Insphero Akura PLUS User manual

Akura™ PLUS Hanging Drop System
Quick Start Guide
Thank you for choosing InSphero's Akura™ PLUS for your 3D cell culture experiments. This Quick Start
Guide contains important information to get you started immediately. For detailed instructions please
refer to the Product Manual and additional resources on shop.insphero.com.
Akura™ PLUS Hanging Drop System Components
A. Akura™ PLUS Plate (frame with 12x8 well
strips) + humidifier pads (in bags with
tweezer)
B. Transparent lid
C. Bottom plate with reservoir
D. Akura™ 96 well plate
Generating Spheroids or Organoids
shop.insphero.cominsphero.com
Figure 1. Akura™ PLUS system components.
Akura™ PLUS
1. Preparation: Prepare a reservoir with 20ml 0.5x PBS, remove one of the humidifier pads using the
tweezer and place it in the reservoir. Wait approx. 5 min until the pad is completely soaked with PBS and
place it in the bottom plate of
the Akura™ PLUS Plate.
2. Cell seeding: Count
the cells and prepare a cell
suspension for seeding, using
a final volume per drop of
40µl. For long-term growth
profiling start with low cell
numbers (250 – 500 cells per
drop). For non-proliferating
cells or rapid production of
larger spheroids start with
higher numbers (from 2,500
cells per drop). Please try
different concentrations for
defining your optimal range.
Ensure tight contact
for SureDrop
TM
inlet
AkuraTM PLUS
AkuraTM 96 Plate
(Spheroid Culture Platform)
Spheroid
culture/assay
Spheroid
transfer
Spheroid
maturation
Cell seeding
!
(Spheroid Formation
Platform)
Figure 2: Delivering cell suspensions
and generating spheroids in Akura™
PLUS Hanging Drop System.

InSphero AG
Schlieren, Switzerland
InSphero Inc.
Brunswick, ME, USA
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please refer to the FAQs section
at shop.insphero.com
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AG. 3D InSight, Akura and InFloat
are trademarks of InSphero AG.
For life science research only. Not
for use in diagnostic procedures.
Transfering 3D Spheroids or Organoids
For long-term cultivation and assays, transfer of the spheroids from the Akura™ PLUS Plate to the Akura™ 96
Spheroid Microplate is required.
Important: In order to prevent inclusion of air bubbles, is is recommended to pre-wet the wells of the Akura™
96 Plate. Apply 40μl of cell line medium in each well. Gently pipette the medium up and down and aspirate the
pre-wetting medium from the plate just prior to seeding.
1. Place the Akura™ PLUS Plate onto the Akura™ 96 Plate by positioning the three pins into the
corresponding holes on the top surface of the Akura™ 96 Plate. The drops under the Akura™ PLUS
Plate will then be perfectly aligned with the wells of the plate underneath.
2. Slowly (<10 µl/sec) add 70µl of medium through the inlet of the Akura™ PLUS Plate wells. The pipette
tips should be in direct contact with the well inlets by simultaneously applying a subtle pressure with
the pipette. The drops will fall into the Akura™ 96 Plate.
3. Sedimentation spin: It is recommended to briefly centrifuge the plate for 2 minutes at 250 RCF to
force all tissues to the bottom of the cavity and to remove air bubbles.
4. To assure defined medium volumes in the wells, replace the solution in the wells by aspiration and
addition of 70µl of fresh medium.
Medium Exchange in the Akura™ 96 Spheroid Microplate
1. Place the pipette tip at the ledge of the well.
2. Remove the medium at low pipetting speed (<30 µl/sec) by
aspirating an excess of volume. A minimal volume of ~5-7 µl
medium will remain in the well.
3. Add 70µl of fresh medium by placing the pipette tip at the
ledge. Use a dispensing rate <50 µl/sec.
4. Place the lid on the Akura™ 96 Plate and place it in a humidified
CO2incubator at 37°C.
InSphero Akura™ PLUS Hanging Drop System
For detailed information, please
refer the Akura™ PLUS System
Product Manual.
Important: To generate spheroids with uniform size and cell composition, it is essential to assure a homogeneous
distribution of cells by gently pipetting up and down prior to seeding.
3. Gently deliver 40µl of cell suspension into each well of the Akura™ PLUS Plate. Make a tight contact
between the pipette tip and the well inlet by applying a slight pressure to form the SureDrop™ seal
(Fig. 2)
4. Place the lid on the Akura™ PLUS Plate and place it in a humidified CO2incubator at 37°C.
5. Assess spheroid formation regularly. After 4 days in culture most cell types re-aggregate and form
a compact spheroid.