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Liu et al. Adipogenesis of obital fat stem cells

2
quantities of fat tissue, minimal patient discomfort, little with obesity (body mass index > 25 kg/m ), orbital
[2]
donor-site morbidity and ease of cell isolation. Thus, disease or endocrine disease were excluded from this
there has been growing interest in applying ASCs as study. The donors were divided into two age groups:
a potential cell source in the field of tissue engineering group A (26.33 ± 6.48 years old, n = 10) and group B
and regenerative medicine during the past decade. (56.44 ± 5.83 years old, n = 10). Patients in group A
had no baggy eye appearance while those in group
The human body can be segmented into various depots B had dermatochalasis with typical OF protrusion in
of fatty tissue based on anatomic region. These the lower eyelids. Fat samples were preserved on ice
[3]
depots vary in the amount of adipose tissue and may under aseptic condition, transported to the laboratory
produce ASCs with different biological characteristics. immediately after surgery, and processed within 6 h.
The orbital cavity contains a highly specialized adipose
depot which differs from the subcutaneous fat (SF) Evaluation of adipocyte morphology
[4]
developmentally and functionally. The existence of After weight measurement, four specimens in each
human orbital adipose-derived stem cells (OASCs) also group were fixed in 10% formalin overnight, embedded
has been confirmed, which can differentiate into the in paraffin, sectioned in 5-µm thickness and processed
corneal epithelial lineage, smooth muscle lineage and for routine hematoxylin and eosin (HE) staining. Images
neuronal lineage under specific inductive conditions, were taken in triplicate for each specimen using an
showing great therapeutic potential in treating orbital optical microscope (IX70, Olympus, Tokyo, Japan). The
and ocular diseases. [4-7] photographs were processed with the green channel
to enhance edge prominence using the Image-Pro
The location of orbital fat (OF) may change dramatically Plus software (v6.0, Media Cybernetics, Silver Spring,
from young to old age, especially in the lower eyelids. MD, USA). Three main descriptive parameters were
Normally the OF is located behind the septum and measured for evaluation of adipocyte morphology as
in front of the aponeurosis, occupying most of the follows: cell diameter (length of the longest line joining
orbital cavity. By middle or early old age, it often two points and passing through the centroid), perimeter
[8]
and area. Approximately 300 adipocytes were
migrates forward to the preseptal region, resulting measured for each histological image.
in a baggy appearance of the eyes. An interesting
question therefore arises from this phenomenon: Isolation and expansion of OASCs
will aging influence the functional characteristics of
OASCs? Identifying this issue is of significance when OASCs were isolated and expanded using methods
[4]
autologous OASC-based therapies are designed for previously reported with minor modification. Briefly,
after washing in phosphate buffer solution (PBS,
elderly patients. To date, however, little literature has pH 7.4, Sigma, Shanghai, China) extensively, 6 OF
investigated the relationship between age and the samples from each group were minced with sterile
biological properties of OASCs. scissors and digested with 0.1% type I collagenase
solution (Worthington Biochemical Corp, Lakewood,
In this study, the authors tested whether the proliferative NJ, USA) at 37 C for 60 min with constant agitation.
o
and differentiation potentials of human OASCs are The upper layer of adipocytes was removed by
affected by donor age. First, the OF samples were aspiration and the remaining cells were filtered
harvested from young (with normal OF) and middle- through a 100-µm and then a 40-µm nylon strainer (BD
aged patients (with protruded OF) during routine Bioscience, Franklin Lakes, NJ, USA). Filtered cells
blepharoplasty. Histological evaluation was performed were centrifuged for 5 min at 400 g, and resuspended
to observe the morphological changes of fat cells in in 3 mL of growth medium [GM, containing low-glucose
relation to age. Next, OASCs were isolated from OF Dulbecco’s modified Eagle’s medium (LG-DMEM,
samples and expanded in vitro. The cell yield, surface Gibco, Grand Island, NY, USA) and 10% fetal bovine
marker expression and growth kinetics were assessed. serum (HyClone, Logan, UT, USA) plus 1% antibiotic/
Finally OASCs were cultured under adipogenic condition antimycotic]. The viability of cells was assessed using
to compare their differentiation potential into adipocytes. the trypan blue exclusion method and the single-cell
suspension was plated onto 35-mm culture dishes
METHODS (Falcon, B&D Bioscience, San Jose, CA, USA) at a
density of 50,000 cells/cm . The medium was replaced
2
Harvest of orbital fat samples 48 h after cell plating to remove the non-adherent red
Adipose tissue samples were surgically harvested blood cells and changed twice a week thereafter.
during lower lid blepharoplasty from the central
compartments of the OF in 20 healthy female patients Cell colony forming and growth kinetics
who had previously given informed consent. Patients The total number of fibroblastic colony-forming units
Plastic and Aesthetic Research ¦ Volume 3 ¦ October 25, 2016 323