Overview of ARCTis™ Human Tumor CryoKit Starter
InSphero’s ARCTis™ Oncology plates represent a novel concept that enables a fast and reliable use of 3D cultured microtissues for adoption in drug discovery programs. They are designed to support your testing strategy and help you by being conveniently ready whenever you need them.
Zero development time & costs - ARCTis™ CryoTumors are optimized for reliable formation, uniform size and cell composition, and growth window. From freezer to assay-ready 3D tumor spheroid in 3-5 days, giving your team a head start generating valuable data for your project.
A broad range of cell lines - We are building the largest 3D CryoTumor bank in the world. This ever-expanding cache of tumor models allows you to generate bigger, better, and more predictive data sets that capture the effects of tumor heterogeneity.
Convenient scaffold-free - Formation of spheroids below 500 µm via cellular self-assembly in ultralow attachment (ULA-treated) plates.
Zero spheroid loss during the medium exchange - SureXchange™ tapered ledge and culture chamber facilitates easy medium exchange and prevents spheroid loss during long-term spheroid growth and analysis. The 1 mm diameter flat bottom observation window enables simple spheroid observation, and greater distance between observation windows of adjacent wells reduces well-towell imaging crosstalk compared to standard 96-well plates.
Features of ARCTis™ Human Tumor CryoKit Starter
The ARCTis™ Starter Pack elements are as follows:
• 6 x ARCTis™ plates – 2 plates for each of three different tumor cell lines: HCT116 (AT-NB00-0T0-001 HCT116), A549 (AT-NB00-0T0-001 A549) and T-47D (AT-NB00-0T0-001 T-47D)
• 6 x 30ml Tumor Reaggregation Medium (CS-07-112-04)
• 4 x 125ml Tumor Maintenance Medium (CS-07-112-03)
• 1 x Tilting stand (CS-AG11)
A detailed protocol for the production of spheroids in the Akura™ 96 Spheroid Microplate is provided in the product manual. Below are answers to some frequently asked questions to help get you started.
Q: What improvements did you make to the new Akura™ 96 Plate?
A: Improved optical properties:
- COP (Cyclo-Olefin Polymer, 92% transparency 400-800 nm) as plate material instead of Polystyrene.
- Thinner well bottom of 0.8 mm, before 1.3 mm.
- A reduced skirt height of 0.4 mm. High NA objectives (e.g., 20X and 40X) may be used to image the outer wells of the plate.
- Excellent planarity across the plate (below 80 μm) for reliable spheroid transfer and precise medium exchange
- Optimized distance (200 μm) between the customized low-evaporation lid and plate reduces evaporation in outer and edge wells
Standard SLAS plate height:
- 14.35 mm plate height instead of 11.48 mm
- Maximum volume 280 μl instead of 170 μl
Q: What is the optimal volume per well in the Akura™ 96 Spheroid Microplate?
A: To achieve optimal conditions, gently deliver 70 μl (pipetting speed < 10 μl/sec) of cell suspension into each well of the Akura™ 96 Plate by placing the pipette tips near, but not touching, the bottom of the wells.
Important - For spheroids with uniform size and cell composition it is essential to assure a homogeneous distribution of the cells by gently pipetting up and down prior to seeding into the Akura™ 96 Plate.
Q: Why do you recommend centrifuging the Akura™ 96 Spheroid Microplate after cell seeding?
A: We recommend briefly centrifuging the plate after cell seeding to remove any air bubbles and to force the cells to the bottom of the well to promote cell aggregation and spheroid formation. For that, place the lid on the plate and spin it in a microtiter-plate centrifuge for 2 minutes at 250 RCF.
Afterward, incubate the plate in a humidified CO2 incubator at 37 °C for 2-5 days.
Q: How do I exchange the medium in the Akura™ 96 Spheroid Microplate without disturbing or losing the spheroids?
A: To prevent spheroid/organoid loss during the exchange of media, the SureXchange™ ledge at the inside wall of each well serves as an anchoring point for the pipette tip. Just place the tip at the ledge of the well, see figure below, and remove the medium at a low pipetting speed (>30 μl/sec). A minimal volume of ~5-7 μl will remain in the well. Then, add 70 μl of a fresh medium by placing the pipette tip at the ledge, and use dispensing rate <50 μl/sec.
Important - when using automated liquid handling devices, an off-center alignment of the vertical pipette tip will achieve the same effect.
Q: What is the best way to prevent evaporation in the outer wells of my plates?
A: Evaporation in the outer (perimeter) rows of wells is a phenomenon common to most low-volume culture platforms, and thus requires careful attention to maintaining proper humidity control. If not controlled, pronounced evaporation can result in the concentration or precipitation of media components (serum, salt) that can impact spheroid formation or health and can alter the effective
concentration of a compound/additive in the medium throughout a long-term experiment. To provide maximum humidity control when using the Akura™ Plates, we recommend the following:
- Use an incubator with good humidity control (>95% of rel. humidity), and exercise best practices in maintaining and minimizing loss of humidity (e.g., minimizing incubator door opening and closing).
- For culture in the Akura™ 96 Spheroid Microplate, at least 50-70 μl of the medium in each well is recommended and can be increased to a maximum of 80 μl if incubator humidity control is a persistent issue. Medium exchange frequency can also be increased to every other day or daily if conditions dictate.
- We recommend using the InSphero Incubox™ (CS-10-001-00) (Figure 10) to reduce edge effects when performing long-term culture with the low-frequency medium exchange. The InSphero Incubox™ is available on shop.insphero.com.