This report describes the development of endothelial cell (EC) cultivation devices with different channel widths (60 to 360 μm). Crucial features of the devices include even cell distribution along the channel, seeding reproducibility, and compatibility with microscopy and flow application. The main achievement of this work is the design of chips which allow reproducible HUVEC culture in narrow (&lt; 400 μm) channels.</p

Grajewski, Maciej

Molema, Grietje

Mulder, Patty P.M.F.A.

Verpoorte, Elisabeth

English

Dissertations of the University of Groningen

   University of GroningenEffect of channel width on human umbilical vein endothelial cell (HUVEC) culture inmicrofluidic channelsGrajewski, Maciej; Mulder, Patty P.M.F.A.; Molema, Grietje; Verpoorte, ElisabethPublished in:18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2014Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Grajewski, M., Mulder, P. P. M. F. A., Molema, G., & Verpoorte, E. (2014). Effect of channel width onhuman umbilical vein endothelial cell (HUVEC) culture in microfluidic channels. In 18th InternationalConference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 (pp. 506-508). (18thInternational Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014).Chemical and Biological Microsystems Society .https://www.rsc.org/images/loc/2014/PDFs/Papers/163_0319.pdfCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.EFFECT OF CHANNEL WIDTH ON HUMAN UMBILICAL VEIN ENDOTHELIAL CELL (HUVEC) CULTURE IN MICROFLUIDIC CHANNELS Maciej Grajewski1, Patty P.M.F.A. Mulder1, Grietje Molema2, and Elisabeth Verpoorte1 1Pharmaceutical Analysis, Groningen Research Institute of Pharmacy,  University of Groningen, The Netherlands 2Endothelial Biomedicine and Vascular Drug Targeting, Department of Pathology and Medical Biology, University Medical Center Groningen, The Netherlands   ABSTRACT This report describes the development of endothelial cell (EC) cultivation devices with different channel widths (60 to 360 µm). Crucial features of the devices include even cell distribution along the channel,  seeding reproducibility, and compatibility with microscopy and flow application. The main achievement  of this work is the design of chips which allow reproducible HUVEC culture in narrow (< 400 µm)  channels.  KEYWORDS: microfluidic cell culture,  narrow microchannels, endothelial cells  INTRODUCTION ECs line all blood vessels and play a crucial role in trafficking molecules between the blood stream  and tissues. Therefore, they play a fundamental role in the regulation of drug transport from the blood stream to tissues, tumor neovascularization and inflammatory response [1]. Microchannels mimic the  in vivo environment of ECs better than well-plates due to matching dimensions. The additional possibility  of flow application makes microchannels well suited to endothelial biology studies [3]. However,  channels reported in the literature have large widths (often ≥ 400 µm) [3], whereas our interest lies  in investigating microvasculature. Small blood vessels are characterized by substantial molecular  exchange with blood because of low fluid velocities. The behavior of endothelium is strongly influenced  by its local environment [2]. However, seeding EC into channels as small as 60 µm is difficult, with cells  tending to distribute unevenly along channels. Moreover, the availability of primary endothelial cells from  microvasculature is limited [4], so cell concentrations and amounts used for culture must  be significantly reduced compared to those employed in reported microfluidic devices [3]. Therefore,  we decided to develop new channel designs which address these issues. EXPERIMENTAL The three channel designs tested are summarised in Table 1. PDMS channels were fabricated  using masters prepared in SU-8 negative photoresist. They were irreversibly bonded to the sterile  microscopic glass slides by exposure to O2 plasma. Microchannels were coated with 1% gelatin  (crosslinked with 0.5% glutaraldehyde) as a substitute for extracellular matrix (ECM). The injected  volumes of cell suspension were experimentally adjusted to match internal device volumes. Cells were  cultivated in an incubator at 37°C and an atmosphere of 95% air and 5% CO2.   RESULTS AND DISCUSSION Reversibly bonded straight channels of uniform width and 2.5 cm length (Design 1, Table 1) were  successfully used previously under static conditions [5], but started to leak upon application of flow.  Therefore, new, irreversibly bonded channel designs were tested, which exhibit significant improvement  of cell load and distribution in the channels (Designs 2 and 3, Table 1). Design 2 has decreased reservoir  diameters (2.5 mm down to 1 mm) and length (25 mm down to 10 mm) compared with Design 1.  978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001 506 18th International Conference on MiniaturizedSystems for Chemistry and Life SciencesOctober 26-30, 2014, San Antonio, Texas, USA Table 1: Layout of three channel designs with parameters tested for cell cultivation  Design 1 Design 2 Design 3 Sketch    Cultivation  channel length [mm] 25 10 10 Diameter of the reservoirs [mm] 2.5 1 1 Height of the channel [µm] 100 100 100 Tested widths [µm] 360, 120, 100, 80, 60 360, 120, 100, 80, 60 360, 120, 100, 80, 60      Figure 1: Overview of cell distribution in the three channel designs. Significant improvement of cell number and  distribution is visible for Design 3 compared to Designs 1 and 2 for all presented channel widths. Cells were  seeded at a  concentration of 5000 cells/µL. Cell suspension volumes introduced to the channels were as follows:  8 µL (360 µm wide channels); 6 µL (120 µm wide channels); and 3 µL (60 µm wide channels). All photos were  acquired 5 mm from the beginning of the cultivation channel 2h after cell seeding; capture area of the photos  is 977 x 590 µm. Magnification: 10x; scale bar: 200 µm 507   Figure 2: Overview of the number of cells attached in the channels with different widths (Figure 1) 2h after  seeding at a concentration of 5000 cells/µL. Design 3 shows the highest number of attached cells for all tested widths. Design 1 is unsuitable for cell cultivation in channels narrower than 360 µm; Design 2 shows satisfactory results for channels down to 100 µm wide;  x-axis represents widths of the channels, y-axis represents average  number of cells per mm2 [n=5].  RESULTS AND DISCUSSION Design 2 shows satisfactory results in a range of channel widths from 360 µm to 100 µm; however,  in narrower channels, the cell load and distribution is insufficient to obtain confluent cell culture (Fig. 1).  In Design 3, an inlet and outlet having half the width of the cultivation channel were  implemented to overcome this limit. These additional structures slow down fluid flow during cell  injection and create backpressure at the outlet from the cultivation channel, promoting retention of cells  in this channel. This improves cell adhesion to the ECM significantly compared to other designs (Fig. 2). Cell seeding becomes possible in channels as small as 60 µm wide (Fig. 1).   CONCLUSION Currently, the glass-PDMS chip is compatible with microscopy, which allows the monitoring  of e.g. inflammation processes in different channel widths. Moreover, sensors could be integrated into  devices to allow determination of chemical parameters related to endothelial dysfunction.  ACKNOWLEDGEMENTS The LiPhos project is partly funded by the European Commission (Contract No. 317916).  REFERENCES [1] Langenkamp et al., Cell Tissue Res 335:205–222 (2009) [2] Kuldo et al., Current Vascular Pharmacology, 3, 11-39, 2005. [3] Young EWK, Simons CA, Lab Chip 2010; 10:143-160. [4] Mulder et al., 14th microTAS, Groningen, The Netherlands, 1137 (2010).  [5] Mulder et al., Tranducers Research Foundation, San Diego, USA, 1383-1385 (2005). CONTACT Maciej Grajewski; phone: +31 50 363 6122; m.grajewski@rug.nl 508

Effect of channel width on human umbilical vein endothelial cell (HUVEC) culture in microfluidic channels

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Effect of channel width on human umbilical vein endothelial cell (HUVEC) culture in microfluidic channels

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