Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

Summary

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Abstract

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

Introduction

Shifting from a monolayer (two-dimensional) culture to a three-dimensional culture is motivated by the need to work with culture models that mimic the cellular functions of living tissues^1,2,3. Flat and hard plastic substrates commonly used in cell culture do not resemble most of the extracellular environments in the human body. In fact, many studies demonstrate that three-dimensional culture recreate tissue-specific architecture, mechanical and biochemical cues, and cell-cell communication, which have not been observed in conventional two-dimensional culture

Protocol

1. Fabrication of the Microfluidic Device Mold

1. Design the pattern of the microfluidic device using commercially available software (Clewin5 or AutoCAD 2016, etc.). For the function of the Clewin5, see the user manual (http://manualzz.com/doc/7159150/clewin-user-s-manual).
   NOTE: The design file is available in Supplementary File 1.
2. Transfer the design file to a micro pattern generator and load the tool with a chromium mask coated with the positive photoresist.
3. Expose the positive photoresist in the pattern area using a micro pattern generator.
4. Develop the positive photoresist using the developer....

Results

Figure 1 shows a design and photo of the microfluidic device. It has three parallel channels, in which channel 2 contains the spheroid well. Channels 1 and 3 are used for the HUVEC culture and channel 2 is for the spheroid. Each channel is separated by trapezoidal microposts designed to pattern PDMS. The microposts prevent the hydrogel in channel 2 from leaking into channels 1 and 3 by surface tension and allow exchanging substances between the spheroid and H.......

Discussion

Previous reports show that hLFs secrete a cocktail of multiple angiogenic factors, such as angiopoietin-1, angiogenin, hepatocyte growth factor, transforming growth factor-α, tumor necrosis factor and some extracellular matrix proteins^29,30. This assay relies on the angiogenic secretion from hLFs in a coculture spheroid, which is the limitation of the technique. Therefore, it is critical for a stable vascular formation to set a coculture spheroid at the bott.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AutoCAD 2017	Autodesk	AutoCAD 2017
A chromium mask coated with AZP 1350.	CLEAN SURFACE TECHNOLOGY	CBL2506Bu-AZP
Micro pattern generator	Heidelberg	uPG101
MF CD-26 developer	Rohm and haas electronic materials	-	Developer in protocol 1.4
S-Clean	Sasaki Chemical	S-24	Chromium etchant in protocol 1.5
Aceton	Wako	012-00343
Silicon Wafer	Canosis	SiJ-4
Spin Coater	MIKASA	1H-D7
Hexamethyldisilazane (HMDS)	Tokyo Ohka Kogyo	H0089
SU-8 3050	MicroChem	-	Negative photoresist in protocol 1.9
UV Exposure	Nanometric Technology Inc	LA310s
SU-8 Developer	MicroChem	Y020100	Developer for the negative photoresist in protocol 1.13
2-propanol	Wako	163-04841
Surface profiler	Veeco	Veeco Dektak XT-S
(Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane	Sigma	448931
Polydimethylsiloxane (PDMS)	Dow Corning Toray	184W/C
Biopsy Punch (1.0mm)	Kai Industries	BP-10F
Biopsy Punch (2.0mm)	Kai Industries	BP-20F
Plasma System	Femto Science	COVANCE
Cover glass	MATSUNAMI GLASS	C024241
Culture Dishes	Iwaki	1000-035
RFP Expressing Human Umbilical Vein Endothelial Cell	Angio Proteomie	cAP-0001RFP
Normal Human Lung Fibroblasts	Lonza	CC-2512
Endothelial Cell Growth Medium	Lonza	CC-3162
Fibroblast Growth Media Kits	Lonza	CC-3132
DMEM	Thermo Fisher Scientific	11965092
Fetal Bovine Serum	Thermo Fisher Scientific	26140079
Penicillin-Streptomycin Solution	Wako	168-23191
0.05w/v% Trypsin-0.53mmol/l EDTA• 4Na Solution with Phenol Red	Wako	204-16935
PBS (Phosphate Buffered Salts)	Takara bio	T900
96-well plate	Sumitomo bakelite	631-21031
1000ul Chip	NIPPON Genetics	FG-402
200ul  Chip	NIPPON Genetics	FG-301
10ul Chip	NIPPON Genetics	37650
CO2 incubator	Thermo Fisher Scientific	Model 370
GFP Expressing Human Umbilical Vein Endothelial Cell	Angio Proteomie	cAP-0001GFP
Fibrinogen from bovine plasma	Sigma	F8630
Aprotinin from bovine lung	Sigma	A6279
Collagen I	Corning	354236
Thrombin from bovine plasma	Sigma	T4648
Hoechst 33342	Invitrogen	H21492	Fluorescent dye to stain nuclei in protocol 5.5
Paraformaldehyde Solution	Wako	163-25983
Inverted Fluorescence Microscope	OLYMPUS	IX71
Degital CCD Camera	OLYMPUS	ORCA-R2
Confocal Laser Scanning Biological Microscope	OLYMPUS	FV1000
Inverted Fluorescence Microscope	OLYMPUS	IX-83
Fluorescein isothiocyanate-dextran	Sigma	FD70S