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Introduction: Circulating endothelial cells (CEC) have been associated with vascular injury and are described as potential biomarkers for cardiovascular disease (1). Besides, current optimized methodologies enable the isolation of a well-characterized subtype of endothelial progenitor known as Endothelial colony-forming cells (ECFCs) (2,3). Although ECFC isolation methodologies are well described; some discrepancies remain in relation to their isolation efficiency. Aims: To evaluate the isolation efficacy ECFCs and CEC frequency in human peripheral blood. Methods: The Ethics Research Committee of the University of Campinas approved the experimental procedures and all donors signed the informed consent form. CEC enumeration was assessed by flow cytometry in the peripheral blood of 18 donors. 100 μL of blood (with a leukocyte concentration between 5 and 10 ×10³/μL) was incubated with monoclonal anti-human antibodies (CD45 PerCp, CD133 APC, CD31 FITC, and CD146 PE). FACS lysing solution was used for red blood cell removal. 300,000 events or the total volume of the tube were acquired using a FACSCalibur® flow cytometer (4). To determine the number of cells/μL, the percentage of events was multiplied per number of leucocytes and divided per 100. Cell culture from venous blood was used to isolate ECFCs which were confirmed by their cobblestone morphology and immunophenotyping for endothelial markers (CD31, CD146, CD309, and CD144). Results: ECFC isolation was successful in 9 out of 18 donors (1 male, 8 females; mean age: 35.8 ±11.1 years), and not successful in the other half of donors (2 male, 7 females; mean age: 37.7 ±9.7 years). CEC frequency was significantly higher (p=0.004) in the group with successful ECFC isolation when compared to the group that did not yield ECFCs (2.9 ±1.2 CECs/μL against 1.2 ±0,8 CECs/μL). This suggests that the success in ECFC isolation might be related to the number of CECs, and therefore ECFC mobilization into circulation may also be linked to vascular injury. Conclusion: Increased number of CECs might be associated with the success of ECFCs isolation.

In this chapter, students will be introduced to the challenges of organ transplantation and the ensuing need to find alternative ways to replace failing organs and tissues. The main organ systems are then briefly summarized, followed by examples of how much their cells and vascularization routes can be different. This helps to emphasize to the students the need to know the anatomy of the animal to be dissected and the vascular structure of the chosen organ. Having adequate vascular access to the organ is required for both live cells isolation and organ decellularization – procedures that students will be performing during this course. The sources of animal tissue, instruments used for dissection and organ removal, and use of heparin and collagenase are then briefly discussed. The chapter is concluded by asserting the importance of anesthesia and the ethical treatment of animals.

After euthanasia, testicles were obtained, tunica albuginea was removed with sterile forceps,and seminiferous tubules were treated with collagenase for 10 minutes at 37°C (5 mg/ml ofcollagenase, SIGMA C5138). Using a trans-illumination method, cycles of spermatogenesiswere detected. Sections of seminiferous tubules, developing the Golgi-acrosome complexin stages VI to VIII, were selected and cut under stereoscope. The tubules were placed onslides containing 40 μl PBS and extruded with a cover-slip in order to obtain the spermatogeniccells. Spermatogenic cells were fixed and smeared on slides or transferred to microtubes.

Bronchoalveolar lavage fluid (BALF) is an important tool in experimental toxicology and pharmacology. A number of researchers have utilized BAL in studies involving rodents. However, the detailed procedure of performing BAL in rodents has rarely been reported. In the present article, in situ BAL in rats has been described with technical points that ensure the quality and validity of BALF samples.BAL was performed in rats of Wistar strain. Flow cytometry and microscopy were utilized to analyze the BAL cells.The collected volume of BALF was 84.32 ± 2.7% of instilled volume. Alveolar macrophages were found to be prevalent in normal lungs when analyzed under microscope and by flow cytometry. The main problem that one may face is the accidental contamination of BALF samples with blood of the animal itself. Presence of blood in BALF certainly adds a significant number of cells and other biochemical variables. No blood contamination was detected in BALF.Here, a simple procedure for BAL and collection of alveolar cells (macrophages in the present study) is explained with an emphasis on technical steps and precautions, which ensures the quality of the BALF samples. Data exhibit that there is no blood contamination in the BALF and provide evidence that the technical points considered in the procedure here are successful in maintaining the quality and validity of BALF samples.

The functional cooperation between the oocyte and its surrounding granulosa cells is mandatory to oocyte growth and acquisition of developmental competence in mammalian species. Availability of in vitro methods for culture granulosa cells is important in understanding the biology of these cells and the response of maturing oocyte to in vitro culture and co-culture. Using the ovine as a model the ensuing chapter describes methods for primary culture of ovine granulosa cells and their co-culture with theca cells or oocytes that allow to mimic the molecular interactions between somatic cells and maturing oocyte and improve oocyte quality.

Obesity is a complex, multifactorial disease that has been extensively researched in recent times. Obesity is characterized by excess deposition of adipose tissue in response to surplus energy. Despite the negative connotations of adipose tissue (AT), it serves as a critical endocrine organ. Adipose tissue is a source of several adipokines and cytokines which have been deemed important for both normal metabolic function and disease formation. The discoveries of metabolically active brown AT in adult humans and adipose tissue derived stem cells (ADSC) have been key findings in the past decade with potential therapeutic implications. ADSCs represent an enticing pool of multipotent adult stem cells because of their noncontroversial nature, relative abundance, ease of isolation, and expandability. A decade and a half since the discovery of ADSCs, the scientific community is still working to uncover their therapeutic potential in a wide range of diseases. In this review, we provide an overview of the recent developments in the field of ADSCs and examine their potential use in transplantation and cell-based therapies for the regeneration of diseased organs and systems. We also hope to provide perspective on how to best utilize this readily available, powerful pool of stem cells in the future.

Strategies to promote intervertebral disc (IVD) regeneration have been hindered by the lack of knowledge of IVD fundamental cellular/molecular components. One of the key points to be addressed is the characterization of nucleus pulposus (NP) cell population(s). This study establishes an improved method for bovine NP (bNP) cell isolation, whose procedure is still not consensual among the literature, allowing a thorough characterization of cell (sub)populations that exist in the young NP.bNP was digested with distinct enzymes (collagenase-type-I, collagenase-type-II, and collagenase-type-XI) at different concentrations (0.5, 1.0, and 2.0 mg/mL), for 4 and 19 h. Cell yield, viability/apoptosis, and morphology were analyzed by flow cytometry and imaging flow cytometry. Identification of cell subpopulations within NP and its phenotype was investigated by assessing expression of CD29, CD44, CD45, CD34, CD146, and Brachyury.It was found that bNP cells present a similar morphology independently of the digestive enzyme used. However, cell yield was greatly improved by Coll-XI (2 mg/mL) treatment for a short digestion period. Interestingly, three subpopulations, with different sizes and auto-fluorescence, were consistently identified by flow cytometry. And crucially, differential expression of cell markers was found among these subpopulations.This study demonstrated that collagenase-type-XI is an efficient enzyme that is used for digesting bNP. And most importantly, three phenotypically distinct subpopulations of cells where identified within the bNP. Such knowledge is key for a better understanding of NP cell biology and its potential endogenous regenerative capacity.

Formulation of membrane properties is important prior the successful implantation of encapsulated cells producing therapeutically relevant compounds. The purpose of our study was to specify the methods allowing preliminary evaluation of hollow fibers (HF) chosen for immunoisolation. We have selected as estimates (1) diffusive permeability for small and large solutes, and HF cut off (in vitro), (2) histological evaluation of tissue overgrowth after sc. implantation into mice. It was found that diffusive coefficients were linearly dependent on the particle diameter except that of albumin (2-3 times higher than theoretically estimated). This discrepancy imply that for certain particles the interaction with membrane material may be significant. The histological evaluation showed that siliconized HF implanted for 105 days were accepted (there was thin fibrotic layer on the external surface of the HF, no surrounding haemopoietic cells were found). It is concluded that proposed methods for preliminary evaluation of hollow fibers chosen for immunoisolation seems to be reliable and suitable for testing diffusive permeability of each relevant cell product.

The use of human adipose-derived stem cells (ASC) for research and clinical purposes has become increasingly common over the past years. The advantages of ASC over more traditional sources of adult stem cells such as bone marrow (BM) include it’s ready availability in most people, the ease of significant volume fat removal by liposuction and subsequently the large number of adult mesenchymal stem cells (MSC) able to be isolated from each harvest. This chapter provides clear, focused methods for the isolation of the stromal vascular fraction (SVF) from lipoaspirate, which contains the ASC. Methods for subsequent culture, passage and cryopreservation of ASC are given, to enable other researchers to work with this exciting resource.

Nowadays more and more attention is turned to hepatic stellate cells (HSC) and their role in liver regeneration. Nonetheless there are several methodological questions, for example, methods of HSC isolation, their labeling and ways of transplantation. In this work we compared two different methods of HSC isolation: collagenase-pronase liver perfusion with further Histodenz density gradient centrifugation and method of Seglen for isolation of hepatocytes associated with HSC. We also analyzed diverse methods of cells labeling: membrane fluorescent labels PKH26 and with gene of green fluorescent protein (GFP), that could be get into the cells by electroporation, with chemicals like TurboFect or by adenovirus. Then cells were transplanted into rats in two ways: into lien and into system of portal vein. According to our results, we able to conclude that collagenase-pronase liver perfusion with further cells gradient centrifugation in Histodenz is better for HSC isolation than method of Seglen, the most optimal method for cells labeling is with adenovirus, expressing the GFP gene, for HSC transplantation - Transplantation into system of portal vein. © Human stem cells institute, 2013.

Publisher Summary It is relatively easy to create new cell lines. But their full validation, stability in culture, karyotype, chimera-forming ability, and germline potential take a lot of time and work. It is important to have sufficient characterized stocks of particular passage and to freeze batches to allow useful repeatable studies to be undertaken. Mouse embryonic stem (ES) cells isolation, and the conditions for their growth and maintenance are well described in numerous reviews and methods texts. This chapter discusses the growth and maintenance of cultures of mouse ES cells, because their isolation depends on the secure ability to maintain them. It is recommended that an established ES cell line be used to optimize growth conditions before attempts are made to isolate new ones. ES cells grow well in culture, and are not particularly fastidious about the media, but it must be remembered that ES cell cultures are essentially primary cultures. It is necessary to use conditions of tissue culture that maintain their primary properties, and not select variants. Any growth in nonoptimal conditions will lead to selective pressure, and the appearance of better-growing but worse-differentiating strains. In some cases, these may be recognized as chromosomal variants, but in others there is no gross karyotypic change identifying the altered cell strain.

In this paper the synchronous space hopping based on time reuse and hybrid frequency-time division duplex is proposed. This technique provides intra and inters cells isolation for time slotted scheduling based systems, where resources are shared among the users by means of a scheduling. It's aimed at a high and distributed data rates resulting from a high signal to interference and noise ratio (SINR). In CDMA platform it constitutes a new multiple access technique, which is examined on its downlink and compared to the conventional CDMA. The results are presented in terms of coverage, throughput and latency from system level simulations. A significantly increase in SINR of up to 15 dB, which corresponds to a better coverage, a higher sector throughput and a lower of equal user number based latency compared to the conventional CDMA.

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In order to analyze the RNA populations present in different cells of very early embryos, we have developed a protocol to purify these large blastomeres using counterflow centrifugal elutriation (CCE). This procedure employs ethanol fixation to stabilize the cells against shear forces encountered during CCE. Using this method, we fractionated the three different blastomere types of the 16-cell sea urchin embryo, the micromeres, mesomeres, and macromeres, achieving 96, 94, and 96% mean purities, respectively. We show here that intact RNA is recovered with equal efficiency from each blastomere preparation. Using this method, we have identified several RNAs that are distributed non-uniformly among these cells.

Sperm cells were isolated from pollen of Gerbera jamesonii by gentle grinding in a medium containing buffer, osmoticum, protectants and ions. Sperm nuclei were identified by means of the DNA fluorochrome DAPI. The sperm cells were observed by Nomarski optics and by scanning electron microscopy. Isolated sperm were osmotically sensitive and excluded Evans Blue. The structure of the isolated sperm cells was similar in shape and size to that of sperm cells extruded from fresh pollen and in fixed pollen.

S-Adenosyl-L-homocysteine hydrolase from mouse leukemic cells L1210 was purified to evident homogeneity with no concomitant enzymatic activities. The isoelectric point of the enzyme is 5.65 and the optimum temperatures for the synthesis and hydrolysis are 52 °C and 47 °C, respectively. The respective KM values for adenosine and S-adenosyl-L-homocysteine (SAH) are 3.8 and 4.4 μmol l-1. The optimum pH value for the synthesis of SAH is 6.95-7.20 and for the hydrolysis 7.15-7.80. The enzyme is competitively inhibited by 9-(S)-(2,3-dihydroxypropyl)adenine and inactivated with both enantiomers of eritadenine and 3-(adenin-9-yl)-2-hydroxypropanoic acid.

1. A purified fraction of ribonuclease-resistant RNA isolated from rat liver whole cells and nuclei was chromatographed on cellulose columns, and a double-stranded RNA fraction isolated. 2. The double-stranded structure of the RNA was firmly defined by its behaviour on cellulose, its symmetrical base composition, immunoprecipitin reaction, and its characteristic biphasic heat denaturation curve. 3. A nuclear origin for double-stranded RNA was established. It can be synthesized in the presence of actinomycin D. No significant quantities could be detected in the cytoplasm.

The cytoplasm of cells infected with rovirus contains 8 of the 10 polypeptides coded by this virus in free form in amounts equivalent to 1 to 10% of the progeny virus yield. All of these polypeptides exist in monomeric form except polypeptides μ1 and μ2 which occur as dimers, the form in which they are present in virions. One of these polypeptides, σ3, a constituent of the outer capsid shell, binds strongly to double-stranded RNA; it can be isolated in high yield in pure form by passing extracts of infected cells through columns of poly(IC)-cellulose and eluting. Another polypeptide, σ2A, a nonstructural polypeptide, binds strongly to single-stranded RNA and appears to exist in infected cells complexed with RNA. It can be isolated in high yield in pure form by passing extracts of infected cells, suitably treated to dissociate these complexes, through columns of poly(A)-cellulose and eluting. The possible significance of the highly specific nucleic acid affinities of these two reovirus-coded polypeptides is discussed.

Publisher Summary This chapter discusses isolation, identification, and cultivation of microvascular endothelial cells. The development of successful techniques for the isolation, identification, and culture of large-vessel endothelium has paved the way for the extension of these methods to microvascular endothelial cells. In isolation and culture of retinal microvessel endothelial cells, bovine eyes are obtained from a local slaughterhouse and transported to the laboratory on ice. The culture of heart microvessel endothelial cells has provided an important tool as to the role of the vascular endothelium of cardiac muscle in a number of physiologic and pathophysiologic functions, including prostaglandin synthesis. One of the major difficulties in the isolation and culture of microvascular endothelial cells is the identification of the putative endothelial cells.