Network formation through active migration of human vascular endothelial cellsin a multilayered skeletal myoblast sheet

Eiji Nagamori a, Trung Xuan Ngo b, Yasunori Takezawa b, Atsuhiro Saito c, Yoshiki Sawa c, Tatsuya Shimizu d,Teruo Okano d, Masahito Taya b, Masahiro Kino-oka a,*

aDepartment of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, JapanbDivision of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, JapancDepartment of Surgery, Division of Cardiovascular Surgery, Graduate School of Medicine, Osaka University, 2-15 Yamada-oka, Suita, Osaka 565-0871, Japand Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

a r t i c l e i n f o

Article history:Received 10 August 2012Accepted 23 August 2012Available online 30 October 2012

Keywords:Cell sheetVascular endothelial cellsSkeletal myoblastsAngiogenesisCell migrationImage processing

a b s t r a c t

Autologous transplantation of myoblast sheet has attracted attention as a new technique for curingmyocardial infarction. Myoblast sheet has the ability to secret cytokines that improve heart function viathe facilitation of angiogenesis on affected part. To mimic the in vivo angiogenesis in the myoblast sheetafter transplantation, a five-layered cell sheet of human skeletal muscle myoblasts (HSMMs) was overlaidon human umbilical vein endothelial cells (HUVECs) which enables evaluation of dynamic HUVECbehavior. HUVECs existing initially at the bottom of the sheet changed to be a stretched shape andmigrated upward compared with the surrounding HSMMs in the sheet. Prolonged incubation resulted innetwork formation of HUVECs in the middle of the sheet, although non-networked HUVECs continued tomigrate to the top of the sheet, which meant the spatial habitation of HUVECs in the cell sheet. Imageprocessing was performed to determine the variation in the extent of network formation at differentHUVEC densities. It was found that the extent of formed network depended on the frequency ofencounters among HUVECs in the middle of the sheet. The present system, which can evaluate networkformation, is considered to be a promising in vitro angiogenesis model.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Cell sheet engineering has been proposed to be a promisingtechnique to form plate-shaped aggregates, which are thought tomimic tissues available for transplantation [1]. A temperature-responsive poly-(N-isopropylacrylamide) (PNIPAAm)-graftedsurface can be used to harvest a cell sheet without enzymaticdigestion of the intact extracellular matrix on the detached surface[2] to enable the construction of three-dimensional (3-D) multi-layered tissues without scaffolds [3].

Recently, autologous transplantation of myoblast sheet hasattracted attention as a new technique for curing myocardialinfarction, which is associated with cardiomyocyte dysfunction andirreversible cell loss [4,5]. Skeletal myoblasts, which are relativelyeasy to harvest from patients, can undergo self-renewal anddifferentiation, allowing cardio muscle regeneration upon injury.Myoblast sheet also has the ability to secret cytokines that improve

heart function via the facilitation of angiogenesis and attraction ofprogenitors on affected part. The sheet transplantation method canovercome disadvantages of the myoblast injection method, such asloss of transplanted cells due to poor survival of cells [6] andarrhythmic heart beat due to a global down-regulation of connexin43 in the host heart [7]. Sawa et al. conducted the first clinical trialof the transplantation using myoblast sheet that enables theeffective delivery of a sheet for a large coverage area to providethe improvement of damaged heart function in vivo withoutarrhythmic heart beats [8]. Further development of transplantationusing the multilayered myoblast sheets showed the improvedheart function when compared with monolayer sheet trans-plantation [9].

From a manufacturing point of view, process and qualitycontrols are important for realizing commercialization of cell sheettransplant. Many studies have addressed cell sources, culture,sheet assembling, and in vivo animal tests; however, the methodfor quality control of myoblast sheets, especially for transplantefficacy such as angiogenesis capability, has not been systematized.Although animal tests can be used to estimate the overall efficacyof the transplants, such methods provide an insufficient estimation

* Corresponding author. Tel.: þ81 (0) 6 6879 7444; fax: þ81 (0) 6 6879 4246.E-mail address: kino-oka@bio.eng.osaka-u.ac.jp (M. Kino-oka).

Reproduced from Biomaterials 34: 662-668 (2013).

145

of product quality for autologous transplantation because of thelack of quantitative analysis and non-human estimation as well aspatient dependence. Thus, it is necessary to create a method that issuitable for in vitro quantitative estimation of the transplants.

In a previous study, a five-layered human skeletal musclemyoblast (HSMM) sheet was constructed to determine sheetfluidity by confocal laser scanning microscopy with image pro-cessing [10]. In the present study, an in vitro system based ona multilayered HSMM sheet with human umbilical vein endo-thelial cells (HUVECs) was developed to mimic the in vivoangiogenesis in the HSMM sheet after transplantation. Imageprocessing was performed to evaluate the HUVEC spatial distri-bution and network formation in the HSMM sheet to elucidatethe spatial habitation arising from HUVEC migration andconnection.

2. Materials and methods

2.1. Cell preparation

HSMMs (Lot. No. 4F1618; Lonza Walkersville Inc., Walkersville, MD) andHUVECs (Lot. No. 4F0709; Lonza Walkersville Inc.) were used in the experiments.According to procedures described elsewhere [11], subcultures of HSMMs onlaminin-coated surfaces were conducted at 37 �C in an atmosphere of 5% CO2 inDulbecco’s Modified Eagle’s Medium (DMEM; SigmaeAldrich, St. Louis, MO) con-taining 10% fetal bovine serum (FBS; Invitrogen, Grand Island, NY) and antibiotics(100 U/cm3 penicillin G, 0.1 mg/cm3 streptomycin, and 0.25 mg/cm3 amphotericin

B; Invitrogen). HUVECs were cultured in a commercially available medium (EGM-2;Lonza Walkersville Inc.). The medium depth was set to 2 mm throughout theexperiments.

2.2. Incubation of five-layered HSMM sheet with HUVECs

Five-layered HSMM sheet was fabricated according to a previously reportedmethod [10]. In brief, as shown in Fig. 1A, starter HSMMs prepared by subculturingwere stained with CellTracker� Orange (Invitrogen) to obtain fluorescent orangecells according to a commercially recommendedprotocol (5 mMfor 15min for live cellimaging). The HSMMswere seeded at 2.3�105 cells/cm2 in eachwell (1.9 cm2) of 24-well UpCell� plates (CellSeed, Tokyo, Japan) with a temperature-responsive surfacegraftedwith PNIPAAmand incubated for 24 h at 37 �C in a 5% CO2 atmosphere to formthe monolayer sheet. To stack monolayer sheets to fabricate the multilayered cellsheet, the customized stamps with the gelatin gel (G1890-100G; SigmaeAldrich)were used. To harvest the monolayer sheet, the stamp with the gelatin gel wasoverlaid on themonolayer sheet in awell at 37 �C, and the temperaturewas shifted to20 �C. After 30min, the stampwas lifted together with themonolayer sheet from thebottomsurfaceof thewell. The stepswere then repeated for the sequential harvestsofmonolayer sheets to form themultilayered construct on the stamp. Themultilayeredsheetwith the gelatinwas peeled from the stamp for transfer to a dish containing theprecultured HUVECs. For the preculture, HUVECs were seeded into culture dishes(35 mm in diameter; Corning Inc., NY) and cultured in the EGM-2 medium for 24 h.The initial density of HUVECs (X0) for the subsequent incubation with the sheet wasset in the range of 0.35e3.32 � 104 cells/cm2 by changing the seeding density ofHUVECs (Xs). The relationship between Xs and X0 was determined in advance(Supplementary Table 1). After incubation at 20 �C for 2 h, the sheet was incubatedat 37 �C in DMEM containing 10% FBS for 1 h to remove the gelatin. The mediumcontaining the dissolved gelatin was exchanged with fresh medium. At the givenincubation time (t), triplicate samplings were performed for quantitative analysis.During the incubation period, the mediumwas renewed every day.

37oC

Removal of

gelatin

20o

C, 30 min

HSMMs

37oC, 24 h

Gelatin gel

2.3×105cells/cm2LiftingOverlaying

37oC, 1 h20

oC, 2 h

Repetion of the processes

Pealing

Fluoresce staining

XY Z

UpCell

Thermo-responsive surface

35-mm

culture dish

Five-layeredcell sheet

Observation

Harvesting

Transfer

HUVECs

Incubation

HUVECs

network

A

0.2 to 2.0×105 cells/cm2

37oC, 24 h

Removal of

gelatin

20o

C, 30 min

SMMs

37oC, 24 h

Gelatin gel

2.3×105cells/cm2LiftingOverlaying

Repetion of the processes

Pealing

Fluoresce staining

XY Z

UpCell

mo-responsive surfrr ace

m

Five-layeredcell sheet

Observation

Harvesting

Transfer

HUVECs

Incubation

HUVECs

network

×105 cells/cm2

B

Lowpass Segmentation Skeltonization

Total length, L (mm-1)

Pruning Removal

of noise

Total tip number, NT (tip/mm2)

Fig. 1. Schematic drawings of experimental procedures. A: Construction of multilayered HSMM sheet and incubation with HUVECs. B: Image process procedure to evaluate HUVECnetwork formation and maturation.

146

2.3. Immunostaining for HUVECs

The culture system containing the five-layered HSMM sheet with HUVECs waswashed with phosphate-buffered saline (PBS) and fixed in PBS containing 4%paraformaldehyde (Wako Pure Chemical Industries, Osaka, Japan) overnight. Thefixed specimen was then permeabilized in PBS containing 0.1% Triton X-100 (WakoPure Chemical Industries) for 12min, washed twicewith PBS, and then blocked in 1%bovine serum albumin (BSA; Wako Pure Chemical Industries) in PBS for 1 h. Thespecimen was labeled with a primary antibody (monoclonal mouse anti-humanCD31 antibody; DAKO, Glostrup, Denmark) in 1% BSA solution overnight. Thespecimen was then thoroughly washed with PBS and immersed in 1% BSA solutioncontaining the secondary antibody conjugated with Alexa Fluor� 488 (Invitrogen)for 1 h.

2.4. Evaluation of the HUVEC network formed inside the HSMM sheet

The image capture was carried out using a 10� objective lens of confocal laserscanning microscope (FV-300; Olympus, Tokyo, Japan) at more than 8 positions ineach sample. As shown in Fig. 1B, each image was 8-bit gray scale with a size of256� 256 pixels and covered an area of 942� 942 mm. The imageswere subjected toimage processing (Image-Pro Plus; Media Cybernetics Inc., Bethesda, MD) usinga low-pass filter for primary noise removal and binarization with a certain intensitythreshold. The threshold intensity was determined as the average of the modeintensity and the automatic threshold intensity calculated with an Otsu adaptivethreshold algorithm [12], which chooses the threshold to minimize the intra-classvariance of the thresholded black and white pixels with an exhaustive search. Thebinary images were subjected to skeletonization to produce lined objects, thesecondary noise removal with a size threshold to remove items with a size of lessthan 16 pixels, and the pruning of small branches in the objects. The total length ofthe network per image area (L; cm�1), and the number of total tips of the network(NT; tip/cm2), were measured to estimate the extent of the HUVEC network (L/NT;cm/tip). The tips existing at the edge of the image were not counted.

2.5. Spatial distribution of HUVECs and HSMMs in five-layered HSMM sheets

To determine the vertical distribution of the HUVECs inside the HSMM sheet, thegreen cells (HUVECs labeled with Alexa Fluor 488) and orange cells (HSMMs stainedwith CellTracker� Orange) in each layer were observed and quantitatively analyzedas previously described [10] (Supplementary Fig. 1). The number of colored pixels ineach slice was counted. The green and orange pixels in each slice were normalizedusing the maximum green and orange pixel values, respectively, found in all of theslice images. Slices possessing more than 10% of the colored pixels were regarded toexist inside the cell sheet, fromwhich the vertical positions at the top and bottom ofthe five-layered sheet, and the sheet thickness, h (mm), were determined. The greenpixels inside the sheet were normalized to determine the vertical distribution of thegreen pixels by dividing them into 5 layers. The normalized distribution of the greenpixels was assumed to be equivalent to the distribution of green cells in the sheet,which was determined as the frequency of green cells, fG (e), in each layer. Fordetermination of the vertical distribution of the HSMMs, HSMMs stained withCellTracker� Green (Invitrogen) were placed in the bottom layer of a five-layeredHSMM sheet, and their vertical distribution at 24 h was determined.

3. Results

3.1. HUVEC network formation inside the HSMM sheet

The behavior of HUVECs in the five-layered HSMM sheet wasobserved for 120 h to estimate the growth and network formationaccording to the parameters L, NT, and L/NT. The initial density ofHUVECswas set at X0¼ 1.29� 104 cells/cm2. At the beginning of theincubation period (t ¼ 0), as shown in Fig. 2, the HUVECs wereobserved to be single and round-shaped with podia. At t ¼ 24 h,

A 0 h

24 h

48 h

96 h

120 h

B

Incubation time (h)

L/N

T (c

m/ti

p)N

T (tip

/cm

2 )L (c

m-1

)

0

0.1

0

8000

0

200

300

100

4000

0.2

0.3

20 40 60 80 100 120 140

Fig. 2. Time course of HUVEC network formation inside the five-layered HSMM sheet at an initial HUVEC density (X0) of 1.29 � 104 cells/cm2. A: Horizontal images of HUVECmorphology. Scale bar: 200 mm. B: Evaluation of HUVEC network formation with image processing. L: total length (cm�1), NT: total tip number (tip/cm2), L/NT: extent of networkformation (cm/tip). The bars show the standard deviation (SD) (n ¼ 3).

147

most of the HUVECs were found to have elongated for initiation ofmigration in the sheet, resulting in an increase in L. With furtherprolongation of the incubation period beyond t¼ 48 h, the level of Lwas constant, i.e., the HUVECs did not grow further. In addition, theHUVECs began to encounter and connect with each other, resultingin a decrease in NT until t ¼ 96 h. Smooth connections of theHUVECs appeared at t ¼ 96 h, which suggested the maturation ofthe HUVEC network. These behaviors were reflected by an increasein L/NT although a slight decay of the connections occurred att ¼ 120 h. The maximum value of L/NT was ¼ 0.22 � 0.07 cm/tip att ¼ 96 h, which was 5.6 times higher than the value at t ¼ 24 h.

3.2. Vertical migration of HUVECs during network formation

During network formation, the HUVECs were initially localizedat the bottom of the HSMM sheet and then vertically migrated intothe inner portion of the sheet to form aggregates with a lumenstructure inside the sheet (Supplementary Fig. 2). To estimate the

vertical migration of the HUVECs, their spatial distribution patternwas obtained as shown in Fig. 3. At the beginning (t ¼ 0), thefrequency of green cells (HUVECs) fG was the highest in the firstlayer from the bottom surface, i.e., most of HUVECs were located atthe bottom of the HSMM sheet. A broader distribution of fG wasobtained at t ¼ 24 h, meaning the HUVEC migration toward theupper layers of sheet. In addition, the migration of the HUVECs wasmuch faster than that of HSMMs because of the broader distribu-tion of the HUVECs at t ¼ 24 h (Supplementary Fig. 3). At t ¼ 96 h,HUVECs inhabited the middle layer, with fG ¼ 0.40 and 0.30 in thesecond and third layers, respectively, which were higher than thevalues in the corresponding bottom and top layers. Fig. 3B andSupplementary movie 1 depict the aggregate shape of the HUVECsin the sheet at t ¼ 96 h. The HUVECs in the middle layer formeda net-shaped aggregate to generate the network, whereas those inthe top layer formed an island-shaped aggregate, which indicatesthat the shape of the HUVEC aggregate depended on their locationin the sheet.

Order of sheet layer from bottom (-)

fG

(-)

1 2 3 4 50

0.5

1

1 2 3 4 50

0.5

1

1 2 3 4 50

0.5

1

1 2 3 4 50

0.5

1

1 2 3 4 50

0.5

1

A

B

0 h 24 h 48 h

96 h 120 h

Order of sheet layer from bottom (-)

fG

(-)

Fig. 3. Vertical analysis of HUVEC network formation inside the five-layered HSMM sheet at an initial HUVEC density (X0) of 1.29 � 104 cells/cm2. A: Time course of the spatialdistribution of HUVECs (fG) in each layer of the HSMM sheet. Bars show the standard deviation (SD) (n ¼ 3). B: Three-dimensional image of HSMM sheet showing different shapes ofHUVEC aggregates at different positions in/on the sheet at t ¼ 96 h. The white arrow indicates net-shaped aggregation (network) in the middle layer of the cell sheet. The red arrowindicates island-shaped aggregation in the top layer on the cell sheet. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version ofthis article.)

148

Supplementary video related to this article can be found athttp://dx.doi.org/10.1016/j.biomaterials.2012.08.055.

3.3. Influence of HUVEC seeding density on migration and networkformation

To evaluate the HUVEC connectivity in the sheet, the HUVECswere incubated for t ¼ 96 h with initial densities rangingfrom X0 ¼ 0.35 � 104 to 3.32 � 104 cells/cm2. As shown inFig. 4, with an increase in X0, L/NT increased to a maximum valueof L/NT ¼ 0.484 cm/tip at X0 ¼ 2.20 � 104 cells/cm2, which was2.3 times higher than the value at X0 ¼ 1.29 � 104 cells/cm2.In addition, the length and tip number of the HUVECs wereestimated on the basis of the parameters such as specific length(L/X0) and specific tip number (NT/X0), respectively. The L/X0and NT/X0 decreased with an increase in X0 by L/X0 ¼ 9.0 � 10�3

cm/cell and NT/X0 ¼ 5.0 � 10�2 tip/cell at X0 ¼ 3.32 � 104 cells/cm2, which were 3 and 30 times lower than the values atX0 ¼ 0.35 � 104 cells/cm2, respectively.

The fG value of HUVECs at 96 h in the first, third, and fifth layerswith respect to the initial density is shown in Fig. 5. In the fifth layer(top layer), with an increase in X0, fG decreased to 0.07 at

X0 ¼ 1.29 � 104 cells/cm2, which was half of the value atX0 ¼ 0.35 � 104 and 0.69 � 104 cells/cm2. A further increase in X0resulted in a slight increase of fG to 0.10 at X0¼ 3.32�104 cells/cm2.This trend was contrary to that observed in the third layer (middlelayer).

0.35×104 cells/cm2

0.69×104 cells/cm2

1.29×104 cells/cm2

2.20×104 cells/cm2

3.32×104 cells/cm2

0.69×104 cells/cm

1.29×104 cells/cm

2.20×104 cells/cm

3.32×104 cells/cm

A B

Initial density, X0 (104 cells/cm2)

L/N

T (c

m/ti

p)L/X

0 (c

m/c

ell)

1.0 2.0 3.00

0.5

0

1

0

0.02

0.04

NT/

X0

(tip/

cell)

Fig. 4. Network formation at 96 h with different initial densities of HUVECs (X0) ranging from 0.35 � 104 to 3.32 � 104 cells/cm2. A: Horizontal images of HUVEC morphology. Scalebar: 200 mm. B: Evaluation of HUVEC network formation with image processing. L/X0: specific length (cm/cell), NT/X0: specific tip number (tip/cell), L/NT: extent of networkformation (cm/tip). The bars show the standard deviation (SD) (n ¼ 3).

fG

(-)

Initial density, X0 (104 cells/cm2)1 2 3

0

0.2

0.4

Fig. 5. Spatial distribution of HUVECs at 96 h with different initial densities of HUVECs(X0) ranging from 0.35 � 104 to 3.32 � 104 cells/cm2. Frequency of HUVECs (fG) in thetop (square), middle (triangle) and bottom (circle) layers are shown against X0. Barsshow the standard deviation (SD) (n ¼ 3).

149

4. Discussion

Cell migration in 3-D constructs plays an important role inphysiological and pathological phenomena such as embryonicdevelopment, cell alignment, immune reactivity, angiogenesis, andmetastasis [13]. Determining the mechanisms underlying cellmigration in 3-D tissues having fluidity will be useful for designingbiomimetic structures and functionally engineered tissues.Although the behaviors of cells on two-dimensional culturesurfaces or in static 3-D scaffold have been extensively investigated,cell movement in fluidic 3-D tissues, especially vertical migrationinside the tissue, has not been well addressed because of theabsence of in vitro methods which enable quantitative and repro-ducible measurements. In the present study, a five-layered HSMMsheet was fabricated as a 3-D model to evaluate vertical cellmigration by confocal laser scanning microscopy with image pro-cessing. We also established the mimic system of transplantationconsisting of HUVECs on a culture dish that represents target cellsat the lesion site as well as a five-layered HSMM sheet as thetransplant tissue. This system can be used to model angiogenesisafter transplantation. The HUVECs that were initially localized atthe bottom of the HSMM sheet vertically migrated into the innerportion of the sheet. The migrating HUVECs encountered andconnected with other HUVECs in the middle layer of the sheet toform a network (Figs. 2 and 3). In our previous study [10], the activefluidity by cellular migrations of HSMMs in the horizontaland vertical directions in the five-layered HSMM sheet wasobserved. The results of the present study revealed that the verticalmigration of HUVECs was higher than that of HSMMs(Supplementary Fig. 3), demonstrating the active migration ofHUVECs in a fluidic sheet.

The formation of HUVEC network regarded as a net-shapedaggregate was localized in the middle layer of the sheet owing tothe encountering of HUVECs and their linear connections, while theunconnected HUVECs continued to migrate toward the top layer,forming the island-shaped aggregate (Fig. 3). These results show

that the spatial habitation of HUVECs occurred in the top andmiddle layers of the sheet by their encountering and connection. Inaddition, the shape of the HUVEC aggregates depended on theirlocation in the sheet. The further performance of HUVEC behaviorat different densities supported the habitation (Figs. 4 and 5),revealing that the HUVECs were localized both in the middle andtop layer at the low initial densities (X0 ¼ 0.35�104 and 0.69 � 104

cells/cm2), while only in the middle layer at the higher initialdensities (X0 ¼ 1.29 � 104 and 2.20 � 104 cells/cm2). These resultsled to the mechanism for the spatial inhabitation as follows. Asshown in Fig. 6, the frequency of encountering of HUVECs at a lowconcentration caused less connectivity in the middle layer of thesheet, and unconnected free HUVECs continued tomigrate upward.With an increase in the HUVEC density in the sheet, the HUVECsexhibited a higher frequency of encountering to connect each other(at a moderate concentration). Excessive existence of HUVECs leadsto less sites for their connection although higher frequency ofencountering occurred, resulting in the larger number of HUVECsmigrating toward top layer.

The dependence of the shape of the HUVEC aggregate on thelocation of the cells in the sheet was reported in previous studies.Asakawa et al. reported that HUVECs in multilayered sheet ofhuman dermal fibroblasts formed a network with a tubular struc-ture when the HUVECs were initially set in the middle layer of thesheet [14]. On the other hand, the introduction of HUVECs in the toplayer of the sheet generated the island-shaped aggregates; thus theHUVEC habitation in the top and middle layers depended on theinitial location of the HUVECs in the sheet. In the present study, theHUVECs outside the sheet did not grow in DMEM including 10% FBSdue to no additional cytokines (data not shown). Thus, the networkformation in the sheet could be attributed to the local feeding ofcytokines fromHSMMs. Candidate cytokines secreted fromHSMMsare considered to be vascular endothelial growth factor, hepatocytegrowth factor and fibroblast growth factor [15] which affectendothelial cell division, migration, and connection mediated byVE-cadherin expression [16e18].

Encountering of HUVECs and making connection

Habitation in middle and top layers

Net-shaped aggregate

Island-shaped aggregate

Migration of HUVECs toward top layer due to less encountering each other

Habitation in middle layer

Low concentration of HUVECs (X0

= 0.35 × 104and 0.69 × 10

4cells/cm

2)

Migration of excessive HUVECs toward top layer due to less connection sites

Middle concentration of HUVECs (X0

= 1.29 × 104

and 2.20 × 104cells/cm

2)

High concentration of HUVECs (X0

= 3.32 × 104cells/cm

2)

Elongation of their shape and starting vertical migration

HUVECs

HSMMs

Fig. 6. Schematic illustration showing the probable behaviors of HUVECs in the HSMM sheet with various HUVEC initial densities.

150

The fabrication of aggregates, which is a minimum unit exhib-iting tissue function, has been required to propose angiogenesismodels [19]. A sphere-shaped aggregate is widely used as a plat-form for 3-D models or bio-mimic assay. However, histologicalanalysis is necessary to evaluate the HUVEC network in the sphere-shaped aggregate, leading to a difficulty in observation [20e22].Our system with a widespread HUVEC network in a multilayeredsheet regarded as a plate-shaped aggregate enables the indepen-dent observation of the vertical and horizontal distributions of cellsin 3-D constructs via one-dimensional (Z-axis) and two-dimensional (XY-axis) analyses, being a powerful tool for in vitroangiogenesis assays, which can evaluate the HUVEC behaviors suchas migration, connection, as well as the formation of an endothelialnetwork in a 3-D construct.

5. Conclusion

A five-layered HSMM sheet with HUVECs which mimic thein vivo transplantation system was developed. The HUVECs thatwere initially localized at the bottom of the HSMM sheet, exhibitedvertical migration into the inner portions of the sheet, and formednetworks in the middle layer of the sheet. The quantitative analysisof the vertical distribution of HUVECs in the sheet clarified theprocess of their network formation. Spatial habitation of the HUVECnetwork in the middle layer of the sheet was observed andthe extent of the network formation depended on the frequencyof encountering between HUVECs. Our system including wide-spread formation of HUVEC network in a fluidic scaffold and itsevaluation method might be applicable not only for the in vitroconstruction of pre-vascularized tissue but also for in vitro angio-genesis models.

Acknowledgments

This study was supported by the New Energy and IndustrialTechnology Development Organization (NEDO) of Japan, the JapanSociety for the Promotion of Science (JSPS) through the “FundingProgram for World-Leading Innovative R&D on Science and Tech-nology (FIRST Program),” initiated by the Council for Science andTechnology Policy (CSTP), and Grant-in Aids for Scientific Research(Nos. 21360402 and 24360341) fromMinistry of Education, Culture,Sports, Science and Technology.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2012.08.055.

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