Basic Protocol for the SciFlow™1000 System One Cell Type Adherent Cells Monolayer Culture

System Description

The SciFlow™ 1000 Fluidic Culture System is a benchtop tool for in vitro use to mimic cell, organ, and living systems. The SciFlow™ System operates like a shallow river bed with a series of compartments for cell culture. The design allows for isolated and stagnant culture during cell seeding, and then delivers real-time fluid flow and compartment-to-compartment signaling over time. The entire system is contained within a 96-well formatted culture plate that includes 8 repeatable channels. Each channel has the capacity to connect 1-to-10 cell culture wells in a linear array. As a benchtop tool, the SciFlow™ System is configured for cell and tissue assessments allowing easy access to all culture wells and media streams. Additionally, the SciFlow™ System is compatible with microplate readers, high content imaging platforms, and microscopes.

Benefit: No external pumps, tubes, nor controllers are required.

Protocol Focus: There are many applications of the SciFlow™ 1000 System. This protocol focuses on the culturing and treatment of monolayer (planar), adherent cells in all 9 interconnected cellular compartments of the SciFlow™ System. This protocol will focus on each row containing only 1 cell type, separate rows can contain distinct cultures.

Overview: A general overview of establishing cultures in The SciFlow™ System is outlined below.

  1. Fill sink (well-12) with 350 µL of medium.


  2. Seed the cells in 50 µL of medium per well – static culture conditions.


  3. To connect wells, increase medium volume to 100 µL/well.


  4. Begin fluidics by adding 400 µL of medium to the source well (well-1).

Protocol

  1. Seeding the Cells
    1. Remove the SciFlow™ 1000 plate from the sterile pouch and place in humidified incubator for 2 hours or longer. This pre-incubation aids in wettability of the plate.
    2. Seed cells in wells with 50 µL of medium. Fill wells from right to left (column 11, then 10, then 9, 8, 7, 6, 5, 4, 3). Column 2 is normally left acellular as it is in very rapid equilibrium with the source well.
    3. Allow cells to settle and attach before initiating fluidics (2 hours to overnight).
  2. Connecting Wells and Initiating Fluidics
    1. (Optional) Remove spent medium from wells 3-11; working in a “downhill” direction (left to right).
    2. Add 350 µL of warm 37 °C medium to the sink (well-12).
    3. If step 2a is performed, add 100 µL of warm 37 °C medium to each well working in an “uphill” direction (right to left). If step 2a is omitted, add 50 µL of additional medium to each well.
    4. Add 400 µL of warm 37 °C medium to well-1, the source-well. Flow will begin
    5. To ensure connection of the wells, return the SciFlow™ plate to the incubator for 30 minutes.
    6. Assess well-to-well connections by pipetting small fluid volumes from one well and visualizing volume fluctuations in neighboring wells.
  3. Compound Exposure
    1. To begin gradient formation, gently add 100 µL of a 5× stock solution of the compound of interest (or fluorescent tracer), creating a 1× solution in well-1. Mixing is not suggested as it will disturb the gradient formation. Volume and stock concentration of compound can be determined by experimental need.
    2. For continuing experiments beyond 1-day OR to increase the gradient, remove 150 µL medium from well-12 of each row and add 150 µL of 1× compound to well-1. Repeat feeding at user-defined intervals based on experimental design.

 

Sample Plate Map

Sample plate map


Example of Cell Seeding Parameters
SciFlow™ culture well areas are ½ the size of traditional 96-well culture surface areas (0.167 cm2 or 16.7 mm2).

Cell Seeding Examples, 2D monolayers Number of Cells per SciFlow™ Plate Number of Cells per Well Number of Culture Wells Seed Time Initial Confluence Adjustment
Primary human hepatocytes with collagen coating 2.0 E6 27,500 72 (3 – 11) Overnight Confluent By viewing
Primary rat hepatocytes with collagen coating 1.0 E6 14,000 72 (3 – 11) Overnight Confluent By viewing
Primary mouse hepatocytes with collagen coating 6.5 E5 9,000 72 (3 – 11) Overnight Confluent By viewing
Primary duck hepatocytes with collagen coating 6.5 E5 9,000 72 (3 – 11) Overnight Confluent By viewing
Primary canine hepatocytes with collagen coating 1.0 E6 14,000 72 (3 – 11) Overnight Confluent By viewing
HepG2 2.2 E6 30,000 72 (3 – 11) Overnight 80% By viewing
HepaRG 2.9 E6 40,000 72 (3 – 11) Overnight 80% By viewing
HepaRG (no spin) 2.9 E6 40,000 72 (3 – 11) Overnight 80% By viewing
Cell Line MCF7 7.2 E5 10,000 72 (3 – 11) Overnight 20% By viewing

Tips

Removing Medium (if required): The SciFlow™ System can be emptied by inversion and flicking into an appropriate waste container. Additionally, the entire row can be emptied via vacuum aspiration through the sink well.

Adding Medium (if required): When adding fluid to the SciFlow™ plate, it is best to begin additions at the lower end of the plate (Column 11), followed by columns 10 then 9…. This will result in downstream wells being filled before the upstream wells, and result in a much more controlled fluid flow.

Cell Number: SciFlow™ culture well areas are ½ the size of traditional 96-well culture surface areas. The number of cells seeded in each well should be adjusted accordingly (see cell seeding table).

Compound Addition: When initiating a gradient, the most reproducible method is to add a small volume of a more concentrated stock solution into the source well. When adding the more concentrated stock, do not pipette up and down (to mix) as this will change the flow dynamics. Add gently.

Feeding/Dosing: For experiments requiring incubations longer than 24 hours or for repeated exposures to the compounds of interest, additional medium/compound must be added to the source well. Volumes between 50–250 µL can be used for dosing/feeding. In most cases 150 µL is an appropriate volume. Whatever volume is chosen, the same volume should be initially removed from column 12 (waste) and an equal volume of fresh medium or 1× compound in fresh medium added into the source well.

Tracking Flow: Fluorescein can be used as a tracking dye to monitor the flow during an experiment and to approximate compound concentrations in each well, not accounting for cellular metabolism. See separate protocol for detailed instructions on quantifying fluorescein and compound concentrations.

Moving the SciFlow™ 1000 System: The SciFlow™ System is a fluidic system and tipping the plate can disrupt or modify both flow and any established gradients. Reasonable care should be taken when moving the plate to minimize unintended flow.

Evaporation: Although the SciFlow™ 1000 System does have a lid, evaporation can occur. For experiments over 7 days, a 10–20% greater volume can be added to the source well than is removed from the sink.

Sampling: It is possible to sample from the wells during the experiment. 5–10 µL aliquots can be simultaneously removed from each well in a row using a multichannel pipette.

Running Assays in SciFlow: Cell based assays can be run in the SciFlow™ plate. It is important to remember that once the fluidics are engaged they will stay engaged. Here are a few assay suggestions:

  • For endpoint assays remove all media from the plate by inversion and flicking the plate over an appropriate waste container.
  • Keep volumes of buffer and assay reagents less than 50 µL in order to reduce possible flow from well to well.
  • Shorter incubation times are preferable, minimizing inter-well flow.