Comparison of freshly cultured versus cryopreserved mesenchymal stem cells in animal models of inflammation: A pre-clinical systematic review

Decision letter after peer review:

Thank you for submitting your article "Comparison of Freshly-Cultured versus Cryopreserved Mesenchymal Stem Cells in Animal Models of Inflammation: A Pre-Clinical Systematic Review" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Extend their systematic review to make it more comprehensive and obtain higher power of analysis. If the heterogeneity of studies and parameters available does not allow to perform cross-comparisons, the limitations of the conclusions and interpretations should be acknowledged.

2) Key parameters that were not available for all studies are the methods for cryopreservation and thawing before use. These have a direct impact on cell viability and activity and, if not available, the conclusions should be toned down.

3) Previous studies directly comparing freshly-culture vs cryopreserved MSCs or demonstrating impaired immunosuppressive properties (e.g. 10.3109/14653249.2011.623691 or 10.1002/stem.2415) are not cited or discussed.

4) The authors discuss the differential level of apoptosis (higher in cryopreserved MSCs, compared with freshly cultured cells). The possible role of apoptosis in the immunodulatory effects of MSCs (doi: 10.1126/scitranslmed.aam7828) should be discussed as well.

5) The authors should include studies administering human MSCs using immuno-compromised animals to avoid immune rejection. The rationale for excluding this group is not justified.

6) Further clarification on criteria used to assess the methodological rigor of the included studies is needed.

7) The impact of cryopreservation compared with many other experimental variables, such as route of administration, cell dose, cell sources, etc. should be discussed. The various administration routes should be considered particularly in the context of treatment of inflammatory diseases in pre-clinical animal models. The authors should acknowledge that reporting on differences for a single variable is unlikely to provide a measure of overall animal health and provide clinically meaningful information of the duration and severity of the disease course. The authors should place more emphasis on this point in the discussion and acknowledge pitfalls associated with focusing on any one outcome metric.

8) The impact of the study would be greatly improved if a meta-analysis was performed including a higher number of studies. More recent studies need to be included as well.

Reviewer #2 (Recommendations for the authors):

The manuscript by Dave et al., describes a systemic analysis of existing literature comparing the potency and efficacy of fresh vs. cryopreserved mesenchymal stem/stromal cells (MSCs) in preclinical disease models. The systemic analysis is properly executed and appropriately evaluates biases in reporting. Inclusion and exclusion criteria are well described and appear to be well justified. However, the studies included in the analysis are broad based with respect to disease type, cell source, route of administration, dose, and outcome measures used to evaluate efficacy are also highly variable. Due to this high degree of heterogeneity, a rigorous meta-analysis was not feasible. Therefore, the results of the study are limited in scope, and it is unclear if the conclusions of the study are clinically meaningful. Therefore, aspects of the study require clarification.

1. The literature search was not biased by language and the authors were careful to encompass the broad terminology used to describe MSCs in their search, which ensures all relevant studies were captured. Despite these efforts, only 15 studies were included and these encompassed an array of conditions, such as lung injury, sepsis, allergic airway inflammation, wound healing, neurological and ocular disease. Therefore, while the study addresses a focused question of clinical relevance, the analysis of such heterogeneous studies limits overall confidence in the reliability of the results.

2. The methods section does a good job of defining inclusion and exclusion criteria. However, if xenogeneic, syngeneic, autologous and allogeneic MSCs were included it is unclear why use of immuno-compromised animals was excluded. For example, studies administering human MSCs to mice often employ immuno-compromised animals to avoid immune rejection. The authors should explain their rationale for excluding this group.

3. Bias assessment included criteria related to reporting bias. However, since it is critical to exclude studies that employ inferior methodological approaches, the authors should provide further clarification on criteria used to assess the methodological rigor of the included studies.

4. The authors indicate that the duration of cryopreservation was not reported in half of the studies analyzed, and the remaining studies used varying durations. Since the authors do not rule out cryopreservation duration as a variable that influences potency/efficacy, the inconstancy between studies is a concern. This, coupled with the fact that variable methods were used to thaw cells, and viability of the final product varied significantly (60-97%), raises serious concerns about the reliability of the analysis. These concerns regarding cryopreservation are also relevant for many other variables, such as route of administration, cell dose, cell sources, etc.

5. The impact of the study would be improved if a meta-analysis was performed. However, due to the heterogeneity of the studies evaluated, differences between fresh vs. cryopreserved MSCs were converted to mean and standard deviation for each outcome measure, which is of limited significance. For example, reporting on differences for a single variable is unlikely to provide a measure of overall animal health and provide clinically meaningful information of the duration and severity of the disease course. The authors should place more emphasis on this point in the discussion and acknowledge pitfalls associated with focusing on any one outcome metric.

Reviewer #3 (Recommendations for the authors):

In the present review, Dave et al., provide an innovative and eye-catching comparison on the efficacy between freshly-cultured and cryopreserved mesenchymal stem cells in the pre-clinical treatment of inflammatory diseases. As the authors focused on detailed analysis of both in-vivo pre-clinical and in-vitro MSC potency outcomes from those published studies until June, 2020, no significant difference was detected in pre-clinical efficacy between freshly-cultured and cryopreserved MSCs basing on a massive electronic search and bioinformatics analysis on the on-line literature databases. This review is well-structured and provide evidence for considering a cryopreserved MSC product in further clinical trials.

The various administration routes should be detailed analyzed for treating inflammatory diseases in pre-clinical animal models. Besides, more recent published studies in 2021 should be included.