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

Summary

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.

Abstract

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.

Introduction

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.^4,5 In addition, exposure to NPs containing transformative agents (growth factors, corticosteroids, etc.) at excessively high concentration can cause cytotoxicity and misdirected exposure may induce unintended consequences on non-target cells. Even particulate carriers comprising of "biocompatible" materials [e.g., poly(lactic co-glycolic acid), PLGA] can incite potent immune cell responses under certain conditions as well.⁶ This is especially risky in individuals with impaired immunity (e.g., rheumatoid arthritis) which potentially delays systemic nanoparticle clearance.⁷ Thus, efficient removal of free particles prior to the introduction of particle-engineered cells is of great importance to minimize toxicity profile and reduce misdirected exposure to agent-loaded particles in vivo.

Conventional gradient centrifugation is often used to separate engineered cells from free particles but is laborious and operated in batch mode. Moreover, shear stresses experienced by cells during high-speed centrifugation and the constituents of the density gradient medium may compromise cell integrity and/or influence cell behavior.⁸ Microfluidics is an attractive alternative with several separation technologies including deterministic lateral displacement (DLD)⁹, dieletrophoresis^10,11 and acoustophoresis¹² developed for small particles separation and buffer exchange applications. However, these methods suffer from low throughput (1-10 µl·min⁻¹) and are prone to clogging issues. Active separations such as dielectrophoresis-based methods also require differences in intrinsic dielectrophoretic cell phenotypes or additional cell labeling steps to achieve separation. A more promising approach involves inertial microfluidics — the lateral migration of particles or cells across streamlines to focus at distinct positions due to dominant lift forces (F_L) at high Reynolds number (Re).¹³ Due to its high flow conditions and superior size resolution, it has often been exploited for size-based cell separation^14,15 and buffer exchange applications.^16-18 However, buffer exchange performance remains poor with ∼10−30% contaminant solution as the separated cells usually remain close to the boundary between original and new buffer solutions.^16-18 More importantly, the size distribution of target cells has to be similar to achieve precise inertial focusing and separation from the original buffer solution which poses an issue especially in the processing of heterogeneous-sized cell types such as mesenchymal stem cells (MSCs).¹⁹

We have previously developed a new inertial microfluidics cell sorting technique termed Dean Flow Fractionation (DFF) for isolating circulating tumor cells (CTCs)²⁰ and bacteria²¹ from whole blood using a 2-inlet, 2-outlet spiral microchannel device. In this video protocol, we will describe the process of labeling THP-1 (human acute monocytic leukemia cell line) suspension monocytic cells (~15 µm) and MSCs (10-30 µm) with calcein-loaded NPs, followed by fabrication and operation of the DFF spiral microdevice for efficient recovery of labeled cells and removal of unbound NPs.²² This single step purification strategy enables continuous recovery of labeled suspension and adherent cells suspended in fresh buffer solution without centrifugation. Moreover, it can process up to 10 million cells·ml⁻¹, a cell density amenable for regenerative medicine applications.

Protocol

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) using overnight stirring. Fabricate PLGA-calcein AM (CAM) using a protocol described previously.²²
   1. Dissolve 250 µg CAM and 100 mg PLGA (50:50) in chloroform at 4 °C.
   2. Generate single emulsion NPs using a high speed homogenizer (13,600 x g, 60 sec) at room temperature. Evaporate the chloroform in a chemical hood (≥3 hr) before collection using centrifugation (3,400 x g for 5 min), washing (double distilled water), freeze-drying and storage in -20 °C.
3. Incubate CAM-PLGA NPs (1 mg) or calcein silica NPs (150 µg) in 0.01% poly-L-lysine (PLL) solution at room temperature for 15 - 20 min.
4. Centrifuge at 3,400 x g for 5 min to remove excess PLL supernatant before NPs resuspension in 1 ml of respective culture medium.
5. Incubate the NPs with cells (MSCs or THP-1, ~1 - 2 x 10⁶ cells in total) for approximately 24 hr (0.1 mg·ml⁻¹ labeling concentration).
6. Dissociate and harvest the labeled adherent MSCs using 2 ml of 0.25% trypsin (5 min, 37 °C) and quench with 6 ml of DMEM. Spin down the cells at (1,000 x g, 4 min) and resuspend to a concentration of 10⁵ - 10⁶ cells/ml for microfluidic processing. Use labeled THP-1 cells (10⁵ - 10⁶ cells/ml) directly for microfluidics purification.

2. Microfluidic Device Preparation

1. Device Fabrication
   1. Fabricate the microfluidic spiral device (500 µm (w) × 115 µm (h)) with polydimethylsiloxane (PDMS) from a commercial kit using standard soft lithography steps.²³
   2. Mix 30 g of base prepolymer and 3 g curing agent thoroughly in a weighing boat. De-gas the mixture in a desiccator for 60 min to remove any air bubbles.
   3. Pour the PDMS mixture onto the silicon wafer master mold patterned with the spiral channel design carefully to a height of ~5 - 10 mm.
   4. De-gas the mixture in a desiccator vacuum for 60 min again to remove any air bubbles. Repeat the process until all bubbles are eliminated.
   5. Cure the PDMS mixture in an 80 °C oven for 2 hr until the PDMS is set. Ensure the wafer is not tilted during curing to have a constant device height.
   6. Cut out the PDMS spiral device using a scalpel and carefully peel the PDMS slab from the master mold.
   7. Trim the edges of the device with a scalpel to ensure smooth surface for bonding.
   8. Punch two holes (1.5 mm) for the inlets and two holes (1.5 mm) for the outlets on the PDMS device using a 1.5 mm biopsy puncher.
   9. Wash the device with isopropanol (IPA) to remove any debris and dry the device in an 80 °C oven for 5 min.
   10. Clean the bottom surface of the PDMS device (with channel features) using masking tape.
   11. Clean one side of a glass slide (2" by 3") using masking tape.
   12. Carefully place and expose the cleaned surfaces of the PDMS device and glass slide in the chamber of a plasma cleaner and subject them to vacuum for 60 sec. Next, switch on the plasma power to maximum and lower the chamber pressure until the chamber turns pink in color.
       1. Expose the surfaces to air plasma for 60 sec. The plasma creates reactive species on the exposed surfaces of the PDMS and glass which enables tight bonding when brought into physical contact. Turn off the plasma power and release the pressure from the plasma cleaner to retrieve the device and glass slide.
   13. Bond the PDMS device and glass slide together by pressing the plasma-exposed surfaces tightly and ensuring that no bubbles are trapped between the two surfaces.
   14. Heat the bonded device using a hotplate set at 80 °C for 2 hr to strengthen the bonding.
2. Device Operation
   1. Cut two pieces of tubing (1.52 mm OD) of ~15 - 20 cm for the inlet syringes and attach a syringe tip (gauge 23) on one end of each tubing.
   2. Cut two pieces of tubing (1.52 mm OD) of ~5 - 10 cm for the outlets and attach to the outlet holes of the PDMS device.
   3. Prior to sample running, manually prime the device with a syringe containing 70% ethanol until it flows out of the outlet tubing. Allow the ethanol sit for 30 sec to 1 min to sterilize the device.
   4. Load 30 ml of filtered (0.2 µm pore) sheath buffer (Phosphate-Buffered Saline (PBS) with 0.1% Bovine Serum Albumin (BSA)) into the 60 ml syringe and secure the syringe on a syringe pump. Set the pump to the correct settings (Syringe size: 60 ml, Volume: 60,000 µl).
      Note: The addition of BSA to PBS is to minimize cell-cell binding and non-specific binding between cells and the PDMS device.
   5. Load 3 ml of labeled cells into the 3 ml syringe and secure the syringe on a separate syringe pump. Set the pump to the correct settings (Syringe size: 3 ml, Volume: 3,000 µl).
   6. Check that no air bubbles are trapped in the syringes to ensure a stable flow. Remove any air bubbles by gently ejecting few drops of liquid out of the nozzle.
   7. Connect the inlet syringe tips and tubing to the syringes, and insert them into the respective inlets of the device (sheath and sample inlet). Ensure that there are no bubbles along the tubing.
   8. Mount the devices onto an inverted phase-contrast microscope for real time imaging during cell sorting process.
   9. Secure a small waste beaker and two 15 ml tubes close to the device on the microscope stage using adhesives.
   10. Place the outlet tubings into the waste beaker.
   11. Set the flow ratio for sample to sheath buffer to 1:10 and start both syringe pumps to initiate the sorting process (Example: 120 µl/min for sample syringe and 1,200 µl/min for sheath syringe; channel flow velocity ~0.38 m/sec).
   12. Run the device for 1.5 min for the flow rate to stabilize. This can be confirmed by presence of inertially focused cells near the channel inner wall under bright-field with phase contrast using a high-speed camera (~5,000 - 10,000 frames per second (fps), exposure time: 10-50 µsec).
   13. Position the outlet tubings into different tubes to collect the eluents from the cell outlet and waste outlet.

Results

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...

Discussion

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...

Materials

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
Reagents & Materials
0.01% poly-L-lysine (PLL)	Sigma-Aldrich	P8920
3 ml Syringe	BD	302113	Syringe 3 ml Luer-Lock
60 ml Syringe	BD	309653	Syringe 60 ml 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 mm3
Polydimethylsiloxane (PDMS)	Dow Corning	SYLGARD® 184
Scotch tape	3M	21200702044	18 mm x 25 m
Silica NPs (∼200 μm)	Sigma-Aldrich	748161	Pore size 4 nm
Syringe Tip	JEC Technology	7018302	23 G 0.013 x 0.25
Trypsin-EDTA (0.25%)	Life Technologies	25200-056
Tygon Tubing	Spectra-Teknik	06419-01	0.02 x 0.06" 100
Poly (D,L-lactide-co-glycolide) (PLGA; 50:50)	Sigma-Aldrich	P2191
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