A novel in vitro three-dimensional skeletal muscle model

A novel three-dimensional (3D) skeletal muscle model composed of C2C12 mouse myoblasts is described. This model was generated by cultivating myoblasts in suspension using the rotary cell culture system (RCCS), a unique culture environment. Single-cell suspensions of myoblasts were seeded at 5 × 10⁵/ml in growth medium without exogenous support structures or substrates. Cell aggregation occurred in both RCCS and suspension control (SC) conditions within 12 h but occurred more rapidly in the SC at all time intervals examined. RCCS-cultured myoblasts fused and differentiated into a 3D construct without serum deprivation or alterations. Syncitia were quantified at 3 and 6+ d in stained thin sections. A significantly greater number of syncitia was found at 6+ d in the RCCS cultures compared to the SC. The majority of syncitia were localized to the periphery of the cell constructs for all treatments. The expression of sarcomeric myosin heavy chain (MHC) was localized at or near the periphery of the 3D construct. The majority of MHC was associated with the large cells (syncitia) of the 6+-d aggregates. These results show, for the first time, that myoblasts form syncitia and express MHC in the presence of growth factors and without the use of exogenous supports or substrates. This model test system is useful for investigating initial cell binding, myoblast fusion and syncitia formation, and differentiation processes.     

模拟微重力条件下对WB-F344肝干细胞表征分子表达的影响

研究大鼠WB-F344肝干细胞在旋转式细胞培养系统（RCCS）中培养进行细胞大规模扩增并保持干细胞的特性的可能性,为干细胞治疗疾病及肝组织工程提供理想的细胞来源。以WB-F344肝干细胞在RCCS中培养,以平面单层培养为对照,在培养后不同时间分别进行形态观察、流式细胞仪测细胞周期、逆转录-聚合酶链反应（RT-PCR）检测肝干细胞特异性基因甲胎蛋白（AFP）和白蛋白（ALB）的表达, 免疫荧光染色检测 AFP、ALB蛋白的表达。结果表明,RCCS培养的WB-F344细胞粘附在Cytodex-3微载体上状态生长良好,细胞增殖较平面培养有明显增加;RT-PCR和免疫荧光染色检测结果一致：模拟微重力培养组AFP的mRNA表达强度及AFP阳性细胞均显著高于平面培养组,而ALB mRNA表达强度和ALB阳性细胞均低于对照组。说明模拟微重力 培养条件下,能较好的维持肝干细胞特性,进一步证明我们建立的这种培养体系是成功的,是一种理想的肝干细胞培养模式。     

RCCS enhances EOE cell proliferation and their differentiation into ameloblasts

In this article we report on the culturing of dental enamel organ epithelia (EOE) using a rotary cell culture system (RCCS) bioreactor associated with a cytodex-3 microcarrier. This culture system enhanced the proliferation and differentiation of the EOE into ameloblasts. Primary dental EOE trypsinized from 4-day old post-natal rat pups were cultured in the RCCS associated with Cytodex-3. The results were analyzed in comparison to a conventional plate system (control). Cells grown in RCCS have shown higher viabilities (above 90%) and final cell densities in terms of cells/ml than in the control system. In the case of RCCS, 46 ± 2 manifold increases were obtained, while significantly lower yields of 10.8 ± 2.5 manifod were obtained for control plates. Throughout the experiments, glucose levels were maintained within the accepted physiological range. In this case, LDH levels are kept low (below 150 mmol/ml), which is in accordance with the low cell death observed in the RCCS. Scanning electron microscopy revealed cells that were spread and forming three dimensional aggregates on the surface of cytodex-3. Cells cultured in the RCCS exhibited a stronger positive immunofluorescence staining for ameloblastin than those in control plates. RT-PCR results revealed that cells cultured in RCCS have higher amelogenin mRNA levels compared to controls. We have done an exploratory study on biological characteristics and self-assembling of epithelium cellula intersitialis, which demonstrated that the special 3D environment enhanced the rat dental EOE cell proliferation and differentiation into ameloblasts. The study has revealed that RCCS could be used to study the reaction of the EOE cells, tooth enamel organ cells and mesenchymal cells under the spacial 3D culture system, which will also provide a novel hypothesis for dental regeneration.     

Different roles of ERK and p38 MAP kinases during tube formation from endothelial cells cultured in 3-dimensional collagen matrices

In a two-dimensional (2D) culture dish, the major activity of endothelial cells is proliferation with limited morphological change. When cultured in a three-dimensional (3D) collagen gel matrix, endothelial cells undergo a series of morphological changes starting with development of intracellular vacuoles and followed by cell elongation. Adjacent cells then coalesce to form tube-like structures. This process mimics the steps of capillary formation during angiogenesis. Using this model, we investigated the roles of extracellular signal-regulated kinase (ERK) and p38 MAP kinase (p38) in the tube formation from human umbilical vein endothelial cells (HUVEC). Proliferating HUVEC gradually lost their ability to divide after being transferred to 3D collagen matrices, where differentiation became the dominant cellular activity. The transition from proliferation to the differentiation state was accompanied by a drastic reduction of cyclin-dependent kinases CDC2, CDK4, and retinoblastoma (Rb) protein, but the expression of cyclin-dependent kinase inhibitor, p27kip1, was increased. Inhibition of p38 by SB203580 partially prevented these changes and increased the proliferation rate of HUVEC. However, cells under this condition exhibited unusually elongated cell bodies, and they were unable to coalesce to form tube structures. Inhibition of ERK neither affected the cell proliferation rate nor the expression levels of cell cycle regulators, but it completely blocked tube formation by inducing apoptosis, a finding different from the best-known role of ERK in cell proliferation in the 2D cell culture systems. We conclude that the major function of ERK is to maintain cell viability while p38 plays multiple roles in controlling cell proliferation, viability, and morphogenesis during tube formation.     

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.     

Recapitulation of Tumor Heterogeneity and Molecular Signatures in a 3D Brain Cancer Model with Decreased Sensitivity to Histone Deacetylase Inhibition

Introduction

Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS).

Methods

CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat.

Results

Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches.

Conclusions

Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system.     

In vitro three-dimensional modelling of human ovarian surface epithelial cells

Objectives:  Ninety percent of malignant ovarian cancers are epithelial and thought to arise from the ovarian surface epithelium (OSE). We hypothesized that biological characteristics of primary OSE cells would more closely resemble OSE in vivo if established as three-dimensional (3D) cultures.
Materials and methods:  OSE cells were cultured as multicellular spheroids (MCS) (i) in a rotary cell culture system (RCCS) and (ii) on polyHEMA-coated plastics. The MCSs were examined by electron microscopy and compared to OSE from primary tissues and cells grown in 2D. Annexin V FACS analysis was used to evaluate apoptosis and expression of extracellular matrix (ECM) proteins was analysed by immunohistochemical staining.
Results:  On polyHEMA-coated plates, OSE spheroids had defined internal architecture. RCCS MCSs had disorganized structure and higher proportion of apoptotic cells than polyHEMA MCSs and the same cells grown in 2D culture. In 2D, widespread expression of AE1/AE3, laminin and vimentin were undetectable by immunohistochemistry, whereas strong expression of these proteins was observed in the same cells grown in 3D culture and in OSE on primary tissues.
Conclusions:  Physiological and biological features of OSE cells grown in 3D culture more closely resemble characteristics of OSE cells in vivo than when grown by classical 2D approaches. It is likely that establishing in vitro 3D OSE models will lead to greater understanding of the mechanisms of neoplastic transformation in epithelial ovarian cancers.     

模拟微重力条件下 WB-F344细胞的三维培养

与传统的单层平面培养相比,细胞三维培养可更好地模拟生物体内细胞的生长状态和微环境.以Cytodex-3微载体为支持物,利用旋转式细胞培养系统（RCCS）模拟微重力条件,悬浮培养法构建大鼠WB-F344细胞微重力三维培养模型.并通过细胞计数、光学显微镜、透射电镜、逆转录-聚合酶链反应（RT-PCR）和流式细胞术等方法分析了细胞增殖、显微结构、粘附分子及钙粘蛋白（E-cadherin）表达情况.结果表明,模拟微重力三维培养条件下WB-F344细胞增殖块,呈紧密多层排列、可见丰富的微绒毛和线粒体、胞间有桥粒和紧密连接形成,细胞粘着力加强、表现出良好的三维生长特征;与静置三维培养相比,纤粘连蛋白（Fn）mRNA表达呈上调趋势,细胞内E-cadherin表达量增加,这可能是微重力效应下细胞粘附力增强的部分机制.该培养体系可能有利于细胞之间,细胞与胞外基质之间相互作用及其作用机制的研究.     

模拟微重力条件下WB-F344细胞的三维培养

与传统的单层平面培养相比,细胞三维培养可更好地模拟生物体内细胞的生长状态和微环境。以Cytodex-3微载体为支持物,利用旋转式细胞培养系统(RCCS)模拟微重力条件,悬浮培养法构建大鼠WB-F344细胞微重力三维培养模型。并通过细胞计数、光学显微镜、透射电镜、逆转录-聚合酶链反应(RT-PCR)和流式细胞术等方法分析了细胞增殖、显微结构、粘附分子及钙粘蛋白(E-cadherin)表达情况。结果表明,模拟微重力三维培养条件下WB-F344细胞增殖块,呈紧密多层排列、可见丰富的微绒毛和线粒体、胞间有桥粒和紧密连接形成,细胞粘着力加强、表现出良好的三维生长特征;与静置三维培养相比,纤粘连蛋白(Fn)mRNA表达呈上调趋势,细胞内E-cadherin表达量增加,这可能是微重力效应下细胞粘附力增强的部分机制。该培养体系可能有利于细胞之间,细胞与胞外基质之间相互作用及其作用机制的研究。     

Microarray analysis of genes differentially expressed in hepG2 cells cultured in simulated microgravity: Preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.     

应用旋转生物反应器批量制备拟胚体的研究

以小鼠胚胎干细胞(ES-D3)为模型,应用新型细胞培养系统——STLV型旋转生物反应器(rotarycellculturesystem,RCCS)建立一种批量制备拟胚体(embryoidbodies,EBs)的新方法,研究不同细胞接种密度及培养时间对RCCS内EBs产生效率的影响。为了进一步研究该制备方法是否对EBs的分化潜能产生影响,对照传统方法制备的EBs,利用形态学及RT-PCR方法测定经旋转生物反应器制备的EBs在自发性或诱导条件下(1%DMSO)向心肌细胞的分化能力。结果表明:ES-D3在RCCS内能够高效形成EBs,与传统的直接悬浮法比较,其EBs的形成效率可达到后者的2倍。1×104个/ml为最佳细胞接种密度,培养时间也是在RCCS制备EBs过程中的重要因素之一,培养第4~5天为最佳收获EBs的时间。与悬滴法制备的EBs比较,该方法制备的EBs分化为心肌细胞的潜能未改变。由此,应用旋转生物反应器可以高效制备EBs,该方法制备的EBs可以用于发育生物学等基础及应用领域的相关研究。     

Comparison of different culture modes for long-term expansion of neural stem cells

Neural stem cells (NSCs) can be cultured in two modes of suspension and monolayer in vitro. The cultured cells are different in both the ability to proliferate and heterogeneity. In order to find the appropriate methods for large-scale expansion of NSCs, we systematically compared the NSCs cultured in suspension with those cultured in monolayer. The forebrain tissue was removed from embryonic day 14 (E14) mice, then the tissue was dissociated into single-cell suspension by Accutase and mechanical trituration. The cells were cultured in both suspension and monolayer. The NSCs cultured in suspension and in monolayer were compared on viability, ability to proliferate and heterogeneity by fluorescent dyes, immunofluorescence and flow cytometry on DIV21 (21 days in vitro), DIV56 and DIV112, respectively. The results indicated that the NSCs cultured in both suspension and monolayer represented good viability in long-term cultures. But they displayed a distinct ability to proliferate in long-term cultures. The NSCs cultured in monolayer preceded those cultured in suspension on the ability to proliferate on DIV21 and DIV56, but no obvious difference on DIV112. The NSCs population cultured in suspension displayed more nestin-positive cells than those in monolayer during the whole process of culture. The NSCs population cultured in monolayer, however, displayed more βIII tubulin-positive cells than those in suspension in the same period. The suspension culture mode excels the monolayer culture mode for large-scale expansion of NSCs.     

DMSO 对悬浮培养的东北红豆杉细胞凋亡和紫杉醇合成的影响

研究了不同浓度的DMSO对悬浮培养的东北红豆杉(Taxus cuspidata)细胞的增殖能力、细胞活性以及紫杉醇合成和释放等方面的影响,同时应用荧光指示剂双染法检测了细胞凋亡的发生情况.结果显示2%的DMSO处理能显著降低细胞活性,抑制细胞的增殖能力,使细胞核内DNA含量减少,培养中、后期在荧光显微镜下可见部分细胞核出现典型的凋亡形态,同时伴有紫杉醇产量的明显增加;对照组及1%以下浓度组未出现上述改变.结果表明一定浓度的DMSO能诱导细胞凋亡,促进细胞紫杉醇合成能力的提高.     

利用微载体悬浮培养人脐带间充质干细胞

随着干细胞研究的不断深入,干细胞功能分化研究和临床应用转化的需求日益提升。人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs)来源广泛,不仅自我更新能力强、能够分化成多种类型的成体细胞,而且其自身具有免疫调节能力,不易引发免疫排斥反应,在干细胞功能分化研究和临床应用中具有巨大应用前景和应用潜力。目前,传统的细胞培养方式培养效率低、细胞活性较差,不能满足日益增长的研究和应用需求。本研究利用微载体结合旋转瓶的悬浮培养方法,通过优化细胞接种量及转速等影响因素,快速获得大量高质量的人脐带间充质干细胞。经悬浮培养总细胞量可高达到7×10 ⁸个细胞/L,而且细胞活性较高,MSC 特异性标记物表达良好,在恢复平面培养后仍能维持MSC的正常细胞形态和增殖能力。高效脐带间充质干细胞悬浮培养体系的初步建立,为未来的干细胞功能分化研究和临床应用奠定了基础。     

Actomyosin is Involved in the Organization of the Microtubule Preprophase Band in Arabidopsis Suspension Cultured Cells

The microtubule preprophase bands （PPBs） participate in the sequence of events to position cell plates in most plants. However, the mechanism of PPB formation remains to be clarified. In the present study, the organization of PPBs in Arabidopsis suspension cultured cells was investigated by confocal laser scanning microscopy combined with pharmacological treatments of reagents specific for the cytoskeleton elements. Double staining of F-actin and microtubules （MTs） showed that actin filaments were arranged randomly and no colocalization with cortical MTs was observed in the interphase cells. However, cortical actin filaments showed colocalization with MTs during the formation of PPBs. A broad actin band formed with the broad MT band in the initiation of PPB and narrowed down together with the MT band to form the PPB. Nevertheless, broad MT bands were formed but failed to narrow down in cells treated with the F-actin disruptor latrunculin A. In contrast, in the presence of the F-actin stabilizer phalloidin, PPB formation did not exhibit any abnormality. Therefore, the integrity, but not the dynamics, of the actin cytoskeleton is necessary for the formation of normal PPBs. Treatment with 2, 3-butanedine monoxime, a myosin inhibitor, also resulted in the formation of broad MT bands, indicating that actomyosin may be involved in the rearrangement of MTs to form the PPBs. Double staining of MTs and myosin revealed that myosin concentrated on the PPB region during PPB formation. It is suggested that the actin cytoskeleton at the PPB site may serve as a rack to transport cortical MTs by using myosin when the broad MT band narrows down to form the PPB.     

Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.     

Growth arrest of vascular smooth muscle cells in suspension culture using low-acyl gellan gum

The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27^Kip1 expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.     

An anchorage-dependent locus in the cell cycle for the growth of 3T3 cells

Mouse fibroblasts, 3T3 cells, require a solid surface for continuous growth, but when 3T3 cells, during their exponential phase in Petri dishes, were transferred to a suspension culture, the number of cells roughly doubled by 30 h. During the suspension culture the number of pairing cells (c₂) increased, but that of the single cells decreased. When cells synchronized at mitosis or at the G1-S boundary were transferred to the suspension culture, the number of pairing cells peaked at 30 min and at 10 h, respectively. DNA synthesis began immediately after the cells, which were cultured for 16 h in the suspension, had settled onto the surface of the Petri dishes. When cells in a confluent culture were arrested at an early G1 period and were suspended, the number of pairing cells did not increase. These results indicate that the most important locus for anchorage growth seems to be at a late G1 period of the cell cycle.     

Induction of three-dimensional assembly of human liver cells by simulated microgravity

Summary The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.