Microfluidic Bioreactors for Cellular Microarrays
Abstract
:1. Introduction
2. Construction Methods of Cellular Array Microfluidic Chips
- Conventional MEMS (microelectromechanical systems) processing (photo lithography, electroplating, deposition, and etching) are used to create structures on silicium or glass substrates and soft lithography or etching is used to fabricate channels with PDMS or glass. The microstructered layer is bonded with the microchannel layer to form the microfluidic device [12,13,14,15,16].
- Multilayered PDMS based microfluidic systems that are fabricated with one or multiple uses of soft lithographic process: (a) The PDMS layer with the microstructures and the channel are separately fabricated and bonded with alignment [17,18,19,20]; (b) The channel layer containing the structures is fabricated on a multilayered master mold and is bonded with a flat PDMS or glass substrate [21,22,23].
- Micro/nanostructures are formed on a polymer layer or Si substrate by nanoimprinting, soft lithography or reactive etching following a suitable lithographic process. Next, a microchannel layer of PDMS or glass is bonded to form a microfluidic channel [31].
Materials | Fabrication Methods | Array Spots | Cell Type | Refs | ||
---|---|---|---|---|---|---|
Channel | Micro-Structures | Channel | Micro-Structures | |||
PDMS, glass | Si, glass, PDMS 1 | Replica moulding, etching | Photo lithography, deposition, etching | 1 | HL60 | [12] |
PDMS | PDMS | Replica moulding | Replica moulding | 1 16 100 440 8 100/mm2 3600 | Jurkat, U937 C2C12 myoblasts Hela U937, FL 60 HepG2 Hela S. cerevisiae | [18] [19] [22] [23] [32] [33] [20] |
PDMS, glass | Hydrogel PEG | Replica moulding, etching | Photo-polymerization | >300 | Mouse embryonic stem cells, NIH-3T3 fibroblasts | [24,26,27] |
PDMS | PDMS, hydrogel, PMMA | Replica molding | Soft lithography | 25,000 | Hybridoma | [29] |
PDMS, glass Glass, PDMS | Si, hydrogel, PMMA PUA 2 | Replica molding, etching Replica molding | Nanoimprinting, soft lithography, reactive ion etching Capillary molding | 4 | MCF10A, MCF7 | [31] |
3. Cell Arraying Methods
3.1. Cell Patterning on Adhesive Micropatterns
3.2. Mechanical Cell Patterning
Material | Diameter/Width | Cell Type | Characteristics | Refs |
---|---|---|---|---|
PDMS | 20–40 µm | NIH3T3 1, RBL-1 2 | Single-cell screening | [111] |
25 µm | Hepatocyte | Single cell oxidative stress screening | [131] | |
Photoresist 1002F | 50 µm | HeLa | Viability assessment | [132] |
50 µm | H1299, MEF 3 | RhoA GTPase biosensor | [133] | |
Poly(ethylene glycol) | 40–150 µm | Murine embryonic stem cells | The formation of homogeneous embryoid bodies | [134] |
Polystyrene | 10 µm | B-cells | Detecting activated cells | [135] |
Optical imaging fibers | 7 µm | NIH 3T3 mouse fibroblast cells | Observation of cell fluorescence | [136] |
Etched glass | 20 µm | Non-adherent cell lines | Individual cell based assays | [137] |
SU-8 on coverslip | 90 µm2 squares | Adult neural stem cell | Culturing and dynamic monitoring of stem cell proliferation | [138] |
PDMS | 25–50 µm | BCE 4 | PDMS wells coated with fibronectin | [139] |
160 µm | HSC 5 | HSC proliferation control at the single-cell level | [119] | |
70 µm | HeLa | Single cell analysis | [140] | |
Poly(ethylene glycol) diacrylate | 229–442 µm | Murine ES cells 6 | Wiping technique to localize cells in microwells | [141] |
3.3. Robotic Cell Printing
3.4. Selection of Cell Arraying Method
Method | Advantages | Limitations |
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Adhesive micropatterns |
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Mechanical cell patterning
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Robotic printing
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4. Operation and Applications of Microfluidic Cell Microarrays
4.1. Microfluidic Operation
4.2. Single-Cell Measurement/Monitoring in Microfluidic Devices
4.3. Microfluidic Cell Array Culture Applications
Scope | Measurement | Cell Type | Cell-Array Type/Methodology | Cell-Array Size | Fluidic Conditions | Refs |
---|---|---|---|---|---|---|
Adherent Cell Types | ||||||
Drug screening: high-content analysis of cell signaling | On chip off-line immune-cytochemistry. | Mouse fibroblasts (NIH-3T3) | Mechanical cell patterning (PDMS microchambers) | 32 (4 × 8) 4 groups of 8 parallel channel chambers; 8 chambers for each parallel channel | 4 common inlets for 32 parallel channel chambers and 1 additional inlet for a group of 8 channels | [118] |
Drug screening: screening of chemotherapeutic compounds | Cell imaging with fluorescence microscopy | Human breast cancer (MCF-7) and HepG2 cell lines | Mechanical cell patterning (PDMS microchambers) | 24 (4 × 6) 4 channels rows containing 6 microchambers columns | 1 inlet (from gradient generator) for each column of 4 microchambers | [253] |
Cytotoxicity evaluation | Real-time microscopy, albumin concentration determination in the effluent (ELISA assay) | Rat hepatocytes in coculture with fibroblasts (3T3-J2) | Cell patterning on adhesive micropatterns (collagen) in a culture chamber (glass bottom, PDMS walls) | 64 (8 × 8) 8 rows and 8 columns of culture chambers | 1 medium inlet for each row of, culture chambers and 1 gas perfusion inlet for each column of chambers | [173] |
Cytotoxicity evaluation | Real-time cell viability screening by fluorescence microscopy | Human lung carcinoma (A549) cell line | Mechanical cell patterning in microchamber (PDMS with glass bottom) | 25 (5 × 5) 5 parallel channels containing 5 chambers in series | 2 inlets into a gradient concentrator that feeds each parallel channel | [269] |
Cytotoxicity evaluation | Fluorescent measurement (microscopy) of cytosolic calcium | Mouse leukemic monocyte macrophage (RAW) cell line | Mechanical cell trap barrier (glass) | 1 chamber | 1 inlet | [270] |
Cytotoxicity evaluation | Fluorescent measurement (microscopy) of cell viability (ethidium homodimer-1) | Human HeLa cancer cell line | Mechanical cell patterning (PDMS microwells) | 3000 (500 × 6) 500 microwells in each parallel channel | 1 chemical inlet followed by a gradient generator, which feeds a different concentration in each parallel channel | [271] |
Cell biology research: analysis of cell proliferation and migration | Cell number and position (microscopy) | Human breast cancer cells (MDA-MB-231) | Mechanical cell patterning in microchambers (PDSE1 and AZ4562 photoresist) | 1600 (40 × 40) Each chamber is linked together by channels | 1 inlet for all chambers | [272] |
Cell biology research: long-term cellular monitoring for high-throughput cell-based assays | Fluorescence localization of calcein AM (microscopy) | Human HeLa cancer cell line | Mechanical cell trap barrier (PDMS) | 100 (10 × 10) 10 culture chambers row and per column | 1 inlet per column (from a gradient generator) and 1 inlet for all rows | [22] |
Cell biology research: continuous monitoring of gene expression | Fluorescence microscopy of eGFP-tagged reporter gene | Human HeLa cancer cell line | Mechanical cell patterning in microchambers (PDMS) | 40 (8 × 5) 5 microchambers for each of the 8 parallel channels | 1 inlet per parallel channel (from a gradient generator) | [273] |
Cell biology research: adherent cell culture over a logarithmic range of flow rates | Cell number and morphology (microscopy) | Fibroblasts 3T3 cell line | Mechanical cell patterning in microchambers (PDMS with glass bottom) | 16 (4 × 4) 4 groups of 4 chambers in parallel (including the logarithmic diluter) | 1 input per group of 4 chambers | [274] |
Cell biology research: fully automated cell culture screening system | Differentiation and proliferation; cell number/morphology, cell nucleus staining, alkaline phosphatase activity (microscopy) | Human mesenchymal stem cells | Mechanical cell patterning in microchambers (PDMS) | 96 (2 × 48) 2 rows of 48 parallel chambers | 16 inlets per row | [116] |
Cell biology research: real-time gene expression monitoring | Fluorescence microscopy of eGFP-tagged genes | Hepatocytes (H35) cell line | Mechanical cell patterning in microchambers (PDMS) | 256 (8 × 8 × 4) 8 rows and 8 columns; each matrix point is composed of a 2 × 2 subarray | 8 inlets for each row and 8 inlets for each column | [117] |
Non-Adherent Cell Types | ||||||
Drug screening: antifungal evaluation | Effect of antifungal on viability (fluorescence microscopy) | Saccharomyces cerevisiae | Mechanical cell patterning in microwells (glass) | 44,000 (2 × 22,000) Two arrays of 22,000 microwells | Digital microfluidic platform: microwell seeding by shuttling a cell-containing droplet; droplet of antifungal on the array. | [252] |
Drug screening: real-time screening of anticancer drugs | Online fluorescence imaging | Human histiocytic leukaemia (U937) and promyelocytic leukaemia (HL60) cell lines | Mechanical cell trap barrier (PDMS) | 440 All traps in a triangular chambers | 1 inlet and 6 outlets (base of the triangle). | [23] |
Cell biology research: High-throughput analysis of single hematopoietic stem cell proliferation | Proliferation; cell number/morphology; live-cell immunostaining; microscopy | Hemapoietic stem cells (HSC) (clonal population) Preleukemic mouse (ND13) | Mechanical cell patterning in microchambers (PDMS) | 6144 (64 × 96) 64 parallel channels (rows); each channels flows over 96 wells (columns) | 1 inlet for each channel; up to 6 different medium conditions can be loaded | [119] |
Microbiology research: long-term monitoring of bacteria undergoing programmed population control in a microchemostat | Cell number (microscopy) | Escherichia coli | Mechanical cell patterning in microchambers (PDMS) | 6 | Multiple inlets for each culture chamber. Culture chambers are independently operated (including an on chip peristaltic pump). | [112] |
Microbiology research: microfluidic chemostat growth to high cell densities | Cell number (microscopy) | E. coli, S. cerevisiae | Mechanical cell patterning in microchambers (PDMS) | 320 (16 × 20) 16 parallel channels containing 20 chambers | 1 inlet branching into the array of 16 parallel channels | [114] |
Microbiology research: high-throughput time-course analysis of single cell responses | Cell number, fluorescent reporter proteins (microscopy) | S. cerevisiae | Mechanical cell patterning in microwells (polyurethane acrylate) | 3906 wells per mm2 (microwell diameter of 8 µm) | 1 inlet to channel that flows over the wells | [275] |
Microbiology research: microscopic observation of cell behavior at high resolution | Cell number/morphology and single molecule fluorescence imaging (microscopy) | Schizosaccharo-myces pombe | Mechanical cell trap barrier (PDMS) | 7728 (4 × 1932) 4 trapping regions, each with 1932 mechanical cell traps | 3 inlets feeding into 4 trapping regions | [120] |
Microbiology research: spatio-temporal analysis of growing bacterial microcolonies in perfusion reactor | Cell number and morphology (microscopy) | E. coli Corynebacterium glutamicum | Mechanical cell trap barrier (PDMS) | 30 (6 × 5) 6 parallel channel containing each 5 cell trap barriers | 2 inlets into a gradient generator, which feeds each parallel channel | [265,276] |
Scope | Measurement | Cell Type | Populationsize | Cell-Array Type/Methodology | Cell-Array Size | Fluidic Conditions | Refs |
---|---|---|---|---|---|---|---|
Adherent Cell Types | |||||||
Cell biology research: cell culture device (multiple cycles of growth and trypsinization) | Microscopic observation of cell morphology and viability | Murine embryonic fibroblast (BALB/3T3) | 1 to 8 | Mechanical cell patterning (8 mechanical cell trap barriers in PDMS/chamber) | 64 (8 × 8) 8 parallel independent rows; 8 cultivation chambers in series per row | 1 inlet for 1 parallel channel | [277] |
Non-Adherent Cell Types | |||||||
Microbiology research: studying signaling network dynamics | Time-lapse cell imaging; heme expression by genetically encoded GFP reporter and protein localization (GFP-tagged protein) | S. cerevisiae | 8 strains | Mechanical cell patterning (PDMS microchambers) | 128 (8 × 16) | 8 chemical inlets for 16 parallel rows | [195] |
Microbiology research: spatio-temporal analysis of the proteome | Time lapse imaging of GFP-tagged strains | S. cerevisiae | 1152 strains | Direct cell printing and mechanical trapping in microchemostat chambers (PDMS) | 1152 (3 × 384) 3 independent sections of 384 chambers | 1 inlet per chamber section | [202] |
Microbiology research: monitor biofilm formation under near-native conditions | Quantitative cell analysis by bio-impedance measurement and respiration activity measured by electrochemical microelectrodes | Candida albicans | 2 | Mechanical cell patterning (PDMS microchambers) | 2 | 1 inlet per chamber | [278] |
Microbiology research: detect cellular dynamics in response to drugs and chemicals | Cell number and fluorescent imaging of protein tagged proteins (microscopy) | S. cerevisiae | 2 | Direct cell printing on agarose-coated glass | 10,000 (100 × 100) Spot sizes of around 200 µm | 1 inlet, channel over all spots | [56] |
5. Conclusions and Outlook
Acknowledgments
Conflicts of Interest
References
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Willaert, R.G.; Goossens, K. Microfluidic Bioreactors for Cellular Microarrays. Fermentation 2015, 1, 38-78. https://doi.org/10.3390/fermentation1010038
Willaert RG, Goossens K. Microfluidic Bioreactors for Cellular Microarrays. Fermentation. 2015; 1(1):38-78. https://doi.org/10.3390/fermentation1010038
Chicago/Turabian StyleWillaert, Ronnie G., and Katty Goossens. 2015. "Microfluidic Bioreactors for Cellular Microarrays" Fermentation 1, no. 1: 38-78. https://doi.org/10.3390/fermentation1010038