SSR 4月节点 2024

Passive samplers are useful novel tools for assessing contaminant exposure and evaluating its potential for adverse environmental effects. Passive equilibrium samplers enable the quantification of freely dissolved concentrations (C_free) of organic contaminants in sediments. C_free allows a direct assessment of (1) contaminant exchange and equilibrium status between sediment and overlying water, (2) benthic bioaccumulation, and (3) potential toxicity to benthic organisms. Thus, the use of equilibrium samplers improves the mechanistic understanding of fate and transport processes in sediments. Equilibrium samplers can be applied for hazard and risk assessment and management of contaminated sediments of aquatic environments. The objective of this chapter is to provide introductory guidance on the use of passive equilibrium samplers for sediments contaminated with hydrophobic organic contaminants, whereby three different approaches are recommended: (1) silicone-coated glass jars, (2) SPME fibers that can be used ex situ with sediments that had been sampled in the field, and (3) SPME fibers that can be exposed directly in the field (in situ) when they are installed in a protective housing that is buried in the sediment. After reading this chapter, users will have a fundamental understanding of equilibrium passive samplers and their potential applications to sediments.

Non-enzymatically isolated primary dermal progenitor fibroblasts derived from fetal organ donations are ideal cell types for allogenic musculoskeletal regenerative therapeutic applications. These cell types are differentiated, highly proliferative in standard in vitro culture conditions and extremely stable throughout their defined lifespans. Technical simplicity, robustness of bioprocessing and relatively small therapeutic dose requirements enable pragmatic and efficient production of clinical progenitor fibroblast lots under cGMP standards. Herein we describe optimized and standardized monolayer culture expansion protocols using dermal progenitor fibroblasts isolated under a Fetal Transplantation Program for the establishment of GMP tiered Master, Working and End of Production cryopreserved Cell Banks. Safety, stability and quality parameters are assessed through stringent testing of progeny biological materials, in view of clinical application to human patients suffering from diverse cutaneous chronic and acute affections. These methods and approaches, coupled to adequate cell source optimization, enable the obtention of a virtually limitless source of highly consistent and safe biological therapeutic material to be used for innovative regenerative medicine applications.

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Hematopoietic stem cells (HSCs) are ultimately responsible for the lifelong renewal of all blood cell lineages. In the bone marrow (BM), HSCs reside in specialized microenvironments referred to as the “niche.” HSC niche consists of complex components including heterogeneous cell populations, growth factors, and extracellular matrix molecules. The crosstalk between HSCs and their niche is essential to regulate the survival, self-renewal, migration, quiescence, and differentiation of HSCs. The application of mice models with endogenous ablation of specific cell types, advanced imaging technologies, high-throughput single-cell RNA sequencing, and single-cell mass cytometry methods have provided deep insights into communications between HSCs and niche cells. In this chapter, we have focused on three important cell types in the BM niche: mesenchymal stem cells (MSCs), osteoblasts (OBs), and endothelial cells (ECs). In order to address the interaction between HSCs and these three cell populations in BM niche, we have described methodology for (1) collecting total BM from femur and tibia of C57BL/6 mice; (2) analyzing or sorting of MSCs, OBs, and ECs based on the selection of surface markers CD45, Ter119, CD31, Sca1, and CD51 with flow cytometry; and (3) co-culturing the sorted cells with purified HSCs for further functional assays of HSCs.

Genotoxic pollutants are a major concern nowadays, since they can interact with the genetic material of exposed organisms, which may initiate a cascade of damages that may lead to endangerment from molecular to the community levels. The comet and the micronucleus assays performed with fish erythrocytes can be used to identify extrachromosomal DNA adducts in situ resulting from strand breaks, chromosomal breaks, or aneugenic effects. These two assays, performed as both in vitro and in situ, have been successfully applied to detect genotoxic potentials of environmental samples in several monitoring studies. In situ bioassays reflect the real exposure situation of an environment; thus, they may be useful to confirm or falsify the potential toxicity revealed by in vitro bioassays. Hence, this chapter presents a general guideline to perform in situ comet and micronucleus assays, using fish erythrocytes, as well as some important observations that can be used to minimize possible technical errors.

Recently, gene therapy as one of the most promising treatments can apply genes for incurable diseases treatment. In this context, vectors as gene delivery systems play a pivotal role in gene therapy procedure. Hereupon, viral vectors have been increasingly introduced as a hyper-efficient tools for gene therapy. Adenoviral vectors as one of the most common groups which are used in gene therapy have a high ability for humans. Indeed, they are not integrated into host genome. In other words, they can be adapted for direct transduction of recombinant proteins into targeted cells. Moreover, they have large packaging capacity and high levels of efficiency and expression. In accordance with translational pathways from the basic to the clinic, recombinant adenoviral vectors packaging must be managed under good manufacturing practice (GMP) principles before applying in clinical trials. Therein, in this chapter standard methods for manufacturing of GMP-compliant Adenoviral vectors for gene therapy have been introduced.

Although human malignant melanoma is a highly immunogenic cancer, both the endogenous antitumor immune response and melanoma immunotherapy often fail to control neoplastic progression. Accordingly, characterizing melanoma cell subsets capable of evading antitumor immunity could unravel optimized treatment strategies that might reduce morbidity and mortality from melanoma. By virtue of their preferential capacity to modulate antitumor immune responses and drive inexorable tumor growth and progression, malignant melanoma-initiating cells (MMICs) warrant closer investigation to further elucidate the cellular and molecular mechanisms underlying melanoma immune evasion and immunotherapy resistance. Here we describe methodologies that enable the characterization of immunoregulatory effects of purified MMICs versus melanoma bulk populations in coculture with syngeneic or allogeneic lymphocytes, using [³H]thymidine incorporation, enzyme-linked immunosorbent spot (ELISPOT), or ELISA assays. These assays were traditionally developed to analyze alloimmune processes and we successfully adapted them for the study of tumor-mediated immunomodulatory functions.

Serine hydrolases are important in a variety of physiological processes and can be important pharmacological and toxicological molecular targets. A modified photometric method suitable for evaluating inhibition of the serine hydrolases acetylcholinesterase, butyrylcholinesterase, and monoacylglycerol lipase is described. The same reagents and protocol are used for each of the three different enzymes, with the only difference being the substrate. Kinetic considerations for reversible or irreversible inhibitors are discussed. This versatile assay can be used to quickly characterize inhibitory actions (IC₅₀) of test compounds on the three hydrolases, with simple modifications allowing kinetic characterizations (K_i, k₂, k_i).

An antibody microarray (DotScan^TM) has been developed for profiling clinical melanoma specimens. Immobilized antibodies capture live cells expressing corresponding antigens to produce a dot pattern that represents the surface profile or immunophenotype. The unique signatures obtained may correlate with disease subtype, tumor progression, and clinical outcome. Here we describe the rapid analysis of surgically resected metastatic melanoma. Leukocytes are separated from tumor cells using CD45 antibody-conjugated magnetic beads and separated cell populations are profiled on the microarray. This antibody microarray may be extended to include additional antibodies for cell surface biomarkers and therapeutic antibodies.

CRISPR/Cas9-mediated gene editing has been rapidly and widely applied in many organisms for delicate genetic manipulation, including human-induced pluripotent stem cells (iPSCs). Gene editing in human iPSCs is promising for genetics and biomedical research due to that gene-edited iPSC still possesses the potential to be differentiated into any cell lineages. In many cases, the generation of Cas9 expressing cell lines is a prerequisite toward performing successful editing of multiplex genes of interest. Here, we describe a simple, effective method to generate stable Cas9 expressing human iPSCs with high Cas9 activity. In this method, stable Cas9 expressing monoclonal human iPSC lines were generated through lentiviral transduction of Cas9 cassette, followed by blasticidin selection and subcloning with low seeding density. After colonies isolation and expansion, a BFP-GFP reporter assay was applied to validate the Cas9 activities of multiple monoclonal lines by flow cytometry (FACS). These Cas9 expressing human iPSCs generated by our method are single cell-derived monoclonal lines with homogenous population and Cas9 activity of up to 99%.