Yonsei Med J.  2011 Nov;52(6):999-1007. 10.3349/ymj.2011.52.6.999.

Rapid Isolation of Adipose Tissue-Derived Stem Cells by the Storage of Lipoaspirates

• Eom YW
• Lee JE
• Yang MS
• Jang IK
• Kim HE
• Lee DH
• Kim YJ
• Park WJ
• Kong JH
• Shim KY
• Lee JI
• Kim HS

• KMID: 1058822
• DOI: http://doi.org/10.3349/ymj.2011.52.6.999

Abstract

PURPOSE
This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs).
MATERIALS AND METHODS
Aliquots (10 g) of the lipoaspirates were stored at 4degrees C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed.
RESULTS
When the lipoaspirates were stored at 4degrees C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1x1014 fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells.
CONCLUSION
ASCs isolated from lipoaspirates and stored for 24 hours at 4degrees C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4degrees C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.

Keyword

Lipoaspirates; adipose tissue; mesenchymal stem cell; proliferation; differentiation

MeSH Terms

5'-Nucleotidase/metabolism
Adipose Tissue/*cytology
Adult
Antigens, CD/metabolism
Antigens, CD44/metabolism
Antigens, Thy-1/metabolism
Cell Differentiation/physiology
Cells, Cultured
Female
Humans
Immunoblotting
Immunohistochemistry
Immunophenotyping
Mesenchymal Stem Cells/metabolism
Muscle Development/genetics/physiology
Osteogenesis/genetics/physiology
Receptors, Cell Surface/metabolism
Reverse Transcriptase Polymerase Chain Reaction
Stem Cells/*cytology/metabolism
Young Adult

Figure

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  Fig. 1 Isolation of ASCs from lipoaspirates preserved at 4℃. (A) Yields of mononuclear cells isolated from lipoaspirates. Mononuclear cells were isolated from lipoaspirates preserved at 4℃ for the indicated times as described in the Materials and Methods section, and then viable mononuclear cells were counted by staining with trypan blue. (B) Recovery of cells at day 7. Each set of mononuclear cells was isolated at the indicated times and cultured in 100-mm culture dishes at a density of 5×106 cells. On day 7, the expanded cells were counted. (C) Clonogenic ability of mononuclear cells isolated from lipoaspirates. Five thousand mononuclear cells were plated in 6-well plates. They were cultured for two weeks, and then colony forming unit-fibroblasts (CFU-F) were stained with crystal violet. A colony consisting of more than 100 cells was counted microscopically. Representative histograms of three independent experiments are shown, and error bars represent the standard deviation. ASCs, adipose tissue-derived stem cells.

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  Fig. 2 Expression of cell surface markers of ASCs isolated from lipoaspirates preserved for 24 hours. Expression of cell surface markers of ASCs isolated from lipoaspirates preserved for 24 hours was determined by flow cytometry as described in the Materials and Methods section. CD14-FITC and CD105-PE, CD44-FITC and CD133-FITC, CD45-FITC and CD34-PE, and HLA-ABC-FITC and HLA-DR-PE were double stained. ASCs, adipose tissue-derived stem cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

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  Fig. 3 Proliferation potentials of ASCs isolated from lipoaspirates preserved for 24 hours. ASCs were cultured in 6-well plates at a density of 1000 cells/cm2. When the cells grew confluent, the total cell numbers were counted and then re-cultured at the same density. These procedures were repeated until cell growth stopped. Numbers of cells (-▪-) and population doubling times (histogram) were represented at each passage. Representative histograms of three independent experiments are shown, and error bars represent the standard deviation. ASCs, adipose tissue-derived stem cells.

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  Fig. 4 Differentiation potentials of ASCs isolated from lipoaspirates preserved for 24 hours. Passage 1 cells were seeded and differentiated into adipocytes, osteoblasts, muscle cells, or neuronal cells as described in the Materials and Methods section. Osteogenic (A) or adipogenic differentiation (B) were evaluated by assaying the alkaline phosphatase activity or Oil-Red O staining, respectively. To evaluate differentiation potentials of ASCs into muscle (C and D) or neuronal cells (E and F), we investigated the expression of several markers (myogenic; MyoD, Myogenin, Dystrophin, MCK and neurogenic; NeuroD, NF-L, Nestin) by RT-PCR, immunoblotting (myogenic; Myogenin and MyHC), and immucocytochemistry (neurogenic; NF-L). ASCs, adipose tissue-derived stem cells; MyoD, class I myosin; MCK, muscle creatine kinase; NeuroD, neurogenic differentiation; NF-L, neurofilament light polypeptide; DAPI, 4', 6-diamino-2-phenylindole; MyHC, myosin heavy chain.

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