Cost-effective Method For Isolation Research Articles

Isolation of fresh endothelial cells from porcine heart for cardiovascular studies: a new fast protocol suitable for genomic, transcriptomic and cell biology studies

BackgroundEndothelial cells (ECs) play a key role in tissue homeostasis, in several pathological conditions, and specifically in the control of vascular functions. ECs are frequently used as in vitro model systems for cardiovascular studies and vascular biology. The porcine model is commonly used in human clinical cardiovascular studies. Currently, however, there is no robust protocol for the isolation of porcine heart ECs. We have developed a fast isolation protocol, which is cost effective, takes only 1–2 h, and produces EC purity of over 97%. This protocol is optimized for porcine hearts but can be adapted for use with other large animals.MethodsHeart is washed by flushing with PBS, whereafter endothelial cells are detached by collagenase incubation and the cells can then be collected immediately after the incubation and plated within an hour after the heart is isolated from a pig.ResultsThe swiftness of the protocol limits changes in the phenotype and RNA expression profile of the cells. Cells were identified as ECs with CD31 (PECAM-1) antibody immunostaining. Functionality of ECs were ensured with in vitro angiogenesis assay. The purity of the ECs was verified by using fluorescence assisted cell sorting (FACS) with the CD31 antibody.ConclusionWe developed a new, fast, and cost-effective isolation method for pig heart ECs. Successful isolation of pure ECs is a prerequisite for several cardiovascular and vascular biology studies.

Intelligent image-based in situ single-cell isolation

Quantifying heterogeneities within cell populations is important for many fields including cancer research and neurobiology; however, techniques to isolate individual cells are limited. Here, we describe a high-throughput, non-disruptive, and cost-effective isolation method that is capable of capturing individually targeted cells using widely available techniques. Using high-resolution microscopy, laser microcapture microscopy, image analysis, and machine learning, our technology enables scalable molecular genetic analysis of single cells, targetable by morphology or location within the sample.

A simple and cost-effective method for isolation and expansion of human fetal pancreas derived mesenchymal stem cells.

Previous studies have suggested mesenchymal stem cells (MSCs) as a suitable source for cell replacement therapy in diabetes. MSCs have successfully isolated from different adult and fetal tissues, including the pancreas. In vitro studies have shown that human fetal pancreatic stem cells could be extensively expanded and differentiated into islet-like structures. Here, we introduce a simple and cost-effective method for the generation of MSCs from the human fetal pancreas (FPMSCs). To isolate FPMSCs, pancreata from four aborted fetuses (second trimester) were processed with short collagenase digestion. The resulting tissue fragments were transferred to a basic media (DMEM+15%FBS) without adding any growth factor. After 10 to14 days, fibroblast-like cells were harvested and passaged six times for further evaluations. Flow cytometry analysis and three-lineage differentiation capacity have demonstrated that these cells have MSC-like properties. We also continuously passaged samples of FPMSCs and found no evidence for chromosomal instability and morphological changes until 10th subculture. Moreover, our cell culture protocol can be easily modified and translated into a GMP-compliant one. The results of current study demonstrated that our simple and inexpensive method could yield a pure population of FPMSCs that might be suitable for transplantation.

Development and extraction optimization of baicalein and pinostrobin from Scutellaria violacea through response surface methodology.

Objective:To develop a process that involves optimization of the amount of baicalein and pinostrobin from the hydro-methanolic extract of the leaves of Scutellaria violacea by response surface methodology (RSM).Materials and Methods:The combinatorial influence of various extraction parameters on the extraction yield was investigated by adopting Box–Behnken experimental design. Preliminary experiments carried out based on the traditional one variable at a time optimization revealed four such operational parameters to play a crucial role by influencing the yield. These four process parameters at three levels were considered to obtain the Box–Behnken experimental design.Results:RSM based model fitted to the resulting experimental data suggested that 52.3% methanol/water, 12.46:1 solvent-solid ratio, 285 rpm agitation and 6.07 h of extraction time are the optimal conditions which yielded a maximized amount of baicalein and pinostrobin of 2.9 and 4.05 mg/g DM. Analysis of variance revealed a high correlation coefficient (R2 = 0.999 for baicalein and 0.994 for pinostrobin), signifying a good fit between the regression model (second order) and the experimental observations.Conclusion:The present study signifies that both the metabolites have been extracted from S. violacea for the first time. Further, this study developed an optimized extraction procedure to obtain maximum yield of the metabolites, which is unique and better than conventional extraction methodology. The operational parameters under optimized conditions accounts for the lowest cost in extraction process thus, providing an efficient, rapid and cost-effective method for isolation and scale up of these commercially vital flavonoids.

Establishment of a simple and effective isolation method for cyanophycin from recombinant Saccharomyces cerevisiae

An efficient, time-saving, and cost-effective method for isolation of the polyamide cyanophycin from recombinant Saccharomyces cerevisiae was established. Due to its simple procedure, this isolation method may be also applicable at industrial scale and also to other intracellular compounds in this yeast. Production of cyanophycin gained preferential interest in the past, as degradation products thereof are of pharmaceutical and technical interest. Recently, it was shown that Saccharomyces cerevisiae represents a putative candidate for cyanophycin synthesis at industrial scale. For identification of optimal isolation procedures, several parameters such as heat stress, freeze drying, and freeze/thaw cycles of transgenic yeast cells were compared for their effectiveness of cyanophycin isolation. Additionally, optimal resuspension solutions for the applied cells and minimal required materials or chemicals were determined to make the process most environmentally and economically friendly. Maximal cyanophycin granule polypeptide yields of 21% (w/w) were obtained after incubation of dry cells at 70 degrees C or 80 degrees C and precipitation of the polymer with two volumes of ethanol.

A simple, cost effective method for isolation and transportation of

Isolation and propagation of Leishmania parasites using conventional culture methods are difficult, especially under field conditions. Transportation of live parasites requires the maintenance of low temperatures, which increases the cost considerably. The present study demonstrates that monophasic micro capillary culture is a simpler, more economical and better alternative to using conventional Evan's modified Tobie's medium to isolate and transport the Leishmania parasite over long distances without the use of temperature control methods.

Identification and quantification of gossypol in cotton by using packed micro-tips columns in combination with HPLC.

Self-packed micro-tip columns containing a C18-bonded silica stationary phase, based on the same principles as solid-phase extraction methods, were used to obtain gossypol and related sesquiterpenoid aldehyde-enriched fractions. The enriched metabolite fractions were then analyzed by optimized high-performance liquid chromatography (HPLC) with a C18 column (4.6 mm x 25 cm) eluted with the binary mobile phase acetonitrile-0.1% aqueous TFA solution (80:20). This method has proven to be highly reproducible. The precision and accuracy, as %RSD and %RME values, were determined to be less than 15% for the method. The minimum detection limit of gossypol was determined to be 10 ng (absolute gossypol). Absolute recovery was greater than 94% with a standard deviation of +/-3.68%. This is a simple, fast, and cost-effective method for isolation, identification, and quantification of gossypol and related secondary metabolites. Comparative analysis of gossypol content was performed on different parts of the cotton plant (seeds, stems and leaves) of two different cultivars of Gossypium hirsutum L. (Acala(1517-70) and OR19). The results indicate that the OR19 cv naturally contains higher gossypol levels than the Acala cv. It was also found that treatment of leaves with a Verticillium dahliae-derived elicitor induced production of deoxyhemigossypol rather than gossypol.