NanoSurface Cytostretcher-LV User manual

NanoSurface Cytostretcher-LV
Cell Stretching Instrument with Nanopatterned Chambers
User Manual
Last Revised 5 October 2018
(HWrCSLV2.0)
Copyright ã2018
NanoSurface Biomedical, Inc.
4000 Mason Road, Suite 304
Seattle, WA 98195
United States of America
www.nanosurfacebio.com
[email protected]
[phone]

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INTRODUCTION ....................................................................................................................................3
SECTION 1: SYSTEM OVERVIEW........................................................................................................4
A. THE NANOSURFACE CYTOSTRETCHER-LV ............................................................................................................ 4
B. REMOVING THE GLASS WINDOW ....................................................................................................................... 5
C. CYTOSTRETCHER CULTURE CHAMBER ...................................................................................................................... 6
Two Chamber Sizes ......................................................................................................................................... 6
Two Surface Options for Each Chamber Size .................................................................................................. 6
D. SOFTWARE ......................................................................................................................................................... 6
SECTION 2: USING THE CYTOSTRETCHER ......................................................................................7
A. PREPARATION OF THE CYTOSTRETCHER .................................................................................................................... 7
B. SOFTWARE INSTALLATION ..................................................................................................................................... 7
C. FOCUS TILT ADJUSTMENT ................................................................................................................................. 8
Procedure ........................................................................................................................................................ 8
D. SOFTWARE OPERATION .................................................................................................................................... 9
Overview: ........................................................................................................................................................ 9
Before starting: ............................................................................................................................................. 10
Working with Stretch Segments .................................................................................................................... 10
Offsets: .......................................................................................................................................................... 10
Calibration: ................................................................................................................................................... 10
Example- Symmetrical Repetitive Sine Stretch with no fixed time: 10%, 1Hz using a 12 mm chamber. ....... 11
Calculating Strain .......................................................................................................................................... 11
SECTION 3: CULTURING CELLS IN THE CYTOSTRETCHER CHAMBERS ...................................12
A. CHAMBER STERILIZATION .................................................................................................................................... 12
B. CELL ATTACHMENT ............................................................................................................................................ 12
Plasma Treatment (Recommended) ............................................................................................................. 12
Coating with ECM proteins without plasma treatment ................................................................................ 12
Chemical Modification .................................................................................................................................. 13
SECTION 4: WARRANTY & SPECIFICATIONS ................................................................................. 14
WARRANTY .......................................................................................................................................................... 14
SPECIFICATIONS ..................................................................................................................................................... 14
APPENDIX A: INSTALLING THE FONT PACKAGE ..........................................................................15
REFERENCES .....................................................................................................................................16

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Introduction
Thank you for purchasing the NanoSurface Cytostretcher-LV. This device is designed to stretch NanoSurface’s
proprietary in vitro culture chambers on top of an inverted optical microscope.
The Cytostrecher-LV is compatible with NanoSurface Biomedical’s Environmental Control Unit (ECU). The ECU
is an optional accessory that controls the temperature and CO2content of the environment inside the imaging
chamber. Operation of the ECU is covered in a separate manual. If you need to add the ECU accessory to your
Cytostretcher-LV, please email [email protected] or your local distributor.
Please read this manual thoroughly. It contains important information to ensure safe and proper use of the
instrument. Critical information that could result in injury to you or in damage to the instrument will be highlighted
with a warning triangle. Do not ignore these important warnings!
This manual and the product described herein is subject to change. Changes to the product may not be accurately
reflected in this manual. Images of the product may not be identical to the latest shipping product. If there are any
questions, please contact us.
**This product is intended for research use only**
Please follow the instructions in this manual carefully. Failure to do
so could result in injury to the user or damage to the instrument.
There are no user serviceable parts inside the Cytostretcher-LV.
Opening the Cytostretcher-LV case or removing mechanical
components could result in damage to the instrument. Opening the
Cytostretcher-LV will immediately void the warranty. For service,
email [email protected] or call [phone].

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Section 1: System Overview
A. The NanoSurface Cytostretcher-LV
A. Control button: pause and resume stretch. Hold this button to turn your Cytostretcher on and off.
B. AC power port and USB communication cable port.
C. Offset/Pre-tension thumbscrew: allows you to pre-stretch the membrane, if desired.
D. Actuating Bracket: this component holds part of the chamber and moves back and forth to provide stretch.
The actuating bracket moves back and forth when the Cytostretcher is
operating. Keep objects and fingers clear of this bracket, as it can result
in injury to the user or damage to the Cytostretcher.
E. Chamber: Location of the chamber
F. Imaging Chamber Cover: removable glass window for maintaining chamber CO2and Temperature control with
the optional ECU.
G. Cover Connector: Magnetic connector to interface with the optional ECU.
H. Z Focus Shift Micrometer: adjusts the Z-plane of the chamber as it stretches in order to maintain focus under
stretch.

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The following features are shown on the image below:
I. Standard Universal Type-K microscope mount (inquire about other styles)
J. Glass window
B. Removing the Glass Window
If the glass window needs to be removed or replaced, you can access it by removing the four bolts that hold the
window frame in place (Figure A, below). After removing the bolts, gently lift the glass out with a small tool or pair
of tweezers (Figure B, below). If the window is beyond cleaning or damaged, please contact
[email protected]

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C. Cytostretcher Culture Chamber
Two Chamber Sizes
The NanoSurface Cytostretcher is designed to work with NanoSurface’s small and medium-sized cell culture
chambers. They are not compatible with the large (50 x 50 mm) chambers. Compatible chambers are available in
two sizes. The small chamber has a 25 mm2culture area in a square well that measures 5 x 5 mm. The medium
chamber has a total culture area of 144 mm2, in a square well that measures 12 x 12 mm. Dimensional details are
presented in the figure below. The chamber material is silicone based and can be prepared for cell culture using
standard coating techniques. Additional information about treatment with ECM proteins is noted in a subsequent
section of this manual.
To mount the chamber, insert the included stainless-steel rods through the round holes in the sides of the chamber.
Use these rods to mount the chamber into the Cytostretcher.
Two Surface Options for Each Chamber Size
All of the Cytostretcher chambers come with either flat (unpatterned control; left image) or nanopatterned (center
image) surfaces. For the nanopatterned surfaces, the horizontal parts of the ridges and the grooves are both 800
nm. The depth of the grooves is 600 nm. See Figure 1 for details.
Figure 1. Left: A scanning electron micrograph of the unpatterned surface. Center: A scanning electron
micrograph of the nanopattern showing the surface and cross section. Right: A schematic showing the
approximate dimensions of the nanopattern cross-section.
D. Software
The Cytostretcher is controlled by NanoSurface Operational Mechanics Interface (NaOMI). Please email
[e-mail] for the latest version. NaOMI will run on either a PC or a Mac. Operation of the
software will be detailed in the next section.

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Section 2: Using the Cytostretcher
A. Preparation of the Cytostretcher
1- Before using the Cytostretcher, it is recommended that you sterilize it. We recommend wiping the unit
down with 70% ethanol and ultrapure water. Avoid getting liquid into the power or USB ports. Ensure that
the unit is dry before connecting the power or USB cables. Note that the inner motors and electronics are
sealed from liquid intrusion but using excessive amounts of liquid or immersing parts of the Cytostretcher
will damage the instrument.
2- Sterilize and coat the chamber as described in Section 3.
3- Connect the power cable to the Cytostretcher and plug in to the power source. The Cytostretcher will begin
calibration and get ready for stretching. Note that many cell culture incubators have ports located on the
back wall; many of our customers choose to route their USB and power cables through this port.
B. Software Installation
Important Note: NanoSurface’s NaOMI software requires the use of the “Roboto” font/typeface. If your system
does not already have this installed, please see Appendix A for instructions. If this is not installed on your computer,
the software window will not display properly, and some buttons may be missing.
1- Step 1: Download the software
a. NanoSurface’s NaOMI software is made to work on Windows 10. For the latest software version,
please contact [email protected]
b. The software will be a ZIP file. De-compress this. You will see two folders: one for the USB driver,
and one for the Software.
2- Step 2: Install the USB Driver
a. The Cytostretcher requires a specific USB driver. The driver is included with the ZIP archive that
contains the software. You can also download it here: http://www.ftdichip.com/Drivers/D2XX.htm
b. This driver will not affect other devices attached to your computer.
c. The driver is in the “Cytostretcher USB driver” folder.
d. Once the driver is installed, restart your computer.
3- Step 3: Install NaOMI (Cytostretcher Software)
a. Double-click the Setup icon.
b. Depending on your windows security settings, you may have a warning that this software is from
an unverified publisher. Simply override this warning to continue installation.
c. Proceed with the installation. The installer should leave a shortcut on your desktop.

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C. Focus Tilt Adjustment
NanoSurface recommends adjusting the angle of the Cytostretcher chamber to ensure that the pre-stretch and
post-stretch states remain in the same focal plane. This is accomplished by adjusting the four M3-hex head bolts
located adjacent to the chamber rods. To ensure cell viability, this can be performed before cells are cultured on
to the chamber. Note that different chambers may require individual adjustment.
Procedure
1. Start with the test chamber that shipped with your instrument. If the test chamber does not have ink markings
on the surface, use a sharpie to deposit some ink on the cell culture surface.
2. Load the chamber into the Cytostretcher-LV. Make sure you clamp the rods down with the thumbscrews.
3. Turn the Z focus adjust micrometer until the adjustment stage is at its lowest position. You should be able
to look at the chamber and see that the left side is lower than the right.
4. Using the NaOMI software, program a stretch routine that has the same amount of stretch that you plan to
use. Program a cyclical stretch with a delay of a few seconds in between stretch and relax. This delay should
be long enough to allow you to focus the optical microscope between stretch-and relax phases.
5. Use the microscope to focus on the surface of the chamber. The edges of the inkblot can be helpful focusing
targets.
6. Start the stretch routine.
7. When the sample is fully stretched, you will have to adjust the microscope focus knob to keep the sample in
focus.
8. Iteratively adjust the Z micrometer in steps while re-checking focus. You may not see a big difference in how
much you need to focus even after several Z adjustments.
9. Over several adjustment cycles you will find that the focus positions become much closer to each other; at
this point continue the iterative adjustment with smaller steps until the focus at stretch versus relaxation is
the same.
10. Replace the test chamber with your sample. You may have to make some minor adjustments with the Z
micrometer to maintain exact focus.

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D. Software Operation
Overview:
The Cytostretcher is controlled by the NanoSurface Operational Mechanics Interface (NaOMI). NaOMI
allows you to design a particular stretch protocol and run the instrument. The instrument does not need
to be connected to the computer during the actual stretch protocol, but you will need to connect to the
instrument via USB in order to program it with your protocol. Once the protocol has been sent to the
instrument, it no longer needs to be connected to the computer.
Protocols are built as a series of steps or “Stretch Segment”. Your protocol can be one or many Stretch
Segments. Stretch Segments are added, removed, duplicated, or re-ordered under the “Edit” section on
the left of the panel. Each Stretch Segment will appear as a line in the “Protocol Segments” window.
Double-clicking the Stretch Segment will open up the “Stretch Segment Editor” window, which enables
you to change various aspects of the stretch.
Once a protocol is built, the graph at the bottom of the software window will show you an approximate
representation of stretch vs. time.
When you are happy with your protocol, you will send it to the device before starting.

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Before starting:
Do not load your chamber into the instrument until you are ready to stretch.
Before loading the chamber, power on the instrument. Connect to the Cytostretcher
by pressing the “NaOMI Connection” button. Re-set the zero point of the instrument
by pressing the “Calibrate” button. Set your program and send it to the instrument.
Each of these steps may result in an actuation of the device. After doing this, load
your chamber.
Working with Stretch Segments
Each Stretch Segment has its own parameters. Double-clicking
the segment will open the Stretch Segment Editor (image on the
right). This is where you will change the segment parameters.
• First, define the amount of stretch (“Stretch distance”).
The table below shows you how distance relates to %
stretch.
• You can define whether the Cytostretcher will stretch with
a linear (“ramp”) or sinusoidal (“sine”) waveform under
the “Stretch Type” parameter.
• If you like, you can apply a “pre-hold”, which is a pause
that is made before the stretch is applied.
• Last, you can define the speed at which the stretch is
made (“Speed A”).
If you’d like an asymmetrical stretch, where the relaxation has different parameters, check the box next
to “relaxation type”.
• The waveform of the relaxation type can be set to Ramp or Sine
• a “post-hold” time can be added- this will be a holding time between the maximum stretch and
the onset of relaxation. if you do not want any delay between stretch and relax, set this to “0”.
• Specify the speed of relaxation in the “Speed B” parameter.
Offsets:
This can be used to pre-tension your chamber. The offset will be used as the starting point for any stretching
routines, and all stretch distances will be relative to the offset point. You can adjust this by pressing the arrow
buttons or moving the slider.
Calibration:
This button re-sets the zero point of the stretcher. NanoSurface recommends calibrating the instrument before
each use. Note that pressing this button will actuate the stretcher. If any cells are in the device, they will be
stretched to a potentially dangerous degree.