Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens
Open accessAbstract
Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications.
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Written from the paper’s full text.
TL;DR
Direct electroporation of clonal organoids encapsulated in Matrigel microbeads achieves high siRNA transfection efficiency, up to 80% in RWPE-1 organoids, and enables functional gene silencing in 3D. The authors validate p63 and PTEN as key tumor suppressors in breast and prostate acinar development using a morphological read-out.
Key findings
- Electroporation of RWPE-1-GFP organoids in Matrigel microbeads reached almost 80% GFP silencing, compared with ≈5% for Lipofectamine/Oligofectamine, up to 15% for TransIT/3DFect/3DFectIn, and 25% for lentiviral transduction at MOI 10.
- siRNA penetrated to the center of organoids, with almost 90% of RWPE-1 cells integrating labeled siRNA; plasmid GFP expression reached more than 40% but was heterogeneous between experiments.
- MCF10A organoid transfection was lower than RWPE-1 (31% in microbeads vs 57% in 2D), but decreasing microbead size from 450 to 250 μm and Matrigel concentration from 100 to 70% increased extinction efficiency to 57%, comparable to 2D.
- p63 or PTEN knockdown decreased protein levels by ∼50% and increased spheroids/invasive spheroids at the expense of acini in RWPE-1 and MCF10A organoids, supporting their roles in acinar development.
Methods
- Single RWPE-1 or MCF10A cells were encapsulated in Matrigel microbeads using a flow-focusing microfluidic chip, cultured into organoids, and transfected by electroporation (AMAXA, program X-001) with siRNA or plasmid at 20 nM.
- Transfection efficiency was measured by GFP mean fluorescence intensity via flow cytometry after cell extraction; viability was assessed by trypan blue and Caspase-3/7 staining.
- Functional validation used siRNA against p63 or PTEN, western blot, immunofluorescence for polarity/Golgi, and morphological classification into acini, round, spheroid, and invasive spheroid categories; statistics used Mann–Whitney–Wilcoxon tests.
Limitations
- Stated: morphological analysis was performed by eye counting under a phase microscope, which remains time-consuming and laborious; an automatic image analysis software is under development.
- Stated: plasmid transfection showed high heterogeneity between experiments, likely due to plasmid size and physical barriers, and MCF10A organoids required optimization of microbead size and Matrigel concentration to reach 2D-comparable efficiency.
- Evident from the text: the study demonstrates feasibility in selected epithelial cell lines and does not itself perform a high-throughput genetic screen; broader screening applications are proposed rather than shown.
Model-written summary. Check it against the paper before relying on it.
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Cites
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Claims the paper makes
26 of 48 cited sources checked · 3 supported · 1 flagged · accuracy not yet measured
- Findingcitation
An increased proportion of spheroids and invasive spheroids was observed in all tumor cell lines (PC3, DU145, LNCaP, WPE1-NB26, MCF7) compared with control cell lines (RWPE-1 and MCF10A).
“As expected, we observed an increased proportion of spheroids and invasive spheroids in all tumor cell lines (PC3, DU145, LNCaP, WPE1-NB26 and MCF7) compared with the control cell lines (RWPE-1 and MCF10A) (30).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [30] Härmä, 2014, Quantification of dynamic morphological drug responses in 3D organotypic cell cultures by automated image analysis, PLoS One source
The Results (Experimental validation; page not provided) contradict the claim that all listed tumor lines had more invasive spheroids than the controls: LNCaP and DU145 typically formed round spheroids lacking invasive properties, while the control line RWPE-1 formed branching structures. The paper does not report the claimed comparison of spheroid proportions across these lines. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured
- Premisecitation
Organoids are embedded in extracellular matrix, which constitutes a barrier for efficient transfection.
“One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques.”
- Also stated in: § INTRODUCTION · plain
“Indeed, one limitation is that organoids are embedded in ECM, which constitutes a barrier for efficient transfection.”
Quote in the paperSubstantial claim2 of 3 sources checked- cites [7] Kostarelos, 2004, Binding and interstitial penetration of liposomes within avascular tumor spheroids, Int. J. Cancer source
The paper’s introductory passage (indexed under Abstract; page not supplied) says tumor spheroids contain extracellular matrix, and its Abstract and Discussion report barriers to liposome penetration into those spheroids. It does not study organoids or establish that extracellular matrix is the barrier to their transfection; the barrier it identifies for strongly cationic liposomes is electrostatic binding at the spheroid surface. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [8] Canatella, 2004, Tissue electroporation: quantification and analysis of heterogeneous transport in multicellular environments, Biophys. J. source
The Introduction describes densely packed tumor spheroids surrounded by extracellular matrix, and the Conclusions report lower molecular uptake during electroporation than in isolated cells. But the study examines spheroids, not organoids, and does not establish that extracellular matrix itself is the barrier to transfection. (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [9] Zhang, 2012, High-throughput transfection of interfering RNA into a 3D cell-culture chip, Small Weinh. Bergstr. Ger. source
- Also stated in: § INTRODUCTION · plain
- Premisereasoning
Direct 3D transfection on already formed organoids remains challenging.
“However, direct 3D transfection on already formed organoids remains challenging.”
- Also stated in: § INTRODUCTION · plain
“Thus, direct 3D transfection on already formed organoids remains challenging.”
Quote in the paperSubstantial claim2 of 4 sources checked- cites [7] Kostarelos, 2004, Binding and interstitial penetration of liposomes within avascular tumor spheroids, Int. J. Cancer source
The Abstract says poor liposome penetration into tumor tissue remains a problem, and the Results show that some liposomes remained at the surface of already formed 3D tumor spheroids. This supports a delivery challenge, but the paper measures liposome binding and penetration in spheroids, not transfection success in organoids; its findings do not establish the claim at that broader level. (LLM, conf=0.85)
Experimental — accuracy not yet measured - cites [8] Canatella, 2004, Tissue electroporation: quantification and analysis of heterogeneous transport in multicellular environments, Biophys. J. source
The Results and discussion section reports that cells in already formed multicellular spheroids took up less calcein during electroporation than isolated cells, particularly deep within the spheroids. This supports a challenge with direct delivery into a 3D structure, but the paper tests calcein uptake in spheroids, not transfection of organoids. (LLM, conf=0.85)
Experimental — accuracy not yet measured - cites [9] Zhang, 2012, High-throughput transfection of interfering RNA into a 3D cell-culture chip, Small Weinh. Bergstr. Ger. source
- cites [10] Mellor, 2006, Optimising non-viral gene delivery in a tumour spheroid model, J. Gene Med. source
- Also stated in: § INTRODUCTION · plain
- Premisecitation
Organoids grow into dense and compact structures that impede diffusion, penetration and cellular accumulation of genetic material, making transfection via traditional techniques difficult.
“Moreover, organoids grow into dense and compact structures that impede diffusion, penetration and cellular accumulation of genetic material, which makes transfection via traditional techniques difficult (7–9).”
Quote in the paperSubstantial claim2 of 3 sources checked- cites [7] Kostarelos, 2004, Binding and interstitial penetration of liposomes within avascular tumor spheroids, Int. J. Cancer source
The Abstract and Discussion (page numbers unavailable) describe three-dimensional tumor spheroids and show that some liposomes penetrate only their outer cell layers; the paper also identifies cationic liposomes as gene-delivery vectors. It studies tumor spheroids, not organoids, and does not show that genetic material fails to accumulate in cells or that traditional transfection techniques are difficult to use in organoids. (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [8] Canatella, 2004, Tissue electroporation: quantification and analysis of heterogeneous transport in multicellular environments, Biophys. J. source
The Results and discussion (page not available) report a diffusional delay into spheroid interiors and lower calcein uptake by interior cells during electroporation. The experiments concern multicellular spheroids and calcein, not organoids or genetic material; they do not establish that traditional transfection techniques are difficult in organoids. The paper also says the diffusion delay did not explain its uptake results after the longer incubation used for most experiments. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [9] Zhang, 2012, High-throughput transfection of interfering RNA into a 3D cell-culture chip, Small Weinh. Bergstr. Ger. source
- Interpretationcitation
The heterogeneity in plasmid transfection is probably due to the size of the plasmid (~5000 bp) compared to siRNA (20 bp).
“This is probably due to the size of the plasmid (∼5000 bp) when compared to siRNA (20 bp), as previously described (6).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [6] Kim, 2010, Mammalian cell transfection: the present and the future, Anal. Bioanal. Chem. source
The Methodological diversity section mentions variable transfection efficiency by cell type or condition, and Transfection of RNAs discusses siRNA delivery. Neither passage attributes heterogeneity in plasmid transfection to plasmid size relative to siRNA, or gives the claimed ~5000 bp and 20 bp figures. Searches for that comparison and both sizes found no supporting passage; page numbers were unavailable in our copy. (LLM, conf=0.73)
Experimental — accuracy not yet measured
- Premisecitation
The morphology-based classification allows quantification of the tumorigenic potential of different cell lines.
“This morphology-based classification allows the quantification of the tumorigenic potential of different cell lines and the quick visualization of different conditions.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [27] Taubenberger, 2016, 3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments, Acta Biomater. source
Section 3.2 classifies spheroid morphologies across MCF-7, LNCaP and PC-3 cell lines and quantifies the categories, identifying category IV as an invasive, aggressive phenotype. This supports using the classification to compare morphology and invasion, but the paper does not establish it as a measure of overall tumorigenic potential. (LLM, conf=0.82)
Experimental — accuracy not yet measured
- Premisecitation
Morphological classification can be used as a quick and reliable read-out to discriminate organoid features and gene functions, as previously described.
“Here, we used morphological classification as a quick and reliable read-out to discriminate organoid features and gene functions, as previously described (27).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [27] Taubenberger, 2016, 3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments, Acta Biomater. source
Section 3.2 classifies tumour spheroids by morphology and uses the categories to distinguish invasive from non-invasive phenotypes. The paper does not establish morphological classification as a quick and reliable read-out for organoid features or gene functions; it studies tumour spheroids and ECM conditions instead. Page numbers were not available in our copy. (LLM, conf=0.86)
Experimental — accuracy not yet measured
- Premisecitation
A viable strategy to transfect ECM-embedded organoids is to use inducible systems.
“A viable strategy to transfect ECM-embedded organoids is to use inducible systems (41).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [41] Herr, 2011, A novel MCF-10A line allowing conditional oncogene expression in 3D culture, Cell Commun. Signal. source
The Results describe transfecting MCF-10A cells with a tetracycline-inducible system and inducing transgene expression after the cells formed 3D acini. This supports inducible expression in embedded structures, but not transfection of organoids while they are ECM-embedded; the cells were transfected before 3D culture. The indexed passages provide section labels but no page numbers. (LLM, conf=0.88)
Experimental — accuracy not yet measured
- Premisecitation
These studies were performed on incomplete 3D models, cell aggregates such as MCTS or dissociated organoids devoid of any exogenous ECM.
“However, these studies were performed on incomplete 3D models, cell aggregates such as MCTS or dissociated organoids devoid of any exogenous ECM (40).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [40] Picollet-D’hahan, 2016, A 3D toolbox to enhance physiological relevance of human tissue models, Trends Biotechnol. source
The Microfabrication section says hanging-drop microtissues form by artificial cell aggregation and usually lack matrices; the 3D Toolbox to Create Artificial Scaffolded Organs section also describes gel-free spheroid models. The paper does not establish that the particular studies referred to used those models, or that they used dissociated organoids devoid of exogenous ECM. Page numbers were not available in our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measured
- Findingown data
In contrast with a previous study demonstrating low plasmid transfection efficiency (about 6%) in 3D cultures, the present system reached plasmid extinction efficiency of more than 40%.
“In contrast with a previous study demonstrating low plasmid transfection efficiency (about 6%) in 3D cultures (30), we managed to reach plasmid extinction efficiency of more than 40%.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [30] Härmä, 2014, Quantification of dynamic morphological drug responses in 3D organotypic cell cultures by automated image analysis, PLoS One source
The citation is attached to the claim that a previous study found about 6% plasmid transfection efficiency in 3D cultures; the citing authors' own result of more than 40% is not attributed to this source. The Materials and Methods section describes 3D culture preparation, but neither it nor the retrieved passages report plasmid transfection efficiency (searched: 6% plasmid transfection, plasmid gene delivery). (LLM, conf=0.72)
Experimental — accuracy not yet measured
- Findingown data
The method successfully transfects tumorigenic cell lines that form full tumor-like spheroids in the beads.
“The versatility of this method was also showed by successfully transfecting tumorigenic cell lines which form full tumor-like spheroids in the beads (Supplementary Figure S7).”
Quote in the paperSubstantial claim - Findingown data
Lentiviral transduction at MOI 10 leads to a 25% decrease in GFP fluorescent signal.
“The best results were obtained with an MOI (estimated number of virus per cell) of 10, which led to a 25% decrease in GFP fluorescent signal (Supplementary Figure S5).”
Quote in the paperNot a substantial claim- Rests on an assumption: GFP knock-down (extinction efficiency) is a valid surrogate for siRNA transfection/delivery efficiency in organoids.
- Findingown data
In other experiments, a not significant decrease in cell viability (from 85 to 74%, P=0.1) was observed.
“In other experiments, a not significant decrease in cell viability was also observed (from 85 to 74%) (ns, P = 0.1) (Supplementary Figure S6).”
Quote in the paperNot a substantial claim - Findingreasoning
In each microbead, one single cell gives rise to an organoid derived from clonal origin.
“In each microbead, one single cell will give rise to an organoid derived from clonal origin.”
Quote in the paperSubstantial claim - Findingown data
Almost 90% of RWPE-1 cells have integrated the labeled siRNA after electroporation (P=0.05).
“We further used flow cytometry analysis on cells that were extracted from transfected organoids, which demonstrated that almost 90% of RWPE-1 cells have integrated the labeled siRNA (P = 0.05) (Supplementary Figure S8A).”
Quote in the paperSubstantial claim - Findingown data
Direct electroporation of encapsulated organoids reaches up to 80% transfection efficiency.
“We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency.”
- Also stated in: § Direct transfection of organoids in Matrigel microbeads · plain
“Finally, the highest results were obtained using electroporation, with GFP silencing efficiencies of almost 80% (P = 0.029).”
Quote in the paperSubstantial claim- Rests on an assumption: GFP knock-down (extinction efficiency) is a valid surrogate for siRNA transfection/delivery efficiency in organoids.
- Also stated in: § Direct transfection of organoids in Matrigel microbeads · plain
- Findingown data
Electroporation of GFP-expressing plasmid into RWPE-1 organoids results in homogeneous distribution of GFP-expressing cells, including in cells at the center of 3D structures.
“We observed a homogeneous distribution of GFP-expressing cells in organoids, even in cells that are located at the center of 3D structures (Figure 3B).”
Quote in the paperSubstantial claim - Findingown data
Electroporation of RWPE-1 organoids in microbeads causes a small but not significant decrease in cell viability (from 91 to 88%, P=0.1).
“A small but not significant (ns) decrease in cell viability (from 91 to 88%) (P = 0.1) was observed in cells subjected to electroporation or to lentiviral transduction (Figure 2C).”
Quote in the paperSubstantial claim - Findingown data
350 μl of Matrigel for one well of a Lab-Tek 4-chambers slide allows production of 42802 microbeads with a diameter of 250 μm.
“For example, 350 μl of Matrigel for one well of a Lab-Tek 4-chambers slide allows to produce 42802 microbeads with a diameter of 250 μm (Figure 1B–b).”
Quote in the paperNot a substantial claim - Findingown data
siRNA alone without any vectors is not sufficient to transfect organoids.
“As expected, siRNA alone without any vectors was not sufficient to transfect organoids.”
Quote in the paperSubstantial claim - Findingown data
Electroporation of Alexa Fluor 546-labeled siRNA into RWPE-1 organoids results in homogeneous distribution of siRNA, including in cells at the center of 3D structures.
“We observed a homogeneous distribution of siRNA in organoids, even in the cells that are located at the center of 3D structures.”
Quote in the paperSubstantial claim - Findingcitation
The microfluidic setup allows automatic generation of homogeneous organoids embedded within microbeads of controlled size, composition and cell distribution.
“This setup allows the automatic generation of homogeneous organoids that are embedded within microbeads of controlled size, composition and cell distribution (Figure 1B–a) (20).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [20] Dolega, 2015, Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development, Biomaterials source
- Findingown data
RWPE-1 and MCF10A cell lines gave rise to polarized hollow acini in both controls (untreated and siRNA AllStars), as shown by absence of cells at the center of acini and apical localization of the Golgi apparatus.
“RWPE-1 and MCF10A cell lines gave rise to polarized hollow acini in both controls (untreated and siRNA AllStars), as shown by the absence of cells at the center of acini and by the apical localization of the Golgi apparatus.”
Quote in the paperSubstantial claim - Findingown data
In RWPE-1 organoids, transfection with siRNA against p63 decreased acini from 52% to 30% (P = 0.008) compared with siRNA AllStars.
“However, when transfecting RWPE-1 organoids with siRNA directed against p63, we observed an increase in both spheroids (from 14% to 31%) (P = 0.008) and invasive spheroids (from 2% to 6%) (P = 0.056) at the expense of acini (from 52 to 30%) (P = 0.008) compared with transfection with siRNA AllStars.”
Quote in the paperSubstantial claim - Findingown data
Cell viability was more than 85% under all tested conditions.
“Cell viability was found to be more than 85% under all conditions (Figure 4B).”
Quote in the paperSubstantial claim - Findingown data
In RWPE-1 organoids, transfection with siRNA against PTEN decreased acini from 52% to 36% (P = 0.016) compared with siRNA AllStars.
“When performing transfection with siRNA directed against the PTEN gene, we also observed an increase in spheroids (from 14 to 27%) (P = 0.008) and invasive spheroids (from 2 to 4%) (P = 0.095) at the expense of acini (from 52 to 36%) (P = 0.016).”
Quote in the paperSubstantial claim - Findingown data
In MCF10A organoids, PTEN inhibition decreased acini from 78% to 65% (P = 0.008).
“This was accompanied by a decrease in the percentage of acini from 78 to 60% for p63 inhibition (P = 0.008) and from 78 to 65% for PTEN inhibition (P = 0.008).”
Quote in the paperSubstantial claim - Findingown data
In RWPE-1 organoids, transfection with siRNA against p63 increased spheroids from 14% to 31% (P = 0.008) compared with siRNA AllStars.
“However, when transfecting RWPE-1 organoids with siRNA directed against p63, we observed an increase in both spheroids (from 14% to 31%) (P = 0.008) and invasive spheroids (from 2% to 6%) (P = 0.056) at the expense of acini (from 52 to 30%) (P = 0.008) compared with transfection with siRNA AllStars.”
Quote in the paperSubstantial claim - Findingown data
Electroporation was very effective for transfecting RWPE-1 cells.
“As previously observed, electroporation was found to be very effective for transfecting RWPE-1 cells.”
Quote in the paperNot a substantial claim - Findingown data
MCF10A cells displayed higher acini-forming capability than RWPE-1 cells (80% versus 50%; P = 0.004).
“MCF10A cells displayed a higher acini-forming capability compared with RWPE-1 cells (respectively 80 and 50%) (P = 0.004).”
Quote in the paperNot a substantial claim - Findingown data
In MCF10A organoids, p63 inhibition increased round structures from 19% to 33% (P = 0.008).
“Similar results were obtained using MCF10A organoids, with an increase in round structures (from 19 to 33% for p63 inhibition (P = 0.008) and 19 to 29% for PTEN inhibition (P = 0.008)), spheroids (from 3 to 7% for p63 inhibition (P = 0.008) and 3 to 6% for PTEN inhibition (P = 0.008)) and invasive spheroids (from 0.07 to 0.18% for p63 inhibition (P = 0.016) and 0.07 to 0.24% for PTEN inhibition (P”
Quote in the paperSubstantial claim - Findingown data
In RWPE-1 organoids, transfection with siRNA against PTEN increased invasive spheroids from 2% to 4% (P = 0.095) compared with siRNA AllStars.
“When performing transfection with siRNA directed against the PTEN gene, we also observed an increase in spheroids (from 14 to 27%) (P = 0.008) and invasive spheroids (from 2 to 4%) (P = 0.095) at the expense of acini (from 52 to 36%) (P = 0.016).”
Quote in the paperSubstantial claim - Findingown data
In RWPE-1 organoids, transfection with siRNA against PTEN increased spheroids from 14% to 27% (P = 0.008) compared with siRNA AllStars.
“When performing transfection with siRNA directed against the PTEN gene, we also observed an increase in spheroids (from 14 to 27%) (P = 0.008) and invasive spheroids (from 2 to 4%) (P = 0.095) at the expense of acini (from 52 to 36%) (P = 0.016).”
Quote in the paperSubstantial claim - Findingreasoning
In RWPE-1 cells, no significant difference in transfection efficiency was observed when varying Matrigel concentration from 70% to 50%.
“In RWPE-1 cells, no significant difference was observed when varying microbead size (from 450 to 250 μm) or Matrigel concentration (from 70 to 50%) (Figure 4C).”
Quote in the paperNot a substantial claim - Findingown data
No significant difference in organoid size was observed between different conditions in RWPE-1 and MCF10A models.
“Interestingly, we observed no significant difference when comparing the size of organoids between the different conditions in RWPE-1 and MCF10A models (Figure 6C), which shows the relevance of morphological classification used here.”
Quote in the paperNot a substantial claim - Findingown data
In MCF10A organoids, PTEN inhibition increased spheroids from 3% to 6% (P = 0.008).
“Similar results were obtained using MCF10A organoids, with an increase in round structures (from 19 to 33% for p63 inhibition (P = 0.008) and 19 to 29% for PTEN inhibition (P = 0.008)), spheroids (from 3 to 7% for p63 inhibition (P = 0.008) and 3 to 6% for PTEN inhibition (P = 0.008)) and invasive spheroids (from 0.07 to 0.18% for p63 inhibition (P = 0.016) and 0.07 to 0.24% for PTEN inhibition (P”
Quote in the paperSubstantial claim - Findingown data
In MCF10A organoids, transfection efficiency increased from 23% to 38% when decreasing Matrigel concentration from 100% to 70%.
“In contrast, transfection efficiency increased from 23 to 36% when decreasing microbead size (from 450 to 250 μm) and from 23 to 38% when decreasing Matrigel concentration (from 100 to 70%) in MCF10A organoids.”
Quote in the paperSubstantial claim - Findingown data
MCF10A cells had a lower percentage of spheroids than RWPE-1 cells (3% versus 13%; P = 0.004).
“This was accompanied by a lower percentage of round structures (17 versus 34%) (P = 0.048) as well as spheroids (3 versus 13%) (P = 0.004) and invasive spheroids (0 versus 3%) (P = 0.004).”
Quote in the paperNot a substantial claim - Findingown data
In MCF10A organoids, p63 inhibition increased spheroids from 3% to 7% (P = 0.008).
“Similar results were obtained using MCF10A organoids, with an increase in round structures (from 19 to 33% for p63 inhibition (P = 0.008) and 19 to 29% for PTEN inhibition (P = 0.008)), spheroids (from 3 to 7% for p63 inhibition (P = 0.008) and 3 to 6% for PTEN inhibition (P = 0.008)) and invasive spheroids (from 0.07 to 0.18% for p63 inhibition (P = 0.016) and 0.07 to 0.24% for PTEN inhibition (P”
Quote in the paperSubstantial claim - Findingown data
p63 and PTEN protein levels were effectively decreased by ~50% in both RWPE-1 and MCF10A models after siRNA transfection.
“The results demonstrate no difference between untreated controls and organoids that are transfected with siRNA AllStars, while p63 and PTEN protein levels were effectively decreased by ∼50% in both RWPE-1 and MCF10A models.”
Quote in the paperSubstantial claim - Findingreasoning
High heterogeneity was observed between experiments when using plasmids for transfection.
“Despite these promising results, a high heterogeneity was observed between experiments when using plasmids.”
Quote in the paperSubstantial claim - Findingown data
The 57% extinction efficiency reached in MCF10A organoids by decreasing both microbead size and Matrigel concentration is equal to that previously observed in 2D-cultured MCF10A cells.
“This extinction efficiency is equal to the one we previously observed in 2D-cultured MCF10A cells (Figure 4A).”
Quote in the paperSubstantial claim - Findingown data
Transfection efficiency was higher in encapsulated RWPE-1 organoids than in 2D-cultured RWPE-1 cells (77% versus 65%; ns, P = 0.114).
“Interestingly, extinction efficiency was even higher in encapsulated organoids compared with 2D-cultured cells (77 versus 65%) (ns, P = 0.114) (Figure 4A).”
Quote in the paperNot a substantial claim - Findingreasoning
In RWPE-1 cells, no significant difference in transfection efficiency was observed when varying microbead size from 450 to 250 μm.
“In RWPE-1 cells, no significant difference was observed when varying microbead size (from 450 to 250 μm) or Matrigel concentration (from 70 to 50%) (Figure 4C).”
Quote in the paperNot a substantial claim - Findingown data
No difference in p63 and PTEN protein levels was observed between untreated controls and organoids transfected with siRNA AllStars.
“The results demonstrate no difference between untreated controls and organoids that are transfected with siRNA AllStars, while p63 and PTEN protein levels were effectively decreased by ∼50% in both RWPE-1 and MCF10A models.”
Quote in the paperSubstantial claim - Findingown data
Electroporation efficiency in MCF10A cells further decreased when performing direct 3D organoid transfection compared with 2D culture (57% versus 31%; P = 0.024).
“and further decreased when performing direct 3D organoid transfection (from 57% in 2D to 31% in microbeads) (P = 0.024).”
Quote in the paperSubstantial claim - Findingown data
In MCF10A organoids, p63 inhibition decreased acini from 78% to 60% (P = 0.008).
“This was accompanied by a decrease in the percentage of acini from 78 to 60% for p63 inhibition (P = 0.008) and from 78 to 65% for PTEN inhibition (P = 0.008).”
Quote in the paperSubstantial claim - Findingown data
MCF10A cells had a lower percentage of round structures than RWPE-1 cells (17% versus 34%; P = 0.048).
“This was accompanied by a lower percentage of round structures (17 versus 34%) (P = 0.048) as well as spheroids (3 versus 13%) (P = 0.004) and invasive spheroids (0 versus 3%) (P = 0.004).”
Quote in the paperNot a substantial claim - Findingown data
In MCF10A organoids, p63 inhibition increased invasive spheroids from 0.07% to 0.18% (P = 0.016).
“Similar results were obtained using MCF10A organoids, with an increase in round structures (from 19 to 33% for p63 inhibition (P = 0.008) and 19 to 29% for PTEN inhibition (P = 0.008)), spheroids (from 3 to 7% for p63 inhibition (P = 0.008) and 3 to 6% for PTEN inhibition (P = 0.008)) and invasive spheroids (from 0.07 to 0.18% for p63 inhibition (P = 0.016) and 0.07 to 0.24% for PTEN inhibition (P”
Quote in the paperSubstantial claim - Findingown data
MCF10A cells had a lower percentage of invasive spheroids than RWPE-1 cells (0% versus 3%; P = 0.004).
“This was accompanied by a lower percentage of round structures (17 versus 34%) (P = 0.048) as well as spheroids (3 versus 13%) (P = 0.004) and invasive spheroids (0 versus 3%) (P = 0.004).”
Quote in the paperNot a substantial claim - Findingown data
In MCF10A organoids, PTEN inhibition increased round structures from 19% to 29% (P = 0.008).
“Similar results were obtained using MCF10A organoids, with an increase in round structures (from 19 to 33% for p63 inhibition (P = 0.008) and 19 to 29% for PTEN inhibition (P = 0.008)), spheroids (from 3 to 7% for p63 inhibition (P = 0.008) and 3 to 6% for PTEN inhibition (P = 0.008)) and invasive spheroids (from 0.07 to 0.18% for p63 inhibition (P = 0.016) and 0.07 to 0.24% for PTEN inhibition (P”
Quote in the paperSubstantial claim - Findingown data
No difference in the proportion of different morphological categories was observed between untreated organoids and organoids transfected with siRNA AllStars.
“We observed no difference in the proportion of different morphological categories between untreated organoids or organoids transfected with siRNA AllStars (Figure 6B).”
Quote in the paperSubstantial claim - Findingown data
In RWPE-1 organoids, transfection with siRNA against p63 increased invasive spheroids from 2% to 6% (P = 0.056) compared with siRNA AllStars.
“However, when transfecting RWPE-1 organoids with siRNA directed against p63, we observed an increase in both spheroids (from 14% to 31%) (P = 0.008) and invasive spheroids (from 2% to 6%) (P = 0.056) at the expense of acini (from 52 to 30%) (P = 0.008) compared with transfection with siRNA AllStars.”
Quote in the paperSubstantial claim - Findingown data
A small but not significant decrease (~5%) in cell viability was observed after 3D transfection compared with 2D transfection in both RWPE-1 and MCF10A cells.
“A small but not significant decrease (∼5%) in cell viability was observed after 3D transfection compared with 2D transfection in both RWPE-1 and MCF10A cells.”
Quote in the paperNot a substantial claim - Findingown data
In MCF10A organoids, transfection efficiency increased from 23% to 36% when decreasing microbead size from 450 to 250 μm.
“In contrast, transfection efficiency increased from 23 to 36% when decreasing microbead size (from 450 to 250 μm) and from 23 to 38% when decreasing Matrigel concentration (from 100 to 70%) in MCF10A organoids.”
Quote in the paperSubstantial claim - Findingown data
Electroporation efficiency was lower in MCF10A cells than in RWPE-1 cells under 2D culture conditions (65% versus 57%; ns, P = 0.5).
“In contrast, electroporation efficiency was found to be lower in MCF10A cells compared with RWPE-1 cells under 2D culture conditions (from 65 to 57%) (ns, P = 0.5)”
Quote in the paperNot a substantial claim - Findingown data
More than 40% of RWPE-1 cells were expressing GFP protein after electroporation of GFP-expressing plasmid (P=0.004).
“We further used flow cytometry analysis on cells that were extracted from transfected organoids and demonstrated that more than 40% of RWPE-1 cells were expressing the GFP protein (P = 0.004) (Supplementary Figure S8B).”
Quote in the paperSubstantial claim - Findingown data
In MCF10A organoids, PTEN inhibition increased invasive spheroids from 0.07% to 0.24% (P = 0.016).
“Similar results were obtained using MCF10A organoids, with an increase in round structures (from 19 to 33% for p63 inhibition (P = 0.008) and 19 to 29% for PTEN inhibition (P = 0.008)), spheroids (from 3 to 7% for p63 inhibition (P = 0.008) and 3 to 6% for PTEN inhibition (P = 0.008)) and invasive spheroids (from 0.07 to 0.18% for p63 inhibition (P = 0.016) and 0.07 to 0.24% for PTEN inhibition (P”
Quote in the paperSubstantial claim - Findingown data
In MCF10A organoids, decreasing both microbead size and Matrigel concentration reached up to 57% extinction efficiency (P = 0.05).
“Finally, we were able to reach up to 57% extinction efficiency when decreasing both microbead size and Matrigel concentration in MCF10A organoids (P = 0.05).”
Quote in the paperSubstantial claim - Findingown data
Direct electroporation of encapsulated organoids reaches up to 80% transfection efficiency in RWPE-1 organoids.
“we demonstrate that direct electroporation of encapsulated organoids reach up to 80% transfection efficiency in RWPE-1 organoids.”
Quote in the paperSubstantial claim - Findingreasoning
The designed automatic microfluidic platform provides incremental improvements toward high-throughput production of well-controlled organoids embedded within microbeads (~2000 microbeads/min).
“we designed an automatic and robust microfluidic platform that provides incremental improvements toward a high-throughput production of well-controlled organoids that are embedded within microbeads (∼2000 microbeads/min).”
Quote in the paperSubstantial claim - Findingown data
Down-regulation of p63 and PTEN led to failure of acinar development as evidenced by lack of cell polarization, lumen clearance and generation of spheroids consisting of loosely associated disordered cells.
“We observed that down-regulation of p63 and PTEN led to the failure of acinar development as evidenced by the lack of cell polarization, lumen clearance and the generation of spheroids that consist of loosely associated disordered cells.”
Quote in the paperSubstantial claim - Premisecitation
Quiescent cells located at the center of 3D structures are often refractory to transfection.
“In addition, quiescent cells that are located at the center of 3D structures are often refractory to transfection (10).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [10] Mellor, 2006, Optimising non-viral gene delivery in a tumour spheroid model, J. Gene Med. source
- Interpretationown data
The microbead setup is less expensive than traditional 3D culture because it requires two to three times less Matrigel to obtain the same amount of organoids.
“This setup would be compatible with high-throughput screening applications and is less expensive than traditional 3D culture because it requires two to three times less Matrigel to obtain the same amount of organoids.”
Quote in the paperSubstantial claim - Premisecitation
3D epithelial organoid culture in Matrigel recapitulates numerous features of glandular tissues, including fully differentiated acini that maintain apico-basal polarity by enclosing a central lumen.
“3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen.”
- Also stated in: § INTRODUCTION · plain
“For example, epithelial organoid culture in Matrigel recapitulates numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity by enclosing a central lumen (5).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [5] O’Brien, 2002, Opinion: building epithelial architecture: insights from three-dimensional culture models, Nat. Rev. Mol. Cell Biol. source
- Also stated in: § INTRODUCTION · plain
- Interpretationown data
Absence of either p63 or PTEN expression during organoid development leads to failure of cell polarization and lumen clearance, generating spheroids of loosely associated disordered cells.
“This marks a failure of cell polarization and lumen clearance, which leads to the generation of spheroids that consist of loosely associated disordered cells in the absence of either p63 or PTEN expression during organoid development.”
Quote in the paperSubstantial claim - Interpretationreasoning
The small decrease in cell viability after 3D transfection is probably due to the cell extraction process combining prolonged trypsin incubation and vortex steps needed to extract cells from Matrigel microbeads before viability assessment.
“However, this is probably due to the cell extraction process, which combines prolonged trypsin incubation and several vortex steps (‘Materials and Methods’ section) that are needed to extract cells from Matrigel microbeads before assessing their viability.”
Quote in the paperNot a substantial claim - Interpretationown data
The lack of size difference shows the relevance of the morphological classification used here.
“which shows the relevance of morphological classification used here.”
Quote in the paperSubstantial claim - Interpretationown data
Morphological criteria can be used to systematically discriminate glandular organoids from tumor-like spheroids.
“Altogether, these results support the use of morphological criteria to systematically discriminate glandular organoids from tumor-like spheroids.”
Quote in the paperSubstantial claim - Interpretationown data
It is possible to increase extinction efficiency in organoids refractory to electroporation by optimizing microbead size and Matrigel concentration to decrease Matrigel quantity around 3D structures.
“Altogether, these results demonstrate the possibility of increasing extinction efficiency in organoid refractory for electroporation by optimizing parameters such as microbead size and Matrigel concentration to decrease Matrigel quantity around 3D structures.”
Quote in the paperSubstantial claim - Interpretationown data
By optimizing microbead size and Matrigel concentration, extinction efficiencies comparable between 2D cultures and 3D organoids were obtained.
“By doing so, we obtained extinction efficiencies that are comparable between 2D cultures and 3D organoids.”
Quote in the paperSubstantial claim - Interpretationown data
The transfection method does not affect acini development in either RWPE-1 or MCF10A models.
“This demonstrates that the transfection method does not affect acini development in either RWPE-1 or MCF10A models.”
Quote in the paperSubstantial claim - Interpretationreasoning
Close contacts between cells may act as physical barriers that limit diffusion of plasmid DNA and therefore its access to cells present in the core of the spheroid.
“Indeed, close contacts between cells may act as physical barriers that limit the diffusion of plasmid DNA and therefore its access to cells present in the core of the spheroid.”
Quote in the paperNot a substantial claim - Interpretationreasoning
This phenomenon would likely not be reflected at first by an increase in overall size of organoids because of cell proliferation that occurs at the center of 3D structures such as for in situ carcinoma.
“This phenomenon would likely not be reflected at first by an increase in the overall size of organoids because of cell proliferation that occurs at the center of 3D structures such as for in situ carcinoma.”
Quote in the paperSubstantial claim - Interpretationown data
The present system reached siRNA transfection efficiencies comparable between 2D cultures and 3D organoids for the first time.
“Strikingly, we managed for the first time to reach siRNA transfection efficiencies that are comparable between 2D cultures and 3D organoids.”
Quote in the paperSubstantial claim - Premisecitation
ECM impedes virus-mediated gene delivery since its digestion dramatically improves extinction efficiency.
“While viral transduction systems usually allow higher transfection efficiencies, it has been demonstrated that ECM impedes virus-mediated gene delivery since its digestion dramatically improves extinction efficiency (12), which explains the limited effect on Matrigel microbeads.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [12] Koo, 2011, Controlled gene expression in primary Lgr5 organoid cultures, Nat. Methods source
- Interpretationcitation
These results suggest that limited access to central regions of MCTS remains a significant barrier to gene delivery.
“These results suggest that limited access to central regions of MCTS remains a significant barrier to gene delivery.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [28] Wasungu, 2009, A 3D in vitro spheroid model as a way to study the mechanisms of electroporation, Int. J. Pharm. source
- Interpretationreasoning
Direct organoid electroporation is particularly useful for transfecting long-term cultured organoids and at different development stages.
“Direct organoid electroporation is particularly useful for transfecting long-term cultured organoids and at different development stages.”
Quote in the paperSubstantial claim - Interpretationown data
The reduced amount of Matrigel surrounding organoids constitutes a permissive 3D environment that facilitates transfection.
“In addition, the reduced amount of Matrigel surrounding organoids constitutes a permissive 3D environment that facilitates transfection.”
Quote in the paperSubstantial claim - Interpretationreasoning
The difference in spheroid proportion without size difference could be explained by an increase in cell proliferation and/or a loss of apoptosis at the center of acini, leading to an increase in cell number progressively filling the lumen.
“This difference could be explained by an increase in cell proliferation and/or a loss of apoptosis, which occurs at the center of acini, leading to an increase in cell number progressively filling the lumen.”
Quote in the paperNot a substantial claim - Interpretationown data
The morphological classification contains better criteria compared with 3D structure size to discriminate healthy organoids from tumor spheroids.
“We believe that this classification contains better criteria compared with 3D structure size to discriminate healthy organoids from tumor spheroids.”
Quote in the paperSubstantial claim
- May combine two claims. 29
- Classified as probably not a substantial claim. 15
- The quote only partly states this claim. 1
May combine two claims.
PremisecitationLipofection and electroporation have been proven relatively inefficient in transfecting 3D cultures, with transfection efficiencies lower than 5% and 20%, respectively.
“However, they have been proven to be relatively inefficient in transfecting 3D cultures with transfection efficiencies lower than 5 and 20%, respectively (7–9).”
Quote in the paperSubstantial claim2 of 3 sources checked- cites [7] Kostarelos, 2004, Binding and interstitial penetration of liposomes within avascular tumor spheroids, Int. J. Cancer source
The Results and Discussion examine how liposomes bind to and penetrate prostate tumor spheroids, not how efficiently lipofection or electroporation transfects 3D cultures. Searches for electroporation and the stated 5% and 20% transfection efficiencies found no passage supporting those figures in our copy. (LLM, conf=0.75)
Experimental — accuracy not yet measured - cites [8] Canatella, 2004, Tissue electroporation: quantification and analysis of heterogeneous transport in multicellular environments, Biophys. J. source
The Results and discussion section reports that cells in multicellular spheroids took up fewer molecules after electroporation than isolated cells, supporting a reduced-delivery effect in a 3D culture model. The study measured calcein uptake, not transfection efficiency, and does not establish the claimed below-20% figure or the lipofection figure. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [9] Zhang, 2012, High-throughput transfection of interfering RNA into a 3D cell-culture chip, Small Weinh. Bergstr. Ger. source
May combine two claims.
PremisecitationGene-activated matrices maintain permanent transfection with 60-90% siRNA-mediated gene knock-down during 3D organoid development but do not allow transfection at different time points.
“While this system maintains a permanent transfection with 60–90% of siRNA-mediated gene knock-down during 3D organoid development, it does not allow transfection at different time points.”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [18] Zoldan, 2011, Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture, Biomaterials source
The Introduction reports 60–90% silencing in a 3D hESC matrix, and the Results report siRNA-mediated knockdown during differentiation. But the Results describe knockdown measured after three days, not permanent transfection or 3D organoid development. The paper does not establish that transfection cannot occur at different time points. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [19] Sapet, 2013, 3D-fection: cell transfection within 3D scaffolds and hydrogels, Ther. Deliv. source
Classified as probably not a substantial claim. May combine two claims.
Premisecitationp63 and PTEN are tumor suppressors that play a key role in breast and prostate tissue development.
“we inhibited p63 and PTEN expression, two genes that are described as tumor-suppressors that play a key role in breast and prostate tissue development (32–35).”
Quote in the paperSubstantial claim3 of 4 sources checked- cites [32] Shore, 2016, PTEN is required to maintain luminal epithelial homeostasis and integrity in the adult mammary gland, Dev. Biol. source
The Introduction (page not provided) identifies PTEN as a tumor suppressor and describes its role in mammary epithelial development; it also discusses effects of PTEN loss in the prostate. The paper does not establish the corresponding claim about p63, so it supports only the PTEN portion of the statement attributed to the citation group. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [33] Wang, 2003, Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer, Cancer Cell source
The Introduction (page not specified) calls PTEN a tumor suppressor and links PTEN loss to mammary tumors and prostate cancer. The Results (page not specified) show that deleting Pten causes prostate tumors. Our copy does not address p63 or establish that either gene plays a key role in normal breast and prostate tissue development. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [34] Tucci, 2012, Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer, Proc. Natl. Acad. Sci. U.S.A. source
- cites [35] Yoh, 2016, Repression of p63 and induction of EMT by mutant Ras in mammary epithelial cells, Proc. Natl. Acad. Sci. U.S.A. source
The paper’s introductory passage (page not provided) says p63 is a master regulator of epithelial cells and that mice lacking it have no mammary glands, supporting a role in breast development. It discusses p63’s cancer role as tissue-dependent and mentions PTEN loss in breast cancer, but does not establish that both genes are tumor suppressors with key roles in breast and prostate tissue development. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductcitationThe selected siRNA dose (20 nM) is used in routine in the authors' lab and is consistent with other works from the literature working with MCF10A or RWPE1.
“This selected dose is used in routine in our lab (22) and is consistent with other works from the literature working with MCF10A (23) or RWPE1 (24).”
Quote in the paperNot a substantial claim3 of 3 sources checked- cites [22] Benzina, 2015, A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation, Cell. Mol. Life Sci. source
The citation (22) is attached to the claim that the citing authors routinely use the selected 20 nM dose; the MCF10A and RWPE1 comparisons have separate citations. The cited paper’s Materials and methods (Standard transfection; siRNA microarray printing; page unavailable) describes siRNA use, including a 20 µM stock solution, but does not report routine use of a 20 nM dose. Searched for the final concentration and routine use. (LLM, conf=0.74)
Experimental — accuracy not yet measured - cites [23] Martin, 2007, Multiplexing siRNAs to compress RNAi-based screen size in human cells, Nucleic Acids Res. source
The citation to this paper is attached to the MCF10A comparison, not the citing authors’ routine laboratory use or the RWPE1 comparison. The Methods (‘Transfection of synthetic siRNAs’; page unavailable) report 20 nM for paired siRNAs and controls, and report MCF10A transfections, but do not establish that the MCF10A experiments used a 20 nM total dose. The Results (‘Molecular validation of multiplexed siRNAs’; page unavailable) describe the illustrated MCF10A multiplex experiment at 60 nM total. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [24] Ezponda, 2013, The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial-mesenchymal transition and invasive properties of prostate cancer, Oncogene source
Probably not a substantial claim: may not pass S3 (load-bearing).
InterpretationcitationThese results are consistent with a previous study demonstrating that PC3 cells probably retain some differentiation capabilities.
“These results are consistent with a previous study, which demonstrates that PC3 cells probably retain some differentiation capabilities (31).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [31] Härmä, 2010, A comprehensive panel of three-dimensional models for studies of prostate cancer growth, invasion and drug responses, PLoS One source
May combine two claims.
InterpretationcitationIn a recent study, gas plasma-facilitated cellular transfection reached siRNA extinction efficiency of about 20% and plasmid transfection efficiency of less than 5% in a 3D culture system, compared with more than 80% and more than 40% in the present system.
“Moreover, in a recent study (36) demonstrating the benefits of a novel gas plasma-facilitated cellular transfection method, siRNA extinction efficiency of about 20% (as compared to more than 80% in our system) and plasmid transfection efficiency of less than 5% (as compared to more than 40% in our system) were reached in a 3D culture system.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [36] Xu, 2016, Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures, Sci. Rep. source
The Results (page unavailable) report gas plasma-facilitated delivery of siRNA in 3D cultures and relatively poor plasmid transfection in both 2D and 3D cultures. They do not state about 20% siRNA extinction or less than 5% plasmid transfection; the Methods describe measuring siRNA uptake, not siRNA extinction. The figures cited in the Results do not supply those percentages in our text copy. (LLM, conf=0.83)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOther studies achieved high transfection efficiencies using polyplex nanomicelles, flexible nanocarriers or polyamine nanoparticles on MCTS.
“In other studies, researchers managed to achieve high transfection efficiencies using polyplex nanomicelles (37), flexible nanocarriers (38) or polyamine nanoparticles (39) on MCTS.”
Quote in the paperNot a substantial claim1 of 3 sources checked- cites [37] Han, 2007, Transfection study using multicellular tumor spheroids for screening non-viral polymeric gene vectors with low cytotoxicity and high transfection efficiencies, J. Control. Release source
- cites [38] Jung, 2017, Development of flexible nanocarriers for siRNA delivery into tumor tissue, Int. J. Pharm. source
- cites [39] Zamora, 2014, Photochemical internalization-mediated nonviral gene transfection: polyamine core-shell nanoparticles as gene carrier, J. Biomed. Opt. source
May combine two claims.
InterpretationcitationWhile ensuring controlled and efficient transfection, inducible systems are hardly compatible with high-throughput analysis.
“Nonetheless, while ensuring a controlled and efficient transfection, this system is hardly compatible with high-throughput analysis.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [41] Herr, 2011, A novel MCF-10A line allowing conditional oncogene expression in 3D culture, Cell Commun. Signal. source
The Results section describes tightly controlled, efficient gene expression, and Methods (Cell lines and tissue culture; page unavailable) reports improved transfection efficiency with nucleofection. The paper does not say the system is hardly compatible with high-throughput analysis; the Discussion instead says its protocol can generate several sublines in parallel. (LLM, conf=0.82)
Experimental — accuracy not yet measured
May combine two claims.
Findingown datap63 and PTEN are key genes in acinar development in breast and prostate tissues.
“Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataLipofectamin and Oligofectamin yield low extinction efficiencies (≈5%), while TransIT, 3DFect and 3DFectIn reach up to 15% extinction efficiency.
“Lipofectamin and Oligofectamin demonstrated low extinction efficiencies (≈5%), while TransIT, 3DFect and 3DFectIn reached up to 15% extinction efficiency (Figure 2B).”
Quote in the paperSubstantial claim- Rests on an assumption: GFP knock-down (extinction efficiency) is a valid surrogate for siRNA transfection/delivery efficiency in organoids.
May combine two claims.
Findingown data5% of PC3-derived organoids differentiated into hollow acini, while this was rarely observed for other cancer cell lines.
“Interestingly, 5% of the PC3-derived organoids were able to differentiate into hollow acini, while this phenomenon was rarely observed for other cancer cell lines.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingnone givenOrganoids in microbeads are easy to handle and can be stored for long periods by using cryopreservation procedures.
“Finally, these organoids are easy to handle, allowing us to store them for long periods by using cryopreservation procedures.”
Quote in the paperNot a substantial claimMay combine two claims.
FindingreasoningThe process of droplet formation is highly repeatable under laminar flow conditions, making it possible to produce thousands of identical bioreactors.
“Because the process of droplet formation is highly repeatable under laminar flow conditions, it is possible to produce thousands of identical bioreactors.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataOrganoids transfected with siRNA targeting p63 or PTEN failed to develop into fully differentiated acini, demonstrating a tumor-like phenotype, evidenced by loss of polarity and presence of cells at the center of 3D structures.
“In contrast, organoids transfected with siRNA that targeted p63 or PTEN failed to develop into fully differentiated acini, which demonstrates a tumor-like phenotype, as evidenced by the loss of polarity and the presence of cells at the center of 3D structures.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe approach enables and improves acini culture homogeneity, recovery of 3D structures, throughput of 3D culture preparation, and high-throughput analysis.
“As previously demonstrated, our approach enables and improves acini culture homogeneity, recovery of 3D structures for further analysis such as fundamental genomics, throughput of the 3D culture preparation and most importantly high-throughput analysis, as we have shown by applying large-particle FACS.”
Quote in the paperSubstantial claimMay combine two claims.
FindingreasoningMicrobead electroporation is generally applicable to transfect siRNA and, to a lower extent, plasmids, even into cells located at the center of organoids.
“However, these results clearly demonstrate the general applicability of microbead electroporation to transfect siRNA and to a lower extent plasmids, even into cells that are located at the center of organoids.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe present results demonstrate high levels of siRNA molecule penetration through ECM and increased efficacy in quiescent cells at the center of organoids afforded by electroporation.
“In contrast, our results demonstrate high levels of siRNA molecule penetration through ECM and increased efficacy in quiescent cells at the center of organoids afforded by electroporation, which makes it a promising strategy for gene silencing studies.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationViral vectors provide the highest transfection efficiency but have serious limitations such as the size of DNA carried, intrinsic biosafety issues, concern for viral insertion mutagenesis, and an inability to diffuse through ECM.
“Viral vectors provide the highest transfection efficiency but have serious limitations such as the size of DNA carried in the vector, intrinsic biosafety issues, concern for viral insertion mutagenesis (11) and an inability to diffuse through ECM (12).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [11] Salimzadeh, 2013, Non-viral transfection methods optimized for gene delivery to a lung cancer cell line, Avicenna J. Med. Biotechnol.
- cites [12] Koo, 2011, Controlled gene expression in primary Lgr5 organoid cultures, Nat. Methods source
May combine two claims.
Interpretationown dataElectroporation is efficient and innocuous for transfecting RWPE-1 organoids in microbeads compared with other techniques.
“These results highlight the efficiency and inocuity of electroporation to transfect RWPE-1 organoids in microbeads compared with other techniques.”
Quote in the paperSubstantial claim- Rests on an assumption: GFP knock-down (extinction efficiency) is a valid surrogate for siRNA transfection/delivery efficiency in organoids.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll data are represented as a mean of at least three separate experiments.
“All data are represented as a mean of at least three separate experiments.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationRWPE-1 cells are a model for normal prostate epithelial cell behavior, characterized by a polarized acinar morphology in 3D cultures.
“RWPE-1 cells were previously used as a model for normal prostate epithelial cell behavior, as characterized by a polarized acinar morphology in 3D cultures (20).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [20] Dolega, 2015, Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development, Biomaterials source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationreasoningDifferential GFP expression may be related to large molecules such as plasmid DNA facing physical barriers (nuclear envelope in addition to cytoplasmic membrane) before reaching the nucleus, while small molecules like siRNA freely cross the electropermeabilized membrane.
“This differential GFP expression may also be related to the fact that large molecules such as plasmid DNA face physical barriers (nuclear envelope in addition to cytoplasmic membrane), before reaching the nucleus, while small molecules like siRNA freely cross the electropermeabilized membrane and have a free access to the”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningThe four-category morphological classification reflects different stages of tumor development and can be used to quantify tumorigenic potential of cells.
“We believe that this classification reflects the different stages of tumor development and can thus be used to quantify tumorigenic potential of the cells.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningThe combination of organoid microencapsulation and efficient siRNA delivery methods developed here encompasses existing techniques and allows transfection of physiological organoid cultures embedded in ECM that mimic complex tissue development steps.
“Thus, the combination of organoid microencapsulation and efficient siRNA delivery methods developed here encompasses existing techniques, and allows the transfection of physiological organoid cultures embedded in ECM that mimic complex tissue development steps.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningIn contrast to static 3D cultures in Lab-Tek chambers, organoids grown in Matrigel microbeads are easy to handle and can be manipulated at will to perform electroporation.
“In contrast to static 3D cultures in Lab-Tek chambers, organoids that are grown in Matrigel microbeads are easy to handle and can be manipulated at will to perform electroporation.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTransfected cells were heterogeneously distributed and appeared to be only localized in the outer layers of the spheroids.
“A striking observation is that transfected cells were heterogeneously distributed and appeared to be only localized in the outer layers of the spheroids.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [28] Wasungu, 2009, A 3D in vitro spheroid model as a way to study the mechanisms of electroporation, Int. J. Pharm. source
May combine two claims.
Interpretationown dataThese results validate the roles of p63 and PTEN as tumor-suppressor genes that play a key role in acinar development in both breast and prostate tissues.
“These results validate the roles of p63 and PTEN as tumor-suppressor genes that play a key role in acinar development in both breast and prostate tissues.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenMorphological analysis of 3D structures was performed by eye counting under a phase microscope, which remains time consuming and laborious, although more rapid than conventional immunostaining and confocal z-stack analysis.
“In this study, morphological analysis of 3D structures was performed by eye counting under a phase microscope, which remains time consuming and laborious, although more rapid that the conventional immunostaining and confocal z-stack analysis.”
Quote in the paperNot a substantial claimThe quote only partly states this claim.
Interpretationown dataThe high siRNA penetration and efficacy in central quiescent cells makes electroporation a promising strategy for gene silencing studies.
“which makes it a promising strategy for gene silencing studies.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationUptake efficiency of macromolecules such as plasmid DNA was inefficient; only a few cells from colon cancer MCTS were transfected using a plasmid coding for GFP.
“However, the uptake efficiency of macromolecules such as plasmid DNA was found to be inefficient. Indeed, only a few cells from colon cancer MCTS were transfected using a plasmid that codes for the GFP.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [28] Wasungu, 2009, A 3D in vitro spheroid model as a way to study the mechanisms of electroporation, Int. J. Pharm. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationRetroviral infection allowed sustained expression of GFP in more than 90% of cells from organoid fragments removed from Matrigel.
“For example, retroviral infection allowed the sustained expression of GFP in more than 90% of cells from organoid fragments that were removed from Matrigel (12).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [12] Koo, 2011, Controlled gene expression in primary Lgr5 organoid cultures, Nat. Methods source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationMCTS from mouse sarcoma were transfected via electroporation in the presence of propidium iodide; practically the entire cell population was permeabilized, as measured by propidium iodide uptake.
“In another study, MCTS from mouse sarcoma were transfected via electroporation in the presence of propidium iodide. The method was highly efficient since practically the entire cell population was permeabilized, as measured by propidium iodide uptake (28).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [28] Wasungu, 2009, A 3D in vitro spheroid model as a way to study the mechanisms of electroporation, Int. J. Pharm. source
May combine two claims.
Interpretationown dataDespite lower transfection efficiencies in MCF10A organoids, gene silencing can be improved by tuning parameters such as microbead size or Matrigel concentration.
“Despite reaching lower transfection efficiencies in MCF10A organoids, we also showed that gene silencing can be improved by tuning parameters such as microbead size or Matrigel concentration.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationNo difference was detected between cells at the periphery and cells located deeper within the spheroid's core in that electroporation study.
“In addition, no difference was detected between cells at the periphery and the cells located deeper within the spheroid's core.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [28] Wasungu, 2009, A 3D in vitro spheroid model as a way to study the mechanisms of electroporation, Int. J. Pharm. source
Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.
Interpretationown dataThe results highlight the potential of microencapsulated organoid electroporation to study tissue development and tumorigenesis and provide valuable tools for fundamental research and organoid/spheroid-based drug assays.
“Our results highlight the potential of microencapsulated organoid electroporation to study tissue development and tumorigenesis and provide valuable tools that should find versatile applications in fundamental research and organoid/spheroid-based drug assays.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
InterpretationreasoningThe siRNA effect would be totally lost if performing 2D transfection before 3D culture using MCF10A cells because they need a minimum of 14 days to fully develop into differentiated acini.
“For example, the siRNA effect would be totally lost if performing 2D transfection before 3D culture using MCF10A cells because they need a minimum of 14 days to fully develop into differentiated acini.”
Quote in the paperNot a substantial claim- Rests on an assumption: MCF10A cells need a minimum of 14 days to fully develop into differentiated acini.
May combine two claims.
Interpretationown dataElectroporation of organoids grown in Matrigel microbeads is efficient in decreasing p63 and PTEN expression and leads to failure of acini development accompanied by transition toward a tumor-like phenotype.
“Altogether, these results demonstrate that electroporation of organoids that are grown in Matrigel microbeads is efficient in decreasing p63 and PTEN expression and leads to the failure of acini development accompanied by the transition toward a tumor-like phenotype.”
Quote in the paperSubstantial claim
The paper relies on this without support.
GFP knock-down (extinction efficiency) is a valid surrogate for siRNA transfection/delivery efficiency in organoids.
“GFP was used as a surrogate RNAi target in determining siRNA knock-down efficiency with the variety of methods.”
“Actually, we measured the siRNA transfection efficiency because, at the same time, we monitored the siRNA transfection and target gene (GFP) knock-down.”
The paper relies on this without support.
Dissociating organoids into single cells or small groups of cells before transfection causes loss of natural self-organization, spatial architecture and polarity.
“However, under these conditions, the natural self-organization of organoids is lost along with their spatial architecture and polarity, ultimately going back to a 2D cell transfection.”
The paper relies on this without support.
Progeny cells that colonize the matrix after 2D transfection will not be transfected and will gradually lose the desired effect.
“However, progeny cells that colonize the matrix will not be transfected and will gradually lose the desired effect.”
The paper relies on this without support.
Current analysis of organoids uses confocal microscopy and is limited by time-consuming acquisitions and the need for human assistance, which hampers its applicability for pharmaceutical studies.
“Current analysis of organoids uses confocal microscopy and is thus limited by time-consuming acquisitions and the need for human assistance, which hampers their applicability for pharmaceutical studies.”
The paper relies on this without support.
Direct 3D transfection remains challenging and limits high-throughput genetic screening studies in 3D cultures.
“Even though 3D cultures are widely used as relevant models to study physiological and pathological processes, direct 3D transfection remains challenging and limits high-throughput genetic screening studies in 3D cultures.”
The paper relies on this without support.
Current 3D culture systems rely on multiwell plates where cells embedded in ECM scaffolds develop into heterogeneous and overlapping organoids, limiting experiment reproducibility, analysis and gene delivery.
“To date, current 3D culture systems rely on multiwell plates where cells that are embedded in ECM scaffolds develop into heterogeneous and overlapping organoids, limiting experiment reproducibility, analysis and gene delivery.”
The paper relies on this without support.
MCF10A cells need a minimum of 14 days to fully develop into differentiated acini.
“For example, the siRNA effect would be totally lost if performing 2D transfection before 3D culture using MCF10A cells because they need a minimum of 14 days to fully develop into differentiated acini.”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
MCF10A organoids were transfected on day 10 or day 11
MinorThe gene-silencing results say MCF10A organoids developed for 10 days before transfection, while the caption for the same experiments says 11 days.
“prostate RWPE-1 and breast MCF10A organoids were allowed to develop in microbeads for 4 and 10 days, respectively, before performing transfection.”
“Encapsulated RWPE-1 or MCF10A cells were allowed to develop into organoids for respectively 4 and 11 days and were then transfected with indicated siRNA at 20 nM for 3 days”
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Binding and interstitial penetration of liposomes within avascular tumor spheroids
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ResultSelf-citationHan, M., Bae, Y., Nishiyama, N., Miyata, K., Oba, M., & Kataoka, K. (2007) · Journal of Controlled Release · doi:10.1016/j.jconrel.2007.05.012 · Cited by 82 · cited 1× · see citing works
“In other studies, researchers managed to achieve high transfection eficiencies using polyplex nanomicelles (37), lexible nanocarriers (38) or polyamine nanoparticles (39) on MCTS.”
BackgroundRepression of p63 and induction of EMT by mutant Ras in mammary epithelial cells
Yoh, K. E., Regunath, K., Guzman, A., Lee, S.-M., Pfister, N. T., Akanni, O., Kaufman, L. J., Prives, C., & Prywes, R. (2016) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1613417113 · Cited by 75 · see citing works
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“While this method might appear long and laborious, it requires less time and efforts compared to immunoluorescence staining and confocal imaging that remain the gold standard to analyze organoid fate in 3D cultures (25,26).”
MethodA 3D toolbox to enhance physiological relevance of human tissue models
Picollet-D’hahan, N., Dolega, M. E., Liguori, L., Marquette, C., Le Gac, S., Gidrol, X., & Martin, D. K. (2016) · Trends in Biotechnology · doi:10.1016/j.tibtech.2016.06.012 · Cited by 65 · see citing works
Self-citationA 3D in vitro spheroid model as a way to study the mechanisms of electroporation
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“Even in spheroid models produced by cell aggregation and devoid of ECM, direct 3D transfection is usually limited to the outer layer of cells (28), which reduces its relevance and leads to heterogeneous results.”
BackgroundMethodOptimising non-viral gene delivery in a tumour spheroid model
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Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture
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“Gene-activated matrices that combine scaffolds or hydrogels and lipid-based gene delivery reagents were developed to allow direct in situ transfection of cells that grow in 3D matrices (18,19).”
BackgroundIntracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures
Xu, D., Wang, B., Xu, Y., Chen, Z., Cui, Q., Yang, Y., Chen, H., & Kong, M. G. (2016) · Scientific Reports · doi:10.1038/srep27872 · Cited by 46 · cited 1× · see citing works
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BackgroundCryopreservation and Thawing of Cells
Yokoyama, W. M., Thompson, M. L., & Ehrhardt, R. O. (2012) · Current Protocols in Immunology · doi:10.1002/0471142735.ima03gs99 · Cited by 40 · cited 1× · see citing works
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MethodMultiplexing siRNAs to compress RNAi-based screen size in human cells
Martin, S. E., Jones, T. L., Thomas, C. L., Lorenzi, P. L., Nguyen, D. A., Runfola, T., Gunsior, M., Weinstein, J. N., Goldsmith, P. K., Lader, E., et al. (2007) · Nucleic Acids Research · doi:10.1093/nar/gkm141 · Cited by 40 · cited 1× · see citing works
“This selected dose is used in routine in our lab (22) and is consistent with other works from the literature working with MCF10A (23) or RWPE1 (24).”
ResultA novel MCF-10A line allowing conditional oncogene expression in 3D culture
Herr, R., Wöhrle, F. U., Danke, C., Berens, C., & Brummer, T. (2011) · Cell Communication and Signaling · doi:10.1186/1478-811x-9-17 · Cited by 37 · cited 1× · see citing works
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“In other studies, researchers managed to achieve high transfection eficiencies using polyplex nanomicelles (37), lexible nanocarriers (38) or polyamine nanoparticles (39) on MCTS.”
BackgroundHigh-throughput transfection of interfering RNA into a 3D cell-culture chip
Zhang, H., Lee, M., Hogg, M. G., Dordick, J. S., & Sharfstein, S. T. (2012) · Small · doi:10.1002/smll.201102205 · Cited by 16 · see citing works
PTEN is required to maintain luminal epithelial homeostasis and integrity in the adult mammary gland
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BackgroundBenzina, S., Pitaval, A., Lemercier, C., Lustremant, C., Frouin, V., Wu, N., Papine, A., Soussaline, F., Romeo, P.-H., & Gidrol, X. (2015) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-015-1901-7 · Cited by 10 · cited 1× · see citing works
“This selected dose is used in routine in our lab (22) and is consistent with other works from the literature working with MCF10A (23) or RWPE1 (24).”
ResultSelf-citationKong, Q., Wu, G., Han, L., Zhang, Z., Du, J., Sun, W., & Cao, L. (2015) · Cancer Gene Therapy · doi:10.1038/cgt.2015.22 · Cited by 7 · cited 1× · see citing works
“A common strategy to circumvent this issue is to transfect cells that are grown in 2D and to transfer them into 3D culture, which limits the biological issues that we can address (15,17).”
BackgroundDevelopment of flexible nanocarriers for siRNA delivery into tumor tissue
Jung, H., Shimatani, Y., Hasan, M., Uno, K., Hama, S., & Kogure, K. (2017) · International Journal of Pharmaceutics · doi:10.1016/j.ijpharm.2016.11.042 · Cited by 6 · cited 1× · see citing works
“In other studies, researchers managed to achieve high transfection eficiencies using polyplex nanomicelles (37), lexible nanocarriers (38) or polyamine nanoparticles (39) on MCTS.”
BackgroundHuman-Organoid Models: Accomplishments to Salvage Test-Animals
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“While traditional 2D cultures on rigid surfaces fail to reproduce in vivo cell behavior, 3D matrices are becoming increasingly popular supports for cell cultures because they allow mimicking the complex environment that supports cell physiological functions (1) to better predict in vivo responses (2,3) and thus to lim…”
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“Moreover, this process may be emphasized by a slower diffusion of plasmids through the matrix and may contribute to reduce transfection eficiency (29).”
Background
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Hofer, M., & Lutolf, M. P. (2021) · Nature Reviews Materials · doi:10.1038/s41578-021-00279-y · Cited by 1,184 · see citing works
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Advances in Spheroids and Organoids on a Chip
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Copper Transport and Disease: What Can We Learn from Organoids?
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“In addition, while they are still more difficult than in cell culture, gene introduction and other molecular manipulations are possible [33].”
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“Interestingly, parallel studies also demonstrated that encapsulation systems can be successfully used to store and cryopreserve organoids without altering cell growth and differentiation ability, as well as to perform post-treatments, such as direct transfection, and post-genetic screen and organoid analysis [83].”
BackgroundAdvancing Organoid Engineering for Tissue Regeneration and Biofunctional Reconstruction
Jin, H., Xue, Z., Liu, J., Ma, B., Yang, J., & Lei, L. (2024) · Biomaterials Research · doi:10.34133/bmr.0016 · Cited by 53 · see citing works
“The application of a high-throughput, automated approach to generating cell-containing matrix gel beads [171] provided many microcapsules of uniform size, cell distribution, and composition, offering the potential to enable high-throughput characterization.”
MethodRapid Microfluidic Formation of Uniform Patient-Derived Breast Tumor Spheroids
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Liu, J., Du, C., Chen, J., Tang, B., Liu, S., Tan, J., Luo, X., Zhan, J., Liu, J., Chen, Z., et al. (2025) · Small · doi:10.1002/smll.202504563 · Cited by 31 · see citing works
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Khurana, A., Sayed, N., Singh, V., Khurana, I., Allawadhi, P., Rawat, P. S., Navik, U., Pasumarthi, S. K., Bharani, K. K., & Weiskirchen, R. (2022) · Journal of Cellular Biochemistry · doi:10.1002/jcb.30329 · Cited by 22 · see citing works
“This has proved to be a great tool but many researchers pointed out that this could better be used to validate the studies at the transcriptome level and was not a robust method to probe the implications of a gene dysfunction in various biological studies.(42) Before CRISPR, scientists tried to use nucleases such as Z…”
BackgroundPolysarcosine functionalised cationic polyesters efficiently deliver self-amplifying mRNA
Bayraktutan, H., Kopiasz, R. J., Elsherbeny, A., Martínez-Espuga, M., Gümüş, N., Öz, U. C., Polra, K., McKay, P. F., Shattock, R. J., Ordóñez‐Morán, P., et al. (2024) · Polymer Chemistry · doi:10.1039/d4py00064a · Cited by 18 · see citing works
Yoo, K. H., Hennighausen, L. G., & Shin, H. Y. (2018) · Journal of Mammary Gland Biology and Neoplasia · doi:10.1007/s10911-018-9417-z · Cited by 18 · see citing works
Human Endometrial Organoids: Recent Research Progress and Potential Applications
Lou, L., Kong, S., Sun, Y., Zhang, Z., & Wang, H. (2022) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2022.844623 · Cited by 17 · see citing works
Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases
Clément, F., Nougarède, A., Combe, S., et al. (2021) · Journal of Crohn s and Colitis · doi:10.1093/ecco-jcc/jjab129 · Cited by 13 · see citing works
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Organoids as Complex In Vitro Models for Studying Radiation-Induced Cell Recruitment
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“…[184], tumor [185, 186], hair follicle [187], kidney [188], lacrimal gland [189], dental pulp [190], testis [151, 191]– [193], tongue epithelium [194], inner ear [195], cornea [196], sweat gland [197], mammary gland [198], taste bud [199], prostate [200], epithelial cells [201], blastocyst [202] 2.”
BackgroundStructural materials meet synthetic biology in biomedical applications
Zhang, X., & Fussenegger, M. (2023) · Materials Today · doi:10.1016/j.mattod.2023.12.008 · Cited by 12 · see citing works
Development of in vitro hair pigmentation model using hair follicle organoids
Tu, S., Kageyama, T., Seo, J., Zhou, Y., & Fukuda, J. (2024) · Journal of Bioscience and Bioengineering · doi:10.1016/j.jbiosc.2024.11.003 · Cited by 11 · see citing works
Transfer of small interfering RNA by electropermeabilization in tumor spheroids
Pélofy, S., Bousquet, H., Gibot, L., Rols, M., & Golzio, M. (2021) · Bioelectrochemistry · doi:10.1016/j.bioelechem.2021.107848 · Cited by 10 · see citing works
DoE Analysis of Approaches for Hydrogel Microbeads’ Preparation by Millifluidic Methods
Nastruzzi, A., Pitingolo, G., Luca, G., & Nastruzzi, C. (2020) · Micromachines · doi:10.3390/mi11111007 · Cited by 9 · see citing works
“More recently, an approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads, using a microfluidic device and electroporation, was proposed by Laperrousaz and colleagues [16].”
PAI-1 regulates AT2-mediated re-alveolarization and ion permeability
Ali, G., Zhang, M., Chang, J., Zhao, R., Jin, Y., Zhang, J., & Ji, H. (2023) · Stem Cell Research & Therapy · doi:10.1186/s13287-023-03414-4 · Cited by 8 · see citing works
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“Additionally, biomaterial scaffolds present challenges for viral transduction, as thick constructs and crosslinked filaments can hinder virus diffusion, which can result in uneven gene delivery [19].”
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A novel perspective on bone tumors: advances in organoid research
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Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeads
Kim, J. J., Hebisch, M., Kwak, S. S., Zheng, M., Nuli, S. S., Bae, J., Brand, E., Tanzi, R. E., Irimia, D., & Kim, D. Y. (2025) · Scientific Reports · doi:10.1038/s41598-025-94810-8 · Cited by 8 · see citing works
Organoid-based platforms in livestock: Current advances and future prospects
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“…Laperrousaz et al. were found by direct transfection of clonal organoids in matrigel microbeads, which achieved 80% transduction efficiency by electroporation of matrigel-encapsulated organoids using this technique and a morphological study that summarizes the different stages of tumor growth [24].”
Reconstructing tumor tissues in 3D: From organoids to bioengineered niches
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Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts
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Current Insights and Perspectives on High-Throughput BiFCPreprint
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An Optimized Method for Single Cell Cloning of Human CAR-T Cells based on FBS-coated Plates
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“9 .”
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“Organoids embedded in dense matrices such as Matrigel can severely restrict liposome diffusion, as demonstrated by Laperrousaz et al. [27], who developed a microfluidic approach using Matrigel microbeads and electroporation because traditional lipofection failed to transfect mature organoids…”
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“Therefore, our results suggest that the phenotypic resistance observed in OV90 spheroids is not a delivery artifact caused by the limited penetration of lipid-based vehicles, which frequently exhibit low efficiency in 3D models [41].”
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“Accordingly, workflows frequently implement the delivery of siRNAs at the dissociated/single-cell stage prior to re-embedding (Aloia et al., 2019), whereas knockdown in intact 3D structures is generally more challenging and less standardized (Laperrousaz et al., 2018; Morgan et al., 2018).”
The Application of 3D Cell Culture in Thyroid Cancer
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Ultrasound‐Actuated Gene Editing in Human Kidney Organoids
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“To circumvent this, organoids are often dissociated into single cells or cell clusters, non-virally transfected, and then re-embedded into solid media [ 9, 10 ].”
Influential
- Authors
- Laperrousaz, B., Porte, S., Gerbaud, S., Härmä, V., Kermarrec, F., Hourtane, V., Bottausci, F., Gidrol, X., & Picollet-D’hahan, N.
- Affiliations
- Inserm (FR)
- University of Grenoble Alpes, CEA, INSERM, BIG-BGE, 38000 Grenoble, France
- University of Grenoble Alpes, CEA, LETI, F-38000 Grenoble, France
- Commissariat à l'Énergie Atomique et aux Énergies Alternatives (FR)
- Corresponding author
- Gidrol, Xavier
- Picollet-D’hahan, Nathalie
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2018-01-31
- Volume
- 46
- Issue
- 12
- Pages
- e70-e70
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC6158603
- OpenAlex
- W2790915881
- Semantic Scholar
- 1c2161e82a679620509ce6d9e068a04c98d575d5
- Cited by
- 54
- References
- 41
- Citations by year
- 2026: 7 · 2025: 8 · 2024: 7 · 2023: 7 · 2022: 3 · 2021: 11 · 2020: 5 · 2019: 5 · 2018: 1
- FWCI
- 2.08
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (acceptedVersion)
- Europe PMC (PubMed Central) (submittedVersion, cc-by-nc)
- PubMed Central (submittedVersion, cc-by-nc)
- HAL (Le Centre pour la Communication Scientifique Directe) (submittedVersion)
- Funders
- Agence Nationale de la Recherche (ANR-11-NANB-0002)
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Microbial Inactivation Methods
- Keywords
- organoids, single-cell encapsulation, microfluidics, electroporation, p63, PTEN, breast tissue, prostate tissue, high-throughput genetic screening, extracellular matrix
- MeSH terms
- Breast, Cell Line, Collagen, Drug Combinations, Female, Humans, Laminin, Male, Microspheres, Organoids, Prostate, Proteoglycans, Transcription Factors, Transfection, Electroporation, Spheroids, Cellular, Tumor Suppressor Proteins, RNA Interference, RNA, Small Interfering, Cell Line, Tumor, PTEN Phosphohydrolase, Lab-On-A-Chip Devices
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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