Accelerated Production of Brain Organoids Using RNA Transfections and Microfluidics as a Model for Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide, with limited effective treatment options. Traditional TBI models using animal systems have provided valuable insights but face limitations due to fundamental differences in brain architecture and physiology between humans and animals. Brain organoids, which closely mimic human brain structure and function, represent a transformative tool for studying TBI pathogenesis and therapeutic interventions. Here, we present a protocol for the accelerated production of brain organoids using RNA transfections and microfluidics. This method enables the cost-effective, high-throughput generation of human-induced pluripotent stem cells in xeno-free conditions, providing a scalable platform for modeling brain injury. Our approach integrates advanced microfluidic device fabrication, optimized cell reprogramming using low-dose RNA transfections, and controlled differentiation into brain organoids. Additionally, we outline various injury modeling techniques, including static mechanical forces, fluid shear stress, and dynamic mechanical injury, to simulate TBI in organoid systems. This innovative platform offers a novel strategy to study TBI pathogenesis, screen therapeutics, and develop personalized treatment approaches.

 

MissingLinkText

Keywords

brain organoid - microfluids - modeling injury - RNA transfection - traumatic brain injury

Introduction

Millions of patients worldwide suffer a traumatic brain injury (TBI) every year. Moderate to severe TBI (msTBI), defined as Glasgow Coma Scale (GCS) score of 3 to 12 on admission, carries the largest burden of morbidity and mortality.[1] To find a rational treatment plan for TBI, a large number of in vitro cellular and in vivo animal models have been established to study the pathogenesis of TBI. Different animal models have been established to replicate different types of TBI. Although larger animals are more similar to humans in size and physiology, rodents have been widely used in TBI models because of their small size, low cost, and easy quantification.[2] [3] Early TBI models mainly simulated the biomechanical changes of brain injury, while the models created in recent years have been used to study molecular mechanisms and molecular cascades triggered by head trauma.[3] [4] Using the animal model for a TBI has many limitation like their lissencephalic nature, which is not perfect as it is different from the human brain. The brain organoid model can be helpful in simulating brain injury and used as a TBI model, as it has many advantages such as the following: it mimics human brain architecture in 3D and allows experimental manipulation for long-term surgery.[5]

The human brain organoid is a novel technique that can be used as a TBI model in which human somatic cells can be reprogrammed to pluripotency. We aimed to develop a protocol for the cost-effective production of quality-controlled human pluripotent stem cells in nonintegrating and chemically defined xeno-free culture conditions. The method we developed enables reprogramming of a limited number of human somatic cells with high efficiency, high throughput, and low costs to produce human-induced pluripotent stem cells (hiPSCs) without genomic integration.

 

MissingLinkText

Materials and Methods

Microfluidic Device Fabrication

The following methods were used in the fabrication processes: Sylgard 184 polydimethylsiloxane (PDMS; Dow Corning), chrome photomasks (Photo Sciences), and transparency photomasks produced using a high-resolution printer (5,060 dpi). CAD software, including Adobe Illustrator and Autodesk AutoCAD, was utilized for pattern design. Fabrication of masters employed photolithography and electron beam lithography (EBL). For microcontact printing (µCP) and replica molding (REM), substrates included silicon wafers and glass slides.

 

MissingLinkText

Experimental Design

The soft lithography process was conducted in four major steps: pattern design, mask and master fabrication, elastomeric stamp fabrication, and micro-/nanostructure application. A clean room environment was used where required, particularly during photolithography and EBL processes.

 

MissingLinkText

Pattern Design

Patterns were designed using vector-based CAD tools (Adobe Illustrator) for transparency masks and advanced CAD software (AutoCAD) for chrome masks. Patterns were optimized for feature sizes ranging from 1 µm to submicron. High-resolution transparency photomasks were prepared by exporting designs as EPS files and printing using a commercial high-resolution printer.

 

MissingLinkText

Fabrication of Mask and Master

Photolithography was used to fabricate masters with feature sizes above 1 µm, while EBL was employed for submicron patterns. Both methods were performed in a clean room facility. Chrome photomasks were obtained from a commercial supplier for high-precision applications. Transparency masks were used for proof-of-concept experiments to reduce costs.

 

MissingLinkText

Fabrication of PDMS Stamp

Sylgard 184 PDMS was prepared by mixing the base and curing agent at a 10:1 ratio. The liquid precursor was poured onto the master, degassed in a vacuum chamber, and cured at 60°C for 2 hours. For sub-500 nm features, composite stamps consisting of a stiff h-PDMS layer supported by Sylgard 184 were fabricated. The cured PDMS was peeled off the master to obtain the patterned elastomeric stamp.

 

MissingLinkText

Application of Micro- and Nanostructures

Soft lithographic techniques were employed to transfer patterns onto substrates:

• Microcontact printing (µCP): Alkanethiols were used as “ink,” and thin gold films served as substrates. The PDMS stamp was inked, dried, and brought into conformal contact with the substrate to form self-assembled monolayers (SAMs).

• Replica molding (REM): Liquid prepolymers, such as UV-curable polyurethane, were cast against the PDMS mold and cured to produce replicas of the original master.

• Solvent-assisted micro-contact molding (SAMIM): A PDMS mold was wetted with a polymer–solvent mixture and pressed onto a polymer film. The solvent dissipated, leaving behind patterned relief structures ([Fig. 1]).

Fig. 1 Model of the microfluidic device for printing.

 

MissingLinkText

Brain Organoid Formation

On Day 1

1. Prepare the 10-chamber microfluidic devices.

2. Seed human fibroblasts to be reprogrammed in microfluidics and place them in an incubator set to hypoxic conditions (37°C, 5% (vol/vol) CO₂, 5% (vol/vol) O₂) overnight. The incubator used should be set to hypoxic conditions for the whole duration of the experiment.

3. Replace the medium with 12 µL of supplemented Pluriton medium per chamber. Alternatively, the E7 medium (E6 supplemented with FGF2 at 100 ng/mL) can be used in place of the supplemented Pluriton medium. The medium can be supplemented with penicillin–streptomycin, but this is optional.

4. Prepare a low-dose transfection solution at room temperature (25°C), using the StemMACS mRNA transfection kit, keeping the RNA on ice.

5. Incubate the mixture at room temperature for 20 minutes.

6. Add the transfection solution to supplemented Pluriton medium, prewarmed to room temperature, and gently mix. Alternatively, the E7 medium can be used in place of the supplemented Pluriton medium. The medium can be optionally supplemented with penicillin–streptomycin.

7. Take the cells out of the incubator, empty the reservoirs of the microfluidic device, and gently pipette 12 µL of transfection solution into each chamber.

8. Incubate the cells overnight at 37°C in the hypoxic incubator.

 

MissingLinkText

Days 2 to 8

• 9. Repeat steps 3 to 8 with the following RNA doses: day 2, low dose; days 3 to 5, medium dose; and days 6 to 8, high dose. On days 1 to 8, the medium can be supplemented with an LSD1 inhibitor at a final concentration of 1 µM to further promote the mesenchymal-to-epithelial transition.

 

MissingLinkText

Days 5 to 8

The medium can be supplemented with Human iPS Reprogramming Boost Supplement II (containing sodium butyrate, PS48, and TGF-β RI Kinase Inhibitor IV) to further enhance reprogramming efficiency.

 

MissingLinkText

Days 9 to 15

Replace the medium in the morning (∼9 a.m.) and at night (∼7 p.m.) with 12 µL per chamber of hiPSC expansion medium (e.g., StemMACS iPS-BREW XF or E8 medium).

• 10. When hiPSC colonies start to be visible (∼days 10–14), verify the expression of the pluripotency marker TRA-1–60 by live staining. To do this, first prepare a solution of Stemgent StainAlive TRA-1–60 antibody at 5 µg/mL and inject 12 µL per chamber.

• 11. Incubate the cells for 30 minutes in the incubator.

• 12. Inject 12 µL of medium per chamber and incubate for 5 minutes.

• 13. Repeat step 12 twice.

• 14. Observe the culture chambers by fluorescence microscopy (excitation/emission: 488/525).

• 15. Continue culture as described in step 10 ([Fig. 2]).

Fig. 2 Schematic representation of the culturing of brain organoid using RNA transfection in the microfluidic device.

 

MissingLinkText

 

MissingLinkText

Various Methods for Modeling Injury

Static Mechanical Injury

A physical damage given to the brain organoid formed by applying sustained and nonmechanical forces.

 

MissingLinkText

Fluid Shear Forces

In this process, mechanical force is exerted on the organoid cells by flowing fluid, impacting their development, differentiation, and function.

 

MissingLinkText

Dynamic Mechanical Injury

It is done by moving the plate on which the organoid is growing.

 

MissingLinkText

 

MissingLinkText

 

MissingLinkText

Conclusion

The development of brain organoids using RNA transfections and microfluidics represents a significant advancement in TBI research. By leveraging their ability to closely mimic human brain architecture, brain organoids overcome many limitations of traditional animal models, offering a more physiologically relevant system for studying TBI pathogenesis. Our protocol enables the efficient, cost-effective, and high-throughput production of hiPSCs under chemically defined xeno-free conditions, paving the way for scalable organoid generation. Furthermore, the application of various injury modeling techniques, such as static, fluid shear, and dynamic mechanical forces, provides a versatile platform to investigate the acute and chronic effects of TBI in a controlled environment. This approach not only facilitates a deeper understanding of TBI mechanisms but also supports high-throughput screening of potential therapeutic interventions, advancing personalized medicine for TBI patients. Future refinements in organoid technology and integration with advanced bioengineering tools could further enhance its utility as a powerful model for TBI and other neurodegenerative conditions.

 

MissingLinkText

 

MissingLinkText

Conflict of Interest

None declared.

• References

• 1 Centers for Disease Control and Prevention. Get the Facts: TBI Data. 2021. Accessed September 30, 2021 at: https;//www.cdc.gov/traumaticbraininjury/data/index/html
• 2 Ebrahimi H, Kazem Nezhad S, Farmoudeh A. et al. Design and optimization of metformin-loaded solid lipid nanoparticles for neuroprotective effects in a rat model of diffuse traumatic brain injury: a biochemical, behavioral, and histological study. Eur J Pharm Biopharm 2022; 181: 122-135
• 3 Feeney DM, Boyeson MG, Linn RT, Murray HM, Dail WG. Responses to cortical injury: I. Methodology and local effects of contusions in the rat. Brain Res 1981; 211 (01) 67-77
• 4 Khellaf A, Khan DZ, Helmy A. Recent advances in traumatic brain injury. J Neurol 2019; 266 (11) 2878-2889
• 5 Jgamadze D, Johnson VE, Wolf JA. et al. Modeling traumatic brain injury with human brain organoids. Curr Opin Biomed Eng 2020; 14: 52-58

Publication History

Article published online:
02 July 2025