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Method Article

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

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DOI:

10.3791/66229

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April 18th, 2025

 ,  ,  ,  ,  , 
1Department of Biomedical Engineering, University at Buffalo (State University of New York), 2Clinical and Translation Research Center (CTRC), University at Buffalo (State University of New York), 3Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), 4Center for Cell, Gene and Tissue Engineering, University at Buffalo, State University of New York, 5Khufu Therapeutics, Inc.

In This Article

Summary

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Abstract

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Introduction

Liver cell migration plays a significant role in liver organogenesis, disease, and cell therapy. During liver organogenesis (E8.5-9.0, mouse), the ventral foregut pre-hepatic epithelium begins to express liver genes due to inductive signals emanating from the surrounding mesenchyme and heart. At E9.0, the foregut epithelium thickens as the cells transition from a cuboidal to a pseudostratified columnar morphology, forming the liver diverticulum1,2. At this critical stage, the liver diverticulum is comprised of only ~1,500 cells. Next, the hepatic endoderm lining the liver diverticulum thickens, delaminates, and forms cords of migrating hepatoblasts (HBs). These HBs co-migrate with endothelial cells and mesenchymal cells, branching into the surrounding mesenchymal tissue, initiating three-dimensional collective cell migration to form the liver bud3,4. In fact, during this stage, cells collectively undergo (1) co-migration, or movement together with other cell types, (2) branching morphogenesis or formation of branching tube-like structures, and (3) interstitial migration, or migration on top of, or between, other cells. By E11.5, migration ceases, and the primitive liver has formed, expanding 103 fold3. Liver cell migration may also be required in later stages of liver organogenesis, as rat fetal HBs expression has shown evidence of highly upregulated genes associated with 3D collective cell migration, morphogenesis, and extracellular matrix remodeling5. In addition to its role in early liver organogenesis, 3D collective migration is intricately linked to the local spread and metastasis of advanced hepatocellular carcinoma (HCC), ultimately leading to worsened prognosis and increased treatment resistance6. Adult and fetal hepatocytes also employ collective migration when moving from the spleen to within the liver during liver repopulation. In vivo imaging studies have demonstrated that transplanted hepatocytes enter the portal vein and then the capillaries within hours, migrating across the liver sinusoids and through the liver tissue7,8,9. Finally, recent studies demonstrate that migrating HBs arise during murine and human liver regeneration, with some evidence of movement in sheets10. Overall, liver collective migration, capable of multiple modes of morphogenesis, plays a significant role in organogenesis, cancer, hepatocyte cell therapy, and liver regeneration.

Numerous genetic studies have explored the molecular pathways underlying 3D liver collective cell migration. These studies reveal that the absence of hepatic cords hinders liver formation, underscoring the necessity of hepatic cord formation and their interactions with supporting cells for liver formation1,11,12,13. These studies also demonstrate that liver growth is initiated by various factors, including FGF-2 secreted from the cardiac mesoderm, BMP4 secreted from the surrounding mesenchyme, HGF, and endothelial cell interactions. Additionally, migration-associated transcription factors, including HEX, PROX1, and TBX3, play crucial roles2,14. In conclusion, genetic studies provide evidence that soluble factor signaling, leading to transcription factor expression, drives migration, signaling, and molecular interactions between HBs and their surrounding mesenchyme.

While cell migration in early liver organogenesis has been extensively investigated, current in vitro studies of hepatic migration frequently employ 2D assays utilizing highly migratory HCC cells alongside in vivo tumor models15. These studies have provided insight into several factors influencing hepatic migration, including TGFβ-116, c-Myc17, YAP18, goosecoid19, actopaxin20, and miRNAs21,22,23. Although noteworthy progress has been made in understanding the molecular mechanisms in 3D hepatic cell migration, the distinct disparities between 2D and 3D cellular migration suggest that 2D assays possess inherent limitations. Moreover, these models often lack the inclusion of mesenchymal cell types, which are essential for both migration and growth. Although there has been advancement in the development of 3D models for liver migration that integrate the supporting mesenchyme, these models primarily emphasize co-migration rather than exploring the diverse modes of collective migration.

The ability to generate tissues from spheroids through various self-assembly and morphogenetic processes facilitates the scientific exploration of synthetic tissues. These tissues have broad applications in drug development and screening, disease modeling, therapy, and other biomedical and biotechnological applications. The methodological details of several 3D in vitro cultivation systems engineered to exhibit different modes of liver 3D collective migration are provided. These systems comprise (1) co-spheroid culture with HEP and MES spheroids in a matrix, (2) HEP spheroid matrix droplet cultured with M-CM, (3) HEP-MES mixed spheroids, and (4) HEP spheroid matrix cultured with a high density of MES cells. These systems enable robust modeling of liver collective migration in a 3D environment, thereby advancing our understanding of molecular and cellular processes underlying liver organogenesis, cancer, and therapy.

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Protocol

The details of the reagents and the equipment used in the study are listed in the Table of Materials. The abbreviations used in this article are defined in Supplementary File 1.

1. Preparation of 1% low electroendosmosis (EEO) agarose solution

  1. Weigh 2.5 g of low EEO agarose powder and transfer it to a beaker.
    NOTE: Ensure the beaker is at least twice the size of the desired volume to accommodate bubbling during the solution preparation.
  2. Use a graduated cylinder to measure 250 mL of distilled water and pour it into the beaker, aiming for a final concentration (w/v) of 1%.
  3. Cover the beaker's opening with plastic wrap, leaving a small hole for ventilation. Heat the beaker in the microwave for 30 s.
  4. Carefully remove the beaker and swirl the contents. Repeat this process every 30 s until complete dissolution.
    CAUTION: Handle microwaved glassware carefully, using proper gloves. Monitor the solution closely to prevent overheating or boiling over.
  5. Once a homogeneous solution is achieved, remove the beaker from the microwave. Gently swirl the solution before transferring it to a pre-sterilized bottle. Autoclave the solution and store the agarose solution at room temperature (RT) until ready to use.

2. Coating 96-well plate

NOTE: 384-well ultra-low attached spheroid microplates can be utilized in these and further experiments.

  1. Loosen the cap of the bottle containing the sterile 1% agarose solution. Warm the solution in the microwave in 30 s intervals until it transitions to the liquid phase; then tighten the cap.
    CAUTION: Handle microwaved glassware carefully, using proper gloves. Monitor the solution closely to prevent overheating or boiling over.
    NOTE: Perform these steps under a sterile tissue-culture laminar flow hood.
  2. Transfer 55-65 µL of the 1% agarose solution per well to coat the 96-well tissue-cultured plate and immediately rotate the plate.
  3. Once the 1% agarose solution has been transferred to the desired number of wells, place the plates in a 4 °C fridge for 20-30 min to allow the agarose to solidify. Before use, bring the plate to room temperature (Figure 1A).

3. Preparation of HEP spheroids

  1. Cultivate HepG2-WT cells in a T-75 flask using completed growth medium (cDMEM) comprising of Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (Pen-Strep). Maintain the cell culture at 37 °C and 5% CO2, changing the growth medium daily.
  2. Upon reaching 80% confluency, rinse the cells with 1x phosphate-buffered saline (PBS) and discard the rinse. Add 5 mL of 0.05% of Trypsin-EDTA to the flask and incubate at 37 °C and 5% CO2 for 5-10 min.
  3. Add equal amounts of cDMEM and wash the cells off the flask. Once cells detach, transfer the mixture to a 15 mL sterile conical centrifuge tube and centrifuge the cell suspension at 300 x g for 5 min at RT.
    NOTE: All centrifugation steps outlined in this protocol were performed at room temperature (RT). However, these centrifugation procedures can alternatively be carried out at cold temperatures.
  4. Re-suspend the cell suspension in fresh cDMEM and count the cells to determine the final concentration in cells/mL (Figure 1B).
  5. Dye-labeling of the cells
    NOTE: This is an optional step.
    1. Transfer the desired amount of cell suspension to a 15 mL sterile conical centrifuge tube and centrifuge the cell suspension at 300 x g for 5 min.
    2. Re-suspend the cell pellet in a serum-free growth medium to achieve a final concentration of 1.0 x 106 cells/mL. Add 5 µL of fluorescent cell-labeling solution per mL of cell suspension and incubate the cell suspension at 37 °C and 5% CO2 on rotation for 20 min.
      NOTE: The serum-free growth medium is DMEM and is only supplemented with 1% pen-strep. Adjust the incubation time for uniform staining based on the cell suspension density.
    3. Once the incubation is completed, centrifuge the cell suspension at 450 x g for 5 min and re-suspend the cell pellet in fresh cDMEM. Repeat this wash process two more times (Figure 1C).
  6. Spheroid formation
    1. Suspend the cells in fresh cDMEM to achieve a final concentration of 5.0 x 104 cells/mL based on the cell count. Mix the cell suspension well and transfer 100 µL of cell suspension per well to the agarose-coated 96-well plate.
      NOTE: The cell suspension density is set to achieve 5,000 cells per well in the agarose-coated 96-well plate. For the use of 384-well plates, cells are suspended at a final concentration of 2.0 x 105 cells/mL, and 50 µL of cell suspension is transferred per well to achieve a concentration of 10,000 cells per well.
    2. Centrifuge the plate at 340 x g for 10 min and incubate at 37 °C and 5% CO2 for 5-9 days (Figure 1D).
      NOTE: Change the growth medium every other day after plating, gently removing 50% of cDMEM and replacing it. HEP spheroids are suitable for the HEP-MES assembloid model or M-CM model.

4. Preparation of MES spheroids

  1. Cultivate HFF/MRC-5 cells in a T-175 flask using cDMEM comprising of DMEM, supplemented with 10% FBS and 1% Pen-Strep. Maintain the cell culture at 37 °C and 5% CO2, changing the medium every other day.
  2. Upon reaching 80% confluency, perform a rinse of the cells with 1x PBS and discard the rinse. Add 5 mL of 0.25% of Trypsin-EDTA to the flask and incubate at 37 °C and 5% CO2 for 5-10 min.
  3. Add equal amounts of cDMEM and wash the cells off the flask. Once cells detach, transfer the mixture to a 15 mL sterile conical centrifuge tube and centrifuge the cell suspension at 300 x g for 5 min.
  4. Re-suspend the cell suspension in fresh cDMEM and count the cells to determine the final concentration in cells/mL (Figure 1B).
  5. Dye-labeling of cells
    NOTE: This is an optional step.
    1. Transfer the desired amount of cell suspension to a 15 mL sterile conical centrifuge tube and centrifuge the cell suspension at 300 x g for 5 min.
    2. Re-suspend the cell pellet in a serum-free growth medium to achieve a final concentration of 1.0 x 106 cells/mL. Add 5 µL of fluorescent cell-labeling solution per mL of cell suspension and incubate the cell suspension at 37 °C and 5% CO2 on rotation for 20 min.
      NOTE: The serum-free growth medium is DMEM and is only supplemented with 1% pen-strep. Adjust the incubation time for uniform staining based on the cell suspension density.
    3. Once the incubation is completed, centrifuge the cell suspension at 450 x g for 5 min and re-suspend the cell pellet in fresh cDMEM. Repeat this wash process two more times (Figure 1C).
  6. Spheroid formation
    1. Suspend the cells in fresh cDMEM to achieve a final concentration of 1.0 x 105 cells/mL based on the cell count. Mix the cell suspension well and transfer 100 µL of cell suspension per well to the agarose-coated 96-well plate.
      NOTE: The cell suspension density is set to achieve a density of 10,000 cells per well in the agarose-coated 96-well plate.
    2. Centrifuge the plate at 340 x g for 10 min and incubate at 37 °C and 5% CO2 for 5-9 days (Figure 1D).
      NOTE: Change the growth medium every other day after plating, gently removing 50% of cDMEM and replacing it. MES spheroids are suitable for the HEP-MES assembloid model.

5. HEP-MES assembloid formation

NOTE: Refer to step 3 and step 4 for the formation of HEP (Figure 2A) and MES spheroids (Figure 2B).

  1. Collect HFF/MRC-5 spheroids individually using a pipette from the 96-well plate. Transfer them to a 15 mL sterile conical centrifuge tube containing fresh cDMEM. Allow the spheroids to settle in the tube for 5 min, and then gently rinse with warm cDMEM.
    NOTE: Perform the rinsing process gently and with care.
  2. Gently transfer a single HFF/MRC-5 spheroid using a pipette to a well containing a HepG2-WT spheroid. Add ice-cold growth factor reduced (GFR) Matrigel (MG) or rat tail Collagen (CG) in a 1:1 and 1:5 dilution and incubate at 37 °C at 5% CO2 for 3 h (Figure 2C).
    NOTE: Thaw MG overnight on ice in a 2 °C to 8 °C refrigerator. Ensure the use of pre-cooled pipettes, tips, and tubes when handling MG, as it forms a gel above 10 °C.
  3. After incubation, add 75 µL of fresh cDMEM to each well and incubate at 37 °C at 5% CO2 for 2-3 days.
    NOTE: Change the growth medium every other day after plating, gently removing 50% of cDMEM and replacing it. Assembloids will still form without media changes for up to 3-4 days. HEP-MES assembloid formation can occur without the use of the matrix.

6. HEP spheroid droplet formation

NOTE: Refer to step 3 for the formation of HEP spheroids (Figure 3A). Two different materials can be used for suspending the HEP spheroids in droplets. The two methods are provided below.

  1. GFR MG droplets
    1. Collect HepG2-WT spheroids individually using a pipette from the 96-well plate, allowing them to settle in a 15 mL sterile conical centrifuge on ice.
    2. In a separate 15 mL sterile conical centrifuge tube, mix 1 mL of ice-cold GFR MG and ice-cold control growth medium at a 1:1 dilution. Aspirate the medium using a pipette from the 15 mL tube containing the HepG2-WT spheroids and place it on ice.
      NOTE: Thaw MG overnight on ice in a 2 °C to 8 °C fridge. Ensure pre-cooled pipettes, tips, and tubes when handling MG, as it forms a gel above 10 °C.
    3. Add the MG solution to HepG2-WT spheroids, ensuring even spheroid distribution inside the MG solution.
    4. Using a 200 µL pipette, collect a 15 µL volume of a single spheroid in the MG solution and slowly seed it onto a 60 mm Petri dish (Figure 3B).
      NOTE: If more than one spheroid is seeded per droplet, remove and reseed properly. Spheroids are repositioned if needed to ensure they are at the center of the droplet. For optimal spheroid seeding, consider using reverse pipetting to minimize air bubble formation.
  2. Rat tail CG droplets
    NOTE: All CG preparation should be done on ice.
    1. Collect HepG2-WT spheroids individually using a pipette from the 96-well plate, allowing them to settle in a 15 mL sterile conical centrifuge tube on ice (Figure 3B).
    2. In a microcentrifuge tube, add 358.8 µL of de-ionized water, 100 µL of 10x PBS, 12.1 µL of 1 N NaOH, and 529.1 µL of stock rat tail CG for a total volume of 1 mL.
      NOTE: Stock rat tail CG must always be added at the end.
    3. Add the CG solution to HepG2-WT spheroids, ensuring even spheroid distribution inside the CG solution.
    4. Using a 200 µL pipette, collect a 15 µL volume of a single spheroid in the CG solution and slowly seed it onto a 60 mm Petri dish.
      NOTE: If more than one spheroid is seeded per droplet, remove and reseed properly. Spheroids are repositioned to ensure they are at the center of the droplet. For optimal spheroid seeding, consider using reverse pipetting to minimize air bubble formation.
  3. Incubate the droplets at 37 °C and 5% CO2 for 60 min before adding the growth medium.
  4. Slowly add 5 mL of the desired growth medium to the petri dish and incubate at 37 °C, and 5% CO2 with growth medium changes every three days (Figure 3C).
    NOTE: M-CM was used in the HEP droplet formation assay.

7. Preparation of M-CM

  1. Harvest HFF/MRC-5 cells and seed them into a T-75 tissue culture-treated flask at a seeding density of 5,000 cells/cm2 and incubate the flask at 37 °C and 5% CO2 for 72 h in 15 mL of cDMEM.
    NOTE: Monitor the flask during this period to ensure optimal cell health.
  2. After the 72 h incubation, collect the M-CM in a 15 mL sterile conical centrifuge tube. Centrifuge the M-CM at 290 x g for 5 min for debris removal and filter using a 0.2 µm filter for sterilization (Figure 4A).
  3. Dilute the M-CM with complete growth medium at a 1:1 to 1:7 dilution ratio and add to the desired experiment (Figure 4B).
    NOTE: Utilize M-CM in the HEP droplet formation assay.

8. HEP-MES mixed spheroid formation

NOTE: Refer to steps 3.1-3.5. for HepG2-WT cell suspension preparation and steps 4.1- 4.5 for HFF/MRC-5 cell suspension preparation.

  1. Suspend the HepG2-WT cells in fresh cDMEM to achieve a final concentration of 1.0 x 105 cells/mL for each cell suspension.
  2. Suspend the HFF/MRC-5 cells in fresh cDMEM to achieve a final concentration of 1.0 x 105 cells/mL for each cell suspension.
  3. Transfer HepG2-WT and HFF/MRC-5 cell suspensions to a 15 mL sterile conical centrifuge tube at a 1:1 ratio to obtain a final concentration of 2.0 x 105 cells/mL. Thoroughly mix the cell suspension and transfer 100 µL of cell suspension per well to the agarose-coated 96-well plate.
    NOTE: The cell suspension density is set to obtain 20,000 cells per well in the agarose-coated 96-well plate.
  4. Centrifuge the plate at 340 x g for 10 min and incubate at 37 °C and 5% CO2 for 1-2 days (Figure 5C).
  5. Addition of fibrin hydrogels
    NOTE: This is an optional step that is done in 72 h after plating HEP-MES mixed spheroids.
    1. In a 15 mL sterile conical centrifuge tube, mix fibrinogen (3.25 mg/mL) and thrombin (12.5 units/mL) at a 4:1 ratio for the formation of fibrin and put the tube on ice.
  6. Transfer the fibrin to each well with a pre-existing mixed spheroid at a 1:1 dilution with current cDMEM in the well. Incubate the plate at 37 °C and 5% CO2 for 30 min.
  7. After incubation, add 75 µL of fresh cDMEM to each well and incubate at 37 °C at 5% CO2 for 1-2 days.
    NOTE: Change the growth medium every other day after plating, gently removing 50% of cDMEM and replacing it.

9. High-density MES migratory model

NOTE: Refer to step 3 for HepG2-WT spheroid preparation (Figure 6A) and steps 4.1-4.5 for HFF/MRC-5 cell suspension preparation (Figure 6B).

  1. Suspend the HFF/MRC-5 cells in fresh cDMEM to achieve a final concentration of 6.0 x 105 cells/mL and put them on ice.
  2. Mix the HFF/MRC-5 cell suspension and ice-cold MG/CG at a 1:1 dilution (Figure 6C).
    NOTE: The cell suspension density is set to obtain 30,000 cells per well in the agarose-coated 96-well plate. Thaw MG overnight on ice in a 2 °C to 8 °C fridge. Ensure pre-cooled pipettes, tips, and tubes when handling MG, as it forms a gel above 10 °C.
  3. Transfer 100 µL of HFF/MRC-5 and MG solution using a pipette to a well containing a HepG2-WT spheroid. Incubate the plate at 37 °C and 5% CO2 for 30 min.
  4. After incubation, add 75 µL of fresh cDMEM to each well and incubate at 37 °C at 5% CO2 for up to 12 days.

10. Statistical analysis

  1. Review and analyze the data collected using Microsoft Excel or any statistical/graphing software.
    NOTE: For statistical assessment between two groups, Student's t-test was done (type II with two tails), and p-values equal to or less than 0.05 were considered to have statistical significance and are defined within the text and figures/figure legends.

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Results

Interest in synthetic tissues for a range of biomedical applications, such as disease modeling, drug discovery, and tissue engineering (Figure 7), is on the rise. Within this field, human pluripotent stem cell (hPSC)-derived organoids, spheroids, and cells are being transformed into more synthetic, complex, larger tissues. This transformation involves the application of principles of morphogenesis, utilization of tools such as microfabrication, and the integration of biomatrices, enabling pr...

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Discussion

The present protocol encompasses multiple methods for cultivating both simple and complex assembloids, as well as techniques for inducing 3D collective cell migration in early liver organogenesis24,25. The outlined protocols highlight several critical steps, with spheroid formation being a central aspect across all methodologies. Microwells (96- or 384-wells) are utilized with either non-adherent or agarose-coated plates to achieve spheroid formation. This method...

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Disclosures

NP is the founder of Khufu Therapeutics, an organoid engineering company that develops treatments for acute and chronic liver disease.

Acknowledgements

The authors would like to express sincere gratitude to Ogechi Ogoke and Osama Yousef for their invaluable contributions to the data used in this paper. NP was supported by the UB CBE startup funds, a Mark Diamond Fellowship, the New York State Stem Cell Science C024316, the UB Stem Cells in Regenerative Medicine (ScIRM) center, the University at Buffalo Center for Cell, Gene, Tissue Engineering (CGTE).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
384-well Black/Clear Round Bottom Ultra-Low Attachment Spheroid MicroplateCorning3830
96-wl Cell Culture Plate Flat,TC,STRL,1/pk,100/CSLaboratory Products Sales701001
AgaroseSigma-AldrichA0576Low EEO
Axiovision SoftwareZEISSversion 4
Cell Culture Dish, 60mm, TC, STRL, 20/pk, 500/CSLaboratory Products Sales705001
Cell Culture Flask,175cm², Vent,TC,STRL,5/pk,40/CSLaboratory Products Sales709003
Cell Culture Flask,75cm², Vent,TC,STRL,5/pk,100/CSLaboratory Products Sales708003
Centrifuge Tubes, 15mL,PP,STRL,grad,50/bg,500/CSLaboratory Products SalesL226010
Collagen I, Rat Tail, 100 mgCorning354236
DMEM, high glucose, GlutaMAX Supplement, pyruvateThermoFisher Scientific10569010
EVOS Fluorescent, phase-contrast microscopeEVOSAMEFC4300R1,360 x 1,024 pixel density
Fetal Bovine Serum, qualified, United StatesThermoFisher Scientific26140079
Fibrinogen from human plasmaSigma-AldrichF4883-500MG500 mg
Hep G2 [HEPG2]ATCCHB-8065
HERAcell vios 160iThermoFisher Scientific51033557
HFF-1ATCCSCRC-1041
ImageJIJ1.46r; https://imagej.net/ij/
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, 10 mLCorning354230
MRC-5ATCCCCL-171
MSCGMT Human Mesenchymal Stem Cell Growth BulletKitLonzaPT-3001
Penicillin-Streptomycin (10,000 U/mL)ThermoFisher Scientific15140122
Thrombin from human plasmaSigma-AldrichT6884-100UN100 units
Trypsin-EDTA (0.05%), phenol redThermoFisher Scientific25300062
Trypsin-EDTA (0.25%), phenol redThermoFisher Scientific25200072
Vybrant DiD Cell-Labeling SolutionThermoFisher ScientificV22887
Vybrant DiI Cell-Labeling SolutionThermoFisher ScientificV22885
Vybrant DiO Cell-Labeling SolutionThermoFisher ScientificV22886
Zeiss Axio fluorescence microscopeZEISSSE64, 1,344 × 1,024 pixel density

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Liver AssembloidsHepatic SpheroidsMesenchymal SpheroidsBranching MorphogenesisCollective MigrationMesenchymal Conditioned Media
In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines
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