Pilot Study for Isolation of Stromal Vascular Fraction with Collagenase Using an Automated Processing System.

There are many potential therapeutic applications for autologous adipose-derived stromal cells. These cells are found in a heterogeneous population isolated from adipose tissue called the stromal vascular fraction (SVF). Closed automated systems are available to release cells from the adherent stroma. Here, we test one system to evaluate the heterogeneous output for yield, purity, cellular characterization, and stemness criteria. The SVF was isolated from three donors using the Automated Cell Station (ACS) from BSL Co., Ltd., Busan, Republic of Korea. The SVF cellular output was characterized for cell yield and viability, immunophenotyping analysis, pluripotent differentiation potential, adhesion to plastic, and colony-forming units. Additionally, the SVF was tested for endotoxin and collagenase residuals. The SVF yield from the ACS system was an average volume of 7.9 ± 0.5 mL containing an average of 19 × 106 nucleated cells with 85 ± 12% viability. Flow cytometry identified a variety of cells, including ASCs (23%), macrophages (24%), endothelial cells (5%), pericytes (4%), and transitional cells (0.5%). The final concentrated product contained cells capable of differentiating into adipogenic, chondrogenic, and osteogenic phenotypes. Furthermore, tests for SVF sterility and purity showed no evidence of endotoxin or collagenase residuals. The ACS system can efficiently process cells from adipose tissue within the timeframe of a single surgical procedure. The cellular characterization indicated that this system can yield a sterile and concentrated SVF output, providing a valuable source of ASCs within the heterogeneous cell population.

A Multivariate Meta-Analysis for Optimizing Cell Counts When Using the Mechanical Processing of Lipoaspirate for Regenerative Applications.

Lipoaspirate has become the preferred source for regenerative cells. The mechanical processing of lipoaspirate has advantages over enzymatic processing but has a lower yield of regenerative cells. A review of the literature shows different techniques of extraction, but the ideal method or combination has not been determined. METHODS: A comprehensive literature search was focused on the mechanical processing of lipoaspirate, without the use of enzymes. Data from the articles were integrated by utilizing a multivariate meta-analysis approach and used to create a statistical-based predictive model for a combination of multiple variables. RESULTS: Starting with 10,000 titles, 159 articles were reviewed, and 6 met the criteria for inclusion and exclusion. The six studies included data on 117 patients. Sixteen factors were analyzed and six were identified as significant. The predictive profilers indicated that the optimal combination to maximize the cell yield was: a centrifuge force of 2000× g, a centrifuge time of 10 min, a cannula diameter of 2 mm, and an intra-syringe number of passes of 30. The optimal patient factors were a higher BMI and younger age. CONCLUSIONS: The novelty of the method used here was in combining data across different studies to understand the effect of the individual factors and in the optimization of their combination for mechanical lipoaspirate processing.

Controlling the release rate of topotecan from PLGA spheres and increasing its cytotoxicity towards glioblastoma cells by co-loading with calcium chloride.

It has been suggested that local administration of topotecan (TT) could increase its efficacy in the treatment of glioblastoma. In this context, a PLGA implant model in the form of spheres with a porous core and stiff surface, loaded with TT and CaCl2 was developed. An array of formulations differing from each other by the type of PLGA used, the integrity of the surface, the concentrations of TT and CaCl2 added during the preparation, and the volume of water in the PLGA mix, was prepared, screened and explored by computerized multifactorial analysis. This analysis enabled the simultaneous identification of the most influential experimental factors on the experimental responses, which were pre-determined as the efficiency of TT loading and the TT % cumulative release at 14 days. The multifactorial analysis also revealed how the interactions among the experimental factors affect the performance of the various formulations. Thus, TT concentration and its factorial interaction with the concentration of CaCl2 added during the spheres' preparation were identified as most prominent on the loading efficiency, while the surface integrity (intact or punctured) and CaCl2 amount in the spheres were identified as most prominent on the TT % cumulative release from the spheres. TT was found to be cytotoxic towards glioblastoma U87 MG cells, an activity which was enhanced, synergistically, in the presence of CaCl2 (the relative viability was reduced from 36 to 28% with combination indices of 1.0, 0.37, 0.13 and 0.06 for EC50, EC75, EC90 and EC95, respectively). Interestingly, dividing the TT dose into 3 equal portions, replenished daily to the incubation medium, increased TT cytotoxicity. The relative viability was then reduced from 35 to 7% and in the presence of CaCl2 - from 28 to 1.9%, suggesting that a local, slow input of TT could be effective in the treatment of glioblastoma by an adjacent TT implant. The increased effect of CaCl2 on cytotoxicity was also observed when it was co-loaded into the TT spheres. In that case, the cells' viability was reduced from 72 to 27%. It is suggested that the PLGA spheres could be used for tunable local delivery of TT in post-resection adjuvant therapy of glioblastoma.