体外肝细胞培养及在药物筛选中的应用

肝脏是人体内最复杂的器官之一,负责执行多种功能,是药物毒性检测的重要靶器官。体外培养肝细胞是进行药物毒性检测的重要途径。传统的体外培养主要是让细胞在不同成分的培养基中生长,或将细胞接种于主要由体内ECM成分如胶原或基质胶组成的基底上成层生长,但易快速丧失肝特异性功能。为解决此问题,学者们研究设计了多种能更好地模拟肝脏体内微环境特征的精加工技术,以进行肝脏细胞的体外培养。在三维支架上培养肝细胞,如球状聚集体和细胞片层,可促进细胞-细胞以及细胞-基质间的相互作用和肝细胞分化,维持肝细胞特异性功能,并形成类似体内的结构。最近,脱细胞基质已被用作支持理想的细胞-基质相互作用的细胞培养支架。本文就体外二维和应用球状聚集体、细胞片层、脱细胞基质进行三维培养肝细胞的具体技术进行了综述,并简要介绍其在药物筛选中的应用。     

Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen

Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals. A biodegradable, biocompatible and non-toxic polymer chitosan was employed for 3D culture of MCF-7 cell lines. Cells grown on chitosan scaffold produce more lactate from glucose in comparison to that secreted by cells grown on tissue culture plate, thus indicating the suitability of chitosan scaffold as an in vitro model resembling cancer tissue growth in vivo. Cytotoxic effect of tamoxifen at different concentrations was evaluated for MCF-7 breast cancer cell lines grown on tissue culture plate as well as on 3D chitosan scaffold. At a tamoxifen concentration of 10(-6) M, 50% reduction in cell growth was observed in tissue culture plate-grown cells where 15% reduction in cell growth was observed when cells were grown in chitosan scaffold. Higher tamoxifen concentrations were required to achieve comparable cytostatic action in 3D culture, supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. Carbohydrate metabolism of MCF-7 cells in terms of glucose utilization and lactate production in 3D and monolayer culture were unaffected by tamoxifen treatment. Cathepsin D activity, an autocrine growth factor in breast cancer cells was monitored in all experiments. In 3D culture, addition of tamoxifen promoted cathepsin D secretion but inhibited its uptake by cells. Growth of cells in 3D chitosan scaffold indicated that action of tamoxifen on estrogen positive cancer cells is also mediated through inhibition of cathepsin D uptake from the culture medium.     

Encapsulation and osteoinduction of human periodontal ligament fibroblasts in chitosan-hydroxyapatite microspheres

Periodontal ligament cells play a crucial role in the regeneration of periodontal tissues and an undifferentiated mesenchymal cell subset is thought to exist within this population. The aim of this study was to assess the osteogenic differentiation potential of human periodontal ligament fibroblasts (hPDLFs) in three dimensional (3D)-osteogenic culture environment following encapsulation in chitosan-hydroxyapatite (C/HA) microspheres with the size range of 350-450 microm. Human PDLF cultures were established and three experimental groups were formed: (i) two-dimensional (2D)-culture as single cell monolayer, (ii) 3D-static culture of C/HA encapsulated hPDLFs, and (iii) 3D-dynamic culture of C/HA encapsulated hPDLFs in a rotating wall vessel bioreactor. The cells were cultured in standard culture medium supplemented with beta-glycerophosphate, dexamethasone, and ascorbic acid. After 21 days, immunohistochemistry was performed using antibodies against osteonectin, osteopontin, bone-sialoprotein, and osteocalcin as osteogenic differentiation markers. Phase-contrast and scanning electron microscopy observations were used for histological and morphological evaluation. The combined effects of osteoinductive medium and HA-containing composite microsphere material on encapsulated hPDLFs resulted in the transformation of a considerable portion of the cells into osteoblastic lineage at the end of the experiments. Results demonstrate the ability of hPDLFs to undergo osteogenic differentiation upon induction in vitro, both under 2D and 3D culture conditions. C/HA microspheres in microgravity bioreactor may serve as a suitable 3D environment to support the osteogenic differentiation of human PDLFs, in vitro.     

A porous 3D cell culture micro device for cell migration study

Cell migration under chemoattractant is an important biological step in cancer metastasis that causes the spread of malignant tumor cells. Porous polymeric materials are widely used to mimic the extracellular matrix (ECM) environment for applications such as three dimensional (3D) cell culturing and tissue engineering. In this paper we report a novel 3D cell culture device based on porous polymeric material to study cancer migration. We fabricated a porous channel on a polymeric chip using a selective ultrasonic foaming method. We demonstrate that a chemical concentration gradient could be established through the porous channel due to the slow diffusion process. We show that significant cell migration could be observed through the porous channel within 1–2 weeks of cell culturing when metastatic M4A4-GFP breast cancer cells were induced by 20% fetal bovine serum (FBS).We also developed a mathematical model to evaluate the diffusivity and concentration gradient through the fabricated porous structure.     

Enhanced in vitro maturation of subcultivated fetal human hepatocytes in three dimensional culture using poly-L-lactic acid scaffolds in the presence of oncostatin M

Fetal human liver cell fractions, which contain large numbers of hepatocyte progenitors, have high proliferation potential in vitro. To create an engineered liver tissue equivalent of a clinically significant size, however, repeated subcultivation and functional maturation are necessary in vitro. A commercially available human fetal liver cell fraction that was cultivated for some time in vitro has been reported to lose liver specific functions almost completely. We therefore investigated the effects of oncostatin M (OSM) and hepatocyte growth factor (HGF) in long-term three-dimensional (3D) culture using macroporous poly-L-lactic acid (PLLA) scaffolds on the restoration of such liver-specific functions of the fraction. 3D culture using PLLA scaffolds with OSM remarkably enhanced the albumin production and cytochrome P450 1A1/2 capacity with the culture time. HGF alone had no preferable effect on these functions even in 3D culture. Alpha-fetoprotein production was consistently suppressed in the 3D culture compared with that in monolayers. This suppression was not observed in the same types of culture of hepatocarcinoma Hep G2 cells. Despite these favorable observations on the 3D culture with OSM, the final attained functional levels at the 5th week were still over ten-times lower than those of Hep G2 cells when standardized with a cellular DNA amount. Although further improvement is needed for the complete functional restoration and maturation in vitro, these results demonstrate that a combination of 3D culture using PLLA scaffolds and OSM offers promising culture conditions for in vitro maturation of human hepatocyte progenitors.     

Elastic cartilage engineering using novel scaffold architectures in combination with a biomimetic cell carrier

Tissue engineering of an elastic cartilage graft that meets the criterion for both structural and functional integration into host tissue, as well as allowing for a clinically tolerable immune response, is a challenging endeavour. Conventional scaffold technologies have limitations in their ability to design and fabricate complex-shaped matrix architectures of structural and mechanical equivalence to elastic cartilage found in the body. We attempted to investigate the potential of conventionally isolated and passaged chondrocytes (2D environment) when seeded and cultured in combination with a biomimetic hydrogel in a mechanically stable and biomimetic composite matrix to form elastic cartilage within ectopic implantation sites. In vitro cultured scaffold/hydrogel/chondrocytes constructs showed islets of cartilage and mineralized tissue formation within the cell-seeded specimens in both pig and rabbit models. Specimens with no cells seeded showed only vascularized fibrous tissue ingrowth. These studies demonstrated the potential of such scaffold/hydrogel/cell constructs to support chondrogenesis in vivo. However, it also showed that even mechanically stable scaffolds do not allow regeneration of a large mass of structural and functional cartilage within a matrix architecture seeded with 2D passaged chondrocytes in combination with a cell biomimetic carrier. Hence, future experiments will be designed to evaluate an initial 3D culture of chondrocytes, effect on cell phenotype and their subsequent culture within biomimetic 3D scaffold/cell constructs.     

Multi-parametric hydrogels support 3D in vitro bioengineered microenvironment models of tumour angiogenesis

Tumour microenvironment greatly influences the development and metastasis of cancer progression. The development of three dimensional (3D) culture models which mimic that displayed in vivo can improve cancer biology studies and accelerate novel anticancer drug screening. Inspired by a systems biology approach, we have formed 3D in vitro bioengineered tumour angiogenesis microenvironments within a glycosaminoglycan-based hydrogel culture system. This microenvironment model can routinely recreate breast and prostate tumour vascularisation. The multiple cell types cultured within this model were less sensitive to chemotherapy when compared with two dimensional (2D) cultures, and displayed comparative tumour regression to that displayed in vivo. These features highlight the use of our in vitro culture model as a complementary testing platform in conjunction with animal models, addressing key reduction and replacement goals of the future. We anticipate that this biomimetic model will provide a platform for the in-depth analysis of cancer development and the discovery of novel therapeutic targets.     

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.     

Comparing predictive drug nephrotoxicity biomarkers in kidney 3-D primary organoid culture and immortalized cell lines

The cellular microenvironment is recognized to play a key role in stabilizing cell differentiation states and phenotypes in culture. This study addresses the hypothesis that preservation of in vivo-like tissue architecture in vitro produces a cell culture more capable of responding to environmental stimuli with clinically relevant toxicity biomarkers. This was achieved using kidney proximal tubules in three-dimensional organoid hydrogel culture, with comparisons to conventional monolayer kidney cell cultures on plastic. Kidney proximal tubule cultures and two immortalized kidney cell line monolayer cultures exposed to known nephrotoxic drugs were evaluated for inflammatory cytokines, nephrotoxicity-associated genes, Kim-1 protein, cytochrome enzymes, and characteristic cellular enzyme shedding. Significant similarities are shown for these traditional biomarkers of kidney toxicity between in vivo and 3-D organoid endpoints of drug toxicity, and significantly, a consistent lack of clinically relevant endpoints produced by traditional 2-D kidney cell cultures. These findings impact both in vitro bioreactor-based kidney functional and regenerative medicine models, as well as high-throughput cell-based drug screening validations.     

体外构建胚胎干细胞生长分化的三维研究模型

目的以液态胶原为支架,小鼠胚胎干细胞(ESCs)为细胞模型,构建ESCs胶-原复合体,旨在尝试建立一种能够实现干细胞生长、分化的三维培养体系。方法提取鼠尾胶原,观察ESCs在自制胶原条带内增殖的形态特征,并测定葡萄糖/乳酸活性。以ESCs源心肌细胞为目的细胞,利用免疫组化、RT-PCR及电镜技术评价胶原条带内ESCs进行自发性分化的能力。结果ESCs在胶原条带所提供的三维培养体系内生长、增殖状态良好,且彼此间能够建立细胞连接。胶原条带内部分ESCs能够自发性分化为心肌细胞。该心肌细胞均可表达心肌蛋白cTn-T,心肌转录因子NKX2.5及肌球蛋白轻链MLC-2 vmRNA,且肌小节结构发育成熟。结论以液态Ⅰ型胶原为主体的支架材料可以为ESCs提供良好的生长基质,促进其组织化发育。该实验初步明确了体外构建干细胞生长分化三维模型的可行性。     

体外构建三维肝肿瘤模型及药物筛选

背景：体外构建三维肿瘤模型替代现有二维平面肿瘤细胞模型用于药物筛选是肿瘤药筛技术发展的必然趋势。 目的：体外构建三维肝肿瘤模型体系,并用于抗肿瘤药物的敏感性研究。 方法：以人肝癌细胞HepG2作为模型细胞,以壳聚糖/胶原混合材料制备水凝胶支架,体外构建肝(肿瘤)细胞的三维培养体系,表征三维肝(肿瘤)细胞聚集体的形态、生长、细胞骨架分布等,并以二维平面培养的肝肿瘤细胞为对照,研究三维肝肿瘤模型对临床常用的化疗药物的敏感性。 结果与结论：①肝细胞在壳聚糖/胶原水凝胶支架中培养10 d后形成三维的聚集细胞团。②肝细胞在水凝胶支架中生长速度略慢于二维平面培养,但在三维体系下肝细胞能长时间保持细胞活性。③在水凝胶支架中肝细胞三维生长后,纤维蛋白骨架发生重排,结构与在体肝组织更接近。④在水凝胶支架中的三维肝肿瘤细胞模型对化疗药物的敏感性降低。由此可见,在壳聚糖/胶原水凝胶支架中形成的三维肝(肿瘤)模型,其细胞骨架结构更接近体内肝组织,因此可用于体外药筛模型研究。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

Enhancement of viability of muscle precursor cells on 3D scaffold in a perfusion bioreactor

The aim of this study was to develop a methodology for the in vitro expansion of skeletal-muscle precursor cells (SMPC) in a three-dimensional (3D) environment in order to fabricate a cellularized artificial graft characterized by high density of viable cells and uniform cell distribution over the entire 3D domain. Cell seeding and culture within 3D porous scaffolds by conventional static techniques can lead to a uniform cell distribution only on the scaffold surface, whereas dynamic culture systems have the potential of allowing a uniform growth of SMPCs within the entire scaffold structure. In this work, we designed and developed a perfusion bioreactor able to ensure long-term culture conditions and uniform flow of medium through 3D collagen sponges. A mathematical model to assist the design of the experimental setup and of the operative conditions was developed. The effects of dynamic vs static culture in terms of cell viability and spatial distribution within 3D collagen scaffolds were evaluated at 1, 4 and 7 days and for different flow rates of 1, 2, 3.5 and 4.5 ml/min using C2C12 muscle cell line and SMPCs derived from satellite cells. C2C12 cells, after 7 days of culture in our bioreactor, perfused applying a 3.5 ml/min flow rate, showed a higher viability resulting in a three-fold increase when compared with the same parameter evaluated for cultures kept under static conditions. In addition, dynamic culture resulted in a more uniform 3D cell distribution. The 3.5 ml/min flow rate in the bioreactor was also applied to satellite cell-derived SMPCs cultured on 3D collagen scaffolds. The dynamic culture conditions improved cell viability leading to higher cell density and uniform distribution throughout the entire 3D collagen sponge for both C2C12 and satellite cells.     

A 3-D organoid kidney culture model engineered for high-throughput nephrotoxicity assays

Cell-cell and cell-matrix interactions control cell phenotypes and functions in vivo. Maintaining these interactions in vitro is essential to both produce and retain cultured cell fidelity to normal phenotype and function in the context of drug efficacy and toxicity screening. Two-dimensional (2-D) cultures on culture plastics rarely recapitulate any of these desired conditions. Three dimensional (3-D) culture systems provide a critical junction between traditional, yet often irrelevant, in vitro cell cultures and more accurate, yet costly, in vivo models. This study describes development of an organoid-derived 3-D culture of kidney proximal tubules (PTs) that maintains native cellular interactions in tissue context, regulating phenotypic stability of primary cells in vitro for up to 6 weeks. Furthermore, unlike immortalized cells on plastic, these 3-D organoid kidney cultures provide a more physiologically-relevant response to nephrotoxic agent exposure, with production of toxicity biomarkers found in vivo. This biomimetic primary kidney model has broad applicability to high-throughput drug and biomarker nephrotoxicity screening, as well as more mechanistic drug toxicology, pharmacology, and metabolism studies.     

Novel approach to fabricate porous gelatin-siloxane hybrids for bone tissue engineering

Porous and bioactive gelatin–siloxane hybrids were successfully synthesized by using a combined sol–gel processing, post-gelation soaking, and freeze-drying process to provide a novel kind of materials in the developments and optimization of bone tissue engineering. The pore sizes of the hybrids can be well controlled by varying the freezing temperature. The scaffolds were soaked in a simulated body fluid (SBF) up to 14 days to evaluate the in vitro bioactivity. The Ca²⁺-containing scaffolds showed in vitro bioactivity as they biomimetically deposited apatite, but the Ca²⁺-free scaffolds failed. Cytotoxicity and cytocompatibility of those scaffolds and their extracts were monitored by the MC3T3-E1 cell responses, including the cell proliferation and the alkaline phosphatase activity. It was demonstrated that appropriate incorporation of Ca²⁺ ions stimulated osteoblast proliferation and differentiation in vitro.     

Perfusion culture enhanced human endometrial stromal cell growth in alginate-multivalent integrin α5β1 ligand scaffolds

A method to functionalize alginate by introducing monomeric or self-assembling (tetrameric) fibronectin (FN) domains is described, leading to a functional scaffold, which is used for three dimensional (3D) culture of human endometrial stromal cells (EnSCs). EnSCs encapsulated in the functional alginate were cultured under perfusion using the TissueFlex? platform, a multiple parallel microbioreactor system for 3D cell culture. The effect of the novel scaffold and the effect of perfusion were examined. Cell viability, proliferation, and extracellular matrix (ECM) deposition were determined and the results compared with those obtained with cells encapsulated in non-functionalized alginate, and also those without perfusion. Staining for focal adhesions and actin showed maximal cell adhesion only for alginate-tetrameric FN scaffolds under perfusion, associated with a significant increase in cell number over 7 days culture; in contrast to poor cell adhesion and a decrease in cell number for non-functionalized alginate scaffolds (irrespective of perfused/static culture) and 3D static culture (irrespective of the scaffold). Conjugation of alginate to FN was an absolute requirement to attenuate the loss of cell metabolic activity over 7 days culture. ECM deposition for blank alginate and alginate-monomeric FN was similar, but increased around 2-fold and 3-fold for alginate-tetrameric FN under static and perfusion culture, respectively. It is concluded that the requirement for EnSC engagement with multivalent integrin α5β1 ligands and perfused culture are both essential as a first step toward endometrial tissue engineering.     

Non-linear material models for tracheal smooth muscle tissue

The aim of this study is to investigate the hyperelastic material models to describe the non-linear stress-strain behavior of tracheal smooth muscle tissue. Specifically, the goal is to validate the material model with experimental data using different finite element models and discuss the trends in stress-strain behavior of smooth muscle tissue. Both 2D and 3D finite element analyses were carried out to estimate the stress-strain behavior of the smooth muscle tissue. The results obtained indicate that the developed Ogden material model is valid and useful in explaining the stress strain behavior of tracheal smooth muscle tissue under different conditions. Finite element simulation results of the stress-strain behavior in the transverse direction are presented.     

Advances in 3D cell culture technologies enabling tissue‐like structures to be created in vitro

Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co‐culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue‐like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two‐dimensional (2D) substrates and review the popular approaches currently available that enable the development of three‐dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold‐free or scaffold‐based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science.     

MicroCT evaluation of three-dimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects

Numerous growth factors, peptides, and small molecules are being developed for bone tissue engineering. The optimal dosing, stability, and bioactivity of these biological molecules are likely influenced by the carrier biomaterial. Efficient evaluation of various formulations will require objective evaluation of in vitro culture systems and in vivo regeneration models. The objective of this paper is to examine the utility of microcomputed tomography (microCT) over conventional techniques in the evaluation of the bone morphogenetic protein-2 (BMP-2) dose response effect in a three-dimensional (3D) in vitro culture system and in an established calvarial defect model. Cultured MC3T3-E1 osteoblasts displayed increased cellular density, extracellular matrix (ECM) production, and mineralization on 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds in a BMP-2 dose dependent manner. MicroCT revealed differences in shape and spatial organization of mineralized areas, which would not have been possible through conventional alizarin red staining alone. Additionally, BMP-2 (doses of 30 to 240 ng/mm(3)) was grafted into 5 mm critical sized rat calvarial defects, where increased bone regeneration was observed in a dose dependent manner, with higher doses of BMP-2 inducing greater bone area, volume, and density. The data revealed the utility of microCT analysis as a beneficial addition to existing techniques for objective evaluation of bone tissue engineering and regeneration.     

Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-beta 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-beta 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.