In vitro models最新文献

[This corrects the article DOI: 10.1007/s44164-022-00015-y.].

[This corrects the article DOI: 10.1007/s44164-022-00037-6.].

The translational potential of promising anticancer medications and treatments may be enhanced by the creation of 3D in vitro models that can accurately reproduce native tumor microenvironments. Tumor microenvironments for cancer treatment and research can be built in vitro using biomaterials. Three-dimensional in vitro cancer models have provided new insights into the biology of cancer. Cancer researchers are creating artificial three-dimensional tumor models based on functional biomaterials that mimic the microenvironment of the real tumor. Our understanding of tumor stroma activity over the course of cancer has improved because of the use of scaffold and matrix-based three-dimensional systems intended for regenerative medicine. Scientists have created synthetic tumor models thanks to recent developments in materials engineering. These models enable researchers to investigate the biology of cancer and assess the therapeutic effectiveness of available medications. The emergence of biomaterial engineering technologies with the potential to hasten treatment outcomes is highlighted in this review, which also discusses the influence of creating in vitro biomimetic 3D tumor microenvironments utilizing functional biomaterials. Future cancer treatments will rely much more heavily on biomaterials engineering.

[This corrects the article DOI: 10.1007/s44164-022-00030-z.].

Cancer therapeutics are often highly toxic to the patient, and they often elicit rapid resistance in the tumour. Recent advances have suggested a potential new way in which we may improve on this, through two important concepts: (1) that multitudinous pathway alterations converge on a limited number of cancer cellular phenotypes, and (2) that these cancer cellular phenotypes depend on reactivation of developmental processes that are only minimally active in adult tissues. This provides a rationale for pursuing an approach of 'drugging the phenotype' focussed on targeting reactivated cellular processes from embryonic development. In this concepts paper, we cover these recent developments and their implications for the development of new cancer therapeutics that can avoid patient toxicity and acquired resistance. We then propose that in vitro tumour and developmental models can provide an experimental approach to identify and target the specific developmental processes at play, with a focus on the reactivation of developmental processes in the cancer stem cells that drive tumour progression and spread. Ultimately, the aim is to identify cellular processes that are specific to developmental phenotypes, are reactivated in cancer stem cells, and are essential to tumour progression. Therapeutically targeting these cellular processes could represent a new approach of 'drugging the phenotype' that treats the tumour whilst avoiding patient toxicity or the acquisition of therapeutic resistance.

Purpose: This 3D in vitro cancer model for propagation of patient-derived cells, using a synthetic self-assembling peptide gel, allows the formation of a fully characterised, tailorable tumour microenvironment. Unlike many existing 3D cancer models, the peptide gel is inert, apart from molecules and motifs deliberately added or produced by cells within the model.

Methods: Breast cancer patient-derived xenografts (PDXs) were disaggregated and embedded in a peptide hydrogel. Growth was monitored by microscopic examination and at intervals, cells were extracted from the gels and passaged on into fresh gels. Passaged cells were assessed by qPCR and immunostaining techniques for the retention of characteristic markers.

Results: Breast cancer PDXs were shown to be capable of expansion over four or more passages in the peptide gel. Contaminating mouse cells were found to be rapidly removed by successive passages. The resulting human cells were shown to be compatible with a range of common assays useful for assessing survival, growth and maintenance of heterogeneity.

Conclusions: Based on these findings, the hydrogel has the potential to provide an effective and practical breast cancer model for the passage of PDXs which will have the added benefits of being relatively cheap, fully-defined and free from the use of animals or animal products. Encapsulated cells will require further validation to confirm the maintenance of cell heterogeneity, genotypes and phenotypes across passage, but with further development, including the addition of bespoke cell and matrix components of the tumour microenvironment, there is clear potential to model other cancer types.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-023-00048-x.

Purpose: Alzheimer's disease (AD) early pathology needs better understanding and models. Here, we describe a human induced pluripotent stem cells (iPSCs)-derived 3D neural culture model to study certain aspects of AD biochemistry and pathology.

Method: iPSCs derived from controls and AD patients with Presenilin1 mutations were cultured in a 3D platform with a similar microenvironment to the brain, to differentiate into neurons and astrocytes and self-organise into 3D structures by 3 weeks of differentiation in vitro.

Results: Cells express astrocytic (GFAP), neuronal (β3-Tubulin, MAP2), glutamatergic (VGLUT1), GABAergic (GAD65/67), pre-synaptic (Synapsin1) markers and a low level of neural progenitor cell (Nestin) marker after 6 and 12 weeks of differentiation in 3D. The foetal 3R Tau isoforms and adult 4R Tau isoforms were detected at 6 weeks post differentiation, showing advanced neuronal maturity. In the 3D AD cells, total and insoluble Tau levels were higher than in 3D control cells.

Conclusion: Our data indicates that this model may recapitulate the early biochemical and pathological disease features and can be a relevant platform for studying early cellular and biochemical changes and the identification of drug targets.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00038-5.

Background: Advanced cell culture techniques such as 3D bioprinting and hydrogel-based cell embedding techniques harbor many new and exciting opportunities to study cells in environments that closely recapitulate in vivo conditions. Researchers often study these environments using fluorescence microscopy to visualize the protein association with objects such as cells within the 3D environment, yet quantification of concentration profiles in the microenvironment has remained elusive.

Objective: Demonstrate an assay that enables near real-time in situ biomarker detection and spatiotemporal quantification of biomarker concentration in 3D cell culture.

Methods: A distributed bead-based immuno-assay was used in 3D cell culture to continuously measure the time-dependent concentration gradient of various biomarkers by sequestering soluble target molecules and concentrating the fluorescence intensity of these tagged proteins. Timelapse confocal microscopy was used to measure the in situ fluorescence intensity profile and a calibration curve was separately generated. Application of a calibration transfer function to in situ data is used to quantify spatiotemporal concentration.

Results: Example assays utilize an osteosarcoma spheroid as a case study for a quantitative single-plexed gel encapsulated assay, and a qualitative multi-plexed 3D-bioprinted assay. In both cases, a time-varying cytokine concentration gradient is measured. An estimation for the production rate of the IL-8 cytokine per second per osteosarcoma cell results from fitting an analytical function for continuous point source diffusion to the measured concentration gradient and reveals that spheroid production approaches nearly 0.18 fg/s of IL-8 after 18 h in culture.

Conclusions: Theoretical and experimental demonstration of bead-based immunoassays in diffusion-limited environments such as 3D cell culture is shown, and includes example measurements of various cytokines produced by an osteosarcoma spheroid. Proper calibration and use of this assay is exhaustively explored for the case of diffusion-limited Langmuir kinetics of a spherical adsorber.

Cancer cell spheroids are the simplest 3D in vitro cancer models and have been extensively used for cancer research. More recently, models have been becoming complex, with the introduction of a matrix and non-cancer cell types to mimic specific tumour aspects. However, applying drugs or agents in matrix-embedded cancer spheroids can be problematic. Most matrices can impede and also bind drugs or visualizing agents non-specifically, in the vicinity of the embedded spheroids. This may interfere with imaging or further analysis without breaking apart the 3D model into its constituents. Here, we developed a combined gelatin-carboxymethyl cellulose (G-CMC) hydrogel for initiating cancer spheroids that enabled intact harvesting pre/post treatment for further investigation, such as targeting and imaging. We combined CMC (1.25%) and gelatin (2.5%) at 25 °C and initiated polymerisation after autoclaving (121 °C) to obtain a mechanical strength (sheer stress) of 38 Pas versus 1.28 Pas for CMC alone. These matrix conditions facilitated separation of the spheroids from the G-CMC, using low centrifugation (100 g). We described growth of colorectal and breast cancer spheroids within the G-CMC matrix (with average diameters of 220 mm and 180 μm for representative cell lines HT29 and MCF7 at 10 days, respectively). As the cancer cells express the surface biomarker calreticulin (CRT), we manufactured anti-calreticulin IgG (anti-CRT) conjugated to fluorescent gold nanoclusters (anti-CRT-AuNC) as a probe. We harvested cancer spheroids and incubated live with the nanoclusters. Imaging demonstrated strong binding of CRT-targeted AuNCs compared to control AuNCs. This novel model preserves cancer spheroid integrity upon isolation and is well suited for targeted imaging and drug delivery of cancer in 3D.