Technology

The major issue in conventional 3D culture of sensitive cells is the presence of strong shear forces induced by the mixing system (wheel, helix, turbine or bubbles). 

Our innovating bioreactor permit to get a homogeneous mixing of nutrients and gases with a minimum shear.

Fluids in a precessing cylinder

Precessing containers are known to create an efficient mixing of the inner fluid.
It is the case for example of the Earth whose slow precession creates energetic flows of the liquid iron in its core. 
It is also the case of cylindrical paint pots when introduced inside biaxial mixers (also called gyroscopic mixers) achieving precessing motion of the pot with an angle of 90 degrees. 
 
The presence of 2 rotating axes for a precessing motion highly complexifies the experimental set-up. However, it is possible to create the same flow using a simpler device: a partly filled cylinder rotating around its axis tilted with respect to the vertical (Thompson, 1970). The free surface, which remain horizontal, is precessing in the frame rotating with the cylinder, as in the case of the experiment of Mc Ewan (1970). This moving boundary condition forces inertial waves inside the rotating fluid that interfere to create a global mode with a wavelength equal to approximately 2 diameters, as in the case of a precessing cylinder.

Turbulent mixing

If the free surface is located at half a wavelength, the forcing is precisely at the node of the global mode and generate a resonant flow inside the cylinder (Meunier, 2020). 
This is similar to a guitar string with one end vibrating at the node of the standing wave. 
The large amplitude motion of the fluid further destabilize thanks to a triadic resonance instability, also called parametric instability (Lagrange, 2011). 
 
It leads to pulsing motions that are homogeneously distributed in the height of the cylinder, in contrast with the local motion created by a rotating turbine.

Shearing forces inside the cylinder

We have shown theoretically and verified experimentally that the instability creates at mid-height 6 positive and 6 negative vortices (Meunier, 2020). 
They are less intense than the vortices created by a rotating turbine for the same velocity. 
The shear induced by these 12 vortices is thus much smaller compared to the shear created by a rotating turbine. 
 
For example, for 1 liter of water in a cylinder rotating at 60rpm, the shear stress is only of the order of 2mPa whereas it would be of the order of 100mPa for a turbine rotating at 60rpm. 
This particularity of the flow makes it an ideal candidate for the 3D culture of  shear sensitive cells.

Homogeneous mixing

We have measured the mixing property of the chaotic flow by introducing a fluorescent dye in the tilted rotating cylinder. 
 
The characteristic time to reach mixing is equal between the soft mixer and the rotating turbine. 
 
However, the mixing is more homogeneous in the case of the soft mixer.  
This is ideal for cells living in the bioreactor since it provides them a constant and homogeneous rate of oxygen.  
It is the guaranty of an optimal control of culture conditions and a reduced variability.

Benefits

Cost effective: 

Conventional bioreactors use detrimental stirrer tools which complexify the biomanufacturing process, generate foam, and prevent the scale up.

In our scalable bioreactors, you have a full control on the shear within a very simple biomanufacturing process, preserving for example the self-renewal capacities of pluripotent stem cells.

This translates in a very cost-effective process whatever the scale, from R&D to clinical needs.

Scalability:

In conventional bioreactors, the size of the stirring device (turbine, wheel or helix) must increase with the size of the container. 

The shear stress thus increases strongly with the volume (red curve). 

In our bioreactors, for the same mixing rate, the shear stress does not increase with the volume of the bioreactor (blue curve). 
The scale up of the process is thus much easier.
Shear-stress level (mPa) in our bioreactor (blue) or turbine-stirred bioreactor depending on the volume.

Maximal mass transfer:

Finally, we have measured the flux of gas such as oxygen or carbon dioxyde that can penetrate or exit at the free surface.

This flux is larger by 2 orders of magnitude compared to the case of a static cylinder.

This is essential for an efficient bioreactor. It means that the number of living cells in the bioreactor can be increased by 2 orders of magnitude.

Characteristic diffusion time through the membrane τ = Hδ/κ as a function of the Ekman number for a diffusive bottom layer of thickness δ. (LeFranc et al., 2023)

Cell models

Our technology was successfully used on several cell models from human cell to microalgae.

Stem cells

Induced Pluripotent Stem cells (iPSc) are genetically reprogrammed cells capable of dividing in culture with unlimited self-renewal. The emergence of these cells represents an opportunity to create a potentially unlimited cell source for differentiation into specialized cell types.

However, their amplification remains delicate. Current 3D technologies with stirrer tools can easily lead to premature cell differentiation, or damage them with excessive shearing forces, reducing yields and leading to high costs.

In our bioreactor, we could amplify iPSc during 2 weeks without passage. We started with a 10^5 cell/ml up to 10^7 cell/ml.

Natural Killer

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Their excellent immune tolerance make them great candidates for allogeneic therapies, requiring large scale manufacturing. 

Successful scale-up of NK cells critically depends on a manufacturing process sustaining their expansion while preserving their cytotoxic functionality.

Colorized scanning electron micrograph of a natural killer cell from a human donor. Credit: National Institutes of Allergy and Infectious Diseases, National Institutes of Health

Dinoflagellates

Dinoflagellates are a family of microalgae which is particularity shear-sensitive. They are really promising in drug development (Detournay et al., palytoxine, 2023) because they produce potent bioactive small molecules showing high affinity for ion channels, the most important pharmaceutical targets.  

However, no technology exists to cultivate dinoflagellates at high yield (only 2D culture is possible) leading to high production costs and as a consequence pure molecule price on the market ranging from 10 to 90 k€/mg.

Thanks to our bioreactors (1- 200L), we have proved that it is possible to cultivate these shear-sensitive microalgae in 3D conditions at high yield level, making the biomanufacturing process affordable and scalable.

These results pave the way to the industrial production of microalgae of interest for pharmaceutical purposes in a wide range of applications (Lefranc et al., 2023)

These molecules were demonstrated to have a high tropism for ion channels the second therapeutic target.  However, no technology exists to cultivate dinoflagellates at high yield.

Only 2D culture is possible leading to molecule of interest with prices about 30k€/mg and more.

With our technology, we proved that it is possible to cultivate these shear-sensitive microalgae at high yield and good reliability.

These results indicate that is possible to produce high potential molecules from dinoflagellates at affordable price, which could address the lack of new bioactive molecules for drug discovery. 

Dinoflagellates harvesting on our 200L Softmixer

Publications & Patents

Two patent has been filed on this bioreactor : The first (WO2017149034A1) and the second (FR3117505A1), thus we decided to share it with scientific community.

Our technology came from seven years of development leading to  numerous publications and a PhD in physics and computational fluid dynamics, mainly managed by Patrice Meunier (IRPHE, Aix Marseille Université) Learn more.

Articles :

Natural Killer cultivation in a rotating bioreactor, in prep.  

Gas injection into a tilted rotating cylinder, Lefranc et al., 2023

Cellura is a french biotech company commited in the development of solution to unlock cell expansion issues.

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