Katsujiro Sato

Cell-Assisted Lipotransfer for Cosmetic Breast Augmentation: Supportive Use of Adipose-Derived Stem/Stromal Cells

BackgroundLipoinjection is a promising treatment but has some problems, such as unpredictability and a low rate of graft survival due to partial necrosis.MethodsTo overcome the problems with lipoinjection, the authors developed a novel strategy known as cell-assisted lipotransfer (CAL). In CAL, autologous adipose-derived stem (stromal) cells (ASCs) are used in combination with lipoinjection. A stromal vascular fraction (SVF) containing ASCs is freshly isolated from half of the aspirated fat and recombined with the other half. This process converts relatively ASC-poor aspirated fat to ASC-rich fat. This report presents the findings for 40 patients who underwent CAL for cosmetic breast augmentation.ResultsFinal breast volume showed augmentation by 100 to 200 ml after a mean fat amount of 270 ml was injected. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Cyst formation or microcalcification was detected in four patients. Almost all the patients were satisfied with the soft and natural-appearing augmentation.ConclusionsThe preliminary results suggest that CAL is effective and safe for soft tissue augmentation and superior to conventional lipoinjection. Additional study is necessary to evaluate the efficacy of this technique further.

Characterization of Freshly Isolated and Cultured Cells Derived From the Fatty and Fluid Portions of Liposuction Aspirates

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose‐derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non‐cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+CD45− cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood‐derived cells (CD45+), ASCs (CD31−CD34+CD45−CD90+CD105−CD146−), endothelial (progenitor) cells (CD31+CD34+CD45−CD90+CD105lowCD146+), pericytes (CD31−CD34−CD45−CD90+CD105−CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10–20 weeks. These results suggest that liposuction‐derived cells may be useful and valuable for cell‐based therapies. J. Cell. Physiol.

Cell‐Assisted Lipotransfer for Facial Lipoatrophy: Efficacy of Clinical Use of Adipose‐Derived Stem Cells

BACKGROUND Lipoinjection is a promising treatment, but its efficacy in recontouring facial lipoatrophy remains to be established. OBJECTIVE The objective was to evaluate the efficacy and adverse effects of lipoinjection and supplementation of adipose-derived stem/stromal cells (ASCs) to adipose grafts. METHODS To overcome drawbacks of autologous lipoinjection, we have developed a novel strategy called cell-assisted lipotransfer (CAL). In CAL, stromal vascular fraction containing ASCs was freshly isolated from half of an aspirated fat sample and attached to the other half of aspirated fat sample with the fat acting as a scaffold. This process converts relatively ASC-poor aspirated fat into ASC-rich fat. We performed conventional lipoinjection (non-CAL; n=3) or CAL (n=3) on six patients with facial lipoatrophy due to lupus profundus or Parry-Romberg syndrome. RESULTS All patients obtained improvement in facial contour, but the CAL group had a better clinical improvement score than did the non-CAL patients, although the difference did not reach statistical significance (p=.11). Adipose necrosis was found in one non-CAL case who took perioperative oral corticosteroids. CONCLUSION Our results suggest that CAL is both effective and safe and potentially superior to conventional lipoinjection for facial recontouring.

Progenitor-Enriched Adipose Tissue Transplantation as Rescue for Breast Implant Complications

Abstract:  Breast enhancement with artificial implants is one of the most frequently performed cosmetic surgeries but is associated with various complications, such as capsular contracture, that lead to implant removal or replacement at a relatively high rate. For replacement, we used transplantation of progenitor‐supplemented adipose tissue (cell‐assisted lipotransfer; CAL) in 15 patients. The stromal vascular fraction containing adipose tissue progenitor cells obtained from liposuction aspirates was used to enrich for progenitor cells in the graft. Overall, clinical results were very satisfactory, and no major abnormalities were seen on magnetic resonance imaging or mammogram after 12 months. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Surviving fat volume at 12 months was 155 ± 50 mL (Right; mean ± SD) and 143 ± 80 mL (Left) following lipoinjection from an initial mean of 264 mL. These preliminary results suggest that CAL is a suitable methodology for the replacement of breast implants.

Influences of centrifugation on cells and tissues in liposuction aspirates: optimized centrifugation for lipotransfer and cell isolation.

Background: Although injective autologous fat transplantation is one of the most attractive options for soft-tissue augmentation, problems such as unpredictability and fibrosis resulting from fat necrosis limit its universal acceptance. Centrifugation is one of most common methods for overcoming these difficulties. This study was performed to investigate quantitatively the effects of centrifugation on liposuction aspirates to optimize centrifugal conditions for fat transplantation and isolation of adipose-derived stem cells. Methods: Liposuction aspirates, obtained from eight healthy female donors, were either not centrifuged or centrifuged at 400, 700, 1200, 3000, or 4200 g for 3 minutes. The volumes of the oil, adipose, and fluid portions and numbers of blood cells and adipose-derived cells in each portion were examined. The processed adipose tissues (1 ml) were injected into athymic mice, and grafts were harvested and weighed at 4 weeks. Morphologic alterations were observed using light and scanning electron microscopy. Results: Centrifugation concentrated adipose tissues and adipose-derived stem cells in the adipose portion and partly removed red blood cells from the adipose portion. Centrifugation at more than 3000 g significantly damaged adipose-derived stem cells. Centrifugation enhanced graft take per 1 ml centrifuged adipose but reduced calculated graft take per 1 ml adipose before centrifugation. Conclusions: Excessive centrifugation can destroy adipocytes and adipose-derived stem cells, but appropriate centrifugation concentrates them, resulting in enhanced graft take. The authors tentatively recommend 1200 g as an optimized centrifugal force for obtaining good short- and long-term results in adipose transplantation.

Preserved Proliferative Capacity and Multipotency of Human Adipose-Derived Stem Cells after Long-Term Cryopreservation

Background: Human adipose-derived stem (stromal) cells are promising as a regenerative therapy tool for defective tissues of mesenchymal lineage, including fat, bone, and cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation could be an indispensable fundamental technology, as has occurred in other fields involving cell-based therapies using hematopoietic stem cells and umbilical cord blood cells. Methods: The authors examined the proliferative capacity and multipotency of human adipose-derived stem cells isolated from lipoaspirates of 18 patients in total before and after a 6-month cryopreservation following their defined protocol. Proliferative capacity was quantified by measuring doubling time in cell culture, and multipotency was examined with differentiation assays for chondrogenic, osteogenic, and adipogenic lineages. In addition, expression profiles of cell surface markers were determined by flow cytometry and compared between fresh and cryopreserved adipose-derived stem cells. Results: Cryopreserved adipose-derived stem cells fully retained the potential for differentiation into adipocytes, osteoblasts, and chondrocytes and for proliferative capacity. Flow cytometric analyses revealed that surface marker expression profiles remained constant before and after storage. Conclusions: Adipose-derived stem cells can be cryopreserved at least for up to 6 months under the present protocol without any loss of proliferative or differentiation potential. These results ensure the availability of autologous banked adipose-derived stem cells for clinical applications in the future.

Rapid expansion of human adipose-derived stromal cells preserving multipotency

BACKGROUND Adipose-derived stromal (stem) cells (ASC) have been shown to be of great therapeutic use in pre-clinical studies in diverse fields, but a standard expansion method has not been established. We investigated the effects of an endothelial growth medium (EGM-2) on ASC, focusing on proliferation and differentiation potentials. METHODS ASC were cultured in EGM-2 and DMEM. Doubling time and total cell number were compared between the two media. The proliferative effect of each growth factor supplemented in EGM-2 was also examined. Cultured cells in each medium were examined for surface marker expression using flow cytometry. Differentiation into the adipogenic, chondrogenic and osteogenic lineages was analyzed after culture in each medium. RESULTS ASC cultured with EGM-2 proliferated much more rapidly (10(5) times in 2 weeks) and reached the stationary phase earlier than those cultured with DMEM. Among the supplements contained in EGM-2, only fibroblast growth factor-2 (FGF-2) significantly promoted proliferation of ASC, although the proliferative effect of FGF-2 was much less than that of EGM-2, suggesting a synergism among other supplement factors. Flow cytometry and differentiation assays suggested that ASC cultured in EGM-2 preserved immunophenotype and differentiation capacity for at least three mesenchymal lineages (adipogenic, chondrogenic and osteogenic), similar to those cultured with DMEM. DISCUSSION The present expansion method markedly accelerates proliferation of ASC, preserving their multipotent differentiation capacities, and lays the groundwork for establishing a practical route to mega-expansion of ASC for clinical applications.

Influences of preservation at various temperatures on liposuction aspirates.

Background: Aspirated fat is not only a filler material but also an abundant source of adipose-derived stem cells. The aim of this study was to assess degeneration of aspirated fat during preservation and optimize the preservation method for lipoaspirates. Methods: Aspirated fat was preserved at room temperature for 1, 2, 4, and 24 hours (n = 10 each); at 4°C for 1, 2, and 3 days (n = 14 each); or at –80°C for 1 month (n = 3). Morphologic changes were assessed with scanning electron microscopy. Adipose-derived stem cell yield was measured after 1 week of culture. For aspirated fat preserved at room temperature, damaged adipocytes were assessed by measuring the oil volume ratio after centrifugation (n = 6) and glycerol-3-phosphate-dehydrogenase activity in washing solution (n = 4). Cell surface marker expression was examined by flow cytometry (n = 3). Results: Although the scanning electron microscopic assay indicated no remarkable anatomical changes based on preservation methods, oil volume significantly increased in fat preserved at room temperature for 4 hours. Adipose-derived stem cell yield was significantly reduced by preservation at room temperature for 24 hours and by preservation at 4°C for 2 or 3 days. Flow cytometric analysis suggested that the biological properties of adipose-derived stem cells did not significantly change at 4°C up to 3 days. The cells were isolated from cryopreserved fat, but the yield was much less than that from fresh aspirated fat. Conclusions: Aspirated fat should be transplanted as quickly as possible if it is preserved at room temperature. For adipose-derived stem cell isolation, aspirated fat can be stored or transported overnight if it is preserved at 4°C without adipose-derived stem cell yield loss or changes in biological properties.

Ectopic fibrogenesis induced by transplantation of adipose-derived progenitor cell suspension immediately after lipoinjection.

Adipose tissue has its own tissue-specific progenitor cells, a subpopulation of which has been shown to have multipotency and called adipose stem cells or adipose-derived mesenchymal stem cells (1,2). During a clinical trial in which soft tissue is augmented by transplantation of progenitor-enriched

Pretreatment with topical all-trans-retinoic acid is beneficial for wound healing in genetically diabetic mice

Abstract Objective: Topical pretreatment with all- trans -retinoic acid (atRA) is known to improve healing of cutaneous wounds. We tested the effect of atRA on wound healing of genetically diabetic db/db mice. It is known that cutaneous wounds of db/db mice show delayed wound healing due to impaired wound contraction, delayed granulation tissue formation and underexpression of keratinocyte growth factor (KGF). Methods: 0.1% atRA in 100 mg aqueous gel was applied to the back skin of db/db mice as well as to their normal heterozygous littermates, db/+ mice, for five consecutive days, and 2 days after completion of the atRA treatment, two round excisional wounds were created down the panniculus carnosus with a 6-mm punch biopsy on the back skin of each mouse. Results: After 5 days treatment with 0.1% atRA, significant hypertrophy of the epidermis and dermis, neovascularization, and inflammatory cell invasion were seen in the skin of the db/db mice, but these effects were seen only weakly in db/+ mice. Wounds in atRA-treated db/db mice closed more rapidly than those in vehicle-treated db/db mice. KGF mRNA expression, which is usually significantly lower in db/db mice than in normal mice, in wounds of atRA-treated db/db mice on day 1 of treatment was as strong as in db/+ mice. Conclusion: Pretreatment with atRA reversed the impaired wound healing in db/db mice.