A Chemically Defined Hydrogel for Human Liver Organoid Culture

Abstract

End-stage liver diseases are an increasing health burden, and liver transplantations are currently the only curative treatment option. Due to a lack of donor livers, alternative treatments are urgently needed. Human liver organoids are very promising for regenerative medicine; however, organoids are currently cultured in Matrigel, which is extracted from the extracellular matrix of the Engelbreth-Holm-Swarm mouse sarcoma. Matrigel is poorly defined, suffers from high batch-to-batch variability and is of xenogeneic origin, which limits the clinical application of organoids. Here, a novel hydrogel based on polyisocyanopeptides (PIC) and laminin-111 is described for human liver organoid cultures. PIC is a synthetic polymer that can form a hydrogel with thermosensitive properties, making it easy to handle and very attractive for clinical applications. Organoids in an optimized PIC hydrogel proliferate at rates comparable to those observed with Matrigel; proliferation rates are stiffness-dependent, with lower stiffnesses being optimal for organoid proliferation. Moreover, organoids can be efficiently differentiated toward a hepatocyte-like phenotype with key liver functions. This proliferation and differentiation potential maintain over at least 14 passages. The results indicate that PIC is very promising for human liver organoid culture and has the potential to be used in a variety of clinical applications including cell therapy and tissue engineering.

Keywords: hepatocyte differentiation; human liver organoids; polyisocyanopeptides; synthetic hydrogels; tissue engineering.

Figure 1. An optimized PIC hydrogel supports liver organoid expansion.

Single organoid cells were seeded at day 0 and cultured in human organoid expansion medium (EM) supplemented with the Rho kinase inhibitor Y-27632 for 14 days. Four different donors were analyzed in independent experiments (N = 4). a) Light microscopy images of organoids at day 7 after single cell seeding. Organoids did not proliferate in PIC-plain and PIC-RGD, but showed a morphology comparable to Matrigel in PIC supplemented with 3 mg mL⁻¹ LEC (PIC-LEC). b) Lower concentrations of PIC improve organoid proliferation. c) Light microscopy pictures reveal a dose-dependent effect of LEC on organoid proliferation. Quantification of organoid proliferation in d) 5k PIC and e) 1k PIC. Organoids from five different donors were cultured in Matrigel or PIC with different concentrations of LEC. Relative cell numbers were determined by an Alamar blue assay every 2-3 days and cell expansion relative to day 0 was calculated. Each dot represents the mean of the four donors with standard deviation.

Figure 2. Soft PIC hydrogels enhance organoid expansion.

Mechanical properties of Matrigel and PIC hydrogels measured at constant frequency (1 rad s⁻¹) and strain (1%). a) Time trace of storage modulus G′ showing hydrogel gelation with the temperature raising from 4 to 37 °C (+7 °C min⁻¹) and holding at 37 °C. For all conditions, the starting modulus at time = 0 min were much lower because all the samples were liquid at 4 °C; the modulus almost reached plateau at time = 6 min when the temperature was held at 37 °C (N = 3). b) Stiffness of hydrogels showing the storage modulus G′ of Matrigel and PIC hydrogels after 10 min incubation at 37 °C (N = 3). c) Time trace of storage modulus G′ and loss modulus G″ of Matrigel and PIC-LEC hydrogels through a heating and cooling cycle. At time = 0 min, G′ and G″ were close to each other in all three conditions showing a liquid state; at time = 5 min, G′ reached a peak higher than G″ after heating from 4 to 37 °C (+7 °C min⁻¹); during time = 5–10 min, all gels remained plateaued when the temperature was held at 37 °C; after time = 15 min, both PIC hydrogels showed a sharp decrease of G′ when the cooling cycle started from 37 to 4 °C (-7 °C min⁻¹), and G′ of both PIC hydrogels dropped to less than 1 Pa after being held at 4 °C for 10 min. However, G′ of Matrigel was still higher than 50 Pa after cooling for 15 min (N = 2).

Figure 3. Organoids in PIC retain a stem/progenitor phenotype and are highly proliferative.

Single organoid cells were seeded at day 0 and organoids were expanded in the different hydrogels for 14 days. Three different donors were analyzed in independent experiments (N = 3). a) Immunofluorescent analysis of paraffin-embedded organoids confirmed that the organoids in all gels have an epithelial progenitor phenotype and are highly proliferative. Human liver tissue was used as a control. Whole transcriptome sequencing analysis of organoids from two independent donors at day 7 of expansion in Matrigel, 1k PIC-LEC, and 5k PIC-LEC (N = 2). b) Pearson correlation map of the organoids in different hydrogels. Note that the correlation of cultured organoids is more than 99%. c) Heatmap of markers for stem/progenitor cells, epithelial cells, and mature hepatocytes selected from the mRNA sequencing data.

Figure 4. Organoids differentiated into functional hepatocyte-like cells in PIC.

Organoids were differentiated in Matrigel, 1k PIC-LEC, or 5k PIC-LEC for 8 days (N = 4). a) Light microscopy images of organoids showing that differentiated organoids became denser and darker compared to EM organoids in all hydrogels. b) Gene expression of stem cell and hepatocyte markers in differentiated organoids from four independent donors. Transcriptional levels of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), albumin (ALB), cytochrome p450 3A4 (CYP3A4), and multidrug resistance-associated protein 2 (MRP2) were determined by qRT-PCR and compared to cryopreserved human hepatocytes. Hepatocyte functionality of differentiated organoids in the different hydrogels was assessed. Four different donors were analyzed in independent experiments. d) Rh123 transport was determined as read-out for MDR1 activity. Verapamil was added as an inhibitor of MDR1 function. c) Albumin concentrations and e) GLDH levels in cell lysates were measured after incubation in DM for 24 h. Comparison was normalized to total protein concentrations. Graphs indicate mean ± SD. f) Ammonium elimination from the culture medium was determined as read-out for hepatocyte functionality. *indicates p-value ≤ 0.05.

Figure 5. Long-term expansion of organoids in PIC.

Organoids from two different donors were cultured for 12 weeks in Matrigel, 1k PIC-LEC, and 5k PIC-LEC with weekly passaging. a) Light microscopy pictures show that organoids maintained their morphological phenotype during all passages in Matrigel, 1k PIC-LEC, and 5k PIC-LEC. b) qRT-PCR analysis of the stem cell marker LGR5 shows stable expression levels at passage 2, 6, 10, and 14 in all different hydrogels. c) mRNA sequencing on organoids from two independent donors in EM and after differentiation (day 8 in DM). The 100 most significant differentially-expressed genes in DM versus EM are displayed in a heatmap. Note that EM samples from both donors and all three hydrogels show a very similar expression pattern and that all DM samples from both donors and all three different hydrogels are similar. A full list of genes is provided as Table S1, Supporting Information. d) Gene expression of hepatocyte markers in differentiated organoids from two independent donors during long-term culture in Matrigel or PIC hydrogels. Transcriptional levels of hepatocyte markers ALB, CYP3A4, and MRP2 were determined by qRT-PCR and compared to their respective EM controls (N = 2).

Figure 6. Human recombinant laminin-111 can substitute LEC.

a) Organoids from three different donors were cultured for 7 days in Matrigel, PIC-plain, or 1k PIC supplemented with either LEC or human recombinant Laminin (hrlaminin-111). Light microscopy pictures show a similar morphological phenotype in LEC and hrlaminin-111, with increased organoid size and number at higher laminin concentrations. (N = 3) b) Immunofluorescent analysis of paraffin-embedded organoids confirmed that the organoids in all gels have an epithelial progenitor phenotype and are highly proliferative. Organoids cultured in Matrigel, 5k PIC with 3 mg mL⁻¹ LEC or 5k PIC with 2 mg mL⁻¹ hrlaminin-111 are displayed. Human liver tissue was used as a control (N = 1).