Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2025.12.09.693181

Understanding the physiological behaviour of disc cells in an in vitro imitation of the healthy and degenerated disc niche

Snuggs, J.·Basatvat, S.·Kanelis, E.·Binch, A.·Alexopoulos, L. G.·Tryfonidou, M. A.·Le Maitre, C. L.
View original source ↗Export metadata ↓

Abstract

INTRODUCTIONThe natural environment within the nucleus pulposus (NP) is hypoxic, acidic, low in nutrition and exerts a high osmotic-pressure. NP cells have adapted to this harsh environment, however, in vitro conditions often fail to recapitulate this environment. Hence, this study aimed to develop media to mimic the conditions of the native NP, with regards to pH, osmolality, glucose, combined with culture in physioxia (5% O2), and determine their effects upon human NP cells and tissue. METHODSAll media utilised low glucose DMEM/serum free conditions with supplements supporting matrix synthesis, based on media recommended for alginate culture (standard media). Healthy media modulated osmolarity (425mOsm/Kg), and pH7.2, degenerate media consisted of 325mOsm/kg and pH6.8. The latter was further supplemented with 100pg/ml IL-1{beta} (degenerate+IL-1{beta} media). NP cells in 3D alginate and NP tissue explants were cultured in these media for up to 2 weeks in physioxia (5% O2) to determine effects on viability, mitochondrial activity, protein expression and secretome. RESULTSMedia osmolarity and pH remained stable and cell viability was not altered by any media composition. Mitochondrial activity was increased during short term cultures, whilst a decrease was seen following 14 days in degenerate media. The secretome of NP cells was differentially affected in healthy or degenerate media, with most increases in catabolic cytokines observed following the addition of IL-1{beta}. Tissue explants showed stability of protein expression of matrix components in both healthy and degenerate+IL-1{beta} media, with limited effects seen on the secretome. DISCUSSIONThe media formulations developed here can provide more appropriate environmental conditions in vitro, mimicking more closely the in vivo conditions observed within healthy and degenerate IVDs. The application of which can provide more appropriate culture conditions to test potential therapeutic approaches and understand more fully the pathogenesis of disease using in vitro and ex vivo models.

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Snuggs, J., Basatvat, S., Kanelis, E., Binch, A., Alexopoulos, L. G., Tryfonidou, M. A., Le Maitre, C. L.. 2025-12-11. Understanding the physiological behaviour of disc cells in an in vitro imitation of the healthy and degenerated disc niche. https://doi.org/10.64898/2025.12.09.693181

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

The nuclear membrane protein Samp1 links peripheral genome organization to the myogenic transcriptional program

Samp1 is an inner nuclear membrane protein required for myogenic differentiation and involved in chromatin organization at the nuclear periphery. Here, we investigated whether these functions are connected by studying the effects of Samp1 depletion during C2C12 myogenic differentiation using immunofluorescence microscopy, RNA sequencing, FRIC, and chromosome-positioning analysis. Samp1-depleted cells showed strongly reduced MyHC expression and virtually abrogated multinucleated fiber formation. Although cell-cycle withdrawal was not prevented, the transcriptional program driving differentiation was drastically perturbed, with reduced muscle-associated transcripts and incomplete repression of genes normally downregulated during myogenesis. Samp1 depletion also disrupted peripheral chromatin organization and prevented the accumulation of peripheral heterochromatin typically seen during differentiation. In addition, radial chromosome distribution was disrupted, evidenced by the failure of chromosome 8 to reposition to the nuclear periphery during differentiation. Together, these findings link the requirement for Samp1 in myogenic differentiation to its role in genome organization at the nuclear periphery.

cell biology↗
bioRxiv2026

Unraveling the metabolic landscape of alkaptonuria through a human-relevant in vitro liver disease model

Alkaptonuria (AKU) is a rare inherited metabolic disorder of tyrosine catabolism caused by a deficient homogentisate 1,2-dioxygenase (HGD) enzyme. This results in the accumulation of homogentisic acid (HGA), driving a progressive multisystem pathology characterized by debilitating early-onset osteoarthritis due to connective tissue degeneration. While previous in vitro studies have primarily relied on exogenous HGA exposure in osteoarticular cell models, the direct metabolic consequences of endogenous HGD deficiency within its native hepatic context remain poorly understood. Here, we established the first human-relevant HGD knockout hepatic in vitro model using a universal in-house-developed homology-directed repair approach. Integrative multi-omic analysis revealed that HGD deficiency induces widespread metabolic rewiring extending beyond disrupted tyrosine catabolism. HGD-deficient hepatocytes exhibited elevated oxidative stress accompanied by impaired mitochondrial respiration and a pseudohypoxic metabolic adaptation toward increased glycolytic dependency. Despite this glycolytic shift, the cells displayed reduced anabolic and translational activity alongside attenuated proliferation, consistent with a chronic stress-adaptive survival state rather than a proliferative metabolic phenotype. This study provides systems-level insights into the pathophysiology of AKU and establishes a versatile platform for mechanistic and therapeutic investigation.

cell biology↗
bioRxiv2026

The circadian clock regulates KCNH2 (hERG) promoter activity through daily temperature rhythms.

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.

cell biology↗