Basic Protocol for the SciFlow™ 1000 System Collagen Coating of Well Surfaces Pre-Coat of a Thin Protein Layer 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 collagen coating of well surfaces prior to plating monolayer type cells. Adjustments for specific coatings should be explored by users. This protocol is for coating each well surface with collagen type-1. Separate wells can contain distinct surface coats.

Overview
SciFlow™ culture well areas are ½ the size of traditional 96-well culture surface areas (0.167 cm2 or 16.7 mm2). As purchased, all culture wells are tissue culture treated.

Surface images of linear aligned culture wells

Protocol:

To achieve a 10 µg/cm2 surface collagen coating - Add 1.67 µg of collagen to each well. Use a 30 µL volume per well to avoid surface tension variations and climbing of liquid up culture walls.


Preparation of Rat Tail Collagen Type-1

  1. Dilute collagen type-1 (Product Number C3867 or equivalent) to 0.056 mg/mL using base medium without serum (e.g., DMEM or Williams E) in 0.1 N acetic acid.
  2. Keep the solution cold on wet ice.
  3. Add 30 µL of prepared diluted collagen solution to each well.
  4. Spread evenly by rocking the plate in a figure-eight pattern. For best results, set plate on a paper towel on top of a bed of wet ice prior to applying collagen.
  5. Place in humidified 37 °C incubator for >1 hour, providing enough time for protein adhesion.
  6. Remove excess fluid.
  7. Rinse plate twice with base cell culture medium or PBS prior to plating cells.

 

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.

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 caused by tipping the plate.

Evaporation: Alhough 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.