BackgroundStromal vascular fraction (SVF) can easily be obtained from a mini-lipoaspirate procedure of fat tissue and platelet rich plasma (PRP) can be obtained from peripheral blood. The SVF contains a mixture of cells including ADSCs and growth factors and has been depleted of the adipocyte (fat cell) population. We evaluated the safety and efficacy of administering SVF and PRP intra-discally into patients with degenerative disc disease.MethodsA total of 15 patients underwent a local tumescent liposuction procedure to remove approximately 60 ml of fat tissue. The fat was separated to isolate the SVF and the cells were delivered into the disc nucleus of patients with degenerative disc disease. The subjects were then monitored for adverse events, range of motion, visual analog scale (VAS), present pain intensity (PPI), Oswestry Disability Index (ODI), Beck Depression Inventory (BDI), Dallas Pain Questionnaire and Short Form (SF)-12 scores over a period of 6 months. Safety events were followed for 12 months.ResultsNo severe adverse events (SAEs) were reported during a 12 month follow up period with no incidences of infection. Patients demonstrated statistically significant improvements in several parameters including flexion, pain ratings, VAS, PPI, and short form questionnaires. In addition, both ODI and BDI data was trending positive and a majority of patients reported improvements in their Dallas Pain Questionnaire scores.ConclusionsOverall, patients were pleased with the treatment results. More importantly, the procedure demonstrated a strong safety profile with no severe adverse events or complications linked to the therapy. Trial registration NCT02097862. Name of registry: www.clinicaltrials.gov. https://clinicaltrials.gov/ct2/show/NCT02097862?term=bioheart&rank=6. Date of registration: March 25, 2014; Date of enrollment: March 2014

BackgroundStromal vascular fraction (SVF) can easily be obtained from a mini-lipoaspirate procedure of fat tissue and platelet rich plasma (PRP) can be obtained from peripheral blood. We evaluated the safety and preliminary efficacy of administering SVF and PRP intra-articularly into patients with osteoarthritis grade 1 and 2.MethodsA total of ten patients underwent a local tumescent liposuction procedure to remove approximately 100 ml of fat tissue from the abdomen. SVF was isolated using an enzyme digestion and resuspended in PRP for intra-articular injection in the knee. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score and six-minute walk distance (6MWD) were used to evaluate clinical effects and included measure of patient’s subjective assessment of pain, joint mobility, and physical disability. WOMAC score, 6MWD and laboratory tests were repeated at 3 and 6 months and 1, 1.5 and 2 years. XRAY and MRI were completed at 1 year.ResultsThe average total WOMAC score was 64 at baseline and significantly reduced to 52 at 3 months, 46 at 6 months, 42 at 1 year, 38 at 1.5 years, and 41 at 2 years. Patients walked an average of 1310 feet at baseline and demonstrated a statistically significant improvement at 3 and 6 months and 1, 1.5, and 2 years post treatment. Cartilage thickness as determined by MRI improved by at least 0.2 mm in six patients, was unchanged in two patients and decreased by at least 0.2 mm in two patients.ConclusionsOverall, all of the patients were pleased with the treatment results. They reported a reduction in pain levels, especially after 3 months. More importantly, the procedure demonstrated a strong safety profile with no severe adverse events or complications reported. Trial registration NCT03089762; Name of registry: http://www.clinicaltrials.gov

We have developed a quadrupole magnetic flow sorter (QMS) to facilitate high-throughput binary cell separation. Optimized QMS operation requires the adjustment of three flow parameters based on the immunomagnetic characteristics of the target cell sample. To overcome the inefficiency of semiempirical operation/optimization of QMS flow parameters, a theoretical model of the QMS sorting process was developed. Application of this model requires measurement of the magnetophoretic mobility distribution of the cell sample by the cell tracking velocimetry (CTV) technique developed in our laboratory. In this work, the theoretical model was experimentally tested using breast carcinoma cells (HCC1954) overexpressing the HER-2/neu gene, and peripheral blood leukocytes (PBLs). The magnetophoretic mobility distribution of immunomagnetically labeled HCC1954 cells was measured using the CTV technique, and then theoretical predictions of sorting recoveries were calculated. Mean magnetophoretic mobilities of (1-3) x 10(-4) mm(3)/(T A s) were obtained depending on the labeling conditions. Labeled HCC1954 cells were mixed with unlabeled PBLs to form a "spiked" sample to be separated by the QMS. Fractional recoveries of cells for different flow parameters were examined and compared with theoretical predictions. Experimental results showed that the theoretical model accurately predicted fractional recoveries of HCC1954 cells. High-throughput (3.29 x 10(5) cells/s) separations with high recovery (0.89) of HCC1954 cells were achieved.

BackgroundStromal vascular fraction (SVF) can easily be obtained from a mini-lipoaspirate procedure of fat tissue. The SVF contains a mixture of cells including ADSCs and growth factors and has been depleted of the adipocyte (fat cell) population. We evaluated the safety and efficacy of administering SVF intra-myocardially into patients with chronic ischemic cardiomyopathy.MethodsA total of 28 patients underwent a local tumescent liposuction procedure to remove approximately 60 ml of fat tissue. The fat was separated to isolate the SVF and the cells were delivered into the akinetic myocardial scar region using a transendocardial delivery system (MyoCath®) in patients who had experienced a previous myocardial infarct. The subjects were then monitored for adverse events, ejection fraction via echocardiogram and six-minute walk test (6MWT) over a period of 6 months.ResultsThe average EF was 29 % at baseline and significantly increased to 35 % at both 3 and 6 months. Patients walked an average of 349 m at baseline and demonstrated a statistically significant improvement at 3 and 6 months’ post treatment of more than 80 m.ConclusionsOverall, patients were pleased with the treatment results. More importantly, the procedure demonstrated a strong safety profile with no severe adverse events or complications linked to the therapy.Trial registration NCT01502514Name of registry: http://www.clinicaltrials.govURL: https://www.clinicaltrials.gov/ct2/show/NCT01502514?term=adipose+cells+heart&rank=4Date of registration: December 27, 2011Date of enrollment: January 2012

Magnetic cell separation methods commonly utilize paramagnetic materials conjugated to antibodies that target specific cell surface molecules. The amount of magnetic material bound to a cell is directly proportional to the magnetophoretic mobility of that cell. A mathematical model has been developed which characterizes the fundamental parameters controlling the amount of magnetic material bound, and thus, the magnetophoretic mobility of an immunomagnetically labeled cell. In characterization of the paramagnetic labeling, one of the parameters of interest is the increase in magnetophoretic mobility due to the secondary antibody binding to multiple epitopes on the primary antibody, referred to as the "secondary antibody binding amplification," Psi. Secondary antibody-binding amplification has been investigated and quantitated by comparing the mobilities of lymphocytes directly labeled with anti-CD4 MACS (Miltenyi Biotec, Auburn, CA) magnetic nanoparticle antibody with the mobilities of lymphocytes from the same sample labeled with two different indirect antibody-labeling schemes. Each indirect labeling scheme incorporated a primary mouse anti-CD4 FITC antibody that provides both FITC and mouse-specific binding sites for two different secondary antibody-magnetic nanoparticle conjugates: either anti-FITC MACS magnetic nanoparticle antibody or anti-mouse MACS magnetic nanoparticle antibody. The magnetophoretic mobilities of the immunomagnetically labeled cells were obtained using Cell Tracking Velocimetry (CTV). The results indicate that an average of 3.4 anti-FITC MACS magnetic nanoparticle antibodies bind to each primary CD4 FITC antibody, Psi(1,2f) = 3.4 +/- 0.33, and that approximately one, Psi(1,2m) = 0.98 +/- 0.081, anti-mouse MACS magnetic nanoparticle antibody binds to each primary mouse CD4 FITC antibody on a CD4 positive lymphocyte. These results have provided a better understanding of the antibody-binding mechanisms used in paramagnetic cell labeling for magnetic cell separation.

Background:The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. Methods: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. Results: From the results of these studies, two different labeling protocols were used to separate CD56ϩ cells from human, peripheral blood by a Miltenyi Biotech Mini-MACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. Conclusions: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process. Cytometry 45: 285-293, 2001.

BackgroundStromal vascular fraction (SVF) is a mixture of adipose-derived stem cells/mesenchymal stem cells, endothelial/progenitors, pericytes, fibroblasts, and other cells obtained from fat tissue. A small sample of fat or adipose tissue can be obtained under local anesthesia using a cannula. After an enzymatic digestion and centrifugation, the adipocytes (fat cells) are removed to obtain an SVF. Here, we describe the rationale and, to our knowledge, the first clinical implementation of SVF intravenously in a patient with severe psoriasis.MethodsAdipose tissue (60 mL) was collected under local anesthesia via a mini-lipoaspirate procedure. The SVF was separated from the adipocytes via centrifugation after an enzymatic digestion. The cells were resuspended in normal saline and injected via bolus push intravenous. The subject was monitored over a period of 12 months for safety (adverse events), medication changes, and quality of life parameters.ResultsThe patient did not report any safety concerns and did not experience any severe adverse events. The patient demonstrated a significant decrease in symptoms with a noticeable difference in skin quality appearance. Psoriasis area and severity index score went from 50.4 at baseline to 0.3 at 1 month follow-up.ConclusionOverall, the patient reported improved quality of life and willingness to continue treatments. This successful initial case study demonstrates that this may be a feasible treatment plan for patients suffering from psoriasis.

We investigated the effectiveness of integrating tissue engineered cartilage derived from human bone marrow derived stem cells (HBMSCs) to healthy as well as osteoarthritic cartilage mimics using hydroxyapatite (HA) nanoparticles immersed within a hydrogel substrate. Healthy and diseased engineered cartilage from human chondrocytes (cultured in agar gels) were integrated with human bone marrow stem cell (HBMSC)-derived cartilaginous engineered matrix with and without HA, and evaluated after 28 days of growth. HBMSCs were seeded within photopolymerizable poly (ethylene glycol) diacrylate (PEGDA) hydrogels. In addition, we also conducted a preliminary in vivo evaluation of cartilage repair in rabbit knee chondral defects treated with subchondral bone microfracture and cell-free PEGDA with and without HA. Under in vitro conditions, the interfacial shear strength between tissue engineered cartilage derived from HBMSCs and osteoarthritic chondrocytes was significantly higher (p < 0.05) when HA nanoparticles were incorporated within the HBMSC culture system. Histological evidence confirmed a distinct spatial transition zone, rich in calcium phosphate deposits. Assessment of explanted rabbit knees by histology demonstrated that cellularity within the repair tissues that had filled the defects were of significantly higher number (p < 0.05) when HA was used. HA nanoparticles play an important role in treating chondral defects when osteoarthritis is a co-morbidity. We speculate that the calcified layer formation at the interface in the osteoarthritic environment in the presence of HA is likely to have attributed to higher interfacial strength found in vitro. From an in vivo standpoint, the presence of HA promoted cellularity in the tissues that subsequently filled the chondral defects. This higher presence of cells can be considered important in the context of accelerating long-term cartilage remodeling. We conclude that HA nanoparticles play an important role in engineered to native cartilage integration and cellular processes.