In vitro models最新文献

Purpose: Alterations to the strength and stiffness of the human extracellular matrix (ECM) are the underlying pathology manifest in a wide range of diseases. These include inherited conditions, such as Ehlers Danlos syndrome, as well as acquired diseases such as fibrosis, which remains a major unmet medical need. To evaluate promising therapies, new models are needed that can measure the strength and stiffness of the human ECM.

Methods: Cultured human fibroblasts were seeded into circular troughs of agarose that had been molded into a 24 well plate and equilibrated with cell culture medium. The cells settled by gravity, aggregated and formed 3D ring-shaped tissues 5 mm in diameter without the aid of added exogenous scaffold material. The ECM proteins synthesized de novo by the rings were characterized by immuno-staining. The response of the rings to drug and growth factor treatments were assessed by measuring changes to the dimensions of the rings and by measuring levels of collagen. A tensile test was used to quantify drug and growth factor induced changes to the strength and stiffness of the rings.

Results: Ring-shaped tissues readily formed in the molds and synthesized de novo a circumferentially aligned collagen-rich fibrous ECM network positive for collagen type I, collagen type III and fibronectin. Low dose treatment with incyclinide, an inhibitor of matrix metalloproteinases (MMPs), increased strength and stiffness, whereas as a high dose decreased tensile properties, likely due to a toxic effect. Treatment with TGF-β1, a well-known driver of fibrosis, increased levels of collagen and tensile properties and mimicked the fibrotic environment in vitro. Treatment with PAT-1251, an inhibitor of the collagen crosslinking enzyme lysyl oxidase-like protein 2 (LOXL2), had no effect on levels of collagen but significantly reduced the strength and stiffness of the ring even when elevated by treatment with TGF-β1.

Conclusion: Human fibroblasts will self-assemble a 3D ring-shaped tissue and synthesize a fibrous network of ECM proteins whose tensile properties can be measured. The fibrotic environment can be mimicked by addition of TGF-β1, which increases levels of collagen as well as the strength and stiffness of the rings. Treatment with two drugs, incyclinide and PAT 1251 that were developed as potential treatments for diseases of the ECM, altered the strength and stiffness of the rings, thereby demonstrating the utility of the model for testing new therapies that target the biomechanics of the ECM.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-025-00081-y.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths, and perineural invasion (PNI), in which cancer cells infiltrate nerves, enables metastasis in most patients. PNI is largely attributed to Schwann cells (SC) that, when activated, accelerate cancer cell migration towards nerves. However, this cancer-associated reprogramming is generally under-appreciated. Additionally, tumor extracellular vesicle (EV) facilitation of cancer aggravation is well documented, but more investigation is required to better understand their role in PNI. Here, we assessed whether PDAC EVs mediate PNI via SC activation using tissue-engineered in vitro platforms and PANC-1 and HPNE human cell lines as models.

Methods: NanoSight, Luminex®, and proteomic-pathway analyses characterized tumor (PANC-1) and healthy cell (HPNE) EVs. Human Schwann-like cells (sNF96.2) were embedded in decellularized nerve matrix hydrogels and then treated with EVs and a cargo-function-blocking antibody. Immunofluorescence and Luminex® multiplex assays assessed Schwann cell activation. Subsequently, sNF96.2 cells were co-cultured with EVs and either PANC-1 or HPNE cells; Transwell® invasion assays with SC-conditioned media were also conducted to establish a mechanism of in vitro PNI.

Results: PANC-1 EVs contained higher levels of interleukin-8 (IL-8) signaling-associated proteins than HPNE EVs. Within nerve-mimetic in vitro testbeds, PANC-1 EVs promoted sNF96.2 activation per cytoskeletal marker alterations and secretion of pro-tumorigenic cytokines, e.g., chemokine ligand-2 (CCL2), via IL-8 cargoes. Furthermore, the IL-8/CCL2 axis heightened PANC-1 invasiveness.

Conclusion: These findings highlight the potential role of PDAC EVs in PNI, which necessitates continued preclinical assessments with increased biodiversity to determine the efficacy of targeting IL-8/CCL2 for PNI.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-025-00083-w.

Organoids are three-dimensional, miniaturized tissue-like structures derived from either stem cells or primary cells, emerging as powerful in vitro models for studying developmental biology, disease pathology, and drug discovery. These organoids more accurately mimic cell-cell interactions and complexities of human tissues compared to traditional cell cultures. However, challenges such as limited nutrient supply and biomechanical cue replication hinder their maturation and viability. Microfluidic technologies, with their ability to control fluid flow and mimic the mechanical environment of tissues, have been integrated with organoids to create organoid-on-chip models that address these limitations. These models not only improve the physiological relevance of organoids but also enable more precise investigation of disease mechanisms and therapeutic responses. By combining microfluidics and organoids, several advanced organoids-on-chip models have been developed to investigate mechanical and biochemical cues involved in disease progression. This review discusses various methods to develop organoids-on-chip and the recently established organoids-on-chip models with their advanced functions. Finally, we highlighted potential strategies to enhance the functionality of organoid models, aiming to overcome current limitations and bridge the gap between current cell culture models and clinical applications, advancing personalized medicine, and improving therapeutic testing.

Patient-derived organoids (PDO) have the potential to be used as preclinical cancer models for testing anti-cancer drug efficiency. Cancer-associated fibroblasts (CAFs), which have been closely linked with colorectal carcinoma (CRC) progression and drug resistance, however, are generally not included (or gradually lost during culture) in the PDO models, leading to a major limitation in this cancer model. In this study, we established a new in vitro model with CRC organoids and co-cultured with CAFs and compared it with the organoid-only model. Through testing with anti-cancer drug, we demonstrated a significant difference in drug sensitivity between the two models, and the co-culture model showed higher drug resistance. RNA and whole exome sequencing were performed to reveal gene expression profiles in organoids and organoids co-culture with CAFs to assess interactions between drug sensitivity and gene copy number variation. We found that the expression levels of several pathway protein genes, which are highly expressed in original surgical specimens of colorectal carcinomas, were downregulated in organoids but restored in organoids by co-culturing with CAFs. In summary, the PDO-CAF joint model for CRC can recapitulate a more biomimetic tumor microenvironment and the drug resistance lead by changes in multiple signaling pathways that we discovered; thus, it could be a suitable model for future usage in drug discovery and precision medicine research.

Over the years, significant progress has been made in developing cost-effective and ethical in vitro bioengineered skin substitutes to study cutaneous wound healing processes. Rodents and small animal models are not optimal due to physiological differences in their skin compared to human skin. The generation of reproducible and precise wounds is key to obtaining comparable results. We created a three-dimensional skin wounding model by growing a fully differentiated, stratified squamous epithelium from human keratinocytes at an air-liquid interface on a type I collagen scaffold concealed with human dermal BJ fibroblasts. To generate the wounds, a stamp with incorporated needles with a length of 250 µm was used to puncture the epidermis to produce standardized wounds. The stamping needle technique is a practical and inexpensive method for creating length-tailored wounds on three-dimensional skin models. The effectiveness of this technique in treating 3D skin models was demonstrated, accompanied by an evaluation of the model's functionality in terms of cell proliferation, differentiation, and immunological characteristics.

The cryopreservation of neonatal testicular tissue containing gonocytes is crucial for preserving genetic diversity, advancing research, and developing reproductive technologies. In this study, we investigated three cryopreservation techniques, slow freezing (in which the rate of freezing was controlled or uncontrolled) and vitrification, using neonatal bovine testicular tissues containing gonocytes, followed by in vitro culture to evaluate cell functionality. Vitrification resulted in a significantly lower proportion (19.15 ± 1.82%) of seminiferous tubules with > 70% attachment to the basement membrane in comparison to both the controlled slow freezing group (47.89 ± 10.98%) and the uncontrolled slow freezing group (39.05 ± 4.15%) (P < 0.05). No significant differences were observed in the proportion of seminiferous tubules containing PGP9.5-positive germ cells when compared between the three methods. Comparable densities of germ cells per unit area were observed in the controlled/uncontrolled slow freezing groups and the vitrification group (7.89 ± 1.83, 7.75 ± 1.75, and 7.92 ± 1.23/104 µm², respectively). In addition, the proportions of Sertoli cells (vimentin-positive) and proliferating cells (Ki67-positive) were similar across the three cryopreservation methods. There were no significant differences in cell membrane integrity and the expression of selected genes when compared between the three cryopreservation groups. Compared to fresh tissue, the uncontrolled slow freezing groups exhibited significantly higher levels of apoptosis (P < 0.05); there was no significant change in the controlled slow freezing and vitrification group. Notably, all in vitro cultures of testicular cells, from both fresh and freeze/thawed tissues, displayed the formation of germ cell colonies. Our data demonstrate that vitrification effectively preserves neonatal bovine testicular tissues containing gonocytes, safeguarding cell membrane integrity, promoting proliferation, and protecting against apoptosis. Collectively, these findings propose vitrification as a promising alternative cryopreservation method for immature testicular tissue (ITT) in clinical applications.

Purpose: For optimization of respiratory drug delivery, the selection of suitable in vitro cell models plays an important role in predicting the efficacy and safety of (bio)pharmaceutics and pharmaceutical formulations. Therefore, an in-depth comparison of different primary and permanent in vitro cellular airway models was performed with a focus on selecting a suitable model for inhalative antibodies.

Methods: Primary cells isolated from the porcine trachea were compared with the established human cell lines CaLu3 and RPMI 2650. The in vitro models were characterized for different epithelial markers by real-time quantitative polymerase chain reaction, which provides insight into the cellular composition of each model. For a few selected markers, the results from RT-qPCR were confirmed via immunofluorescence. Barrier integrity was assessed by transepithelial electrical resistance measurements and FITC-dextran permeability.

Results: Primary cell models retain key features of the respiratory epithelium, e.g., the formation of a tight epithelial barrier, mucin production, and the presence of club/basal cells. Furthermore, the expression of Fc receptors in the primary cell models closely resembles that in respiratory mucosal tissue, an essential parameter to consider when developing therapeutic antibodies for inhalation.

Conclusion: The study underlines the importance of selecting wisely appropriate in vitro models. Despite the greater effort and variability in cultivating primary airway cells, they are far superior to permanent cells and a suitable model for drug development.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-024-00079-y.

Background: Extracellular matrix (ECM) is a three-dimensional (3D) structure found around cells in the tissues of many organisms. It is composed mainly of fibrous proteins, such as collagen and elastin, and adhesive glycoproteins, such as fibronectin and laminin-as well as proteoglycans, such as hyaluronic acid. The ECM performs several essential functions, including structural support of tissues, regulation of cell communication, adhesion, migration, and differentiation by providing biochemical and biomechanical cues to the cells. Pancreatic β-cells have been previously shown to be responsive to the surrounding mechanical stress, impacting their insulin-secreting function.

Purpose: We aimed to derive a physiologically relevant in vitro model of pancreatic tissue by using an innovative synthesised porous ECM that mimics the native tissue microenvironment and mechanical properties.

Methods: Here we performed mechanical, physico-chemical and functional characterisation of a synthetic hydrogel ECM, composed of hyaluronic acid cross-linked with collagen types I and VI and modified with fibronectin. The hydrogel was used as a 3D cell culture scaffold for the MIN6 insulinoma cell line. Cell proliferation, viability, gene expression, and insulin secretion in response to glucose stimulus were assessed and contrasted with classic monolayer culture.

Results: The biomaterial exhibited a shear modulus of 815.37 kPa and a distinctive viscoelastic response. MIN6 cells showed a higher proliferation and viability rates and maintained insulin secretion in response to glucose stimulus and β-cell identity gene expression when cultured in the 3D hydrogel compared to monolayer culture.

Conclusion: Our study demonstrated the potential of this biomimetic hydrogel scaffold as an innovative matrix enabling better in vitro models to study disease physiopathology.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-024-00078-z.

Obesity is associated with several comorbidities that cause high mortality rates worldwide. Thus, the study of adipose tissue (AT) has become a target of high interest because of its crucial contribution to many metabolic diseases and metabolizing potential. However, many AT-related physiological, pathophysiological, and toxicological mechanisms in humans are still poorly understood, mainly due to the use of non-human animal models. Organ-on-chip (OoC) platform is a promising alternative to animal models. However, the use of adipose-derived human mesenchymal stem cells (hASCs) in these models is still scarce, and more knowledge on the effects properties of culturing hASCs in OoC models is needed. Here, we present the development of an OoC using hASCs to assess adipogenic differentiation. The device capability to increase hASC differentiation levels was confirmed by Nile red staining to verify lipid droplets inside cells after 10 days of culture and fluid flow of 10 µL/h. The Adipo-on-a-chip system increases hASC proliferation and differentiation area compared with the standard culture method under static conditions (96-well plates) verified in hASCs from different donors by image analysis of cells stained with Nile red. The expression of the gene FABP4 is lower in the MPS, which calls attention to different homeostasis and control of lipids in cells in the MPS, compared with the plates. An increase of hASC proliferation in the MPS related to the 96-well plate was verified through protein Ki-67 expression. Cell and nuclei morphology (area, roundness, perimeter, width, length, width to length rate, symmetry, compactness, axial and radial properties to nuclei, and texture) and dominant direction of cells inside the MPS were evaluated to characterize hASCs in the present model. The presented microphysiological system (MPS) provides a promising tool for applications in mechanistic research aiming to investigate adipogenesis in AT and toxicological assessment based on the hASC differentiation potential.

Zinc (Zn) and its alloys have been the focus of recent materials and manufacturing research for orthopaedic implants due to their favorable characteristics including desirable mechanical strength, biodegradability, and biocompatibility. In this research, a novel process involving additive manufacturing (AM) augmented casting was employed to fabricate zinc-magnesium (Zn-0.8 Mg) artifacts with surface lattices composed of triply periodic minimal surfaces (TPMS), specifically gyroid. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analysis confirmed that Zn-Mg intermetallic phases formed at the grain boundary. Micro indentation testing resulted in hardness value ranging from 83.772 to 99.112 HV and an elastic modulus varying from 92.601 to 94.625 GPa. Results from in vitro cell culture experiments showed that cells robustly survived on both TPMS and solid scaffolds, confirming the suitability of the material and structure as biomedical implants. This work suggests that this novel hybrid manufacturing process may be a viable approach to fabricating next generation biodegradable orthopaedic implants.

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