**8. Microbiological controls**

(at 10–40% concentrations), with the increasingly high viscosities at low temperature and avoiding that water molecules form ice crystals [52]. In a study reported by Renzi et al. [57], several cryopreservation solutions for MSCs isolated from equine, ovine, rodent bone marrow, and equine adipose tissue were compared: the best results regarding cell viability were obtained using a solution of fetal bovine serum added with 10% of DMSO. Conversely, in a previous study, Ock and Rho [58] reported that the survival and number of colonies formed by porcine MSCs were significantly decreased following short‐term storage (less than a month) into liquid nitrogen (−196°C) and the amount of this decrease was inversely proportional to the DMSO concentration. Those data strongly suggest the use of 5% DMSO instead of conventional 10% DMSO for the cryopreservation of porcine MSCs, for minimizing the CPA toxicity on cells. However, slow freezing with reduced concentration of CPAs has gained much interest in order to decrease the effect of the osmotic shock and chemical toxicity. Nevertheless, the commonly used CPAs are highly toxic at 37°C (body temperature) and could not be applied to patients. For this reason, multistep washing is required to completely remove the highly toxic, cell membrane‐permeable cryoprotectants from cryopreserved cells for clinical use, though this procedure is often associated with significant loss of precious cells (~10% during each washing step). Therefore, it is important to achieve cell cryopreservation with nontoxic CPAs. Recently, Rao et al. [59] demonstrate that nanoparticle‐mediated delivery of trehalose into mammalian cells has great potential for cryopreserving the human primary adipose derived stem cells (hADSCs) and possibly other types of stem cells to facilitate their ready availability for clinical use. In fact, successful results on cryopreservation of hADSCs using only trehalose as cryoprotectant has been achieved with high survival and undamaged

As well as cooling, optimizing the thawing method of frozen MSCs is also important. Furthermore, in clinical transplantation applications the post‐thaw viability assessment has shown to be of paramount importance. Several techniques have already been suggested for thawing frozen sample. A procedure of thaw and wash allows to remove DMSO and cell fragments, but may cause cell loss or cellular aggregation during centrifugation. Thaw, dilution, and wash procedure avoids the problem due to the centrifugation, allowing an osmolar equilibration, but the untoward effects of DMSO and cell debris infusion are not prevented. Currently, the standard method for thaw frozen MSCs, either from slow freezing or vitrification, is to warm them rapidly (>100°C/min) in a water bath at 37°C, until all ice crystals disappear. This method generally results in high post‐thaw recovery of viable cells without using high‐cost equipment, but it is safer to thaw cells using a dry warming procedure, due to the potential microbiological contaminations of the water bath [60]. Literature suggests that rapid thawing rates (>100°C/min) that can prevent damaging ice crystals during recrystallization are optimal choice and generally results in the best post‐ thaw recovery and viability of cells [61]. High post‐thaw viability of MSCs, comparable to those thawed with the standard method, were obtained by Thirumala et al. [62] with a thawing procedure in a controlled‐rate freezing/thawing chamber at 10°C/min. For evaluating the cryopreservation outcomes in terms of post‐thaw cell quality and quantity,

function post cryopreservation.

44 Cryopreservation in Eukaryotes

**7.3. Thawing and viability assessment**

Biosafety assessment of cryopreserved MSCs is necessary to ensure the safe use of the cells prior to clinical applications. Specific tests for the detection of bacteria, yeast, fungi, myco‐ plasmas, and viruses should be used as a part of routine and regular quality control screening procedures. To detect low levels of contamination, samples from the cell cultures and their products may be inoculated in either liquid tryptic soy broth (TSB) for the detection of aerobes, facultative anaerobes, and fungi, fluid thioglycollate medium (FTM) for the detection of aerobic and anaerobic bacteria, or onto solid (trypticase soy agar, blood agar, Sabouraud's dextrose agar, and malt extract agar) growth media. These inoculated media may be incubated at different temperatures, reflecting conditions for pathogen culture (37°C) and environmental organisms with lower growth temperature optimal (25°C) in microbiological culture incuba‐ tors, depending on the specific testing standards used. Mycoplasmas competes with the cells for the nutrients in the culture medium, typical signs of contamination consist in a reduction of the rate of cell proliferation, and changes in cellular physiology including gene expression, metabolism, and phenotype. Among the wide variety of techniques that have been developed to detect mycoplasma contamination of cell cultures, Uphoff and Drexler [64] recommended the PCR analysis for the screening, as it considered the most reliable and useful detection method. The presence of viral agents could be evaluated by a panel of tests to detect pathogens and adventitious viruses. Usually, this panel of tests includes: electronic microscopy, reverse transcriptase detection (as a general test for retroviruses), and other tests to find specific agents, depending on the animal species of the sample.

### **9. Storage of MSCs**

MSCs should be preserved without direct exposure to liquid nitrogen, to reduce the risk of pathogenic cross‐contamination. This issue enforces the stem cells banks to store materials at vapor phase of liquid nitrogen. However, recent evidence suggests that storage in vapor phase above liquid nitrogen still carries the risk of cross‐contamination [65]. Potentially, infective agents may also enter storage directly from the facility atmosphere, contaminated surfaces, or leaking samples, and they can be accumulated in viable condition. Stem cell banks should also maintain secure liquid nitrogen storage equipment in cryogenic tanks monitored by a specific control and alarm system (−196°C), in order to avoid catastrophic loss of cryopreserved samples. Furthermore, proper storage requires the use of cryovials and labeling systems that will withstand the intended storage conditions: labels and bar codes or other printing systems are chosen for extended storage periods.
