A method for constructing an in-vitro liver regeneration model and application thereof

Abstract

The application belongs to the technical field of cell biology and biomedical engineering, and particularly relates to a method for constructing an in-vitro liver regeneration model and application. The method for constructing the in-vitro liver regeneration model comprises the following steps: placing adult liver cells in a cell culture system, applying a fluid static pressure of 10 kPa higher than the atmospheric pressure to the culture system for pressure culture, and obtaining a liver cell population as an in-vitro model of liver regeneration. The in-vitro model constructed by the method of the application not only realizes cell proliferation, but also has multi-dimensional characteristics related to liver regeneration, and reappears the liver cell state of in-vivo liver regeneration. The in-vitro model of liver regeneration constructed by the method of the application can be used for evaluating candidate drugs related to liver regeneration, and for researching the mechanism of liver regeneration after liver resection.

Description

Technical Field

[0001] This invention belongs to the field of cell biology and biomedical engineering technology, specifically relating to a method for constructing an in vitro liver regeneration model and its application, particularly a method and its application for promoting liver cell proliferation and reproducing the liver regeneration state in vitro by applying hydrostatic pressure higher than atmospheric pressure to liver cells for pressurized culture. Background Technology

[0002] The liver is one of the few solid organs in mammals with a strong regenerative capacity. In clinical situations such as partial hepatectomy, living donor liver transplantation, and acute liver failure, the body relies on the regeneration of the remaining liver tissue to restore liver function. Therefore, elucidating the initiation and regulation mechanisms of liver regeneration is of great significance to liver surgery, liver transplantation, and regenerative medicine.

[0003] Regarding the initiation signals of liver regeneration, the classic view holds that it is mainly driven by growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor (EGF), as well as cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Recent studies have further demonstrated that hemodynamics and mechanical signals are important early initiating factors for liver regeneration: after partial hepatectomy (PHx), portal vein blood, which originally flowed through the entire liver, now flows through the significantly reduced residual liver, causing a sudden increase in portal vein pressure and intrahepatic shear stress (reportedly, in rats, portal vein pressure increased from approximately 8 mmHg to approximately 16 mmHg after 70% hepatectomy). These increased mechanical cues are sensed by intrahepatic cells through mechanotransduction pathways such as the Piezo1 mechanosensitive ion channel, the YAP / Hippo pathway, and KLF2-eNOS, thereby prompting hepatocytes to re-enter the cell cycle and initiate regeneration (Wu Y, et al. Biomechanics in liver regeneration after partial hepatectomy. Front Bioeng Biotechnol. 2023;11:1165651. PMID: 37214300).

[0004] Regarding the in vitro reproduction of the aforementioned mechanical signals, the closest existing technology to this invention is: applying mechanical force directly to hepatocytes—including interstitial flow during ex vivo liver perfusion and fluid shear stress in an in vitro “liver sinusoidal chip”—can initiate hepatocyte proliferation in a duration- and amplitude-dependent manner via the β1 integrin / FAK / YAP-Hippo pathway (Wang L et al., Direct mechanical exposure initiates hepatocyte proliferation. JHEP Rep. 2023;5(12):100905. PMID: 37920845).

[0005] However, the above-mentioned existing technologies have the following shortcomings: (1) They rely on fluid flow or shear stress and require complex devices such as ex vivo organ perfusion systems or microfluidic blood sinus chips to achieve the operation. They are cumbersome, have low throughput, poor batch consistency, and are difficult to standardize and promote; (2) Existing in vitro culture of hepatocytes is mostly carried out under normal pressure and static conditions, which cannot easily reproduce the microenvironment of the residual liver after liver resection with increased mechanical load; (3) Existing technologies have not yet established an "in vitro liver regeneration model" that is easy to operate, can be standardized, and has been verified at the transcriptome level to correspond to the state of regenerated liver parenchymal cells after liver resection in vivo. Therefore, it is difficult to use it as a stable platform for studying the mechanism of postoperative liver regeneration.

[0006] Therefore, there is an urgent need in this field for a simple, controllable, and standardized method that can reproduce the biomechanical activation state of liver regeneration in vitro, promote hepatocyte proliferation, and reliably simulate the liver regeneration process after liver resection. Summary of the Invention

[0007] The problem this invention aims to solve is that existing technologies struggle to easily and systematically reproduce the biomechanical microenvironment after hepatectomy and simulate liver regeneration in vitro. To address this, this invention employs pressurized culture of hepatocytes under hydrostatic pressure exceeding atmospheric pressure to promote hepatocyte proliferation, thereby reproducing the molecular state of liver regeneration after hepatectomy in vitro. This provides a simple, controllable, and standardized in vitro model for studying the mechanism of liver regeneration after hepatectomy.

[0008] The principle of this invention lies in the fact that the increased mechanical load (blood flow, pressure, and shear stress) experienced by the residual liver after hepatectomy is a crucial signal for initiating liver regeneration. This invention recreates this "mechanical activation" state in vitro using controllable hydrostatic pressure (i.e., applying a continuous positive gas pressure approximately 10 kPa higher than atmospheric pressure above the cell culture system, which is conducted through the gas phase to the culture system, subjecting hepatocytes to increased static pressure), thereby triggering hepatocyte proliferation signals and simulating liver regeneration. It should be noted that this method aims to qualitatively reproduce the increased postoperative mechanical load, rather than precisely replicating the specific value of portal vein pressure.

[0009] The technical solution adopted in this invention is:

[0010] A method for constructing an in vitro liver regeneration model includes: placing adult hepatocytes in a cell culture system, applying a hydrostatic pressure 10 kPa higher than atmospheric pressure (i.e., approximately 10 kPa gauge pressure and approximately 110 kPa absolute pressure) to the culture system for pressurized culture, and using the resulting hepatocyte population as an in vitro model for liver regeneration.

[0011] In this invention, it is preferred that the culture system be subjected to a hydrostatic pressure higher than atmospheric pressure for 12-48 hours of pressurized culture. More preferably, the pressurized culture is carried out for 24 hours.

[0012] In this invention, the conditions of the culture system are the same as those of conventional atmospheric pressure culture, except for the pressure. For example, the gas and temperature environment is: CO2 volume fraction of about 5%, O2 volume fraction of about 20%, and temperature of about 37°C, in order to maintain the pH of the culture medium and the normal survival conditions of hepatocytes.

[0013] In this invention, the adult hepatocytes are preferably primary hepatocytes, more preferably mouse primary hepatocytes; they can also be rat hepatocyte lines such as BRL-3A. Primary hepatocytes are used as the main model, and hepatocyte lines are used to verify the universality of the proliferative effect in hepatocyte-derived cells.

[0014] Through processes such as control setup, proliferation determination, and model verification, this invention demonstrates that the model constructed by the method of this invention does not simply increase the number of cells, but rather constructs an in vitro model with multidimensional features related to liver regeneration, thus reproducing the hepatocyte state of in vivo liver regeneration.

[0015] Control group: A normal pressure culture control group is preferably set up. Except that no hydrostatic pressure is applied, the other culture conditions are the same as those of the pressurized group to determine the promoting effect of pressurization on proliferation.

[0016] Proliferation determination: Preferred methods include the expression of proliferation-related proteins PCNA and Cyclin D1, the mRNA expression of proliferation-related genes Ccna2, Ccnb1, Ccnd1, Ccne1, Pcna, and Mki67, and the positive rate of EdU incorporation.

[0017] Model validation: Transcriptomic analysis was performed on hepatocytes from the pressurized group and the control group, and the results were projected / mapped onto the transcriptomic atlas of regenerated hepatocytes after hepatectomy. When the pressurized group fell into the regeneration activation state region, it indicated that the model reproduced the hepatocyte state of in vivo liver regeneration at the transcriptomic level.

[0018] This invention also provides an application of the aforementioned in vitro liver regeneration model construction method. Specifically, an in vitro simulated liver regeneration model is constructed using the method of this invention for the evaluation of candidate drugs related to liver regeneration. Adult hepatocytes are divided into a stress model group and a stress model plus candidate drug group. After culture, EdU positivity rate, PCNA, Cyclin D1, and liver regeneration-like gene scores are detected to evaluate the promoting or inhibiting effects of candidate drugs on hepatocyte regeneration-like state.

[0019] The present invention also provides the application of hydrostatic pressure stimulation above atmospheric pressure in constructing an in vitro liver regeneration model or in promoting hepatocyte proliferation in vitro; the in vitro liver regeneration model can be used to study the mechanism of liver regeneration after hepatectomy.

[0020] The beneficial effects of this invention are reflected in:

[0021] This invention utilizes a hydrostatic pressure approximately 10 kPa higher than atmospheric pressure to pressurize and culture hepatocytes, significantly promoting proliferation in both primary mouse hepatocytes and rat BRL-3A cells (fold increases are approximate values ​​based on quantitative results; the original data should be considered the definitive measure).

[0022] (1) Upregulation of proliferation-related protein expression: Compared with the normobaric control, the PCNA / ACTIN ratio of mouse primary hepatocytes increased from about 1.0 to about 2.1 and the Cyclin D1 / ACTIN ratio increased from about 1.0 to about 1.7 after pressure culture (p<0.05); the Cyclin D1 / ACTIN ratio of BRL-3A cells increased from about 1.0 to about 2.9 (p<0.001) and the PCNA / ACTIN ratio increased from about 1.0 to about 1.5 (p<0.05).

[0023] (2) Upregulation of proliferation-related gene mRNA expression: After pressure culture, Ccna2, Ccnb1, Pcna, and Mki67 in mouse primary hepatocytes were significantly upregulated (approximately 2.2, 2.6, 1.7, and 3.2 times that of the control, respectively), while Ccnd1 and Ccne1 showed no significant changes; in BRL-3A cells, the mRNA of Ccna2, Ccnb1, Ccnd1, Ccne1, Pcna, and Mki67 was all significantly upregulated (p<0.0001).

[0024] (3) EdU incorporation detection showed that the proportion of proliferating (EdU positive) cells in the pressurized group was significantly higher than that in the normobaric control, providing direct evidence that pressurization promotes hepatocyte proliferation.

[0025] (4) Transcriptome projection showed that the transcriptome features of hepatocytes cultured under pressure fell into the activated state region of regenerated hepatocytes after liver resection, while the normobaric control fell into the relatively resting state region, indicating that this method reproduced the state of hepatocytes regenerated in vivo at the transcriptome level.

[0026] In summary, the method of this invention is simple to operate and the conditions are controllable. It does not require complex devices such as in vitro perfusion or microfluidic sinusoidal chips, and can stably promote proliferation in liver-derived cells and reproduce the molecular characteristics of liver regeneration. It provides a standardized in vitro model for the study of the mechanism of liver regeneration after hepatectomy. Attached Figure Description

[0027] Figure 1 mRNA expression of proliferation-related genes Ccna2, Ccnb1, Ccnd1, Ccne1, Pcna, and Mki67 in mouse primary hepatocytes after culture under control and pressure (qRT-PCR). * indicates p<0.05, **** indicates p<0.0001.

[0028] Figure 2 Western blot bands of PCNA and CCND1 proteins in mouse primary hepatocytes after normal and pressurized culture, with ACTIN as an internal control.

[0029] Figure 3 : Figure 2 Quantification of PCNA and CCND1 protein expression relative to ACTIN. * indicates p<0.05.

[0030] Figure 4 Representative images of EdU incorporation detection in primary mouse hepatocytes after normal and pressurized culture (EdU, DAPI, and combined images); scale bar = 100 μm.

[0031] Figure 5 : Figure 4Quantification of the proportion of EdU-positive cells in the middle. ** indicates p<0.01.

[0032] Figure 6 Volcano plot of differentially expressed genes between the pressure group (Pressure) and the normobaric control group (Control). The horizontal axis represents the log2 fold change (Pressure / Control), and the vertical axis represents the -log10 corrected P-value. Orange, blue, and gray dots represent genes that are significantly upregulated, significantly downregulated, and not significantly different, respectively. The dashed line represents the differential screening threshold. Upregulated genes include cell cycle and proliferation-related genes such as Top2a, Ccnb1, Tdo2, Mki67, Ccna2, Ube2c, and Pcna; downregulated genes include genes related to hepatocyte maturation and metabolic function such as Ttr, Apoa1, Cyp2e1, Cyp1a2, Fapp1, Pck1, and Hnf4a.

[0033] Figure 7 Gene set enrichment analysis (GSEA) results based on Hallmark gene sets from primary hepatocytes of mice in the pressurized and normobaric groups (top 20 pathways by enrichment level). The horizontal axis represents the normalized enrichment score (NES); orange indicates positive enrichment in the pressurized group, blue indicates negative enrichment, and the size of the dot indicates the degree of enrichment.

[0034] Figure 8 The results of projecting the transcriptomes of mouse primary hepatocytes cultured under pressure and normal pressure onto a reference atlas of regenerated hepatocytes after hepatectomy. The gray and orange dots in the background represent quiescent hepatocytes and proliferating hepatocytes, respectively; the blue circles and orange squares represent samples cultured under normal pressure and pressure, respectively; and the black arrows indicate culture-induced shift of the transcriptome state caused by pressure culture.

[0035] Figure 9 mRNA expression of proliferation-related genes Ccna2, Ccnb1, Ccnd1, Ccne1, Pcna, and Mki67 in rat BRL-3A cells after culture under control and pressure conditions (qRT-PCR). **** indicates p<0.0001.

[0036] Figure 10 Western blot bands of PCNA and CCND1 proteins in rat BRL-3A cells after normal and pressurized culture, with ACTIN as an internal control.

[0037] Figure 11 : Figure 10Quantification of PCNA and CCND1 protein expression relative to ACTIN. * indicates p<0.05, *** indicates p<0.001. Detailed Implementation

[0038] In the following examples, pressurized cultures were conducted in a pressurized culture system that provided approximately 5% CO2, 20% O2, and a temperature of approximately 37°C, subjecting the culture system to a hydrostatic pressure approximately 10 kPa higher than atmospheric pressure. The normostatic control group was placed under the same gas and temperature conditions as the conventional culture system, without the aforementioned additional hydrostatic pressure. Cells in each group were analyzed approximately 24 hours after pressurized / normative-pressure culture.

[0039] Example 1: Pressure culture of primary mouse hepatocytes promotes proliferation and recreates liver regeneration at the transcriptome level.

[0040] (a) Proliferation detection

[0041] Freshly isolated primary mouse hepatocytes were seeded into culture plates using standard methods and then randomly divided into a control group (atmospheric pressure) and a pressure group (pressure). The pressure group was placed in the aforementioned pressure culture system and cultured under a hydrostatic pressure approximately 10 kPa higher than atmospheric pressure for about 24 hours; the control group was cultured at atmospheric pressure. After culture, samples were collected and analyzed by Western blot, qRT-PCR, and EdU.

[0042] Results: Compared with the control group, the mRNA levels of proliferation-related genes Ccna2, Ccnb1, Pcna, and Mki67 in primary hepatocytes of mice in the pressurized group were significantly upregulated (approximately 2.2, 2.6, 1.7, and 3.2 times that of the control group, respectively, p<0.05), while no significant differences were observed in Ccnd1 and Ccne1 (see...). Figure 1 The expression of proliferation-related proteins PCNA and Cyclin D1 was significantly upregulated (PCNA / ACTIN increased from approximately 1.0 to 2.1, p < 0.05, Cyclin D1 / ACTIN increased from approximately 1.0 to 1.7-fold; Western blot bands are shown). Figure 2 Quantitative data Figure 3 The proportion of EdU-incorporated positive cells increased significantly (see representative figure). Figure 4 Quantitative data Figure 5 The above results indicate that hydrostatic pressure approximately 10 kPa higher than atmospheric pressure can significantly promote the proliferation of primary mouse hepatocytes.

[0043] (II) Transcriptome Validation

[0044] To further verify whether the in vitro model constructed by the above-mentioned pressurized culture can reproduce the in vivo liver regeneration state, transcriptome sequencing was performed on primary mouse hepatocytes from the pressurized group and the normobaric group in this embodiment (I), and differential expression analysis and gene set enrichment analysis (GSEA) were performed. At the same time, using the transcriptome data of liver parenchymal cells regenerating after hepatectomy (partial hepatectomy model) (Xu J, Guo P, Hao S, et al., A spatiotemporal atlas of mouse liver homeostasis and regeneration, Nature Genetics, 2024, 56:953–969, DOI: 10.1038 / s41588-024-01709-7; Publication Data Number CNP0002310.) as a reference, the samples from the pressurized group and the normobaric group were projected / mapped onto this reference atlas.

[0045] Results: (1) Differential expression analysis showed that, compared with the normobaric control group, the genes significantly upregulated in the pressurized group were enriched in genes related to cell cycle and proliferation (such as Top2a, Ccnb1, Ccna2, Mki67, Pcna, Ube2c, etc.), while the genes significantly downregulated were mainly genes related to hepatocyte maturation and metabolic function (such as Cyp1a2, Hnf4a, Pck1, Apoa1, etc.) (see Figure 6 (2) GSEA showed that the pressurized group was positively enriched in proliferation-related pathways such as the G2 / M checkpoint, E2F target genes, mitotic spindle, and Myc target genes (see [link to GSEA]). Figure 7 (3) After projecting the samples onto the reference atlas of regenerated hepatocytes after hepatectomy, the pressurized group fell into the regeneration activation state region, while the normobaric control group fell into the relatively resting state region (see Figure 8 The above results consistently demonstrate that the pressure culture method of the present invention not only promotes the proliferation of primary mouse hepatocytes, but also reproduces the state of hepatocytes in liver regeneration after hepatectomy at the transcriptomic level, thus proving that it can serve as an in vitro model for simulating liver regeneration after hepatectomy and for related mechanism studies.

[0046] Example 2: Pressure culture of rat BRL-3A cells promotes proliferation

[0047] Normal rat hepatocytes (BRL-3A) were used, and the grouping and culture methods (pressurized / normal pressure) were the same as in Example 1 (I). The cells were cultured under pressure for approximately 24 hours. Western blot and qRT-PCR were performed after the culture period.

[0048] Results: Compared with the control group, the expression of Cyclin D1 and PCNA proteins in BRL-3A cells of the pressurized group was significantly upregulated (Cyclin D1 / ACTIN approximately 1.0 → 2.9, p < 0.001; PCNA / ACTIN approximately 1.0 → 1.5, p < 0.05; Western blot bands were observed). Figure 10 Quantitative data Figure 11 The mRNA levels of proliferation-related genes Ccna2, Ccnb1, Ccnd1, Ccne1, Pcna, and Mki67 were all significantly upregulated (p < 0.0001, see below). Figure 9 The results showed that pressure culture also significantly promoted the proliferation of the rat hepatocyte line BRL-3A, indicating that the effect of pressure-induced proliferation is universal in hepatocyte-derived cells.

[0049] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for constructing an in vitro liver regeneration model, comprising culturing primary mouse hepatocytes in a cell culture system, characterized in that, The culture system was subjected to a hydrostatic pressure 10 kPa higher than atmospheric pressure for 12-48 hours, and the resulting hepatocyte population was used as an in vitro model for liver regeneration.

2. The method for constructing an in vitro liver regeneration model according to claim 1, characterized in that, Incubate under pressure for 24 hours.

3. The method for constructing an in vitro liver regeneration model according to claim 2, characterized in that, The gas and temperature environment of the culture system was: 5% CO2 volume fraction, 20% O2 volume fraction, and 37℃.

4. The application of the method for constructing an in vitro liver regeneration model according to any one of claims 1-3, characterized in that, An in vitro simulated liver regeneration model is constructed using the in vitro liver regeneration model construction method described in any one of claims 1-3, and is used for the evaluation of candidate drugs related to liver regeneration.

5. The application of the in vitro liver regeneration model construction method according to any one of claims 1-3 in studying the liver regeneration mechanism after hepatectomy.

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