Isolation of Human Adipose-derived Stem Cells from Biopsies and Liposuction Specimens

Pennington Biomedical Research Center, Baton Rouge, LA, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 01/2008; 449:69-79. DOI: 10.1007/978-1-60327-169-1_5
Source: PubMed


Adipose tissue has proven to serve as an abundant, accessible, and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Here, we describe a detailed method for the isolation and expansion of adipose-derived stem cells (ASCs). We present a large scale procedure suitable for processing >100 mL volumes of lipoaspirate tissue specimens and a small scale procedure suitable for processing adipose tissue biopsy specimens of < 0.5 g. Although we have focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.

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    • "To facilitate tissue disruption, constant shaking is usually applied during the digestion process [28]. High collagenase concentrations can be cost prohibitive and result in cell damage as can prolonged digestion times [59,60]. Alternative enzymes like trypsin tend to have lower cell yields than collagenase [60]. "
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    ABSTRACT: Low yield of adult adipose-derived multipotent stromal cells (ASC) can limit autologous cell therapy in individuals with minimal adipose tissue. In this study, ASC isolation was optimized from approximately 0.2 g of feline epididymal adipose tissue for a treatment dose of 10(6)-10(7) ASCs/kg. The ASC yield was determined for three digestions, 0.1 % collagenase in medium for 30 min (Classic), 0.3 % collagenase in buffer for 30 min (New) and 0.3 % collagenase in buffer for 1 h (Hour). After isolation by the new tissue digestion, continuously cultured ASCs (fresh) and cells recovered and expanded after cryostorage at P0 (revitalized) were characterized up to cell passage (P) 5. Outcomes included CD9, CD29, CD44, CD90 and CD105 expression, cell doublings and doubling times, fibroblastic, adipogenic and osteogenic colony forming unit (CFU) frequency percentages and lineage-specific target gene expression after induction. The New digestion had the highest CFU yield, and about 7x10(6) ASCs/kg were available within three cell passages (P2). Compared to earlier passages, target surface antigen expression was lowest in fresh P5 cells, and fresh and revitalized P3-5 cells had slower expansion. Fresh and revitalized P1 ASCs had higher CFU frequency percentages and lineage-specific gene expression than P3. The New method described in this study was most efficient for feline epididymal ASC isolation and did not alter in vitro cell behavior. Fresh and revitalized P0-P2 feline ASCs may be most effective for preclinical and clinical trials. This study offers a potential option for ASC isolation from limited adipose tissue resources across species.
    Stem Cell Reviews and Reports 05/2014; 10(4). DOI:10.1007/s12015-014-9508-1 · 2.77 Impact Factor
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    • "Omental and subcutaneous adipose tissue was taken during bariatric surgery. Adipose tissue was rapidly processed after surgery using a modified protocol [32]. Briefly, connective tissue, blood vessels and fibrous materials were removed from the adipose tissue samples. "
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    ABSTRACT: DNA methylation at specific CpG sites within gene promoter regions is known to regulate transcriptional activity in vitro. In human adipose tissue, basal transcription of the aromatase (CYP19A1) gene is driven primarily by the I.4 promoter however the role of DNA methylation in regulating expression in ex vivo mature adipocytes is unknown. This observational study reports the correlation of DNA methylation within the I.4 promoter region of human mature subcutaneous and omental adipocytes with aromatase expression and body composition measures. Omental and subcutaneous adipose tissue were collected from 25 obese subjects undergoing bariatric surgery and the mature adipocyte fraction purified. DNA methylation status of 5 CpG sites within a 550 base pair region encompassing the transcription start site (TSS) of promoter I.4 was determined using pyrosequencing. Relative aromatase and I.4 promoter specific mRNA expression was determined by qRT-PCR and whole body DXA performed in 25 participants. Site-specific DNA methylation varied from 21±10% to 81±11%. In omental adipocytes percentage methylation at the I.4.1 and I.4.2 CpG sites, but not other nearby sites, was negatively correlated with relative aromatase mRNA expression (R=- 0.52, P=0.017 and R=- 0.52, P=0.015). In contrast subcutaneous adipocytes percentage DNA methylation at the I.4.3 and I.4.5 sites were positively correlated with relative aromatase mRNA expression (R=0.47, P=0.022 and R=0.55, P=0.004). In a small subset of patients DNA methylation at the I.4.5 site was also positively correlated with whole body lean mass, bone mineral content and density. In conclusion in mature adipocytes, the primary source of estradiol after menopause, increasing DNA methylation was correlated with aromatase mRNA expression and thus estradiol biosynthesis. These findings support a tissue-specific epigenetic regulation of the basal promoter activity in mature adipocytes; the mechanisms influencing this regulation and its physiological role remain to be elucidated.
    BMC Medical Genetics 08/2013; 14(1):87. DOI:10.1186/1471-2350-14-87 · 2.08 Impact Factor
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    • "The tissue was acquired from elective procedures in local plastic surgery offices, with the patient’s informed consent as approved by the Pennington Biomedical Research Center Institution Review Board. The primary cultures were prepared as described in Dubois et al., 2008 [54]. The tissues used were from 11 female donors of ages between 28 and 61 with a mean ± SD of 41.5 ± 8.61 years. "
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    ABSTRACT: Obesity is associated with a higher risk of developing cancer and co-morbidities that are part of the metabolic syndrome. Adipose tissue is recognized as an endocrine organ, as it affects a number of physiological functions, and contains adipose tissue-derived stem cells (ASCs). ASCs can differentiate into cells of multiple lineages, and as such are applicable to tissue engineering and regenerative medicine. Yet the question of whether ASC functionality is affected by the donor's body mass index (BMI) still exists. ASCs were isolated from patients having different BMIs (BMI-ASCs), within the ranges of 18.5-32.8. It was hypothesized that overweight BMI-ASCs would be more compromised in early adipogenic and osteogenic potential, and ability to form colonies in vitro. BMI was inversely correlated with ASC proliferation and colony forming potential as assessed by CyQUANT proliferation assay (fluorescence- based measurement of cellular DNA content), and colony forming assays. BMI was positively correlated with early time point (day 7) but not later time point (day 15) intracytoplasmic lipid accumulation as assessed by Oil-Red-O staining. Alizarin red staining and RT-PCR for alkaline phosphatase demonstrated that elevated BMI resulted in compromised ASC mineralization of extracellular matrix and decreased alkaline phosphatase mRNA expression. These data demonstrate that elevated BMI resulted in reduced ASC proliferation, and potentially compromised osteogenic capacity in vitro; thus BMI is an important criterion to consider in selecting ASC donors for clinical applications.
    BMC Cell Biology 08/2013; 14(1):34. DOI:10.1186/1471-2121-14-34 · 2.34 Impact Factor
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