Preventing tissue loss during liquid exchange
Ensuring that spheroids are intact during liquid exchange is not fun and certainly not easy (contrary to what you will see in this video). That’s why we developed our Akura™ Microplates with a special feature to make your life much easier: The SureXchange™ well design enables optimal protection of the spheroids in a cavity at the bottom of the well during liquid exchange.
What else do you need to know to prevent tissue loss? Our Senior Product Manager, Dr. Frauke Greve, asked InSphero's 3D in vitro experts about their tips and tricks.
Tips for preventing tissue loss in Akura™ Spheroid Microplates
What kind of pipettes shall I use to perform medium exchange?
You can use single- or multi-channel pipettes as well as semi- or fully automated liquid handlers. For the Akura™ 96 Plate, we usually use multi-channel pipettes when we work with a lower number of plates, and semi-automated systems when handling larger amounts of plates. For the Akura™ 384 Plates, we only work with semi- or fully automated liquid handlers.
Where shall I place the pipette tip in the well to prevent tissue loss?
Place the pipette tip at the rim specifically designed for pipetting, which is called the SureXchange ™ ledge. In the Akura™ 96 Plate, the well has an axial symmetry (Image 1) whereas in the Akura™ 384 Plate the ledge is only located at one side of the well (asymmetric design - Image 2). Thus, your pipette tip is located as far away as possible from the spheroids to prevent accidental aspiration, and the spheroids are safely located in a small protective cavity at the bottom of the well.
Image 1: Preventing tissue loss - Medium exchange in Akura™ 96 Spheroid Microplate
Image 2: Preventing tissue loss - Medium exchange in Akura™ 384 Spheroid Microplate
How much medium is left after aspiration?
By placing the tip on the ledge, you can aspire medium without hesitance. During aspiration, surface tension leaves a defined minimal residual volume at the bottom of the well. The spheroid is protected from aspiration and high shear forces. This way, > 90% of medium can be reproducibly removed without losing spheroids. For the Akura™ 96 Plate, the remaining volume is approximately 5-7 µl, for the Akura™ 384 Plate approximately 3-5 µl medium is left in the lower cavity of the well.
What speed shall I use when aspirating and dispensing medium?
Remove the medium at low pipetting speed (< 20 µl/sec) by aspirating an excess of volume (i.e. 75 µl when using 70 µl medium per well). A minimal volume will remain in the well.
Add fresh medium (70 µl for the Akura™ 96, 50 µl for the Akura™ 384) at low pipetting speed by placing the pipette tip at the ledge. Use a dispensing rate of < 30 µl/sec to prevent turbulences in the wells and accidental floating of spheroids.
How often do you exchange medium?
Although we use an incubator with good humidity control (>95% of rel. humidity), and exercise best practice in maintaining and minimizing loss of humidity (e.g., minimize incubator door opening and closing), we can only reduce but not prevent evaporation of medium in the outer (perimeter) wells. Therefore, we exchange medium every 2-3 days, but that can be increased to every day if conditions require.