The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: a systematic review

Mesenchymal stem cells (MSCs) represent an invaluable asset for the field of cell therapy. Human Bone marrow-derived MSCs (hBM-MSCs) are one of the most commonly used cell types in clinical trials. They are currently being studied and tested for the treatment of a wide range of diseases and conditions. The future availability of MSCs therapies to the public will require a robust and reliable delivery process. Cryopreservation represents the gold standard in cell storage and transportation, but its effect on BM-MSCs is still not well established. A systematic review was conducted to evaluate the impact of cryopreservation on BM-MSCs and to attempt to uncover the reasons behind some of the controversial results reported in the literature. Forty-one in vitro studies were analysed, and their results organised according to the cell attributes they assess. It was concluded that cryopreservation does not affect BM-MSCs morphology, surface marker expression, differentiation or proliferation potential. However, mixed results exist regarding the effect on colony forming ability and the effects on viability, attachment and migration, genomic stability and paracrine function are undefined mainly due to the huge variabilities governing the cryopreservation process as a whole and to the lack of standardised assays.


Background
Bone marrow non-hematopoietic stem cells represent a fraction of the bone marrow cell population. They may arise from the constituents of the bone marrow structure and they can differentiate into mesenchymal tissues such as adipose, cartilage and bone. Bone marrow nonhematopoietic stem cells were first mentioned by Julius Cohnheim in 1867 and later cultured and characterized by Freidenstein et al. in the 1970s [1][2][3][4]. Friedenstein demonstrated that bone marrow non-hematopoietic stem can be selected by adherence to culture flask and exhibit the following characteristics: fibroblast morphology, colony-forming ability and in vitro proliferation and differentiation potentials [5]; all of which were indicative of 'stemness' properties [6]. With that said, it must be noted that within the scientific community, there is still an ongoing discussion about the true nature of these cells. Two names propagated for these cells "Stromal Stem Cells" [7] and "Mesenchymal Stem Cells" [8,9].
The then newly discovered source of stem cells has attracted great interest in medical research. In addition to the characteristics listed above, isolating mesenchymal stem cells from bone marrow was surrounded with minimal ethical issues and could substitute embryonic stem cells [6]. Therefore, hBM-MSCs became the subject of intense research and in 1995 the first autologous intravenous infusion of these cells in cancer patients was performed [10]. Later, MSCs have been shown to have widespread immunomodulatory effects [11] as well as an angiogenic induction ability [12]. Taken together these characteristics enlarged the scope of application of hMSC-based therapies. As of April 2019, a search on the U.S. National Library of Medicine (ClinicalTrials.gov) using the term 'bone marrow mesenchymal stem cells' retrieved 368 clinical trials aiming to treat conditions like stroke, graft versus host disease, osteoarthritis, crohn's disease, ischemic heart disease and multiple sclerosis.
The future availability of cell therapies to the public will be dependent on easy and quick logistics as well as robust and reliable delivery process. Abazari et al. [13] suggested that if cell therapies "cannot be delivered clinically and logistically then their benefit is irrelevant". Cryopreservation remains the cell therapy industry "standard" for biopreservation [14] as well as the primary option of storage for hMSC-based products [15]. In fact, cryostorage has evolved from being a marginal process in the cell therapy manufacturing process to become a tool widening the availability of stem cell therapy in particular and regenerative medicine in general. However, despite its evolving role, cryobiology is lagging behind the speed at which the cell therapy industry is growing.
Cryopreservation is particularly crucial for a successful cell therapy for various reasons. It facilitates cell transport, it enables the generation of cell banks with indefinite shelf-life thus ensuring off-the-shelf steady supply, access and availability and it gives time for quality control testing and in vitro assays [14,16,17]. In addition, cryostoring therapeutic doses of cells in hospitals and clinics could make cell therapy a treatment choice for many diseases and conditions including acute conditions [18]. Furthermore, cryopreserved cells are ideal for sequential treatments such as the case of chronic heart failure or ischemic heart disease to ensure the consistency of the treatment [19]. Banking cells is also an appropriate option from an economical and a regulatory aspect [20]. The logistics of administration of MSC in many immunotherapy trials were simply described as cryopreserving cells, thawing them when needed and administering them within a couple of hours. This scenario would only be feasible if thawed cells preserved their viability, safety and potency [20].
Cryopreservation of cells is associated with several injuries; physical and molecular. A controversy still exists about the efficacy of fresh cells versus cryopreserved and whether viability implies functionality [21]. In early MSC-based clinical trials, using cryopreserved cells was hypothesised to be the source of failure [21]. In addition, the variability in the outcome of MSC-based clinical trials was proposed to mainly be due to the functional alterations that the freeze-thaw process provokes in MSCs rather than the freezing method itself [17].
Human Bone marrow-derived MSCs (hBM-MSCs) are the most commonly used source of MSCs in clinical trials [22] and have been deployed across 17 European centres manufacturing MSCs [23]. The effects of cryopreservation on this type of cells are not well defined. The aim of this review is to assess whether rigorous data exist regarding the impact of the freeze-thawing process on BM-MSCs phenotypic and functional traits. To our knowledge, this is the first review to factor numerous aspects of the freezing process (freezing solution composition, the freezing protocol, the duration of storage, the concentration of cells at freezing, the passage number at freezing as well as the thawing method) in one analysis, for studies conducted over about 20 years. Such detailed analysis may allow firm conclusions to be drawn regarding BM-MSCs performance after the freeze-thawing process as well as help uncover possible reasons behind some of the controversial existing results and highlight areas which require further investigation.

Methods
The inclusion criteria for this review were: Articles or conference papers assessing the impact of cryopreservation by slow freezing on BM-MSCs in suspension. There was no restriction on the species from which cells were derived. Studies where bone marrow itself was frozen, where freezing of BM-MSCs was done by vitrification or using a 3D structure and where cryopreservation impact was only assessed in vivo, were excluded. A systematic literature search was conducted using PubMed, Science direct and Google Scholar (last search performed April 2019). Two combinations of search terms were used 'cryopreservation mesenchymal' and 'freezing mesenchymal' . The output of each search was first scanned for the relevance of title. Articles were excluded if the topic is unrelated or when an eligibility criterion is not met. The retained articles were then screened for the relevance of abstracts (and in few cases materials and methods) and retained when meeting all the eligibility criteria ( Fig. 1). From the 41 retained studies, information regarding the freezing solution composition, the freezing protocol, the duration of storage, the concentration of cells at freezing, the passage number at freezing as well as the thawing method was extracted and tabulated. Next, studies were grouped in tables according to the "hMSC checklist" proposed in [24]. Cell surface marker expression, differentiation potential, proliferation and growth, attachment and migration potential, genomic stability and paracrine function were examined. In addition, post-thaw viability and morphology information was also collated because they are primary evaluators of cryopreservation.
Across the retained studies, various freezing media formulations were used. 20% of studies (17% human) used commercially available freezing solution such as CELL-BANKER and CRYOSTOR while the rest used "in-lab" homemade formulations. 66% of studies (41% human) used or tested various amounts of serum in the freezing media with the serum principally being animal-derived, 20% of studies (12% human) froze cells in serum-free media while 5% used freezing media containing plasma or human platelet lysates (all of which are human studies). For 17% of the studies, not enough information was included about FBS content and/or about the composition of the freezing medium (12% human). Of the serum-free studies, one study assessed the efficiency of Sericin as a substitute  Fig. 1 Schematic representation of the Bone-marrow derived mesenchymal stem cell cryopreservation search strategy. Diagram of the current systematic search analysis. Studies of bone-marrow derived mesenchymal stem cells aligned to cryopreservation and/or freezing were identified using a combination of two search terms 'cryopreservation mesenchymal' and 'freezing mesenchymal' using PubMed, Science direct and Google scholar. The output of each search was scanned at the title level, then at the abstract level and articles were retained when meeting eligibility criteria, both inclusion and exclusion (see boxes titled inclusion criteria and exclusion criteria in this figure). In specific, for the term 'cryopreservation and mesenchymal' in PubMed, 695 studies were retrieved. By checking the titles against the eligibility criteria, only 65 studies were retained. The abstracts of these 65 articles were then read and checked against the eligibility criteria and only 36 of the 65 articles were retained. For the subsequent searches, these steps of retaining and eliminating articles were followed but preceded by eliminating duplicates i.e. articles which appeared in previous searches   to FBS [25]. Across all of the studies, 13 assessed the freezing in xeno-free media. More than 90% of studies used dimethylsulfoxide (DMSO) at a concentration ranging from 1 to 20% with 10% being the most commonly used. Carboxylated poly-l-lysine (COOH-PLL) was investigated as a cryoprotectant to replace DMSO [26] and hydroxyethyl starch was added to freezing solution as a strategy to reduce the percentage of DMSO [27]. Two studies tested various freezing solutions containing polyethylene glycol (PEG), trehalose and 1,2 propanediol in order to develop a well-defined, serum-free and reduced-DMSO freezing solution [28,29]. Only two studies utilized strategies to prevent post-thaw apoptosis through the addition of Rho-associated kinase inhibitor [30] and Caspase inhibitor z-VAD-fmk [31] in the freezing media. Concerning freezing protocols, two procedures prevail. The first involves incubating the cells at a freezing rate of − 1 °C/min in a − 80 °C freezer for several hours (up to 24 h) then moving the cells to liquid nitrogen (LN 2 ). The second is based on a two to seven-step sequential freezing process using a programmable freezing device to freeze the cells prior to − 150 °C freezer or LN 2 storage. Four studies reported whether cells were stored in liquid phase [32] or vapour phase [30,33,34]  Seven studies (3 human and 4 animal) did not specify the passage number at which cells were frozen. Six studies (3 human and 3 animal) used cells at passage 1, one study (monkey) used cells at passage 9 and the rest (20 human and 6 animal) used cells at passages ranging from 1 to 6. The concentration of cells at freezing was very variable ranging from 1 * 10 5 to 1 * 10 7 cells/mL with 1 * 10 6 cells/mL being the most frequently used (17 studies; 9 human and 8 animal). There was only one study in which human-derived cells were frozen in cryopreservation bags at a concentration of 1.8 * 10 8 [40]. There was a huge variation regarding the duration of storage of cells in the frozen state; the shortest period being 1 h [30] and the longest 10 or more years [37].
Seven studies (human) did not include information about their thawing protocols. Two studies (1 human and 1 animal) just mentioned 'quickly thawed' [20,39], one in α-MEM [41], one at room temperature [37] and one in a 37 °C incubator [42]. For all the rest (16 human and 13 animal), there was some consistency; cells were typically thawed at 37 °C, most likely in a water bath with or without gentle agitation for 1-4 min. it shows that viability and differentiation potential are the only attributes assessed in over half of the retained studies.

Viability
Post-thaw viability was the most assessed cell attribute (37 studies). Table 1 lists the studies which assessed viability immediately post-thaw (37 studies) or after a period of post-thaw culture, which was monitored in five studies (post-thaw time point from 4 h to 3 passages). Three main methods for viability assessment were used; trypan blue exclusion, flow cytometry and fluorescent microscopy. The immediately post-thaw viability varied from about 50% to 100% which is noteworthy. Sixteen studies reported no change in viability immediately after thawing while 10 studies reported significantly lower viability. In some studies where a new freezing formulation was tested, viability was compared to 10% DMSO which is still considered the gold standard for freezing BM-MSCs. The timing of viability measure is crucial due to the induction of apoptotic events some time post-thaw [43]. As mentioned above only five studies assessed the long-term effect (up to 3 passages post-thaw of freezing) with two reporting lower viability and three reporting no effect. It was also noted that variability in the level of viable cells within the same study differed when using different methods of measurement such as trypan blue exclusion compared to flow cytometry [44,45]. Table 2 summarises the studies which assessed post-thaw cell morphology. This attribute was mainly assessed using microscopy. Irrespective of all the variables considered in this data analysis and the time post-thaw at which cell morphology was checked, 13 of the 16 studies agree that cryopreservation itself has no effect on post-thaw cell morphology. The addition of Rho-associate kinase (ROCK) inhibitor Y-27632 was reported to give BM-MSCs a web-like appearance which indicated some neuronal differentiation [30]. In addition, several cell shapes were observed at day 2 and day 5 post-thaw [46] and cell shrinkage was detected using flow cytometry [38].

Immunophenotyping
Marker expression is one of the International Society for Cellular Therapy (ISCT) criteria for defining MSCs [47] so it is of real importance to check BM-MSCs phenotype before freezing and/or after thawing. Table 3 lists the studies which assessed BM-MSCs marker expression post-thaw. Despite its importance, less than half of the 41 studies retained assessed post-thaw marker expression retention and despite all the variables taken into consideration in this investigation, there was a consensus regarding the methodology used (flow cytometry) and the results; cryopreservation does not affect BM-MSCs immunophenotype.

Differentiation potential
Tri-lineage differentiation (adipogenic, osteogenic and chondrogenic) is another criterion listed by the ISCT guide for defining MSCs [47]. Hence, more than half of the studies [24] assessed BM-MSCs post-thaw differentiation potential and these are listed in Table 4. Osteogenesis is the most frequently assessed differentiation pathway (20 studies) qualitatively through Alizarin red staining and/or quantitatively through measurement of alkaline phosphatase activity. There was an agreement among 18 studies that cryopreservation did not affect BM-MSCs osteogeneic potential. One study reported lower osteogenesis [39] and one reported improved osteogenesis post-thaw [48]. Adipogenesis was next in terms of frequency of testing using Oil Red O staining as a qualitative assessment and no effect of cryopreservation was observed in 12 studies. Only one study provided a quantitative assessment of adipogenesis and it was the only one reporting lower differentiation level [49]. Chondrogenesis presented as the least studied tri-linage differentiation pathway. Only five studies differentiated thawed BM-MSCs into chondrocytes with a qualitative assessment made via Alcian Blue staining. It was concluded that cryopreserved cells did not lose the ability to chondrogenic differentiation (of note thawed BM-MSCs were also able to commit to neuronal and endothelial lineages). Table 5 lists the 20 studies which examined post-thaw BM-MSCs proliferation potential. Various methods were used to determine proliferation rate such as population doublings and DNA quantification. The majority of results agree that cryopreservation does not affect post-thaw BM-MSCs proliferation potential, nonetheless lower proliferation rate was obtained by two studies [34,39] and higher proliferation rate was obtained by one study [50]. Colony-forming unit ability, a traditional measure of BM-MSCs proliferation, was assessed by three studies with mixed results [25,32,51]. Table 6 lists the six studies which examined post-thaw BM-MSCS metabolic activity. Three methods were equally used to determine cell metabolic activity; AlamarBlue, Presoblue and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) reduction-based assays. From the data collated it could be seen that two-thirds of experiments performed reported impaired metabolic activity post-thaw.

Apoptosis and senescence levels
Typically assessed using flow cytometry, the induction of apoptosis is evident when considering the six studies entered in Table 7. However, cryopreservation does not seem to induce senescence (refer Table 7) although more studies are needed to draw a firm conclusion.

Attachment and migration
Only four studies assessed BM-MSCs attachment ability post-thaw, and these are recorded in Table 8. This table indicates that frozen cells have lower adherence capability post-thaw. Only one study assessed post-thaw cell migration [52]. It concluded that cryopreservation has no effect on post-thaw cell migration ability.

Paracrine function
Paracrine function is related to two main MSCs activities namely immunomodulation and angiogenesis. In total, 10 studies (Refer Table 9) assessed BM-MSCs paracrine function with immunomodulation being the most frequently assessed with eight studies. The results of these studies are equally balanced with four of them reporting no effect of cryopreservation on BM-MSCs post-thaw immunomodulatory potential and four reporting an impaired potential. Angiogenesis potential and secretion of growth factors were only assessed by one study each with no effect of cryopreservation reported.

Discussion
A recent analysis of MSC-based clinical trials showed that although no safety concerns surround MSC infusion, the translation from bench to bedside is still confronted by what the authors called the ' Achilles heel'; donor heterogeneity, ex vivo expansion, immunogenicity and cryopreservation [53]. There is no doubt that cryopreservation is essential for MSC therapy translation, both autologous and allogeneic, and is still one the limitations to be addressed. Cryopreservation by slow freezing can cause two types of cell damage; physical and molecular. Physical injuries were the first to be identified and include ice nucleation, solution effects, osmotic shock, cold shock as well as cryoprotectant toxicity [14]. Molecular injuries encompass the effect of cryopreservation on gene expression, protein levels, cell functionality, the induction of stress response as well as post-thaw epigenetic changes [54,55].

Table 2 Bone-marrow derived mesenchymal stem cell studies assessing post-thaw cell morphology
The key results on bone-marrow derived mesenchymal stem cell morphology are presented in this table. For further details on the cryopreservation experimental details refer to either Table 1   In the case of MSCs, studying the effect of molecular injuries and how to mitigate them is a twofold problem. Firstly, investigating molecular injuries is still a developing branch of cryobiology. In fact, immediately post-thaw cell viability has always been the most assessed cell attribute in cryopreservation studies. However, it has been shown that signs of cellular damage may take some time to manifest (cryopreservation-induced delayed-onset cell death [54]). Leading to viability and functional losses which are compounded by a lack of detection and reporting in immediate post-thaw analysis. Approaches to tackle molecular injuries, intracellular-like freezing solutions and anti-apoptotic compounds, can be deployed yet research in this area is still at an early stage [54].
Secondly, establishing and standardising potency markers and assays to characterise MSCs is still a challenge [24]. In fact, MSCs possess variability in their gene expression profiles, differentiation and expansion potential and phenotype depending on tissue origin, cell isolation and expansion procedures [56] as well as donor characteristics [57]. In 2006, the ISCT published a guideline on minimal criteria to define MSC; plastic adherence, expression of certain surface markers and lack of others and tri-lineage differentiation [47]. In 2013, these criteria were expanded to include a fourth parameter, quantification of MSC immune functional potency [58]. In 2016, the society suggested "a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of the secretome" in order to fulfil the fourth criterion [59].
Currently, there are no standard markers or potency assays to typify MSCs or evaluate their post-thaw potency despite much discussion within the scientific community [24,60]. Therefore, research laboratories follow differing protocols which makes data evaluation complex. As both research areas (freezing molecular injuries and MSC characterisation) develop so will the methodology of evaluating MSC cryopreservation.
There are profound variabilities in the whole cryopreservation process from freezing media formulation, method of freezing and thawing and duration of storage

Table 3 Bone-marrow derived Mesenchymal Stem Cell studies evaluating surface marker expression post-thaw
The main results on bone-marrow derived mesenchymal stem cell surface marker expression are presented in this table. For further details on the cryopreservation experimental details refer to either Table 1    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   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 to passage number and cell concentration at freezing. However, despite all these variabilities and all the species included, there was evidence showing that four BM-MSCs attributes are stable and unaffected by the stresses imposed by freezing and thawing and these are: cell morphology, marker expression, proliferation potential and tri-lineage differentiation capability (although chondrogenesis was only assessed by five independent studies). The four other attributes viability, attachment and migration, genomic stability and paracrine function were governed by either conflicting results or by low assessment frequency.
All studies have employed a strategy to evade freezing and thawing physical damage. Freezing BM-MSCs at a slow cooling rate (at least at the start of the cooling process) and thawing at a high rate was followed by almost all the studies in this review. In terms of media formulation, DMSO remains the most commonly used cryoprotectant to protect BM-MSCs (used by 90% of studies analysed). However, DMSO is associated with adverse effects such as cardiac side effects [61,62] and severe neurotoxicity [63,64] when infused in patients. Consequently, reducing or eliminating DMSO, depending on clinical outcome, may become a requisite.
Mixing other potential cryoprotectants with DMSO at percentages < 10% indicates that this traditionally held protectant and percentage can both be altered [26][27][28]. This is an interesting result which indicates that eliminating DMSO from freezing solution is a viable option and is worthy of further evaluation based on a wider postthaw MSC functional characteristics. Another important aspect of MSC cryopreservation is the fact that FBS is commonly added to freezing solutions for its benefits to stabilise the cell membrane and adjust cell osmotic pressure [65]. The main issue in using FBS is that it is animal-derived and may cause a xenogenic reaction if infused in patients [65]. Cryopreserving hBM-MSCs in xeno-free media was assessed across 13 studies [18, 26, 34, 38, 40, 48-50, 52, 66-69]. Verdanova et al. reported using Sericin (a protein derived from silkworm cocoon) as an FBS substitute for preserving human BM-MSCs [25]. The possibility of overlapping xeno-free and low or no DMSO exists in one study where monkey BM-MSCs were successfully frozen in xeno-free solution composed of methylcellulose, poloxamer, α-tocopherol and only 2.5% DMSO [49]. Such a freezing solution would be ideal because it is not only xeno-free but also contains a very low DMSO concentration.
Cell concentration at freezing and the duration of storage are not well studied, and it is not well documented if it plays a role in the performance of the end-product. The only study which tested cell viability after freezing at three different cell concentrations found no significant variation [52]. In regard to duration of storage, of note is the study where BM-MSCs were stored for more than 10 years without losing multipotency [37]. This could well be a suggestion that once cells enter a quiescent state, its duration is not of great significance.

Table 6 Bone-marrow derived mesenchymal stem cell studies evaluating post-thaw metabolic activity
The key results on bone-marrow derived mesenchymal stem cell metabolic activity after cryopreservation are presented in this table. For further details on the cryopreservation experimental details refer to either Table 1   The evaluation of the cryopreservation process is certainly an existing challenge. Our understanding of the biology of MSCs is evolving and so are the possibilities of the application of these cells in the medical field. Yet, as stated recently "Significant challenge remains the development of a relevant potency assay" [34]. These assays must be quick, easy and should not require trained personnel if they are to be used to release each cell batch in a clinical setting and if they are to fit with operation theatre logistics (thawing, testing and infusing within a couple of hours). In theory, potency assays could be therapy-specific and must indicate cell functionality; in other words, "mechanism(s) of action" [70]. How much these assays correlate with the in vivo niche is also of great importance. In addition, "the assay should be able to differentiate between sufficiently potent and sub-potent batches, a (semi-)quantitative assay is required" [56]. This thawing-infusing scenario would be realistic only if cryopreservation methods have improved to give an optimal product immediately after thawing.
Cell morphology (shape and size) can give indication on cell's health as well as whether they have committed to differentiate or not. The absence of change in BM-MSCs morphology after cryopreservation can indicate that the freeze-thaw process does not cause differentiation or Table 7 The induction of apoptosis in post-thaw BM-MSCs The key results on bone-marrow derived mesenchymal stem cell apoptotic activity post-thaw 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 change in cell phenotype. This conclusion is further evidenced by the absence of effect of cryopreservation on BM-MSCs marker expression post-thaw. No such firm conclusion can be drawn when it comes to viability. This is of real importance given that viability is one of the release criteria for cell therapies. In fact, viability has and will always be the primary indicator on cryopreservation success. It is an easy, cheap and fast measurement but is has some limitations. The various methods used across labs to measure viability and the lack of a unified reporting structure makes it hard to compare results. Although ≥ 90% viability for fresh MSC product and ≥ 70% viability for cryopreserved MSC product are generally considered the benchmark for clinical application [57], different labs report viability maintenance or loss of viability based on comparison with pre-freeze viability or on comparison with freezing with 10% DMSO (refer Table 1). In addition, the common measurement time-point, only immediately post-thaw could be misleading due to the late manifestation of the effect of current cryopreservation protocols on cells.
In fact, the initiation of apoptotic events is evident according to the data in Table 7. Despite the importance of this cryopreservation-related cell death, it is surprising how limited the investigation of this molecular pathway in thawed BM-MSCs is and the strategies to reduce it are. Only two studies utilized strategies to prevent post-thaw apoptosis (molecular injury). The addition of Rho-associated kinase inhibitor Y-27632 in the freezing medium and in the post-thaw culture medium did not improve hBM-MSCs viability immediately but recorded enhanced recoveries at 24-h post-thaw [30]. However, the addition of Caspase inhibitor z-VAD-fmk in the freezing media did not prove to be beneficial for equine, ovine and rodent BM-MSCs as assessed by viability immediately, at 24 and 48 h post-thaw [31].
An expert workshop on preservation and stability of cell therapy products was held in May 2015 [71]. Assessing post-thaw viability was one of the topics discussed and the limitations/conflicts mentioned above were identified. The group advised that assessing cell viability post-thaw should "go beyond simple enumeration of cell numbers to facilitate a greater understanding of the cell system in question". In addition, the group realised the need for more advanced and accurate methods to assess cell viability that can be linked to cell function [71].
Interestingly, impaired cell metabolism can be closely associated with apoptotic pathways through the Bcl-2 family proteins which initiates apoptosis in metabolically stressed cells through utilization of autophagy as a nutrient source before ultimately undergoing necrosis [72,73]. From the data presented in Table 6 some impairment of BM-MSCs metabolic activity post-thaw is evident. Therefore, a link between defective metabolic activity in thawed BM-MSCs and a higher level of apoptosis postthaw could be postulated. This may indicate new pathways to mitigate against these post-thaw phenomena.

Table 8 Bone-marrow derived Mesenchymal Stem Cell studies evaluating cellular attachment post-thaw
The effects of cryopreservation on bone-marrow derived mesenchymal stem cell attachment 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 No effect on migration potential Trans-well plates by Baust et al. [76] who stated, "unstudied but of concern is the potential for the preservation process to select for increased resistance to preservation stresses". From Table 5, it can be concluded that there is a strong agreement that cryopreservation does not affect BM-MSCs differentiation potential. This conclusion is of value to fulfil one of the 2006 ISCT criteria for MSCs. However, differentiation potential may become less important for cell therapy. For example, in heart disease, MSCs' initial mechanism of regeneration was outlined as differentiation into cardiomyocytes and incorporation into the host tissue. Recently, this outline has been updated to shed a light on a more effective regeneration mechanism and that is their associated paracrine signalling [75]. In fact, paracrine signalling is not confined to cardiac regeneration but is now generally considered as the MSCs main mode of action. Caplan [77] suggested that it is "time to change the name" of MSCs to "Medicinal Signalling Cells" in order to better describe the secretion of trophic factors. According to Gonzalez et al. [78] "80% of the therapeutic effect of stem cells is attributed to paracrine actions".
The MSCs secretome is composed of growth factors and cytokines either soluble or engulfed in exosomes and/or vesicles [75,79,80]. MSC paracrine signalling is described as exerting plethora of effects including induction of angiogenesis, regulation of immune response and inflammation, modulation of cell differentiation and proliferation, extracellular matrix formation, neuroprotective and neurotrophic effects, anti-apoptotic, anti-tumour and anti-microbial activities [81,82]. Yet despite such a compelling list of activities no comprehensive evaluation of the MSC secretome, and or its cell-free utility, has been conducted [83]. Data on the BM-MSCs secretome upon thawing is very limited and so is the data on angiogenic potential. There are only two studies which concluded that no effect of cryopreservation was observed (Table 9).
At an injury site, MSC contribute to the creation of an anti-inflammatory environment by suppressing the activation and proliferation of pro-inflammatory cells and promoting anti-inflammatory cells [84]. This modulation of both the innate and adaptive immune system can be accomplished in two ways: via cell-cell interaction and cell-cell communication through an array of soluble factors including indoleamine 2,3-dioxygenase (IDO) and/ or extracellular vesicles [85]. These characteristics were the main contributors in raising the MSC profile as therapeutically valuable. As discussed above, MSC immune function has now become an essential indicator on MSC function. In view of this, it is expected that more studies on MSC immunomodulatory properties, and assays to evaluate them will emerge. Recently, Chinnadurai et al. [86] tested BM-MSCs potency using a matrix approach and concluded that cryopreservation negatively affects cells' secretome with T cell proliferation. According to Table 9, eight studies assessed BM-MSCs immune function post-thaw with a balance of four concluding damaging effect and four concluding no effect.

Conclusion
This systematic review has highlighted areas of agreement and deviation that currently exist around BM-MSCs properties and functions after cryopreservation (Fig. 4).
With MSC-based therapies expected to offer treatment choices for several conditions and diseases that are currently incurable and presently no MSC drug yet approved by the FDA the biological community must work towards reproducible and reliable data sets to achieve regulatory accepted Fig. 4 Effects of cryopreservation on BM-MSC in terms of cellular attributes and function. From the systematic analysis performed, cryopreservation appears to influence viability and apoptosis, cellular attachment, immunomodulation and metabolism (cell schematics shown in blue on right-hand side). Whereas, no common significant effects mediated by cryopreservation have been documented in proliferation, morphology, differentiation or immunophenotyping (cell schematics given in green, red, orange and black on the left-hand side)