Thermo Scientific gibco CTS Rotea User manual

For Research Use or Manufacturing of Cell, Gene, or Tissue- Based
Products.
CTS™Rotea™Counterflow Centrifugation
System Process Design
USER GUIDE
Catalog Number A44769
Publication Number MAN0018754
Revision A.0

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The information in this guide is subject to change without notice.
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ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0018754
Revision Date Description
A.0 20 October 2020 New document. User guide for process design workflow.
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©2020 Thermo Fisher Scientific Inc. All rights reserved.

Contents
■CHAPTER 1 Product information .................................................. 7
Product description ............................................................. 7
System overview ................................................................ 8
CTS™Rotea™Counterflow Centrifugation Instrument ............................ 8
CTS™Rotea™Single-Use Kit ................................................. 9
CTS™Rotea™Graphical User Interface (GUI) .................................. 10
Rotea Protocol Builder ..................................................... 11
■CHAPTER 2 Software installation ................................................ 12
Software installation ............................................................ 12
■CHAPTER 3 Rotea Protocol Builder Application ................................ 17
Create or open a protocol ....................................................... 17
Define Kit ..................................................................... 18
CTS™Rotea™Single-Use Kit diagram ........................................ 19
Cell Type ................................................................. 21
Description ............................................................... 21
Kit Type .................................................................. 22
Long Description .......................................................... 22
Step view ..................................................................... 23
Define fluid path and direction of flow ........................................ 24
Step Details .............................................................. 25
Step Type ................................................................ 27
Trigger ........................................................................ 32
Volume ................................................................... 33
Timer .................................................................... 35
Pressure 1 and Pressure 2 ................................................. 36
Bubble Sensor Input and Bubble Sensor Output .............................. 37
Data Entry .................................................................... 38
CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 3

List view ...................................................................... 39
Step summary ............................................................. 40
Edit using List view ........................................................ 42
Loops ........................................................................ 42
Repeat Count ............................................................. 43
Bubble Sensor Not Triggered ............................................... 44
Process Counter .......................................................... 44
Timer (Seconds) and Timer (Minutes) ........................................ 45
Skip Button Triggered ..................................................... 45
Editing loops .............................................................. 46
■CHAPTER 4 Protocol Building Blocks ........................................... 47
Priming ....................................................................... 47
Overview ................................................................. 47
Bubbles .................................................................. 47
Priming volumes ........................................................... 48
Basic priming steps ........................................................ 49
Prime remote sources ...................................................... 52
Manage air in the system ................................................... 53
Establish a fluidized bed ........................................................ 53
Overview ................................................................. 53
The seven parameters for establishing a fluidized bed .......................... 55
Optimize G force and flow rate .............................................. 56
Cell size (d)............................................................... 56
Media density (ρ) .......................................................... 57
Cell concentration ......................................................... 57
Establish a bed in recirculation .............................................. 57
Establish a bed without recirculation ......................................... 59
What a good bed looks like ................................................. 60
Establish a bed: Troubleshooting ............................................ 62
Cell loading ................................................................... 64
Overview ................................................................. 64
Cells loaded into the CFC Chamber .......................................... 64
Bed disruption during loading ............................................... 65
Cell separation during loading ............................................... 65
Re-use input bags ......................................................... 66
Refill the Bubble Trap ...................................................... 69
Connect to bioreactors ..................................................... 70
Wash cells and buer exchange ................................................. 71
Overview ................................................................. 71
Media selection ........................................................... 72
Wash to similar density media ............................................... 72
Wash to more dense media ................................................. 72
Wash to lower density media ................................................ 73
Defensive washing strategies ................................................ 73
Contents
4CTS™Rotea™Counterflow Centrifugation System Process Design User Guide

Elutriation ..................................................................... 77
Overview ................................................................. 77
Improve cell viability ....................................................... 79
Leukopak processing ...................................................... 79
Typical leukaphoresis composition ........................................... 80
Lentivirus clarification ...................................................... 81
Separate cells from microcarriers ............................................ 81
Elutriation techniques ...................................................... 82
Concentrate Recovery ......................................................... 85
Overview ................................................................. 85
Normal step ............................................................... 86
Speed settings ............................................................ 86
Pause step ................................................................ 87
Harvest step .............................................................. 88
Harvest using OD Sensor ................................................... 90
Multi-bite processing ........................................................... 92
Overview ................................................................. 92
Determine the number of cells per bite ....................................... 92
Maximize throughput ....................................................... 93
Process integrity ............................................................... 94
Overview ................................................................. 94
CTS™Rotea™Single-Use Kit ................................................ 94
Check manual clamps ...................................................... 95
Verify reagent connected and not clamped ................................... 95
Check for closed clamps ................................................... 95
■CHAPTER 5 Rotea™Process Model ............................................. 96
Counter flow centrifugation forces ................................................ 96
Predict process settings ........................................................ 98
Select and format media .................................................. 100
Create custom media ..................................................... 102
Model particle behavior ................................................... 103
Change and delete particles from a model .................................. 106
Create custom particles ................................................... 107
Edit custom particles and custom media .................................... 107
■CHAPTER 6 Rotea™Simulation software ...................................... 109
Rotea™Simulation software .................................................... 109
■CHAPTER 7 Design new protocols ............................................. 115
Plan the kit configuration ....................................................... 115
Plan the protocol ............................................................. 115
Prepare for the first trial run .................................................... 116
Contents
CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 5

Water runs ................................................................... 116
First run with cells ............................................................. 116
Analyze the outcome .......................................................... 116
Refine the protocol to achieve target cell outcomes ............................... 116
Add process integrity features .................................................. 117
■APPENDIX A Safety .............................................................. 118
Symbols on this instrument .................................................... 118
Standard safety symbols .................................................. 119
Control and connection symbols ........................................... 119
Conformity symbols ...................................................... 119
Safety information for instruments not manufactured by Thermo Fisher Scientific ..... 121
Instrument safety ............................................................. 121
General ................................................................. 121
Physical injury ............................................................ 122
Electrical safety .......................................................... 122
Cleaning and decontamination ............................................. 123
Safety and electromagnetic compatibility (EMC) standards ......................... 123
Safety standards ......................................................... 123
EMC standards ........................................................... 124
Environmental design standards ............................................ 124
Chemical safety .............................................................. 125
Biological hazard safety ....................................................... 126
■APPENDIX B Documentation and support .................................... 127
Customer and technical support ................................................ 127
Limited product warranty ...................................................... 127
Contents
6CTS™Rotea™Counterflow Centrifugation System Process Design User Guide

Product information
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
The Gibco™CTS™Rotea™Counterflow Centrifugation System is an automated, closed-flow benchtop
instrument that can perform a wide variety of cell processing steps across dierent cell types.
This CTS™Rotea™Counterflow Centrifugation System Process Design User Guide provides the user
with an overview of the Rotea Protocol Builder application and describes some of the commonly used
features and strategies to assist the user with the development of protocols.
Note: The user is responsible for the development, optimization, qualification, storage and
management of protocols used on the CTS™Rotea™Counterflow Centrifugation System.
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CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 7

System overview
CTS™Rotea™Counterflow Centrifugation Instrument
The CTS™Rotea™Counterflow Centrifugation Instrument is an automated benchtop instrument that
incorporates several important features that enable the user to develop and optimize protocols while
ensuring the integrity of the process.
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Figure 1 Instrument – Front View
1Instrument Door
2Pinch Valves (J & K)
3Kit Location Button
42D Barcode Reader
5CFC Chamber Detector
6CFC Chamber Carrier
7Camera
8Moisture Sensor
9Pushbutton Controls
10 Optical Density Sensor & Pressure Sensor (P1)
11 Peristaltic Pump
12 Pump Clamp Arm
13 Pinch Valve (H)
14 Kit Location Button & Pressure Sensor (P2)
15 Door Latch
16 Pinch Valves (A, B, C, D, E, F, & G)
17 Bubble Sensors
18 Hanger Posts
Chapter 1 Product information
System overview
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8CTS™Rotea™Counterflow Centrifugation System Process Design User Guide

CTS™Rotea™Single-Use Kit
The CTS™Rotea™Single-Use Kit can be flexibly configured by the user to perform a range of cell
processing applications such as separation, isolation, buer exchange, wash and concentrate. The
Single-Use Kit comprises eight sterile weldable tubes to which the user connects input and output
bags/vessels to suit their specific process. The Single-Use Kit also includes a 2D Barcode for kit
identification and labelling to aid kit assembly.
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Figure 2 CTS™Rotea™Single-Use Kit
1Valve Hole & Tube ID
2Carrier Frame
3Bubble Trap
4Location Hole
5CFC Chamber
6Rotary Coupling
72D Barcode
8Pump Tubing
9Tube Retainer
10 Valve H Hole
11 Location Hole
12 Kit Tubing (Input and Output)
Chapter 1 Product information
System overview 1
CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 9

CTS™Rotea™Graphical User Interface (GUI)
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
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The CTS™Rotea™Graphical User Interface (GUI) enables users to download and run protocols on the instrument. The
Rotea™GUI also includes the ability to make in-process adjustments to the G force and flow rate and has an integrated
high-resolution color camera to visualize the behavior of cells in the CFC Chamber in real-time. This provides the user with
an interactive environment for protocol development and optimization.
Chapter 1 Product information
System overview
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10 CTS™Rotea™Counterflow Centrifugation System Process Design User Guide

Rotea Protocol Builder
The Rotea Protocol Builder is a Windows-based application that enables users to download, create, modify and save
protocols.
Chapter 1 Product information
System overview 1
CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 11

Software installation
Software installation
1. Prior to installing a new release of the Rotea Protocol Builder application, first un-install any
previous versions.
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12 CTS™Rotea™Counterflow Centrifugation System Process Design User Guide

2. Locate the latest release of the Rotea Protocol Builder Setup on your PC or accessible directory
(in this example a USB Drive). Double click it to begin installation.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
1 1
1
1
3. Select whether the software will be installed for the current user or all users on the device, and
click Next.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
1 1
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Chapter 2 Software installation
Software installation 2
CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 13

4. Choose where you want the software to be installed. By default, a folder called Rotea Protocol
Builder is created in Program Files on your C:\ drive. Then click Install.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
1 1
1
1
Chapter 2 Software installation
Software installation
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14 CTS™Rotea™Counterflow Centrifugation System Process Design User Guide

5. Wait for the installation to complete.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
1 1
1
1
Chapter 2 Software installation
Software installation 2
CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 15

6. Choose whether you want Rotea Protocol Builder to open once installation has finished, and click
Finish to close the installer.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
1 1
1
1
Chapter 2 Software installation
Software installation
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16 CTS™Rotea™Counterflow Centrifugation System Process Design User Guide

Rotea Protocol Builder Application
Create or open a protocol
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
1 1
1
1
This is the home screen of the Rotea Protocol Builder. Select Create New to create a new protocol, or
Select a Protocol to open an existing protocol from an accessible local or external folder.
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CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 17

Define Kit
Select Create New, to go to the Define Kit screen.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
1 1
1
1
1CTS™Rotea™Single-Use Kit diagram
2Cell Type
3Description
4Kit Type
5Long Description
Chapter 3 Rotea Protocol Builder Application
Define Kit
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18 CTS™Rotea™Counterflow Centrifugation System Process Design User Guide

CTS™Rotea™Single-Use Kit diagram
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
2. Edit number and/or line-length, as needed.
1 1
1
1
On the left of the Define Kit screen is a diagram of a CTS™Rotea™Single-Use Kit, with no bags attached. The Single-Use
Kit configuration is defined by the user by adding bags to the various lines required to perform the protocol.
Chapter 3 Rotea Protocol Builder Application
Define Kit 3
CTS™Rotea™Counterflow Centrifugation System Process Design User Guide 19

Bag Configuration
To create a new bag, select the to open the Bag Configuration window.
1REQUIRED: Enter a label for the bag here (maximum 20 characters). The name can be anything you want but should
be a logical reference for the operator of the instrument. This label will appear in multiple places throughout both the
Rotea Protocol Builder Application and the Rotea™GUI as the primary way of identifying the function of the bag. It
is very important that the name given is clear and provides the user with an adequate understanding of the bags
function and/or contents.
2REQUIRED: Select which valve the bag will be connected to for the protocol from the dropdown menu.
3OPTIONAL: Set the volume of fluid that should be in the bag at the beginning of the protocol. This is used as a
reference so that the user can ensure there is enough fluid to run the protocol.
4OPTIONAL: Enter the maximum volume the bag can contain, for user reference.
5OPTIONAL: In the Description box you can enter a more detailed description for the bag (maximum 50 characters).
This is for reference only and can be anything you wish.
6If the bag has a second connection, you can select one from the dropdown menu. Only valid options will be
presented.
7Click Confirm to save the bag, or Cancel to return to the Define Kit screen.
Chapter 3 Rotea Protocol Builder Application
Define Kit
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20 CTS™Rotea™Counterflow Centrifugation System Process Design User Guide