Abstract

Chronic liver diseases are becoming more frequent and can be fatal. Due to a shortage of liver donors, not all patients can receive a transplant. Testing drugs on organoids grown from stem cells could help many patients. The effect of APAP on liver organoids in different gels and media was investigated, by looking at the number of living and dead cells and the excretion of ATP. Although damage was seen in all cells that had been in contact with APAP, most damage was visible in cells grown in differentiation medium.

Introduction

Chronic liver diseases are becoming more common and are sometimes fatal. Liver transplantation is usually the only solution for deadly liver diseases. This works very well, but is not widely applicable due to the lack of donors. 10% of patients die before a donor liver becomes available ^[[1]]. As a solution to this problem, stem cell transplantation into the liver is currently being researched, so that donor livers are no longer needed and liver diseases can be treated on a much larger scale. There are already results of research into mesenchymal stem cell (MSC) transplantation. These stem cells can develop into a number of cell types that occur in the liver and can thus replace parts of a damaged liver. This treatment has shown an improvement in liver functions in patients suffering from liver fibrosis, cirrhosis of the liver, hepatitis B virus, hepatitis C virus and damage due to alcohol consumption ^[[2]].

The use of stem cells, as part of regenerative medicine, may in the future offer a solution for patients for whom liver transplantation is not an option. In the long run it might work even better than liver transplantation ^[[3]].

One of the most common cell types in the liver is the hepatocyte ^[[4]]. Hepatocytes fulfill a large number of essential functions of the liver. The most important of these are the production of bile, the production of essential proteins in the blood plasma (plasma proteins), the balance of the metabolism (metabolic homeostasis), the detoxification of a large number of molecules and the storage of vitamins and minerals ^[[51]].

About 80 mass-% of the liver consists of hepatocytes ^[[61]]. Research shows that with a loss of 70% of the tissue, the liver could regrow within a few weeks ^[[72]]. Hepatocytes have a large replication capacity: when liver damage occurs, these cells can multiply very quickly and regrow. In practice, however, regeneration is slower. Research by Ribeiro has shown that adult hepatocytes, despite their enormous replication capacity, often only multiply once or twice and thus grow back more slowly ^[[85]]. Research into the effect of hepatocytes can contribute much to the development of liver damage treatments based on stem cells.

Research into hepatocytes in the liver is difficult because hepatocytes do not last long ex vivo and lose their functions quickly ^[[91]]. A solution to this problem is the use of hepatocyte-like cells (HLCs) that survive longer ex vivo. These are not real hepatocytes, because the processes that influence the development of hepatocytes cannot be properly replicated in vitro. These HLCs therefore lack a number of functions, but resemble adult hepatocytes enough to be suitable for research. Investigating the extent to which the functions of HLCs, in the form of organoids, correspond in vitro to adult hepatocytes in vivo can be done by a toxicity test of frequently used medication.

\”Overdoses of the analgesic and antipyretic acetaminophen represent one of the most common pharmaceutical product poisonings in the United States today\” ^[[106]]. The toxicity of the active ingredient of paracetamol, acetaminophen, to hepatocytes can cause paracetamol overdoses to have serious effects on the liver. For example, it can cause strong damage to the liver, but in some cases it can also lead to acute liver failure ^[[117]], in which the liver ceases to function.

When paracetamol is administered to the body, acetaminophen (APAP) can be converted in two different ways. One of these ways causes 5 to 10% of the dose to oxidize by certain enzymes, in which N-acetyl-p-benzoquinone imine (NAPQI) is formed ^[[12]8^]. NAPQI is harmful to the liver: it is hepatotoxic⁠^[[139]]. \”With normal therapeutic doses (…), NAPQI is generally inactivated sufficiently rapidly, so as not to cause liver damage\” ^[[149]]. An overdose has an overwhelming effect on the normal metabolism of acetaminophen, as a result of which a larger part of the substance is converted to NAPQI.

Cultured HLCs in the form of organoids were used during this study. Organoids can be grown in various media with growth factors. Two of these media are expansion medium and differentiation medium. Organoids are grown in gels. In this study Matrigel was used, which is an animal gel. In addition, the effectiveness of the animal-friendly GelMA, which is an organic gel, was investigated.

This study has been carried out to research to what extent HLCs in the form of organoids can develop towards adult hepatocytes in different conditions. The question to be researched was: \”What is the cell damage to organ stemmed liver stem cells in different media (both expansion medium and differentiation medium in Matrigel and differentiation medium in GelMA) after 72 hours of exposure to acetaminophen?\”. An answer to this question can provide insight into the development of stem cell treatments for patients with severe liver diseases.

The research consisted of two parts. In the first part, the effect of APAP on liver organoids was investigated. After exposure to APAP, the number of living and dead cells and the excretion of adenosine triphosphate (ATP), a marker for cell damage, was observed. When liver cells are damaged, they excrete ATP ^[[1510]]. ATP is broken down after a few seconds outside the cell ^[[16]11^]. Because there is an increased secretion of ATP in case of cell damage, a measurement of the amount of ATP outside the cells can give an indication of how much damage has occurred. This can be measured using an ATP assay.
The second part shows the extent to which the organoids have differentiated towards adult hepatocytes. This was achieved by performing staining on the cells (and liver tissue for control). Adult hepatocytes can be distinguished on the basis of these colourings, which demonstrate the presence of certain characteristic proteins. This shows that the results of the research come from hepatocytes and not from other types of cells. This method uses antibodies from mice and rabbits, which target specific proteins of the cells that are unique to hepatocytes. Fluorescent secondary antibodies are attached to these antibodies. Since they fluoresce, different parts of the cells become clearly visible.

In addition, it was determined whether the animal gel (Matrigel), in which the organoids are normally grown, could be replaced by a more animal-friendly organic gel (GelMA).

 

The study is an exploratory study. This means that it has never been implemented in exactly the same way before. Therefore, the hypothesis cannot be fully associated with any existing research. It is expected that cells exposed to APAP will have more damage than cells not exposed to APAP. Damaged cells secrete ATP. It is therefore logical to assume that a higher concentration of ATP can be measured near the cells exposed to APAP. There should also be visible damage in pictures taken after the cells’ exposure to APAP.

Furthermore, in comparison to expansion medium, more cell damage should be visible in organoids growing in differentiation medium. The organoids in differentiation medium should be further differentiated towards adult hepatocytes and should therefore be more able to convert APAP into NAPQI. The higher concentration of NAPQI will cause more damage to the cells in differentiation medium. No prediction can be made for GelMA, since this gel is still under development. Therefore, not enough research has yet been done.
One of this research’s aims is to compare the difference in the amount of damage to cells in GelMA and Matrigel.

Method

The methods used are based on protocols developed by the Hubrecht Institute.

APAP on liver organoids

A 30 mM APAP solution (in expansion medium Matrigel, differentiation medium Matrigel and GelMA) was added to organoids in the wells (all in triplicate). Control wells were also made (also in triplicate). In the control wells, the media were added without APAP. Finally, wells without organoids were filled with medium. The well plate was placed in the incubator at 37℃. After 5 hours, 24 hours and 72 hours, 10 µl of medium was collected per well.

Of all measuring points, a live/dead staining was performed per well. For this Hoechst (1:1000), calcein (1:500) and ethidium bromide (EtBr) (1:500) were dissolved in a basal medium. 250 µl of this was then added per well. After 15 minutes, photos were taken with an EVOS fluorescence microscope.

An ATP assay was performed on all collected media. A standard line (100 µM, 10 µM, 1 µM, 100 nM, 10 nM, 1 nM, 100 pM, 0 M) was also made from an ATP solution for comparison. 90 µl per well of the ATP assay standard reaction solution was added in a white well plate. This plate was placed in the luminometer to measure the background noise. Then 10 µl per collected sample was added to the plate. This plate was then placed under the luminometer.

First, the data from the background measurement were subtracted from the data with sample. The extreme values were then filtered out. The average value of all triplicates was then calculated.

A graph was made of the standard line in which the concentration was plotted against the measured value. A trend line was plotted and the coefficient of determination (R²) was calculated. The direction coefficient of the trend line was then used to convert the measured values to the concentration of ATP per well. For this, the measured values were divided by the direction coefficient. Line charts were then made from these tables: one from the Matrigel, one from the GelMA and one combined.

Liver and liver organoid staining

4 µm thick slides of liver organoids and human liver were deparaffinized and rehydrated. An antigen retrieval was then performed. For this, the slides that were to be treated with anti-multidrug resistance-associated protein 2 (anti-MRP2) and anti-Villin were put in a TE buffer. These slides with buffer were then placed in a water bath (98℃) for 30 minutes. The slides that were to be treated with anti-Ki67 were placed in a pepsin buffer and placed in the incubator at 37 ℃ for 20 minutes. The slides were then washed 2 times for 2 minutes in a PBS/T buffer and stored for 20 minutes in a PBS buffer with 20 mM NH₄Cl. The slides were then washed again for 2 minutes in a PBS/T buffer. The slides were sprinkled with 10% goat serum and stored in a PBS buffer for 30 minutes. Then, to the slides treated with the TE buffer, 100 µl of antibody dissolved in Bright Diluent was added (anti-MRP2 2.5 mg/ml in the ratio 1:1000 and anti-Villin in the ratio 1:500). Anti-Ki67 (dissolved in Bright Diluent in a ratio of 1:200) was added to the slides treated with pepsin buffer. Control slides were also made, with only Bright Diluent. These slides were then put away overnight in the refrigerator at 4℃.

The following morning, the slides were washed 3 times for 5 minutes in a PBS/T buffer. As of this moment, the slides were protected against light. Secondary antibody was dropped on the slides (AF568 goat-anti-rabbit and AF488 goat-anti-mouse, both 1:200 dissolved in antibody diluent). The slides were set aside for 1 hour in the dark. The slides were then washed 3 times 5 minutes in a PBS buffer. To all slides, 4′,6-diamidino-2-phenylindole (DAPI) was added (1:2000) to stain the cell nuclei. The slides were left in the dark for 7 minutes. The slides were then washed 3 times for 5 minutes in PBS buffer. A microscope slide was then stuck to all slides with fluorsave and the slides were stored in the refrigerator at 4℃. The next day the slides were placed under the fluorescence microscope to take pictures.

Results

This chapter contains the results that are important for the conclusion. The following abbreviations have been used:

A = APAP

Z = without APAP

EM = expansion medium Matrigel

DM = differentiation medium Matrigel

GM = GelMA (differentiation medium)

A

	EM A	EM Z	DM A	DM Z	GM A	GM Z
5 hours	0.004977	0.002123	0.0064408	0.008673	0.013687	0.090171
24 hours	0.009515	0.002928	0.0073191	0.010174	0.088122	0.016248
72 hours	0.005709	0.005709	0.0053795	0.008783	0.007978	0.022726

B

C

D

E

Fig. 1: Measured ATP values 5 hours, 24 hours and 72 hours after addition of APAP.

(A). The relationship between the measured values of the ATP measurement in RLU and the ATP concentration in µM. This relationship has been calculated on the basis of ATP measurements with known concentrations of ATP.

(B). ATP concentration in different conditions in µM. Everything was measured in triplicate. Here the averages are taken. The concentrations were calculated on the basis of the relationship shown in graph (A).

(C). The data from table (B) was processed in a graph, with the ATP concentration plotted against time.

(D). The measurement values of EM A, EM Z, DM A and DM Z are very close to each other in graph (C). These four conditions were therefore taken separately in graph (D). The ATP concentration was also plotted against time, but with greater accuracy to more clearly show the differences.

(E). The two other conditions, GM A and GM Z, are shown separately in the graph, in which the ATP concentration is also plotted against time. The measured values in graph (C) differed greatly from the other four conditions, which can be seen in (D).

There is a high peak in ATP concentration at GM Z after 5 hours and a high peak at GM A after 24 hours (see (C)). The ATP concentration increases for all conditions, except GM Z, between 5 hours and 24 hours (see (C) and (D)). For all conditions, except for EM Z and GM Z, the ATP concentration decreases between 24 hours and 72 hours (see (C) and (D)).

In Figure 2, Ki67 expression is visible in (A) (green colour). This means that there are dividing cells. In (C) and (E) there are no dividing cells. In (E) and (F) very few organoids are visible, and these consist of few cells. MRP2 expression is visible in human liver tissue (red colour, see (G)). In the organoids, no MRP2 expression can be seen (see (B), (D) and (F)). The red colour in the image is noise. The organoids lack the structural protein MRP2 that occurs in human liver tissue. In addition, Villin expression can be seen in both (G) and (D) and (F) (green colour). The protein Villin found in human liver tissue is therefore present in organoids in DM.

There are no images of the live/dead colouring. The cause of this is explained in the discussion. The BrightField and Hoechst photos clearly show that the organoids exposed to APAP, both in EM and in DM, are dead or damaged (see (B), (D) and (F)). The organoids that have not received APAP are still alive

at both stages (see (A), (C) and (E)). Small organoids in EM APAP and DM APAP are also still alive (see (B) and (D)).

Conclusion

After 72 hours, cell damage can be seen in all cells exposed to APAP. There are, however, major differences. For example, the photos of the microscope show that cells in the differentiation media were more damaged than cells in expansion media. The ATP measurement also indicates this. It can be concluded that cells in the differentiation media have converted more APAP to NAPQI and therefore have more damage.

In addition, it appears that cells in GelMA do not grow optimally. Under the microscope, a large amount of damage can be seen in the cells that were cultivated in GelMA. The ATP measurements from these conditions are also much higher than those from the cells cultivated in Matrigel. This indicates a high amount of cell damage in GelMA. After 72 hours, differences can be seen under the microscope between the organoids in GelMA with and without APAP. Small organoids are visible in the condition not exposed to APAP. In the condition that was exposed to APAP, there were no living organoids left.

The aim of the cell staining was to demonstrate that organoids in differentiation medium differentiated into adult hepatocytes. However, this is not apparent from the results of the staining with anti-MRP2. What did become clear is that just like adult hepatocytes, cells in the differentiation medium condition no longer divide much. In expansion medium, there are significantly more cell divisions. The staining clearly shows dividing cells in this condition. In addition to that, there is Villin expression in organoids in differentiation medium, just as in human liver tissue. Cells in expansion medium do not yet exhibit Villin expression. Just like adult hepatocytes, cells in differentiation medium are more damaged and therefore must have converted more APAP to NAPQI than the cells in expansion medium.

The hypothesis that the cells exposed to APAP have more damage than cells not exposed to APAP seems to be correct. The hypothesis that organoids grown in differentiation medium suffer more damage than organoids in expansion medium appears to be correct too.

Discussion

There are several uncertainties that could have influenced the results of the study. One of the most influential uncertainties was that after the study had been carried out, it was not possible to conclude with certainty whether the cell damage observed and measured in organoids exposed to APAP had been caused entirely by the conversion of APAP to NAPQI. Especially with organoids grown in GelMA, this is uncertain. In both the cells that were not exposed to APAP and those that were, a large concentration of extracellular ATP was measured. GelMA could have a negative influence on the condition of cells, which could be an explanation for the large concentration of extracellular ATP. If GelMA actually causes cell damage, a possible explanation for the increased concentration of extracellular ATP could be that the cells secrete more ATP after growth in GelMA to prevent an inflammatory response. A follow-up study could investigate whether there is a way to better differentiate cells in GelMA or the same experiment could be repeated with another animal-friendly gel.

In addition, the results showed that 72 hours is a late time point to measure representative ATP concentrations. Since the degradation of extracellular ATP takes place within a few seconds and the cells probably died well before 72 hours, the measured ATP concentrations at 72 hours do not provide a reliable picture of the point at which the cells died. A follow-up study should include measurements at more different time points to achieve results that are more reliable. For example, these measurement points could be 1 hour, 2.5 hours, 5 hours, 10 hours, 24 hours, 36 hours and 48 hours.
In regard to the live/dead staining, measurements at other time points could also provide a clearer picture of the moment at which the cells died. During this study, photos were taken only after 72 hours. Photos after 5 hours and 24 hours could help determine a more precise time for when the cells died.

The luminometer had a major negative impact on the reliability of the ATP assay. The results showed that this device sometimes had such a large noise that the background noise was greater than the measured ATP concentration. Some extreme values could not be explained. As a result, the photos taken for the research formed a more reliable and clearer result than the measurements from the ATP assay.

Another problem that had arisen is that the live/dead stainings were not successful. An EVOS fluorescence microscope was used. This turned out to be the wrong microscope. Ethidium bromide and calcein were used for live/dead staining. These substances are only visible under a UV microscope, which should have been used instead of the fluorescence microscope. The structure of the organoids made it possible to distinguish between living and dead cells in the photos that were taken with the fluorescence microscope.

Furthermore, the fact that it was uncertain whether NAPQI was actually formed by the cells exposed to APAP was a major bottleneck. The cell damage was measured indirectly by an ATP assay, which measured the extracellular ATP concentration. However, this does not yet prove that the cells had converted the APAP to NAPQI and whether they had died from the NAPQI. The chance that the conversion from APAP to NAPQI was the cause of cell damage and death is very high, but this is not certain. To clarify this uncertainty, during a follow-up study the NAPQI concentration could be measured instead of the extracellular ATP concentration.

An accuracy error may also have occurred during cell staining by means of anti-Ki67. Due to the absence of this antibody in the laboratory, anti-Ki67 was borrowed from another laboratory. The anti-Ki67 that was meant to be used originated from a mouse, but the borrowed anti-Ki67 originated from a rabbit. This may have led to different results than originally expected. It is also possible that the antibodies did not work properly.

Finally, this study was conducted on a relatively small scale. Samples of six different conditions were used in triplicate. This has yielded a relatively large amount of results, but a larger number of samples and more control measurements would lead to results that are more reliable. To involve these results even more directly in research into personalized medicine, one or more control groups, consisting of adult hepatocytes from the (human) liver, should be formed in a follow-up study. This would allow the influence of APAP on cultured hepatocytes to be compared to the influence of APAP on adult hepatocytes from the liver under the same conditions. This can clarify whether a patient\’s liver stem cells can be cultured in such a way that they develop into adult hepatocytes that have virtually the same effect as adult hepatocytes from the liver of the same patient.

Previous research shows similarities with the results in this study on several points. Firstly, the article by James (2003) shows that the concentration of ATP in mice decreases considerably after 24 hours. This is also clearly visible in our research. Figures 1.D and 1.E show that all organoids that were exposed to APAP also show a peak of ATP at 24 hours.

Fig. 4: ATP in mouse livers after administration of APAP.

In his study (2019), Ramachandran showed that hepatocytes die after being exposed to a large dose of NAPQI converted from APAP. In our research, the photos also show a clear difference between cells that differentiated and cells that did not differentiate toward adult hepatocytes. Between Figure 3.A in combination with 3.B and Figure 3.C in combination with 3.D, a difference is visible in the extent to which the cells were affected by APAP.

Acknowledgement

We would like to thank the Hubrecht Institute in Utrecht and U-Talent for their help with this research. In particular, Loes Oosterhoff and Bart Spee from the Hubrecht Institute for research supervision and Arnoud van Zoest from U-Talent for his help in writing this article.

References

1. Chen, Chen, Alejandro Soto-Gutierrez, Pedro M. Baptista, and Bart Spee. ‘Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells’. Gastroenterology 154, no. 5 (2018): 1258–72. https://doi.org/10.1053/j.gastro.2018.01.066.
2. Alwahsh, Salamah M., Hassan Rashidi, and David C. Hay. ‘Liver Cell Therapy: Is This the End of the Beginning?’ Cellular and Molecular Life Sciences 75, no. 8 (2018): 1307–24. https://doi.org/10.1007/s00018-017-2713-8.
3. Rabelink, Ton, Ineke Slaper-Cortenbach, and Hanneke De Kort. ‘Kennisagenda Regeneratieve Geneeskunde’, 2018. https://www.nfu.nl/img/pdf/18.2851_NFU_Kennisagenda_Regeneratieve_Geneeskunde_def_online.pdf
4. Huch, Meritxell, Helmuth Gehart, Ruben Van Boxtel, Karien Hamer, Francis Blokzijl, Monique M.A. Verstegen, Ewa Ellis, et al. ‘Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver’. Cell 160, no. 1–2 (2015): 299–312. https://doi.org/10.1016/j.cell.2014.11.050.
5. Ribeiro, Marcelo Af. ‘Liver Regeneration, Stem Cells and Beyond.’ World Journal of Gastrointestinal Surgery 1, no. 1 (2009): 6–7. https://doi.org/10.4240/wjgs.v1.i1.6.
6. James, Laura P, Laura W Lamps, Sandra McCullough, and Jack A Hinson. ‘Interleukin 6 and Hepatocyte Regeneration in Acetaminophen Toxicity in the Mouse’. Biochemical and Biophysical Research Communications 309, no. 4 (3 October 2003): 857–63. https://doi.org/10.1016/J.BBRC.2003.08.085.
7. Ramachandran, Anup, and Hartmut Jaeschke. ‘Acetaminophen Hepatotoxicity’. Seminars Liver Disease 1, no. 212 (2019): 221–34.
8. Bateman, D. Nicholas, and Allister Vale. ‘Paracetamol (Acetaminophen)’. Medicine (United Kingdom) 44, no. 3 (2016): 190–92. https://doi.org/10.1016/j.mpmed.2015.12.014.
9. Twycross, Robert, Victor Pace, Mary Mihalyo, and Andrew Wilcock. ‘Acetaminophen (Paracetamol)’. Journal of Pain and Symptom Management 46, no. 5 (1 November 2013): 747–55. https://doi.org/10.1016/j.jpainsymman.2013.08.001.
10. González, Leiva, and Fernanda Daniela. ‘Highlight Report: Role of the ATP-Releasing Panx Channels in Liver Fibrosis’. EXCLI Journal 18 (22 January 2019): 8–9. https://www.ncbi.nlm.nih.gov/pubmed/30956634.
11. Fitz, J. G. (2007). Regulation of cellular ATP release. Transactions of the American Clinical and Climatological Association, 118, 199–208. https://www.ncbi.nlm.nih.gov/pubmed/18528503

 

Appendix

Appendix A. ATP assay

Appendix B. Protocol APAP on liver organoids

Appendix C. Protocol colouring cells and organoids

Appendix A. ATP assay

1

2

3

4

5

6

7

8

9

10

11

12

A

Standard line 1

5 EM A neg

5 EM Z neg

5 DM A neg

5 DM Z neg

5 EM A1

5 EM A2

5 EM A3

5 EM Z1

5 EM Z2

5 EM Z3

5 DM A1

B

Standard line 2

5 DM A2

5 DM A3

5 DM Z1

5 DM Z2

5 DM Z3

5 GM A1

5 GM A2

5 GM A3

5 GM Z1

5 GM Z2

5 GM Z3

C

Standard line 3

24 EM A neg

24 EM Z neg

24 DM A neg

24 DM Z neg

24 EM A1

24 EM A2

24 EM A3

24 EM Z1

24 EM Z2

24 EM Z3

24 DM A1

D

Standard line 4

24 DM A2

24 DM A3

24 DM Z1

24 DM Z2

24 DM Z3

24 GM A1

24 GM A2

24 GM A3

24 GM Z1

24 GM Z2

24 GM Z3

E

Standard line 5

72 EM A neg

72 EM Z neg

72 DM A neg

72 DM Z neg

72 EM A1

72 EM A2

72 EM A3

72 EM Z1

72 EM Z2

72 EM Z3

72 DM A1

F

Standard line 6

72 DM A2

72 DM A3

72 DM Z1

72 DM Z2

72 DM Z3

72 GM A1

72 GM A2

72 GM A3

72 GM Z1

72 GM Z2

72 GM Z3

G

Standard line 7

H

Neg

Fig. 5: Plate set-up ATP assay

Explanation A = APAP

Z = without APAP

EM = expansion medium Matrigel

DM = differentiation medium Matrigel

GM = GelMA (differentiation medium)

5 = 5 hours

24 = 24 hours

72 = 72 hours

Neg = negative control

Standard line: see protocol ‘APAP on liver organoids’

The plate has been put into the luminometer the wrong way around.

	1	2	3	4	5	6	7	8	9	10	11	12
A	316	286	266	217	197	207	177	148	148	138	138	128
B	108	108	99	89	99	99	99	79	79	89	69	49
C	49	69	69	59	69	59	69	59	59	59	69	69
D	39	59	49	59	49	49	49	49	39	39	39	49
E	49	49	59	49	20	59	49	39	39	49	30	49
F	39	69	79	39	49	39	49	30	49	39	30	30
G	49	39	49	39	39	39	30	39	39	30	49	39
H	39	49	49	39	30	30	30	39	39	39	49	30

Fig. 6: Raw data ATP assay: first measurements of the luminometer. Shown is the amount of light emitted by ATP reaction with luciferase (in relative light units (RLU)).

	1	2	3	4	5	6	7	8	9	10	11	12
A	286	256	256	237	197	168	177	158	158	177	158	128
B	108	128	138	108	99	69	99	99	69	69	69	59
C	4635	138	187	79	79	89	79	444	79	59	59	49
D	39	49	59	69	59	69	49	39	49	39	39	138
E	11668	118	108	306	128	917	148	69	49	69	69	1164
F	79	49	49	59	69	89	89	39	39	30	39	10583
G	661	345	325	128	79	79	89	2307	59	59	79	96077
H	49	49	39	59	39	49	79	39	49	49	148	450357

Fig. 7: Raw data ATP assay: second measurements of the luminometer. Shown is the amount of light emitted by ATP reaction with luciferase (in relative light units (RLU)).

Because the plate was inserted into the luminometer the wrong way around, the table is mirrored horizontally and vertically. Therefore, in figure 6 and figure 7, the measured value in box A1 constitutes the results for the content of H12 (et cetera) of figure 5.

In figure 7 the extreme values are highlighted in yellow. These values are not included in the calculations because they differ too much from the other measurements.

Appendix B. Protocol APAP on liver organoids

Necessities

• Liver organoids:
  • 6 times in expansion condition after 7 days in Matrigel
  • 6 times in differentiation condition after 7 days in Matrigel
  • 6 times in differentiation condition after 7 days in GelMA
• 10 ml expansion medium (EM)
• 10 ml differentiation medium (DM)
• Read-outs:
  • Live/dead-staining
  • ATP determination kit
• Scale (accuracy ± 0.0001 g)
• Pipettes (5 ml, 1 ml, 250 µl, 100 µl, 10 µl) with corresponding pipette tips
• Multititer plate
• Cell culture cabinet
• 70% ethanol
• Incubator
• Eppendorf tubes
• Marker
• 5 ml Basal medium
• 5 µl Hoechst
• 10 µl Ethidium bromide (EtBr)
• 10 µl Calcein
• EVOS fluorescence microscope
• 50 µl reaction buffer (20X)
• Demineralised water
• 100 µl DTT solution (100 mM)
• 2 µl ATP solution (5 mM)
• 2.5 µl Firefly luciferase 5 mg/ml solution

Steps

Preparation

1. Add 4.5349 mg/ml APAP solution
   1. 5 ml EM
   2. 5 ml DM
2. Take a solution for the negative controls: EM and DM without APAP
3. Filter and sterilize the solutions mentioned above

Protocol for cell culture

1. Heat the APAP media and the media for the negative controls
2. Switch on the cell culture cabinet and clean it with 70% ethanol
3. Remove the old medium from the liver organoids
4. Note which wells with cells receive APAP and which are the negative controls (in triplicate)
5. Add the correct media to the wells (205 µl / well)
6. Place the plate in the incubator at 37 ℃ with 5% CO2

Collect medium

Collect 20 µl of the medium in separate Eppendorf tubes at the following time points after exposure to APAP: 5 hours, 24 hours and 72 hours. Write the times and contents down on the tubes. Use a new pipette tip each time new medium is collected.

Live/dead-staining

1. Make a solution with Hoechst, Live (Calceine) and Dead (EtBr). To 5 ml basal medium, add: 5 µl Hoechst, 10 µl Calceine and 10 µl EtBr
2. Add 250 µl of the solution made per well and incubate for 10 minutes at room temperature
3. View and photograph the wells with an EVOS fluorescence microscope

ATP assay

1. Unpack the ATP assay package
2. Prepare 1 ml of 1X reaction buffer: add 50 µl of 20X reaction buffer (component E) to 950 µl of demineralised water
3. Make 1 ml of the 10 mM D-Luciferin stock solution (component with the blue cap): add 1 ml of the 1X reaction buffer to 1 vial D-Luciferin
4. Defrost the 100 mM DDT stock solution
5. Make a standard line (see below) from the 5 mM ATP solution (green cap)

	Concentration	ATP	Demineralised water
S1	100 µM	2 µl ATP	98 µl
S2	10 µM	10 µl S1	90 µl
S3	1 µM	10 µl S2	90 µl
S4	100 nM	10 µl S3	90 µl
S5	10 nM	10 µl S4	90 µl
S6	1 nM	10 µl S5	90 µl
S7	100 pM	10 µl S6	90 µl
Negative	0	–	100 µl

1. Defrost all collected samples
2. Make the standard reaction solution
   1. 8.9 ml of demineralised water
   2. 500 µl 20X reaction buffer (component E)
   3. 100 µl DTT
   4. 500 µl Luciferin
   5. 2.5 µl Firefly luciferase 5 mg/ml stock solution
3. Swivel the solution and protect it from light
4. Make the arrangement for all samples on paper, as well as the standard line and the negative control
5. Pipette 90 µl of the standard reaction solution per well into a white plate
6. Add 10 µl of each sample in the correct well
7. Measure the plate in the luminometer (Settings: endpoint: kinetic, counting time: 0.5 s, emission filter slot: A5, Mean operation: By well)
8. Extract the background measurement from the measurement of the sample in a data processing program (for example Excel) and calculate the ATP concentration per sample on the standard curve. Clearly display the data in a graph.

Appendix C. Protocol staining cells and organoids

Necessities

• Slides (4 µm thickness) with:
  • Human liver
  • Cultured organoids
• 2 ml TE
• 2 ml of pepsin
• 500 ml of PBS buffer
• 500 ml of PBS/T buffer
• 100 ml of 20 mM NH₄Cl
• 2 µl MRP antibody 2.5 mg/ml (rabbit)
• 4 µl anti-Villin (mouse)
• 8 µl anti-Ki67 (rabbit)
• 4 ml of bright diluent
• AF568 goes anti-rabbit 1: 200 in antibody diluent
• AF488 goes anti-mouse 1: 200 in antibody diluent
• AF488 goes anti-rabbit 1: 200 in antibody diluent
• 2 ml of DAPI 1: 2000
• 2 ml of fluorosave
• Pipettes (1 ml, 250 µl, 100 µl, 10 µl) with corresponding pipette tips
• Slides
• Fluorescence microscope
• 200 ml of xylene
• 200 ml of ethanol solution (98%, 95%, 90%, 70%)
• 2 ml goat serum

Steps

1. Deparaffinise and rehydrate the slides with xylene and then the ethanol series (98%, 95%, 90%, 70%)
2. Perform an antigen retrieval:
   • 1. Place the slides in a TE buffer (for the slides that are treated with anti-MRP2 and anti-Villin). Place them in a 98 ℃ water bath for 30 minutes and allow to cool back to room temperature
     2. Place the slides in a pepsin buffer (for the slides that will be treated with anti-Ki67). Place them in a 37 ℃ incubation cabinet for 20 minutes
3. Wash all slides 2 times for 2 minutes with a PBS/T buffer
4. Place the slides in a PBS buffer with 20 mM NH₄Cl for 20 minutes
5. Wash all slides with a PBS / T buffer for 2 minutes
6. Drizzle all slides with 10% goat serum. Then put them in a PBS buffer for 30 minutes
7. Make an antibody solution
   • 1. Anti-MRP2 2.5mg / ml 1: 1000 and villin 1: 500 in Bright diluent
     2. Anti-Ki67 1: 200 in Bright diluent
     3. Also make a solution for the negative control with only Bright diluent
8. Drop 100 µl of the solutions per slide and note the correct solution on the slide
9. Leave the slides for 1 night at 4 ℃
10. Wash the slides 3 times for 5 minutes with PBS/T buffer

Limit the amount of light on the slides as of this step

1. On the secondary antibody slides, drop: AF568 goat-anti-rabbit 1: 200 in antibody diluent and AF488 goat-anti-mouse 1: 200 in antibody diluent. Let it stand in the dark for 1 hour
2. Wash 3 times for 5 minutes with PBS buffer in the dark
3. Colour the cores of the slides with DAPI. Drop DAPI 1: 2000 on the slides and leave in the dark for 7 minutes.
4. Wash the slides 3 times for 5 minutes with a PBS buffer in the dark
5. Stick the slides on a microscope slide and save the slides in the refrigerator

Microscope

Place the slides under the fluorescence microscope one by one and take pictures.

 

Eline Hoogreef, 17, the Netherlands

Eline Hoogreef is 17 years old and is currently in her last year at the Johan van Oldenbarnevelt high school in the Netherlands. In addition to biology and medicine, her interests include photography and travelling.

Timo Klabbers, 18, the Netherlands

Timo Klabbers is 18 years old and is currently in his last year at the Johan van Oldenbarnevelt high school in the Netherlands. He loves numbers (mathematics, physics) and biology. Next year he will study in Rotterdam. He does athletics and his biggest hobby is scouting.

Irina Kramers, 17, the Netherlands

Irina Kramers is 17 years old and is currently in her last year at the Johan van Oldenbarnevelt high school in the Netherlands. Her favourite subjects are mathematics, biology and art. In her free time, she plays piano. Irina has no idea what the future holds for her.