The research team, working at the University of Minnesota under protocols approved by the institution’s animal care committee, isolated primary hepatocytes from Sprague–Dawley rats using a two-step perfusion method. Livers were first flushed with University of Wisconsin solution, then perfused ex vivo with Krebs–Henseleit buffer followed by collagenase to digest the tissue architecture, after which gentle mechanical disruption released the cells. Trypan blue exclusion confirmed viabilities between 95 and 98 percent, ensuring that the starting material was healthy enough for the demanding aggregation process ahead. Each experimental condition drew on hepatocytes from a single isolation, a design choice that minimized inter-isolation variability and allowed the investigators to attribute differences in spheroid morphology specifically to the mechanical and seeding parameters under test.

The formation method itself is strikingly simple. Cells were plated at either 1.0 or 1.5 million cells per milliliter in 20 milliliters of spheroid formation media in rectangular Mayo dishes, which were then placed on rocker platforms housed inside a standard humidified carbon dioxide incubator at 37 degrees Celsius. The team tested five rocking speeds—7, 10, 12, 15, and 18 cycles per minute—over seven days of culture, and to rule out instrument-specific artifacts they ran matched conditions on two different commercial rocker platforms. After modest adjustment of speed, tilt angle, and inoculation density, the two platforms produced comparable spheroid size distributions, suggesting that the underlying physics of the system, rather than any particular device, governs the outcome. Within 24 hours, media was exchanged from spheroid-forming to spheroid culture conditions, and fluorescent live-dead staining with acridine orange and propidium iodide revealed smooth-bordered spherical aggregates with nearly complete viability.

The headline finding is a clean inverse relationship between rocking speed and spheroid size. At day 7, mean spheroid diameters measured 199 micrometers at 7 cycles per minute, 173 at 10, 129 at 12, 90.9 at 15, and just 80.2 micrometers at 18 cycles per minute. Median spheroid areas told the same story, falling from roughly 29,400 square micrometers at the slowest speed to 4,300 square micrometers at the fastest. Statistical analysis using the Kruskal–Wallis test confirmed significant effects of both culture day and a speed-by-day interaction on spheroid area, with post hoc Mann–Whitney comparisons showing the inverse speed-size relationship held at every time point from day 1 through day 7. The physical explanation is intuitive: faster rocking imposes greater shear forces and reduces the contact time available for cells to find and bind one another, while slower motion permits extended cell-cell interaction and larger aggregate growth.

There is, however, a floor to the relationship. At 7 cycles per minute, consistent spheroids did not appear until day 3, and in some dishes they never formed at all, presumably because the kinetic energy available for aggregation fell below a critical threshold. The team also observed a characteristic sinusoidal pattern in spheroid size across the week: aggregates grew for the first several days, peaked around days 3 to 5, and then contracted. At 10 cycles per minute, for example, mean area rose from about 13,750 square micrometers on day 1 to a peak of 26,730 on day 4 before settling back to roughly 22,500 by day 7. The authors attribute this shrinkage not to cell loss but to compaction, as maturing intercellular junctions and cytoskeletal remodeling draw the aggregate into a tighter, denser sphere.

Inoculation density provided the second, independent control axis. Holding speed constant at 15 cycles per minute, cultures seeded at 1.5 million cells per milliliter produced significantly larger spheroids than those seeded at 1.0 million at every time point measured, with day 7 mean areas of 7,203 versus 5,627 square micrometers. A significant density-by-day interaction indicated the magnitude of the effect shifted over the culture period, but the overall temporal pattern of early expansion followed by stabilization was preserved. The mechanism again follows from simple probability: more cells per milliliter means more frequent cell-cell encounters, greater aggregate stability, and ultimately larger and more numerous spheroids. Together, speed and density give labs a two-dimensional parameter space in which a desired spheroid size can be targeted rather than left to chance.

Critically, the team showed that size tuning is biologically meaningful, not merely a morphological curiosity. Urea production normalized to DNA content peaked at intermediate rocking speeds, with a median of about 3.47 micrograms per microgram at 12 cycles per minute, while the slowest condition at 7 cycles per minute produced almost none—consistent with diffusion-limited metabolism in oversized aggregates—and production declined again at the highest speeds, possibly reflecting shear stress or insufficient cell-cell signaling. Albumin and urea output both climbed steadily over ten days of culture, with albumin rising from a median of roughly 1,087 nanograms per microgram DNA on day 1 to about 1,534 by day 10. When converted to per-cell terms, these spheroids secreted an estimated 7 to 9 micrograms of albumin per million cells per day, several-fold higher than reported values for static two-dimensional hepatocyte cultures, and urea output by day 7 exceeded that of conventional hepatocyte monolayers.

Efficiency and variability metrics rounded out the practical picture. Volumetric analysis on day 4 estimated that a median of 86 percent of seeded hepatocyte volume had been incorporated into spheroids, with four of five datasets clustering between 73 and 95 percent, the lower values likely reflecting occasional cell adherence to dish walls and the tendency of compacting spheroids to be undersized by diameter-based volume estimates. Size dispersion, quantified by the coefficient of quartile variation, was widest on day 1—as expected during initial aggregation—and converged to a stable 21 to 24 percent by day 7 across all speeds, a range the authors note is acceptable for bioartificial liver applications even though static microwell methods can achieve tighter distributions on the order of plus or minus 10 to 20 micrometers.

The authors are candid about limitations. All experiments used primary rat hepatocytes, and interspecies differences in cytochrome P450 expression, metabolic capacity, and cell-cell dynamics mean extrapolation to human cells requires caution, though the underlying physical principles should generalize. Size was quantified from two-dimensional images assuming ellipsoidal geometry, and long-term culture beyond seven days was not assessed. Absolute parameter values may also need recalibration on different rocker platforms. Still, the study’s contribution is a framework rather than a recipe: by demonstrating that two universally available variables reproducibly govern spheroid dimensions while preserving metabolic function, the work hands bioartificial liver engineers, toxicologists, and regenerative medicine researchers a scalable, inexpensive tool for standardizing one of the field’s most important building blocks—no specialized equipment required.

Subject of Research: Tunable control of hepatic spheroid size through rocking speed and cell inoculation density

Article Title: The effect of rocking speed and cell inoculation density on hepatic spheroid size: A simple method for size modulation

Article References: Megaly, M. G., Tobolt, D., Chen, A. A.-F. D., Namsrai, B.-E., Fisher, B., Rao, J. S., Ramesh, S., Chimedtseren, A., Nyberg, S. L., Clemens, M., Lee, C. Y., Bischof, J. C., & Finger, E. B. (2026). The effect of rocking speed and cell inoculation density on hepatic spheroid size: A simple method for size modulation. Bioengineering & Translational Medicine, 11(5), Article e70154. https://doi.org/10.1002/btm2.70154

Image Credits: AI Generated

DOI: 10.1002/btm2.70154

Keywords: hepatic spheroids, bioartificial liver, hepatocytes, rocking speed, inoculation density, spheroid size, drug toxicity testing, regenerative medicine, 3D cell culture, urea production, albumin secretion, shear stress

The construct, which the team calls SPHEGaT — spheroids of endocrine granulosa and theca cells — was built from primary human cells with careful attention to spatial organization. Granulosa cells, harvested from patients undergoing in vitro fertilization, expressed the aromatase enzyme CYP19A1 and the follicle-stimulating hormone receptor, marking them as the estrogen-producing core. Theca-like cells were differentiated from ovarian stromal cells isolated from postmenopausal donors, and they expressed CYP17A1 and CD13, consistent with their role in producing the androgens that granulosa cells then convert into estradiol. When the granulosa cells were seeded into low-attachment plates, they spontaneously self-aggregated into compact spheres within about three days. Theca-like cells were then added and progressively organized around the granulosa core, and a thin layer of collagen type I was deposited between the two compartments to mimic the follicular basement membrane. The result was a multilayered structure that echoes the compartmental arrangement of a native follicle.

Architecture, it turns out, is not merely cosmetic. After nine days of culture, the spheroids were remarkably healthy: quantitative analysis showed that 97.45 percent of the spheroid area remained viable, with no detectable focal necrosis. Dense three-dimensional aggregates often suffer from oxygen starvation in their cores, a problem the team checked directly by staining for HIF-2α, a marker of hypoxic stress. Only about 3 percent of nuclei were positive, and three-dimensional reconstruction confirmed the absence of a hypoxic core. Critically, the spheroids were functionally competent: the culture medium contained endogenous levels of estradiol at roughly 65 picograms per milliliter and progesterone at roughly 186 nanograms per milliliter. The spatially organized assembly had produced a living, breathing endocrine unit — one that secretes hormones the way a follicle does, rather than sitting passively in a bath of supplied steroids.

The decisive experiment came when the researchers coupled these spheroids to endometrial organoids, miniature gland-like structures grown from endometrial biopsies of women in their late twenties. Over six days of co-culture, the organoids underwent a visible transformation. Monocultured organoids retained mostly spherical, cyst-like shapes, but organoids exposed to the ovarian spheroids progressively developed folded, lobulated, and optically denser architectures. The proportion of folded organoids rose from about 2.4 percent in monoculture to nearly 13.7 percent in co-culture by day six, a highly significant difference. Circularity and solidity — geometric measures of how round and how smooth an organoid is — both declined over time in the co-cultured condition, indicating the emergence of irregular, invaginated epithelial structures reminiscent of glandular remodeling in the cycling endometrium.

What makes this remodeling striking is that it was not driven by proliferation. Ki67 staining, a standard marker of dividing cells, showed no significant difference between monocultured and co-cultured organoids, and total organoid area grew comparably in both conditions. The ovarian spheroids were not making the tissue grow faster; they were teaching it to reorganize. This mirrors in vivo physiology, where epithelial proliferation peaks during the estrogen-dominated proliferative phase and then stabilizes as progesterone steers the tissue toward secretory maturation. A trend toward reduced vimentin expression in co-cultured organoids further hinted at a dampening of epithelial-mesenchymal plasticity, a shift associated with secretory differentiation. In short, the ovarian signal was instructing architecture, not cell division.

At the molecular level, the co-culture produced clear evidence of endocrine reprogramming. Progesterone receptor expression surged in co-cultured organoids: only about 4.7 percent of nuclei were progesterone receptor-positive in monoculture, compared with roughly 40.5 percent after exposure to the spheroids. Estrogen receptor levels, by contrast, remained unchanged. The co-cultured organoids also upregulated a selective set of progesterone-responsive, secretory-phase genes, including PAEP, which encodes pregnancy-associated endometrial protein; SPP1, or secreted phosphoprotein 1; and HSD17B2, an enzyme involved in steroid metabolism. PAEP secretion, measured by ELISA, rose progressively in co-culture. Other receptivity-associated genes such as LIF, SOX17, GATA2, and AREG did not change significantly, indicating that the organoids entered a partial, progesterone-driven secretory state rather than acquiring the full implantation-window program — a limitation the authors attribute in part to the absence of an estrogen-priming phase in the current protocol.

One of the most intriguing observations was a hint of bidirectional communication. In the co-culture system, progesterone levels in the medium dropped markedly — to about 68 nanograms per milliliter compared with roughly 239 in spheroid monocultures — while estradiol levels stayed stable. This hormone-specific reduction suggests the endometrium may be actively metabolizing, taking up, or otherwise modulating ovarian progesterone output, echoing the known ability of uterine prostaglandins and other mediators to regulate luteal steroid production in vivo. The platform, in other words, does not just deliver hormones to the endometrium; it establishes a coupled endocrine axis in which hormone availability reflects ongoing integration between the two tissue compartments.

To test whether the morphogenic effects depended on steroids alone, the team performed an elegant depletion experiment. They treated spheroid-conditioned medium with dextran-coated charcoal, stripping out roughly 59 percent of the estradiol and 90 percent of the progesterone, and then compared three conditions: intact conditioned medium, depleted medium, and depleted medium with exogenous hormones added back. The results were unambiguous. Untreated conditioned medium induced the greatest epithelial folding over time. Depleted medium still supported a reduced but significant increase in folding — hinting at residual steroid activity or non-steroidal factors — but static hormone add-back failed entirely to restore the morphogenic phenotype and actually produced the lowest folding response of all. Organoids in the add-back condition grew larger but stayed smoother, suggesting that supraphysiologic or imbalanced steroid exposure biases tissue toward expansion rather than differentiation.

The implication is profound: the ovary’s instructive power over the endometrium cannot be reduced to a prescription of estradiol and progesterone. The intact follicle is a complex endocrine and paracrine factory, co-secreting inhibins and activins, growth factors such as EGF-like ligands and basic FGF, cytokines, metabolites, carrier proteins, and extracellular vesicles loaded with proteins, lipids, and regulatory RNAs. Many of these factors have been implicated in endometrial remodeling and implantation, and the temporal fluctuations, concentration gradients, and local metabolism inherent to an intact ovarian unit may be as important as any single molecule. Static hormone replacement, the study suggests, fundamentally cannot reproduce that context.

The researchers are candid about the limitations. The system models a progesterone-dominant environment without a preceding estrogen-priming phase, so it captures only part of the menstrual cycle’s proliferative-to-secretory transition. The co-culture lacks stromal decidualization, immune cells, vascular elements, and embryo-derived signals, all of which contribute to true uterine receptivity. The theca-like cells derive from postmenopausal donors, raising questions about age-related epigenetic carryover, and charcoal-dextran treatment may have altered non-steroidal components of the conditioned medium. Yet these caveats do not diminish the central achievement. The team has built the first spatially organized, fully human ovarian endocrine construct capable of directing endometrial morphogenesis in vitro, and shown that its effects exceed anything achievable with hormones alone. The platform opens a tractable window onto infertility, implantation failure, progesterone resistance, and endocrine disruption — and points toward a future generation of reproductive models built not on hormone recipes, but on reconstructed, talking tissues.

Subject of Research: Engineering spatially organized human ovarian spheroids to model ovarian-endometrial endocrine communication in vitro

Article Title: Spatially Organized Human Ovarian Spheroids Instruct Endometrial Morphogenesis

Article References: Sousa, M. J., Vriendt, S. D., Liu, L., Ruiz, T. F. R., Vankelecom, H., & Amorim, C. A. (2026). Spatially Organized Human Ovarian Spheroids Instruct Endometrial Morphogenesis. Advanced Science, 13(55), Article e76538. https://doi.org/10.1002/advs.76538

Image Credits: AI Generated

DOI: 10.1002/advs.76538

Keywords: ovarian spheroids, endometrial organoids, granulosa cells, theca-like cells, progesterone signaling, estradiol, steroidogenesis, reproductive biology, organ-on-a-chip, endometrial morphogenesis, infertility modeling, 3D cell culture

The researchers, led by corresponding authors Seok Chung of Korea University and Hyun Jik Kim of Seoul National University College of Medicine, obtained primary nasal mucosal tissue from healthy donors undergoing nasal surgery, with institutional ethical approval and written informed consent. From these samples they isolated nasal epithelial cells and embedded them in a Matrigel-based three-dimensional culture system, a matrix-rich environment that allows epithelial stem and progenitor cells to self-organize into hollow, polarized structures. Over successive culture cycles, the organoids expanded and could be maintained through multiple passages, a property that distinguishes them from conventional two-dimensional nasal epithelial cultures, which the team found could not be carried reliably beyond passage two. The organoids, by contrast, sustained successful expansion up to passage five, giving laboratories a substantially longer working window from each donor sample.

Characterizing what the organoids actually contained was a central task of the study. The team applied a battery of complementary techniques: histological staining to examine tissue architecture, immunostaining to identify specific cell populations, scanning and transmission electron microscopy to resolve surface features and ultrastructure, and quantitative real-time polymerase chain reaction to measure gene expression. Together these analyses showed that the organoids recapitulated the structural characteristics of the human nasal epithelium and harbored seven distinct cell types. Single-cell RNA sequencing, which profiles gene expression in individual cells, confirmed this cellular diversity and allowed the researchers to map differentiation trajectories, tracing how progenitor populations give rise to the mature cell types that populate the nasal lining.

The cellular composition was not, however, a perfect mirror of the tissue in situ. The organoids contained a markedly higher proportion of basal progenitor cells, the self-renewing population from which the epithelium regenerates, while ciliated cells, the hair-like projections that sweep mucus and trapped pathogens along the airway, were less dominant than in native tissue. This skew toward a progenitor-rich state is a common feature of organoid systems, which favor proliferative cells under culture conditions. It also reflects the biology of the source: basal cells are the engine of epithelial renewal, and their enrichment is precisely what allows the organoids to be passaged repeatedly. For infection studies, the presence of the relevant target cells matters more than an exact census, and the organoids retained the differentiated lineages needed to model virus-host interaction.

To test whether the platform could support respiratory virology, the team exposed the organoids to influenza A virus, using the H1N1 strain A/WS/33. Infection assays measured viral nucleoprotein, a structural component of the virus whose detection marks infected cells, and tracked the course of infection over days post-infection. The organoids proved permissive to influenza A virus, supporting viral entry and replication in a way that made them, in the authors’ assessment, an adequate in vitro model for influenza infection. This is a nontrivial result: many epithelial culture systems either fail to support productive infection or do so inconsistently, undermining their usefulness for reproducible experiments on viral pathogenesis.

The most consequential findings concerned the innate immune response. Following infection, the organoids showed a sharp induction of messenger RNA for interferons, the signaling proteins that constitute the body’s first molecular alarm against viral invaders, and for interferon-stimulated genes, the downstream antiviral effectors that interferons switch on. This interferon-driven cascade is the epithelium’s principal intrinsic defense, and its faithful reproduction in vitro is what turns a static model of tissue architecture into a dynamic model of virus-host biology. With the organoids, the researchers could observe both halves of the interaction: the virus exploiting host cells for replication, and the host cells responding with a coordinated antiviral transcriptional program.

Methodologically, the study also compared the organoid system with air-liquid interface culture, a widely used technique in which epithelial cells are grown on a permeable membrane with their apical surface exposed to air, encouraging differentiation into the full repertoire of airway cell types. Air-liquid interface cultures remain valuable, but the passage limitation the team observed, with quality degrading after passage two, constrains how many experiments a single donor’s cells can support. The organoids’ ability to reach passage five while sustaining differentiation potential addresses a practical constraint that has limited sample throughput in airway epithelial research, particularly for studies requiring paired comparisons across multiple donors or repeated infection experiments from the same genetic background.

The single-cell RNA sequencing component adds a layer of analytical depth that extends beyond simple characterization. By resolving cellular heterogeneity within the organoids and reconstructing differentiation trajectories, the technique allows researchers to ask which cell populations are infected, which respond with interferon production, and how progenitor cells mature along defined pathways. This kind of resolution matters for influenza biology, because the virus shows preferences for particular cell types within the airway, and the strength of the interferon response can vary with the infected population. A model whose cellular composition is mapped at single-cell resolution gives virologists a framework for interpreting such differences rather than treating the epithelium as a uniform sheet of cells.

The broader significance of the work lies in the gap it fills. Respiratory viral research has relied heavily on immortalized cell lines, such as the Madin-Darby canine kidney cells that remain a standard substrate for propagating influenza viruses, and on animal models whose airway biology differs from that of humans in ways that complicate translation. Human airway organoids, including those derived from nasal tissue, have emerged over the past decade as an intermediate option, closer to native physiology than cell lines and more ethically tractable than animal experimentation. The Korean team’s contribution is to demonstrate that nasal organoids specifically can be established reproducibly from surgical specimens, expanded through multiple passages, differentiated into the major epithelial lineages, and used to study influenza infection with a quantifiable innate immune readout.

There are, of course, limits to what any in vitro system can claim. The organoid composition, with its basal-cell enrichment and reduced ciliated fraction, differs from native nasal epithelium, and the study used a single laboratory strain of influenza A virus, H1N1 A/WS/33, rather than the circulating or avian strains that drive public health concern. The authors frame the platform as a tool for exploring interferon-related innate immune responses following influenza infection, a scope that future work can broaden to other respiratory pathogens, including SARS-CoV-2 and respiratory syncytial virus, and to patient-derived samples from individuals with allergic rhinitis, chronic rhinosinusitis, or other conditions that alter nasal epithelial biology. As such extensions accumulate, the nasal organoid system described here could become a standard bridge between donor tissue and antiviral research, allowing the first hours of a respiratory infection to be watched, measured, and manipulated entirely within the walls of a laboratory dish.

Subject of Research: Human nasal epithelial organoids as an in vitro model for airway differentiation and influenza virus innate immune responses

Article Title: Human nasal organoids sustain reproducible airway epithelial differentiation and influenza virus-host innate immune interaction

Article References: Jin, S., Kim, S., Kim, M., Cha, H., Chung, S., & Kim, H. J. (2026). Human nasal organoids sustain reproducible airway epithelial differentiation and influenza virus-host innate immune interaction. Journal of Translational Medicine. https://doi.org/10.1186/s12967-026-08908-2

Image Credits: AI Generated

DOI: 10.1186/s12967-026-08908-2

Keywords: organoids, nasal epithelium, influenza A virus, innate immunity, interferon, air-liquid interface, single-cell RNA sequencing, 3D cell culture, respiratory virology, basal progenitor cells, ciliated cells, in vitro model

The tumor microenvironment, or TME, is far more than a passive backdrop for malignant cells. It is a dense, dynamic ecosystem of endothelial cells, immune cells, neurons, stromal cells, and secreted signaling molecules, all woven into an extracellular matrix composed of structural proteins such as collagen and elastin, adhesive glycoproteins like fibronectin and laminin, and charged polysaccharide chains. This matrix does not merely hold cells in place; it actively determines how fast tumors progress, how readily they metastasize, and how they respond to therapy. Proteoglycans within the matrix, for example, can remodel the biochemical landscape to open pathways through which cancer cells invade the bloodstream, a metastatic step known as intravasation. Once malignant cells reach distant tissues, they may even enter a quiescent, dormant state in the G0 phase of the cell cycle, evading both immune surveillance and chemotherapy until conditions favor renewed growth.

Hydrogels, the focus of the new review, are three-dimensional polymer networks formed by chemically or physically crosslinking hydrophilic building blocks such as hydroxyl, carboxyl, sulfonic acid, and amine groups. Their capacity to absorb large quantities of water gives them a softness and fluidity remarkably similar to soft biological tissue, which is precisely why tissue engineers have embraced them as extracellular matrix mimics. The choice of polymer backbone and crosslinking strategy directly dictates the mechanical strength, porosity, and degradation rate of the resulting material, allowing researchers to tune stiffness and biochemistry with a precision that animal models and plastic dishes cannot match. Hydrogels enable cells to interact with their surroundings on all sides, preserving the dynamic cell-matrix conversations that drive proliferation, migration, and intercellular communication.

The review classifies hydrogels into natural, synthetic, smart, and hybrid categories, each with distinct trade-offs. Natural polymers, including collagen, fibrin, alginate, hyaluronic acid, and chitosan, offer inherent bioactivity and enzymatic biodegradability because they resemble the extracellular matrix the body already knows how to process. Collagen, the most abundant structural protein in skin, cartilage, and bone, remains the workhorse of natural hydrogel matrices. Synthetic polymers such as polyethylene glycol, polyvinyl alcohol, and polyacrylic acid, by contrast, provide adjustable stiffness, reproducible composition, and regulatory pedigree, but they lack natural cell-binding sites and must be decorated with adhesive proteins. Concerns also persist about cytotoxic degradation products; PLGA, for instance, breaks down into acidic by-products that can lower local pH and provoke inflammation, while PEG can yield reactive species under long-term oxidative stress.

Hybrid systems attempt to capture the best of both worlds. PEG-heparin hydrogels, which combine a synthetic backbone with an anionic glycosaminoglycan, have emerged as strong alternatives to Matrigel, the widely used but compositionally ill-defined protein mixture derived from tumor cells. Compared with Matrigel, PEG-heparin platforms offer defined composition, greater reproducibility, and easier modification, and several have already advanced into preclinical and early clinical studies for drug delivery and tissue regeneration. Crosslinking chemistry adds another layer of control: physical crosslinking produces reversible, stimuli-responsive, and cytocompatible networks with modest mechanical strength, whereas covalent strategies such as photopolymerization, Michael addition, click chemistry, and enzymatic crosslinking deliver superior structural integrity and more predictable degradation kinetics.

The practical payoff of these materials is already visible across the five major cancer types examined in the review. In lung cancer, which claimed roughly one hundred twenty thousand American lives in 2024, a temperature-sensitive hydrogel integrating realgar arsenic sulfide with Fe3O4 magnetic nanoparticles created a combined imaging and thermotherapy platform, the first hydrogel formulation of its kind for that compound. Separately, an injectable hydrogel delivering the anti-angiogenic drug anlotinib through a hyaluronic acid-tyramine matrix inhibited endothelial cell proliferation and produced tumor suppression rates ten to twenty percent higher than the free drug across all tested doses, while reducing systemic toxicity. Matrigel-based cultures have even been used to test combined MEK inhibition and anti-PD-L1 immunotherapy in patient-derived three-dimensional spheroids, probing the tumor immune landscape outside the body.

Liver cancer models show a similar leap in physiological relevance. A three-dimensional culture system built from decellularized liver extracellular matrix hydrogel enhanced the self-renewal, migration, and drug resistance of HepG2 cells, unmasking cancer stem cell properties that flat cultures conceal and opening a route to stem cell-targeted drug discovery. A thermosensitive Pluronic F127 hydrogel co-delivering resveratrol microspheres and cisplatin enabled localized intraperitoneal chemotherapy, with resveratrol counteracting cisplatin’s side effects and prolonging drug retention in the abdominal cavity. Synthetic PEG-cysteine hydrogels and organic-inorganic PDMS-TEOS scaffolds, meanwhile, boosted albumin synthesis and urea production in hepatocyte-like cultures, markers of genuine liver function that monolayer systems routinely fail to sustain.

In breast cancer, a chitosan-based thermosensitive hydrogel seeded with 4T1 mouse mammary carcinoma cells produced tumors in mice that grew approximately 2.5-fold larger and weighed twice as much as those from conventional two-dimensional injections, while markedly elevating the cancer stem cell markers CD44 and CD24, demonstrating that the hydrogel platform genuinely replicates the in vivo niche. pH-sensitive chitosan nanocarriers extended the bioactivity of curcumin, a polyphenol crippled by poor solubility, and an L-alanine-derived hydrogel released doxorubicin specifically at acidic pH values characteristic of tumor tissue. An injectable, temperature-sensitive hydrogel loaded with titanium carbide nanoparticles even enabled laser-triggered photothermal therapy, heating tumors locally to kill cancer cells while sparing surrounding tissue.

Prostate and colon cancer applications extend the technology from disease modeling to clinical devices and microbiome engineering. Injectable hydrogel spacers such as SpaceOAR, placed between the prostate and rectum during radiotherapy, physically shield the bowel from radiation for three to six months before dissolving harmlessly, reducing rectal pain, bleeding, and other treatment toxicities. Matching hydrogel stiffness to the bone-like mechanical environment of prostate metastases, around 25 to 40 kilopascals, has proven essential for studying invasion and drug resistance. In colorectal cancer, pH-sensitive alginate beads reinforced with hydroxyapatite and magnetic nanoparticles protected the chemotherapy drug 5-fluorouracil from stomach acid and released it in the intestine, while hyaluronan-based organoid co-cultures combining patient-derived tumor cells with cancer-associated fibroblasts delivered personalized drug-testing platforms. Perhaps most strikingly, a living hydrogel embedded with the bacterium Thiobacillus denitrificans metabolizes immunosuppressive hydrogen sulfide in the colon tumor microenvironment into harmless sulfate, normalizing tumor vasculature and amplifying the efficacy of co-delivered camptothecin.

The review’s authors are candid about the obstacles that stand between these laboratory triumphs and routine clinical use. Natural hydrogels suffer from batch-to-batch variability, weak mechanical strength, and rapid, uneven degradation; synthetic ones raise questions about long-term biodegradation and residual monomer toxicity. Smart, stimuli-responsive hydrogels depend on environmental triggers whose values shift between individuals, disease states, and tissue locations, making therapeutic outcomes difficult to predict. Reproducibility, standardization, and clinical validation remain stubborn hurdles, and manufacturing dual-responsive systems under Good Manufacturing Practice conditions is still rare. Yet the trajectory is unmistakable: 3D bioprinting and photopolymerization are giving researchers precise spatial control over tumor architecture, artificial intelligence is accelerating polymer design, and injectable hydrogels are proving they can localize chemotherapy, immunotherapy, and even engineered bacteria directly at tumor sites. As the authors conclude, hydrogels have evolved from simple extracellular matrix mimics into powerful platforms for decoding tumor biology, and the models they enable may well define the next generation of precision oncology.

Subject of Research: Hydrogel-based three-dimensional models of the tumor microenvironment in cancer research

Article Title: Hydrogel applications in tumor microenvironment modeling

Article References: Karakaya, M., Karataş, R. B., Ayaz, F., & Aydemir, E. (2025). Hydrogel applications in tumor microenvironment modeling. Discover Biotechnology, 2(1), Article 21. https://doi.org/10.1007/s44340-025-00029-8

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00029-8

Keywords: hydrogels, tumor microenvironment, 3D cell culture, extracellular matrix, drug delivery, cancer models, organoids, bioprinting, immunotherapy, biomaterials, precision oncology, spheroids

Replicative aging is the progressive decline that cells undergo as they divide again and again during in vitro expansion. It is distinct from the abrupt, irreversible state of full senescence; instead, it is a gradual erosion of function that unfolds long before cell division stops completely. The hallmarks are familiar to anyone who works with cultured cells: rising levels of the cell cycle inhibitors p16, p21, and p53, increased activity of the senescence-associated beta-galactosidase enzyme, and a slow decay of mitochondrial performance. Mitochondria sit at the center of this decline. As cells keep dividing, their respiratory machinery falters, ATP output drops, and reactive oxygen species spilling from the electron transport chain damage mitochondrial DNA and oxidize mitochondrial proteins. Quality control systems such as mitophagy struggle to keep pace, lesions accumulate, gene expression profiles shift, and eventually proliferation halts altogether. For researchers hoping to manufacture enough limbal niche cells for clinical use, this aging bottleneck has been a persistent barrier.

A new study published in Aging Cell suggests a way around it. A research team based at Tongji Hospital in Wuhan, China, reports that growing limbal niche cells in a three-dimensional Matrigel-based culture system, rather than on the conventional flat, two-dimensional plastic surface, effectively reverses their replicative aging. The work, which combines classic cell biology with single-cell RNA sequencing and genetic gain- and loss-of-function experiments, identifies the transcription factor FOSL1 as a pivotal mediator of this rejuvenation. The findings not only illuminate how three-dimensional architecture preserves cellular youth, but also point toward FOSL1 as a potential target for revitalizing aged cells destined for regenerative therapies.

The rationale for the three-dimensional approach grew out of earlier work. Flat culture surfaces fail to reproduce the rich microenvironment of living tissue, and stem cells grown on them for extended periods tend to drift away from their native identity, losing function as they go. Mesenchymal stem cells grown in three dimensions, by contrast, spontaneously form spheroids and display stronger secretory profiles, better adaptive capacity, and enhanced immunomodulatory behavior, and several studies have suggested that such conditions can ameliorate aging phenotypes. The Tongji group had previously observed that limbal niche cells reseeded onto a Matrigel scaffold in the fourth generation showed a striking rebound in embryonic stem cell marker expression. The new study set out to test whether three-dimensional culture could do more than preserve youthfulness, namely whether it could actually turn back the aging clock.

To build the three-dimensional system, the researchers mixed limbal niche cells with Matrigel at a fifty percent volume ratio and deposited the suspension as small hemispherical droplets in culture plates, which were inverted briefly to encourage gelation and prevent cells from settling. Within this soft, matrix-rich environment, the cells did something remarkable: they self-assembled into spheroids whose diameters grew steadily over six days of culture, a sign of active proliferation and matrix remodeling. The cells used in the experiments were isolated from corneal tissue donated by individuals aged sixty to eighty, digested from limbal tissue with collagenase, and purified through serial passaging until they expressed mesenchymal markers such as vimentin, CD73, and CD90 while losing epithelial markers.

When the team compared aged limbal niche cells maintained in standard two-dimensional culture with counterparts of the same passage grown as three-dimensional spheroids, the differences were unmistakable. The three-dimensionally cultured cells showed markedly higher expression of the proliferation marker Ki67 and stronger growth in CCK-8 proliferation assays across seventy-two hours. Their expression of the stemness markers SOX2, OCT4, and NANOG, measured both by immunofluorescence and quantitative PCR, rebounded significantly. Meanwhile, the aging indicators moved in the opposite direction: far fewer cells stained positive for senescence-associated beta-galactosidase, and the levels of p16, p21, p53, and the DNA damage marker gamma-H2AX all fell. In short, the cells grown in three dimensions looked and behaved younger than their flat-grown siblings.

To understand how this rejuvenation operated at single-cell resolution, the researchers performed single-cell RNA sequencing on early-passage and late-passage cells grown in both formats. Unsupervised clustering revealed ten distinct transcriptional populations. Two of these, clusters C2 and C3, were enriched for cell cycle genes but also carried strong signatures of cellular senescence and p53 signaling, suggesting they were in a precarious transitional state between proliferation and stress-induced aging. These senescence-prone clusters were abundant in two-dimensional cultures but shrank dramatically in the three-dimensional environment. Conversely, a cluster designated C5, defined by enrichment in DNA replication, cell cycle progression, and mitotic nuclear division, expanded significantly under three-dimensional conditions. The rejuvenation, in other words, was not a uniform shift across all cells but a subpopulation remodeling: three-dimensional culture pruned the aging-prone cells and selectively amplified a proliferative, DNA-replication-active fraction.

The search for the molecular driver of this remodeling led to FOSL1, an AP-1 family transcription factor previously known for roles in proliferation, differentiation, and stress adaptation, and for maintaining the stemness of limbal epithelial stem cells in the cornea. By intersecting genes that decline as cells age in two dimensions with genes that rise in three-dimensional culture, the team identified twenty-one candidate rejuvenation regulators, with FOSL1 at the center of a protein interaction network that included fellow AP-1 members FOS and JUN as well as stress-responsive factors MAF and EGR1. FOSL1 expression fell sharply between early and late passages, and functional experiments confirmed its importance: silencing FOSL1 in young cells accelerated aging, increasing senescence staining and aging markers while suppressing proliferation, whereas overexpressing FOSL1 in aged cells did the reverse, restoring proliferative capacity and reducing senescence markers.

Mitochondria emerged as a key venue of FOSL1’s anti-aging activity. When FOSL1 was knocked down in early-passage cells, intracellular reactive oxygen species and mitochondrial superoxide rose, transmission electron microscopy revealed swollen mitochondria with disrupted cristae and vacuolization, and JC-1 staining showed a collapse of mitochondrial membrane potential. Overexpressing FOSL1 in aged cells improved mitochondrial ultrastructure, restored more ordered cristae, and partially recovered membrane potential. The authors suggest that FOSL1 may coordinate a dual defense, enhancing antioxidant capacity through pathways such as NRF2 while preserving mitochondrial health to curb oxidant generation at its source, thereby breaking the vicious cycle in which reactive oxygen species and mitochondrial damage feed each other.

The clinical implications are considerable. Limbal niche cell therapies for limbal stem cell deficiency have been constrained by the limited expansion capacity of conventional two-dimensional culture, which cannot yield sufficient cell numbers before aging sets in. A scalable three-dimensional system that keeps cells youthful, combined with genetic or pharmacological activation of FOSL1, could provide abundant, standardized cell products for bioengineered corneal constructs or direct subconjunctival injection. The authors caution, however, that the findings rest on in vitro experiments and still require validation in animal models of corneal injury, that the downstream targets of FOSL1 remain to be fully mapped, and that FOSL1’s well-documented oncogenic role in epithelial tumors demands careful, locally confined delivery strategies. Within the limbal niche, where the factor is physiologically expressed and even reduced in diseases such as keratoconus, it appears to act as a homeostatic guardian rather than a tumor driver. If future work confirms its safety, FOSL1 could become a molecular lever for turning aged eye-support cells young again.

Subject of Research: Reversal of replicative aging in limbal niche cells through three-dimensional culture and FOSL1 upregulation

Article Title: 3D Culture Reverses Limbal Niche Cell Replicative Aging via FOSL1 Upregulation

Article References: Wang, X., Li, S., Liu, Z., Guo, X., Shen, J., Zhou, T., Liao, S., Huang, X., Wang, W., Xu, L., Zang, X., & Li, G. (2026). 3D Culture Reverses Limbal Niche Cell Replicative Aging via FOSL1 Upregulation. Aging Cell, 25(9), Article e70705. https://doi.org/10.1111/acel.70705

Image Credits: AI Generated

DOI: 10.1111/acel.70705

Keywords: limbal niche cells, cornea, replicative aging, 3D cell culture, FOSL1, mitochondria, senescence, limbal stem cell deficiency, single-cell RNA sequencing, AP-1 transcription factor, regenerative medicine, reactive oxygen species

Traction force microscopy, the foundational method behind CFM, has been a workhorse of mechanobiology for nearly two decades. In its classical form, cells are plated on a flat, elastic gel embedded with fluorescent beads. As a cell pulls and pushes on the gel, the beads shift, and by tracking those displacements researchers can computationally reconstruct the traction stresses the cell applies to its substrate. The approach has revealed how cancer cells turn mechanically aggressive, how stem cells sense the stiffness of their niche, and how fibroblasts remodel wound tissue. But its central limitation has always been geometry: real cells rarely live on open flat surfaces, and their force-generating machinery behaves differently when confined on multiple sides.

Confinement force microscopy addresses this gap with a microconfiner that can be dynamically tuned, positioned with precision and held stable over long imaging periods. The device brings a controlled, deformable ceiling down over cells resting on a soft elastic substrate, effectively sandwiching them in a defined three-dimensional space that mimics the physical confinement of a living tissue. Because the confining element is itself compliant and instrumented, the forces cells exert against it can be measured alongside the traction stresses they generate on the bottom surface. The result is a fuller, more faithful accounting of a single cell’s mechanical output under realistic compressive boundary conditions.

The dynamic character of the platform is one of its most significant advances. Earlier confinement approaches tended to be static, locking cells into a fixed geometry and leaving researchers unable to probe how cells respond as their mechanical environment changes. With CFM, the degree of confinement can be adjusted during an experiment. A cell can be observed in a relatively open state, then progressively constrained, then released, allowing researchers to map how traction stresses evolve as confinement tightens or relaxes in real time. This turns the microconfiner from a passive chamber into an active perturbation tool, capable of asking causal questions about how spatial restriction drives mechanotransduction.

Precision and stability, the other two pillars highlighted by the developers, matter because traction measurements are exquisitely sensitive to boundary conditions. Any drift in the position or compliance of a confining surface contaminates the displacement field on the underlying gel, corrupting the reconstructed force map. The CFM architecture maintains a stable confining geometry over the timescales of live-cell imaging, from minutes to hours, so that force reconstructions remain quantitatively reliable. The precision of the confinement also allows single cells and multicellular aggregates alike to be studied under well-defined conditions, which is essential for comparing mechanical phenotypes across genetic backgrounds, drug treatments or differentiation states.

The applications span an unusually broad swath of biology. In cancer research, one of the central puzzles is how tumor cells invading through dense stiffer tissue generate and reorient forces in tight, crowded spaces. Conventional two-dimensional traction assays cannot reproduce this compressive regime, and invasion assays in organoids or tissue slices typically sacrifice quantitative force readouts for biological realism. CFM offers a bridge between the two: cells or small aggregates can be confined in tissue-like geometries while their complete traction signature is measured with subcellular resolution. Similar logic applies to immune cells squeezing through endothelial barriers, epithelial sheets closing wounds, and stem cells differentiating under the compressive loads of developing organs.

Cell aggregates add a further layer of biological complexity that the platform is specifically designed to handle. Multicellular clusters, such as spheroids used to model early tumors or embryonic tissues, generate forces collectively, coordinating contractility across many cells through junctions and extracellular matrix. Measuring aggregate-scale traction under confinement reveals how mechanical load is distributed across the cluster, where regions of high stress concentrate, and how the collective output changes as the group is compressed. These are questions that neither single-cell assays on flat gels nor bulk rheology of tissue can answer, positioning CFM as a tool for probing emergent mechanics in multicellular systems.

Technically, the method builds on the mature computational machinery of traction force microscopy while adding a second force channel from the confining interface. Displacements of fluorescent markers in the deformable substrate are converted into surface traction fields through regularized inverse algorithms, a process well established in the field. The confining element contributes complementary measurements of the forces transmitted vertically and laterally onto the cell. Combining these datasets requires careful mechanical calibration and modeling of the microconfiner’s own elasticity, but the payoff is a genuinely three-dimensional force balance for a living cell, something the field has pursued for years with more complex and less accessible systems.

The broader significance of the work lies in what it says about the maturation of mechanobiology as a discipline. The field has moved decisively from observing that cells are mechanical objects to quantifying precisely how they sense, generate and transmit force in contexts that resemble the body. Tools such as atomic force microscopy, micropillar arrays, optical tweezers and droplet microfluidics each probe one corner of the mechanical landscape, and CFM fills a conspicuous gap: the confined, compressive, three-dimensional regime that characterizes nearly every tissue. By making that regime dynamic and measurable, the platform opens experimental doors that were previously accessible only through indirect inference.

Looking ahead, the researchers and the field at large anticipate that confinement-resolved traction mapping will become a standard component of mechanobiological phenotyping, much as flow cytometry became standard for molecular phenotyping. Because the technique is compatible with live-cell imaging, it can be paired with fluorescent reporters of cytoskeletal dynamics, focal adhesion turnover and mechanosensitive signaling pathways, linking the force maps it produces directly to the molecular machinery that generates them. For cancer biologists, stem cell engineers and developmental biologists alike, the ability to watch and measure how cells push back against a crowded world represents a meaningful step toward understanding mechanics where it actually matters: inside tissue.

Subject of Research: A tunable microconfinement technique for measuring traction forces of single cells and aggregates under tissue-mimicking spatial confinement.

Article Title: CFM: confinement force microscopy—a dynamic, precise and stable microconfiner for traction force microscopy in spatial confinement

Article References: CFM: confinement force microscopy—a dynamic, precise and stable microconfiner for traction force microscopy in spatial confinement. (n.d.). https://doi.org/10.1038/s41592-026-03216-5

Image Credits: AI Generated

DOI: 10.1038/s41592-026-03216-5

Keywords: confinement force microscopy, traction force microscopy, mechanobiology, cell mechanics, spatial confinement, cell aggregates, 3D cell culture, traction stresses, microconfiner, Nature Methods, cellular forces, tissue mechanics