C.B.S. Scientific FCU-1000 User Manual

Instruction Manual

3-D Flow Chamber Device

TABLE OF CONTENTS

Section 1: General Information

1.1 Flow rate chart. . . . . . . . . . . . . . . . . . . . . . . . . . . 3

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.2

1.3 System Overview

System Contents . . . . . . . . . . . . . . . . . . . . . . . . . .4-5

1.4

Section 2: Cell Culture on Upper and Lower Inserts

2.1 Coating the Membrane disk

2.2 Growing the Cells

Section 3: Flow Chamber Set-Up & Use

3.1 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3.2 Setting up the Tubing . . . . . . . . . . . . . . . . . . . . . . . .8-9

3.3 Priming the Tubing . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.4 Filling the Reservoirs and Assembly of 3D Chamber . . . . . . . . . . . 11-13

3.5 Introducing Circulating Cells to Flow Chamber. . . . . . . . . . . . . . . 13

3.6 Recovery of Cells from Chamber . . . . . . . . . . . . . . . . . . .14-16

3.7 Cleaning the Chmaber after Use . . . . . . . . . . . . . . . . . . . . 17

Section 4: Tips and Troubleshooting

4.1 Media Considerations . . . . . . . . . . . . . . . . . . . . . . . . 18

4.2 Pump Controller

4.2 Flow Rate Chart . . . . . . . . . . . . . . . . . . . . . . . . . . 23

. . . . . . . . . . . . . . . . . . . . . . . . . . 4

. . . . . . . . . . . . . . . . . . . . . . 6

. . . . . . . . . . . . . . . . . . . . . . . . .6-7

. . . . . . . . . . . . . . . . . . . . . . . . .19-22

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SECTION 1 GENERAL INFORMATION

1.1 Flow rate chart for Pump Controller for Quick Reference

ml/min dyn/cm2

0.2 0.7

0.3 1

0.4 1.3

0.5 1.6

0.6 1.9

0.7 2.3

0.8 2.6

0.9 2.9
1 3.3
2 6.6

3 9.8
5 16.3
10 35.7
20 71.6
30 107
44 157.4

1.2 References

Please go to this link: http://www.jove.com/video/50959/a-novel-three-dimensional-ow-cham­ber-device-to-study-chemokine

44ml/min is the maximum ow rate for the Pump Controller.

3-D Flow Chamber Device Instructions April 2014

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1.3 System Overview

The 3-D Flow Chamber Device is a specialized laminar ow chamber that enables the unique ability
to quantitate cellular motility through a semi-permeable membrane in the presence of shear stress.

Relevant applications for the 3-D Flow Chamber Device include transmigration, chemotaxis, vascular
permeability, and complex in vitro modeling of 3D cell cultures such as the blood brain barrier. Fur-

ther, it can be used to hold material coupons in place of the membranes for analysis of biolm activity.

1.4 System Contents

The following components ship with the 3-D Flow Chamber Device.

Complete System Includes:

• Flow Chamber Device with 3 reservoirs

• 3 upper membrane inserts

• 3 lower membrane inserts

• Tubing and connectors

• Carrying tray

• Respirator to allow gas exchange and bubble escape

4

Respirator (Bubble Trap)Pump Controller

Carrying Tray (optional)

Flow Cell (disassembledl)

Reagent Reservoir (3x)

3-D Flow Chamber Device Instructions April 2014

Upper Inserts

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Lower Inserts

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SECTION 2 CELL CULTURE ON UPPER & LOWER INSERTS

2.1. Coat the membrane disk with the relevant substrate to support cell adhesion

a. Using aseptic technique (sterile forceps) place the dry membrane disk on a dry sterile petri dish

The top membrane is positioned so that the membrane support (clear ring) is on the bottom and the

membrane is facing up.

The bottom membrane is positioned so that the membrane support is facing up – the membrane disk

will look like a little cup.

b. Carefully dispense the coating of interest (example: bronectin, poly-l-lysine) onto the membrane

surface. The membrane is hydrophobic, be sure that the liquid covers the entire surface. It will
bead up. It will take approximately 500μl to cover the top membrane and the same amount will ll

the bottom membrane cup.

c. Cover up the petri dish and incubate at the desired temperature for the desired duration.
d. Wash the coating as necessary. The membrane is now ready for use with cells.

2.2 Growing cells on the coated membrane

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a. Remove any liquid remaining from the coating and dispense the cells onto the membrane as shown above. The

liquid will bead up as it did when coating.

b. Place the covered petri dish in the incubator and incubate for the desired amount time – most cells will attach

within 1 hour.

c. Fill the dish with medium so that the membranes are oating. The top membrane is prone to lling up

with air – if this happens you can pick the membrane up with forceps and pipet some of the media
into the underside of the membrane and then slowly lay it down onto the media so that it is wet from
below.

d. Place the dish in the incubator. The cells can be stained with a live cell dye to facilitate viewing them

on the membranes (example: calcein AM or cell tracker dyes).

e. Observe cells and plan to use immediately when they reach the desired density. Conuent HUVEC

cells stained with calcein AM are shown below.

3-D Flow Chamber Device Instructions April 2014

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SECTION 3 FLOW CHAMBER SET-UP & USE

3.1 Preparation

a. Assemble all of the needed components in the area where you will set up the chamber

(the chamber, the tubing, forceps, pump, respirator, the clamps, and connectors)

b. Prepare chemokines, buffers, etc.

c. If you are going to perform your experiments at 37˚C, pre-warm all the media to avoid

spontaneous bubble formation.

3.2 Setting Up the Tubing

The 3D Flow Chamber Device is intended to be connected in a closed loop for most

experiments - the loop will consist of the chamber, tubing, pump tubing (is a xed length

and has color coded stops attached) and the respirator.

a. Set up the pump tubing and clip tubing into the pump.
b. Complete the tubing loop

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