NEURON CULTURE PACK - LOW SHEAR CELL CULTURE

Cell culture system for shear-sensitive cell lines

SAFE NEURON CULTURE UNDER FLOW

Advantages of dynamic cell culture even with shear-sensitive cells

HIGHLY CONTROLLED MICROENVIRONMENT

Continuous and controlled supply of nutrients in a stable flow

UP TO 3-WEEK LONG CELL CULTURES

Less manual work and more accuracy for neuron culture experiments

Neuron culture pack

Neuron culture and shear stress

Unlike other cells in the body, the physiological microenvironment of neurons is usually in tissues that do not have continuous fluid flow. Therefore, neuron culture under media perfusion, exposed to shear stress, can significantly affect their viability [1].

However, cell perfusion has marked advantages that can benefit neuron cell culture. For example, gradients can be formed to study the orientation of axons [1], or chemical cues can be precisely added to induce neural stem cell differentiation [2, 3]. On the other hand, the continuous flow of media ensures a constant supply of nutrients and waste removal, improving the microenvironment conditions [4].

Flow rate and shear stress

Shear stress is a direct effect of flow rate and system dimensions. For a system with defined dimensions, the lower the flow rate, the lower the shear stress. However, there is a limit to how low one can go with the flow rate and still have a stable flow. Thus, to have a system with a steady flow and low shear stress, it is necessary to re-think the dimensions and geometry of the culturing chamber.

Low shear bioreactor for neuronal culture

The Microfluidics Innovation Center partnered with IVTech, a biotechnology company specializing in advanced cell culture chambers, to refine in vitro models. MultiDyn has been designed and produced by IVTech SRL, an evolution of the LiveBox1 chamber. The system is a rack of 4 wells in parallel, compatible in dimension with the column of a standard 24-well plate.

MultiDyn assures the opportunity to recreate all the experiments performed with LiveBox1, replicating the data in 4 parallel tests and reducing the complexity of the chamber setup. The rack is monitorable in real-time, using an inverted microscope, assuring the possibility of developing a 3D in-vitro model in dynamic conditions. Moreover, two or more racks can be joined in series to establish a multi-organ model, where each tissue communicates with the others due to a liquid exchange.

References

1. C. Joanne Wang, X. Li, B. Lin, S. Shim, G. L. Ming, and A. Levchenko, ‘A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues’, Lab Chip, vol. 8, no. 2, pp. 227–237, Jan. 2008.

2. T. Barra et al., ‘Neuroprotective Effects of gH625-lipoPACAP in an In Vitro Fluid Dynamic Model of Parkinson’s Disease’, Biomed. 2022, Vol. 10, Page 2644, vol. 10, no. 10, p. 2644, Oct. 2022.

3. B. G. Chung et al., ‘Human neural stem cell growth and differentiation in a gradient-generating microfluidic device’, Lab Chip, vol. 5, no. 4, pp. 401–406, Mar. 2005.

4. M. L. Coluccio et al., ‘Microfluidic platforms for cell cultures and investigations’, Microelectron. Eng., vol. 208, pp. 14–28, 2019.

Low shear and automated cell culture pack

The Microfluidics Innovation Center and IVTech combined their strengths to develop an automated pack for low-shear neuron cell culture. We used our flow control expertise to create an automated and secure cell perfusion system, integrating IVTech’s bioreactor for low-shear cell culture.

The neuron culture pack includes:

Flow controller
Automated cell culture platform (comprising flow sensor eg. Galileo; recirculation loops, and level sensors)
IVTech MultiDyn bioreactor
Software (Galileo user interface)
User guide

Neuron culture in low-shear bioreactors

Barra et al. [1] used IVTech’s LiveBox 1 bioreactors to create an in vitro Parkinson’s disease (PD) model based on a neuron cell culture. They cultured a neuroblastoma cell line, SH-SY5Y, differentiated with retinoic acid as a model for P. Then, they tested and demonstrated the neuroprotective effects of a neuropeptide (pituitary adenylate cyclase-activating polypeptide, PACAP) in a fluid dynamic model.

neuron culture spheroid barra — Representative image of spheroids obtained by culturing human neuroblastoma 3D SH-SY5Y/RA cells in the LB1 after 24 h of fluid condition (100 μL/min). Spheroids were immunoreacted with PAC1-R primary antibody (GFP, (a)), VPAC1 primary antibody (GFP, (b)), VPAC2 primary antibody (GFP, (c)). Nuclei, DAPI. Images were acquired as Z stack with the JuLI Stage fluorescence recorder and maximum intensity projection was shown. Scale bar: 250 μm. Reproduced from Barra et al., 2022 [1].

In another study to develop a blood-brain barrier model, the neuronal culture aimed to grow spheroids in the bioreactor [2]. According to the authors, “immunofluorescence experiments showed that fluid dynamic conditions have no appreciable influence on spheroids health, morphology, and organization.”

barra et al 2022 bbb spheroids — Representative image of spheroids obtained by culturing human neuroblastoma SH-SY5Y SH-SY5Y cells in the lower chamber of LB2/bEnd.3 cells. Spheroids were immunoreacted with Ki-67 antibody, (GFP, A,B), ZO1 (RFP,C), and β-tubulin III (RFP,D). Nuclei, DAPI. Images were acquired as Z stacks with the JuLI Stage fluorescence recorder, and the maximum intensity projection was shown. The scale bar was 250 µm. reproduced from Barra et al., 2022 [2].

References

1. T. Barra et al., ‘Neuroprotective Effects of gH625-lipoPACAP in an In Vitro Fluid Dynamic Model of Parkinson’s Disease’, Biomed. 2022, Vol. 10, Page 2644, vol. 10, no. 10, p. 2644, Oct. 2022.

2. T. Barra et al., ‘gH625-liposomes deliver PACAP through a dynamic in vitro model of the blood–brain barrier’, Frontiers in Physiology, vol. 13. 2022.

Neuron culture pack specifications

The cell culture system comprises a perfusion platform and a pressure splitter. An OB1 flow controller (Elveflow) controls the fluid flow.

The table summarizes the main specifications of the cell culture system.

Component	Technical specification
OB1 MK4 (Elveflow)	4 Channels (0 to 2000 mbar)
Microfluidic Flow sensor (Galileo)	Range from 0.5 to 10,000 µL/min
Recirculation loop	4 check valves, polycarbonate, male Luer; compatible with 1/4″-28 standard microfluidic fittings
Pressure splitter	Valve controller (Elveflow) with integrated Solenoid valves (Elveflow) for compressed air.
Cell culturing chamber	MultiDyn bioreactor (4 parallel bioreactors, 1,5 ml internal volume, IVTECH SRL)

The cell culture system is compatible with standard incubator conditions (37^oC, 5% CO_2, and 100% humidity); electronic components (except flow sensors) should be kept outside the incubator.

Customize your pack

Our instruments are compatible with standard commercialized chips from different brands.

Our Packs can be modified depending on your specific needs. Our microfluidic specialists will advise you to provide the best instruments and accessories, depending on your needs. They will accompany you during the setup of the microfluidic platform.

– Check our other Packs for various applications –

Can I order a pack?

Since Packs are products that are still being developed, we have a few eligibility criteria to maximize their success rate. A discussion with our experts is needed to determine your specific needs to offer you a personalized response.

What can be placed inside the incubator?

The automated cell culture platform and the embedded flow sensors can be placed inside the CO₂incubator. The OB1 and the pressure splitter should be kept outside.

What type of cells can be cultured with the system?

Although shown here with a neuron culture, the system can be used to culture any cell type, as the culture media can be adjusted according to the cells’ needs, and the cell culturing chamber can be replaced by the required design.

Can the system be sterilized/autoclaved?

The components of the automated cell culture platform can be sterilized. Our user guide provides detailed information on how to do it.

Is the system compatible with microscopes/live cell imaging?

Yes, the MultiDyn bioreactors from IVTech are compatible with live cell imaging.