细胞生物学

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME’s intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche.

The eye is a complex organ composed of multiple tissues in anterior and posterior eye segments. Malfunctions of any of these tissues can lead to ocular diseases and loss of vision. A detailed understanding of the ocular anatomy and physiology in animal models and humans contributes to the development of ocular drugs by enabling studies on drug delivery and clearance routes, pharmacokinetics, and toxicity. This protocol provides step-by-step instructions for the extraction and homogenization of ocular tissues for enzymatic and proteomics analyses.

Streamlined procedures for processing and cryopreservation of cell therapies using good laboratory practices are integral to biomanufacturing process development and clinical applications. The protocol herein begins with the preparation of human cell types cultured as adherent (i.e., mesenchymal stromal cells, MSCs) or suspension cells (i.e., peripheral blood mononuclear cells, PBMCs) to comprehensively demonstrate procedures that are applicable to commonly used primary cell cultures. Cell processing steps consist of preparing high yields of cells for cryopreservation using instruments routinely used in cell manufacturing, including the Finia^® Fill and Finish System and a controlled-rate freezer. The final steps comprise the storage of cells at subzero temperatures in liquid nitrogen vapor phase followed by the analysis of cell phenotypes before and after processing and cryopreservation, along with cell quality metrics for validation. Additionally, the protocol includes important considerations for the implementation of quality control measures for equipment operation and cell handling, as well as Good Laboratory Practices for cell manufacturing, which are essential for the translational use of cell therapies.

Key features

• The protocol applies to small- or large-scale manufacturing of cell therapy products.

• It includes streamlined procedures for processing and cryopreservation of cells cultured as adherent cells (MSCs) and suspension cells (PBMCs).

• Provides temperature control and rapid partitioning of sample in cryopreservation solution to maintain high viability of a range of cell types throughout the procedures.

• This protocol employs the Finia^® Fill and Finish System and a controlled-rate freezer.

Graphical overview

Satellite glial cells (SGCs) are a type of glial cell population that originates from neural crest cells. They ultimately migrate to surround the cell bodies of neurons in the ganglia of the peripheral nervous system. Under physiological conditions, SGCs perform homeostatic functions by modifying the microenvironment around nearby neurons and provide nutrients, structure, and protection. In recent years, they have gained considerable attention due to their involvement in peripheral nerve regeneration and pain. Although methods for culturing neonatal or rat SGCs have long existed, a well-characterized method for dissociating and culturing adult SGCs from mouse tissues has been lacking until recently. This has impeded further studies of their function and the testing of new therapeutics. This protocol provides a detailed description of how to obtain primary cultures of adult SGCs from mouse dorsal root ganglia in approximately two weeks with over 90% cell purity. We also demonstrate cell purity of these cultures using quantitative real-time RT-PCR and their functional integrity using calcium imaging.

Key features

• Detailed and simplified protocol to dissociate and culture primary satellite glial cells (SGCs) from adult mice.

• Cells are dissociated in approximately 2–3 h and cultured for approximately two weeks.

• These SGC cultures allow both molecular and functional studies.

Graphical overview

Dissociation and culture of mouse satellite glial cells

Fertilized teleost fish eggs are a complex formation, in which dividing cells arelocated in a small point in the entire volume of eggs. Isolating embryonic cellscan be considered a necessary step in the research of developmentalpeculiarities of fish cells at the earliest stages of embryogenesis beforeembryo formation. The main advantages of the offered protocol are rapidisolation, no enzymes, and overall low cost compared to other protocols. Theprotocol is suitable for the isolation of embryonic cells from medium-sized eggsat the stages of blastula or gastrula, for studies in a variety of applications(e.g., microscopy, flow cytometry, and other methods). Fertilized nelma eggs(Stenodus leucichthys nelma) are used in the protocol as a model.

Key features

• Fast and cheap isolation of cells from fish eggs at early stages (blastula orgastrula).

• Applicable for most of the methods for cell study (any staining, microscopy, flowcytometry, etc.).

• Can be applied to other teleost fish eggs with medium egg diameter of 3–4mm.

Graphical overview

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the ROSA26^mT/mG reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence. We also provide a method to culture the purified melanoblasts for further analysis. This method yields > 99% purity melanoblasts specifically targeted, and can be used for a variety of studies, including gene expression, clonogenic experiments, and biological assays, such as viability, capacity for directional migration, or differentiation into melanin-producing melanocytic cells.

Graphical overview

Cardiac fibroblasts are one of the major constituents of a healthy heart. Cultured cardiac fibroblasts are a crucial resource for conducting studies on cardiac fibrosis. The existing methods for culturing cardiac fibroblasts involve complicated steps and require special reagents and instruments. The major problems faced with primary cardiac fibroblast culture are the low yield and viability of the cultured cells and contamination with other heart cell types, including cardiomyocytes, endothelial cells, and immune cells. Numerous parameters, including the quality of the reagents used for the culture, conditions maintained during digestion of the cardiac tissue, composition of the digestion mixture used, and age of the pups used for culture determine the yield and purity of the cultured cardiac fibroblasts. The present study describes a detailed and simplified protocol to isolate and culture primary cardiac fibroblasts from neonatal murine pups. We demonstrate the transdifferentiation of fibroblasts into myofibroblasts through transforming growth factor (TGF)-β1 treatment, representing the changes in fibroblasts during cardiac fibrosis. These cells can be used to study the various aspects of cardiac fibrosis, inflammation, fibroblast proliferation, and growth.

The wing imaginal discs in Drosophila larvae are a pair of sac-like structures that later form the wings of the adult fly. During the past decades, wing discs have been used as a simple and accessible model system, for identifying genes and deciphering signaling cascades that play crucial roles in many aspects of development. In this protocol, we describe a simple method for preparing a cell suspension from wing discs (see Graphical abstract). This method can also be applied to the preparation of single-cell suspensions from other types of Drosophila tissues. When combined with genetic labeling, the dissociated cells are suitable for downstream analysis, such as flow cytometry. This method was recently used to isolate different populations of cells from Drosophila imaginal discs (Yang et al., 2022).

Graphical abstract:

Procedures to prepare a single-cell suspension from Drosophila imaginal discs. Illustration of the main steps to dissect, temporarily store, and dissociate imaginal discs from Drosophila larvae. Refer to the Procedure section for detailed description of each step.

Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have the capability of determining the fate of limbal epithelial progenitor cells (LEPC), which are responsible for the homeostasis of corneal epithelium. However, the isolation of these LMSC has proven to be difficult due to the small fraction of LMSC in the total limbal population, and primary cultures are always hampered by contamination with other cell types. We recently published the efficient isolation and functional characterization of LMSC from the human corneal limbus using CD90 as a selective marker. We observed that flow sorting yielded a pure population of LMSC with superior self-renewal capacity and transdifferentiation potential, and supported the maintenance of the LEPC phenotype. Here, we describe an optimized protocol for the isolation of LMSC from cadaveric corneal limbal tissue by combined collagenase digestion and flow sorting with expansion of LMSC on plastic.

Graphical abstract:

Superovulation is a method used to reduce the number of mice used per experiment by increasing the egg number. Conventionally, superovulation for obtaining mouse eggs involves the use of equine chorionic gonadotropin (eCG) for stimulation and human CG for induction. Female mice of the C57BL/6 inbred strain spontaneously ovulate approximately 10 eggs. The average number of eggs ovulated using the conventional superovulation method is approximately twice as high as that obtained by spontaneous ovulation. Here, we describe the conventional and non-conventional methods of intraperitoneal injection of superovulation reagents in mice and subsequent egg collection. The non-conventional superovulation method combining anti-inhibin serum (AIS) plus eCG for stimulation is more efficient than conventional superovulation. Appropriate intervals from each injection to sampling induce large numbers of high-quality eggs. Immediately after ovulation, eggs are surrounded by cumulus cells, forming an egg-cumulus complex. These cumulus cells are then removed from the egg-cumulus complex by treatment with hyaluronidase to obtain the exact number of eggs. This protocol is suitable for further manipulations such as intracytoplasmic sperm injection and cryopreservation of eggs, as well as for the analyses of responsivity to superovulation reagents in genetically modified mice obtained by genome editing.