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.

Regenerative MRL/MpJ Tendon Cells Exhibit Sex Differences in Morphology, Proliferation, Mechanosensitivity, and Cell-Matrix Remodeling

“…[36][37][38] Hussien et al 39 reported that human tendon-derived stromal cells undergo a dramatic phenotypic drift on conventional tissue-culture plastic (TCP) substrates compared to transcriptional activation of tendon-specific signatures on stiffer substrates. Other efforts to maintain tendon cell behavior have focused on standardizing primary cell isolation methods 40 , applying controlled mechanical stimulation using bioreactor designs [41][42][43][44] , and incorporating biochemical cues through exogenous growth factor supplementation and hypoxic (which was not certified by peer review) is the author/funder. All rights reserved.…”

“…[36][37][38] Hussien et al 39 reported that human tendon-derived stromal cells undergo a dramatic phenotypic drift on conventional tissue-culture plastic (TCP) substrates compared to transcriptional activation of tendon-specific signatures on stiffer substrates. Other efforts to maintain tendon cell behavior have focused on standardizing primary cell isolation methods 40 , applying controlled mechanical stimulation using bioreactor designs [41][42][43][44] , and incorporating biochemical cues through exogenous growth factor supplementation and hypoxic culture 45,46 . When considering 3D culture approaches, synthetic scaffolds offer an attractive approach over cell-encapsulated collagen and/or fibrin constructs given their capacity for surface functionalization using ECM proteins and more precise control over the fiber architecture to direct cell attachment, growth, differentiation.…”

Wnt3a-Modified Nanofiber Scaffolds Facilitate Tendon Healing by Driving Macrophage Polarization during Repair

“…Mouse tendon-derived cells were isolated and cultured as described in the previous literature. , In brief, the mice were first intraperitoneally injected with 30 mg/kg 0.3% pentobarbital sodium (Sigma-Aldrich, St. Louis, MO, USA) for anesthesia. Then, the tendons were isolated under sterile conditions, carefully dissected and minced, followed by digestion in 0.1% Col I (Gibco, Grand Island, NY, USA).…”

Epitenon-derived cells comprise a distinct progenitor population that contributes to both tendon fibrosis and regeneration following acute injury

“…Fluorescent images were quantified with VIS Image Analysis Software (v.6.7.0.2590, Visiopharm, Hørsholm, DK) as previously described. 65, 66 Briefly, a region of interest (ROI) was drawn around the scar area (excluding tendon stubs). Individual nuclei were then identified by automatic segmentation and were subsequently assigned a specific identity based on intensity thresholds of each color channel.…”