Stem cell transplantation
Evaluation of Bone Marrow Mesenchymal Stem Cell Standard Cryopreservation Procedure Efficiency

https://doi.org/10.1016/j.transproceed.2008.03.004Get rights and content

Abstract

Introduction

Mesenchymal stem cells are obtained from a variety of sources, particularly bone marrow. These cells have great potential for clinical research due to their potential to regenerate tissue. As is well known, the cryopreservation process can store any cell type, particularly blood cells, for an indeterminate time.

Objective

The aim of this study was to analyze the efficiency of standard cryopreservation procedures for adult mesenchymal stem cells from bone marrow.

Methods

Mononuclear stem cells isolated from 10 Wistar male rats were cultivated for 4 weeks to obtain mesenchymal stem cells. The parameters considered in this study were trypan blue exclusion test and annexin V conjugated with 7-amino-actinomycin for flow cytometry before cryopreservation in liquid nitrogen vapor phase for 1 month and after thawing.

Results

The viabilities determined by the trypan blue exclusion test were 94.76% and 90.58%, and the flow cytometry assay (annexin V conjugated with 7-amino-actinomycin) were 85.52% and 66.25%, before cryopreservation and after thawing, respectively.

Conclusions

Standard procedures for cryopreservation were not efficient for those cells. The flow cytometry assay was more sensitive than the trypan blue exclusion test to demonstrate nonviability.

Section snippets

Materials and Methods

All experiments were performed in accordance with principles of treatment of laboratory animals of the Brazilian College of Experiments in Animals (COBEA). Ten male Wistar rats (250 to 300 g) were euthanized for collection of bone marrow.

Results

Viability determinations by the trypan blue exclusion test showed 94.76% and 90.58% viability before versus after cryopreservation, while the values with the flow cytometry assay (annexin V conjugated with 7-amino-actinomycin) were 85.52% and 66.25%, respectively (Fig 1, Table 1).

Discussion

The standard cryopreservation procedure did not appear to be efficient for BMMeSCs. The flow cytometry assay was more sensitive than the trypan blue exclusion test to demonstrate viability loss of BMMeSCs. Significant cell death could be explained by shrinkage with decreased nucleus to cytoplasm ratio with adherence characteristics, which could increase the risk of freezing damage. The trypan blue exclusion test is acceptable for the determination of the integrity of the plasma membrane before

Acknowledgments

We would like to thank Faculdades Pequeno Príncipe for support.

References (12)

There are more references available in the full text version of this article.

Cited by (41)

  • Effect of cryopreservation on therapeutic potential of canine bone marrow derived mesenchymal stem cells augmented mesh scaffold for wound healing in guinea pig

    2020, Biomedicine and Pharmacotherapy
    Citation Excerpt :

    During the present study, cryopreserved BM MSCs augmented meshes could be revived successfully post-thaw. A variety of different cryopreservation solutions has been used for the preservation of different types of stem cell in different species like rat [26], human [27] and caprine [3]. Culture characteristics of both pre- and post- thaw canine BM MSCs were compared and found no significant difference between the pre- and post-thaw culture and expansion.

  • Cryopreservation of stem cells

    2019, Comprehensive Biotechnology
  • Effect of cryopreservation on viability and growth efficiency of stromal-epithelial cells derived from neonatal human thymus

    2017, Cryobiology
    Citation Excerpt :

    It could lead to some loss of cell proliferation and hence increased monolayer formation time by adherent cells. However, it was not critical for the cell growth or cell function compared to non-frozen samples [2,7,13,33,37,38]. In general, this corresponds to our observations with thymic samples, but we can also conclude that freshly prepared thymic cell suspensions from non-frozen thymic tissue form a stromal-epithelial monolayer in a shorter time under the same conditions.

  • The effects of cryopreservation on cells isolated from adipose, bone marrow and dental pulp tissues

    2014, Cryobiology
    Citation Excerpt :

    However, it cannot be ruled out that ultrastructural changes occurred, as was recently shown by James et al. who described changes in ADC morphology following cryopreservation [10]. ADCs, BMCs and DPCs harvested at passage 1 all exhibited >95% viability before being place in cryostorage, which correlated with a previous study [3] (Fig. 1b). Following cryostorage the proportion of viable cells were not statistically (P < 0.05) lower than when harvested, >90% for each cell type (Fig. 1b).

  • Effectiveness of human mesenchymal stem cells derived from bone marrow cryopreserved for 23-25 years

    2012, Cryobiology
    Citation Excerpt :

    However, the outcomes of this method are not very satisfactory. Carvalho et al. reported that MSC viability decreased significantly after cryopreservation [5]. In our study, we found that the adherence rate of cells in the initial cultures after thawing the BMCs was significantly low in the cryopreserved group and that the relative recovery rate of the MSCs in the primary culture was approximately 50% of that of the control, which is consistent with previous studies [45].

  • Evaluation of a low cost cryopreservation system on the biology of human amniotic fluid-derived mesenchymal stromal cells

    2012, Cryobiology
    Citation Excerpt :

    Multiple studies have investigated the influence of cryopreservation on the biological characteristics of human mesenchymal stromal cells (hMSC). However, most of these studies have failed to perform a systematic follow-up of the cultures after their thawing [20–22]. Furthermore, in many of these studies the cell lines subjected to each assay before and after cryopreservation were not identical or derived from the same donor, as suggested by Gonda et al. [23].

View all citing articles on Scopus

Financial support from SETI-PR and CAPES.

View full text