Reproducible Method For Isolation Research Articles

Observation of the Effects of Different Enzyme Digestion Time and Enzyme Digestion Methods on the Extraction of Primary Renal Tubular Epithelial Cells from Sprague-Dawley Suckling Mice.

Renal tubular epithelial cells are one of the essential functional cells in the kidney. Optimizing the isolation and culture method of primary renal tubular epithelial cells from SD mammary rats provides better experimental materials for renal tubule-related studies, which is essential for studying the pathogenesis of renal diseases, especially diabetic nephropathy and drug screening. SD rat renal tubular epithelial cells were isolated and purified by 2.5-mg/ml collagenase II or 2mg/ml trypsin + 2.5mg/ml collagenase II enzymatic digestion. The isolation and purification were observed at different time points (15min, 30min, 45min, and 60min) to determine the optimal extraction time for the enzymatic digestion method. After comparing the two enzymatic methods, it was determined that the trypsin + collagenase II enzymatic method was more effective. The primary renal tubular epithelial cells extracted by the trypsin + collagenase II digestion method were identified by the marker Cytokeratin 18 of renal tubular epithelial cells at 45min of digestion with high purity. We established a simple, efficient, and reproducible method for isolation and culture of renal tubular epithelial cells in SD mammary gland rats.

A Reproducible Method for Isolation and In Vitro Culture of Functional Human Lymphoid Stromal Cells from Tonsils.

The stromal compartment of secondary lymphoid organs is classicaly known for providing a mechanical scaffold for the complex interactions between hematopoietic cells during immune activation as well as for providing a niche which is favorable for survival of lymphocytes. In recent years, it became increasingly clear that these cells also play an active role during such a response. Currently, knowledge of the interactions between human lymphoid stroma and hematopoietic cells is still lacking and most insight is based on murine systems. Although methods to isolate stromal cells from tonsils have been reported, data on stability in culture, characterization, and functional properties are lacking. Here, we describe a reproducible and easy method for isolation and in vitro culture of functional human lymphoid stromal cells from palatine tonsils. The cells isolated express markers and characteristics of T cell zone fibroblastic reticular cells (FRCs) and react to inflammatory stimuli by upregulating inflammatory cytokines and chemokines as well as adhesion molecules, as previously described for mouse lymphoid stroma. Also, cultured tonsil stromal cells support survival of human innate lymphoid cells, showing that these stromal cells can function as bone fide FRCs, providing a favorable microenvironment for hematopoietic cells.

Recommendations for Short Term Culturing of Viable Rod Shaped Rat Cardiomyocytes

In vitro primary cultures of isolated adult rat cardiomyocytes are becoming an increasingly popular model to study heart muscle stressors. This model can easily be manipulated in a controlled environment and results obtained can provide valuable insights into the pathophysiology of heart disease. Over the past years several improved methodologies have been described in the development of a robust technique to help maintain cardiomyocytes in culture. However, despite these advances, culturing of primary cardiomyocytes remains a challenge. In this study, we present a simple yet reproducible method for isolation and culture of viable rod shaped cardiomyocytes. Cardiomyocytes were maintained in supplemented Media 199 with or without foetal bovine serum. Their viability was assessed using trypan blue while the metabolic activity was measured using an Adenosine 5’-triphosphate assay. Results obtained in this study were used to provide general guidelines to evade pitfalls related to low cardiomyocyte yields and subsequent poor culturability of cardiomyocytes. Isolated cardiomyocytes cultured in the presence of foetal bovine serum maintained their in vitro striated rod shaped morphology for 72 hours in culture, after which, they flattened and spread out. Whereas, cardiomyocytes cultured in the absence of foetal bovine serum remained rod shaped for up to 120 hours.

Comparative Analysis of Highly Defined Proteases for the Isolation of Adipose Tissue-Derived Stem Cells

Before the potential of adipose tissue-derived stem cells can fully be exploited for a broad scope of tissue-engineering and cell-based therapeutical applications, an effective and reproducible method for isolation is needed. To comparatively analyze five highly defined protease formulations, Blendzyme 1-4, liberase H1 and a crude collagenase mixture in the course of digestion that consisted of three 1-h intervals. The resulting digests of human adipose tissue aspirates were evaluated for the yield of nucleated cells, viability and frequency of specific lineages, in particular CD90, CD34 and CD45, by flow cytometry. The functionality of the cells was assessed as to the colony-forming capacity in limiting dilution assays. Based on all evaluation criteria, Blendzymes 1 and 2 and liberase H1 demonstrated a superior performance and highest consistency. Blendzyme 3 clearly underperformed compared with all other enzymes, and the performance of the rest of enzymes appeared erratic. As for the length of digestion, a 2-h interval appeared optimal when weighing both the yield and functionality of the cells in the stromal vascular fractions obtained from different adipose tissue samples. Our results demonstrate that the highly purified proteases provide a valuable alternative to crude collagenase preparations, especially in scenarios where a high definition and reproducibility of the digestion process is of importance.

Isolation and Purification of RNA from Tissues Rich in Polyphenols, Polysaccharides, and Pigments of Annatto (Bixa orellana L.)

The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.

Interactions of butyric acid- and acetic acid-treated Salmonella with chicken primary cecal epithelial cells in vitro.

In vitro studies of the interaction between pathogenic bacteria and the chicken intestinal epithelium are hampered by the lack of a host- and tissue-specific in vitro model. Therefore, a reproducible method for isolation and cultivation of chicken primary cecal epithelial cells was developed. Cecal crypts were isolated and cultured in vitro to form a semiconfluent layer of epithelial cells. Incubation of Salmonella enteritidis with these cells resulted in invasion. Pretreatment of the Salmonella bacteria with butyric acid resulted in a significant decrease of invasion of the bacteria in the chicken cecal epithelial cells, whereas pretreatment with acetic acid increased invasiveness. These interactions of S. enteritidis with primary chicken cecal epithelial cells were similar to the interactions with other epithelial cell types.

Determination of menthol and menthone in food and pharmaceutical products by solid-phase microextraction–gas chromatography

In the current contribution the optimization of menthol and menthone isolated from food and pharmaceutical samples by solid-phase microextraction (SPME) will be presented. Extraction efficiencies for commercial (polydimethylsiloxane) and laboratory-made (ethoxypolydimethylsiloxane) coated quartz fibers were compared. The results show, that SPME coupled with GC–flame ionisation detection is a reproducible method for isolation and for qualitative and quantitative determination of menthol and menthone at ppm as well as ppb levels. The described procedure can be recommended for routine food and pharmaceutical analyses.

The biology of the glomerulus: Endothelial cells

Endothelial cells are active participants in processes controlling coagulation, inflammation and immune processes and an aberration in the controlling mechanisms may contribute to the development of disease within the glomerulus. The major goal of the next few years will be to develop reliable and reproducible methods for isolation and culture of glomerular endothelial cells so that their properties, and interactions with other glomerular cells, may be studied in vitro.