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Bioengineering

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Published: July 10th, 2016

DOI:

10.3791/54327

1Lee Kong Chian School of Medicine, Nanyang Technological University, 2School of Chemical and Biomedical Engineering, Nanyang Technological University, 3NTU-Northwestern Institute of Nanomedicine, Nanyang Technological University

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (>90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving >95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

Engineering cells by agent-loaded micro/nanoparticles (NPs) is a simple, genomic integration-free, and versatile method to enhance bioimaging capability and augment/supplement its native therapeutic properties in regenerative medicine.1-3 Cellular modifications are achieved by labeling the plasma membrane or cytoplasm with an excess concentration of agent-loaded NPs to saturate the binding sites. However, a major drawback of this method is the significant quantities of unbound particles remaining in solution after cell labeling processes, which can potentially confound precise identification of particle-engineered cells or complicate therapeutic outcomes.

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1. Nanoparticles (NPs) Labeling of Mesenchymal Stem Cells and Monocytes

  1. Culture mesenchymal stem cells (MSCs) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics to ≥80% confluency prior to labeling. Similarly, culture THP-1 cells (ATCC) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS to a density of ~106 cells/ml.
  2. Load silica NPs (~500 µm) with calcein dye solution (200 µM) usi.......

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After labeling the cells with bio imaging agent-loaded NPs overnight, the labeled cells (containing free particles) are harvested and purified by DFF spiral microdevice to remove free NPs in a single step process (Figure 1A). The 2-inlet, 2-outlet spiral microchannel is designed by engineering software and microfabricated using SU-8 photoresist. The patterned silicon wafer is then used as a template for PDMS replica molding using soft lithography techniques (Figur.......

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The DFF cell purification technology described herein enables rapid and continuous separation of labeled cells in a high throughput manner. This separation approach is ideal for large sample volume or high cell concentration sample processing, and is better than conventional membrane-based filtration which is prone to clogging after extended use. Similarly, affinity-based magnetic separation requires additional cell labelling steps which are laborious and expensive. The purified cells are shown to retain their labeled ag.......

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Kind gift of THP-1 cells from Dr. Mark Chong and assistance in microfabrication from Dr. Yuejun Kang and Dr. Nishanth V. Menon (School of Chemical and Biomedical Engineering, Nanyang Technological University) were greatly acknowledged. This project was funded by NTU-Northwestern Institute of Nanomedicine (Nanyang Technological University). H.W.H. was supported by Lee Kong Chian School of Medicine (LKCMedicine) postdoctoral fellowship.

....

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Name Company Catalog Number Comments
Cell lines & Media
Mesenchymal Stem Cells (MSCs) Lonza PT-2501
Dulbecco’s modified Eagle’s medium (DMEM) Lonza 12-614F
Fetal Bovine Serum (FBS) Gibco 10270-106
THP-1 monocyte cells (THP-1) ATCC TIB-202
Roswell Park Memorial Institute (RPMI) 1640 media Lonza 12-702F
Name Company Catalog Number Comments/Description
Reagents & Materials
0.01% poly-L-lysine (PLL) Sigma-Aldrich P8920
3 mL Syringe BD 302113 Syringe 3ml Luer-Lock
60 ml Syringe BD 309653 Syringe 60ml Luer-Lock
Bovine Serum Albumin (BSA) Biowest P6154-100GR
Calcein, AM (CAM) Life Technologies C1430
Calcein Sigma-Aldrich C0875
Isopropanol Fisher Chemical #P/7507/17 HPLC Grade 2.5 L
Phosphate-Buffered Saline (PBS) Lonza 17-516Q/12
Plain Microscope Slides Fisher Scientific FIS#12-550D 75 X 25 X 1 mm
Polydimethylsiloxane (PDMS) Dow Corning SYLGARD® 184
Scotch tape 3M 21200702044 18mm x 25m
Silica NPs (∼200 μm) Sigma-Aldrich 748161 Pore size 4 nm
Syringe Tip JEC Technology 7018302 23GA .013 X .25
Trypsin-EDTA (0.25%) Life Technologies 25200-056
Tygon Tubing Spectra-Teknik 06419-01 .02X.06" 100
Name Company Catalog Number Comments/Description
Equipment
Biopsy punch Harris Uni-Core 69036-15 1.50 mm
Dessicator Scienceware 111/4 IN OD
High-speed Camera Phantom  V9.1
Inverted phase-contrast microscope  Nikon Eclipse Ti
Plasma cleaner Harrick Plasma PDC-002
Syringe Pump Chemyx CX Fusion 200

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