Optimization and validation of a fat-on-a-chip model for non-invasive therapeutic drug discovery.

Obesity is a significant public health concern that is closely associated with various comorbidities such as heart disease, stroke, type II diabetes (T2D), and certain cancers. Due to the central role of adipose tissue in many disease etiologies and the pervasive nature in the body, engineered adipose tissue models are essential for drug discovery and studying disease progression. This study validates a fat-on-a-chip (FOAC) model derived from primary mature adipocytes. Our FOAC model uses a Micronit perfusion device and introduces a novel approach for collecting continuous data by using two non-invasive readout techniques, resazurin and glucose uptake. The Micronit platform proved to be a reproducible model that can effectively maintain adipocyte viability, metabolic activity, and basic functionality, and is capable of mimicking physiologically relevant responses such as adipocyte hypertrophy and insulin-mediated glucose uptake. Importantly, we demonstrate that adipocyte size is highly dependent on extracellular matrix properties, as adipocytes derived from different patients with variable starting lipid areas equilibrate to the same size in the hyaluronic acid hydrogel. This model can be used to study T2D and monitor adipocyte responses to insulin for longitudinally tracking therapeutic efficacy of novel drugs or drug combinations.

Intramuscular Nanofat Injection Promotes Inflammation-Induced Gastrocnemius Regeneration in a Syngeneic Rat Sciatic Nerve Injury Model.

BACKGROUND: Mechanical emulsification of adipose tissue to concentrate protein and stromal cell components (ie, nanofat) has gained considerable interest in clinical practice. Although the regenerative potential of nanofat has largely been used in aesthetic applications, these effects have considerable potential in reconstruction as well. Here, the authors investigated the therapeutic properties of nanofat injected directly into the denervated gastrocnemius after a sciatic nerve injury in Lewis rats. METHODS: Muscle denervation was induced by transecting and immediately repairing the sciatic nerve. Inguinal and subcutaneous adipose was harvested from donor rodents, processed into nanofat, and then injected intramuscularly into the gastrocnemius. Gait analysis was performed weekly. Rodents were euthanized at 9 and 12 weeks, after which tetanic contraction force was measured, and gene expression, histology, and cytokine multiplexing were performed. RESULTS: Intramuscular injection of nanofat significantly increased maximum tetanic force generation at 9 and 12 weeks. The forces of the nanofat-injected gastrocnemii were better correlated to their contralateral gastrocnemii relative to controls. Muscle repair-associated inflammatory gene expressions were significantly up-regulated in nanofat-injected gastrocnemii. Cytokines interleukin (IL)-1ß, IL-18, vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, and tissue inhibitor of metalloproteinase-1 were significantly higher in nanofat-injected gastrocnemii relative to control gastrocnemii, and the tetanic force was linearly and significantly correlated to IL-1ß and IL-18 and their interacting effects. CONCLUSIONS: Intramuscular injection of emulsified adipose tissue (nanofat) significantly increased gastrocnemii contraction force after sciatic nerve injury, with prolonged reconstructive inflammation by means of CD68, inducible nitric oxide synthase, IL-1ß, and IL-18 all being potential mechanisms for this recovery. This application could potentially increase the therapeutic breadth of nanofat to include muscular recovery after nerve injury. CLINICAL RELEVANCE STATEMENT: The authors' study investigates a clinically translatable therapy to mitigate muscle atrophy after nerve injury.

Active Vitamin D3 (Calcitriol) Increases Adipose Graft Retention in a Xenograft Model.

BACKGROUND: Autologous fat grafting, although broadly indicated, is limited by unsatisfactory retention and often requires multiple procedures to achieve durable outcomes. Graft survival is strongly influenced by the magnitude and duration of post-engraftment ischemia. Calcitriol is a pleiotropic, safe nutrient with cell-specific influence on viability and metabolic flux. OBJECTIVES: Evaluate the efficacy of activated vitamin D3 (calcitriol) in improving grafting outcomes and examine its mechanisms. METHODS: Lipoaspirate was collected for ex vivo culture (7 unique donors), in vitro bioenergetic analysis (6 unique donors), and in vivo transplantation (5 unique donors). Ex vivo samples were incubated for up to 2 weeks before extraction of the stromal vascular fraction (SVF) for viability or flow cytometry. SVF was collected for Seahorse (Agilent; Santa Clara, CA) analysis of metabolic activity. Human endothelial cell lines were utilized for analyses of endothelial function. In vivo, samples were implanted into athymic mice with calcitriol treatment either (1) once locally or (2) 3 times weekly via intraperitoneal injection. Grafts were assessed photographically, volumetrically, and histologically at 1, 4, and 12 weeks. Hematoxylin and eosin (H&E), Sirius red, perilipin, HIF1α, and CD31 tests were performed. RESULTS: Calcitriol-treated lipoaspirate demonstrated dose-dependent increases in SVF viability and metabolic reserve during hypoxic stress. Calcitriol treatment enhanced endothelial mobility ex vivo and endothelial function in vitro. In vivo, calcitriol enhanced adipocyte viability, reduced fibrosis, and improved vascularity. Continuous calcitriol was sufficient to improve graft retention at 12 weeks (P < .05). CONCLUSIONS: Calcitriol increased fat graft retention in a xenograft model. Calcitriol has potential to be a simple, economical means of increasing fat graft retention and long-term outcomes.

Automated Decellularization of the Rodent Epigastric Free Flap: A Comparison of Sodium Dodecyl Sulfate-Based Protocols.

BACKGROUND: Free tissue transfer to cover complex wounds with exposed critical structures results in donor-site morbidity. Perfusion decellularization and recellularization of vascularized composite tissues is an active area of research to fabricate complex constructs without a donor site. Sodium dodecyl sulfate (SDS)-based protocols remain the predominant choice for decellularization despite the deleterious effects on tissue ultrastructure and capillary networks. We aimed to develop an automated decellularization process and compare different SDS perfusion times to optimize the protocol. METHODS: A three-dimensional-printed closed-system bioreactor capable of continuously perfusing fluid through the vasculature was used for decellularization. The artery and vein of rat epigastric fasciocutaneous free flaps were cannulated and connected to the bioreactor. Protocols had varying durations of 1% SDS solution (3, 5, and 10 days) followed by 1 day of 1% Triton X-100 and 1 day of 1x phosphate-buffered saline. The residual DNA was quantified. Microarchitecture of the constructs was assessed with histology, and the vascular network was visualized for qualitative assessment. RESULTS: The structural integrity and the microarchitecture of the extracellular matrix was preserved in the 3- and 5-day SDS perfusion groups; however, the subcutaneous tissue of the 10-day protocol lost its structure. Collagen and elastin structures of the pedicle vessels were not compromised by the decellularization process. Five-day SDS exposure group had the least residual DNA content (p < 0.001). Across all protocols, skin consistently had twice as much residual DNA over the subcutaneous tissues. CONCLUSION: A compact and integrated bioreactor can automate decellularization of free flaps to bioengineer regenerative constructs for future use in reconstruction of complex defects. A decellularization protocol with 5 days of 1% SDS exposure was the most successful to keep the residual DNA content at a minimum while preserving the structural integrity of the tissues.

Use of wound edge inversion (epibole) to generate recalcitrant and inflamed diabetic wounds.

Robust and predictive pre-clinical models of recalcitrant diabetic wounds are critical for advancing research efforts toward improving healing. Murine models have logistic and genetic benefits versus larger animals; however, native murine healing inadequately represents clinically recalcitrant wounds in humans. Furthermore, current humanization techniques employing devices, deleterious mutations or chemical agents each carry model-specific limitations. To better replicate human wounds in a mouse, we developed a novel wound-edge inversion (WEI) technique that mimics the architecture of epibole and mitigates contracture, epithelialization, and consequently wound closure. In this study, we evaluated the reliability and durability of the WEI model in wild-type and obese diabetic mice and compared to healing after (i) punch biopsy, (ii) mechanical/silicone stenting or (iii) exogenous oxidative stressors. In wild-type mice, WEI demonstrated favourable closure characteristics compared to both control and stented wounds, however, wounds progressed to closure by 4 weeks. In contrast, diabetic WEI wounds persisted for 6-10 weeks with reduced contracture and epithelialization. In both diabetic and wild-type mice, WEI sites demonstrated persistence of inflammatory populations, absence of epithelialization, and histologic presence of alpha-SMA positive granulation tissue when compared to controls. We conclude that the WEI technique is particularly valuable for modelling recalcitrant diabetic wounds with sustained inflammation and dysfunctional healing.

Engineering a 3D Vascularized Adipose Tissue Construct Using a Decellularized Lung Matrix.

Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as larger defect sizes are correlated with higher rates of complications and failure due to insufficient vascularization following implantation. Our study demonstrates, for the first time, a method to engineer perfusable, pre-vascularized, high-density adipose grafts that combine patient-derived adipose cells with a decellularized lung matrix (DLM). The lung is one of the most vascularized organs with high flow, low resistance, and a large blood-alveolar interface separated by a thin basement membrane. For our work, the large volume capacity within the alveolar compartment was repurposed for high-density adipose cell filling, while the acellular vascular bed provided efficient graft perfusion throughout. Both adipocytes and hASCs were successfully delivered and remained in the alveolar space even after weeks of culture. While adipose-derived cells maintained their morphology and functionality in both static and perfusion DLM cultures, perfusion culture offered enhanced outcomes over static culture. Furthermore, we demonstrate that endothelial cells seamlessly integrate into the acellular vascular tree of the DLM with adipocytes. These results support that the DLM is a unique platform for creating vascularized adipose tissue grafts for large defect filling.

The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction.

BACKGROUND: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. METHODS: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell-cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. RESULTS: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell-cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. CONCLUSION: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations.

Clinical Evaluation of an Off-the-Shelf Allogeneic Adipose Matrix for Soft Tissue Reconstruction.

Biomaterials derived from human adipose extracellular matrix have shown promise in vitro and in animal studies as an off-the-shelf adipogenic matrix for sustained volume replacement. Herein, we report the results of a randomized prospective study conducted with allograft adipose matrix (AAM) grafted into the pannus of presurgical abdominoplasty patients 3 or 6 months before scheduled surgery. This is the first report of a longitudinal histologic analysis of AAM in clinical use. METHODS: Ten healthy patients undergoing elective abdominoplasty were recruited to receive AAM before surgery. Enrolled subjects were randomized into either a 3-month follow-up cohort or a 6-month follow-up cohort. Subjects were monitored for adverse events associated with AAM grafting in addition to undergoing serial biopsy. Following surgical excision of the pannus, representative samples from the AAM surgical sites were stained and evaluated with hematoxylin and eosin for tissue morphology, Masson's trichrome for collagen, and perilipin for adipocytes. RESULTS: All subjects tolerated AAM with no severe adverse events reported. At 3 months following implantation, AAM remained visible within the confines of the subjects' native surrounding adipose tissue with sparse adipocytes apparent within the matrix. By 6 months, AAM had remodeled and was primarily composed of perilipin-positive adipocytes. Histologic analysis confirmed tissue remodeling (hematoxylin and eosin), adipogenesis (perilipin), and angiogenesis (Masson's trichrome) occurred with the presence of AAM. CONCLUSIONS: AAM is a safe, allogeneic, off-the-shelf regenerative matrix that is adipogenic and noninflammatory and promotes angiogenesis.

Injectable Allograft Adipose Matrix Supports Adipogenic Tissue Remodeling in the Nude Mouse and Human.

BACKGROUND: Adipose tissue reaches cellular stasis after puberty, leaving adipocytes unable to significantly expand or renew under normal physiologic conditions. This is problematic in progressive lipodystrophies, in instances of scarring, and in soft-tissue damage resulting from lumpectomy and traumatic deformities, because adipose tissue will not self-renew once damaged. This yields significant clinical necessity for an off-the-shelf de novo soft-tissue replacement mechanism. METHODS: A process comprising separate steps of removing lipid and cellular materials from adipose tissue has been developed, creating an ambient temperature-stable allograft adipose matrix. Growth factors and matrix proteins relevant to angiogenesis and adipogenesis were identified by enzyme-linked immunosorbent assay and immunohistochemistry, and subcutaneous soft-tissue integration of the allograft adipose matrix was investigated in vivo in both the athymic mouse and the dorsum of the human wrist. RESULTS: Allograft adipose matrix maintained structural components and endogenous growth factors. In vitro, adipose-derived stem cells cultured on allograft adipose matrix underwent adipogenesis in the absence of media-based cues. In vivo, animal modeling showed vasculature formation followed by perilipin A-positive tissue segments. Allograft adipose matrix maintained soft-tissue volume in the dorsal wrist in a 4-month investigation with no severe adverse events, becoming palpably consistent with subcutaneous adipose. CONCLUSIONS: Subcutaneous implantation of allograft adipose matrix laden with retained angiogenic and adipogenic factors served as an inductive scaffold for sustaining adipogenesis. Tissue incorporation assessed histologically from both the subcutaneous injection site of the athymic nude mouse over 6 months and human dorsal wrist presented adipocyte morphology residing within the injected scaffold.

Oncologic Safety of Fat Grafting for Autologous Breast Reconstruction in an Animal Model of Residual Breast Cancer.

BACKGROUND: Clinical outcomes suggest that postoncologic reconstruction with fat grafting yields cumulative incidence curves of recurrence comparable to those of other breast reconstruction procedures; however, results from experimental research studies suggest that adipose stem cells can stimulate cancer growth. In this study, a novel animal model of residual cancer was developed in mouse mammary pads to test whether lipofilling impacts the probability of locoregional recurrence of breast cancer after breast conserving surgery. METHODS: Mammary fat pads of female NOD-SCID gamma mice were each injected with MCF-7 cells in Matrigel. Tumors were allowed to engraft for 2 weeks, after which time either sterile saline (n = 20) or human fat graft (n = 20) was injected adjacent to tumor sites. After 8 weeks, tumors were assessed for volume measurement, histologic grade, Ki67 positivity, and metastatic spread. RESULTS: Animals receiving lipofilling after tumor cell engraftment had lower tumor volume and mass (p = 0.046 and p = 0.038, respectively). Macroscopic invasion was higher in the saline group. Histologic grade was not significantly different in the two groups (p = 0.17). Ki67 proliferation index was lower in tumors surrounded by fat graft (p = 0.01). No metastatic lesion was identified in any animal. CONCLUSIONS: Adipose transfer for breast reconstruction performed in the setting of residual breast tumor in a clinically relevant animal model did not increase tumor size, proliferation, histologic grade, or metastatic spread. This study supports the oncologic safety of lipofilling as part of the surgical platform for breast reconstruction after cancer therapy.

An exploratory study on the preparation and evaluation of a "same-day" adipose stem cell-based tissue-engineered vascular graft.

OBJECTIVE: Tissue-engineered vascular grafts containing adipose-derived mesenchymal stem cells offer an alternative to small-diameter vascular grafts currently used in cardiac and lower-extremity revascularization procedures. Adipose-derived, mesenchymal stem cell-infused, tissue-engineered vascular grafts have been shown to promote remodeling and vascular homeostasis in vivo and offer a possible treatment solution for those with cardiovascular disease. Unfortunately, the time needed to cultivate adipose-derived mesenchymal stem cells remains a large hurdle for tissue-engineered vascular grafts as a treatment option. The purpose of this study was to determine if stromal vascular fraction (known to contain progenitor cells) seeded tissue-engineered vascular grafts would remain patent in vivo and remodel, allowing for a "same-day" process for tissue-engineered vascular graft fabrication and implantation. METHODS: Stromal vascular fraction, obtained from adult human adipose tissue, was seeded within 4 hours after acquisition from the patient onto poly(ester urethane)urea bilayered scaffolds using a customized rotational vacuum seeding device. Constructs were then surgically implanted as abdominal aortic interposition grafts in Lewis rats. RESULTS: Findings revealed patency in 5 of 7 implanted scaffolds at 8 weeks, along with neotissue formation and remodeling occurring in patent tissue-engineered vascular grafts. Patency was documented using angiography and gross inspection, and remodeling and vascular components were detected using immunofluorescent chemistry. CONCLUSIONS: A "same-day" cell-seeded, tissue-engineered vascular graft can remain patent after implantation in vivo, with neotissue formation and remodeling occurring by 8 weeks.

Inflammatory biomarker in adipose stem cells of women with endometrial cancer.

AIM: To explore inflammatory biomarkers secreted by adipose stem cells (ASCs) in omental, retroperitoneal and subcutaneous adipose tissues of women with endometrial cancer. PATIENTS & METHODS: ASCs were collected from 22 women, aged 35-83 years, undergoing hysterectomy for endometrial cancer. Angiopoietin-2, EGF, IL-8, leptin, VEGFA, VEGFC and VEFGD levels in the ASC-conditioned media were analyzed by Luminex. RESULTS: We found a significant difference between the three depots for IL-8 (p < 0.0001), with the highest levels of IL-8 in the omental depot. VEGFA levels were highest in the retroperitoneal depot. CONCLUSION: This is one of the first studies to explore biomarker expression in ASC-conditioned media in adipose tissue. ASC characteristics may be important to evaluate in relation to cancer risk.

Optimization and Standardization of the Immunodeficient Mouse Model for Assessing Fat Grafting Outcomes.

BACKGROUND: Animal models are often used to assess interventions that might improve fat grafting outcomes; however, there is great variability in the models. The authors sought to determine the predictive value of the immunocompromised mouse model for fat grafting so that experiments could be standardized and optimized. METHODS: Human lipoaspirate injections at different volumes and time points were assessed in a nude mouse model and compared with control injections of nonviable fat. Volume retention and explant histologic score were compared. In a separate study, interanimal reproducibility was determined by implanting a highly consistent hydrogel and measuring variability in volume retention. RESULTS: Injection volume significantly affects adipose resorption kinetics at 6 and 12 weeks. Masson trichrome staining revealed that macrophages were unable to infiltrate large (1 ml) grafts, and oil cysts were not absorbed by 18 weeks, which interfered with interpretation of volume retention data. Nonviable tissue was resorbed when grafts were 0.3 ml, and quantification of graft histologic viability correlated well with graft retention at all study time points. Interanimal variability was measured to be 8.44 percent of the mean retention volume for small graft volumes. CONCLUSIONS: Human fat graft retention in the immunodeficient mouse correlates with graft viability in small, 0.3-ml-volume grafts. However, centralized oil cysts in nonviable 1.0-ml grafts were not resorbed by 18 weeks and thus volume measurements were confounded and not significantly different from viable samples. In addition, tissue injury scores increased in initially healthy fat grafts at 18 weeks, possibly because of a delayed immune reaction.

Adipose Stem Cell Function Maintained with Age: An Intra-Subject Study of Long-Term Cryopreserved Cells.

Background: The progressive decline in tissue mechanical strength that occurs with aging is hypothesized to be due to a loss of resident stem cell number and function. As such, there is concern regarding use of autologous adult stem cell therapy in older patients. To abrogate this, many patients elect to cryopreserve the adipose stromal-vascular fraction (SVF) of lipoaspirate, which contains resident adipose stem cells (ASC). However, it is not clear yet if there is any clinical benefit from banking cells at a younger age. Objectives: We performed a comparative analysis of SVF composition and ASC function from cells obtained under GMP conditions from the same three patients with time gap of 7 to 12 years. Methods: SVF, cryobanked under good manufacturing practice (GMP) conditions, was thawed and cell yield, viability, and cellular composition were assessed. In parallel, ASC proliferation and efficiency of tri-lineage differentiation were evaluated. Results: The results showed no significant differences existed in cell yield and SVF subpopulation composition within the same patient between harvest procedures 7 to 12 years apart. Further, no change in proliferation rates of cultured ASCs was found, and expanded cells from all patients were capable of tri-lineage differentiation. Conclusions: By harvesting fat from the same patient at two time points, we have shown that despite the natural human aging process, the prevalence and functional activity of ASCs in an adult mesenchymal stem cell, is highly preserved. Level of Evidence: 5.

Imaging the Stromal Vascular Fraction during Soft-Tissue Reconstruction.

BACKGROUND: Although fat grafting is an increasingly popular practice, suboptimal volume retention remains an obstacle. Graft enrichment with the stromal vascular fraction has gained attention as a method of increasing graft retention. However, few studies have assessed the fate and impact of transplanted stromal vascular fraction on fat grafts. In vivo imaging techniques can be used to help determine the influence stromal vascular fraction has on transplanted fat. METHODS: Stromal vascular fraction was labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), a near-infrared dye, and tracked in vivo. Proliferation and differentiation of labeled cells were assessed to confirm that labeling did not adversely affect cellular function. Different doses of labeled stromal vascular fraction were tracked within fat grafts over time using the in vivo imaging system. RESULTS: No significant differences in differentiation and proliferation were observed in labeled versus unlabeled cells (p > 0.05). A pilot study confirmed that stromal vascular fraction fluorescence was localized to fat grafts and different cell doses could be distinguished. A larger-scale in vivo study revealed that stromal vascular fraction fluorescence was statistically significant (p < 0.05) between different cell dose groups and this significance was maintained in higher doses (3 × 10(6) and 2 × 10(6) cells/ml of fat graft) for up to 41 days in vivo. CONCLUSIONS: DiR labeling allowed the authors to differentiate between cell doses and confirm localization. This article supports the use of DiR labeling in conjunction with in vivo imaging as a tool for imaging stromal vascular fraction within fat grafts.

A review of adipocyte lineage cells and dermal papilla cells in hair follicle regeneration.

Alopecia is an exceedingly prevalent problem effecting men and women of all ages. The standard of care for alopecia involves either transplanting existing hair follicles to bald areas or attempting to stimulate existing follicles with topical and/or oral medication. Yet, these treatment options are fraught with problems of cost, side effects, and, most importantly, inadequate long-term hair coverage. Innovative cell-based therapies have focused on the dermal papilla cell as a way to grow new hair in previously bald areas. However, despite this attention, many obstacles exist, including retention of dermal papilla inducing ability and maintenance of dermal papilla productivity after several passages of culture. The use of adipocyte lineage cells, including adipose-derived stem cells, has shown promise as a cell-based solution to regulate hair regeneration and may help in maintaining or increasing dermal papilla cells inducing hair ability. In this review, we highlight recent advances in the understanding of the cellular contribution and regulation of dermal papilla cells and summarize adipocyte lineage cells in hair regeneration.

Particle size in fat graft retention: A review on the impact of harvesting technique in lipofilling surgical outcomes.

Fat grafting popularity continues to rise among plastic surgeons. As a soft tissue filler, adipose tissue had many desirable attributes: it is easy to obtain, autologous, and may reintegrate into recipient sites. However, fat grafting is clinically plagued by unpredictable resorption rates, thus there is much interest in optimizing the procedure of fat grafting for consistent graft volumes. Fat harvesting, a part of fat transfer surgery, involves the removal of adipose tissue from the donor site. Different harvest procedures, such as whole fat excision or liposuction cannulas, result in a range of fat particle volumes, which may play a role in the cellular stability of grafts. The ideal harvesting technique and fat particle diameter is not currently known. This study aims to review the literature on the impact of fat particle size and clinical fat grafting outcomes, to present overarching conclusions, and to provide future directions for study. Current evidence supports excisional methods and larger bore cannulas to minimize cellular damage, preserve the native architecture, and maximize the number of cells within fat particles.