Abstract
Adipose derived multipotent stromal cells (ASCs) isolated from brown versus white adipose tissues, may have distinct in vitro properties, including response to cryopreservation, due to differences in tissue physiology. This study was designed to determine the ultrastructure, immunophenotype, in vitro expansion capabilities and multipotentiality of paired canine ASCs harvested from subcutaneous (SUB) and infrapatellar (IFP) adipose tissue up to cell passage (P) 3 before and after cryopreservation. Adipocyte and ASC ultrastructure from the same tissue were distinct, and morphologies of both differed between tissue sources and with cryopreservation. Cell expansion and colony forming unit frequencies were similar between ASCs from both tissue sources before and after cryopreservation. Most fresh cells were CD29+, CD44+, CD90+ and CD34− up to P3. Cryopreserved P1 and P3 cells had lower percentages of CD29+ and 44+ cells, respectively, compared to fresh. Peroxisome proliferator-activated receptor γ (PPAR-γ) gene expression and sex determining region Y-box 2 (SOX2), CD29 and CD44 protein expression was lower in cryopreserved versus fresh P3 ASCs. Both PPAR-γ and osteopontin (OPN) protein expression increased in fresh and cryopreserved P3 ASCs cultured in adipogenic and osteogenic induction medium, respectively, while SOX2 decreased. Based on the study findings, in vitro expansion and multipotentiality are not distinct among canine SUB and IFP ASCs before or after cryopreservation. However, cryopreservation alters ASC ultrastructure, immunophenotype and transcription factor expression from both tissue sources. Future studies are necessary to determine the impact of cryopreservation on cell potential for therapy and de novo tissue generation.
Highlights
Tissue engineering with adult multipotent stromal cells (MSCs) harvested from adipose tissue, adipose derived multipotent stromal cells (ASCs), is rapidly emerging as a therapeutic reality in human and veterinary medicine [1, 2]
This study was designed to determine in vitro expansion capabilities and plasticity of paired canine ASCs harvested from subcutaneous (SUB) and infrapatellar (IFP) adipose tissues before and after cryopreservation to test the hypothesis that IFP ASCs have the highest in vitro expansion rates, plasticity and MSC immunophenotypes that are sustained over multiple cell passages
IFP ASCs had many small lipid vacuoles and few mitochondria in the cytoplasm compared to few lipid vacuoles in the cytoplasm and mitochondria clustered around the nucleus in SUB ASCs (Fig. 1)
Summary
Tissue engineering with adult multipotent stromal cells (MSCs) harvested from adipose tissue, adipose derived multipotent stromal cells (ASCs), is rapidly emerging as a therapeutic reality in human and veterinary medicine [1, 2]. One rationale for observed differences is varying proportions of brown and white adipose tissue among harvest sites [6]. Intra-articular and visceral adipose tissue are predominantly white while subcutaneous adipose tissues are mostly brown [7]. Use of subcutaneous adipose tissue ASCs may reduce morbidity and augment tissue resources for generation of intra-articular structures. A recent study highlighted differences between ASCs from intra-articular and subcutaneous adipose tissues in and around the human knee [12]. This study was designed to determine in vitro expansion capabilities and plasticity of paired canine ASCs harvested from subcutaneous (SUB) and infrapatellar (IFP) adipose tissues before and after cryopreservation to test the hypothesis that IFP ASCs have the highest in vitro expansion rates, plasticity and MSC immunophenotypes that are sustained over multiple cell passages
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