Formulation and Characterization of Bioactive Agent Containing Nanodisks.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle's lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin B as the hydrophobic bioactive agent.

A Method for Isolation of Stromal Vascular Fraction Cells in a Clinically Relevant Time Frame.

There is increasing interest in the clinical applications of adipose-derived stem cells (ASCs) and the stromal vascular fraction (SVF) based on promising preclinical data. As adipose-derived therapeutics begin to translate into the clinical setting, it is important to maintain patient safety as well as uniformity in technique. Here, we describe a method for isolation of stromal vascular fraction cells in a clinically relevant time frame. Analytical laboratory techniques are mentioned, but respective protocols are not provided here.

Use of Freshly Isolated Human Adipose Stromal Cells for Clinical Applications.

The clinical use of adipose-derived cells is being explored very actively around the world for various human diseases. Adipose tissue is an abundant tissue source that can be easily harvested using liposuction. Human lipoaspirates contain a significant amount of mesenchymal stromal cells, as well as other progenitors and terminally differentiated cell types. This review covers the isolation of adipose stromal vascular fraction (SVF), the quality control and safety analysis of freshly isolated cell suspensions. The comparison between freshly isolated stromal cells and culture expanded cells from adipose tissue samples is also highlighted. This article provides a brief but comprehensive review about SVF isolation in the clinical setting, cell characterization, and biological potency of freshly obtained adipose stromal cells.

Skin Perfusion Pressure and Wound Closure Time in Lower Extremity Wounds.

INTRODUCTION: Peripheral Arterial Disease (PAD) affects approximately 8 million patients in the United States. We investigate the relationship of Skin Perfusion Pressure (SPP) and wound closure time in lower extremity wounds. METHODS: We conducted a retrospective study of 1125 lower extremity wounds in 998 patients between June 2006 and October 2014 in our wound clinic. We analyzed the relationship between SPPand wound closure time. SPP was measured using a Laser Doppler instrument. RESULTS: Patients with SPP values over 30 mmHg had shorter wound closure times, while patients with SPP values below 30 mmHg had a significantly longer wound closure time. Diabetic patients took longer to achieve wound closure compared to non-diabetics. No significant relationship was observed between SPP and wound closure time in relation to age or gender. CONCLUSION: SPP is a useful tool in estimating time to wound closure and assessing the necessity of vascular interventions in lower extremity wounds.

Adipose Stromal Vascular Fraction Isolation: A Head-to-Head Comparison of 4 Cell Separation Systems #2.

INTRODUCTION: With stromal vascular fraction (SVF) cell and adipose-derived stem cell-based technologies translating into the clinical setting, numerous isolation systems have been developed for the point of care isolation of SVF cells from adipose tissue. A relative lack of performance data on these systems can make objective assessment difficult for prospective clinicians. This study compared the performance of 4 SVF cell isolation systems. METHODS: Four isolation systems were compared: the MultiStation by PNC International, the LipoKit by MediKhan, the GID SVF-2 platform by GID Europe Ltd, and the StemSource 900/MB system by Cytori Therapeutics, Inc. Identical lipoaspirate samples for 5 separate donors were used. Stromal vascular fraction output was compared in terms of nucleated cell yield, viability, residual collagenase activity, sterility of the output, colony-forming unit-fibroblast frequency, frequency of CD31-/CD34+/CD45- cells, and operating statistics. RESULTS: Mean process time ranged from 65.4 to 120.8 minutes. Mean nucleated cell yield per milliliter of tissue processed ranged from 1.01 × 10 cells/mL to 6.24 × 10 cells/mL. Mean cellular viability ranged from 50.3% to 84.02%. Residual collagenase activity was negligible across all systems. Observed colony-forming unit-fibroblast frequency ranged from 0.495% to 1.704%. No significant difference was observed in frequency of CD31-/CD34+/CD45- cells. Results of the anaerobic/aerobic cultures were mixed. CONCLUSIONS: There was considerable variability between the outputs of each system. The system used by a clinician should be tailored to the individual needs of the practice. There is a range of cost options available. This study may help clinicians make more educated decisions when choosing an isolation system to meet their clinical needs.

Lateral Intercostal Artery Perforator Flap in Breast Reconstruction: A Simplified Pedicle Permits an Expanded Role.

BACKGROUND: The lateral intercostal artery perforator (LICAP) flap is a versatile second-tier option in breast reconstruction. The flap is rotated from redundant lateral chest fold on an easily dissected skin bridge pedicle without microsurgery in an outpatient setting. This series illustrates safety and effectiveness of the LICAP flap for prosthesis coverage when a muscle flap is not available or desired. In some cases, it even provides adequate soft tissue to reconstruct the breast mound without an implant. METHODS: Lateral intercostal artery perforator flaps performed for breast reconstruction at an ambulatory surgery center were reviewed. RESULTS: A total of 39 flaps were performed on an outpatient basis for a variety of breast reconstruction indications. One immediate reconstruction with bilateral LICAP flaps was performed after mastectomy. All remaining flaps were for delayed breast reconstruction. Mean operative time for each flap was 65 minutes, and concomitant procedures were performed in 25 of 27 patients. Follow-up was 5 to 96 months. There was 1 major complication (2.5%) and 5 minor (12.8%) complications. CONCLUSIONS: This series demonstrates unique advantages of the LICAP flap for a variety of breast reconstruction problems, including outpatient setting, no muscle sacrifice, flap reliability, and low donor site morbidity. These results confirm previous reports in post bariatric augmentation that the LICAP flap reliably supplies a large skin/adipose flap from the redundant tissue of the lateral chest fold with minimal morbidity even after radiation. The LICAP flap warrants closer consideration in breast reconstruction.

Mechanical versus enzymatic isolation of stromal vascular fraction cells from adipose tissue.

Clinical use of adipose-derived stem cells (ASCs) for a variety of indications is rapidly expanding in medicine. Most commonly, ASCs are isolated at the point of care from lipoaspirate tissue as the stromal vascular fraction (SVF). The cells are immediately administered to the patient as an injection or used to enrich fat grafts. Isolation of ASCs from adipose tissue is a relatively simple process performed routinely in cell biology laboratories, but isolation at the point of care for immediate clinical administration requires special methodology to prevent contamination, ensure integrity of clinical research and comply with regulatory requirements. A lack of practical laboratory experience, regulatory uncertainty and a relative paucity of objective published data can make selection of the optimum separation method for specific indications a difficult task for the clinician and can discourage clinical adoption. In this paper, we discuss the processes which can be used to separate SVF cells from fat tissue. We compare the various mechanical and enzymatic methods. We discuss the practical considerations involved in selecting an appropriate method from a clinical perspective. Studies consistently show that breakdown of the extracellular matrix achieved with proteolytic enzymes affords significantly greater efficiency to the separation process. SVF isolated through mechanical methods is equally safe, less costly and less time consuming but the product contains a higher frequency of blood mononuclear cells and fewer progenitor cells. Mechanical methods can provide a low cost, rapid and simple alternative to enzymatic isolation methods, and are attractive when smaller quantities of ASCs are sufficient.

Clinical Safety of Stromal Vascular Fraction Separation at the Point of Care.

INTRODUCTION: Pluripotential cells in adipose tissue may be important in long-term volume retention and regenerative effects of fat grafting. Unfortunately, graft harvest with lipoaspiration significantly depletes the population of stromal vascular cells, which includes adipose stem cells. Stromal vascular fraction (SVF) cells may be isolated from excess lipoaspirate at the point of care and used to replenish fat grafts, a technique termed cell-assisted lipotransfer (CAL). Preclinical and clinical evidence supports the rationale of CAL but clinical adoption of the strategy requires evidence of clinical safety. This prospective, level 1 study reports clinical safety of SVF-enhanced fat grafting using a manual, collagenase-based separation process to isolate autogenous progenitor cells from lipoaspirate at the point of care. METHODS: One hundred sixty-four subjects underwent 174 SVF-enhanced autologous fat grafting procedures at the university stem cell center between August 2009 and November 2014 for a variety of cosmetic and reconstructive indications. RESULTS: Cell-assisted lipotransfer was performed for a variety of cosmetic and reconstructive indications. The mean time of the SVF isolation process was 91 minutes. Because of the frequent concomitant procedures, the average operating room time increased by only 11 minutes. Mean follow-up was 19.9 months. There were no major complications and 6 minor complications. No collagenase or neutral protease related complications were observed. CONCLUSIONS: This series of 174 CAL cases demonstrates that SVF cell isolation using a standardized, manual, collagenase-based process at the POC is equivalent in safety compared to nonenhanced fat grafting. These results support expanded use of CAL in the clinical research setting.