Development of a biomimetic liver tumor-on-a-chip model based on decellularized liver matrix for toxicity testing

The DLM-GelMA-based liver tumor-on-a-chip, compared with GelMA only, enhanced hepatocyte functions under flow conditions.

“The biomimetic liver tumor-on-a-chip based on the integration DLM components with GelMA, as opposed to GelMA only, had an increased capability to maintain cell viability and to enhance hepatocyte functions under flow conditions.”

The DLM-GelMA-based tumor-on-a-chip exhibited linear dose-dependent drug responses to sorafenib toxicity.

“Furthermore, this DLM-GelMA-based tumor-on-a-chip exhibited linear dose-dependent drug responses to the toxicity of acetaminophen and sorafenib.”

Liver decellularization was successfully obtained through physical treatment and chemical perfusion based on an optimized decellularization protocol.

“Liver decellularization was successfully obtained through physical treatment and chemical perfusion based on the optimization of a decellularization protocol as reported before 27 .”

• cites [27] 27. B. E. Uygun, A. Soto-Gutierrez, H. Yagi, M.-L. Izamis, M. A. Guzzardi, C. Shulman, J. Milwid, N. Kobayashi, A. Tilles, F. Berthiaume, M. Hertl, Y. Nahmias, M. L. Yarmush and K. Uygun, Nature Medicine, 2010, 16, 814-U120.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The DLM-GelMA-based tumor-on-a-chip demonstrates dose-dependent responses to the toxicity of acetaminophen and sorafenib.

“Moreover, this DLM-GelMA-based tumor-on-a-chip demonstrates dose-dependent responses to the toxicity of acetaminophen and sorafenib.”

The DLM-GelMA-based liver tumor-on-a-chip promoted hepatocyte functions such as albumin and urea production in HepG2 cells, as opposed to GelMA only, in a pathologically relevant 3D microenvironment.

“As demonstrated, this DLM-GelMA-based liver tumor-on-a-chip promoted the cell viability and hepatocyte functions (e.g., production of albumin and urea) of HepG2 cells, as opposed to the use of GelMA only, in a pathologically relevant 3D microenvironment.”

The DLM-GelMA-based liver tumor-on-a-chip promoted HepG2 cell viability, as opposed to GelMA only, in a pathologically relevant 3D microenvironment.

“As demonstrated, this DLM-GelMA-based liver tumor-on-a-chip promoted the cell viability and hepatocyte functions (e.g., production of albumin and urea) of HepG2 cells, as opposed to the use of GelMA only, in a pathologically relevant 3D microenvironment.”

The DLM-GelMA-based tumor-on-a-chip exhibited linear dose-dependent drug responses to acetaminophen toxicity.

“Furthermore, this DLM-GelMA-based tumor-on-a-chip exhibited linear dose-dependent drug responses to the toxicity of acetaminophen and sorafenib.”

The 2:3 DLM-GelMA hydrogels had an average pore size of 100.34 ± 8.56 μm.

“Additionally, the 2:3 DLM-GelMA hydrogels had an average pore size 100.34 ± 8.56 µm, which could promote cellular spreading and cell-to-cell interaction, and thus facilitate the formation of extensive 3-D cellular network.”

HGF concentration in the DLM solution before UV exposure was 4.27 ± 0.05 pg/mg dry weight.

“The concentration of HGF in the native liver was 6.70 ± 0.08 pg/mg, whereas it decreased to 4.27 ± 0.05 pg/mg (before UV exposure) and 4.37 ± 0.04 pg/mg dry weight (after UV exposure).”

The dry concentration of collagen in the DLM was increased by approximately 18-fold compared to native liver tissue.

“Compared to the collagen concentration in native liver tissue, the dry concentration of collagen in the DLM was increased by approximately 18-fold (Fig. 3B and Table S4).”

Other ECM-affiliated proteins, ECM glycoproteins, and ECM regulators are preserved along with some secreted factors after decellularization and enzymolysis.

“In addition, other ECM-affiliated proteins, ECM glycoproteins, and ECM regulators are preserved along with some secreted factors after decellularization and enzymolysis (Fig. 3E).”

UV exposure did not seem to affect the concentration of GAG within the DLM solution.

“Similarly, the UV exposure did not seem to affect the concentration of GAG within the DLM solution.”

The incorporation of bioactive extracellular components derived from DLM and growth factors with GelMA provided a similar biomechanical ECM (e.g., Young's modulus) to native liver tissues.

“The incorporation of bioactive extracellular components derived from DLM and growth factors with GelMA not only provided a similar biomechanical ECM (e.g., Young's modulus) with native liver tissues (Fig. 4), but also increased the viability and function of HepG2 cells (e.g., secretion of albumin and urea) in the liver tumor-on-a-chip (Fig. 5).”

The collagen concentration in the device (0.0591 μg/μL) closely resembles the collagen content in the native liver (0.06275 μg/μL).

“Although the collagen dry weight of the DLM solution is 18-fold that of the native liver (Fig. 3B), the collagen concentration in the device (0.0591 µg/µL) closely resembles the collagen content in the native liver (0.06275 µg/µL) (Detailed calculation can be found in Supporting Information in Table S1).”

bFGF concentration in the DLM solution was 4.58 ± 0.11 pg/mg.

“The concentration of bFGF the native liver was 5.29 ± 0.09 pg/mg, whereas it was 4.58 ± 0.11 pg/mg in the DLM solution, suggesting a significant loss of bFGF during the decellularization and enzymolysis processes.”

Linear dose-dependent drug responses were observed for both acetaminophen and sorafenib in terms of cell viability, urea secretion, and albumin secretion.

“Furthermore, linear dose-dependent drug responses were observed for both model drugs in terms of cell viability, and urea and albumin secretion (Fig. 6B-G).”

• Also stated in: § 4. Discussion and conclusion · plain

  “Furthermore, linear dose-dependent drug responses were observed in cell viability and hepatocyte function such as secretion of urea and albumin for both acetaminophen and sorafenib (Fig. 6B-G).”

Collagen concentration from the DLM solution before UV irradiation was 144.33 ± 50.74 ng/mg.

“The concentration of collagen from the DLM solution before and after UV irradiation was 144.33 ± 50.74 ng/mg, and 147.84 ± 23.71 ng/mg, respectively, indicating that the collagen was not affected by UV exposure (p > 0.05).”

Collagen in the DLM solution was not affected by UV exposure (p > 0.05).

“The concentration of collagen from the DLM solution before and after UV irradiation was 144.33 ± 50.74 ng/mg, and 147.84 ± 23.71 ng/mg, respectively, indicating that the collagen was not affected by UV exposure (p > 0.05).”

Collagen concentration from the DLM solution after UV irradiation was 147.84 ± 23.71 ng/mg.

“The concentration of collagen from the DLM solution before and after UV irradiation was 144.33 ± 50.74 ng/mg, and 147.84 ± 23.71 ng/mg, respectively, indicating that the collagen was not affected by UV exposure (p > 0.05).”

The DLM-GelMA biomaterial exhibited porous microstructures under SEM.

“Following UV exposure, the mixture of DLM with GelMA solution became gellified, and the obtained DLM-GelMA biomaterial exhibited porous microstructures under SEM (Fig. 4A).”

UV exposure did not seem to affect the concentration of HGF.

“These data indicate that the decellularization and enzymolysis process reduced the HGF content, and the UV exposure did not seem to affect the concentration of HGF.”

The majority of HGF and bFGF sustained after UV exposure.

“Nevertheless, the majority of HGF and bFGF were preserved within the DLM solution, and the majority of them sustained after UV exposure.”

Via sequential perfusion of SDS and Triton X-100, translucent acellular liver matrices were obtained in 9 h.

“Via sequential perfusion of SDS and Triton X-100, translucent acellular liver matrices were obtained in 9 h, offering a more efficient way than the method outlined by Uygun et al., which takes almost 72 h to obtain a decellularized liver 27 .”

The absence of cells in the DLM was demonstrated by the lack of blue color following DAPI staining.

“The absence of cells in the DLM was demonstrated by the lack of blue color following the DAPI staining (Fig. 2B).”

HepG2 viability was significantly elevated by the addition of DLM components in hybrid DLM-GelMA hydrogels for up to 7 days.

“More importantly, HepG2 viability was significantly elevated due to the addition of DLM components in the hybrid DLM-GelMA hydrogels for up to 7 days (Fig. 5B).”

HGF concentration in native liver was 6.70 ± 0.08 pg/mg.

“The concentration of HGF in the native liver was 6.70 ± 0.08 pg/mg, whereas it decreased to 4.27 ± 0.05 pg/mg (before UV exposure) and 4.37 ± 0.04 pg/mg dry weight (after UV exposure).”

The Young's modulus of 2:3 DLM-GelMA was 16.91 ± 0.54 kPa.

“The Young's modulus of 2:3 DLM-GelMA was determined to be 16.91± 0.54 kPa (Table S5), which better resembles the Young's modulus of a fibrotic liver (> 12kPa) 31 , a pre-condition for liver cancer, and which falls in the range of liver stiffness (ranging approximately from 5 to 60kPa) 15,32 .”

The majority of HGF and bFGF were preserved within the DLM solution.

“Nevertheless, the majority of HGF and bFGF were preserved within the DLM solution, and the majority of them sustained after UV exposure.”

Immunofluorescence and H&E staining confirmed that all cells were completely removed from the liver tissues.

“Immunofluorescence (Fig. 2B) and H&E staining results (Fig. 2C) confirmed that all the cells were completely removed from the liver tissues.”

HepG2 cells encapsulated in 2:3 DLM-GelMA hydrogels were cultured over 21 days without reducing cell viability, as measured by live/dead staining with confocal imaging.

“Since the cells were grown in 3D hydrogels with continuously flow of media, HepG2 cells were able to be cultured over a period of 21 days without reducing cell viability, which was measured via live/dead staining with the aid of confocal imaging (Fig. 5A).”

bFGF concentration within the DLM solution reduced to 4.07 ± 0.02 pg/mg after UV exposure.

“Furthermore, the concentration of bFGF within the DLM solution reduced to 4.07 ± 0.02 pg/mg, as opposed to 4.58 ± 0.11 pg/mg, after UV exposure, suggesting that UV exposure might damage bFGF.”

Sorafenib treatment at 10.0, 15.0, and 20.0 μM produced a more evident toxic effect on HepG2 cells in the liver tumor-on-a-chip.

“A more evident toxic effect was observed for 10.0, 15.0, 20.0 µM of sorafenib treated HepG2 cells in the liver tumor-on-a-chip (Fig. 6A).”

Total DNA after decellularization was 21.99 ± 1.34 ng/mL.

“DNA quantification results showed the total of DNA after decellularization was 21.99 ± 1.34 ng/mL, suggesting that DNA from the native liver were removed by decellularization and DNase treatment (Fig. 2E).”

There was no significant difference in cell viability during the 3D dynamic culture until Day 21.

“There was no significant difference in cell viability during the 3D dynamic culture till Day 21.”

Albumin secretion from HepG2 cells encapsulated in 2:3 DLM-GelMA hydrogels was significantly higher than in the GelMA-only control until Day 14.

“In contrast, the secretion of albumin from HepG2 cells encapsulated in the 2：3 DLM-GelMA hydrogels showed significantly higher performance till Day 14 (Fig. 5D).”

GAG concentration from the DLM solution was increased by 2.5-fold compared to native liver tissues.

“The concentration of GAG from the DLM solution was also increased by 2.5-fold (Fig. 3C and Table S4), compared to its counterpart in native liver tissues.”

Acetaminophen treatment at 5.0, 10.0, and 20.0 mM resulted in significant cell death after 24 h compared to the untreated group in the liver tumor-on-a-chip.

“The overlaid confocal images of live/dead staining showed that the acetaminophen treatment at 5.0, 10.0 and 20.0 mM resulted in significant cell death after 24 h, compared to the untreated group (Fig. 6A).”

bFGF concentration in native liver was 5.29 ± 0.09 pg/mg.

“The concentration of bFGF the native liver was 5.29 ± 0.09 pg/mg, whereas it was 4.58 ± 0.11 pg/mg in the DLM solution, suggesting a significant loss of bFGF during the decellularization and enzymolysis processes.”

Compared to GelMA only, 2:3 DLM-GelMA consistently had higher performance regarding cell viability and hepatocyte function in the liver tumor-on-a-chip.

“Compared to GelMA only in the liver tumor-on-a-chip, the performance of 2:3 DLM-GelMA consistently had higher performance regarding cell viability and hepatocyte function (Fig. 6B-G).”

A similar trend of toxicity from acetaminophen and sorafenib was observed in the liver tumor-on-a-chip with GelMA only.

“A similar trend of toxicity arising from acetaminophen and sorafenib was also observed in the liver tumor-on-a-chip with GelMA only (Fig. S3).”

The difference in supporting cell growth tapers off during long-term culture from Day 10 to Day 21.

“However, it should be noted that the difference in supporting cell growth tapers off during long-term culture from Day 10 to Day 21.”

Urea secretion from HepG2 cells encapsulated in 2:3 DLM-GelMA hydrogels was consistently higher than in the GelMA-only control over the entire 21 days of 3D dynamic cell culture on-chip.

“In addition, the secretion of urea from HepG2 cells encapsulated in the 2:3 DLM-GelMA hydrogels was consistently higher than that in the control group with GelMA only over the entire 21 days of 3D dynamic cell culture on-chip (Fig. 5C).”

The acellular scaffolds preserved the four major structural proteins (collagen I, collagen IV, fibronectin, laminin) of ECM and growth factors.

“More importantly, acellular scaffolds preserved the four major structural proteins (i.e., collagen I, collagen IV, fibronectin and laminin) of ECM and growth factors (Fig. 3).”

HGF concentration in the DLM solution after UV exposure was 4.37 ± 0.04 pg/mg dry weight.

“The concentration of HGF in the native liver was 6.70 ± 0.08 pg/mg, whereas it decreased to 4.27 ± 0.05 pg/mg (before UV exposure) and 4.37 ± 0.04 pg/mg dry weight (after UV exposure).”

The proportion of protein components in the DLM solution was 43% ECM-affiliated proteins, 29% glycoproteins, 14% collagens, 7% ECM regulators, and 7% secreted factors.

“As revealed in the pie chart (Fig. 3F), the proportion of protein components in the DLM solution was determined to be 43% ECM-affiliated proteins, 29% glycoproteins, 14% collagens, 7% ECM regulators, and 7% secreted factors.”

SEM imaging revealed a mesh-like structure of the DLM.

“SEM imaging revealed a mesh-like structure of the DLM (Fig. 2D).”

A large proportion of cellular proteins and debris were removed from the native liver during the decellularization and enzymolysis processes.

“The results showed that a large proportion of cellular proteins and debris were removed from the native liver, as shown in the heatmap (Fig. 3E), during the decellularization and enzymolysis processes.”

The DLM-GelMA hydrogel has a thickness of 1 mm.

“The fluid flow is of particularly importance for the DLM-GelMA hydrogel, which has a thickness of 1 mm, since 200 micrometers is reported to be the maximal diffusion distance of oxygen 33 .”

The amount of double-stranded DNA (dsDNA) on the decellularized liver was less than 50 ng/mg of ECM dry weight.

“Moreover, the amount of double stranded DNA (dsDNA) found on the decellularized liver was less than 50 ng/mg of the ECM dry weight (Fig. 2E), which is low enough to meet decellularization requirements 34 .”

The ECM proteins fibronectin, laminin, collagen type I, and collagen type IV were preserved after the decellularization process.

“The immunostaining of the four ECM proteins (i.e., fibronectin, laminin, collagen type I, and collagen type IV) demonstrated that these ECM basement components were preserved after the decellularization process (Fig. 2B).”

Bioactive substances such as collagens, ECM-associated proteins and glycoproteins, as well as growth factors, were successfully preserved in the DLM solution.

“The proteomic analysis revealed that bioactive substances such as collagens, ECM-associated proteins and glycoproteins, as well as growth factors were successfully preserved in the DLM solution.”

The DLM-GelMA-based liver tumor-on-a-chip maintained a relatively higher level of cell viability and hepatocyte function in the presence of increasing concentrations of acetaminophen and sorafenib compared to GelMA only.

“In addition, this biomimetic tumor-on-a-chip with integration of DLM-GelMA, as opposed to GelMA only, maintained a relatively higher level of cell viability and hepatocyte function in the presence of increasing concentrations of acetaminophen and sorafenib (Fig. 6).”

Following a 9-h decellularization process, rat livers became transparent.

“Following a 9-h decellularization process, rat livers became transparent (Fig. 2A).”

Cell viability of HepG2 cells in DLM-GelMA groups, regardless of volume ratio, was significantly elevated compared to GelMA only.

“Evidently, the cell viability of HepG2 cells in the group of DLM-GelMA, regardless of the volume ratio, showed significantly elevated cell viability than the group with GelMA only.”

Protein abundance in the DLM solution did not differ substantially before and after UV exposure.

“In addition, the protein abundance in the DLM solution did not differ substantially before and after UV exposure.”

The 2:3 DLM-GelMA hydrogels showed superior performance.

“Taken together, DLM-GelMA hydrogels showed great biocompatibility with 3D culture of HepG2 cells, and the 2:3 DLM-GelMA hydrogels showed the superior performance.”

Multiple ECM-associated proteins and collagens are preserved after decellularization, as revealed by mass spectrometry.

“Furthermore, multiple ECM-associated proteins and collagens are preserved, as revealed by mass spectrometry analysis (Fig. 3F).”

The level of cell viability and hepatocyte functions in the 2:3 DLM-GelMA hydrogels were elevated compared to GelMA only in the biomimetic tumor-on-a-chip.

“More importantly, the level of cell viability and hepatocyte functions in the 2:3 DLM-GelMA hydrogels were elevated, compared to GelMA only, in the biomimetic tumor-on-a-chip, suggesting that this DLM-GelMA based biomimetic liver tumor-on-a-chip better recapitulate the in vivo liver 3D TME for more accurate toxicity assessment.”

There was no significant difference in Young's modulus among hybrid hydrogels with different mixing ratios and GelMA only.

“The results showed that the Young's modulus of the 2:3 DLM-GelMA hydrogels was 16.91 ± 0.54 kPa, which showed no significant difference among the hybrid hydrogels with different mixing ratios and GelMA only (Fig. 4C and Table S5), indicating that the integration of DLM does not significantly alter the Young's modulus of GelMA.”

The largest pore sizes were observed in DLM-GelMA with mixing ratios of 2:3 and 1:1, ranging from 100.34 ± 8.56 to 101.54 ± 5.97 μm.

“Notably, the largest pore sizes were observed in DLM-GelMA with a mixing ratio of 2:3 and 1:1, ranging from 100.34 ± 8.56 to 101.54 ± 5.97 µm (Fig. 4B and Table S5).”

Cell viability decreased in the presence of increasing concentrations of both acetaminophen and sorafenib.

“Indeed, the cell viability decreased in the presence of incresing cencentrations of both model drugs as indicated in confocoal images of live/dead staining (Fig. 6A and Fig. S3), which is also supported by a previous study 35 .”

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The 2:3 volume ratio showed superior cell viability of HepG2 to the second best ratio of 1:1 during the period of 8 days.

“This volume ratio showed superior cell viability of HepG2 to the second best ratio of 1:1 during the period of 8 days of”

The cell viability of HepG2, as indicated by CCK-8 absorbance at 450 nm, was highest on day 1, 4, and 8 for DLM-GelMA hydrogels at a volume ratio of 2:3.

“As shown in Fig. 4E, the cell viability of HepG2, as indicated by the absorbance of CCK-8 reagent at 450 nm, was the highest on day 1, 4 and 8，for the DLM-GelMA hydrogels at a volume ratio of 2:3.”

DLM allows for better maintenance of hepatocyte phenotypes such as secretion of albumin and urea in vitro.

“Although a decellularized liver matrix (DLM) allows for a better maintenance of hepatocyte phenotypes such as section of albumin (ALB) and urea in vitro 21,[25][26][27] , the incorporation of DLM components into a perfusion-based tumor-on-a-chip platform for better recapitulation of 3D liver tumor microenvironment (TME) has not yet been reported.”

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A close simulation of 3D ECM with native growth factors would be more accurate for assessing drug toxicity.

“Thus, a close simulation of 3D ECM with native growth factors would be more accurate for assessing drug toxicity.”

High risks exist in Stage 1 clinical trials when drug candidates pass animal studies, as unknown side effects are often observed.

“As a result, high risks still exist in Stage 1 clinical trials, when drug candidates even pass animal studies, as unknown side effects are often observed 7 .”

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Tumor-on-a-chip technologies offer a feasible approach to drug toxicity testing.

“Although tumor-on-a-chip technologies offers a feasible approach to drug toxicity testing, their capability to mimic the native extracellular matrix (ECM) is still, to a certain extent, limited 12 .”

• cites [12] 12. H. F. Tsai, A. Trubelja, A. Q. Shen and G. Bao, Journal of the Royal Society, Interface, 2017, 14.Not matched

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Hepatocytes are significantly affected by bioactive molecules such as HGF and bFGF preserved in liver acellular scaffolds.

“Furthermore, hepatocytes can attach to the ECM components of an acellular scaffold, and they are significantly affected by bioactive molecules (e.g., hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF)) preserved in the liver acellular scaffolds 24 .”

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Hepatocytes can attach to the ECM components of an acellular scaffold.

“Furthermore, hepatocytes can attach to the ECM components of an acellular scaffold, and they are significantly affected by bioactive molecules (e.g., hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF)) preserved in the liver acellular scaffolds 24 .”

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Effective compounds screened by traditional 2D culture systems often fail in animal studies or clinical trials.

“Despite the clinical severity, few new anti-cancer drugs are readily available, since effective compounds screened by traditional two-dimensional (2D) culture system often fail in animal studies or clinical trials 2,3 .”

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In vitro 3D culture systems using GelMA scaffolds lack appropriate native growth factors that promote cell growth and sustain cell function.

“Although GelMA scaffolds closely resemble the mechanical characteristics of ECM, the in vitro 3D culture systems still lack appropriate native growth factors that promote cell growth and sustain cell function 16,19,20 .”

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Animal models cannot reliably predict safety and efficacy in clinical studies.

“Although animal models are used as the gold standard for drug testing, these models are expensive and time-consuming, and more importantly they cannot reliably predict the safety and efficacy in clinical studies 2,6 .”

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The DLM-GelMA-based biomimetic liver tumor-on-a-chip better mimics the in vivo TME.

“Taken together, our study demonstrates that the DLM-GelMA-based biomimetic liver tumor-on-a-chip better mimics in vivo TME and holds great promise for a breadth of pathological and pharmacological studies.”

The improved performance of the DLM-GelMA-based tumor-on-a-chip may be attributed to provision of biochemical factors, preservation of scaffold proteins, and reestablishment of biophysical cues for better recapitulation of the 3D liver TME.

“The improved performance of the DLM-GelMA-based tumor-on-a-chip may be attributed to the provision of biochemical factors (e.g., growth factors), the preservation of scaffold proteins, and the reestablishment of biophysical cues (e.g., stiffness and shear stress) for better recapitulation of 3D liver TME.”

Decellularized liver scaffolds have been developed for the preservation of major collagens and growth factors.

“To establish an ECM with native growth factors, decellularized liver scaffolds have been developed for the preservation of major collagens and growth factors 21 .”

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A decellularized liver scaffold with retained growth factors enhances the culturing of hepatic cells.

“For example, a decellularized liver scaffold with retained growth factors enhances the culturing of hepatic cells [21][22][23] .”

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The biomimetic liver tumor-on-a-chip platform with essential collagens and growth factors derived from DLM can better recapitulate the 3D liver TME.

“Clearly, this biomimetic liver tumor-on-a-chip platform, with the addition of essential collagens and growth factors derived from DLM, can better recapitulate the 3D liver TME, thus offering an important testing platform for assessing drug toxicity.”

• AssumptionRests on an assumption: The incorporation of DLM components into a perfusion-based tumor-on-a-chip platform for better recapitulation of the 3D liver tumor microenvironment has not yet been reported.

The biomimetic liver tumor-on-a-chip better recapitulates the 3D liver TME via DLM-GelMA hydrogels with essential scaffold proteins and growth factors, as well as biophysical cues such as stiffness and shear stress.

“This biomimetic liver tumor-on-a-chip better recapitulates the 3D liver TME via DLM-GelMA hydrogels with essential scaffold proteins and growth factors, as well as biophysical cues such as stiffness and shear stress.”

• AssumptionRests on an assumption: The incorporation of DLM components into a perfusion-based tumor-on-a-chip platform for better recapitulation of the 3D liver tumor microenvironment has not yet been reported.

Organ-on-a-chip models have been used as drug testing platforms due to their close simulation of drug-induced toxicity in vivo.

“Recently, organ-on-a-chip models have been used as drug testing platforms due to their close simulation of drug-induced toxicity in vivo, a major reason for the withdrawal of drugs [8][9][10][11] .”

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Tumor-on-a-chip technologies' capability to mimic the native ECM is limited.

“Although tumor-on-a-chip technologies offers a feasible approach to drug toxicity testing, their capability to mimic the native extracellular matrix (ECM) is still, to a certain extent, limited 12 .”

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The protein components derived from DLM play an important role in supporting cell growth and sustaining hepatocyte function.

“The protein components derived from DLM are believed to play an important role in supporting cell growth and sustain hepatocyte function (Fig. 4E and Fig. 5), since addition of growth factors can boost cell growth and functions 22,30 .”

A dsDNA amount of less than 50 ng/mg ECM dry weight is low enough to meet decellularization requirements.

“Moreover, the amount of double stranded DNA (dsDNA) found on the decellularized liver was less than 50 ng/mg of the ECM dry weight (Fig. 2E), which is low enough to meet decellularization requirements 34 .”

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The method outlined by Uygun et al. takes almost 72 h to obtain a decellularized liver.

“Via sequential perfusion of SDS and Triton X-100, translucent acellular liver matrices were obtained in 9 h, offering a more efficient way than the method outlined by Uygun et al., which takes almost 72 h to obtain a decellularized liver 27 .”

• cites [27] 27. B. E. Uygun, A. Soto-Gutierrez, H. Yagi, M.-L. Izamis, M. A. Guzzardi, C. Shulman, J. Milwid, N. Kobayashi, A. Tilles, F. Berthiaume, M. Hertl, Y. Nahmias, M. L. Yarmush and K. Uygun, Nature Medicine, 2010, 16, 814-U120.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Integration of DLM-based liver-specific growth factors and collagens in a microfluidic system significantly enhanced the viability and function of HepG2 cells.

“Via the technical advances mentioned above, a major biological finding from this study is that the integration of DLM-based liver-specific growth factors and collagens in a microfluidic system significantly enhanced the viability and function of HepG2 cells.”

The biomimetic liver tumor-on-a-chip with integrated structural proteins and growth factors derived from DLM, being more similar to in vivo situations, might have more accurate assessment of drug toxicity.

“More importantly, this biomimetic liver tumor-on-a-chip with integrated structural proteins and growth factors derived from DLM, which is more similar to in vivo situations, might have more accurate assessment of drug toxicity.”

DLM-GelMA hydrogels showed great biocompatibility with 3D culture of HepG2 cells.

“Taken together, DLM-GelMA hydrogels showed great biocompatibility with 3D culture of HepG2 cells, and the 2:3 DLM-GelMA hydrogels showed the superior performance.”

The 3D DLM-GelMA cell culture in a microfluidic system reestablishes similar biophysical cues such as stiffness and shear stress as in vivo, thereby simulating the 3D liver TME.

“We also simulated the 3D liver TME by reestablishing similar biophysical cues such as stiffness and shear stress as in vivo with the aid of 3D DLM-GelMA cell culture in a microfluidic system.”

• AssumptionRests on an assumption: The fluidic flow at 2 µL/h in the microfluidic device provides physiologically relevant shear stress to liver cells.

The 9-h decellularization protocol is more efficient than the method of Uygun et al.

“Via sequential perfusion of SDS and Triton X-100, translucent acellular liver matrices were obtained in 9 h, offering a more efficient way than the method outlined by Uygun et al., which takes almost 72 h to obtain a decellularized liver 27 .”

Addition of growth factors may be needed to maintain high viability and hepatocyte function in the 2:3 DLM-GelMA hydrogels over a long period of time.

“These data suggest that addition of grow factors may be needed to maintain high viability and hepatocyte function in the 2:3 DLM-GelMA hydrogels over a long period of time.”

200 micrometers is the maximal diffusion distance of oxygen.

“The fluid flow is of particularly importance for the DLM-GelMA hydrogel, which has a thickness of 1 mm, since 200 micrometers is reported to be the maximal diffusion distance of oxygen 33 .”

• cites [33] 33. L. Corstorphine and M. V. Sefton, Journal Of Tissue Engineering And Regenerative Medicine, 2011, 5, 119-129.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

DNA from the native liver was removed by decellularization and DNase treatment.

“DNA quantification results showed the total of DNA after decellularization was 21.99 ± 1.34 ng/mL, suggesting that DNA from the native liver were removed by decellularization and DNase treatment (Fig. 2E).”

The biomimetic liver tumor-on-a-chip is capable of toxicity testing.

“These results demonstrated the capability of this biomimetic liver tumor-on-a-chip for toxicity testing.”

The biomimetic liver tumor-on-a-chip would better recapitulate liver 3D TME for more accurate toxicity assessment compared to 3D Transwell or 2D petri dish models.

“Although hepatotoxicity testing in general can be performed in 3D Transwell or 2D petri dish models, the evident technological advances of this biomimetic liver tumor-on-a-chip would be able better recapitulate liver 3D TME for more accurate toxicity assessment.”

• AssumptionRests on an assumption: Existing tumor-on-a-chip technologies have limited capability to highly mimic the native TME.

There was a significant loss of bFGF during the decellularization and enzymolysis processes.

“The concentration of bFGF the native liver was 5.29 ± 0.09 pg/mg, whereas it was 4.58 ± 0.11 pg/mg in the DLM solution, suggesting a significant loss of bFGF during the decellularization and enzymolysis processes.”

Major collagen components of liver scaffolds (collagen I, collagen IV, fibronectin, laminin) play an essential role in tumor growth and metastasis.

“As demonstrated, the major collagen components of liver scaffolds (e.g., collagen I, collagen IV, fibronectin, and laminin) were preserved after decellularization (Fig. 2), which plays an essential role in tumor growth and metastasis 28,29 .”

• cites [28] 28. K. R. Levental, H. M. Yu, L. Kass, J. N. Lakins, M. Egeblad, J. T. Erler, S. F. T. Fong, K. Csiszar, A. Giaccia, W. Weninger, M. Yamauchi, D. L. Gasser and V. M. Weaver, Cell, 2009, 139, 891-906.Not matched

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• cites [29] 29. T. R. Cox, D. Bird, A. M. Baker, H. E. Barker, M. W. Y. Ho, G. Lang and J. T. Erler, Cancer Res., 2013, 73, 1721-1732.Not matched

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The decellularization and enzymolysis process reduced HGF content.

“These data indicate that the decellularization and enzymolysis process reduced the HGF content, and the UV exposure did not seem to affect the concentration of HGF.”

The average pore size of 2:3 DLM-GelMA hydrogels could promote cellular spreading and cell-to-cell interaction, facilitating formation of an extensive 3D cellular network.

“Additionally, the 2:3 DLM-GelMA hydrogels had an average pore size 100.34 ± 8.56 µm, which could promote cellular spreading and cell-to-cell interaction, and thus facilitate the formation of extensive 3-D cellular network.”

The reconstructed 3D liver TME by supplementing GelMA with scaffold proteins and growth factors derived from DLM in a perfusion-based microfluidic chip better recapitulates the 3D liver TME with respect to scaffolds, biochemical factors, and biophysical cues.

“Hence, the reconstructed 3D liver TME by supplementing GelMA with scaffold proteins and growth factors derived from DLM in a perfusion-based microfluidic chip better recapitulate the 3D liver TME with respect to scaffolds, biochemical factors and biophysical cues.”

• AssumptionRests on an assumption: Existing tumor-on-a-chip technologies have limited capability to highly mimic the native TME.
• AssumptionRests on an assumption: The fluidic flow at 2 µL/h in the microfluidic device provides physiologically relevant shear stress to liver cells.

The novel DLM-GelMA-based liver tumor-on-a-chip further improves recapitulation of the liver 3D TME with essential components derived from DLM and mimicry of biophysical cues.

“Therefore, the novel DLM-GelMA-based liver tumor-on-a-chip further improves the recapitulation of the liver 3D TME with essential components derived from DLM and the mimicry of biophysical cues, which holds great potential for more accurate toxicity testing.”

• AssumptionRests on an assumption: Existing tumor-on-a-chip technologies have limited capability to highly mimic the native TME.

UV exposure might damage bFGF.

“Furthermore, the concentration of bFGF within the DLM solution reduced to 4.07 ± 0.02 pg/mg, as opposed to 4.58 ± 0.11 pg/mg, after UV exposure, suggesting that UV exposure might damage bFGF.”

The incorporation of bioactive extracellular components derived from DLM and growth factors with GelMA increased the viability and function of HepG2 cells in the liver tumor-on-a-chip.

“The incorporation of bioactive extracellular components derived from DLM and growth factors with GelMA not only provided a similar biomechanical ECM (e.g., Young's modulus) with native liver tissues (Fig. 4), but also increased the viability and function of HepG2 cells (e.g., secretion of albumin and urea) in the liver tumor-on-a-chip (Fig. 5).”

The Young's modulus of 2:3 DLM-GelMA falls in the range of liver stiffness (approximately 5 to 60 kPa).

“The Young's modulus of 2:3 DLM-GelMA was determined to be 16.91± 0.54 kPa (Table S5), which better resembles the Young's modulus of a fibrotic liver (> 12kPa) 31 , a pre-condition for liver cancer, and which falls in the range of liver stiffness (ranging approximately from 5 to 60kPa) 15,32 .”

• cites [15] 15. D. Dong, T. Hao, C. Wang, Y. Zhang, Z. Qin, B. Yang, W. Fang, L. Ye, F. Yao and J. Li, Biomaterials, 2018, 157, 149-160.Not matched

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• cites [32] 32. E. C. Beck, M. Barragan, M. H. Tadros, S. H. Gehrke and M. S. Detamore, Acta biomaterialia, 2016, 38, 94-105.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The novel DLM-GelMA-based liver tumor-on-a-chip holds great potential for more accurate toxicity testing.

“Therefore, the novel DLM-GelMA-based liver tumor-on-a-chip further improves the recapitulation of the liver 3D TME with essential components derived from DLM and the mimicry of biophysical cues, which holds great potential for more accurate toxicity testing.”

The tapering off of the difference in supporting cell growth from Day 10 to Day 21 is most likely due to consumption or removal of growth factors from the microfluidics system, since the media was changed at an interval of 7 days.

“The major reason is most likely due to the consumption or removal of growth factors from the microfluidics system, since the media was changed at an interval of 7 days.”

It is possible that perfusion slowly removes growth factors from the 3D construct, which can also mitigate cell viability and hepatocyte function over time.

“Additionally, it is possible that the perfusion might slowly remove the growth factors from the 3D construct, which can also mitigate the cell viability and hepatocyte function over time.”

The 2:3 DLM-GelMA hydrogels are superior to the GelMA-only control in supporting cell growth and hepatocyte function of HepG2 cells on the liver tumor-on-a-chip platform.

“Taken together, these results clearly indicated the superiority of the 2:3 DLM-GelMA hydrogels, compared to the control group with GelMA only, in supporting cell growth and hepatocyte function of HepG2 cells on the liver tumor-on-a-chip platform.”

Fluid flow is particularly important for the DLM-GelMA hydrogel because its 1 mm thickness exceeds the maximal oxygen diffusion distance.

“The fluid flow is of particularly importance for the DLM-GelMA hydrogel, which has a thickness of 1 mm, since 200 micrometers is reported to be the maximal diffusion distance of oxygen 33 .”

Essential ECM components and liver-specific factors derived from DLM contribute to the proliferation and functionality of HepG2 cells in the biomimetic liver tumor-on-a-chip with better recapitulation of the 3D liver TME.

“These data altogether demonstrate that essential ECM components and liver-specific factors derived from DLM contribute to the proliferation and functionality of HepG2 cells in the biomimetic liver tumor-on-a-chip with better recapitulation of the 3D liver TME.”

The Young's modulus of 2:3 DLM-GelMA better resembles the Young's modulus of a fibrotic liver (>12 kPa), a precondition for liver cancer.

“The Young's modulus of 2:3 DLM-GelMA was determined to be 16.91± 0.54 kPa (Table S5), which better resembles the Young's modulus of a fibrotic liver (> 12kPa) 31 , a pre-condition for liver cancer, and which falls in the range of liver stiffness (ranging approximately from 5 to 60kPa) 15,32 .”

• cites [31] 31. R. G. Wells, Biochimica Et Biophysica Acta-Molecular Basis Of Disease, 2013, 1832, 884-890.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The DLM-GelMA based biomimetic liver tumor-on-a-chip better recapitulates the in vivo liver 3D TME for more accurate toxicity assessment.

“More importantly, the level of cell viability and hepatocyte functions in the 2:3 DLM-GelMA hydrogels were elevated, compared to GelMA only, in the biomimetic tumor-on-a-chip, suggesting that this DLM-GelMA based biomimetic liver tumor-on-a-chip better recapitulate the in vivo liver 3D TME for more accurate toxicity assessment.”

Integration of DLM does not significantly alter the Young's modulus of GelMA.

“The results showed that the Young's modulus of the 2:3 DLM-GelMA hydrogels was 16.91 ± 0.54 kPa, which showed no significant difference among the hybrid hydrogels with different mixing ratios and GelMA only (Fig. 4C and Table S5), indicating that the integration of DLM does not significantly alter the Young's modulus of GelMA.”