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Introduction
Liver organoids derived from cholangiocytes exhibit regenerative potential and have a unique structure characterized by a bulging spherical lumen, similar to intestinal organoids [1-3]. However, their capacity to synthesize and store various biomolecules, including albumin, as well as to metabolize drugs, was limited. To overcome these limitations, hepatocyte organoids (HOs) were developed from hepatocytes, which constitute the majority of the liver parenchyma. Research involving both human and mouse models has advanced HOs, demonstrating their ability to synthesize proteins and store glycogen, fat, and cholesterol, closely resembling the functions of the native liver [4].
Cholangiocyte-derived organoids demonstrate impressive longevity, with cultures maintaining viability for over 6 months in humans [3]. In contrast, HOs exhibit a notable 2-3-fold reduction in growth rate after 2 to 3 months, resulting in a lifespan that is approximately 3 times shorter than that of cholangiocyte organoids [4]. This discrepancy in longevity reflects the inherent challenges posed by the limited proliferative capacity of hepatocytes in vitro compared to cholangiocytes [5]. Although organoid technology provides a degree of lifespan extension and functional improvement, ongoing efforts to optimize culture techniques are imperative for ensuring their sustained and effective long-term utilization.
From an alternative perspective, it is noteworthy that hepatocytes, unlike fetal hepatocytes, are characterized by short telomeres and limited proliferative capacity [4,6,7]. Our investigation into the extended in vitro culture of HOs derived from newborn swine revealed no observable increase in telomerase activity over the duration of the culture, indicating a persistent constraint on telomere dynamics in these HOs [8].
Liver organoids hold promise for curing severe liver failure and enabling personalized medicine. Although transplantation is currently the standard treatment, the scarcity of donor livers impedes further progress. These challenges underscore the importance of research such as ours in creating functional hepatocyte sources for bioartificial liver devices. These devices provide a promising interim solution for patients awaiting transplants and those experiencing liver failure after hepatectomy [8-11].
Based on our successful in vitro culture of HOs for over 120 days, we previously identified challenges associated with limited telomerase reverse transcriptase expression, which could impact their long-term functionality and lifespan. In this study, we explored the efficacy of cryopreservation for the extended preservation of HOs, assessing their regeneration capacity, survival, and function. Furthermore, in light of earlier findings suggesting that adipose tissue derived mesenchymal stem cell (A-MSC) co-culture improves HO function and generation efficiency, we examined the effects of co-culturing both before and after cryopreservation.
Materials and Methods
Ethics statement: All experiments were approved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science (approval number: NIAS20212195) of the Rural Development Administration, Republic of Korea.
1. Culture medium
All culture media, unless otherwise specified, were purchased from Stem Cell Technologies (STEMCELL Technologies, Vancouver, BC, Canada). All cultures were performed in ultra-low attachment 24-well plate matrices (Corning, Glendale, AZ, USA). Matrigel was used from Corning's Matrigel matrix (Corning).
2. Culturing of swine HOs
Porcine HOs were generated and cultured in the presence and absence of A-MSCs, following the protocol detailed by Ock et al. [8]. The culture period extended for 14 days, after which subculture was performed at a 1:2 ratio. For long-term viability, day-14 fresh HOs (passage 0, p0) were cut into fragments using live digest medium (Gibco, Grand Island, NY, USA) and cryopreserved using CryoStor CS10 (STEMCELL Technologies) following the manufacturer's instructions. After a year of storage in liquid nitrogen (LN2), the cryopreserved HO fragments were thawed in 37℃ water and sub-cultured. Using the same conditions as for fresh HOs, they were maintained for an additional 14 days.
As shown in Fig. 1, the experimental groups were designed as follows: Groups 1 and 2 were freshly cultured with HOs alone or with A-MSCs until p1. Groups 3 and 4 were HOs that were cryopreserved from HOs cultured alone at p0, and then either cultured alone (group 3) or with A-MSCs (group 5) at p1. Groups 5 and 6 were HOs that were cryopreserved from HOs co-cultured with A-MSCs at p0, and then either cultured alone (group 5) or with A-MSCs (group 6) at p1. At p1, all samples were harvested to extract total RNA for real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) experiments.
3. Immunofluorescence staining
HOs cultured in vitro for 14 days were harvested at p0. They underwent immunostaining using a specific albumin antibody, following the procedures outlined in a previous study [8]. The morphological characteristics were assessed under a laser-scanning confocal microscope (Leica; Wetzlar, Hesse, Germany) with red fluorescence indicating albumin-positive staining, and blue fluorescence representing nuclei.
4. Nile red staining
HOs were fixed in 3.7% neutral buffered formalin (Sigma Aldrich, St. Louis, MO, USA) for 30 minutes to preserve their structure. After 2 washes with phosphate-buffered saline (Sigma Aldrich), they were stained with a Nile red staining kit (Abcam, Cambridge, United Kingdom) for 20 minutes following the manufacturer's instructions. This staining specifically targets intracellular lipid droplets, including neutral lipids, enabling their visualization. For counterstaining, DAPI (NucBlue Fixed Cell ReadyProbes reagent; Invitrogen, Eugene, OR, USA) staining was performed for 20 minutes. Red fluorescence indicated a positive reaction for lipids. The samples were observed under a confocal microscope (Nikon AX, Tokyo, Japan).
5. Real-time reverse-transcription quantitative PCR
Total RNA extraction, cDNA synthesis, and RT-qPCR were performed using modifications of previously published methods [8]. Briefly, 100 ng of purified total RNA was reverse-transcribed. Gene-specific primers were adopted from the literature. Data analysis utilized the ΔΔCT method with hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the reference gene. The experiment employed 3 biological replicates, followed by pooling of samples and 5 technical replicates for each pooled sample. The results are presented as mean relative quantification (RQ) values, with error bars representing the observed minimum (RQ min) and maximum (RQ max) RQ values.
6. Statistical analyses
Statistical analyses were conducted using IBM SPSS ver. 25.0 (IBM Corp., Armonk, NY, USA). Data were analyzed using one-way analysis of variance followed by the Tukey honestly significant difference post-hoc test for pairwise comparisons among groups. All experiments were replicated 5 times. Statistical significance was denoted by p<0.05, p<0.01 and p<0.001.
Results
1. Development of HOs
The hepatocytes successfully formed HOs, as depicted in Fig. 2. The recovered hepatocytes differentiated into dark, compact HOs, in contrast to the light, expanded cholangiocyte organoids, which are indicated by a white arrow in Fig. 2A. Notably, after 30 days of continuous culture, some HOs exhibited budding at their edges (Fig. 2B).
2. Function of HOs
The expression of albumin, a protein primarily synthesized in the liver, was examined in day 14 HOs at p0. Immunofluorescence staining revealed strong intracellular localization of albumin within HOs, as shown in Fig. 3. The red fluorescence signal confirmed the predominant cytoplasmic localization of albumin in HOs. This suggests that HOs are capable of producing albumin, a key protein in blood plasma.
To reaffirm the liver’s critical role in nutrient storage, we evaluated the lipid accumulation capacity of HOs using Nile red staining. This technique utilizes a highly specific marker for intracellular lipid droplets, such as cholesterol and neutral lipids. Immunofluorescence analysis revealed red signals in all HOs, showing a subtle, size-dependent increase in signal intensity (Fig. 3B).
3. Effects of cryopreservation on proliferation-related gene expression in HOs
HO fragments frozen for a year exhibited robust regeneration capability, regardless of co-culture with A-MSCs at p0. However, co-culture of A-MSCs after thawing improved the regenerative ability of Hos in groups 4 and 5. Even HOs co-cultured with A-MSCs at p0 showed a decreased HO regeneration rate when not co-cultured with A-MSCs after thawing (group 5) (Fig. 4A).
Genes related to cell growth (TP53/P21) and cell death (CASP8) were analyzed in both fresh and frozen HOs (Fig. 4B). Primary hepatocytes (PHs) served as a control, showing significantly lower expression of these genes compared to any HOs groups. Fresh HOs co-cultured with A-MSCs (group 2) exhibited significantly reduced the expression of TP53, P21, and CASP8. In cryopreserved HOs, P21 expression was significantly upregulated to a greater extent in groups 4 and 6 than in groups 3 and 5 after co-culture with A-MSCs at p1. Notably, HOs co-cultured with A-MSCs at p0 (groups 2 and 5) maintained similar expression levels of TP53, P21, and CASP8 as fresh HOs co-cultured with A-MSCs (group 2), even when A-MSCs were not present at p1 in group 5. However, group 6, which received A-MSCs at p1, exhibited significantly higher expression of these genes.
The pronounced reduction in the expression of TP53, P21, and CASP8 expression in fresh HOs co-cultured with A-MSCs suggests a preconditioning effect for enhanced stress resistance, potentially due to A-MSC-mediated anti-apoptotic signaling, leading to improved cell viability and early organoid formation.
4. Effects of cryopreservation on liver function-related genes in HOs
Compared to PHs, all HO groups exhibited relatively high expression of ALB, CYP3A29, and epithelial cell adhesion molecule (EPCAM), as shown in Fig. 5A. Regardless of cryopreservation, co-culture with A-MSCs at p1 significantly increased ALB expression, as shown in groups 2, 4, and 6. However, ALB expression was lowest in group 5, with a minimum value of 1.15 and a maximum value of 1.46. This suggests that the change in expression was relatively small. ALB expression levels did not change after cryopreservation for groups cultured under the same conditions, such as group 1 compared to group 3, and group 2 compared to group 6.
For CYP3A29, co-culture with A-MSCs (groups 2, 4, and 6) at p1 resulted in decreased expression in all groups compared to the HO-alone groups (groups 1, 3, 5), regardless of cryopreservation. Group 5, which was co-cultured with A-MSCs at p0, showed similar gene expression levels to group 2 at p1, despite the absence of co-culture with A-MSCs.
Compared to the fresh groups (groups 1 and 2), all cryopreserved groups (groups 3-6) displayed significantly elevated EPCAM expression, especially group 3. Notably, cryopreservation itself appeared to be the main factor driving this upregulation. Within the shared culture environment, group 3 showed a 3.6-fold increase compared to group 1 and group 6 exhibited a 3.4-fold increase compared to group 2. After cryopreserving HOs cultured alone at p0, co-culturing them with A-MSCs (group 4) at p1 resulted in a reduced expression of EPCAM compared to HOs cultured alone without A-MSCs (group 3). However, after cryopreservation of HOs co-cultured with A-MSCs at p0, co-culture with or without A-MSCs did not significantly affect the expression level of EPCAM.
Fig. 5B presents a microscopic image of HOs at p1 after cryopreservation and Matrigel removal. Strikingly, co-culturing with A-MSCs led to a significant increase in healthy HOs, suggesting their potential to promote tissue regeneration.
The most notable finding was that while cryopreservation had minimal effects on ALB and CYP3A29 expression in HO, it significantly increased EPCAM expression. Additionally, post-cryopreservation co-culture with A-MSCs led to a slight increase in ALB expression and a slight decrease in CYP3A29 compared to the non-co-cultured group.
Discussion
The cryopreservation of porcine HOs for a year was explored with the goal of facilitating extended storage and subsequent application. Our results revealed the remarkable regenerative potential of HOs, surpassing that of cryopreserved PHs. Additionally, crucial hepatic functions, such as albumin synthesis and CYP3A29 activity, persisted after cryopreservation, confirming the effectiveness of this method for prolonged storage and future utilization.
Liver organoids, derived from pluripotent stem cells (PSCs) and cholangiocytes, are renowned for their potent regenerative abilities. However, these organoids exhibit reduced liver function compared to mature livers when cultured in the laboratory, as demonstrated by various studies [12,13]. To address this issue, Hans Clevers’ team successfully engineered HOs using hepatocytes, effectively preserving liver-like functions. Despite their initial success, they reported a growth delay after 2 to 3 months of in vitro culture. In our independent research, we achieved a significant advancement by developing functional porcine HOs. Our results indicate that their functional lifespan could be extended to over 4 months through co-culturing with A-MSCs [8].
Contrary to prior reports that described prolonged proliferation of liver organoids derived from cholangiocytes or PSCs, our study confirmed a relatively restricted in vitro culture period for HOs. This observation suggests potential variability in the in vitro lifespan of organoids based on cellular origin, emphasizing the substantial influence of cell type on the longevity of cultured organoids. To leverage the utility of functionally superior HOs as models for in vitro hepatotoxicity assessments, alternative methods such as organoid freezing may be employed to extend the lifespan of additional organoids.
We cryopreserved fragmented HOs with 10% dimethyl sulfoxide for a year. After thawing, the fragments retained the ability to form HOs, albeit reduced compared to the fresh HOs. Our results are consistent with previous studies reporting that the cell viability of PSC-liver organoids and intestinal organoids decreases after thawing [13,14]. Building on prior evidence [8,15], our study reveals that co-culture with A-MSCs significantly boosts the regenerative capacity of cryopreserved HO fragments. We analyzed gene activity to investigate the mechanism, and we discovered that co-culturing fresh HOs with A-MSCs reduces the activity of apoptosis and cell cycle genes [16] such as TP53, P21, and CASP8, thereby improving organoid robustness. After thawing the fragmented HOs, the gene expression in the HOs depended on pre-freezing culture condition. Co-culture with A-MSCs before freezing and thawing makes HOs more resilient, preventing reductions in gene activity caused by the freezing and thawing process. This enhancement appears to be driven by factors in co-cultured A-MSCs that promote cell-cell adhesion and extracellular matrix synthesis in hepatocytes. Additionally, MSC-derived extracellular vesicles significantly support hepatocyte survival by activating anti-apoptotic pathways and inhibiting pro-apoptotic mechanisms, primarily through the suppression of tumor necrosis factor-α, interleukin-1β, and collagen-1α production [17,18]. These findings demonstrate that HOs generated through initial co-culture with MSCs can form functional organoids and retain their potential even after cryopreservation.
The impact of cryopreservation on HO functionality and regenerative capacity was assessed by analyzing gene expression levels in organoids regenerated after thawing. The expression of genes related to the liver’s primary function, such as ALB, remained relatively stable, with fold changes ranging from 1.3 to 1.5 across both frozen and co-culture conditions. This stability suggests that freezing and co-culture minimally impact albumin production. However, a consistent decrease in the expression of CYP3A29, a critical drug-metabolizing enzyme [19], was observed in co-cultured organoids, irrespective of their freezing status. This decrease may be attributed to the presence of residual A-MSCs during RNA extraction, which potentially influenced the measured expression levels. EPCAM, a key liver stem cell gene, regulates the growth, proliferation, and differentiation of hepatic progenitor cells [20]. Its increase in HOs after thawing suggests the survival of EPCAM-expressing cells and the increased regenerative capacity of organoids. This finding implies that EPCAM-expressing cells resist freezing stress and contribute to the organoids’ regenerative potential. Both CYP3A29 and EPCAM expression in organoids were influenced by the pre-freezing culture conditions, underscoring the importance of optimizing protocols before freezing to maintain specific functional characteristics within the organoids.
In our preceding research, we successfully reported the development of proliferative porcine liver organoids based on a three-dimensional Matrigel platform. To extend their potential lifespan further, we proposed cryopreservation. Subsequent investigations demonstrated not only these organoids’ ability to be preserved for extended periods but also their maintained functionality. Additionally, co-culturing with A-MSCs was found to increase the resilience of the organoids post-freezing. These findings suggest the applicability of our in vitro model for toxicity testing in various animal species, providing the advantage of readily available models for testing when needed.
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