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PDMS 마이크로 스텐실이 내재된 세포공동배양 미세유체 장치 제작

최진호1, 김동욱2, 김철민3, 김규만2,#orcid

Fabrication of a Microfluidic Device with an Embedded PDMS Microstencil for Co-Culture of Cells

Jin Ho Choi1, Dong Wook Kim2, Chul Min Kim3, Gyu Man Kim2,#orcid
Journal of the Korean Society for Precision Engineering 2022;39(10):731-737.
Published online: October 1, 2022

1 ㈜케이아이플렉스

2 경북대학교 기계공학부

3 경상국립대학교 메카트로닉스공학부

1 KIFLEX Co., Ltd. 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#E-mail: gyuman.kim@knu.ac.kr, TEL: +82-53-950-7570
• Received: July 5, 2022   • Revised: August 25, 2022   • Accepted: August 26, 2022

Copyright © The Korean Society for Precision Engineering

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • In this paper, a microfluidic co-culture system comprising an embedded polydimethylsiloxane (PDMS) microstencil was fabricated. The fabricated co-culture system has two micro-channels separated with a PDMS microstencil membrane. Master molds for microchannels and stencil membranes were fabricated by photolithography, then used for casting of PDMS devices. The stencil membrane was 10 thick, with holes 10-μm large in diameter. The fabricated system co-cultured two types of cells (HepG2, NIH-3T3 Cells) successfully for seven days. The viability and stability of the cells were verified through LIVE/DEAD® staining and analysis. Additionally, albumin secretion of HepG2 cells was measured for seven days, using an HSA ELISA kit. The measured data were analyzed, to compare the activity of HepG2 cells. Results confirmed that cells can be co-cultured in the fabricated microfluidic system.
  • KEYWORDS: PDMS microstencil, Cell culture, Co-culture, Microfluidic device, Porous membrane
  • KEYWORDS: PDMS 마이크로 스텐실, 세포배양, 공동배양, 미세유체 장치, 다공성 막
살아있는 조직으로부터 분리된 세포를 성장, 증식하기 위해서는 제어된 외부 조건에서 세포를 배양한다. 세포, 특히 동물세포의 배양을 통하여 여러 종류의 단백질을 생성하거나 조직(Tissue)을 만들 수 있다. 현탁액과 같이 배양액에 떠다니면서 배양되는 부유 세포가 있는 반면에 대부분의 세포들은 표면에 부착되어서 배양되는데 이러한 부착 세포들의 부착이 용이하도록 표면 처리된 배양 접시가 주로 사용된다. 그리나 이러한 배양 접시를 사용하는 세포배양은 평편한 2차원 표면을 사용함으로써 실제 생물체 내(In-Vivo) 환경에서 세포가 자라는 것과 차이가 존재한다. 따라서 생체 내 환경과 유사한 3차원 환경에서 세포를 배양하기 위한 3차원 배양 시스템에 대한 연구가 진행되고 있다.
세포배양을 위하여 다양한 마이크로 기술이 사용되고 있으며, 그중 하나로 마이크로 스텐실을 들 수 있다. 마이크로 스텐실을 일종의 템플릿으로 사용하여 세포가 표면의 국부적인 영역에 국한되어 부착하도록 유도함으로써 다양한 세포의 패터닝이 가능하다. 또한, 마이크로 유체 칩을 세포배양에 적용할 수 있다. 세포가 배양되는 마이크로 챔버와 배지가 공급되는 마이크로 채널을 다양한 형태로 설계, 제작할 수 있고 세포의 동적 배양이 가능하다. 이러한 마이크로 기술은 세포의 위치 및 배양 환경 등을 정밀하게 제어함으로써 세포에 관한 다양한 In Vitro 연구에서 체계적이고 공학적인 접근을 제공할 수 있다[1-12].
최근에는 마이크로 유체 칩 상에 다양한 장기 세포를 배양함으로써 체내 장기(Organ)를 모사하는 다양한 장기 칩에 대한 관심이 증가하고 있다[13]. 실제 체내에서는 여러 가지 세포가 함께 존재하면서 서로 물질교환을 하고 있으며, 이러한 체내 상태를 재현하기 위해서는 다중 세포를 공동배양하기 위한 시스템 개발의 필요성이 높아지고 있다[13-18].
세포 공동배양 시 이종 세포 사이의 상호영향과 다양한 반응을 분석하기 위하여 이종 세포들은 서로 분리막으로 분리되어 배양되며 분리막을 통한 세포들 사이의 물질교환 및 세포성장에 미치는 영향 등에 대한 연구가 진행되고 있다[1-5,19-21]. 분리막은 세포들의 물리적인 분리와 동시에 물질 교환의 역할을 지닌다. 한편, 앞서 언급한 마이크로 스텐실은 작은 미세 패턴을 지닌 막의 형상이며, 패턴의 크기를 수 마이크로미터 크기로 작게 제작하는 경우 분리막의 특성을 지닐 수 있다. 따라서 본 논문에서는 물질교환이 가능한 미세 구멍 구조를 가지는 PDMS 마이크로 스텐실(Microstencil)을 분리막으로 적용하여 두 종류의 세포인 HepG2와 NIH-3T3 세포를 공동배양하는 시스템을 개발하고, 두 종류의 세포가 공동배양 시스템에서 배양이 가능함을 검증하기 위해 HepG2 세포의 알부민 측정을 통한 활동량과 생존율을 분석하는 연구를 수행하였다. 이러한 연구를 통해 개발된 공동배양 시스템은 세포연구의 비용 절감과 고효율 및 시간 절약으로 동물실험 및 임상시험을 대체할 수 있는 유용한 도구가 될 것으로 예상되며, 향후 바이오 과학(Bioscience) 및 세포 생물학(Cell Biology) 연구로 적용되어 장기 칩(Organ on Chip) 또는 세포 칩(Cell on a Chip) 연구 등으로 응용될 수 있을 것으로 기대된다.
공동배양 시스템은 포토리소그래피(Photolithography) 공정을 이용하여 마스터 몰드를 제작하고 PDMS 캐스팅 공정을 통하여 미세유체 채널과 PDMS 마이크로 스텐실을 제작하였다. Fig. 1은 공동배양 시스템의 모식도를 나타낸다. 시스템은 상하부를 구성하는 두 개의 미세유체 채널과 그 사이를 분리하는 분리막이 있는 구조이며, 분리막인 PDMS 마이크로 스텐실은 작은 공극(Pore) 패턴을 가진 얇은 멤브레인으로 미세유체 채널 간의 물질교환이 가능하다.
Fig. 1

Schematic image of co-culture system

KSPE_2022_v39n10_731_f001.jpg
2.1 마스터 몰드 제작
PDMS 마이크로 스텐실과 미세유체 채널의 제작을 위해 포토리소그래피 공정으로 마스터 몰드를 제작하였다. 미세유체 채널의 몰드 제작을 위하여 300 μm 두께의 SU-8_2150 포토레지스트(Photoresist) (Microchem, USA)를 실리콘 웨이퍼 위에 스핀코팅하였으며, 소프트베이킹(Soft Baking)을 65oC에서 8분, 95oC에서 67분간 수행하였다. 포토마스크를 얹고 UV광 노출(Exposure) 후에 다시 65oC에서 5분, 95oC에서 21분동안 포스트 베이킹을 진행하였다. SU-8 Developer (Microchem, USA)로 현상(Developing)하였으며, 이소프로필 알코올(Isopropyl Alcohol)로 세척(Rinsing)하였다. 이후 질소로 말려서 몰드를 완성하였다. PDMS 마이크로 스텐실의 몰드는 직경 10, 중심 간 피치(Pitch) 30 μm의 원형 패턴의 어레이(Array)로 이루어져 있고 어레이 면적은 15 × 30 mm이다. 모재와 마이크로 어레이 패턴 구조의 접착(Adhesion)을 돕기 위하여 모재 위에 15 μm 두께의 SU-8 레이어를 코팅한 이후 원형 패턴 어레이를 포토리소그래피로 생성하였다. 제작된 SU-8 몰드 표면에 Tridecafluouro-1, 1, 2, 2-Tetrahydrooctyl-1-Trichlorosilane (UCT, USA)로 표면 처리하여 이후 마스터 몰드로부터 PDMS 스텐실을 떼어내는데 용이하게 하였다.
2.2 미세유체 장치(Microfluidic Device) 제작
마스터 몰드로부터 캐스팅(Casting)하여 PDMS 재질의 미세 유체 장치를 제작하였다. 실리콘 엘라스토머(Silicon Elastomer, Sylgard 184)와 결합제(Curing Agent)를 10 : 1 비율로 혼합한 이후에, 진공 데시케이터(Vacuum Desiccator)에 60분 동안 두어 내부 버블(Bubble)을 제거하였다. 그 이후 90oC 오븐(Oven)에서 60분 동안 두어 경화(Curing)하였다. 경화된 PDMS 복제물(Replica)은 마스터 몰드로부터 떼어내고 개별 미세유체 장치로 잘라내었다. 미세유체 장치에 유체 주입구(Inlets), 배출구(Outlets) 등 구멍을 내었다.
2.3 PDMS 마이크로 스텐실 제작
PDMS 마이크로 스텐실(Microstencil)을 제작하기 위하여 에어나이프 시스템(Air Knife System)을 이용하였다[22,23]. 얇은 두께의 스텐실을 만들기 위해서는 PDMS 프리폴리머의 점도를 낮추어야 하는데, 이를 위해서 PDMS 프리폴리머와 헥사데칸(Hexadecane)을 1 : 4 비율로 혼합하였다. 포토리소그래피로 준비한 마스터 웨이퍼 위에, 준비된 PDMS 프리폴리머를 스핀코팅(Spin Coating)하고 에어나이프를 이용하여 질소(N2) 가스를 분사하여 마스터 패턴 위에 남아 있는 잔류 프리폴리머를 제거하였다. 이후에 몰드를 오븐에 넣고 90oC에서 60분 동안 두어 PDMS 프리폴리머를 경화하였다. 이를 통하여 직경 10의 원형 어레이 패턴을 지닌 두께 10 μm의 마이크로 스텐실을 제작하였다.
2.4 공동배양 시스템(Co-Culture System) 제작
제작된 PDMS 마이크로 스텐실과 상부 미세채널 부분과 하부 미세채널 부분을 구성하는 두 개의 PDMS 미세유체 장치를 결합하여 공동배양 시스템을 제작하였다. 우선 상부 미세채널 부분에 바이옵시 펀치(Biopsy Punch)를 사용하여 유체 주입구와 배출구를 형성한 이후에, 플라즈마 본딩(Oxygen Plasma Bonding) (Harrick Plasma, Ithaca, NY, USA)을 이용하여 PDMS 마이크로 스텐실을 결합하였다. PDMS 스텐실은 마스터 웨이퍼 상에서 상부 미세유체 장치와 결합한 후 웨이퍼에서 떼어내는데, 이는 PDMS 스텐실이 매우 얇아 마스터 웨이퍼에서 분리된 이후에는 다루기(Handling)가 어렵기 때문이다. 이후에 하부 미세채널 부분과의 결합은 동일하게 플라즈마 본딩을 이용하였다. 최종적으로 주입구와 배출구에 튜브(Tube)를 삽입하여 공동배양 시스템을 제작하였다(Fig. 2).
Fig. 2

Fabrication process of co-culture device and co-culture system image

KSPE_2022_v39n10_731_f002.jpg
제작된 공동배양 시스템은 직경 3의 중앙 세포배양 챔버(Chamber)가 하부 미세유체 채널에 있고, 상부 미세유체 채널에는 직경 5 mm의 관통형 챔버를 두었다. 상부와 하부 미세유체 채널의 폭은 300 μm이고, 튜브와 연결을 위한 1.5 mm 구멍을 바이옵시 펀치를 이용하여 생성하였다. 공동배양 테스트를 위해 하부 미세유체 채널에 NIH-3T3 세포를 배양하고, 상부 미세유체 채널에는 HepG2 세포를 배양하였다.
2.5 세포 배양 준비
HepG2 세포와 NIH/3T3 세포는 한국세포주은행과 American Type Culture Collection에서 각각 구매하였다. 두 세포들은 모두 Dulbecco’s Eagle’s Medium (DMEM; Gibco, Grand Island, NY)에서 배양하였으며, DMEM 용액에는 10% Fetal Bovine Serum (FBS; Gibco)과 100 units/mL Penicillin-Streptomycin (Gibco)가 첨가되었다. NIH/3T3와 HepG2 세포들은 모두 세포 인큐베이터에서 배양하였고, 배양기에는 37oC 가습된 환경과 5% CO2가 포함된 공기를 일정하게 제공하였다. 세포들의 성장 거동은 광학현미경(Optical Microscopy)과 형광현미경(Fluorescence Microscopy)으로 관찰하였다[19,24].
2.6 공동배양 시스템을 이용한 세포 배양
세포를 시딩(Seeding)하기에 앞서 플라즈마 장치로 표면을 개질하여 미세유체 장치를 결합한 다음, 75% 에탄올을 주입하여 12시간 동안 멸균하면서 공동배양 챔버의 에어버블(Air Bubble)을 제거하였다. 그리고 30분 동안 UV 살균을 진행하고 인산염 버퍼 용액(Phosphate Buffer Saline, PBS)으로 3차례 세척하였다. 미세유체 장치에 미세 채널의 친수성을 유지하기 위하여 세포 배양액(Cell Medium)을 담아 30분간 두었다. 첫 번째 세포로 NIH-3T3 세포를 하부 미세유체 채널(Lower Micro-Channel)에 마이크로피펫(Micropipette)으로 시딩한 다음, 세포의 안착을 위하여 하루 동안 인큐베이터에서 방치하였다. 그 후, 동일한 방법으로 HepG2 세포를 상부 미세유체 채널(Upper Micro-Channel)에 시딩하였다. 공동배양하는 동안, 배양액은 매일 교환하였고 200 μL 배양액 샘플을 채취하여 HepG2 세포의 알부민(Albumin) 분비를 분석하였다(Fig. 3). 두 세포의 공동배양은 7일간 수행하였다.
Fig. 3

Co-culture medium extraction process image for albumin secretion analysis

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2.7 세포 생존력(Cell Viability) 및 HAS ELISA 테스트
LIVE/DEAD® Assay (Invitrogen, USA)를 사용하여 HepG2 세포와 NIH-3T3 세포 생존능(Cell Viability)을 살펴보았다. Calcein AM으로 생존세포를 녹색으로 염색(Stain)하고, Ethidium Homodimer-1으로 죽은 세포를 붉은색으로 염색하였으며, 형광 현미경을 이용하여 가시화하였다.
알부민 분비 분석(Albumin Secretion Analysis)을 위하여 샌드위치 ELISA (Sandwich ELISA) 분석을 진행하였다. 샌드위치 ELISA 방법은 항원-항체반응을 이용하여 측정하고자 하는 단백질의 양을 정량으로 측정하는 방법으로, ELISA 리더(Reader)나 분광광도계(Spectrophotometer)를 이용하여 흡광도(Absorbance)를 측정하여 분석한다. 공동배양(Co-Culture) 배양액(Medium) 샘플을 7일 동안 매일 200 μl 추출하여 분비된 알부민의 양을 ELISA로 분석하였다.
3.1 PDMS Microstencil을 이용한 공동배양 시스템
본 연구에 사용된 공동배양 시스템은 2개의 PDMS 미세유체 장치(Microfluidic Device)와 분리막으로 사용되는 PDMS 마이크로 스텐실으로 구성되며, 분리막을 통해 세포의 이동은 차단하고 물질전달만 가능하도록 설계되었다. 실험에 사용된 마이크로 스텐실은 두께가 10이고, 직경 10 μm의 미세구멍을 가지고 있다.
Fig. 4는 다공성(Porous) 구조를 가지는 분리막의 전자주사현미경 이미지를 보여주고 있으며, Fig. 5는 제작된 분리막이 PDMS 유체채널 사이에 삽입된 공동배양 시스템의 실제 모습이다. 제작된 공동배양 시스템은 상부 미세유체 채널에는 HepG2 세포를 배양하고, 하부 미세유체 채널에는 NIH-3T3 세포를 각각 배양하는 시스템으로 분리막을 통해 세포들의 이동은 차단하면서 서로 간의 물질교환은 가능한 구조를 가지고 있다. Fig. 6은 제작된 공동배양 시스템의 물질교환 여부를 확인하기 위하여 확산(Diffusion) 실험을 진행한 결과를 보여주고 있다. 확산 실험 방법은 실린지 펌프를 공동배양 시스템의 상하부 미세유체 채널에 각각 연결하여 1 ml/h의 유속으로 상부 미세유체 채널에는 물(DIW)을 흘리고, 하부 미세유체 채널에는 청치자(Blue Dye)를 동시에 흘려 약 30분 후 청치자가 확산되는 것을 확인하였으며, 이는 분리막의 미세구멍을 통해 물질교환이 가능함을 검증하였다.
Fig. 4

Scanning Electron Microscope (SEM) images of PDMS microstencil (Thickness: 10, pore size: 10, pitch: 30 μm)

KSPE_2022_v39n10_731_f004.jpg
Fig. 5

Image of the fabricated microfluidic chip with PDMS microstencil

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Fig. 6

Diffusion test of co-culture system image

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3.2 공동배양 및 세포 생존력
제작된 공동배양 시스템의 성능을 검증하기 위해서 두 종류의 세포를 총 7일간 공동배양하여 세포의 생존율을 확인하는 연구를 수행하였다. Fig. 7은 공동배양 시스템으로 HepG2와 NIH-3T3 세포를 공동배양 후 LIVE/DEAD Kit를 이용하여 세포 생존능을 확인한 결과이다. 7일간 공동배양한 2종의 세포를 형광 염색하여 세포 생존능을 분석한 결과 위쪽 유체채널의 PDMS 마이크로 스텐실 위에 HepG2 세포가, 아래 유체채널에는 NIH-3T3 세포가 모두 잘 자라고 있음을 확인하였다. 각 채널에서 살아있는 세포는 녹색으로 죽은 세포는 적색으로 표시되며, 분석 결과 대부분의 세포가 생존해있는 것으로 관찰되어 제작된 공동배양 시스템의 사용성을 검증하였다.
Fig. 7

Optical image and fluorescence image of cell culture using co-culture system. (a) Lower micro-channel - NIH-3T3 cells; Fluorescent image for the cell viability was assessed at day 7 using the LIVE/DEAD assay (Live cell stained green, dead cell stained red, scale bar 200 μm), and (b) Upper micro-channel-HepG2 cells; Fluorescent image for the cell viability was assessed at day 7 using the LIVE/DEAD assay (Live cell stained green, dead cell stained red, scale bar 500 μm)

KSPE_2022_v39n10_731_f007.jpg
제작된 공동배양 시스템의 물질전달(Mass Transfer) 기능을 검증하기 위해서 HepG2/NIH-3T3 세포의 알부민 분석으로 세포의 활성도를 측정하였다. HepG2/NIH-3T3 세포의 활성도에 관한 연구는 이미 다른 연구그룹에 의해 보고가 되었으며, 보고된 내용을 보면 HepG2/NIH-3T3 세포를 공동배양하면 HepG2 세포의 알부민 분비량이 HepG2 세포를 단독배양한 결과보다 공동배양에서 증가하고 있음을 확인할 수 있다[4,14,19]. 이미 보고된 연구 결과를 기반으로 본 연구에서는 기존의 세포배양 방식과 차별화된 새로운 형태의 마이크로 유체 칩 기반 공동배양 시스템을 적용하여 HepG2 세포의 알부민 분비량을 측정하였다(Fig. 8). HepG2 세포의 알부민 분비량 분석은 7일간(매일 200 μl) 배양액을 수집하여 동결저장으로 보관하고, 수집된 샘플은 ELISA Kit를 사용하여 분석하였다. 분석 결과 총 7일간 HepG2 세포가 분비한 알부민의 양은 모두 비슷하게 유지되고 있음을 확인할 수 있었다. 이러한 분석 결과는 제작된 공동배양 시스템을 통해 7일간 HepG2 세포가 분비하는 알부민 양이 비슷하므로 물질교환을 통해 HepG2 세포의 활성도가 유지되고 있음을 보여준다.
Fig. 8

Albumin secretion analysis of HepG2 cell using ELISA Kit

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본 연구를 통해 검증된 공동배양 시스템은 In-Vitro에서 세포들을 공동배양할 수 있었으며, 향후 세포들 사이의 연계성과 다양한 반응을 분석하는 유용한 도구로서 다양한 세포연구에 활용성이 높을 것으로 예상된다.
본 연구에서는 에어나이프 시스템으로 제작된 PDMS 마이크로 스텐실을 가지는 새로운 형태의 공동배양 시스템을 제작하는 방법을 제안하였다. 제안된 새로운 공동배양 시스템은 물질 전달을 위해 미세 다공성 구조와 두께 조절이 가능한 PDMS 마이크로 스텐실이 분리막으로 사용되었으며, 두 개의 미세 유체칩과 결합되어 제작되었다. 제작된 공동배양 시스템은 두 종류의 세포(HepG2, NIH-3T3 cells)를 이용하여 7일간 공동배양 하였으며, LIVE/DEAD 염색과 분석을 통해 세포의 생존율 및 안정성을 확인하였다. 그리고 HSA ELISA Kit를 이용하여 7일간 공동배양된 HepG2 세포의 알부민 분비를 측정하여 HepG2 세포의 활성도를 확인하였다. HepG2 세포의 알부민 분석 결과 알부민 분비량이 7일간 비슷하게 유지됨을 확인할 수 있었으며, 이것은 물질교환을 통해 HepG2 세포의 활성도가 유지되면서 세포가 잘 자라고 있음을 확인할 수 있었다.
세포 공동배양 시스템은 세포 사이의 연계성과 다양한 반응을 분석하기 위하여 생물체내(In-Vivo) 환경과 유사한 배양 시스템의 개발을 위해 세포를 단독으로 배양하는 방식이 아닌 서로 다른 두 가지 이상의 세포를 공동으로 배양할 수 있는 새로운 형태 세포배양 시스템으로 인체 내부에 존재하는 다양한 세포들이 물질교환을 통해 세포성장에 미치는 영향을 확인할 수 있는 유용한 도구로써 활용될 것으로 기대된다.
본 연구는 한국연구재단(Nos. NRF-2020R1A4A1018652, NRF-2020R1A2B5B01002471)과 한국산업기술평가관리원(No. 20010984)의 지원을 받아 수행되었습니다.
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Jin Ho Choi
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Ph.D./Researcher in the KIFLEX CO., Ltd.. His research interest is micro-electro mechanical system and medical device.
Dong Wook Kim
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M.S. student in the School of Mechanical Engineering, Kyungpook National University. His research interests are microfabrication and microsystem for biomedical applications.
Chul Min Kim
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Assistant Professor in the School of Mechatronics Engineering, Gyeongsang National University. His research interests are microfabrication and microfluidic system for tissue engineering and biomedical application.
E-mail: cm@gnu.ac.kr
Gyu Man Kim
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Professor in the School of Mechanical Engineering, Kyungpook National University. His research interests are microfabrication with micro-electro mechanical system, physics and biomedical applications.

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Fabrication of a Microfluidic Device with an Embedded PDMS Microstencil for Co-Culture of Cells
J. Korean Soc. Precis. Eng.. 2022;39(10):731-737.   Published online October 1, 2022
DOI: https://doi.org/10.7736/JKSPE.022.080
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Fabrication of a Microfluidic Device with an Embedded PDMS Microstencil for Co-Culture of Cells
J. Korean Soc. Precis. Eng.. 2022;39(10):731-737.   Published online October 1, 2022
DOI: https://doi.org/10.7736/JKSPE.022.080
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Fabrication of a Microfluidic Device with an Embedded PDMS Microstencil for Co-Culture of Cells
Image Image Image Image Image Image Image Image
Fig. 1 Schematic image of co-culture system
Fig. 2 Fabrication process of co-culture device and co-culture system image
Fig. 3 Co-culture medium extraction process image for albumin secretion analysis
Fig. 4 Scanning Electron Microscope (SEM) images of PDMS microstencil (Thickness: 10, pore size: 10, pitch: 30 μm)
Fig. 5 Image of the fabricated microfluidic chip with PDMS microstencil
Fig. 6 Diffusion test of co-culture system image
Fig. 7 Optical image and fluorescence image of cell culture using co-culture system. (a) Lower micro-channel - NIH-3T3 cells; Fluorescent image for the cell viability was assessed at day 7 using the LIVE/DEAD assay (Live cell stained green, dead cell stained red, scale bar 200 μm), and (b) Upper micro-channel-HepG2 cells; Fluorescent image for the cell viability was assessed at day 7 using the LIVE/DEAD assay (Live cell stained green, dead cell stained red, scale bar 500 μm)
Fig. 8 Albumin secretion analysis of HepG2 cell using ELISA Kit

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Fabrication of a Microfluidic Device with an Embedded PDMS Microstencil for Co-Culture of Cells
JKSPE : Journal of the Korean Society for Precision Engineering

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