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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

Abstract

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

Introduction

Many important biological processes occur in cell and tissue cultures on the timescale of minutes to hours1,2,3. While some of these phenomena may be observed and recorded in an automated fashion using time-lapse microscopy4, bioluminescence1, or other methods, experiments involving the collection of culture supernatant samples for chemical analysis are often performed manually in static cell cultures. Manual sampling limits the feasibility of certain studies due to the inconvenience of frequent or after-hours sampling timepoint....

Protocol

1. Prepare parts for well plate perfusion

  1. Prepare tubing
    1. Cut two lengths of silicone tubing (1.6 mm inner diameter) for each cell culture to be perfused. Ensure that the piece used as the upstream tubing is long enough to reach from the syringe pump to the cell culture inside the incubator, and that the downstream piece can reach from the cell culture to the furthest extended position of the fraction collector.
    2. Give each piece of tubing a unique label on both of its end.......

Representative Results

The perfusion system with multiple medium sources from section 5 of the protocol was used to measure the expression dynamics of a reporter gene driven by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor in human embryonic kidney 293 (HEK293) cells in response to a 1.5 h pulse of tumor necrosis factor alpha (TNF-α). HEK293 cells were stably transduced using lentiviral vectors with a gene construct containing Gaussia luciferase (GLuc), driven by a promoter called NFK.......

Discussion

This work describes the assembly and operation of a perfusion cell culture system with multiple medium sources demonstrated with a specific example in which the dynamics of NF-κB-driven gene expression in response to a transient pulse of TNF-α were measured. The RTDs of the perfusion system components were measured and modeled, and signal convolution was used to predict both the exposure of the cells to the TNF-α pulse and the TNF-α distribution in the collected effluent medium fractions. The cells we.......

Acknowledgements

This research was conducted with support under Grant Nos. R01EB012521, R01EB028782, and T32 GM008339 from the National Institutes of Health.

....

Materials

NameCompanyCatalog NumberComments
18 Gauge 1 1/2- in Disposable Probe Needle For Use With Syringes and Dispensing MachinesGrainger5FVK2
293T CellsATCCCRL-3216HEK 293T cells used in the Representative Results experiment.
96-Well Clear Bottom Plates, CorningVWR89091-010Plates for measuring dye concentrations in RTD experiments and GLuc in representative results experiment.
BD Disposable Syringes with Luer-Lok Tips, 5 mLFisher Scientific14-829-45
BioFrac Fraction Collector Bio-Rad7410002Fraction collector that can be used for a single stream, or modified using our method to enable collection from multiple streams.
Clear High-Strength UV-Resistant Acrylic 12" x 12" x 1/8"McMaster-Carr4615T93This sheet is cut using a laser cutter according to the DXF file in the supplemental materials to produce the multi-head dispenser that can be attached to the BioFrac fraction collector.
Coelenterazine nativeNanoLight Technology303Substrate used in Gaussia luciferase bioluminescence assay in representative results.
Corning Costar TC-Treated Multiple Well Plates, size 48 wells, polystyrene plate, flat bottom wellsMillipore SigmaCLS3548Used to grow and perfuse 293T cells in representative results.
Corning Costar Flat Bottom Cell Culture Plates, size 12 wellsFisher Scientific720081Can be plugged and used as a stirred tank to produce pharmacokinetic profiles in perfusion. Can also contain cells for perfusion.
DMEM, high glucoseThermoFisher Scientific11965126
Epilog Zing 24 LaserCutting Edge SystemsEpilog Zing 24Laser cutter used to produce multi-head dispenser from acrylic sheet. Other laser cutters may be used.
Fisherbrand Sterile Syringes for Single Use, Luer-Lock, 20 mLFisher Scientific14-955-460
Fisherbrand Sterile Syringes for Single Use, Luer-Lock, 60 mLFisher Scientific14-955-461
Fisherbrand Premium Microcentrifuge Tubes: 1.5mLFisher Scientific05-408-129Microcentrifuge tubes for collecting fractions.
Fisherbrand Round Bottom Disposable Borosilicate Glass Tubes with Plain EndFisher Scientific14-961-26Glass tubes for collecting fractions.
Fisherbrand SureOne Micropoint Pipette Tips, Universal Fit, Non-FilteredFisher Scientific2707410300 ul pipette tips that best fit the multi-head dispenser and tubing to act as dispensing tips.
Gibco DPBS, powder, no calcium, no magnesiumFisher Scientific21600010Phosphate buffered saline.
Labline 4625 Titer ShakerMarshall ScientificLabline 4625 Titer ShakerOrbital shaker used to keep stirred tanks mixed.
Masterflex Fitting, Polycarbonate, Four-Way Stopcock, Male Luer Lock, Non-Sterile; 10/PKCole-ParmerEW-30600-04Used to join multiple inlet streams for RTD experiments and cell culture experiments.
Masterflex Fitting, Polycarbonate, Straight, Female Luer x Cap; 25/PKMasterflexUX-45501-28
Masterflex Fitting, Polypropylene, Straight, Female Luer to Hosebarb Adapters, 1/16"Cole-ParmerEW-45508-00
Masterflex Fitting, Polypropylene, Straight, Male Luer Lock to Hosebarb Adapter, 1/16" IDCole-ParmerEW-45518-00
Masterflex Fitting, Polypropylene, Straight, Male Luer Lock to Plug Adapter; 25/PKMasterflexEW-30800-30
Masterflex L/S Precision Pump Tubing, Platinum-Cured Silicone, L/S 14; 25 ftMasterflexEW-96410-14
MATLABMathWorksR2019bVersion R2019b. Newer versions may also be used. Some older versions may work.
NE-1600 Six Channel Programmable Syringe PumpNew Era Pump SystemsNE-1600
Rack Set F1Bio-Rad7410010Racks to hold collecting tubes in the fraction collector.
Recombinant Human TNF-alpha (HEK293-expressed) Protein, CFBio-Techne10291-TA-020Cytokine used to stimulate 293T cells in representative results.
Saint Gobain Solid Stoppers, Versilic Silicone, Size: 00, Bottom 10.5mmSaint GobainDX263015-50Fits 48-well plates.
Saint Gobain Solid Stoppers, Versilic Silicone, Size: 4 Bottom 21mmSaint GobainDX263027-10Fits 12-well plates.
Sodium Hydroxide, 10.0 N Aqueous Solution APHA; 1 LSpectrum ChemicalsS-395-1LT
SolidWorksDassault SystemsSolidWorksCAD software used to create the multi-head dispenser DXF file.
Varioskan LUX multimode microplate readerThermoFisher ScientificVL0000D0Plate reader.
Wilton Color Right Performance Color System Base Refill, BlueMichaels10404779Blue food dye containing Brilliant Blue FCF, used as a tracer in RTD experiments. Absorbance spectrum peaks at 628 nm.

References

  1. Welsh, D. K., Yoo, S. H., Liu, A. C., Takahashi, J. S., Kay, S. A. Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression. Current Biology. 14 (24), 2289-2295 (2004).
  2. Talaei, K., et al.

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