Plastic Surgery Research Council
Members Only  |  Contact  |  PSRC on Facebook
PSRC 60th Annual Meeting
Program and Abstracts

Back to 2015 Annual Meeting Program


Novel Preparation Method for The Therapeutic Micro-spheroids Of Adipose-derived Stromal Cells (ASCs): Three-dimensional Floating Culture In Non-cross-linked Hyaluronic Acid (HA) Gel
Jingwei Feng, MD, Kazuhide Mineda, MD, Kentaro Doi, MD, Shinichiro Kuno, MD, Kahori Kinoshita, MD, Koji Kanayama, MD, Kotaro Yoshimura, MD.
University of Tokyo, Tokyo, Japan.

PURPOSE: Spheroids formation is important to promote stem cell functions and avoid unfavorable migration in regenerative therapies. It is usually performed with non-adherent dish culture, but yielded spheroids have varies sizes. The large size spheroids show central necrosis and limit the therapeutic efficacy. Here we sought to establish a novel method of functional micro-spheroid formation using HA gel with high efficiency and consistency.
METHODS: Monolayer cultured human ASCs were seeded on 2, 3, 4, 5 or 10% of non-cross-linked HA gel from 1 to 7 days to optimize spheroid formation. Spheroids and 2D cultured ASCs were comparatively analyzed with microarray. Pluripotent stem cell markers were compared with immunocytology. ELISA was also performed to assess secretion of angiogenic growth factors when cultured in 1% (hypoxia) or 6% (normoxia) oxygen. Human ASC spheroids, dissociated ASCs, and saline were injected into the inguinal fat pads which underwent ischemia-reperfusion injury in severe combined immune-deficiency (SCID) mice and into normal mice with mitomycin C-induced refractory skin ulcers. Therapeutic effects, tissue regeneration and injected cell differentiation were evaluated.
RESULTS: ASCs cultured in 4-5% HA gel formed proper-sized spheroids with most efficiency and consistency as short as 48 hours after plating: 90% of the spheres were with appropriate diameters (between 20 µm and 100 µm) and only 0.2% of the spheroids were too large (>100 µm). Spheroids developed from classical non-adherent dishes had oversized (>100 µm) ones at 17%, and micro-spheroids with appropriate size was only 41% of those prepared in HA-gel. Immunocytology showed that, 37% of spheroids from HA gel were SSEA-3(+), a marker of pluripotency, and considered multi-lineage differentiating stress enduring (Muse) cell-rich spheroids. They were also positive for other pluripotent markers such as Nanog, Oct3/4 and Sox-2. While cell number did not change after HA-gel culture for spheroid formation, ELISA showed that prepared spheroids secreted higher levels of HGF and VEGF than 2D cultured ASCs in hypoxic condition. Microarray indicated that spheroids showed substantially upregulated expression of genes encoding angiogenesis growth factors including VEGF, HGF, and FGF2; expression of pluripotent markers including Oct3/4, Sox2 and Stat3 also raised, while some proliferation related genes were suppressed. Reperfusion injury mice treated with ASC-spheroids suffered the least fat atrophy (1.6%) comparing with dissociated ASCs-treated group (14.3%) or saline group (20.3%) at 28 days post injury. In the skin ulcer model, immunostaining revealed better in vivo differentiation capacity for spheroids: spheroids-treated group had 2.4 times more of CD34, h-golgi co-localization cells (p=0.009) and 1.8 times more of α-SMA, h-golgi co-localization cells (p=0.037) than dissociated ASCs-treated group on 10 days after treatment.
CONCLUSION: ASC spheroids formed within HA gel had relatively uniform sizes which potentially avoid unpreferable migration. Evaluating the comparative therapeutic efficacy with monolayer cultured ASCs, spheroids prepared with above method exhibited better differentiation capacity and trophic effect in vitro, and demonstrated pro-angiogenesis effects in the process of skin/adipose injury repair in vivo.


Back to 2015 Annual Meeting Program