In vitro safety and efficacy evaluation of a novel hybrid bioartificial liver system with simulated liver failure serum

Abstract

Background:

Acute liver failure (ALF), which can potentially be treated with an artificial liver, is a fatal condition. The purpose of this study was to evaluate the safety and effectiveness of a novel hybrid bioartificial liver system (NHBLS) using simulated liver failure serum in vitro.

Methods:

The bioreactor in experimental group was cultivated with primary porcine hepatocytes, whereas in control group was not. Next, the simulated liver failure serum was treated using the NHBLS for 10 h. Changes in albumin (ALB), total bilirubin (TBIL), ammonia (Amm), total bile acid (TBA), creatinine (Cr), and blood urea nitrogen (BUN) were measured before treatment (0 h) and every 2 h during treatment. In addition, changes in NHBLS pressures, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lidocaine metabolism were also recorded.

Results:

The NHBLS worked steadily without unexpected occurrences during the treatment. Blood culture showed no bacterial growth after 7 days, and the endotoxin level was less than 0.5 EU. The TBIL, TBA, Cr, and BUN levels in both groups were markedly lower than those at 0 h (p < 0.05). The Amm level in experimental group was significantly lower than that in control group (p < 0.05). NHBLS pressures were also stable, and the hepatocytes in the bioreactor functioned well.

Conclusions:

The preparation method for the simulated liver failure serum was optimized successfully, and the safety and effectiveness of the NHBLS in vitro were verified. Furthermore, the NHBLS significantly reduced the levels of Amm which can lead to hepatic encephalopathy.

Graphical Abstract

Keywords

Bioartificial liver system simulated liver failure serum in vitro safety effectiveness

Introduction

Acute liver failure (ALF) is one of the most fatal complications worldwide.¹ Currently, there are no effective drugs for ALF treatment due to the complicated functioning of the liver;² liver transplantation remains the most effective treatment. However, donor liver shortages are a major problem. Therefore, it is necessary to develop an alternative method for treating ALF. The most promising method is the bioartificial liver system.³

An in vitro safety and effectiveness evaluation of an artificial liver system (ALS) is an important process which precedes in vivo and pre-clinical trial safety and effectiveness verification. Presently, a common evaluation method is to use discarded patient plasma obtained by clinical plasma exchange^4
–7 or plasma from animals with liver failure.⁸ However, because of individual differences, the related indicators of discarded plasma or animal serum are not uniform, and liver failure serum has a cytotoxic effect, which affects the results of in vitro function evaluation. A study⁹ has also utilized the Roswell Park Memorial Institute (RPMI) 1640 culture medium to evaluate the function of an ALS in vitro; however, this does not replicate the actual treatment in patients with liver failure.

Based on our previous research, we further optimized the preparation method of the simulated liver failure serum in this study to better simulate the serum status of patients with ALF and to evaluate the safety and effectiveness of the novel hybrid bioartificial liver system (NHBLS) in vitro. In addition, we conducted these investigations to build the foundation for follow-up safety and effectiveness evaluations during in vivo and clinical trial use of the NHBLS.

Methods

Animals

Three male Tibetan miniature pigs, aged 3–6 months and weighing 15–20 kg, were purchased from Dongguan Songshan Lake Pearl Laboratory Animal Technology Co., Ltd (License No. SCXK (Guangdong Province) 2017-0030). The protocol and procedures for the study were reviewed and approved by the Zhujiang Hospital Institutional Review Board. All experimental animals were quarantined and housed in special cages. All animals were fed a special diet, and drinking water was provided freely; animals were adaptively fed for 1 week and fasted for 12 h before the experiment.

Preparation of simulated liver failure serum

Based on our previous research, we optimized the preparation method for the simulated liver failure serum. The preparation process is shown in Supplemental Figure S1.

Experimental grouping

The study was divided into the following two groups: the experimental group (n = 3), where the NHBLS (with primary porcine hepatocytes) was treated for 10 h using the simulated liver failure serum and the control group (n = 3), where the treatment mode was the same as in the experimental group, except the bioreactor did not contain hepatocytes. The circulatory volumes in both groups were equal.

Construction of the NHBLS

A set of circulating pipelines (self-made), two membrane plasma separators (EC-20W and OP-02), four peristaltic pumps, one plasma perfusion adsorber (HA330-II), one bilirubin adsorber (BS-330), and one circulating perfusion contact-type bioreactor (The middle of the bioreactor was a fixed space with fiber scaffolds made of medical-grade polyester microfibers which providing a growth surface maximum of 4 m²) were connected to construct the NHBLS (Figure 1).

Figure 1.

Study design, bioreactor and the schematic diagram of NHBLS: (a) study design; (b) the schematic diagram of NHBLS (1–14: pinch valves; H1/H2: heaters; EC-20W/OP-02: plasma separator; BP: blood pump; FP: points slurry pump; CP: circulating pump; RP: returning pump; HA-330: hemoperfusion cartridge; BS-330: bilirubin absorber; Pa: pre-filter pressure detection port; Pv: venous pressure detection port; Pu: ultrafiltration pressure detection port); C: bioreactor (1: magnetic agitator; 2: external cavity; 3: internal cavity; 4: scaffolds; 5: holder; 6/7/8/12/13/14: ports; 9: temperature detection port; 10: sample port; 11: PH detection port).

Parameter setting of the NHBLS

The parameters of both groups were as follows: the flow rate of the blood pump, separating pump, circulating pump, and returning pump were 50, 10–15, 50, and 10–15 mL/min, respectively. The operating conditions of the peristaltic pumps in the system were checked, and all pipelines and bioreactors were pre-filled with normal saline. Gas was discharged into the pipelines and filters before treatment.

Primary porcine hepatocyte harvest

Details of the liquid preparation methods used for obtaining primary hepatocytes are described in previous literature.^10,11 The specific operation process is shown in Supplemental Figure S2. Cell viability was detected by trypan blue staining and cell counting using a cell counting plate.

Inoculation of primary porcine hepatocytes into a bioreactor

Primary porcine hepatocytes (150 mL) from the Tibetan miniature pigs were successfully inoculated into the circulating perfusion contact-type bioreactor through a liquid storage bottle. The temperature of the bioreactor was maintained at 36°C–37°C, the pH remained at 7.35, and the oxygenation rate was approximately 90%. Serum-free medium (600 mL) was added to the bioreactor after inoculation with primary hepatocytes. Following overnight culture, primary hepatocytes were adequately attached to the carriers in the bioreactor.

Simulated liver failure serum treated by the NHBLS

The prepared simulated liver failure serum was loaded into container G of the NHBLS and placed in a 37°C water bath. The serum was then separated at a flow rate of 10–20 mL/min in the experimental group. Next, the separated plasma was processed by a resin perfusion adsorber and bilirubin adsorber before entering the hepatocyte-containing bioreactor. The serum in the bioreactor was then returned to container G after secondary separation and heating, with a total treatment time of 10 h. The process of the control group was the same as that of the experimental group, except the bioreactor did not contain cells.

Observation indicators

Observation of whether the circulation pipelines exhibited leaks and bubbles occurred during the treatment. Changes in ALB, TBIL, Amm, TBA, Cr, and BUN were measured in container G before treatment (0 h) and every 2 h during treatment. At the same time, changes in arterial, venous, and transmembrane pressures were recorded. Changes in the ALT and AST levels in the bioreactor were determined during treatment. Hepatocytes were counted in the bioreactor before and after treatment in the experimental group. In addition, the return liquid was observed under a microscope to determine whether there were cells and cell fragments. Blood cultures and endotoxin tests were carried out in container G at 0 h and every 2 h during treatment.

Lidocaine metabolism experiment

At the start of the treatment, 30 mg (40 mg/L) lidocaine was added to the bioreactor of the experimental group, and samples were taken at 15 min and 1, 2, 4, 6, 8, and 10 h to test monoethylglycinexylidide (MEGX) levels.

Statistical analysis

Measurement data are expressed as mean ± standard deviation. Variance analysis of repeated measurement data was used for intergroup comparisons, while the t-test was used to compare data between the two groups. SPSS 21.0 statistical software (IBM Corp., Armonk, NY) was used to analyze the data. GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA) and AI software were used to create images. The difference was considered statistically significant for p < 0.05.

Results

Preparation of the simulated liver failure serum

By optimizing our previous research results, we successfully prepared the simulated liver failure serum in which the concentration of Amm was 665.7 ± 28.3 µmol/L, TBIL was 133.3 ± 3.8 µmol/L, BUN was 4.65 ± 0.28 mmol/L, Cr was 442 ± 9.6 µmol/L, ALB was 5.1 ± 0.2 g/L, and TBA was 124.2 ± 9.4 µmol/L.

Primary porcine hepatocyte harvest

We used a two-step perfusion method for collagenase in vitro to obtain primary porcine hepatocytes. The harvest process is detailed in Supplemental Figure S2. Hepatocyte yields were approximately 1.6 × 10⁹–4.5 × 10⁹, and morphological analysis revealed cells that were singular and spherical or aggregated (Figure 2(a) and (b)). The isolated hepatocytes were successfully inoculated into the bioreactor and cultured overnight, the number of cells adhered to the scaffold were about 1.5 × 10⁹–4.0 × 10⁹(Figure 2(c)). Cell viability was maintained at more than 80% in the bioreactor after treatment (Figure 2(d)).

Figure 2.

The conditions of primary hepatocytes under microscope (a, b, × 200); Cell activity in bioreactor before (c) and after (d) treatment (×100); The green means live cells, the red means dead cells.

Condition of the NHBLS

Unexpected occurrences (such as a sudden cease in functioning or an unexpected power failure) were not noted in the NHBLS during the 10 h of treatment. The peristaltic pumps and control system monitoring operated normally. Additionally, the pipeline of the NHBLS displayed sufficient sealing, with no leakage or explosions. The treatment process is illustrated in Supplemental Figure S3. No residual cell fragments were seen in the return fluid at any time point when viewed under the microscope.

Conditions of pollution

Blood cultures and endotoxin tests were performed at 0, 2, 4, 6, 8, and 10 h after treatment. The results showed that no bacterial growth was observed after 7 days of culture, and endotoxin levels were less than 0.5 EU.

Changes in the observation indicators

The levels of Amm, TBIL, TBA, Cr, BUN, and ALB were measured every 2 h during treatment. After 10 h of treatment, the Amm level in the experimental group decreased from 650.5 ± 12.0 µmol/L to 289.5 ± 16.3 µmol/L, and there were significant differences in Amm levels between the experimental and control groups (p < 0.05). In the control group, the Amm level decreased from 650.7 ± 33.2 µmol/L to 577.3 ± 8.0 µmol/L, but there was no significant decrease in the Amm levels from 2 h onwards. Except for the 0 h interval, the Amm levels at the other time points in the experimental group were significantly reduced compared to those of the control group (p < 0.05), as shown in Figure 3(a).

Figure 3.

The changes of observed indicators during treatment. (a) ammonia(Amm); (b) total bilirubin(TBIL); (c) total bile acid(TBA); (d) Creatinine(Cr); (e): blood urea nitrogen(BUN); (f) albumin(ALB). *: p < 0.05 when compared with experimental group; #: p < 0.05 when compared with 0 h.

In both groups, TBIL and TBA gradually decreased with prolonged treatment. After 10 h of treatment, TBIL decreased from 134 ± 4.6 µmol/L to 6.3 ± 3.5 µmol/L in the experimental group, and from 132 ± 2.0 µmol/L to 11.0 ± 3.0 µmol/L in the control group. There was no significant difference between the two groups at any time point (p > 0.05). After 10 h of treatment, TBA decreased from 131.3 ± 3.2 µmol/L to 7.3 ± 1.0 µmol/L in the experimental group, and from 127.2 ± 10.9 µmol/L to 7.9 ± 3.4 µmol/L in the control group. There was also no significant difference in TBIL levels between time points (p > 0.05), as shown in Figure 3(b) and (c).

In both groups, except for the first 2 h, the levels of Cr, BUN, and ALB did not decrease significantly. After 10 h of treatment, the level of Cr decreased from 446.0 ±12.0 µmol/L to 360.7 ± 5.7 µmol/L in the experimental group, and from 440.3 ± 8.5 µmol/L to 361.3 ± 3.1 µmol/L in the control group. There was no significant difference in Cr levels between the two groups at any time point (p > 0.05), as shown in Figure 3(d).

After 10 h of treatment, BUN decreased from 4.7 ± 0.2 mmol/L to 3.8 ± 0.2 mmol/L in the experimental group, and from 4.7 ± 0.3 mmol/L to 3.5 ± 0.1 mmol/L in the control group. The BUN in the experimental group was slightly higher than that in the control group at each time point, but there was no significant difference (p > 0.05), as shown in Figure 3(e).

After 10 h of treatment, ALB decreased from 5.2 ± 0.2 g/L to 4.0 ± 0.3 g/L in the experimental group, and from 5.1 ± 0.2 g/L to 3.1 ± 0.1 g/L in the control group. ALB in the experimental group was slightly higher than that in the control group at each time point, except at 4 h and 6 h, and the difference was statistically significant (p < 0.05), as shown in Figure 3(f).

Conditions associated with hepatocyte injury

In the experimental group, ALT and AST increased slowly as the treatment time increased. ALT increased from 51.5 ± 4.8 U/L to 85.1 ± 1.5 U/L, and AST increased from 66.5 ± 4.1 U/L to 103.2 ± 1.9 U/L, as shown in Figure 4. ALT and AST were not detected in the control group because the bioreactor did not contain primary porcine hepatocytes.

Figure 4.

The changes of ALT and AST during treatment. ALT: alanine transaminase; AST: aspartate transaminase.

Machine pressure changes

During treatment with the NHBLS, the arterial, venous, and transmembrane pressures fluctuated between 20–35, 10–20, and 5–10 mmHg, respectively. There was no sudden increase in arterial pressure caused by blockage of the plasma separator during the treatment or filter breakage caused by excessive transmembrane pressure. There was no significant difference between the experimental and control groups (p > 0.05), as shown in Figure 5.

Figure 5.

The changes of artificial liver machine pressures during treatment: (a) pre-filter pressure; (b) venous pressure, and (c) transmembrane pressure.

Lidocaine metabolism experiment

The MEGX values in the experimental group were 30.7 ± 1.6, 46.4 ± 2.1, 39.9 ± 1.5, 35.3 ± 2.6, 31.3 ± 2.4, 25.1 ± 1.3, and 19.7 ± 1.6 µg/L at 15 min and 1, 2, 4, 6, 8, and 10 h after treatment, respectively. Following treatment, the MEGX values peaked and then gradually decreased, as shown in Figure 6.

Figure 6.

The changes of MEGX in the experimental group during treatment. MEGX: monoethylglycinexylidide.

Discussion

ALF is a major clinical problem that needs to be solved urgently.¹ At present, the most common treatment methods for ALF are medical treatment and liver transplantation. Because the curative effects of medical treatment are limited, liver transplantation remains the most effective treatment method.¹² However, because of the shortage of donor liver sources, wide application of this treatment is limited. The ALS is a new method of ALF treatment, which can create time for liver cell regeneration in patients, recover reversible liver injury, and serve as a bridge to liver transplantation for irreversible ALF.¹³

There are three main types of artificial liver systems: non-bioartificial liver system (NBLS), bioartificial liver system (BLS), and hybrid bioartificial liver system (HBLS). An NBLS is a type of artificial liver technology that is widely used in clinical ALF treatment. It can remove toxic substances, supplement with biologically active substances, and partially replace the detoxification function of the liver. However, the curative effects of the NBLS are limited. A BLS is an in vitro bioreactor device based on cultured hepatocytes, and it has liver-specific functions, such as biotransformation. Bioreactors and seed cells are the most important core components of the BLS. Moreover, a BLS is the closest ALS to the normal liver, and it can completely replace the functions of liver detoxification, biosynthesis, secretion, and metabolism. An HBLS combines the advantages of an NBLS and a BLS, and it can sufficiently replace liver detoxification by performing biosynthetic, secretory, and metabolic functions.¹³ Presently, an HBLS offers a new research direction for artificial livers.

The core elements of an HBLS are the seed cell source and bioreactor. In theory, the ideal cell source for an HBLS is normal human hepatocytes, but these are difficult to obtain because few sources exist. Consequently, porcine hepatocytes are a more suitable option for the artificial liver because they have functions similar to human hepatocytes and are easier to obtain. However, the application of porcine hepatocytes is limited by the emergence of porcine endogenous retrovirus (PERV).¹⁴ With the recent development of gene editing technology, scientists^15,16 have been able to knock out the PERV gene in pigs so that it is no longer expressed, which has greatly improved the available artificial liver cell sources. It is generally believed that a hepatocyte number between 10⁹ and 10¹⁰ can meet the need for liver failure treatment.¹⁷ In this study, the in situ collagenase perfusion method was used to isolate and harvest primary porcine hepatocytes. The cell yields were between 1.6 × 10⁹ and 4.5 × 10⁹, and the metabolic activity of the cells was verified by a lidocaine metabolism experiment. Moreover, we used serum-free medium to culture the primary porcine hepatocytes and secondary plasma separation technology to avoid the possibility of an immune response.

At present, the hollow-fiber tubular bioreactor is most commonly used for culturing hepatocytes. However, cell adhesion in this type of reactor is poor and the cells easily aggregate, thus blocking the hollow fiber gap, which is not conducive to material exchange and oxygenation. In addition, the extracorporeal liver assist device (ELAD) using this bioreactor was unsuccessful in a phase III clinical trial,¹⁸ further proving the shortcomings of this bioreactor. Other bioreactor types include multi-layer radial-flow bioreactors,^19,20 microcapsule-suspension fluidized bed bioreactors,^21,22 and spheroid bioreactors,^23,24 most of which are in the experimental research stage.

In this study, we combined the advantages of non-biological and biological artificial livers using a circulating perfusion cell contact bioreactor, and constructed a new type of NHBLS, which has the characteristics of modularization, miniaturization, intelligence, high reliability, high sensitivity, and safety. A single sequential or mixed treatment mode can be achieved by using a set of pipelines and filters, which can reduce the economic burden on patients and operational difficulties.

We evaluated the safety and efficacy of the NHBLS in vitro using our in-house simulated liver failure serum. The results showed that the NHBLS could significantly reduce high levels of Amm, TBIL, TBA, and other indicators of ALF. During the treatment, the monitoring system and pressure was stable, the air-tight seal was sufficient, and there was no contamination or other abnormalities.

Although the safety and effectiveness of the NHBLS have been verified in vitro, further verification in vivo is needed because the simulated liver failure serum is a static and fixed model, while ALF in vivo is a more complex and dynamic process with indicators that increase with time.

In conclusion, we have demonstrated the safety and effectiveness of the NHBLS in vitro using simulated liver failure serum, which can effectively remove liver failure waste and reduce Amm levels, thus delaying the process of liver failure and benefiting liver cell regeneration. This study also laid a foundation for subsequent in vivo safety and validity verification in large animal experiments and clinical trials.

Supplemental Material

sj-pdf-1-jao-10.1177_03913988221091286 – Supplemental material for In vitro safety and efficacy evaluation of a novel hybrid bioartificial liver system with simulated liver failure serum

Supplemental material, sj-pdf-1-jao-10.1177_03913988221091286 for In vitro safety and efficacy evaluation of a novel hybrid bioartificial liver system with simulated liver failure serum by Lei Feng, Yi Wang, Shusong Liu, Guolin He, Lei Cai, Jiasheng Qin, Xiaoping Xu, Zesheng Jiang, Chenjie Zhou and Yi Gao in The International Journal of Artificial Organs

Footnotes

Authors’ contributions

Yi Gao, Chenjie Zhou and Lei Feng designed research; Lei Feng, Shusong Liu, Lei Cai and Guolin He performed research; Lei Feng, Yi Wang, Jiasheng Qin, Zesheng Jiang, Xiaoping Xu and Chenjie Zhou analyzed data; Lei Feng, Yi Wang and Chenjie Zhou wrote the manuscript; Yi Gao and Chenjie Zhou revised the manuscript.

Availability of data and material

Data will be made available on reasonable request.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was co-supported by National Key R&D Program of China (Grant No.2018YFC1106400), Guangdong Basic and Applied Basic Research Foundation (Grant No.2020A1515111111) and Beijing iGandan Foundation (Grant No. RGGJJ-2021-008).

Consent for publication

All authors have read the final manuscript and consented to publication.

ORCID iD

Yi Gao

Supplemental material

Supplemental material for this article is available online.

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