Cyfuse Regenova Manuale di installazione e funzionamento
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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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: (858) 225 6869
●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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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：+81-3-4455-7872 E-mail：sales@cyfusebm.com 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
Indice
