
==== Front
STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00399-X
10.1016/j.xpro.2024.103234
103234
Protocol
Protocol to create phenotypic primary human hepatocyte cultures using the RASTRUM 3D cell model platform
Yee Christine christine.yee@inventia.life
12∗
Chan Yik Lung 1
Utama Robert 1
Besnier Marie 1
Engel Martin 1
Belfiore Lisa lisa.belfiore@inventia.life
13∗∗
1 Inventia Life Science, Alexandria, NSW 2015, Australia
∗ Corresponding author christine.yee@inventia.life
∗∗ Corresponding author lisa.belfiore@inventia.life
2 Technical contact

3 Lead contact

10 8 2024
20 9 2024
10 8 2024
5 3 103234© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

To improve human hepatotoxicity prediction, in vitro liver cell models replicating hepatocyte function, drug metabolism, and toxicity are required. Here, we present a protocol for creating 3D primary human hepatocyte (PHH) cell models using the RASTRUM Platform. We describe the process for PHH model generation; procedures for characterizing the PHH model, including viability, albumin production, and CYP450 inducibility; and drug treatment using acetaminophen and troglitazone. This protocol has applications in upscaling phenotypic hepatotoxicity applications.

Graphical abstract

Highlights

• Create 3D human hepatocyte models in liver-relevant matrix using the RASTRUM Platform

• Maintain viable primary hepatocytes for 2 weeks to allow for chronic drug exposure

• Measure hepatocyte albumin secretion, CYP450 activity, and drug responsiveness

• Generate 3D cell models in a 384-well plate format for drug screening applications

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

To improve human hepatotoxicity prediction, in vitro liver cell models replicating hepatocyte function, drug metabolism, and toxicity are required. Here, we present a protocol for creating 3D primary human hepatocyte (PHH) cell models using the RASTRUM Platform. We describe the process for PHH model generation; procedures for characterizing the PHH model, including viability, albumin production, and CYP450 inducibility; and drug treatment using acetaminophen and troglitazone. This protocol has applications in upscaling phenotypic hepatotoxicity applications.

Subject areas

Cell Biology
Cell culture
Molecular Biology
Biotechnology and bioengineering
==== Body
pmcBefore you begin

Preclinical animal models have historically failed to predict hepatotoxicity prior to human clinical trials.1 Primary human hepatocytes (PHH) have been evaluated as the most predictive and appropriate in vitro model for hepatotoxicity and cytotoxicity screening compared to immortalized cell lines, such as HepG2, which have impaired drug-metabolizing capabilities.2 Yet, sourcing freshly isolated primary hepatocytes is limited and can only be used for very short-term cultures in 2D (<3 days) due to de-differentiation and associated loss of innate hepatocyte functions such as albumin production and cytochrome P450 (CYP450) enzyme activity.3

The RASTRUM Platform can be used to create matrix-embedded 3D cell culture models in a range of well plate formats using pre-validated protocols. RASTRUM uses drop-on-demand bioprinting technology to deposit small droplets of bioink and activator fluids that chemically crosslink upon contact at room temperature to form pre-designed 3D models. The high precision and resolution of this technology enables the generation of reproducible 3D cell models that can be used for a variety of applications.

Here, we have printed commercially available cryopreserved PHH in an optimized RASTRUM Matrix with peptides and proteins attributed with the extracellular matrix (ECM) components in the liver. The printed PHH models were subsequently characterized for their hepatic characteristics and functions. These models maintain cell viability for two weeks post-printing, which is a significant improvement over traditional 2D models, and maintain functional characteristics of hepatocytes, such as albumin production and cell type-specific marker expression. The models allow simultaneous assessment of numerous hepatotoxicity parameters from the same well, and the 384-well plate format enables the analysis of many replicates. The established protocol provides a streamlined approach for creating standardized and physiologically relevant 3D PHH models for hepatotoxicity testing at scale.

Prepare cell culture media and ROCKi solution

Timing: 2 h

1. Prepare serum-free hepatocyte base media (HBM).a. Defrost hepatocyte complete media (HCM) BulletKit supplement pack singlequots at 25°C.

b. Bring HBM basal media from the HCM BulletKit to 25°C.

c. Add all singlequots from the supplement pack to 500 mL HBM media.

d. Rinse each singlequots vial with 1 mL HBM media and return to the main bottle.

Note: This media is usable up to 1 month after singlequot addition when stored at 4°C.

2. Prepare hepatocyte complete media (HCM).a. Bring serum-free HBM and fetal bovine serum (FBS) to 25°C.

b. Add 40 mL serum-free HBM to a sterile 50 mL centrifuge tube.

c. Add 10 mL FBS and 1.25 mL 1 M HEPES to the tube, and invert to mix.

Note: This media is stable for 1 month at 4°C.

3. Prepare hepatocyte plating media.a. Warm the two bottles (MP100-1 and MP100-2) in a 37°C water bath.

b. Combine the contents of MP100-1 (media) and MP100-2 (supplement).

4. Prepare hepatocyte thawing media.a. Warm the media bottle in a 37°C water bath.

b. Transfer the bottle contents to a sterile 50 mL centrifuge tube.

5. Prepare 5 mM Y-27632 dihydrochloride (ROCKi) stock.a. Resuspend 1 mg Y-27632 dihydrochloride (ROCKi) in 624 μL PBS.

Note: Aliquot and store at −20°C for up to 6 months.

Prepare collagen-coated 6-well plate

Timing: 2 h

6. Reconstitute PureCol (3 mg/mL stock).a. Add 5 mL sterile 0.01 N hydrochloric acid (HCl) to the PureCol serum vial containing 15 mg sterile, lyophilized type I bovine collagen.

b. Mix and agitate the contents at 2°C–10°C for approximately 16 h.

Note: Reconstituted PureCol is stable at 4°C for up to 3 months.

7. Prepare a working solution of PureCol.a. Dilute one part PureCol stock (3 mg/mL) with 30 parts sterile 0.01 N HCl.

b. Add 2 mL of working solution into each well of a 6-well plate.

c. Tilt the plate in a circular motion to coat the entire well surface.

d. Cover plate with lid and incubate coated plate in biosafety cabinet at 25°C for 2 h.

e. Aspirate the remaining working solution 2 h later.CRITICAL: Aspirate carefully to avoid scratching the coated surface.

f. Rinse wells thoroughly with 2 mL sterile PBS.

g. Add 2 mL fresh PBS and leave the plate in a biosafety cabinet (BSC) until required.

Thaw and plate cryopreserved primary human hepatocytes

Timing: 24 h

Thawing and plating hepatocytes 24 h prior to printing minimizes cell death-mediated signaling often observed immediately after reanimation. Typically in 2D, the washes after cell attachment to the plate removes dead cells. It is crucial to have high cell viability (>80%) prior to printing as low starting viability will result in poor viability post-printing.8. Thaw PHH by gently swirling the frozen vial in a 37°C water bath for approximately 90–120 s.

CRITICAL: Only retrieve PHH vial from −80°C/dry ice/liquid nitrogen immediately prior to thawing.

9. Transfer the PHH vial contents into hepatocyte thawing media.a. Gently invert 50 mL conical tube with hepatocyte thawing media and cells once to mix.

b. Centrifuge at 25°C at 100 × g for 8 min.

c. Aspirate the supernatant carefully with a Pasteur pipette for the bulk, then a P1000 pipette for closer to the pellet.

d. Resuspend PHH pellet in 3 mL warm hepatocyte plating media by tapping the tube.CRITICAL: It is very important to avoid pipetting up and down excessively. Minimize this by pouring or pipetting up and down only once with Pasteur pipettes or wide-bore pipettes. This is important, as regular P1000 pipette tips create high shear stress that can damage the cells.

e. Count cells using the desired method.

f. Remove PBS from collagen-coated wells.

g. Plate 2 million viable PHH in each well with total 2 mL hepatocyte plating media.

h. After 60 min, gently replace with 2 mL fresh hepatocyte plating media and incubate at 37°C for 4–6 h.

i. After 4–6 h, replace media in each well with 1.5 mL hepatocyte complete media.

j. Return the plate to 37°C incubator for 24 h.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
Anti-albumin primary antibody (1:100)	Bethyl Laboratories	#A80-229A	
Anti-MRP-2 primary antibody (1:100)	Abcam	#ab172630	
Secondary antibody IgG-488 (1:1,000)	Thermo Fisher Scientific	#A-21202	
Secondary antibody IgG-647 (1:1,000)	Thermo Fisher Scientific	#A-31573	
	
Chemicals, peptides, and recombinant proteins	
	
PureCol	Advanced BioMatrix	#5006	
Hydrochloric acid	Merck	#H9892	
HCM BulletKit, containing HBM basal media and HCM supplement pack SingleQuots	Lonza	#CC-3198	
Fetal bovine serum (FBS)	HyClone	#SH30084.03	
Hepatocyte plating media	Lonza	#MP100	
Hepatocyte thawing media	Lonza	#MCHT50	
HEPES (1 M)	Thermo Fisher Scientific	#15630106	
Y-27632 dihydrochloride (ROCKi)	STEMCELL Technologies	#72302	
Phosphate-buffered saline (PBS) (pH 7.4)	Thermo Fisher Scientific	#14190144	
TrypLE	Thermo Fisher Scientific	#12604013	
Sterile-filtered distilled water	Livingstone	#LV-WATERDIS05L	
Sterile-filtered ethanol	LabTech	#EL70-20	
RASTRUM 1 × 96 WP small single matrix models, plus matrices with matrix Px02.29PH	Inventia Life Science	#PRSP-229	
RASTRUM 1.5 × 24/96 WP or 1 × 384 WP, single matrix models, plus matrices with matrix Px02.29PH	Inventia Life Science	#PRLP-229	
Calcein-AM	Biotium	#30002	
Ethidium homodimer-III	Biotium	#30002	
Hoechst 33342	Invitrogen	#H3570	
4% Paraformaldehyde	ProSciTech	#EMS15735-100	
Triton X-100	Merck	#X100	
Phalloidin CF594	Revvity	#CP25941	
Sodium azide	Merck	#S2002	
Rifampicin	Cayman	#14423	
Troglitazone	Cayman	#71750	
Acetaminophen	Sigma	#A5000	
	
Critical commercial assays	
	
CellTiter-Glo 3D	Promega	#G9681	
P450-GloTM assay	Promega	#V9002	
Albumin ELISA	Abcam	#ab179887	
	
Experimental models: Cell lines	
	
Cryopreserved primary human hepatocytes	Lonza	#HUCPI	
	
Software and algorithms	
	
RASTRUM Cloud	Inventia Life Science	N/A	
RASTRUM App	Inventia Life Science	N/A	
GraphPad Prism v9.0	GraphPad Software, Inc.	N/A	
	
Other	
	
RASTRUM Platform	Inventia Life Science	N/A	
Axio Observer 7 microscope	Zeiss	N/A	
Celldiscoverer7 microscope	Zeiss	N/A	
FLUOstar Omega microplate reader	BMG Labtech	N/A	
Countess II automated cell counter	Thermo Fisher Scientific	N/A	
Nunc cell culture treated 6-well plate	Thermo Fisher Scientific	#140675	
PhenoPlate 96-well microplate, tissue culture treated, black, 96-well with lid	Revvity	#6055300	
PhenoPlate 384-well microplate, tissue culture treated, black, 384-well with lid	Revvity	#6057300	
OptiPlate 384-well plate, white opaque	Revvity	#6007290	

Step-by-step method details

Print primary human hepatocytes using RASTRUM

Timing: 4 h

This section covers the steps required for harvesting PHH cells, printing PHH cells using RASTRUM and maintaining PHH cells in 3D culture post-printing. RASTRUM is a drop-on-demand bioprinter that deposits small droplets of bioink and activator fluids into the well, which covalently crosslink with each other upon contact to form a gel. The substrate concentration and formulation details of RASTRUM fluids are proprietary, but they combine to form a polyethylene glycol-based matrix of approximately 1.1 kPa stiffness. The whole bioprinting process is performed at room temperature, and it takes less than 30 min to print cells as 3D cell models.1. Generate RASTRUM PrintRun file and protocol.a. Open RASTRUM Cloud software and select Design PrintRun.

b. Select cell model Imaging Model (for 96-well plate) or HTP Model (for 384-well plate).

c. Select matrix condition Px02.29PH.

d. In Wellplate Editing, select the well plate type to be used, and highlight all wells of the plate for printing the cell model.

e. Download the RASTRUM PrintRun File and the RASTRUM PrintRun Protocol.

2. Add ROCKi to PHH cells prior to printing.a. Pre-warm HCM in 37°C water bath.

b. Add 4 μL of 5 mM ROCKi to each well of the 6-well plate well with PHH and swirl gently to mix.

c. Incubate cells in 37°C incubator for at least 2 h prior to cell harvest.

3. Start RASTRUM PrintRun.a. Turn on the RASTRUM and compressor.

b. Open the RASTRUM App software and run Start of Day Greenlighting. This process will require a RASTRUM cartridge containing sterile-filtered distilled water and ethanol.

c. Open the RASTRUM PrintRun File in the RASTRUM App software.

d. Follow the steps in the RASTRUM PrintRun Protocol to prime the inert base fluids (Activator F3 and Bioink F32) and biofunctional bioink (Bioink F240), and print the inert base into the selected well plate.

e. While the inert base is printing, begin harvesting PHH cells.

4. Harvest the PHH cells.a. Check cell morphology of PHH under microscope.Note: The best-looking cells will have a cobblestone-like morphology and fill the well surface. They will also detach more readily with TrypLE.

b. Remove media from each 6-well plate well and rinse with 1 mL PBS.

c. Add 500 μL TrypLE to each well, swirl gently and incubate the plate at 37°C for 5 min.

d. Gently tap the plate to detach PHH and observe under the microscope to ensure the successful trypsinization.

e. Add 1.5 mL HCM to neutralize TrypLE.

f. Gently collect the cell suspension with a Pasteur pipette or wide-bore pipette tips by pipetting up once onto a fresh 50 mL centrifuge tube.CRITICAL: Avoid forceful pipetting and washing as it can cause shear stress and cell death.

g. Wash each well with 2 mL HCM and repeat twice to collect with PHH, and pool wells if necessary.

h. Centrifuge at 100 × g for 8 min at 25°C.

i. Aspirate the supernatant carefully with a Pasteur pipette for the bulk, then a P1000 pipette for closer to the pellet.Note: The pellet will be very loose, with dead cells floating at the top.

j. Resuspend PHH pellet in 3 mL warm HCM by tapping the tube.Note: No pipetting should be required as pellet is loose.

k. Count cells using the desired method.Note: Some automated cell counters may encounter issues due to the large diameter of hepatocytes. Manual cell counting may be required.

l. Prepare 6.25 × 10⁶ cells/mL in a fresh 50 mL centrifuge tube and pellet cells at 100 × g for 8 min at 25°C.Note: Refer to the RASTRUM PrintRun Protocol for the total number of cells needed to complete the print.

5. Complete RASTRUM PrintRun using PHH cells.a. Remove supernatant without disturbing the pellet.

b. Resuspend the pellet in the biofunctional activator (Activator F177) by tapping the bottom of the tube gently.

c. Transfer the PHH in the biofunctional activator (Activator F177) to the RASTRUM cartridge.

d. Place the RASTRUM cartridge into the printer and immediately start the cell model printing phase.CRITICAL: There is no cell size filtering step here as the cells are of large size. The cells are clumpy and heavy. It is important that there are no delays during printing.

e. While the cell models are printing, prepare HCM+ROCKi at 10 μM final concentration and keep in a 37°C water bath until use.

f. Immediately after the cell model printing phase has finished, add HCM+ROCKi solution to all wells (150 μL per well for 96-well plate and 50 μL per well for 384-well plate) and transfer the plate to the 37°C incubator.

g. Clean RASTRUM using water and ethanol as per the PrintRun Protocol instructions.

h. Shut down the printer.

6. Maintain the printed PHH cell culture plate.a. At 48 h post-printing, remove all the HCM+ROCKi media from the well and replace with the same volume of HCM without ROCKi.

b. Perform media changes every 2–3 days by removing 100 μL of media per well (for 96-well plate) or 35 μL of media per well (for 384-well plate) using a multichannel pipette and replacing it with the same volume of fresh HCM media.

Perform live/dead staining assay

Timing: 1 h

This step outlines the process for staining PHH models to assess cell viability via microscopy.7. Remove calcein-AM and ethidium homodimer-III (EthD-III) from the −20°C freezer and allow to thaw at 25°C for 20 min.

8. Prepare staining solution by diluting calcein-AM to 1 μM and EthD-III to 2 μM in PBS.

9. Dilute Hoechst to 5 mg/mL in the staining solution.

10. Remove media from wells of PHH models.

11. Add 100 μL staining solution and place the plate in the incubator for 30 min.

Optional: Remove the staining solution and replace with PBS for imaging.

12. Use a fluorescence microscope to capture single focal plane images at the excitation/emission maxima for Hoechst (352/454 nm), calcein-AM (495/515 nm) and ethidium homodimer-III (528/617 nm).

Perform cell viability assay

Timing: 1 h

This step measures the viability of cells over the culture period by endpoint metabolic assay.13. At days 1, 3, 7 and 14 of culture, remove 75 μL of media from the wells containing cell models.

14. Add 75 μL of CellTiter-Glo 3D reagent to each well.

15. Incubate the plate at 25°C for 5 min.

16. Read luminescence from the plate using a plate reader as per manufacturer’s protocol.

Perform in situ antibody staining

Timing: 2–3 days

This step determines the expression of classic hepatocyte-specific markers in the PHH model.17. Fix PHH models in 96-well plate at day 7 post-printing by incubating with 100 μL 4% paraformaldehyde for 30 min at 25°C.

CRITICAL: Paraformaldehyde is toxic by skin contact and inhalation. Ensure appropriate personal protective equipment is used when handling paraformaldehyde. Only use paraformaldehyde in a chemical fume hood.

18. Remove 4% paraformaldehyde and wash wells three times with PBS.

CRITICAL: Dispose of paraformaldehyde as hazardous waste in an appropriate waste container.

19. Permeabilize cell models with 100 μL 0.1% Triton X-100 in PBS for 30 min at 25°C.

20. Remove 0.1% Triton X-100 and wash wells three times with PBS.

21. Incubate cell models with 100 μL 10% donkey serum for 1 h at 25°C to block non-specific antibody binding.

22. Remove blocking solution and incubate cell models with 100 μL anti-albumin primary antibody 1:100 in 1% BSA in PBS or anti-MRP-2 primary antibody 1:100 in 1% BSA in PBS for 48 h at 4°C with gentle rocking.

23. Wash wells three times with 150 μL PBS + 0.1% Tween-20 (PBST).

24. Incubate cell models with 100 μL IgG-488 or IgG-647 secondary antibodies at 1:1000 in 1% BSA for 3 h at 25°C with gentle rocking.

25. Wash wells three times with 150 μL PBST.

26. Add 100 μL 1 μM Hoechst and 1:400 phalloidin CF594 in PBS to each well, and incubate for 20 min at 25°C.

27. Wash wells three times with 150 μL PBS.

28. After the final wash step, add 100 μL PBS + 0.1% v/v sodium azide to each well.

29. Process to imaging within 7 days.

Perform ELISA for secreted albumin

Timing: 2 h

This step outlines the measurement of albumin production by PHH.30. Collect cell model supernatant (150 μL per well) at days 1, 7 and 14 (or every 2–3 days, corresponding with media changes, if preferred) post-printing and store at −80°C until assay.

31. Defrost supernatant samples on ice and centrifuge at 500 × g for 5 min at 4°C to remove cell debris.

32. Dilute supernatant samples 1 in 25 with HCM and assay in duplicate.

33. Perform albumin ELISA as per manufacturer’s protocol.

Perform CYP3A4 activity assay

Timing: 2–3 days

This step measures CYP3A4 inducibility of the PHH model printed into a 384-well plate.34. Induce CYP3A4 in PHH cells.a. At day 3 post-printing, dilute Rifampicin in DMSO to 10 μM in HCM (final concentration of 0.27% DMSO).

b. Prepare a 0.27% DMSO vehicle control diluted in HCM.

c. Remove all media from cell models.

d. Add 50 μL of Rifampicin or vehicle control to PHH models.

e. Incubate the plate for 48 h in a 37°C incubator.

f. After 24 h, replace the media with fresh 10 μM Rifampicin or DMSO control.

35. Perform CYP3A4 activity assay.a. Wash PHH models with PBS for 5 min at 37°C.

b. Add 50 μL of Luciferin-IPA substrate to each well and incubate for 1 h at 37°C.

c. Transfer 25 μL substrate from each well to a fresh opaque white 384-well plate, then put aside the plate containing the cell models.

d. Add 25 μL luminescent detection reagent per well to the 25 μL substrate in the opaque white 384-well plate.

e. Agitate the plate at 150 RPM for 1 min and then incubate for 20 min at 25°C.

f. Read luminescence using a plate reader.

36. Perform CellTiter-Glo assay in parallel for data normalization.a. Add 25 μL of CellTiter-Glo 3D reagent per well to the plate containing the remaining cell models from the above step.

b. Agitate the plate at 150 RPM for 5 min and incubate at 25°C for 25 min.

c. Transfer 40 μL from each well to a fresh opaque white 384-well plate.

d. Read luminescence using a plate reader.

e. Correct each CYP3A4 reading to the CellTiter-Glo 3D reading from the corresponding well.

f. Express CYP3A4 activity as a fold-change relative to the DMSO control group.

Perform drug response studies

Timing: 2–7 days

This step measures the hepatotoxicity of acetaminophen and troglitazone in the PHH model.37. Create the acute toxicity model.a. At day 3 post-printing (once the PHH models are well-established in culture), serially dilute acetaminophen in HCM media 1.5-fold from 37.5 mM (37.5 mM, 25 mM, 16.7 mM, etc.).Note: Ensure that ROCKi is removed from the media 24 h prior to drug addition.

b. Add acetaminophen dilutions (0.43–37.5 mM) or 0.75% DMSO vehicle control to PHH models in 384-well plate and incubate plate for 48 h at 37°C.

38. Create the chronic toxicity model.a. At day 3 post-printing (once the PHH models are well-established in culture), serially dilute troglitazone in HCM media 1.5-fold from 375 μM (375 μM, 250 μM, 167 μM, etc.).Note: Ensure that ROCKi is removed from the media 24 h prior to drug addition.

b. Add troglitazone dilutions (22–375 μM) or 0.75% DMSO vehicle control to PHH models in 384-well plate and incubate plate for 48 h at 37°C.

c. Replace drug and media every 3 days up to day 7.

39. Perform CellTiter-Glo 3D assay at endpoint.a. Remove 25 μL of media from each well.

b. Add 25 μL of CellTiter-Glo 3D reagent to each well.

c. Agitate the plate on a plate shaker at 150 RPM for 5 min at 25°C.

d. Incubate plate for 25 min at 25°C.

e. Transfer 40 μL of solution from each well to an opaque white-walled OptiPlate-384.

f. Read luminescence from the plate using a plate reader as per manufacturer’s protocol.

g. Determine IC50 values using nonlinear regression analysis in GraphPad Prism v9.0.

Expected outcomes

The PHH model printed by RASTRUM maintains high viability and crucial hepatocyte functional characteristics for over 14 days, surpassing conventional 2D models. This expanded assay window allows the same model to be used for both acute and chronic drug toxicity testing, which was previously unattainable. Combined with RASTRUM’s ability to accommodate for high-throughput plate format in a highly standardized and reproducible way, the model can be used for various toxicological applications, including drug screening and high-content imaging.

RASTRUM-printed PHH show high cell viability in culture

Printed PHH show characteristic round-to-cuboid morphology with a high level of viability immediately post-printing (Figure 1A). PHH are able to maintain high viability for over 14 days in vitro with the addition of 10 μM Y-27632 to the culture media (Figure 1B). Without Y-27632, the viability of PHH slowly reduces from day 3 to reach 50% at day 14, in a similar manner to other PHH spheroid models.4,5 Notably, as functional primary hepatocytes do not typically proliferate in vitro,6 cell growth over time is not expected (although proliferation can be influenced by the culture environment). Together with the fact that RASTRUM Matrices are stable for at least four weeks in culture,7 the consistency of the viability signal over time demonstrates the stability of the 3D culture over the two-week culture period.Figure 1 RASTRUM-printed PHH show high cell viability in culture

(A) Primary human hepatocytes (PHH) show characteristic morphology and high cell viability one day after encapsulation in the RASTRUM Imaging Model in Matrix Px02.29PH. Bright-field image taken at 20× magnification. Representative live/dead image taken at 5× magnification one day after printing. Green = live cells and red = dead cells. Scale bars = 50 μm (left) and 500 μm (right).

(B) Longer-term PHH viability is maintained over 14 days as a 3D culture with Y-27632 addition to the media. Viability determined by CellTiter-Glo 3D reagent. Black line = hepatocyte culture media (HCM) only. Pink line = HCM + Y-27632. Data points are means ± SDs from n = 4 technical replicates per time point.

RASTRUM-printed PHH show typical hepatocyte functions

Expression of classic hepatocyte-specific markers such as albumin and multidrug resistance protein 2 (MRP2) are observed in RASTRUM-printed PHH at day 14 post-printing (Figure 2A). This feature is indicative of PHH not de-differentiating,6 by epithelial-to-mesenchymal transition, into a fibroblast-like morphology, which is commonly seen in long-term 2D PHH cultures. Measurement of albumin production by hepatocytes is a gold-standard indicator of in vitro hepatocyte functionality.8 The presence of albumin in the supernatant across 14 days in RASTRUM-printed PHH is indicative of the maintenance of innate hepatocyte functions (Figure 2B). This is observed in Y-27632-treated and non-treated PHH, irrespective of the cell viability difference.Figure 2 RASTRUM-printed PHH show typical hepatocyte functions

(A) Immunofluorescent staining of PHH for albumin (top) and MRP2 (bottom) at day 14 post-printing. Imaging model cultured with hepatocyte culture media with 10 μM Y-27632. Imaged at 40× magnification. Scale bars = 20 μm.

(B) Albumin secretion by PHH was maintained over 14 days in culture. ∗∗p < 0.01 vs. day 1, ∗∗∗∗p < 0.0001 vs. day 1. Data points represent individual wells (n = 3 per time point) and error bars are SDs. Statistical analysis by two-way ANOVA with Dunnett’s post-hoc multiple comparisons test.

RASTRUM-printed PHH demonstrate CYP450 inducibility

The majority of drug metabolism occurs in hepatocytes, mediated mainly by cytochrome P450 family enzymes. CYP450 enzyme activity has been described to be drastically reduced in immortalized cell lines such as HepG2 compared to PHH.9 CYP3A4 enzyme activity, one of the major CYP450 family enzymes responsible for the metabolism of the vast majority of drugs in the market,10 is able to be induced in the RASTRUM PHH model. RASTRUM-printed PHH treated with Rifampicin (a non-toxic but potent CYP3A4 inducer) for two days show a ∼4-fold increase in CYP3A4 induction (Figure 3) as measured by P450-Glo CYP3A4 assay.Figure 3 RASTRUM-printed PHH demonstrate CYP450 inducibility

CYP3A4 activity at day 3 of culture can be induced in RASTRUM-printed PHH by Rifampicin treatment for 48 h ∗∗∗∗p < 0.0001 versus DMSO control. Data points represent individual wells (n = 14 per treatment group) and error bars are SDs. Statistical analysis by non-parametric t-test.

RASTRUM-printed PHH show drug responsiveness

PHH in the RASTRUM HTP model challenged with acetaminophen, a widely-used analgesic associated with hepatotoxicity in doses above therapeutic level,11 show an IC50 of approximately 15.96 mM (Figure 4A), which is achieved using fewer cells per replicate than in 2D12 (6,125 cells versus >10,000 cells per well in 2D). PHH in the RASTRUM HTP model treated with troglitazone, an anti-diabetic drug withdrawn from the market due to hepatotoxicity from chronic dosing,13 show an IC50 of approximately 200 μM (Figure 4B).Figure 4 RASTRUM-printed PHH show drug responsiveness

(A) Acute drug exposure to acetaminophen in RASTRUM-printed PHH achieved an IC50 comparable to other 3D PHH models. Measured by CellTiter-Glo 3D at day 5 post-printing, after 2 days (48 h) of acetaminophen exposure. Data points are means ± SDs from n = 3 technical replicates per concentration.

(B) Long-term viability of PHH in RASTRUM Matrix enabled chronic drug toxicity studies with troglitazone. Measured by CellTiter-Glo 3D at day 10 post-printing and after 7 days of troglitazone exposure. Data points are means ± SDs from n = 3 technical replicates per concentration.

Limitations

This protocol was optimized to generate PHH 3D cell models using cryopreserved primary human hepatocytes (Lonza, #HUCPI), but cell responses are donor-dependent and can vary with disease state. The starting cell number per vial, expected viability and attachment rate in 2D can vary between donors. Printing PHH in RASTRUM Matrix may require optimization of matrix composition, cell density and drug dosing depending on the specific PHH source used.

As PHH do not form spheroid or aggregate structures in RASTRUM Matrix, they are not directly comparable with other 3D PHH spheroid models and their functionality. Generally, 3D cell culture models have higher heterogeneity than 2D cell cultures, which can lead to variability of collected data. This variation can be partially addressed by increasing the number of sample replicates when working in 3D compared to 2D cell culture.

While we have endeavored to provide a starting point for biological validation of the PHH model in this protocol, including albumin production, MRP2 production and CYP3A4 enzyme inducibility, further biological characterization of liver function may be required. This may include functional gene expression studies, indocyanine green metabolism, glycogen synthesis, examining inducibility of additional CYP enzymes, and determining the duration over which these functions are sustained in culture.

Troubleshooting

Problem 1

Low cell viability pre-printing (related to before you begin section, steps 8–9).

Potential solution

When reviving cells, use a water bath, NOT a bead bath. Water baths maintain a more consistent temperature, which is better for revival, as the process needs to be relatively quick. Handling the cells with wide-bore tips or Pasteur pipettes, or minimizing pipetting with regular P1000 tips, will reduce shear stress. Tap tubes to resuspend pellets rather than pipetting. The 2D plating and ROCKi steps are included to reduce the dead cell load.

Problem 2

Low cell number pre-printing (related to step-by-step method details section, step 4).

Potential solution

Do not filter cells before counting. Hepatocytes can be large cells (15–30 μm) and may be lost with filtering. They do not usually have issues with clumping or stickiness. The starting cell number per vial, expected viability and attachment rate in 2D can vary between donors, so this should be checked with the supplier. The number of wells to plate in 2D and/or to be printed should be adjusted accordingly to accommodate starting cell numbers. When plating cells in 2D, ensure that cells are not sparse and have formed a cobblestone-like appearance. Sparse cells may undergo epithelial-to-mesenchymal transition, are more difficult to detach, and are more sensitive to stress and/or death during the TrypLE step. Healthy cultures will detach easily with TrypLE.

Problem 3

High cell death post-printing (related to step-by-step method details section, step 4).

Potential solution

Confirm that the cell viability is high (>80%) prior to printing by checking cell morphology and using viability stains such as Trypan Blue. Low starting viability or the presence of dying cells can trigger cell death in adjacent viable cells. ROCKi addition while cells are in 2D and in the media post-printing can help minimize apoptotic signaling.

Problem 4

Cells do not show expected responses (related to step-by-step method details section, steps 27–29).

Potential solution

CYP activity/inducibility and drug responses are donor-dependent. Check the clinical details of the donor prior to the experiment. Responses can vary with disease state. Commercial suppliers may have checked the inducibility of CYP enzymes for each batch. Use the same donor between experiments to ensure reproducibility of results. Ensure ROCKi has been removed at least 24 h prior to drug addition to minimize unwanted interactions.

Problem 5

Printed cell models do not form consistently within and/or between wells (related to step-by-step method details section, step 5).

Potential solution

Minimize the time that bioink or activator fluids are sitting idle within the printer to maximize printer ejection performance.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Lisa Belfiore (lisa.belfiore@inventia.life).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Christine Yee (christine.yee@inventia.life).

Materials availability

No new materials were generated from this protocol.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to analyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The graphical abstract was created using BioRender.com.

Author contributions

C.Y., Y.L.C., R.U., and M.B. designed the research; C.Y. and Y.L.C. performed the research and analyzed the data; C.Y., M.B., M.E., and L.B. wrote the paper. All authors edited and approved the final manuscript.

Declaration of interests

C.Y., R.U., M.B., M.E., and L.B. are employees and shareholders of Inventia Life Science Pty. Ltd. Inventia has an interest in commercializing the RASTRUM Platform.
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