Maintenance of hepatocyte functions in coculture with hepatic stellate cells

Hepatic stellate cells (HSCs) are a type of nonparenchymal liver cells (NPCs) and are present in the perisinusoidal space of Disse. Hepatocytes were cocultured with HSCs isolated from the NPC fraction with the aim of maintaining differentiated liver functions in vitro. Hepatocytes inoculated directly onto the HSC layer (Co-mix) exhibited lower activity of albumin secretion and higher DNA synthesis activity than hepatocytes of the monoculture control. On the contrary, hepatocytes cocultured with HSCs but separated by a semipermeable membrane (Co-sep) maintained the activities of albumin secretion and urea synthesis. The soluble factor(s) secreted from HSCs had the maintenance effect. Subcultured HSCs were activated to myofibroblast-like cells (MFBs) and decreased the maintenance effect on hepatocyte function. However, the MFBs were found to resume the ability to maintain the hepatocyte function by cultivation on type I collagen. The coculture of hepatocytes and HSCS/MFB could be applied to the development of bioartificial liver support system and liver regenerative medicine.     

表达人肝再生增强因子肝细胞系生物人工肝治疗安全性的动物实验初步研究

目的：评价表达人肝再生增强因子基因的HepG2细胞系的细胞培养上清及细胞裂解物的小鼠急性毒性和近期致瘤性。方法：SPF级昆明种小鼠18只,随机分为空白对照组、细胞培养上清组、细胞裂解物组,每组小鼠各6只,腹腔分别接种空白培养液、细胞培养上清、细胞裂解物0．5ml。连续14天,每天观察记录动物毒性反应,14d后宰杀小鼠,取血测血生化指标。及观察病理改变。结果：各组小鼠均存活。除对照组1例小鼠,细胞培养上清组1例小鼠,细胞裂解物组2例小鼠次日活动稍减少外,均未见异常反应。血液生化检测ALT、AST、AFP、TBIL无明显异常,且各组间无差别。普通光镜下各组动物肝脏病理切片染色均未见明显异常。结论：目的细胞系细胞培养上清、细胞裂解物对实验用昆明小鼠无明确毒副作用及短期致瘤性。可能提供一种安全的可用于生物人工肝新的细胞来源。     

Effect of patient, operative and isolation factors on subsequent yield and viability of human hepatocytes for research use

It is widely accepted that the model of choice for pharmacotoxicological studies are human hepatocytes. There is therefore a demand for these cells, but quality must be maintained for their widespread use. We present a retrospective review of the isolation of hepatocytes from both surgically resected tissue and livers rejected for transplantation, and evaluated patient, operative and isolation variables to ascertain which may affect the viability and yield of cells. Seven clinically rejected whole livers and 60 surgically resected specimens (from two distinct operating centres) were isolated. For surgically resected tissue we found that decreasing age, securing the perfusing cannulae with suture rather than reforming Glissons capsule with glue and steatotic livers improved viability. No significant correlation could be found with pre-operative blood results, disease, type of operation, presence or absence of Pringle manoeuvre, weight of tissue isolated, time of digestion with collagenase and cold ischaemic time. There was a reduction in mean yield and viability when hepatocyte isolations were performed in livers rejected for transplant, compared to surgically resected tissue although this did not reach significance. Human hepatocytes can be successfully and consistently isolated from surgically resected tissue and appear to be superior to those isolated from rejected for transplant livers. From our study, there are few parameters that significantly affect the quality of isolated hepatocytes, which increases the possible pool of tissue that hepatocytes can be isolated from.     

Development of hepatocyte spheroids immobilization technique using alternative encapsulation method

Primary hepatocytes of small animals such as rat and rabbit were often used for the study of extracorporeal liver support systems. Freshly isolated rat hepatocytes form spheroids within two days when cultivated as suspension in spinner vessels. These spheroids showed enhanced liver specific functions and more differentiated morphology compared to hepatocytes cultured as monolayers. However, shear stress caused by continuous agitation deteriorated spheroids gradually. In this work we immobilized spheroids to prolong liver specific activities. First, hepatocyte spheroids were suspended in collagen solution containing calcium chloride and then dropped into alginate solution. A thin layer of calcium alginate was formed around the droplet and then was removed after the inner collagen was gelled by treatment of sodium citrate buffer. Spheroids embedded in collagen-gel bead maintained liver specific functions such as albumin secretion rate longer than hepatocyte spheroids exposed to shear stress. Therefore, we suggest that this immobilization technique may offer an effective long-term hepatocyte cultivation and facilitate the development of a bioartificial liver support device.     

A scale down process for the development of large volume cryopreservation

The process of ice formation and propagation during cryopreservation impacts on the post-thaw outcome for a sample. Two processes, either network solidification or progressive solidification, can dominate the water–ice phase transition with network solidification typically present in small sample cryo-straws or cryo-vials. Progressive solidification is more often observed in larger volumes or environmental freezing. These different ice phase progressions could have a significant impact on cryopreservation in scale-up and larger volume cryo-banking protocols necessitating their study when considering cell therapy applications.This study determines the impact of these different processes on alginate encapsulated liver spheroids (ELS) as a model system during cryopreservation, and develops a method to replicate these differences in an economical manner.It was found in the current studies that progressive solidification resulted in fewer, but proportionally more viable cells 24 h post-thaw compared with network solidification. The differences between the groups diminished at later time points post-thaw as cells recovered the ability to undertake cell division, with no statistically significant differences seen by either 48 h or 72 h in recovery cultures.Thus progressive solidification itself should not prove a significant hurdle in the search for successful cryopreservation in large volumes. However, some small but significant differences were noted in total viable cell recoveries and functional assessments between samples cooled with either progressive or network solidification, and these require further investigation.     

Liquidus Tracking: Large scale preservation of encapsulated 3-D cell cultures using a vitrification machine

Currently, cryo-banking of multicellular structures such as organoids, especially in large volumes at clinical scale >1 L, remains elusive for reasons such as insufficient dehydration and cryoprotectant additive (CPA¹) penetration, slow cooling and warming rates and devitrification processes. Here we introduce the concept of Liquidus Tracking (LT) using a semi-automated process for liquid volumes of up to 450 ml including 130 ml of alginate encapsulated liver cells (AELC) that archived controlled and reversible vitrification with minimized toxicity.First a CPA solution with optimal properties for LT was developed by employing different small scale test systems. Combining sugars such as glucose and raffinose with Me₂SO improved post-exposure (at +0.5 °C) viabilities from 6% ±3.6 for Me₂SO alone up to 58% ±6.1 and 65% ±14.2 respectively (p < 0.01). Other permeating CPAs (e.g. ethylene glycol, propylene glycol, methanol) were investigated as partial replacements for Me₂SO. A mixture of Me₂SO, ethylene glycol and glucose (ratio 4:2:1– termed LTdeg) supported glass-forming tendencies with appropriate low viscosities and toxicities required for LT. When running the full LT process, using Me₂SO alone, no viable cells were recovered; using LTdeg, viable recoveries were improved to 40% ±8 (p<0.001%). Further refinements of improved mixing technique further improved recovery after LT. Recoveries of specific liver cell functions such as synthesis of albumin and alpha-fetoprotein (AFP) were retained in post thaw cultures.In summary: By developing a low-toxicity CPA solution of low viscosity (LTdeg) suitable for LT and by improving the stirring system, post-warming viability of AELC of up to 90% and a AFP secretion of 89% were reached. Results show that it may be possible to develop LT as a suitable cryogenic preservation process for different cell therapy products at large scale.     

Study of porcine hepatocyte-entrapped bioartificial liver in surgery-induced fulminant hepatic failure rabbits

ICP is an important cause of mortality in fulminant hepatic failure. The aim of this study was to prolonged survival in surgery-induced FHF rabbit with BAL. Four hours after induction of FHF, rabbits were connected to a 4-h whole blood perfusion through the BAL containing 1.2 × 10⁹ fresh isolated porcine hepatocytes (estimated 30% of liver volume of rabbit) via an arterial-venous shunt. Survival times in control group, sham-BAL group and BAL group were14.4 ± 4.4 h, 12.6 ± 2.6 h and 22.6 ± 5.2 h, respectively (BAL vs. control, p = 0.026; BAL vs. sham-BAL, p = 0.006). BAL group remain ICP level less than 10 mmHg for near 13 h after FHF induction while 19 and 15 mmHg were observed in control and sham-BAL groups, respectively. BAL containing porcine hepatocytes significantly prolonged survival time by delayed increase of intracranial pressure compared with the control and sham-BAL groups.