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Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture.

ABSTRACT: Tendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell subpopulations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation methods preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior. Statement of clinical significance: The development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing.

SUBMITTER: Nichols AEC

PROVIDER: S-EPMC8232016 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Microglial isolation and culturing methods continue to be explored to maximize cellular yield, purity, responsiveness to stimulation and similarity to in vivo microglia. This study aims to evaluate different microglia isolation methods — 3 variants of microglia isolation from neonatal mice and 2 variants of microglia isolation from adult mice — on transcriptional profile and response to HMGB1. Methods: Microglia from neonatal mice, age 0-3 days (P0-P3) were isolated from mixed glial cultures (MGC). We included three variations of this protocol that differed by use of GM-CSF in culture (No GM-CSF or 500 pg/mL GM-CSF), and days of culture in MGC before microglial separation (10 or 21). Protocols for studying microglia from adult mice age 6-8 weeks included isolation by adherence properties followed by 7 days of culture with 100 ng/mL GM-CSF and 100 ng/mL M-CSF (Vijaya et al., 2023), or acute isolation using CD11b beads (Bordt et al., 2020). Purity, yield, and RNA quality of the isolated microglia were assessed by flow cytometry, hemocytometer counting, Nanodrop, and Bioanalyzer, respectively. Microglial responsiveness to an inflammatory stimulus, HMGB1, was evaluated by measuring TNFα, IL1β, and IFNβ secretion by ELISA and assessing gene expression patterns using bulk RNA sequencing. Results: All five methods demonstrated greater than 90% purity. Microglia from all cultures increased transcription of or secreted TNFα, IL1β, and IFNβ in response to HMGB1. RNA sequencing showed a larger number of differentially expressed genes in response to HMGB1 treatment in microglia cultured from pups than from adult mice, with sparse changes among the 3 MGC culturing conditions. Conclusion: These findings suggest that while all methods provided high purity, the choice of protocol may significantly influence yield, RNA quality, baseline transcriptional profile and response to stimulation. This comparative study provides valuable insights to inform the choice of microglial isolation and culture method.

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Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture.

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