High-yield Isolation Research Articles

Trapping of a Highly Reactive Oxoiron(IV) Complex in the Catalytic Epoxidation of Olefins by Hydrogen Peroxide.

The generation of a nonheme oxoiron(IV) intermediate, [(cyclam)FeIV (O)(CH3 CN)]2+ (2; cyclam=1,4,8,11-tetraazacyclotetradecane), is reported in the reactions of [(cyclam)FeII ]2+ with aqueous hydrogen peroxide (H2 O2 ) or a soluble iodosylbenzene (sPhIO) as a rare example of an oxoiron(IV) species that shows a preference for epoxidation over allylic oxidation in the oxidation of cyclohexene. Complex 2 is kinetically and catalytically competent to perform the epoxidation of olefins with high stereo- and regioselectivity. More importantly, 2 is likely to be the reactive intermediate involved in the catalytic epoxidation of olefins by [(cyclam)FeII ]2+ and H2 O2 . In spite of the predominance of the oxoiron(IV) cores in biology, the present study is a rare example of high-yield isolation and spectroscopic characterization of a catalytically relevant oxoiron(IV) intermediate in chemical oxidation reactions.

Langendorff-Free Isolation and Propagation of Adult Mouse Cardiomyocytes.

Isolation of healthy, intact cardiomyocytes from the adult mouse heart for cardiac research is challenging. Traditional protocols depend upon specialized Langendorff apparatus, which requires extensive training and presents significant technical and logistical barriers. Described here is a much simplified technique, introducing optimized dissociation buffers to the heart by direct needle injection into the left ventricle, allowing deep myocardial perfusion and the isolation of high yields of viable, rod-shaped cardiomyocytes, using only standard surgical and laboratory equipment. This method also permits the concurrent isolation of cardiac non-myocyte cellular populations.

High-yield isolation of menstrual blood-derived endometrial stem cells by direct red blood cell lysis treatment.

ABSTRACTRecently, menstrual blood-derived endometrial stem cells (MenSCs) have become attractive for stem cell based therapy due to their abundance, easy and non-invasive extraction and isolation process, high proliferative capacity, and multi-lineage differentiation potential. MenSC-based therapies for various diseases are being extensively researched. However, the high death rate and poor engraftment in sites of damaged tissues reduce the therapeutic value of these stem cells for transplantation. In theory, periodic stem cell transplantation is an alternative strategy to overcome the challenge of the loss of beneficial stem cell-derived effects due to the rapid disappearance of the stem cells in vivo. However, periodic stem cell transplantation requires sufficient amounts of the desired stem cells with a low number of subculture passages. Our previous results have demonstrated that primary MenSCs mainly reside in the deciduous endometrium, and considerable amounts of deciduous endometrium intertwined with menstrual blood clots were discarded after conventional density gradient centrifugation (DGC). Therefore, the aim of this study was to determine whether primary MenSCs exist in the sedimentation of the deciduous endometrium after DGC and further to evaluate the isolation of MenSCs by direct red blood cell lysis treatment. As expected, our results confirmed that substantial amounts of primary MenSCs still remain in the sedimentation after DGC and indicated that MenSC isolation by directly lysing the red blood cells not only guaranteed substantial amounts of superior MenSCs with a low number of subculture passages, but also was time efficient and economical, providing a solid support for extensive clinical application.

High energy oxidation and organosolv solubilization for high yield isolation of cellulose nanocrystals (CNC) from Eucalyptus hardwood

Cellulose nanocrystals (CNC) have been widely used as responsive materials, chiral templates, and tough nano-composites due to its unparalleled properties. Acid and enzyme hydrolyses are extensively employed to prepare CNC. These traditional approaches exhibit inherent limitations of corrosion hazards, time-consuming process, and/or low yield. Herein, irradiation oxidation and organosolv solubilization are conducted to cause rapid degradation with simultaneous crystallization of cellulose to achieve approx. 87% yield of CNC. The morphology, spectroscopic, and stability properties of the as-prepared CNC are characterized through UV-vis spectroscopy, zetal potential, XRD, TEM, DLS, GPC, FT-IR and TGA techniques. The resultant CNC suspension presents unique property with high stability after 9 months storage at 4 °C. Moreover, CNC liquid crystal phase is successfully generated by addition of anions or cations solution to the CNC aqueous dispersion without stirring. The innovative approach in this work opens an avenue to obtain CNC directly from lignocellulosic biomass through irradiation oxidation and organosolv solubilization without acid hydrolysis and washing procedure.

Efficient and high yield isolation of myoblasts from skeletal muscle

Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10–20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish.

Human endometrial stem cells: High-yield isolation and characterization

BackgroundMenstrual blood is only recently and still poorly studied, but it is an abundant and noninvasive source of highly proliferative mesenchymal stromal cells (MSCs). However, no appropriate isolation method has been reported due to its high viscosity and high content of clots and desquamated epithelium. MethodsWe studied three different isolation approaches and their combinations: ammonium-containing lysing buffer, distilled water and gradient-density centrifugation. We tested the proliferative capacity, morphology, surface markers and pluripotency of the resulting cells. ResultsOur isolation method yields up to four million nucleated cells per milliliter of initial blood, of which about 0.2–0.3% are colony-forming cells expressing standard mesenchymal markers CD90, CD105 and CD73, but not expressing CD45, CD34, CD117, CD133 or HLA-G. The cells have high proliferative potential (doubling in 26 h) and the ability to differentiate into adipocytes and osteocytes. Early endometrial MSCs (eMSCs) express epithelial marker cytokeratin 7 (CK7). CK7 is easily induced in later passages in a prohepatic environment. We show for the first time that a satisfactory and stable yield of eMSCs is observed throughout the whole menstrual period (5 consecutive days) of a healthy woman. DiscussionThe new cost/yield adequate method allows isolation from menstrual blood a relatively homogenous pool of highly proliferative MSCs, which seem to be the best candidates for internal organ therapy due to their proepithelial background (early expression of CK7 and its easy induction in later passages) and for mass cryobanking due to their high yield and availability.

Synthesis of rhodanine-derived enaminones in aqueous medium

ABSTRACTThe synthesis of rhodanine-derived enaminones has been reported by the reaction of ketene S,S-acetal rhodanine and amine in water under reflux condition. The present methodology includes mild reaction conditions, shorter reaction times, high yield and facile isolation of the desired products by simple filtration.

An optimized method for mouse liver sinusoidal endothelial cell isolation

The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions.

Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals.

Despite the structural, load-bearing role of cellulose in the plant kingdom, countless efforts have been devoted to degrading this recalcitrant polysaccharide, particularly in the context of biofuels and renewable nanomaterials. Herein, we show how the exposure of plant-based fibers to HCl vapor results in rapid degradation with simultaneous crystallization. Because of the unchanged sample texture and the lack of mass transfer out of the substrate in the gas/solid system, the changes in the crystallinity could be reliably monitored. Furthermore, we describe the preparation of cellulose nanocrystals in high yields and with minimal water consumption. The study serves as a starting point for the solid-state tuning of the supramolecular properties of morphologically heterogeneous biological materials.

A High-yield Isolation and Enrichment Strategy for Human Lung Microvascular Endothelial Cells

A High-yield Isolation and Enrichment Strategy for Human Lung Microvascular Endothelial Cells

A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro.

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.

Pulmonary venous blood sampling significantly increases the yield of circulating tumor cells in early-stage lung cancer

ObjectiveTo identify circulating tumor cells (CTCs) in the blood of patients with early-stage lung cancer and to show that sampling pulmonary vein (PV) blood using microfluidic chip technology will yield significantly more CTCs. Improving early detection of lung cancer is critical to improving lung cancer survival. Reproducible detection of CTCs is limited currently in early stage tumors. MethodsPatients undergoing pulmonary resection had PV blood drawn before resection. Peripheral blood was sampled at preoperative, intraoperative, and postoperative times. Samples were analyzed on microfluidic chips using antibody-based capture. ResultsA total of 32 patients with primary lung cancer were evaluated. Twenty patients had 1 or more CTCs detected in at least 1 sample (62.5%). The mean number of CTCs from peripheral vein sources at the preoperative, intraoperative, and postoperative time points was 1.3, 1.9, and 0.6 respectively. The average number of CTCs in the PV was 340.0 (range, 0.0-5422.50; P > .01). When PV CTCs were present, the number of CTCs was correlated with pathological tumor size (P = .0236). The number of PV CTCs was not correlated with any other clinical feature (eg, smoking status, preoperative or postoperative stage). Furthermore, the number of PV CTCs was significantly higher when preoperative bronchoscopic biopsy was performed, compared with computed tomography–guided biopsy (P = .0311). Seven patients had evidence of CTC clusters, or microemboli. ConclusionsWith a single vein draining the entire tumor basin, lung cancers are unique, allowing the high-yield isolation of CTCs from the PV. This method may facilitate future studies to improve the detection and analysis of early-stage lung CTCs.

Qualitative and quantitative differences of adipose-derived stromal cells from superficial and deep subcutaneous lipoaspirates: a matter of fat

Background aimsSubcutaneous fat represents a valuable reservoir of adipose-derived stem cells (ASCs) in the stromal vascular fraction (SVF), widely exploited in regenerative medicine applications, being easily harvested through lipoaspiration. The lack of standardized procedures for autologous fat grafting guided research efforts aimed at identifying possible differences related to the harvesting site, which may affect cell isolation yield, cell growth properties and clinical outcomes. Subcutaneous fat features a complex architecture: the superficial fascia separates superficial adipose tissue (SAT) from deep layer tissue (DAT). We aimed to unravel the differences between SAT and DAT, considering morphological structure, SVF composition, and ASC properties. MethodsSAT and DAT were collected from female donors and comparatively analyzed to evaluate cellular yield and viability, morphology, immunophenotype and molecular profile. ASCs were isolated in primary culture and used for in vitro differentiation assays. SAT and DAT from cadaver donors were also analyzed through histology and immunohistochemistry to assess morphology and cell localization within the hypoderm. ResultsLiposuctioned SAT contained a higher stromal tissue compound, along with a higher proportion of CD105-positive cells, compared with DAT from the same harvesting site. Also, cells isolated from SAT displayed increased multipotency and stemness features. All differences were mainly evidenced in specimens harvested from the abdominal region. According to our results, SAT features overall increased stem properties. ConclusionsGiven that subcutaneous adipose tissue is currently exploited as the gold standard source for high-yield isolation of adult stem cells, these results may provide precious hints toward the definition of standardized protocols for microharvesting.

Identification and isolation of new neolignan and sesquineolignan species: Their acid-catalyzed ring closure and specific accumulation in the fruit wall of Cirsium eriophorum (L.) Scop.

In Cirsium eriophorum fruit, the main neolignan and sesquineolignan compounds, denominated prebalanophonin (preBA) and prepicrasmalignan (prePI), were determined for the first time in the plant kingdom using a combination of optimized acid treatments and complementary spectroscopic (HPLC–MS, GC–MS, CD and NMR) methods. Analysis of fruit parts separated via germination, demonstrated the specific accumulation of these compounds, since preBA and prePI were exclusively found in the fruit wall. Based on quantitative approaches obtained by HPLC-UV measurements, the fruit wall was found to contain extraordinarily high amounts of preBA (4.57%) and prePI (2.88%) allowing their high-yield isolation by preparative HPLC. Optimized acidic treatment (2N trifluoroacetic acid, 50°C, 15min) of the wall extract resulted in the quantitative transformation of preBA and prePI into balanophonin (BA) and picrasmalignan (PI), as a result of acid-catalyzed cyclization by the SN2 reaction. Consequently, acid-treated wall extract was found to be the richest raw material containing BA and PI (5.3% and 3.10%), reported to date.

Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. From the Clinical EditorRecent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers.

Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure takes ∼4 h to complete.

Two-coordinate iron(I) complex [Fe{N(SiMe3)2}2](-) : synthesis, properties, and redox activity.

First-row two-coordinate complexes are attracting much interest. Herein, we report the high-yield isolation of the linear two-coordinate iron(I) complex salt [K(L)][Fe{N(SiMe3 )2 }2 ] (L=18-crown-6 or crypt-222) through the reduction of either [Fe{N(SiMe3 )2 }2 ] or its three-coordinate phosphine adduct [Fe{N(SiMe3 )2 }2 (PCy3 )]. Detailed characterization is gained through X-ray diffraction, variable-temperature NMR spectroscopy, and magnetic susceptibility studies. One- and two-electron oxidation through reaction with I2 is further found to afford the corresponding iodo iron(II) and diiodo iron(III) complexes.

Culturing human cells: Optimizing growth conditions for immunotherapy

Growing human cells in culture for research is one thing, but the isolation and culture of high-quality cells that will be re-injected back into the donor is quite another. Manipulation and expansion of cells in a clinical setting carries its own unique requirements and complications. Growth and survival needs to be optimized and quality control is paramount. Often only a single opportunity for successful treatment is possible, so chances of success need to be maximized in all respects, including high-yield isolation of good quality cells from patients, cell culture conditions, cell characterization, and reperfusion back into patients. During this webinar, we will broadly examine the process of immunotherapy as well as examine in more detail some of the most critical steps along the pathway to generating a therapeutic dose of modified cells.

Quali-quantitative differences of adipose tissuederived stromal cells from superficial and deep subcutaneous lipoaspirates: a matter of fat

Subcutaneous fat (or hypoderm) represents a valuable reservoir of adiposederived stem cells (ASCs), residing in the stromal vascular fraction (SVF), widely exploited in regenerative medicine applications, being easily harvested through lipoaspiration. The lack for standardized procedures for autologous fat grafting, guided research efforts aimed at identifying possible differences related to the harvesting site, that may affect cell isolation yield, cell growth properties and clinical outcomes. The hypoderm features a complex architecture: the superficial fascia separates the superficial adipose tissue (SAT) from the deep layer (DAT). Aim of this study was to unravel the differences between SAT and DAT, considering morphological structure, SVF composition and ASCs’ properties. SAT and DAT specimens were collected from three distinct anatomical regions (abdomen, thigh and knee) of female individuals and comparatively analyzed through histology, flow citometry, and qPCR. ASCs were isolated in primary culture and used for in vitro differentiation assays. Our results indicated that liposucted SAT contains a higher stromal tissue compound, along with a higher proportion of CD105-positive cells, compared to DAT from the same anatomical region. Also, cells isolated from SAT displayed increased multipotency and stemness features. All differences were mainly evidenced in specimens harvested from the abdominal region. According to our results, SAT features overall increased stem properties. Given that subcutaneous adipose tissue is currently exploited as the gold standard source for high yield isolation of somatic stem cells, these results may provide precious hints toward defining a prioritization of tissue harvesting site for regenerative medicine applications.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitromodels of the rat blood-brain barrier and blood-spinal cord barrier

BackgroundModelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies.MethodsBrain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting.ResultsUsing a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter.ConclusionsOur techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.