Abstract
We and others have provided evidence that adipose tissue-derived mesenchymal stem cells (ASCs) can mitigate rat cardiac functional deterioration after myocardial ischemia, even though the mechanism of action or the relevance of these findings to human conditions remains elusive. In this regard, the porcine model is a key translational step, because it displays heart anatomic-physiological features that are similar to those found in the human heart. Towards this end, we wanted to establish the cultural characteristics of porcine ASCs (pASCs) with or without long-term cryostorage, considering that allogeneic transplantation may also be a future option. Compared to fresh pASCs, thawed cells displayed 90–95% viability and no changes in morphological characteristics or in the expression of surface markers (being pASCs characterized by positive markers CD29+; CD90+; CD44+; CD140b+; CD105+; and negative markers CD31−; CD34−; CD45− and SLA-DR−; n = 3). Mean population doubling time was also comparable (64.26±15.11 hours to thawed cells vs. 62.74±18.07 hours to fresh cells) and cumulative population doubling increased constantly until Passage 10 (P10) in the entire cell population, with a small and gradual increase in senescence (P5, 3.25%±0.26 vs. 3.47%±0.32 and P10, 9.6%±0.29 vs. 10.67%±1.25, thawed vs. fresh; SA-β-Gal staining). Chromosomal aberrations were not observed. In addition, under both conditions pASCs responded to adipogenic and osteogenic chemical cues in vitro. In conclusion, we have demonstrated the growth characteristics, senescence, and the capacity of pASCs to respond to chemical cues in vitro and have provided evidence that these properties are not influenced by cryostorage in 10% DMSO solution.
Highlights
Adult mesenchymal stem cells have been investigated in preclinical approaches for tissue regeneration [1,2,3,4,5,6]
Expansion, and Freezing of porcine ASCs (pASCs) ex vivo Subcutaneous adipose tissue was obtained by surgical procedures from adult male pigs (MS60 EMBRAPA lineage), after anesthesia and asepsis procedures. pASCs were isolated from 300 g of adipose tissue as previously described [2,19], cultivated in DMEM-Low supplemented with 10% FBS
Expansion, Storage and Viability of pASCs after Thawing pASCs were extracted and their population characteristics and ability to acquire adipose and osteogenic phenotype features were explored. pASCs demonstrated a high capacity for expansion in vitro and exhibited fibroblast-like morphology (Figure 1A)
Summary
Adult mesenchymal stem cells have been investigated in preclinical approaches for tissue regeneration [1,2,3,4,5,6]. Adipose tissue-derived mesenchymal stem cells (ASCs) were first isolated in humans [7] and later from various species, including rodents [8,9] and swine [10]. These cells show low levels of immunogenicity and have immunomodulatory properties [1,6,11,12] and may be useful for allogeneic transplantation. Rodent models are frequently and efficiently used to determine the response and potential mechanism of action underlying novel cardiovascular therapeutic approaches Extrapolation of these types of data directly to humans is limited because of the specific structural and pathophysiological response associated with the model [16]. The porcine model displays heart anatomic-physiological features that are similar to those found in the human heart, but the specific characteristics of porcine ASCs (pASCs) culture and the influence of long-term cryostorage on these cells is poorly understood
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