![]() Within the CNS cellular composite, isolation of neurons and neuronal subtypes appears particularly challenging due to their susceptibility to damage imposed by mechanical stress or chemical treatments, steps that are inevitable for cell dissemination. Likewise, there are well-described aging-associated peculiarities in the lipid profile of CNS plasmalemma, arising from alterations in their biosynthesis, neuronal vesicle uptake, and inter- and intramembrane cholesterol trafficking, as well as the catabolism and content of glycosylated sphingolipids or gangliosides and ceramides. Such multiple age-related changes, which can manifest even independently of a typical signature of senescence, influence the biophysical and biochemical cell and tissue properties and reciprocally interfere with key parameters that are crucial for the efficiency of cell isolation techniques, such as membrane fluidity, cellular viscidity, adherence, and apoptosis resistance. ![]() Several lines of evidence indicate profound aging-related transformations in the molecular composition of neural plasma membranes, in cell-shaping organelle arrangements, and the cytoskeletal architecture affecting micro-/intermediate filaments and microtubule structures. Thus, there is a yet unmet need for CNS cell isolation techniques that allow for a proportionate retrieval of singularized neural cells, including neurons and entity-specific subpopulations, from aging, progeroid, and neurodegenerative CNS, thereby covering isolates both from brain and spinal cord regions. Though innovative TRAP approaches can theoretically assay any cell type of interest at any age category, its application on aged and neurodegenerative conditions is still sparse. Recent highly specialized procedural algorithms achieving, e.g., neural stem cell isolates and their neuronal differentiation on 2D films or in 3D biopolymer scaffolds, or those challenging the retrieval of CNS subpopulations, e.g., via translating ribosome affinity purification (TRAP) requiring artificial chromosomes in BAC transgenic animals, are often cost- and resource-intensive or depend on specific technical equipment and thus might lack accessibility to a broad majority of work facilities. Similarly underrepresented are algorithms that purify anatomically confined cell entities with specific susceptibility to degeneration and death, e.g., from model systems that pinpoint the effect of certain target mutations. However, protocols dedicated to the achievement of single cell isolates encompassing the retrieval of neurons instead of glia only, and which originate from mature, aged, and particularly neurodegenerative conditions are still infrequent. Such a heterogeneous cellular network environment is now scientifically accessible, e.g., at the organoid, slice, neurosphere, primary tissue, and cell culture level, thereby allowing specifications towards the degree of specimen maturation and coverage of embryonic, postnatal, and juvenile stages. The mammalian CNS is composed of several types of projection neurons, interneurons, glial populations-including oligodendrocytes, astrocytes, and microglia-and extracellular matrix molecules. We expect suitability for transfer to other CNS targets and to a broad spectrum of engineered systems addressing aging, neurodegeneration, progeria, and senescence. It is suitable for a variety of downstream applications aiming at cell type-specific interrogations, including cell culture systems, Flow-FISH, flow cytometry/FACS, senescence studies, and retrieval of omic-scale DNA, RNA, and protein profiles. Technically, the protocol is rapid, efficient as for cellular yield and well preserves physiological cell proportions. Following recent, unprecedented evidence of post-mitotic cellular senescence (PoMiCS), the protocol appears suitable for such de novo characterization and phenotypic opposition to classical senescence. Here, we describe a practical workflow for the acquisition and phenomapping of CNS neural cells at states of health, physiological and precocious aging, and genetically provoked neurodegeneration. Though such obstacles are addressed and partially overcome for embryonic premature and mature CNS tissues, procedural adaptations to an aged, progeroid, and degenerative CNS environment are underrepresented. Moreover, changes in cellular interconnections, membrane lipid and cholesterol compositions, compartment-specific biophysical properties, and intercellular space constituents demand technical adjustments for cell isolation at different stages of maturation and aging. Efficient purification of viable neural cells from the mature CNS has been historically challenging due to the heterogeneity of the inherent cell populations.
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