3D细胞培养体系在促进干细胞向肝细胞诱导分化研究中的作用进展

农卡特（综述），艾开兴,2（审校）.上海交通大学附属上海市第六人民医院普外科，上海 200233 2.同济大学附属同济医院普外科，上海 200065

·综 述·

3D细胞培养体系在促进干细胞向肝细胞诱导分化研究中的作用进展

农卡特1（综述），艾开兴1,2（审校）
1.上海交通大学附属上海市第六人民医院普外科，上海 200233 2.同济大学附属同济医院普外科，上海 200065

功能性肝细胞被广泛应用于细胞移植、生物人工肝治疗、肝脏组织工程、药物代谢及毒性等研究，但作为功能性肝细胞主要来源之一的原代肝细胞无法完全满足这些研究需求。在2D或3D细胞培养体系中经干细胞诱导获得的功能性肝细胞可能可作为替代来源。目前，3D细胞诱导体系与2D体系相比，因前者能模拟体内部分生物微环境而备受关注。该文概述3D细胞培养体系在促进干细胞向肝细胞诱导分化研究中的作用进展，重点关注用于研制3D培养支架的材料。

3D培养支架；肝细胞诱导分化；干细胞

1 Introduction

Functional hepatocytes are valuable cell sources forresearches on cell therapy，bio-artificial liver therapy，liver tissue engineering，drug metabolism and toxicity.Although primary hepatocytes(PHs)are generallyemployedinthoseresearches，thesecellshave somedrawbacks，such aslimitation ofsources，difference in variability and functions from batch to batchandde-differentiationinvitroculture[1].Therefore，a renewable supply of functional hepatocytes from an alternative source becomes an important goal in current studies.Stem cells are considered an alternative cell source of functional hepatocytes.There are increasing evidences demonstrates thathepatocytes can be acquired from stem cells by culturing in specificmicroenvironments[2-5].But most of these evidences were achieved from conventional 2D culture systems. Cells differentiated in 2D cultures have in some aspects demonstrated reduced hepatic functionality when compared to PHs.A novel 3D culture system may overcome some of these problems.With respect to 3D culture system，a scaffold is needed to mimic the biological environment in vivo.

Ideally，ascaffoldshouldbedesignedtopossessthe following characteristics：① a biocompatible and biodegradable substrate with controllable degradation rates.② a 3D and highly porous architecture to accommodatecellattachment，penetration，proliferation，and extracellular cell matrix(ECM)deposition.③an interconnected pore network to facilitate nutrient and waste exchange.④a suitable mechanical strength to support regeneration.⑤ a proper surface chemistry and surface topography to promote cellular interactions and tissue development[6].In this review，we will highlight 3D culture systems that have been utilized in studies on hepatic differentiation from various stem cells. This information provides feedback to researchers in the field for their quest to design better culture scaffold for hepatic differentiation and improved cell culture systems.

2 Naturalmaterialscaffolds used in hepatic differentiation

2.1 Single-source natural material scaffolds

In the past decade，most efforts have combined soluble factors with 3D culture systems to induce superiorstructuraland functionalmaturation of hepatocytes.Various natural materials like collagen，matrigel and alginate have been used to make 3D culture scaffold for supporting the differentiation of hepatocytes from embryonic stem cells(ESCs)or mesenchymal stem cells(MSCs).Imamura T and his colleagues developed a 3D collagen scaffold culture system to induce hepatocyte-like cells from mouse ESCs[7].The scaffold provides an optimal structure forthree-dimensionalculturemimicking in vivo microenvironment and is also used to promote cell-tocell and/or matrix interaction[8-9].In their study，embryoid bodies(EBs)were formed from ESCs cultured in polypropylene conical tubes.The EBs was implanted into collagen scaffold and collagen-coated dishes were used as 2D controls.As a result，they successfully induced cells expressing several fetal and mature liver-specific mRNAs，such as alpha fetoprotein(AFP)，albumin(ALB)，glucose-6-phosphate dehydrogenase(G6PD)and tyrosine aminotransferase (TAT).But cells from collagen scaffold formed cordlike structures that were not present in monolayer cultures.Aggregated multicellular structures have been shown to promote extensive cell-cell contact and formation of gap and tight junctions[10].For the further maturation，collagen scaffolds with hepatocyte-like cells derived from EBs were transplanted into the median lobe of the liver after partial hepatectomy. Cells containing both albumin and cytokeratin 18 (CK18)，a marker of mature hepatocytes[11]，were present in the scaffold by 7 days after transplantation and formed aggregates like hepatic lobules by 14 days.Another team led by Baharvand H applied a similar collagen scaffold to compare the differentiation efficiency of hepatocytes from human ESCs in 3D and 2D culture systems[12].In 3D culture，ALB and G6P were detected earlier and higher levels of urea and AFP were produced，when compared with 2D culture.Electron microscopy of differentiated human ESCs showed hepatocyte-like ultrastructure，including glycogen granules，well developed Golgi apparatuses，rough and smooth endoplasmic reticuli and intercellular canaliculi.

Alginate is a non-toxic，biocompatible，hydrophilic and biodegradable natural polymer with a number of peculiar physicochemical properties for which it has wide applications in drug delivery and cell delivery systems.[13]In addition，culture scaffold made from alginate was transparent，thereby allowing cells and scaffold structures to be observed directly and clearly.Lin N and his colleagues used an alginate scaffold as a three-dimensional matrix to differentiate bone mesenchymal stem cells (BMSCs) into hepatocyte-like cells[14].The differentiated BMSCs adhered to the alginate scaffold and formed multicellular aggregates，also have the abilities to synthesisof ALB and urea，storage of glycogen，and expression of CK-18 and cytochrome P450 isoenzyme 7A1 (CYP7A1).Furthermore，they examined the general characteristics of the alginate scaffold and compatibility of the scaffold with the BMSCs.Scanning electron microscope(SEM)analysis revealed that their alginate scaffolds were highly porous structures with interconnected pores，between 300 μm and 500 μm in diameter.According to Pham[15]，optimaltissue developments requires infiltration of seed cells into the scaffold，which requires the scaffold to have a macroporous structure with interconnected pore diameters of at least 10 μm.Thus，the alginate scaffold they had developed provides enough space for the cells.

Another ready-to-use sponge made from lyophilized alginate gel，Algimatrix scaffolds，was used as a 3D culture media for the hepatic differentiation.AlgiMatrix is a chemically defined，highly porous(＞90%)3D scaffold，the pore sizes in Algimatrix scaffolds are 100－200 μm[16].Ramasamy TS reported the sequential application of 2D and 3D culture systems to differentiate human ESCs to hepatocytes[17]. Human ESCs were initially differentiated in a monolayer culture to definitive endoderm(DE)cells，which were then inoculated into Algimatrix scaffolds.Cells from hESC-DE had been further differentiated in both 2D and 3D culture conditions，and expressed the liver-specific genes，albumin， apolipoprotein F (ApoF)， tryptophan dioxygenase(TO)，andcytochromeP450family members，CYP3A4 and CYP7A1.Significantly，the cells differentiated in the Algimatrix 3D scaffold exhibited considerably higher levels of hepatocytespecificmarkersthan the cellsdifferentiated in monolayer，particularly those thatare expressed predominantly in the more mature hepatocytes，such as ApoF，TO，and CYP3A4.

Volvox spheres are another natural material with the ability to provide 3D environment to culture cells. Wu KL and his colleagues cultured MSCs and liver cells in Volvox spheres to evaluate the hepatic differentiation of MSCs[18].Their results shows that the MSCs culture in the 3D Volvox sphere with liver cell can successfully induced into hepatocyte-like cells expressing hepatocyte-specific markers ALB，AFP and CK18 mRNA expressions and producing CK18 and ALB proteins.

2.2 Mixed natural material scaffolds

Collagen scaffold and alginate scaffold we mentioned aboveweremadefrom single-source material，type I collagen and alginate gel.There are 3D scaffolds made from mixed natural materials，such as extracellular matrix (ECM)， small intestinal submucosa(SIS)and synthesized basement membrane (sBM).Liver-specific functions of adult hepatocytes are better maintained when they are plated on a combination of ECM molecules[19].ECM is secreted by cells to form interstitial matrix and basement membrane，which constitutes the framework to which cells are anchored.Basement membrane provides the special orientation and stability required for the organization and development of the characteristic histology of specific tissues[20].A mixed materials 3D scaffold， ECM-enriched matrigele basement membrane，wasmadebyOkumotoK andhis colleagues for differentiation of rat BMSCs into liver cell lineage[21].ECM-enriched Matrigele basement membraneisasolubilized basementmembrane preparation containing laminin，collagen typeⅣ，heparan sulfate，and entactin that is obtained from Engelbreth-Holm-Swarm murine tumor cells，and promotes cell attachment， differentiation and proliferation.This approach enabled them to maintain the BMSCs culture for a longer period，and the cultured cells differentiated into a liver cell lineage and proliferated on an ECM-containing membrane，and they formed aggregation by day 21 of culture. Another study carried by Ong SY differentiated human MSCs into hepatocytes in a pellet configuration，with or without the presence of SIS.[22]After4 weeksofdifferentiation，they obtained hepatocyte-like cellsthatexpressed a subsetof hepatic genes，secreted albumin and urea，stored glycogen，and showed inducible CYP3A4 mRNA levels.When these cells were implanted into livers ofhepatectomized rats，they secreted human albumin into the bloodstream.But，the hepatic differentiation of MSCs was faster in cell pellets without SIS.They attributed the less efficient differentiation in pellets containing SIS to the three following factors：transforming growth factor-β(TGF-β)，an important factor for chondrogenesis，is present in the SIS scaffold[23]，cell density in the pellet with SIS may not be high enough to form aggregation for hepatic differentiation，and possible hypoxia in the cell/SIS pellets confounds the interpretation of the differentiation result.These results may indicated that SIS is not suitable to be a material for making culture media.The BM，on the other hand，has a highly integrated structure composed of ECM molecules. The major components of most BMs are typeⅣcollagen，laminins(LNs)，entactin(nidogen)and heparin sulfate proteoglycans(HSPGs)，such as perlecan.Shiraki N and his colleagues utilized BM to investigate the differentiation of mouse and human ESCs into hepatic lineages[24].Human and mouse ESCs grown on this BM were able to differentiate into hepatic cellexpressing mature hepatocyte markers and secreting albumin.

2.3 Nano-natural material scaffolds

Nano-medicine combined stem cell therapy is a promising new approach to most of the dreadful diseases， including genetic disorder. Nanosize materials are revolutionizing many areas of medicine and technology and they are small enough to enter almost all areas of the body，including cells and organelles，leading to an effective treatmentin clinical practices[25].For a better efficiency of differentiating murine BMSCs into hepatocytes，Pulavendran S and his colleagues introduced nanotechnology to make 3D culture scaffold[26].They made chitosan nanoparticles(CNP)and incorporated hepatocyte growth factor(HGF)into them，for the purpose of controlling the concentration of HGF in the culture medium.Chitosan is a natural cationic polysaccharide and hasbeen welldefined with characteristics thatinclude biocompatibility，low immunogenicity and minimal cytotoxicity[27].Because of its versatility in biodegradability and drug carrying capacity，chitosan-based nanoparticleshave been used to deliver different bioactive peptides and gene products to various organs[28].In Pulavendran’s study，in the presence of HGF，fibroblast growth factor (FGF)，as positive control，and epidermal growth factor(EGF)and HGF-CNP，fibroblastic morphology of MSC was lost after 21 days and in addition，round shaped cells were observed in the HGF-CNP treated as well as in the positive control groups，where the positive control cells outnumbered the experimental hepatocytes.This may be due to the freely available growth factors in the positive controlforthe differentiation compared to HGF being released from CNP.It meant that only incubation without shaking or enzymatic degradation is not enough for the dissociation ofHGF from the tightly bounded nanoparticles.

3 Synthetic material scaffolds used in hepatic differentiation

In general，naturally derived scaffolds have the potential advantage of biological recognition that may positively support cell adhesion and function. However，scaffoldsfabricated purely from these molecules have their innate limitations，including complex structural composition，purification，immunogenicity，and pathogen transmission，difficulty in controlling of pore size and porosity，large batch to batch variations upon isolation from biological tissues and poor biomechanical strength[29].These have driven the development of synthetic polymers for use as scaffolding materials.Synthetic polymers have a higher degree of processing flexibility and no immunological concerns compared to natural ECM proteins.In recent years，different synthetic materials have been applied to fabricated 3D scaffold and used for liver tissue engineering，such as poly L-lactic acid (PLLA)，poly glycolic acid(PGA)，poly lactic acid (PLA)，and their copolymer poly actic acid-coglycolic acid(PLGA)，polyethylene glycol(PEG)，polyurethane (PU)， polycaprolactones， polyorthoesters and polycarbonates[30].Moststudies on hepatic differentiation from stem cells have beencarried on synthetic polymers 3D scaffold.

3.1 PLGA scaffolds

PLGA arethemostwidely used synthetic polymeric materials in tissue engineering[31].These polymers are well characterized and have gained FDA approval for certain human use.To the best of our knowledge，Levenberg S and his colleagues was the first team to utilize PLGA as a material to make the 3D culture scaffold for hepatic differentiation[32].They seeded human ESCs in the PLGA-PLLA polymer scaffolds.Human ESCs in this 3D scaffold were induced into hepatocyte-like cells expressing AFP and ALB with the presence of activin-A and insulin-like growth factor(IGF).Most recently，Li J used collagencoated PLGA (C-PLGA)3D scaffoldsforthe induction of rat BMSCs into hepatocyte-like cells.[33]Cells induced in this natural-Synthetic hybrid materials 3D scaffold expressed hepatocyte-specific markers(e.g.，ALB，AFP，CK18，hepatocyte nuclear factor4 alpha(HNF4-α)，and cytochrome P450)at mRNA and protein levels.Mostmarkerswere expressed in C-PLGA group 1 week earlier than in the control groups.In this study，the scaffold was coated with collagen，a natural material，in order to provide surfaces with enhanced adhesion and proliferation capability for cells.

3.2 PCL scaffolds

Poly e-caprolactone(PCL)is another synthetic material that has been used for tissue engineering applications.PCL has many advantages because of its biocompatibility，low cost，easy processability and slow hydrolytic degradation rate[34]. It can be fabricated into conduits with the required dimensions，optimum porosity，and viscoelasticity.PCL degrades by hydrolysis and the body resorbs the hydrolyzed products with minimalreaction to the tissues. Hashemi SM and his collegues tested the ability of PCL nanofiber scaffold to support and maintain hepatic differentiation of human cord blood-derived unrestricted somatic stem cells(USSCs)in vitro[35]. They showed that hepatocyte-like cells differentiated from USSCs on the PCL nanofiber scaffold can express hepatic-specific gene(ALB，AFP，CK18)， and had the hepatic-specific functions，such as glycogen storage and indocyanine green (ICG) uptake.Another PCL based 3D media was PCL/ collagen/PES nanofiberscaffold.Polyethersulfone (PES)has many fascinating properties including favorable mechanical strength，thermal and chemical resistance， and excellent biocompatibility[36]. Kazemnejad S and his team fabricated PCL/collagen/ PES nanfiber scaffold by the electrospinning technique[29]. Human BMSCs seeded into this complex 3D scaffold differentiated into hepatocytelike cells and expressed hepatocyte specific markers such as albumin，AFP，CK-18，CK-19 and CYP3A4 at mRNA levels.The number of albumin-positive cells cultivated on the scaffold was higher than that on the 2D culture system.

3.3 PLLA scaffolds

The mostcommon synthetic biodegradable polymers employed in the tissue-engineering field are FDA-approved PLLA.It has been fabricated into nano-fibrous scaffolds to mimic the physical architecture of natural collagen[37].The nano-fibrous PLLA scaffolds have been demonstrated to enhance cell adhesion and differentiation[38].Ghaedi M and his colleagues developed a PLLA collagen nanofiber scaffold by the electrospinning technique[39].Human MSCswereseededontothem andinducedto differentiate into a hepatocyte lineage.The results of this study showed that expression levels of liverspecific markers such as albumin，AFP，and CK-8 and CK-18 were higher in differentiated cells on the nanofibers than that cultured on plates.Importantly，liver functioning serum proteins，albumin and α-1 antitrypsin were secreted into the culture medium at higher levels by the differentiated cells on the nanofibers than on the plates.Moreover，compared to the PCL/collagen/PES nanofiber scaffold in their previous study[29]， the modified PLLA collagen nanofiber scaffold fabricated in this study demonstrated superior cell adhesion capabilities and biocompatibility.Before this study，another team made a PLLA-PGA polymerscaffold using a nonwoven textile process[40].Mouse ESCs were mixedwith matrigel and immediately seeded into this PLLAPGA polymer scaffold.The differentiated hepatocytelike cells were able to express several liver-specific markers and proteins，secrete albumin，store glycogen，and allow the uptake of low-density lipoproteins.

3.4 PU scaffolds

With the advantages of excellentphysical properties，relatively good biocompatibility and biodegradation character，polyurethanes(PUs)remain one of the most popular group of biomaterials applied for medicaldevices[41].Matsumoto K made a polyurethane foam(PUF)for inducing mouse ESs into hepatic lineages[42].Mouse ESs spontaneously formed spherical multicellular aggregates in the PUF and differentiated into cells expressed AFP and ALB. Maximum expression levels were comparable to those of primary mouse hepatocytes.Soto-Gutierrez A and his colleagues made a more complex PU-based 3D scaffold，poly-amino-urethane (PAU)-coated nonwoven polytetrafluoroethylene(PTFE)fabric[43]. Human ESs cultured in this scaffold differentiated into hepatocyte-like cells with the abilities in metabolism of ammonia and lidocaine，as well as production of albumin and urea.They also found that the use of this complex scaffold allowed the human ESs to form 3D structure and cell-cell interactions.

4 3D culture systems and bioreactors

In recent years，different 3D culture systems have been investigated for hepatic differentiation of stem cells，including natural materials scaffolds and synthetic materials scaffolds.These 3D culture systems are all static culture systems，in which medium change were discontinuous and gas exchanges were conducted by diffusion from the surrounding.With the introduction of bioreactors，the culture systems can be defined as dynamic culture systems. The bioreactor consists of a cell compartmentperfused in a decentralized way，allowing the cells to be continuously supplied with nutrients and with a sustained gas exchange.

Matsumoto K and his team firstly and successfully introduced bioreactor in hepatic differentiation study，but no comparsion with 2D culture in thisstudy[42].Later，Miki T and his colleagues also utilized bioreactor in hepatic differentiation study.Compared with the conventional 2D static culture，the 3D perfusion culture systems induced more functional maturation to hESC-derived hepatocytes[44].The expressions of liver-specific genes (albumin，CYP3A4，CYP3A7，HNF4-α)in this 3D dynamic culture were higher than that in 2D static cultures. Hepatocyte-specific functions，such as albumin secretion and urea metabolism，were more active in this new system than in the conventional one.Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker-positive cells in the dynamic culture.There is a possibility that physical parameters such as flow，stress forces，and pressure might play a vital role in the development and differentiation of human ESC into lineage committed cells and tissue structures in addition to the 3D topological parameters.

Ji R and his colleagues combined a biomatrix scaffold with the bioreactor in the study of inducing murine MSCs into hepatic cells[45].They defined this culture system as dynamic cultured scaffold(DCS). This biomatrix scaffold was made from decellularized rat liver.It provided structural templates for cell attachment and proliferation，whereas the bioreactors provide environmental control.The data in this study demonstrate that the 3D biomatrix scaffold in dynamic culture with optimal flow rate promoted significantly better cell proliferation compared to the biomatrix scaffoldculturedinstaticorthemonolayerstaticculture system.WangYand his colleagues made a similar study with a different bioreactor.In their study，a complex 3D culture system consisted of biodegradable PLLA-PGA polymer scaffolds，growth-factor-reduced Matrigel，and a rotating microgravity bioreactor(RMB)that allows simulated microgravity，was used to induce mouse ESCs into hepatic-like cells[46].Expressions of ALB，AFP，and CK18 at genes or proteins levels were more visible in 3D culture with RMB than that of without RMB.Hepatocyte-like functions，such as ALB production，cytochrome P450 activity，low-density lipoprotein uptake，were more active in 3D culture with RMB than that of without RMB.The role of RMB in this culture system was served to yield a 3D microgravity-simulating environment with low-shear，optimizing nutrition and gas exchange[47].These may be beneficial for the hepatic differentiation process.

Sivertsson L and hiscolleaguesutilized a perfused，4-compartmentbioreactorwith decentralized nutrientand gas supply to the cells，mimicking the blood flow in the liver.DE from human ESCs was inoculated into bioreactors or seeded in 2D controls[48].The results showed that the differentiation of human ESC in this specific bioreactor 3D culture system generated hepatocytelike cells that expressed a larger number of hepatocyte-specific characters compared to conventional 2D cultures.

Most recently，Vosough M applied a carrier-free suspension stirred bioreactor to promote the hepatic differentiation of human pluripotentstem cells (PSCs)[49].The differentiated hepatocyte-like cells had multiple features of HP，for example，the expression patterns of liver-specific marker genes(HNF4a，AFP，ALB， TAT， CK18)， albumin secretion， urea production，collagen synthesis，indocyanin green and LDL uptake，glycogen storage，and inducible cytochrome P450 activity.But the urea production efficiency was only 1/10 of that of PHs.

5 Summary

The general functions of hepatocyte-like cells differentiated from stem cells in 3D culture systems are better than that in 2D culture.However，those cells are not fully mature compared with the PHs.Therefore，a deeper understanding of the mechanisms that make 3D cultures more efficient than 2D，and the inducing efficiency of different 3D culture systems，may be helpful to develop well-defined and more efficient protocols for the hepatic differentiation of stem cells. Future studies are needed to improve the efficacy and consistency of hepatic differentiation in 3D culture systems，and more importantly，large clinical trials need to be initiated to validate the therapeutic potential of thosehepatocytesdifferentiatedfromstemcells.

Acknowledgment

This work was supported by the Science and Technology Commission of Shanghai Municipality (13JC1407402).

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Developments of 3D culture systems for improving the efficacy of hepatic differentiation from stem cells：a review in current progress

NONG Kate1，AI Kaixing1，2
1.Department of General Surgery，Shanghai Jiao Tong University Affiliated Sixth People’s Hospital，Shanghai 200233，China；2.Department of General Surgery，Tongji Hospital，Tongji University Shool of Medicine，Shanghai 200065，China

Functional hepatocytes have been used in researches on cell transplantation，bio-artificial liver therapy，liver tissue engineering，drug metabolism and toxicity.Primary hepatocytes(PHs)，as a source of functional hepatocytes，cannot meet the growing demands in those researches.Functional hepatocytes differentiated in 2D or 3D culture systems from stem cells are considered an alternative cell source.3D culture systems，mimicking the biological environment in vivo，have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional 2D culture systems.Here，we review the advances in researches of hepatic differentiation from stem cells in different 3D culture systems，focus on the materials used to make the 3D culture scaffolds.

3D culture scaffolds；Hepatic differentiation；Stem cells

R318.08

A

2095-378X（2015）02-0114-09

10.3969/j.issn.2095-378X.2015.02.012

上海市科学技术委员会资助项目（13JC1407402）

农卡特（1983—），男，硕士，研究普外科肝胆胰

艾开兴，电子信箱：akxing8258@126.com