved in 10% PVP with human serum was comparable with (though slightly lower than) cells cryopreserved in DMSO with animal serum. A similar study utilising methylcellulose either alone or in conjunction with reduced levels of DMSO indicated that human serum could replace FCS in standard DMSO mixtures without affecting the recovery of cells and that 1% methylcellulose produced comparable results with DMSO concentrations as low as 2% when an annexin V apoptosis assay was used to analyses cells 24 h post-thaw. Cells also maintained their adipogenic and osteogenic potential . Lui et al. have also adopted a reduced DMSO approach in a recent study to produce a well-defined and xeno-free cryopreservation media for cell therapy with bone marrow-derived MSCs. They replaced FCS with combinations of polyethylene glycol (PEG) and trehalose, with DMSO concentration ranging from 2.5 to 7.5%. The recovery, metabolic activity, proliferative capacity and differentiation potential were measured against the standard slow cooling protocol of DMSO and 10% FCS. Results replacing FCS with PEG in reduced concentrations of DMSO were comparable with those of the control, but this was achieved only in the presence of 2% bovine serum albumin. Moreover, cryopreservation in 10% DMSO with 90% FCS was more effective in their hands than any other combination. Trehalose was ineffective, in contrast to studies on haematopoietic cells derived from cord blood, bone marrow or mobilised peripheral blood where its effectiveness in combination with reduced DMSO concentration has been demonstrated . Nevertheless, these laboratory studies indicate that there is a potential for the development of xeno-free cryoprotectant solutions utilising lowered concentrations of DMSO and slow cooling.

Best

S.H

thank you everyone!