Date Published: February 1, 2018
Publisher: Public Library of Science
Author(s): Artur Gurgul, Joanna Romanek, Klaudia Pawlina-Tyszko, Tomasz Szmatoła, Jolanta Opiela, Zoran Ivanovic.
Cryopreservation is an important procedure in maintenance and clinical applications of mesenchymal stem/stromal cells (MSCs). Although the methods of cell freezing using various cryoprotectants are well developed and allow preserving structurally intact living cells, the freezing process can be considered as a severe cellular stress associated with ice formation, osmotic damage, cryoprotectants migration/cytotoxicity or rapid cell shrinkage. The cellular response to freezing stress is aimed at the restoring of homeostasis and repair of cell damage and is crucial for cell viability. In this study we evaluated the changes arising in the pig mesenchymal stromal cell transcriptome following cryopreservation and showed the vast alterations in cell transcriptional activity (5,575 genes with altered expression) suggesting the engagement in post-thawing cell recovery of processes connected with cell membrane tension regulation, membrane damage repair, cell shape maintenance, mitochondria-connected energy homeostasis and apoptosis mediation. We also evaluated the effect of known gene expression stimulator—Trichostain A (TSA) on the frozen/thawed cells transcriptome and showed that TSA is able to counteract to a certain extent transcriptome alterations, however, its specificity and advantages for cell recovery after cryopreservation require further studies.
Mesenchymal stem cells (MSCs) are highly attractive for tissue engineering and clinical applications because of their inherent regenerative capacity, high proliferation potential, immunomodulatory activity, ability to differentiate into different cell lineages and low immunogenicity . In most methodologies, MSCs are enriched from bone marrow aspirates by density gradient centrifugation , but their amount usually is insufficient for further procedures. Isolation protocols resulting in high initial cell counts are available and desirable for one-step procedures in regenerative medicine [3, 4], however, they are cost and time-consuming. This makes in vitro expansion of undifferentiated MSCs an indispensable procedure for both scientific and application purposes. In vitro expansion, however, carries the risk of contamination by pathogens or malignant transformation and in addition, the multilineage differentiation capacity of stem cells may be lost during long-term expansion . In various application procedures, cryopreservation plays an important role in obtaining off-the-shelf availability for cells. It also separates cell culture from application and prepares cells for long distance transportation and long term storage.
Cryopreservation is currently a standard procedure in most applications involving in vitro cell expansion and their preservation for a further use. Cryopreservation allows the storage of cells for prolonged time periods while maintaining them in adequate condition for use in clinical or research settings. Freezing and storing of cells are also standard procedures in reproductive biotechnology where gametes or embryos are cryopreserved and stored for long time while maintaining their viability and developmental abilities. Conventional cryopreservation systems include slow-freezing and vitrification, both having advantages and disadvantages in terms of cell viability and/or scalability . In this study we used a simplified freezing procedure. Cryovials with cells suspended in a cryoprotectant were stored for 2 h in -20°C and then snap-frozen in liquid nitrogen. We observed that after re-placing in culture the cells maintained their phenotype although part of them did not attach which is a normal observation as around 1/5 of the frozen cells dies during the freezing/thawing procedure . This method of freezing has been routinely performed in our laboratory to freeze somatic cells [44, 45] and mesenchymal stem cells  used as donors of nuclei for somatic cell cloning purposes as well as VERO cells used as co-culture cells for bovine and goat embryo in vitro culture [47, 48]. Presently the cooling protocol of MSCs from various tissues and species is mostly based on controlled freezing  although different approaches of cryopreservation of porcine MSC are rather scarcely described compared to other species. Our experiment, due to extensive results on porcine transcriptome, delivers convincing evidence for the simple 2-step freezing procedure to be effective and efficient. There are reports on successful freezing results in other species where a MSCs cooling protocol was not based on controlled freezing using a freezer: e.g. human MSC [50, 51, 52, 53], monkey MSC  and dog MSC .
Summarizing, our results show that cryopreservation induces vast alterations in the transcriptional activity of MSCs which implies engagement in post-thawing cell recovery of processes connected with cell membrane tension or damage, cell shape maintenance, transcriptional activity and mitochondria-connected energy homeostasis or apoptosis mediation. Administration of TSA in post-thawing culture is able to a certain extent to counteract these alterations but this effect is transient and its specificity and advantages in frozen cells require further studies.