Static Culture Research Articles

PmrAB controls virulence-associated traits and outer membrane vesicle biogenesis in Acinetobacter baumannii

The PmrAB two-component system modulates colistin resistance in Acinetobacter baumannii, but its association with the virulence traits of this bacterium remains uncharacterized. This study explored the role of A. baumannii PmrAB in surface motility, biofilm formation, and outer membrane vesicle (OMV) biogenesis using wild-type (WT) A. baumannii 17978 and ΔpmrA and ΔpmrB mutant strains. The two mutant strains exhibited significantly decreased surface motility compared with that of WT strain by the low expression of abaI, abaR, A1S_0113, A1S_0115, and A1S_0116. Biofilm mass also significantly decreased in the two mutant strains at 12 h of incubation, but restored at 24 h. Under static culture conditions for 12 h, the two mutant strains showed low pgaA expression. However, the other biofilm-associated genes, such as csuC, csuE, ompA, and bap, showed different expression between the two mutant strains. Although the size of OMVs was similar among the three strains, the number of OMVs secreted from the two mutant strains slightly decreased compared with that secreted from the WT strain. Protein concentrations in the OMVs of ΔpmrA mutant significantly decreased compared with those in the OMVs of WT and ΔpmrB strains. Overall, PmrAB modulates virulence traits and OMV biogenesis in A. baumannii.

Inoculation with Biofilm of Bacillus subtilis Promotes the Growth of Lactuca sativa

In Argentina, the excessive use of fertilizers is common in intensively cultivated zones around highly populated areas. Bioinoculants based on plant growth promoting rhizobacteria (PGPR) could be effective for crop production improvement without negative effects on the environment. The objective of this work was to evaluate an alternative inoculation method, namely the application of the biofilm produced by Bacillus subtilis as a growth promoter on seeds of three varieties of Lactuca sativa, and to compare it with the common planktonic approach. Biofilm was obtained under static culture conditions, while planktonic inoculum was produced at 150 rpm. The major biofilm effects were observed with Bacillus subtilis subsp. spizizenii, that showed antifungal activity against phytopathogens, synthesized plant growth regulators (IAA, cytokinin and ABA) and solubilized phosphates. The Grand Rapid variety inoculated with biofilm showed the best results, with 30% and 37% higher aerial and root biomass, respectively, compared to the planktonic form. Moreover, the biofilm positive effects were observed through successive plant development stages until harvest, when the bacterium was recovered from the interior of the roots. The biofilm of B. subtilis subsp. spizizenii behave as a superior growth-promoting inoculant compared to the traditional planktonic inoculation technique.

Population heterogeneity in Mycobacterium smegmatis and Mycobacterium abscessus.

Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in M. smegmatis - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions. M. abscessus has two recognized morphotypes, smooth and rough. Interestingly, M. tuberculosis exists in only a rough form. The shift from smooth to rough in both M. smegmatis and M. abscessus was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle preparations compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among M. smegmatis and M. abscessus variants. Deletion of the global regulator lsr2 shifted the M. smegmatis intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse outcomes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.

Computer controlled expansion of equine cord blood mesenchymal stromal cells on microcarriers in 3L vertical-wheel® bioreactors.

Mesenchymal stromal cells (MSCs) are an ideal cell source for allogenic cell therapy due to their immunomodulatory and differentiation properties. Equine MSCs (eMSCs) have been found to be a promising treatment for equine joint injuries including meniscal injuries, cartilage degradation, and osteoarthritis. Although the use of eMSCs has shown efficacy in preliminary studies, challenges associated with biomanufacturing remain. To achieve the required cell numbers for clinical application, bioreactor-based processes are required. Initial studies have shown that eMSCs can be cultivated in microcarrier-based, stirred suspension bioreactor culture at the laboratory 0.1L scale using a Vertical-Wheel® (VW) bioreactor. However, investigations regarding scale up of these processes to the required biomanufacturing scales are required. This study investigated the scale-up of a equine cord blood MSC (eCB-MSC) bioprocess in VW bioreactors at three scales. This included scale-up from the 0.1-0.5L bioreactor, scale-up from static culture to the 3L computer-controlled bioreactor, and scale-up into the 3L computer-controlled bioreactor using a mock clinical trial process. Results from the various scale-up experiments demonstrated similar cell expansion at the various tested scales. The 3L computer-controlled system resulted in a final cell densities of 1.5 × 105 cells/cm2 on average, achieving 1.5 × 109 harvested cells. Biological testing of the cells showed that cell phenotype and functionality were maintained after scale-up. These findings demonstrate the scalability of an eCB-MSC bioprocess using microcarriers in VW bioreactors to achieve clinically relevant cell numbers, a critical step to translate MSC treatments from research to clinical applications. This study also represents the first known published study expanding any cell type in the 3L VW bioreactor.

Microphysiological Modeling of Gingival Tissues and Host-Material Interactions Using Gingiva-on-Chip.

Gingiva plays a crucial barrier role at the interface of teeth, tooth-supporting structures, microbiome, and external agents. To mimic this complex microenvironment, an in vitro microphysiological platform and biofabricated full-thickness gingival equivalents (gingiva-on-chip) within a vertically stacked microfluidic device is developed. This design allowed long-term and air-liquid interface culture, and host-material interactions under flow conditions. Compared to static cultures, dynamic cultures on-chip enabled the biofabrication of gingival equivalents with stable mucosal matrix, improved epithelial morphogenesis, and barrier features. Additionally, a diseased state with disrupted barrier function representative of gingival/oral mucosal ulcers is modeled. The apical flow feature is utilized to emulate the mechanical action of mouth rinse and integrate the assessment of host-material interactions and transmucosal permeation of oral-care formulations in both healthy and diseased states. Although the gingiva-on-chip cultures have thicker and more mature epithelium, the flow of oral-care formulations induced increased tissue disruption and cytotoxic features compared to static conditions. The realistic emulation of mouth rinsing action facilitated a more physiological assessment of mucosal irritation potential. Overall, this microphysiological system enables biofabrication of human gingiva equivalents in intact and ulcerated states, providing a miniaturized and integrated platform for downstream host-material and host-microbiome applications in gingival and oral mucosa research.

The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 2-Advanced Microfluidic Nasal In Vitro Model for Drug Absorption Testing.

The nasal mucosa, being accessible and highly vascularized, opens up new opportunities for the systemic administration of drugs. However, there are several protective functions like the mucociliary clearance, a physiological barrier which represents is a difficult obstacle for drug candidates to overcome. For this reason, effective testing procedures are required in the preclinical phase of pharmaceutical development. Based on a recently reported immortalized porcine nasal epithelial cell line, we developed a test platform based on a tissue-compatible microfluidic chip. In this study, a biomimetic glass chip, which was equipped with a controlled bidirectional airflow to induce a physiologically relevant wall shear stress on the epithelial cell layer, was microfabricated. By developing a membrane transfer technique, the epithelial cell layer could be pre-cultivated in a static holder prior to cultivation in a microfluidic environment. The dynamic cultivation within the chip showed a homogenous distribution of the mucus film on top of the cell layer and a significant increase in cilia formation compared to the static cultivation condition. In addition, the recording of the ciliary transport mechanism by microparticle image velocimetry was successful. Using FITC-dextran 4000 as an example, it was shown that this nasal mucosa on a chip is suitable for permeation studies. The obtained permeation coefficient was in the range of values determined by means of other established in vitro and in vivo models. This novel nasal mucosa on chip could, in future, be automated and used as a substitute for animal testing.

Heart and kidney organoids maintain organ-specific function in a microfluidic system

Heart and kidney communicate with one another in an interdependent relationship and they influence each other's behavior reciprocally, as pathological changes in one organ can damage the other. Although independent human in vitro models for heart and kidney exist, they do not capture their dynamic crosstalk. We have developed a microfluidic system which can be used to study heart and kidney interaction in vitro. Cardiac microtissues (cMTs) and kidney organoids (kOs) derived from human induced pluripotent stem cells (hiPSCs) were generated and loaded into two separated communicating chambers of a perfusion chip. Static culture conditions were compared with dynamic culture under unidirectional flow. Tissue viability was maintained for minimally 72 h under both conditions, as indicated by the presence of sarcomeric structures coupled with beating activity in cMTs and the presence of nephron structures and albumin uptake in kOs. We concluded that this system enables the study of human cardiac and kidney organoid interaction in vitro while controlling parameters like fluidic flow speed and direction. Together, this “cardiorenal-unit” provides a new in vitro model to study the cardiorenal axis and it may be further developed to investigate diseases involving both two organs and their potential treatments.

Effect of morphological change on the maturation of human induced pluripotent stem cell-derived cardiac tissue in rotating flow culture

IntroductionUnderstanding the critical factors for the maturation of human induced pluripotent stem cell (hiPSC)-derived cardiac tissue is important for further development of culture techniques. Rotating flow culture, where the tissues float in the culture medium by balancing its gravitational settling and the medium flow generated in rotating disk-shaped culture vessels, is one of culture systems used for tissue engineering. It has previously been demonstrated that rotating flow culture leads to the formation of matured cardiac tissue with higher levels of function and structure than the other culture systems. However, the detailed mechanisms underlying the maturation of cardiac tissue remain unclear. This study investigated the maturation process of hiPSC-derived cardiac tissue in rotating flow culture with a focus on morphological changes in the tissue, which is a trigger for maturation. MethodsThe cardiac tissue, which consisted of cardiomyocytes derived from hiPSCs, was cultured on the 3D scaffold of poly (lactic-co-glycolic) acid (PLGA)-aligned nanofibers, in rotating flow culture for 5 days. During the culture, the time profile of projected area of tissue and formation of maturation marker proteins (β-myosin heavy chain and Connexin-43), tissue structure, and formation of nuclear lamina proteins (Lamin A/C) were compared with that in static suspension culture. ResultsThe ratio of the projected area of tissue significantly decreased from Day 0 to Day 3 due to tissue shrinkage. In contrast, Western blot analysis revealed that maturation protein markers of cardiomyocytes significantly increased after Day 3. In addition, in rotating flow culture, flat-shaped nuclei and fiber-like cytoskeletal structures were distributed in the surface region of tissue where medium flow was continuously applied. Moreover, Lamin A/C, which are generally formed in differentiated cells owing to mechanical force across the cytoskeleton and critically affect the maturation of cardiomyocytes, were significantly formed in the tissue of rotating flow culture. ConclusionsIn this study, we found that spatial heterogeneity of tissue structure and tissue shrinkage occurred in rotating flow culture, which was not observed in static suspension culture. Moreover, from the quantitative analysis, it was also suggested that tissue shrinkage in rotating flow culture contributed its following tissue maturation. These findings showed one of the important characteristics of rotating flow culture which was not revealed in previous studies.

A 3D printed plastic frame deeply impacts yeast cell growth.

Solid State Fermentation (SSF) processes have been explored for yeast growth and protein and metabolites production. However, most of these processes lack standardization. In this work, we present a polylactic acid (PLA) 3D printed matrix that dramatically enhances yeast growth when embedded in liquid media compared to equivalent static cultures, and changes yeast expression patterns at the proteome level (data are available via ProteomeXchange with identifier PXD043759). Moreover, differences in sugar assimilation and ethanol production, as the main product of alcoholic fermentation, are observed. Our results suggest that these matrixes may be useful for a vast range of biotechnological applications based on yeast fermentation.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis.

Human organoids have the potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by the lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, these challenges are overcome by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing, and encapsulation techniques are demonstrated on a pillar plate, which is coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels are differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero.

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This settingmaintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces newpossibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

Oxidative Stress Response of Probiotic Strain Bifidobacterium longum subsp. longum GT15.

Bifidobacterium is a predominant and important genus in the bacterial population of the human gut microbiota. Despite the increasing number of studies on the beneficial functionality of bifidobacteria for human health, knowledge about their antioxidant potential is still insufficient. Several in vivo and in vitro studies of Bifidobacterium strains and their cellular components have shown good antioxidant capacity that provided a certain protection of their own and the host's cells. Our work presents the data of transcriptomic, proteomic, and metabolomic analyses of the growing and stationary culture of the probiotic strain B. longum subsp. longum GT15 after exposure to hydrogen peroxide for 2 h and oxygen for 2 and 4 h. The results of the analysis of the sequenced genome of B. longum GT15 showed the presence of 16 gene-encoding proteins with known antioxidant functions. The results of the full transcriptomic analysis demonstrated a more than two-fold increase of levels of transcripts for eleven genes, encoding proteins with antioxidant functions. Proteomic data analysis showed an increased level of more than two times for glutaredoxin and thioredoxin after the exposure to oxygen, which indicates that the thioredoxin-dependent antioxidant system may be the major redox homeostasis system in B. longum bacteria. We also found that the levels of proteins presumably involved in global stress, amino acid metabolism, nucleotide and carbohydrate metabolism, and transport had significantly increased in response to oxidative stress. The metabolic fingerprint analysis also showed good discrimination between cells responding to oxidative stress and the untreated controls. Our results provide a greater understanding of the mechanism of oxidative stress response in B. longum and the factors that contribute to its survival in functional food products.

Microporous Hydroxyapatite-Based Ceramics Alter the Physiology of Endothelial Cells through Physical and Chemical Cues.

Incorporation of silicate ions in calcium phosphate ceramics (CPC) and modification of their multiscale architecture are two strategies for improving the vascularization of scaffolds for bone regenerative medicine. The response of endothelial cells, actors for vascularization, to the chemical and physical cues of biomaterial surfaces is little documented, although essential. We aimed to characterize in vitro the response of an endothelial cell line, C166, cultivated on the surface CPCs varying either in terms of their chemistry (pure versus silicon-doped HA) or their microstructure (dense versus microporous). Adhesion, metabolic activity, and proliferation were significantly altered on microporous ceramics, but the secretion of the pro-angiogenic VEGF-A increased from 262 to 386 pg/mL on porous compared to dense silicon-doped HA ceramics after 168 h. A tubulogenesis assay was set up directly on the ceramics. Two configurations were designed for discriminating the influence of the chemistry from that of the surface physical properties. The formation of tubule-like structures was qualitatively more frequent on dense ceramics. Microporous ceramics induced calcium depletion in the culture medium (from 2 down to 0.5 mmol/L), which is deleterious for C166. Importantly, this effect might be associated with the in vitro static cell culture. No influence of silicon doping of HA on C166 behavior was detected.

Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System.

Two-dimensional (2D)-based cell culture systems, limited by their inherent heterogeneity and scalability, are a bottleneck in the production of high-quality cells for downstream biomedical applications. Finding the optimal conditions for large-scale stem cell culture while maintaining good cellular status is challenging. The aim of this study was to assess the effects of three-dimensional (3D) culture on the viability, proliferation, self-renewal, and differentiation of human induced pluripotent stem cells (IPSCs). Various culture conditions were evaluated to determine the optimal conditions to maintain the viability and proliferation of human IPSCs in a 3D environment: static versus dynamic culture, type of adhesion protein added to alginate (Matrigel™ versus gelatin), and the addition of Y-27632t on long-term 3D culture. The proliferation ability of the cells was evaluated via the MTS proliferation assay; the expression levels of the pluripotency markers Nanog and Oct3/4, PAX6 as an ectoderm marker, and laminin-5 and fibronectin as markers of extracellular matrix synthesis were assessed; and HIF1α and HIF2α levels were measured using quantitative reverse transcription polymerase chain reaction. Using a high-aspect-ratio vessel bioreactor with a gentle, low-sheer, and low-turbulence environment with sufficient oxygenation and effective mass transfer of nutrients and waste, we verified its ability to promote cell proliferation and self-renewal. The findings showed that human IPSCs have the ability to maintain pluripotency in a feeder-free system and by inhibiting ROCK signaling and using hypoxia to improve single-cell viability in 3D culture. Furthermore, these cells demonstrated increased self-renewal and proliferation when inoculated as single cells in 3D alginate beads by adding RI during the culture period. Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs.

P16-54: Comparison of the impact of static and dynamic cultivation conditions on 2D and 3D cell cultures

P16-54: Comparison of the impact of static and dynamic cultivation conditions on 2D and 3D cell cultures

Mathematical modeling and simulation of oxygen mass transfer in rotating biological contactor (RBC) for bacterial cellulose production

The Rotary Biological Contact (RBC) reactor has a unique aeration system that provides a moving bed for easy and efficient oxygen transfer within the system to overcome the limitations of stationary culture in bacterial cellulose (BC) production. In most cases, oxygen mass transfer is the limiting factor of an aerobic process, hence the changes in the oxygen mass transfer coefficient within the system have been given special attention. In this study, the RBC bioreactor producing BC was modeled based on one-dimensional oxygen mass transfer in the direction perpendicular to the surface of the discs. The Monod kinetic model has been used to express cell growth. Simulation of the mathematical model was done based on the numerical solution with finite difference method in MATLAB software. Sensitivity analysis was performed to investigate the effect of changing different system parameters in RBC modeling. The simulation results showed that with the increase in angular velocity, the oxygen transfer coefficient and subsequently the mass transfer rate increase. Also, at a certain angular velocity, the oxygen mass transfer coefficient increases with the increase of immersion depth. The best disk rotation speed considering other parameters as well as the laboratory results was 13 rpm with a 45% indentation percentage and the optimal distance between the disks was 1.5 cm.

Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System.

Neural injuries disrupt the normal functions of the nervous system, whose complexities limit current treatment options. Because of their enhanced therapeutic effects, neurospheres have the potential to advance the field of regenerative medicine and neural tissue engineering. Methodological steps can pose challenges for implementing neurosphere assemblies; for example, conventional static cultures hinder yield and throughput, while the presence of the necrotic core, time-consuming methodology, and high variability can slow their progression to clinical application. Here we demonstrate the optimization of primary neural cell-derived neurospheres, developed using a high-throughput, stress-free, 3D bioreactor. This process provides a necessary baseline for future studies that could develop co-cultured assemblies of stem cells combined with endothelial cells, and/or biomaterials and nanomaterials for clinical therapeutic use. Neurosphere size and neurite spreading were evaluated under various conditions using Image J software. Primary neural cells obtained from the hippocampi of three-day-old rat pups, when incubated for 24 h in a reactor coated with 2% Pluronic and seeded on Poly-D-Lysine-coated plates establish neurospheres suitable for therapeutic use within five days. Most notably, neurospheres maintained high cell viability of ≥84% and expressed the neural marker MAP2, neural marker β-Tubulin III, and glial marker GFAP at all time points when evaluated over seven days. Establishing these factors reduces the variability in developing neurospheres, while increasing the ease and output of the culture process and maintaining viable cellular constructs.

Tendon Extracellular Matrix Promotes Myotendinous Junction Protein Expression in Engineered Muscle Tissue under Both Static and Mechanically Stimulated Culture Conditions

Studying the crosstalk between the muscle and tendon tissue is an important yet understudied area in musculoskeletal research. In vitro models can help elucidate the function and repair of the myotendinous junction (MTJ) under static and dynamic culture conditions using engineered muscle tissues. The goal of this study was to culture engineered muscle tissues in a novel bioreactor in both static and mechanically stimulated cultures and evaluate the expression of MTJ-specific proteins within the muscle-tendon unit(paxillin and type XXII collagen). C2C12 myoblasts were seeded in hydrogels made from type I collagen ortendon-derived extracellular matrix (tECM) and allowed to form around movable anchors. Engineered tissues were allowed to form and stabilize for 10 days. After 10 days in the culture, stimulated cultures were cyclically stimulated for 3 hours per day for 2 and 4 weeks alongside static cultures. Strain values at the maximum displacement of the anchors averaged about 0.10, a target that has been shown to induce myogenic phenotype in C2C12s. Protein expression of paxillin after 2 weeks did not differ between hydrogel materials in static cultures but increased by 62% in tECM when mechanically stimulated. These differences continued after 4 weeks, with 31% and 57% increases in tECM tissues relative to type I collagen. Expression of type XXII collagen was similarly influenced by hydrogel material and culture conditions. Overall, this research combined a relevant microenvironment to study muscle and tendon interactions with a novel bioreactor to apply mechanical strain, an important regulator of the formation and maintenance of the native MTJ.

Bacillus subtilis NDmed, a model strain for biofilm genetic studies

The Bacillus subtilis strain NDmed was isolated from an endoscope washer-disinfector in a medical environment. NDmed can form complex macrocolonies with highly wrinkled architectural structures on solid medium. In static liquid culture, it produces thick pellicles at the interface with air as well as remarkable highly protruding ‘‘beanstalk-like’’ submerged biofilm structures at the solid surface. Since these mucoid submerged structures are hyper-resistant to biocides, NDmed has the ability to protect pathogens embedded in mixed-species biofilms by sheltering them from the action of these agents. Additionally, this non-domesticated and highly biofilm forming strain has the propensity of being genetically manipulated. Due to all these properties, the NDmed strain becomes a valuable model for the study of B. subtilis biofilms. This review focuses on several studies performed with NDmed that have highlighted the sophisticated genetic dynamics at play during B. subtilis biofilm formation. Further studies in project using modern molecular tools of advanced technologies with this strain, will allow to deepen our knowledge on the emerging properties of multicellular bacterial life.

Intervertebral Disc‐on‐a‐ChipMF: A New Model for Mouse Disc Culture via Integrating Mechanical Loading and Dynamic Media Flow

This study aims to develop an ex vivo organ‐on‐a‐chip model, intervertebral Disc‐on‐a‐ChipMF, to investigate integrated effects of mechanical loading and nutrition on disc health. The system consists of a detachable multilayer microfluidic chip, a Computer‐Arduino‐based control system, and a mechanical loading unit, which are optimized for accurate axial force measurement and the maintenance of a 21‐day ex vivo disc culture. To ensure accuracy of axial force, the axial mechanical loading regimen is optimized, using the Computer‐Arduino‐based system and low‐profile force sensors (LPFS) to control the mechanical loading unit, and the force distribution on the disc surface is modeled by computational simulation. A 21‐day ex vivo disc culture is demonstrated using the Disc‐on‐a‐ChipMF system, with optimized mechanical loading (0.02 MPa at 1Hz, 1.5 hr day−1) and flow rate (1 µL min−1). The structural integrity, collagen breakdown, catabolic enzyme activities, and disc cell and collagen alignment reveal that the on‐chip cultured discs exhibit a preferred disc health similar to that of native discs for up to 21 days, while discs in a static culture show degenerative changes. The mouse Disc‐on‐a‐ChipMF system mimics in vivo disc microenvironment and provides a valuable platform for studying the effects of various factors on disc health and degeneration and testing new therapies.