by Cellex | Apr 29, 2025 | bone tissue engineering
Tissue engineering offers excellent opportunities, such as creating mechanically viable structures in vitro that can functionally replace damaged or diseased tissues in vivo.
From this point of view, bone tissue engineering is very promising. Indeed, it is possible to promote the growth of bone-forming cells through seeding systems on three-dimensional scaffolds within bioreactors.
There are several bioreactor technologies for culturing engineered tissues for in vivo implantation. These include spinner flask bioreactors.
Spinner Flask Bioreactors, what they are and how they work
Spinner flask bioreactors are often used to seed cells on scaffolds and obtain constructs for bone tissue regeneration.
This is a type of bioreactor with a very simple design.
Inside the culture chamber, the scaffolds are attached to needles secured to the lid, immersed in a liquid-state culture medium. At the bottom of the chamber is a magnetic stirring rod that, as it rotates, imparts a convective force that allows continuous mixing of the liquid surrounding the scaffolds.
In this way, the cells remain in suspension and the movement of the fluid promotes nutrient transport.
Advantages of Spinner Flask bioreactors for bone tissue engineering
In the study “Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor” (Sikavitsas, Bancroft et Mikos, 2001), promising data were recorded on the use of the spinner flask bioreactor for the development of cartilage and bone tissue equivalents.
This study investigates the cell culture conditions of three-dimensional polymeric scaffolds seeded with rat marrow stromal cells (MSCs) grown in three different bioreactors: spinner flask, rotating wall vessel and static culture.
In particular, compared with static culture, spinner flask culture demonstrated:
- Day 7: 60% increase in cell proliferation;
- Day 14: all cell/polymer constructs showed the highest alkaline phosphatase (AP) activity, with a 2.4 times higher value than the constructs cultured under static conditions;
- Day 18: peak secretion of osteocalcin (OC), which reached a total value 3.5 times higher than that of static culture;
- Day 21: calcium deposition was 6.6 times higher than that of constructs in static culture.
During the experiment, the spinner flask bioreactor also recorded better data than the rotating-wall vessel bioreactors.
On day 21, calcium deposition in the spinner flask culture was more than 30 times higher than that in the rotating-wall vessel culture. Moreover, in the rotating-wall vessel culture, no considerable AP activity and OC secretion were detected throughout the culture period.
Critical issues of Spinner Flask Bioreactors for bone tissue engineering
Does this mean that spinner flask bioreactors are the ultimate answer to bone tissue engineering?
Not exactly. In fact, this culture system has some critical issues.
First, the convective forces generated by the rotation of the magnetic stirrer at the bottom of the chamber induce uneven transport of nutrients and gases to the cellularized scaffolds.
In fact, the generation of convective forces does not extend into the interior of the scaffolds. All this limits the transport of nutrients, causing uneven distribution of cells and mineral deposits in the scaffolds.
A direct consequence is that mineralization is limited only to cells exposed outside the scaffold.
The aforementioned study by Sikavitsas, Bancroft et Mikos also highlights that “the histology sections revealed a dense cell layer on the surface of the scaffolds and a considerably lower cell distribution in the scaffold’s interior.”
In addition, the existence of convective forces in the bioreactors exposes the resident cells on the outer surface of the scaffolds to stressful shear forces.
The bioreactor that solves these critical issues
Despite the promising results obtained from culturing in spinner flask bioreactors, improved culture conditions are needed to enable cell growth through cellular polymer constructs.
The main problem with spinner flasks lies in the difficulty of nutrient and gas transport within the scaffolds, which is crucial to achieve homogeneous distribution of cells “in and out” of the scaffold.
The innovative BioAxFlow bioreactor, developed by the Cellex team specifically for tissue engineering, transports nutrients and oxygen more efficiently, ensuring improved cell culture.
Gentle fluid dynamics ensures the transport of nutrients and gases, allowing permeation into the cellular media. Indeed, in BioAxFlow, cell culture medium is continuously pumped from below gently with the help of an external peristaltic pump.
The flow generated ensures continuous movement of the medium, promoting constant exposure of cells to nutrients and growth factors. This ensures optimal growth conditions both outside and inside the scaffolds, resulting in more homogeneous cell adhesion and proliferation.
This revolutionary fluid dynamics also reduces stress generated by shear forces, adversely affecting cells adhered to the scaffolds.
In support of this, the Cellex team tested BioAxFlow with SAOS-2 (osteosarcoma) cells, and the data revealed excellent results regarding cell adhesion and growth and homogeneous distribution of cells on the scaffolds.
Want to learn more about our tissue engineering bioreactor? Discover all the benefits of BioAxFlow and contact our team.
Sources
“Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor”, Vassilios I. Sikavitsas, Gregory N. Bancroft, Antonios G. Mikos, 2001
“A fluid dynamics-model system for advancing Tissue Engineering and Cancer Research studies: Dynamic Culture with the innovative BioAxFlow Bioreactor”, Giulia Gramigna, Federica Liguori, Ludovica Filippini, Maurizio Mastantuono, Michele Pistillo, Margherita Scamarcio, Antonella Lisi, Giuseppe Falvo D’Urso Labate, Mario Ledda, 2025
by Cellex | Apr 15, 2025 | bone tissue engineering
We have already discussed the use of bioreactors in tissue engineering, particularly bone tissue engineering. After analyzing the contribution of the rotating wall vessel bioreactor and the spinner flask, we will now examine a new type of culture system: the flow perfusion bioreactor.
Let’s find out together what its advantages and critical issues are.
Flow perfusion bioreactor, what it is and how it works
The paper “Design of a Flow Perfusion Bioreactor System for Bone Tissue-Engineering Applications” (Gregory N. Bancroft, Vassilios I. Sikavitsas, Antonios G. Mikos) suggests that the flow perfusion bioreactor is an ideal solution for developing bone tissue equivalents.
According to the paper, the flow perfusion bioreactor would overcome the limitations of the spinner flask bioreactor.
To better understand this point, it is necessary to take a step back.
In our article about spinner flask bioreactors for bone tissue engineering, we had described how this type of bioreactor offered promising performance for cell culture of three-dimensional polymeric scaffolds with MSCs.
However, we also pointed out some critical issues; first, the limitation of nutrient transport in the culture fluid, which was limited only to cells exposed outside the scaffold.
The flow perfusion bioreactor can potentially overcome this limitation. By using a pump that continuously perfuses the culture medium through the porous network of the scaffold, it would indeed be possible to ensure external and internal mixing of the culture fluid.
In other words, in a flow perfusion bioreactor, fluid flow occurs through the scaffold instead of being limited to just the edges, improving nutrient delivery.
To demonstrate this, colleagues Gregory N. Bancroft, Vassilios I. Sikavitsas, Antonios G. Mikos built a flow perfusion bioreactor in order to identify the best design for bone tissue engineering.
The resulting culture system is essentially composed of two parts:
- individual flow chambers, which are the culture chambers within which the scaffolds are placed;
- the flow system circuit consists of reservoirs and a peristaltic pump that is used to pump the liquid medium inside the culture chamber.
Basically, the whole system works like this: fluid is drawn from a first reservoir by the action of the peristaltic pump.
At this point, the culture fluid enters the flow chamber through a top hole and, once pumped inside, flows through the scaffold and out the bottom, with a directional flow from top to bottom.
The outflowing fluid flows to the second reservoir. Then, under the effect of gravity, it returns to the first tank, completing the circuit.
Advantages and critical issues of flow perfusion bioreactors in bone tissue engineering
The flow perfusion bioreactor offers several advantages:
- mitigates external and internal diffusion limitations;
- provides nutrients and gases to the cells in the scaffold;
- ensures a more homogeneous distribution of cells and mineral deposits in the scaffolds than spinner flask bioreactors.
However, flow perfusion bioreactors must meet certain key characteristics to ensure these conditions.
As reported by Gregory N. Bancroft, Vassilios I. Sikavitsas, Antonios G. Mikos, “there are several requirements for a successful flow perfusion system design.”
In particular, the system must distribute flow through the scaffolds, minimizing the nonperfusion flow surrounding each cultured scaffold. If it fails to do so, the entire system offers little advantage over the aforementioned spinner flask bioreactor.
The other critical issue remains the high shear stress applied to the cultured cells, a problem already encountered in spinner flask bioreactors.
Is there an alternative to flow perfusion bioreactors for bone tissue engineering?
For the past several years, the Cellex team has been working to propose an alternative that can mitigate the limitations of flow perfusion bioreactors and spinner flasks for developing tissue engineering constructs.
This alternative is called BioAxFlow, a bioreactor designed specifically for Tissue Engineering.
The bioreactor is based on advanced fluid dynamics based on four main components:
- a base integrating inlet and outlet ports for controlled media flow;
- a cylindrical chamber;
- scaffold stand for scaffold placement;
- a cap with two openings for vent caps and two sampling ports.
In BioAxFlow, during the seeding process, a cell suspension is injected into the main chamber where the scaffolds are located and placed on the corresponding stand. This is done in an automated process that maximizes cell utilization and ensures even scaffold distribution.
The flow generated within it ensures continuous movement of the medium. In this way, the scaffolds are constantly surrounded and perfused by a cell culture medium, being exposed to nutrients and growth factors.
In addition, within BioAxFlow, minimal mechanical shear stress is applied to the cultured cells, which is ensured precisely by advanced fluid dynamics.
Want to learn more about our tissue engineering bioreactor? Learn about all the benefits of BioAxFlow and contact our team.
Sources
“Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor”, Vassilios I. Sikavitsas, Gregory N. Bancroft, Antonios G. Mikos, 2001
“Design of a Flow Perfusion Bioreactor System for Bone Tissue-Engineering Applications”, Gregory N. Bancroft, Vassilios I. Sikavitsas, Antonios G. Mikos, 2003
“A fluid dynamics-model system for advancing Tissue Engineering and Cancer Research studies: Dynamic Culture with the innovative BioAxFlow Bioreactor”, Giulia Gramigna, Federica Liguori, Ludovica Filippini, Maurizio Mastantuono, Michele Pistillo, Margherita Scamarcio, Antonella Lisi, Giuseppe Falvo D’Urso Labate, Mario Ledda, 2025
by Cellex | Dec 20, 2024 | bone tissue engineering
BioAxFlow is designed for 3D cell culture in tissue engineering applications, particularly for bone tissue engineering.
With its advanced fluid dynamics and absence of mechanical components, it overcomes the major drawbacks of reactors currently used in tissue engineering, among which we find Rotating-wall vessels.
Rotating-wall vessels bioreactor, what it is and how it works
NASA developed the Rotating-wall vessels (RWV) as a simulator to mimic and model the effects of microgravity on cells in laboratory studies on Earth. Indeed, the goal of the device was to protect cell cultures from the high shear forces generated during the launch and landing phases of the Space Shuttle.
During the testing phases on Earth, it was discovered that the cells inside the bioreactor aggregated and formed spheroid structures similar to tissue. It was then guessed that the Rotating-wall vessels could be used to develop numerous three-dimensional cellular models.
The bioreactor consists of two concentric cylinders, a growth chamber crossed by an inner cylinder for gas exchange and oxygenation. The chamber is integrally filled with growth medium.
Once the motor is driven, a belt rotates the culture chamber along its horizontal axis; this way, the liquid inside accelerates until the fluid mass rotates at the same angular velocity as the wall. The result is a culture environment with minimal shear forces, in which the cells are uniformly suspended in the culture medium. Due to these conditions, the cells aggregate and initiate three-dimensional growth.
Both microcarriers and scaffolds can be used as supporting matrices within this bioreactor.
The latter turns out to be crucial for bone tissue engineering. However, from this point of view, Rotating-wall vessel bioreactors demonstrate some critical issues.
The main critical issues of a Rotating-wall vessels bioreactor for bone tissue engineering
The culture environment of the Rotating-wall vessels bioreactor has opened up new observations and experiments in tissue engineering. In particular, the bioreactor has been used to develop cartilage and bone tissue equivalents.
However, as reported in “Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor” (Sikavitsas, Bancroft et Mikos, 2001), the Rotating-wall vessels bioreactor has limitations.
The purpose of the study by Sikavitsas, Bancroft, et Mikos was to compare different types of 3D and 2D osteoblast-like cell cultures in terms of morphology, proliferation, and differentiation within Rotating-wall vessels, spinner flask, and static culture bioreactors.
During the experiments, it was noted that when multiple large scaffolds are placed in the Rotating-wall vessels, although free settling can be achieved, the collision of the scaffolds with the reactor walls cannot be avoided.
Such collisions momentarily disturb free settlement and can traumatize cells residing on the scaffold surface due to direct contact with the wall surface.
From this, it can be inferred that the rotating wall bioreactor damages the cells on the surface of the scaffold.
Not only that, at the end of the study’s observations, it was found that cell constructs cultured in the Rotating-wall vessels bioreactor had demonstrated slower proliferation, leading to inferior performance to other bioreactors examined in the study (spinner flask and static culture) about osteoblastic cell differentiation.
How BioAxFlow delivers a solution to these critical issues
Despite the excellent conditions inside the culture chamber, the very operation of the Rotating-wall vessels compromises the effectiveness of three-dimensional cell culture on large scaffolds.
The only solution would seem to be adopting a larger culture chamber. Or use another type of bioreactor that is more functional and versatile.
The critical issue raised by the size of the scaffold prompted the Cellex team to develop an innovative tissue engineering bioreactor that can address the need to use scaffolds of different sizes.
The BioAxFlow culture chamber is specifically designed to use any scaffold, not only in size but also in different geometries.
Individual component modules can be configured to meet the investigator’s needs, particularly regarding scaffold support and the scaffold itself.
BioAxFlow’s innovative fluid dynamics also allow cell seeding on the scaffold without trauma, solving another critical issue raised by Rotating-wall bioreactors.
Want to learn more about our tissue engineering bioreactor? Learn about all the advantages of BioAxFlow and contact our team.
Sources
“Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor”, Vassilios I. Sikavitsas, Gregory N. Bancroft, Antonios G. Mikos, 2001
“Rotating-wall vessels, promising bioreactors for osteoblastic cell culture: comparison with other 3D conditions”, C. Granet N. Laroche L. Vico C. Alexandre M.H. Lafage-Proust, 1998
