A: Should you experience issues with your ClinoStar, you can try and perform a factory reset. All data and settings will be lost and an initial setup needs to be performed. Be aware that all data on the ClinoStar will be permanently and irrecoverably erased. Have the QR code and password sheet ready when setting up the system again, have the sheet has been lost contact support@celvivo.com.

Follow these instructions to do a factory reset of the ClinoStar system.

1. Press the small reset button located on the back side of the ClinoStar with a small pin (see ‘G’ in figure 3 in the ClinoStar manual).
2. ClinoStar front-LED to flash red, green, and blue.
3. Open and close the front door two times while the LED flashes to initiate a factory data reset.

A: usually a restart of the device can resolve many common issues with the tablet and increase performance. Press and hold the power/lock button to switch off the device.

A: Yes, any sterile water will suffice.

A: No. After 10 days of use the membrane in the ClinoReactor will clot and suffocate the cells inside, to continue the experiment the ClinoReactor should be exchanged.  Misuse will void the warranty.

A: Formation of irregular big aggregates during clinostat bioreactor cultures can be related to several factors: 

– the cell/spheroid/organoid population is too dense in relation to available volume. 

– cell conglomerates (spheroids, organoids, or biopsies) are not uniform. 

– “condensation centres” such as bigger agglomerates, fibres or contaminants are present in the vessel. 

– highly proliferative cells with strong migration and attachment properties are being used (e.g. cancer cell lines with high metastatic potential) for creation of clinostat cultures. 

We would recommend following general practice to avoid unintentional agglomeration of spheroid or organoid cultures: 

– use a less dense starting population: This will give more space for individual spheroids/organoids and will make it easier to find the optimal rotation speed.  

– at any given time point the vessel can only have one rotation  speedrotation speed and therefore having a uniformly sized spheroid/organoid population is crucial. This is especially important during the initial phase of clinostat culture (e.g.  usually the first week for immortal cell lines).  

– if the growing population tends to have different sizes, we recommend separating them into different ClinoReactors (by manually sorting into different sizes in different vessels). 

– uneven spheroid/organoid sizes or any other odd size objects will create agglomeration centres (especially during initial phase of culturing – first week) therefore it is important to remove them as soon as they are seen. 

– in some cases, when highly proliferating cells (e.g. some highly malignant cell lines) are used for spheroid/organoid cultures, we would recommend coating the initial spheroids/organoids with biodegradable materials (e.g. we use sodium alginate coating (see the appropriate SOP) to prevent their agglomeration). 

In general, the materials used for our ClinoReactor are very low attachment materials. If cells/spheroids/ organoids are attaching to the ClinoReactor plastic surface, we would speculate that some kind of protein coating has been deposited on the plastic. It could be secreted protein or protein released from damaged cells. Try moving the culture to a fresh ClinoReactor and change them more frequently.

Aggregation of big conglomerates in one spot inside clinostat bioreactor is a result of balance between rotation speed/viscosity of the liquid/ surface of the conglomerate/weight of the conglomerate = lift capacity. The placement of steady spot on left or right side depends on direction of the clinostat rotation. 

When big aggregates are already formed most usually changing of the rotation speed will not result in dispersion of the aggregate. Either discard the aggregates or select good spheroids or organoids and move them to a new ClinoReactor. 

The preferred aggregation method will very much depend on properties of cell type(s) used. In general, for single cells generated from tissue biopsies (cells with relative high oxygen demands and already coming from 3D structures) we would recommend methods which allow quick transfer to the ClinoReactor (e.g. ‘force’-aggregation with use of scaffold/hydrogel materials like sodium alginate) or the direct self-aggregation of single cell suspension in clinostat bioreactor vessel. 

In general, try to use less dense population for starting the clinostat cultures from this type of cell. A too dense population is one of most often causes of spheroid/organoid conglomeration 

Considering the materials used to make the ClinoReactor there is no problem to use the amphotericin B during spheroid culture.  

If we consider the spheroid/organoid population itself, the answer depends on what type of research the spheroids/organoids will be used for. 

Depending on the type of research, as well as lab conditions and routines, the use of certain media additives can be permitted or needed. 

In our practice, we successfully have culture spheroid/organoid population without any additives (antibiotics or fungicides) for extended periods of time (up to a year). For ease of use and for experiments where it is not expected to affect the results, we use an antibiotic cocktail in our standard growth media (Pen/Strep 0.5% v/v). 

If you would like to use the big single spheroid conglomerates for your experiments, we would suggest following normalization methods: 

– non-invasive volume calculation based on conglomerate photography. 

– if the conglomerates are bigger than 3-4 mm in diameter, we have successfully used wet mass weight to normalize the treatment experiments. 

– if the treatment is relatively short and terminating, you can use a known part of sample to perform the biomass calculation by protein determination.