Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.     

Growth induction of rat primary hepatocytes using antisense oligonucleotides

We examined growth control of adult and fetal hepatocytes by regulating the expression of cell-cycle-related proteins using antisense S-oligonucleotides to tumor suppressors retinoblastoma (RB) protein and p53, and cyclin-dependent kinase (CDK) inhibitors p21 and p27. The protein expression in both adult and fetal hepatocytes was significantly suppressed with the addition of corresponding antisense oligonucleotides at a concentration of 2.5 microM. For the evaluation of growth, 3H-thymidine incorporation and DNA content were measured and the results demonstrated that all the antisense oligonucleotides had growth-promoting effects and the promoting potential was equivalent or slightly greater than that with the addition of hepatocyte growth factor (HGF) (10 ng/ml). The growth-promoting effect of the antisense oligonucleotides was enhanced by HGF in both adult and fetal hepatocyte cultures, and the effects on hepatocyte growth were also observed in a suspension culture.     

Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH)

We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.     

Growth factor/heparin-immobilized collagen gel system enhances viability of transplanted hepatocytes and induces angiogenesis

Hepatocyte transplantation is being explored as a treatment strategy for end-stage liver disease; however, the main limitation is the insufficient vascularization of transplanted hepatocytes. To overcome this problem, a suitable 3D microenvironment and the types of transplanted cells must be considered for hepatocyte transplantation. In this study, a growth factor (GF)/heparin-immobilized collagen gel-filled polyurethane foam (PUF) scaffold was developed for angiogenesis induction and hepatocyte transplantation. First, a vascular endothelial growth factor (VEGF)/heparin-immobilized, collagen-gel-filled PUF scaffold was developed to establish a prevascularized cavity in the subcutaneous space in rats. Second, accompanied by 70% partial hepatectomy (PH), hepatocytes were embedded inside heparin-immobilized, collagen-gel-filled PUF scaffolds, and were transplanted into the VEGF-induced prevascularized cavity. The benefits of using this system were confirmed by using three types of hepatocytes, namely single hepatocyte, hepatocyte spheroids, and fetal hepatocytes. The normalized hemoglobin content and live nucleus numbers were determined separately to evaluate the angiogenesis and viability of transplanted hepatocytes. In summary, after PH pretreatment, transplantation of fetal hepatocyte-embedded, heparin-immobilized, collagen-gel-filled PUF scaffold into a VEGF-induced prevascularized cavity appears to be a promising strategy for future liver tissue engineering.     

In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells

A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.     

Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes

A hepatocyte growth factor (HGF)/heparin-immobilized collagen system was used as a synthetic extracellular matrix for hepatocyte culture. The albumin synthesis, nucleus numbers and morphology of the hepatocytes were determined separately to evaluate the hepatocyte number and hepatocyte-specific function under this system. The benefits of the HGF/heparin-immobilized collagen system for hepatocyte culture were confirmed by three types of culture methods in vitro, namely 2D film cultures, 2D gel cultures and 3D gel cultures. In 2D collagen film cultures, hepatocytes exhibited the highest albumin synthesis (1.42 μg/well/day) in HGF/heparin-immobilized collagen films at 7 days of culture. Heparin inhibited hepatocyte adhesion while HGF promoted hepatocyte migration, and spheroid formation was easily detected in HGF/heparin-immobilized collagen films. In 2D collagen gel cultures, albumin synthesis of around 15 μg/well/day was detected and maintained for more than 18 days on HGF/heparin-immobilized collagen gels. Similar findings were obtained in 3D HGF/heparin-immobilized collagen gel cultures, which exhibited albumin synthesis of up to 30 μg/well/day. The albumin synthesis by hepatocytes was two-fold higher in 3D gel cultures compared with 2D gel cultures, and was maintained for over 2 weeks compared with 2D film cultures using the HGF/heparin-immobilized collagen system. Taken together, the HGF/heparin-immobilized collagen system was effective for albumin synthesis by hepatocytes in both 2D film cultures and 3D gel cultures, and therefore shows good potential for tissue engineering use.     

Dynamic seeding and perfusion culture of hepatocytes with galactosylated vegetable sponge in packed-bed bioreactor

A galactose moiety was introduced into the fiber surface of a vegetable sponge by the covalent binding of lactobionic acid. The galactosylated sponge was used as scaffold for the culture of rat hepatocytes in a packed-bed bioreactor. Hepatocytes could be dynamically seeded into and uniformly distributed throughout the scaffold, and the immobilized cells maintained high albumin and urea production rates during long-term perfusion culture. The hepatocytes showed an increasing albumin production rate from 49 to 109 microg/10(6) cells/d over the 7-d culture.     

The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells

Heterospheroids consisting of hepatocytes and bone marrow cells (BMCs) are formed by the mixed coculture of these cells and enhance the expression and maintenance of the liver-specific functions of hepatocytes. Not only the soluble factors derived from these cells, but also functional organoid (heterospheroid) formation, are considered to underlie this coculture effect. Therefore, in the present study, we aimed to clarify the mechanism of this co-culture effect. We performed hepatocyte monoculture with conditioned media prepared from hepatocyte cultures, BMC cultures and a coculture of hepatocytes and BMCs. When using any type of conditioned medium, no hepatocyte spheroids formed, and the hepatocytes formed a monolayer. In addition, an effect for these conditioned media was shown in terms of the albumin production and ammonia metabolism activities of the hepatocytes; conditioned medium from BMCs showed the strongest effect. The monocultured hepatocytes in the conditioned medium derived from BMCs showed equivalent albumin production and ammonia metabolism activities to the cocultured spheroids of hepatocytes and BMCs. Therefore, it was determined that the effect of the coculture of hepatocytes and BMCs was caused by soluble factors derived from BMCs.     

人参多糖对氧化应激损伤肝细胞的保护作用机制研究

目的:以氧化应激损伤模型,通过对人参中物质基础的筛选,研究其对氧化应激造成的肝细胞损伤的保护作用及其可能的作用机制的初步探讨。方法:采用浓度为25 μmol/L的过氧化氢溶液建立肝细胞损伤模型,对人参中总蛋白、总多糖、总皂苷的保护作用进行筛选。在此基础上,采用流式细胞术检测其凋亡程度、线粒体膜电位的改变,活性氧的含量变化。并采用ELISA法测肝糖原,超氧化物歧化酶(SOD)、丙二醛(MDA)、葡萄糖-6-磷酸酶(G6P)、磷酸烯醇式丙酮酸羧激酶(PEPCK)及ATP酶等指标的变化情况。结果:经过氧化氢诱导后的肝细胞,细胞活力极显著降低(P<0.01),通过对比人参中活性成分,发现人参多糖的保护作用较强,且呈现浓度依赖性;与模型组比较,人参多糖高剂量组大鼠肝细胞中MDA含量非常显著降低(P<0.001);中、高剂量组大鼠肝细胞中SOD含量显著升高(P<0.05);低剂量组大鼠肝细胞中G6P含量显著升高(P<0.05),中、高剂量组中G6P含量极显著升高(P<0.01);中剂量组大鼠肝细胞中PEPCK的含量显著升高(P<0.05),高剂量组中PEPCK的含量极显著升高(P<0.01);低、中剂量组大鼠肝细胞中ATP酶含量极显著升高(P<0.01);中、高剂量组大鼠肝细胞中肝糖原含量显著升高(P<0.05)。结论:人参多糖通过提高肝细胞中ATP酶的活力,升高线粒体膜电位来恢复肝细胞线粒体的功能,通过恢复G6P与PEPCK的含量来恢复肝脏糖异生的功能,同时通过升高SOD,降低MDA来减弱氧化应激带来的损伤,为后续研究氧化应激造成肝损伤提供一定的基础。     

Morphologic changes and cytochrome P-450 induction in the liver of the rat after exposure to polychlorinated biphenyls in various diets

In is shown that administration of sovol, a mixture of polychlorinated diphenyls, in a dose of 500 mg/kg (about 1:10 LD50) induces pronounced changes in the rat liver structure, in particular, fat accumulation and diminution of RNA lump number in hepatocyte cytoplasma, alteration of nuclei, decrease in the number of hepatocytes and binuclear cells in the visual field. Structural disorders in the liver are observed during a long period (up to 5 months) and, to a certain extent, depend on the composition of the lipid component of the ration. Certain differences are recorded in the character of the morphological changes in the liver after single and repeated injections of the agent. No distinct relationship is recorded between the manifestation of hepatocyte fat infiltration and other morphological changes in the liver, and the index of microsomal cytochrome P-450 induction during sovol action.     

Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture

The culture of spermatogonial cells under well-defined conditions would be an important method for elucidating the mechanisms involved in spermatogenesis and in establishing tissue regeneration in vivo. In this study, a serum-free culture system was established, with type A spermatogonia isolated from adult vitamin A-deficient mice. At days 1, 3 and 7 of culture, the viability and proliferation of cells were monitored. The viability of the cells decreased by day 7 to 10% of the cells present. Proliferation occurred mainly during day 1, when 1% of the germ cells was proliferating. Co-labelling for a germ cell marker (heat shock protein-90alpha, Hsp90alpha), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed that this proliferation was restricted to germ cells. In an attempt to improve these parameters, medium containing fetal calf serum (FCS) was used. Viability was not influenced by serum, but proliferation was markedly enhanced. However, after day 7 of incubation with FCS, co-immunolocalization for Hsp90alpha and BrdU showed a preferential proliferation of somatic cells. Comparison of cultures of adult cells with cultures of prepubertal germ cells, commonly used in studies of spermatogenesis, showed that prepubertal germ cells are twice as viable. In addition, a different proliferation profile was observed, with a peak at day 3. Here, a distinct proliferation of somatic cells was also noted. The results from the present study indicate that the origin of isolated germ cells partly determines culture outcome and that cultures of prepubertal germ cells may not be representative for adult spermatogenesis. Moreover, adding FCS to the culture medium invokes the risk of profound and undesirable effects on cell composition, also underlining the need for identification of germ cells during culture.     

氧载体生物反应器的研究

本文探讨在深层培养条件下利用全氟化碳作为氧载体的一种新的供氧方法,在一喷射自吸式反应器中氧通过氧载体的循环传递给培养液。采用这种供氧方法,反应器的供氧速率可提高2.3倍。     

Micropatterned organoid culture of rat hepatocytes and HepG2 cells

The culture of liver cell organoids (multicellular aggregates) such as spheroids or cylindroids, which can strongly express liver functions, has been advocated as a useful technique that has advantages over monolayer culture. This paper describes a micropatterning technique for obtaining spheroids and cylindroids by using rat hepatocytes or HepG2 cells. We developed culture chips that comprised multiple, circular or rectangular microwells; the bottom surface of each microwell was modified with collagen to create a cell adhesion area, and the entire microwell, excluding the collagen-coated spots, was modified with polyethylene glycol (PEG) to create a nonadhesive area. Rat hepatocytes and HepG2 cells formed uniform spheroids and cylindroids on the circular and rectangular chips, respectively. Consequently, two-dimensional micropatterned chips containing homogeneous spheroids or cylindroids were generated. The expression of liver functions (protein secretion and ammonia removal) was greater in the spheroids and cylindroids than in the monolayer culture, and this expression was maintained for at least 2 weeks of culture. Thus, this chip technology has potential for use in various applications that involve organoid culture.     

Production of podophyllotoxin by plant cell cultures of Podophyllum hexandrum in bioreactor

Submerged cultivation of Podophyllum hexandrum for the production of podophyllotoxin was carried out in a 3l stirred tank bioreactor fitted with a low-shear Setric impeller. The specific requirements of the medium, such as carbon source (sugar) and light, were established for the growth of and podophyllotoxin production by P. hexandrum in suspension cultures. Substitution of sucrose by glucose resulted in higher growth and podophyllotoxin production. The biosynthesis of podophyllotoxin was favored when plant cells were cultivated in the dark. An agitation speed of 100 rpm was sufficient to mix the culture broth in the bioreactor without causing any significant cell damage. Biomass and podophyllotoxin accumulation in 3 l bioreactor under batch growth conditions were 6.5 g/l and 4.26 mg/l, respectively, in 22 d. This resulted in an overall podophyllotoxin productivity of 0.19 mg/(l.d), which represented an increase of 27% in comparison to its productivity in a shake flask. Podophyllotoxin production was found to be a combined growth-associated and non-growth associated process.     

Morphology of spheroidal hepatocytes within injectable, biodegradable, and thermosensitive poly(organophosphazene) hydrogel as cell delivery vehicle

An aggregate of specific cells is often regarded as a better form of single-cell in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, cultivated as spheroids and entrapped in a synthetic thermoreversible extracellular matrix, were examined for differentiation morphology and enhanced liver-specific functions and compared with a control set (single-cell hepatocytes). A copolymer of poly(organophsophazene) hydrogel, an injectable, biodegradable, and thermosensitive matrix, was used to entrap hepatocytes as spheroids or single cells. In a 28-d culture period, the spheroidal hepatocytes maintained a higher viability and produced albumin and urea at constant rates, whereas the single hepatocytes showed a reduction in the level of albumin secretion in the hydrogel. The culturing of spheroidal hepatocytes is a potentially useful three-dimensional cell system for application in bioartificial liver devices and bioreactors.     

Expression of a carrot invertase gene in tobacco suspension cells cultivated in batch and continuous culture conditions

Plant cells (Nicotiana tabacum) were genetically modified to produce an heterologous protein, the acidic invertase from carrot, and invertase production from suspension tobacco cells was investigated. Suspension cultures were grown in shake flasks and stirred bioreactor. Total invertase activity was growth related. A 75 d continuous culture in 10 l bioreactor was performed. Our study demonstrates the high potential of plant cell cultures for long term production of heterologous protein.     

Triglyceride accumulation and very low density lipoprotein secretion by rat and goat hepatocytes in vitro

Utilization of [1-14C] oleate by freshly isolated rat and goat hepatocytes was compared. Intracellular [14C] triglyceride accumulation by hepatocytes did not differ between species. At 2 h of incubation, rat hepatocytes secreted approximately 25 times more [14C] triglyceride than goat hepatocytes. Very low density lipoprotein secretion was greatest by hepatocytes incubated in media containing 4:1 oleate:bovine serum albumin. Rat hepatocytes converted three to four times more [1-14C] oleate to 14CO2 and acid-soluble products than goat hepatocytes. Rate of 14CO2 formation by both rat and goat hepatocytes increased as incubation time increased and as rate of cellular triglyceride accumulation decreased. The ratio of 14CO2:[14C] acid-soluble products formed was greater for rat than goat hepatocytes, which indicated rat hepatocytes may oxidize fatty acid more completely. Differences in metabolic rate, based on oxygen consumption, between isolated goat and rat hepatocytes were minor and could not account for marked differences in very low density lipoprotein secretion. Goat hepatocytes did not incorporate detectable quantities of labeled fatty acid into low or high density lipoproteins. Ruminants may be susceptible to fatty liver when the liver takes up large amount of nonesterified fatty acid due to an inability to efficiently export fatty acid as very low density lipoprotein triglyceride.     

Bioartificial liver systems: current status and future perspective

Because the liver is a multifunctional and a vital organ for survival, the management of acute liver failure requires the support of a huge number of metabolic functions performed by the organ. Many early detoxification-based artificial liver techniques failed to treat the patients owing to the inadequate support of the many essential hepatic functions. For this reason, a bioartificial liver (BAL) comprising of viable hepatocytes on a mechanical support is believed to more likely provide these essential functions than a purely mechanical device. From 1990, nine clinical studies of various BAL systems have been reported, most of which utilize a hollow fiber technology, and a much larger number of various BAL systems have been suggested to show an enhanced performance. Safety issues such as immunological reactions, zoonosis and tumorgenicity have been successfully addressed for regulatory approval, but a recent report from a large-scale, randomized, and controlled phase III trial of a leading BAL system (HepatAssist) failed to meet our expectation of efficacy in terms of the overall survival rate. In this paper, we review the current BAL systems actively studied and discuss critical issues such as the hepatocyte bioreactor configuration and the hepatocyte source. On the basis of the insights gained from previously developed BAL systems and the rapid progress in stem cell technology, the short-term and long-term future perspectives of BAL systems are suggested.     

Effect of mixing time on taxoid production using suspension cultures of Taxus chinensis in a centrifugal impeller bioreactor

The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active secondary metabolite, were used as a model system for this investigation. The mixing time (t(m)) and volumetric oxygen transfer coefficient (k(L)a) in the bioreactor were characterized at various cell densities and operating conditions. A constant t(m) of 5 s or 10 s was maintained during cultivation by adjusting the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accumulation was significantly increased through culture elicitation using 100 microM methyl jasmonate (MJA). The lower Tc production at the longer t(m) of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing.     

Continuous plant cell perfusion culture: bioreactor characterization and secreted enzyme production

Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody production. Here, we report the development of an efficient continuous perfusion process for the cultivation of plant cell suspensions. The key to this process is a perfusion bioreactor that incorporates an annular settling zone into a stirred-tank bioreactor to achieve continuous cell/medium separation via gravitational sedimentation. From washout experiments, we found that under typical operating conditions (e.g., 200 rpm and 0.3 vvm) the liquid phase in the entire perfusion bioreactor was homogeneous despite the presence of the cylindrical baffle. Using secreted acid phosphatase (APase) produced in Anchusa officinalis cell culture as a model we have studied the perfusion cultures under complete or partial cell retention. The perfusion culture was operated under phosphate limitation to stimulate APase production. Successful operation of the perfusion process over four weeks has been achieved in this work. When A. officinalis cells were grown in the perfusion reactor and perfused at up to 0.4 vvd with complete cell retention, a cell dry weight exceeding 20 g/l could be achieved while secreted APase productivity leveled off at approximately 300 units/l/d. The culture became extremely dense with the maximum packed cell volume (PCV) surpassing 70%. In comparison, the maximum cell dry weight and overall secreted APase productivity in a typical batch culture were 10-12 g/l and 100-150 units/l/d, respectively. Operation of the perfusion culture under extremely high PCV for a prolonged period, however, led to declined oxygen uptake and reduced viability. Subsequently, cell removal via a bleed stream at up to 0.11 vvd was tested and shown to stabilize the culture at a PCV below 60%. With culture bleeding, both specific oxygen uptake rate and viability were shown to increase. This also led to a higher cell dry weight exceeding 25 g/l, and further improvement of secreted APase productivity that reached a plateau fluctuating around 490 units/l/d.