Cyfuse Regenova Installation and operation manual

Regenova®
A Bio 3D Printer
Start up Manual Ver.3
Feb.2016
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
1

Contents
Prior to 3D printing
1.Accessories and tools・・・・・・・・・・・・・・・・・・3
2.Making spheroids・・・・・・・・・・・6
3.Designing your 3D・・・・・・・・・・・・・・・7
4.Sterilizing accessories and tools・・・・・・・・・・・・・・・・・8
3D printing
1.Powering on・・・・・・・・・・・・・・・・・・11
2.Starting the software ・・・・・・・・・・・・・・・・・・13
3.Set up・・・・・・・・・・・・・・・・・・14
4.Setting up Cell Accumu・・・・・・・・・・・・・・・・・・16
5.3D printing・・・・・・・・・・・・・・・・・・・・・20
6.After 3D printing・・・・・・・・・・・・・・・・・25
Maturation
1.Maturation・・・・・・・・・・・・・・・・・・・・・27
2.Removing tissue construct from Kenzan・・・・・・・・・・・・28
FAQ・・・・・・・・・・・・・・・・・・・29
Advanced Operation・・・・・・・・・31
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
2
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.

Before 3D printing with Regenova,
prepare the following accessories and tools.
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
3
Product name Product No
AMUZA Inc.
Included in
Regenova set
Current List
Price
AMUZA Inc.
9X9 Standard Kenzan CY1.001.00 2$1,000
9X9 Kenzan
for tubular construct CY1.002.00 2$1,000
26X26 Kenzan CY1.003.00 2$2,000
Kenzan Base Plate
for 9X9 Kenzan CY0.104.00 1$1,500
Kenzan Base Plate
for 26X26 Kenzan CY0.105.00 1$1,500
Kenzan Container CY0.103.00 1$500
Nozzle 25G CY0.107.00
60 units
$250
Nozzle 26G CY0.102.00 5
Nozzle 27G CY0.108.00 5
Air System CY0.101.00 1$1,500
Waste Bin CY0.106.00 1$150
Cell Culture Plates SB9.096.00 20 plates $300
Perfusion Chamber Type 1 CY1.004.00 $400
PBS Both sterilized and non-sterilized
Micropipette 100uL, 1mL
Peristaltic Pump see page 25
Tweezers see page 5
Cell, culture medium See page 6, 25
To purchase;
AMUZA Inc.
Toll Free: [phone]
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
Prior to 3D Printing 1: Accessories and Tools

９Ｘ９ Kenzan
for tubular construct
９Ｘ９ Standard Kenzan
largest possible: 3.5 x 2.5 x 10 mm
２６Ｘ２６ Kenzan
largest possible: 10.5 x 10.5 x 10 mm
Dimension Width 10 x Depth 20 x Height 18 mm
（Grid plate and base included）
Needle diameter 170 μm
Tip-tip distance 400 μm
Material Stainless Steel, Heat resistant resin
Parts # CY1.003.00
Dimension Width 20 x Depth 30 x Height 18 mm
(Grid plate and base included）
Needle diameter 170 μm
Tip-tip distance 400 μm
Needle quantity 676 needles (26 x 26)
Material Stainless Steel, Heat resistant resin
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
Select the correct Kenzan for the project.
* NEVER remove bottom grid plate, requires our help to put back
Grid Plates (2 plates)
Base
4
CY1.001.00
81 needles CY1.002.00
60 needles
Kenzan for
tubular construct
has a hole in the
middle to
facilitate culture
medium delivery
for maturation.
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
Prior to 3D Printing 1: Accessories and Tools

Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
1 set of accessories comes with the Regenova.
To reduce downtime, it is recommended to purchase
one additional set as autoclaving is required for each
printing.
5
Nozzle
26G (CY0.102.00) is recommended
for regular spheroids
Recommended single usage due to
potential clogging.
Kenzan Base Plate
For 9X9
CY0.104.00
For 26X26
CY0.105.00
Kenzan Container
CY0.103.00
Air System
CY0.101.00
Waste Bin
CY0.106.00
Cell Culture
Plates
PrimeSurface MS-9096UZ
(S-bio) is highly
recommended.
96 well, u-bottom, Ultra-
Low Attachment
SB9.096.00
Tweezers
Need wide tweezer
to handle 26X26
Fine tweezer for
Kenzan removal,
See page 26
4mm
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
Prior to 3D Printing 1: Accessories and Tools

Step 1 : Culture cells
Step 2 : Distribute cell suspension to multi well plates
Step 3 : Cell aggregate formation
PrimeSurface MS-9096UZ (S-bio) plates are critical as the
machine stage and suction pressure are optimized for them
Distribute 100 µL/ well cell suspension to cell culture well
plates:
•5-50 thousands of cells / well*
•Spheroids of single or multiple cell types can be used
Culture for about 48 h in a CO2incubator (37℃, 5% CO2)
Less incubation time may cause loosely formed spheroids
difficult to use for 3D printing.
Check diameters of aggregates with a microscope, they should
be 450-600 μm for optimal 3D printing.
Culture enough cells for your 3D printing project.
It is recommended to use cells in/near a log growth phase.
6
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
Prior to 3D Printing 2:
Making Spheroids (Cell Aggregates )
For optimal 3D printing, spheroids must be in good
condition. Please determine the best protocol for your
cells in advance.
Concentration
(cells/mL)
400,000 200,000 100,000 50,000
Cells / well
(cells/uL)
40,000 20,000 10,000 5,000
Diameter
after 48h
600um 500um 360um 280um
*Seeding condition depends on cell type, please perform a preliminary test
with your cells to find out the best conditions as following this example:
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.

Step1: Install Bio 3D Designer on your own computer
Step2: Design 3D structure
Step3: Save
Select the “csv” file type to transfer the designed data to
Regenova.
You can design 3D structures with your own computer. Please
contact a Cyfuse representative to get the software.
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
Prior to 3D Printing 3:
Designing your 3D structure
7
* For more details, please refer to the manual of B3D software.
Designs recommended for beginners
Tube
Use 9X9 Kenzan for
tubular structure
Recommended to use
perfusion chamber
which supplies culture
medium inside tube:
Perfusion Chamber
Type 1 (CY1.004.00),
see page 27.
Sheet
Use 9X9 standard
Kenzan
Recommended to
use layers in the
middle of the
Kenzan to better
supply culture
medium and for
ease of printing.
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.

Sterilize accessories and tools with an autoclave set
using normal conditions for high-pressure steam
sterilization (121℃ for 20 min).
Air Bottle*
Silicon Tube with
appropriate length*
Nozzle
Waste Bin Kenzan Base Plate
8
Nozzle Adapter*
Kenzan
Kenzan Container
*Air System
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
Prior to 3D Printing 4:
Sterilizing Accessories and Tools
Before sterilization, use a stereo
microscope to check that there
are no foreign materials on, or
damage to the Kenzan needles.
If any needles are bent or
damaged, please use a new
Kenzan.
Also sterilize tweezers together.
Damage on a needle
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.

Use bags for sterilization or tinfoil to wrap each
accessory.
bags for sterilization
A nozzle can be attached to
the connector before
sterilization
Caution: No tubing kinks
Place a Kenzan in a
Kenzan base plate inside a
Kenzan container
Fill the container with PBS
(see the next page)
9
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
Prior to 3D Printing 4:
Sterilizing Accessories and Tools
For the nozzle and Kenzan, it is recommended to
prepare and autoclave a couple of extras as back up.
The back up Kenzans should be autoclaved in MilliQ
w a t e r.
9X9: Place backup
Kenzan sideways.
26X26: Place backup
Kenzan upwards.
Fill with enough MilliQ
to submerge Kenzans
generously.
Covered with tinfoil
Place backup
nozzles in a beaker
and cover with
tinfoil
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.

1. Secure the Kenzan in a Kenzan
base plate inside a Kenzan
container.
2. Fix the Kenzan using the jigs of
the Kenzan base plate
3. Pour approximately 50 mls of PBS
until A=B (see below).
Use tweezers to confirm secure
placement of the Kenzan and
Kenzan base plate.
Sterilizing the Kenzan
To remove air from the Kenzan and the PBS, it is
recommended to sterilize them together in the
following manner. Air bubbles can disrupt camera
images and cause failure to detect needle tips.
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
Prior to 3D Printing 4:
Sterilizing Accessories and Tools
10
A: 8-10mm
B: 8-10mm
4. Wrap whole
container including
PBS with tinfoil
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
PBS: about 50mL

1: Main power
2: Power supply indicator
3: Operation power supply ON/OFF
4: Buzzer
5: Heater temperature
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
3D Printing 1: Powering on
1: START
2: STOP
3: ALARM STOP
4: ABNORMAL RESET
5: EMERGENCY STOP
11
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
START

12
1Turn “air bleed valve” of the
compressor clockwise to close.
2(For the 1st time) power on by
turning the main power knob
clockwise
3Turn on FAN and FL at right hand
inside the clean bench.
4Turn the Regenova operation
power supply to ON, and wait a
minute until it is fully initialized.
5Power the PC on
*Stop alarm with “ALARM STOP”
Main power
operation power
supply ON/OFF
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
3D Printing 1: Powering on
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.

3D Printing 2：Starting the Software
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
Step１:
Open CellAccumu
*Press “ABNORMAL
RESET” (red arrowhead)
after the software started
13
Press “Auxiliary” (①) and
hold down “Maintenance”
(②)until the button turns
to pink. Then the printing
robot arm will move
towards the left to make
space for set up.
Step 2:
Maintenance Mode
Basic guidlines for Regenova
- The sash of the clean bench must be
closed down to the bottom.
-When an alarm is activated, press
“ALARM STOP” and “ABNORMAL
RESET”. Then operation can resume.
-Buttons on the right tab of CellAccumu (③) turn blue when active.
-Buttons on the “Auxiliary” page (④) turn pink when active.
-Several important operation buttons of Cell Accumu, i.e. “Home Return”,
“START”, and others require holding down to activate.
-After changing the mode between Inspect and Stack, opening the front
panel of the clean bench, and other various actions, it is necessary to
hold down “Home Return” to resume operation.
③
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
①
②
④

Step ２: Place and set up the Air System
1. Confirm the 2 small caps and 1 big cap of the Air Bottle are
securely closed.
2. Place the bottle (See photo 2 above)
3. Connect with the Air Tube and secure tight connection 14
123
Waste Bin
Holder
Air Bottle Holder
Nozzle Holder
Kenzan
Container
stage
Air Tube
Step １: Place the Waste Bin
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
3D Printing 3: Set up
Before starting, use an alcohol wipe to clean places
that cells and culture medium may touch, i.e. stages,
nozzle holder, etc.
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.

Step 5：Load well plates into
magazine towers
ABC
3D Printing 3: Set up
Step 4 : Place the Kenzan
Container including Kenzan
Step 1: Placing the Nozzle
1. The silicon tube coming out of the Air Bottle must go below the CCD
camera cable, otherwise the tubing may interfere with the robotic arm.
2. Connect the nozzle to the nozzle adaptor on the silicon tube (if not
initially autoclaved together) and install the nozzle into the holder. Be
sure to avoid touching the tip of the nozzle to keep it clean.
3. Tighten the screw at the nozzle stand
Step 6：Close the front panel of the clean bench
down to the bottom and hold down “Home Return”.
15
２3
１
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ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
Before loading well plates, please check from
the bottom that the spheroids can move in the
wells.If they don’t move, see page 23.
A：Discharge position
B：For Type 1Spheroids
C：For Type 2Spheroids
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.

3D Printing 4：Setting up CellAccumu
Step 2: Selecting Kenzan Type
Press “Needle Array Selection”(①)
Select the type of Kenzan (②) and press “Apply” (③)
①
②
③
Step 4: Inspection Mode
Press “Auxiliary”（⑦）
Hold down “Inspect Mode” (⑧)
until it turns pink.
Hold down “Home Return” (⑤）
until it turns pink.
“Home Return” must be held down
whenever the mode is changed
(Inspect <-> Stack)
16
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
⑥
④
Step 3: Inspecting Kenzan
Make sure “Home Return” (⑤) is pink. If not hold down the button until it is.
Hold down “Needle Array Check” (⑥)
If needle tips are not recognized correctly, adjust with “OffsetZ” (-500～+500㎛)
located directly above “Needle Array Check” (⑥). Or add/subtract PBS.
Press “Position Memory” (④)and select “Area Image” to confirm correct
recognition of the needle tips. Check that green +’s match to the needle tips.
⑤
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
⑦
⑧
④

Step 5: Inspect spheroids
Select “Inspect” mode (①), hold
down “START” (②).
The robotic platform will bring a well
plate to the stage for spheroid
inspection. There will be a notification
alarm when inspection of all spheroids
is completed. （③）
Review the diameters and parameters
of the spheroids at the window (④).
Data in the window will disappear
after completion of inspection but if
you’d like to see the results, go to
D:CAD3¥Cell detect
①
②
③
Press “Initial settings”
Adjust the z interval (Stack z pitch ⑤)
based on the diameters of spheroids
inspection (see below). Press “total (
⑥)” then the system will calculate
how many layers are possible to us
for the print. Then press “Apply (⑦) ”.
Step 6 : Adjust z direction interval ⑥
⑦
17
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
④
④
3D Printing 4：Setting up CellAccumu
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
Recommended z interval for spheroids to optimally touch each other:
Spheroids larger than 500 um: 80% of the average diameter.
Spheroids smaller than 500um: 70% of the average diameter.
⑤
If there is discrepancy
between the diameters
measured by microscope and
the bio 3D printer, defer to
values measured by
microscope.

Step 1 : Needle Array Map
Step 2 : Parameters
Press “Needle Array Map”（①）
Design or load pre-designed
data and press “Apply” (②)
To load a construct, select from
“File”.
To design a construct using 2
types of cells, it is
recommended to design such
that all of one type (“Type 1 (③
)” or “Type 2 (④)” is printed
first for most efficiency due to
limited plate exchanges.
To print using the very top
layer, see page 32, Advanced
operation.
3D Printing 4：Setting up CellAccumu
①
②
③
⑥
The Regenova detects spheroids and measures
them based on defined parameters before
picking them up. After picking up, the Regenova
checks again to confirm successful pick up.
Press “Inspection Parameters” (⑤)
Select “Type 1” or “Type 2” tab（⑥）Adjust/set
parameters, or previous data can be inserted
from “File”. Parameters on “Type 1” tab should
be based upon results of Type 1 spheroid
inspection and “Type 2” tab should be based
upon Type 2 results.
If “Auto set (⑦)” is checked, the threshold will
be set automatically.
Parameters on the “Presence (⑧)” tab are for
checking after the spheroid is picked up. If
there is nothing to meet those parameters, the
machine recognizes a successful pick up.
18
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
③④
⑤
⑦
⑧

Parameters for spheroids
3D Printing 4：Setting up CellAccumu
Distance from center (μm): Distance between the center of the well and a spheroid to be
detected, typically it’s 2,500-3,000.
Diameter (μm): The minimum and maximum diameters of the spheroid to be detected.
Refer to the average diameters derived from spheroid inspection or preliminary
microscopic evaluation.
Roundness (%): Typically set to approximately 70. Refer to average roundness from
spheroid inspection.
•Increase Roundness: the machine only detects perfect circles as spheroids.
•Decrease Roundness: the machine detects more oval or rough circles as spheroids.
Smoothness (%): Typically set to approximately 70. Refer average smoothness from
spheroid inspection.
To adjust light settings, press “Lighting Settings” on
the “Needle Array Map” page and adjust “Ring Light” of
upper camera (①). Press “Apply” to activate (②).
Recommended settings of lights for spot and ring light
are as follows:
9X9 standard: around 100
9X9 tube: around 80
26X26: Area 5 (center) 150-200
Other areas 100-150
19
Cyfuse Biomedical K.K
ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.
Threshold: Range 0 to 255 (dark to bright) for binarization.
Typically use between 60-150. Spheroids gathered loosely like in the
photo on the right, need Threshold adjustment.
Reduce Threshold: the machine detects only darkest area as a
spheroid. Increase Threshold: the machine expands to the
surrounding area. Please find the best conditions for your spheroids.
Threshold: 65 Threshold: 120 Threshold: 170 Green line denotes
the area the machine
recognizes as a
“spheroid”.
Step 3: Lighting Settings
①
②

Step 1 : Stack mode
Step 2 : Check spheroid pick up & penetration
Press ”Auxiliary” (①)and
hold down “Stack” (②) until
the button turns pink
Hold down “Home Return”
until it turns pink
①
④
To check the pick up & penetration of the spheroids, print
a few spheroids on the top layer of the Kenzan before 3D
printing.
20
②
③
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ICN Building-5A 2-27-17, Hongo, Bunkyo-ku, Tokyo, 113-0033, JAPAN
TEL：[phone] E-mail：[e-mail] URL：http://www.cyfusebio.com
3D Printing 5：3D Printing
1. In the “Needle Array Map” page, place 3-4 spheroids on the top layer.
2. Press and hold “START” 3D printing only for those few spheroids.
3. Press and hold “STOP” 3D printing after each penetration and perform
“Needle Array Check” (see page 16).
If spheroids are penetrated successfully you can see them clearly as
pictured above.
Also check that no bubbles come out of the nozzle at penetration. If there
are any bubbles, check that the 2 small caps and 1 big cap are secure on
the Air Bottle, also secure connection of the Air Tube to the Air Bottle (p14).
●This Manual is subject to change without notice. ●Information is as of Feb, 2016.