Tutorials/Protocols for the CellASIC® ONIX Microfluidic Platform

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Ready to take control with dynamic cell culture and analysis? Find all the step-by-step protocols you need to get the most out of your CellASIC® ONIX2 platform.

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Cell Culture Chamber Preparation/Priming
CellASIC® ONIX2 microfluidic plates are pre-primed and ready for pressure-driven cell loading. However, if using capillary-driven cell loading, or if your cell culture requires coating, follow the appropriate plate priming protocol prior to cell loading. Coating of tissue culture surfaces with extracellular matrices (ECMs) or polylysine generally promotes cell attachment and monolayer formation.
Protocol
Description
Priming M04S Plates with Growth Medium Perform this step when using capillary loading to ensure even distribution of cells in the cell chamber.
Coating M04S Plates with 0.1 % Gelatin Gelatin coatings are commonly used for various adherent cell types, such as vascular endothelial cells, embryonic stem cells and F9 teratocarcinoma cells.
Coating M04S Plates with Collagen I Collagen I coating is commonly used to promote the attachment of various cell types, including endothelial cells, fibroblasts, hepatocytes and epithelial cells.
Coating M04S Plates with Fibronectin Fibronectin coating is commonly used for culturing endothelial cells, fibroblasts, neurons and adherent CHO cells.
Coating M04S Plates with Laminin Laminin coating is commonly used for neurons, epithelial cells, leukocytes, myoblasts and CHO cells.
Coating M04S Plates with Matrigel® Matrix (THIN COATING METHOD) Matrigel® matrix is a solution of multiple ECM proteins, growth factors and other proteins. Depending on the cell type, this matrix can induce complex changes in cell behavior, such as stem cell differentiation.
Coating M04S Plates with Poly-L-Lysine Polylysine promotes attachment of virtually all cell types, and is particularly useful for culturing central nervous system neurons.



Cell Loading
Making biologically relevant conclusions by comparing one cell culture chamber to another in a single multichamber plate is only possible if cells are evenly loaded and subject to the same perfusion conditions in space and time. The CellASIC® ONIX2 Microfluidic Platform provides options for pressure-driven loading or capillary-driven loading, which allows the user to simply load cells using a pipette in any sterile laminar flow hood without any external systems.

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Reproducibility of cell loading depends on cell type, chamber coating and cell concentration. In general, the number of cells loaded per chamber is more reproducible at lower cell concentrations and in the presence of ECM coating.


Protocol
Description
Capillary-driven Cell Loading (No ECM) This protocol allows the user to load cells using a pipette, and can be done in a laminar flow hood without any external systems.
Capillary-driven Cell Loading (After ECM Coating) This protocol allows the user to load cells into ECM-coated chambers using a pipette, and can be done in a laminar flow hood without any external systems.
Pressure-driven Cell Loading For even, completely automated cell loading, use this pressure-driven loading protocol using the CellASIC® ONIX2 system and CellASIC® ONIX2 FG software.



Establishing Healthy Cell Cultures
The CellASIC® ONIX2 Microfluidic Platform gives you increased microenvironment control, so that you can tune your culture conditions to mimic your cells’ in vivo environment (Table 1). In addition, automated perfusion protocols enable you to set up your experiment and walk away, requiring only that you empty the waste wells periodically on a schedule dependent on your flow rate (Table 2). Continuous outflow of waste may help your cells stay healthier, longer, without interruption of optical access during live cell imaging.

RepresentativeCell TypeCells Cat. No.ECMECM ConcentrationECM Cat. No.Attachment Time Before PerfusionPSICell Concentration (95% Confluence at Day 4)
Endothelial Cells HUVEC Merck SCCE001 Collagen I  0.67 µg/µL Sigma 3867 0 0.5 1.0 x 106 cells/mL
Epithelial, Cancer, Light Adherence MCF-7 ATCC HTB-22 Fibronectin 0.33 µg/µL Merck 341635 0 0.25 1.5 x 106 cells/mL
Epithelial, Cancer, Robust HeLa ATCC CCL-2 NONE n/a n/a 0 1 1.0 x 106 cells/mL
Fibroblast, Robust NIH 3T3 ATCC CRL-1658 Gelatin  1% Gelatin Solution Merck ES-006-B 0 1 1.0 x 106 cells/mL

Table 1. Optimized conditions obtained by testing four representative cell lines with varying flow rates, durations, loading concentrations and ECM coatings on the CellASIC® ONIX M04S Plate. Given our observations, we recommend that users optimize these conditions for their own cells and experimental perturbations.

Calculating Waste Well Emptying Schedule for M04S Plate
Pressure (psi)Average Flow rate ( μL/h)Empty Waste Wells 7&8 Every:
0.25 0.6 1250 h (~52 days)
0.5 2 375 h (~16 days)
1 7 107 h (~4 days)
2 20 37 h (~1.5 days)
3 32 23 h (~1 day)
4 45 16 h
5 72 10 h
6 95 8 h
7 120 6 h
8 147 5 h
  • Take fastest flowrate, X + 20%
  • Assume max volume of wells 7 & 8 is 900 μL
  • Assume run 1 well/experiment

Table 2. Schedule for emptying waste wells of CellASIC® ONIX2 M04S Microfluidic Plate.


Cell Analysis
Follow these step-by-step guides for cell staining and analysis within the microfluidic plate. Not only do these automated protocols offer significant time savings, but the use of low-volume microfluidics can also conserve precious antibodies and staining reagents.

Protocol
Description

Viability Staining Protocol within M04S Microfluidic Plate

In this in-plate, perfusion-based assay, live cells, stained with Calcein-AM, fluoresce green, and dead cells, stained with EthD-1, fluoresce red.
Automated Immunostaining Within M04S Plate – Surface Marker This protocol uses the CellASIC® ONIX2 system to stain specific extracellular proteins for microscopy while maintaining uninterrupted optical access to cells.
Automated Immunostaining Within M04S Plate – Intracellular Marker This protocol uses the CellASIC® ONIX2 system to stain specific intracellular proteins for microscopy while maintaining uninterrupted optical access to cells.