Organofluidics - Fil Rouge - TRY

• Microfluidic system
• Micropillars: Interface of the cult ure chamber and perfusion channel
• Perfusion channels to provide nutrients and oxygen
• Long-term culture
• Supported by microfluidics expert

Overview (by AutoCAD)

• Macrofluidic system
• Long-term culture
• Mini spinning bioreactor
• Perfusion channels to provide nutrients and oxygen

Organofluidic microchip

Adaptation of the Spin∞ chip

• Increase the nutrient and oxygen supply

REFS.

COLLAB.

ABSTRACT

Sup'Biotech Interactive Scientific Poster ©

• By company ~ 1,500€
• With contributors

• Most expensive part : microchip construction

BUDGET & TIMING

EXPECTEDOUTCOME

EXPERIMENTAL APPROACH

• Fund microchip construction

• Reduce hypoxia and necrotic core

• Long-term culture

CONTEXT

AIMS

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

• Organoid : a 3D multicellular in vitro tissue construct
• Robust advancement in in vitro platforms
• Intrinsic hypoxia jeopardizes the culture sustainability

Organofluidics

Representation of the challenges (via BioRender)

• Proper organ microenvironment is complex and difficult to reproduce
• Current technologies do not allow uniform organoid production
• Formation of a necrotic core within the organoid
• Difficulty to obtain mature organoids in vitro

2. What are the limits ?

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen,TRY Élisabeth & YATES Frank*

Organofluidics

• 3D multicellular in vitro tissue construct
• Able to mimic its corresponding in vivo organ
• Give scientists a detailed view of the formation and growth of the organs
• New insights on human development and models of disease
• In vitro models in the long term

1. Definition of an organoid

CONTEXT

Representation of the existing method (via BioRender)

[1] 1. Quadrato, G.; Nguyen, T.; Macosko, E.Z.; Sherwood, J.L.; Yang, S.M.; Berger, D.R.; Maria, N.; Scholvin, J.; Goldman, M.; Kinney, J.P.; et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 2017, 545, 48–53 [2] 2. Rossi, G.; Manfrin, A.; Lutolf, M.P. Progress and potential in organoid research. Nat. Rev. Genet. 2018, 19, 671–687 [3] 3. Yaqing Wang, Li Wang, Yaqiong Guo, Yujuan Zhu, Jianhua Qin, “Engineering stem cell- derived organoids in a perfusable organ-on-a-ship system”, RSC Adv., 2018, 8, 1677 [4] 4. Romero-Morales, A. I., O’Grady, B. J., Balotin, K. M., Bellan, L. M., Lippmann, E. S., & Gama, V. (2019). Spin∞: an updated miniaturized spinning bioreactor design for the generation of human cerebral organoids from pluripotent stem cells. HardwareX, 6, 20. https://doi.org/10.1016/j.ohx.2019.e00084 .

• Air–liquid interface method [1][2]:
• Cells are cultured on a thin, microporous membrane
• Enhance the uptake of oxygen by the organoid
• Microfluidic approach [3]:
• Inject culture medium into a microfluidic chip
• Improved interaction between organoid and the culture medium
• Mini bioreactors [4]:
• Using motorized helix
• Increased flow of nutrients and an oxygenation of the medium

3. Several methods to overcome hypoxia core issue

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

CONTEXT

Organofluidics' logo

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen,TRY Élisabeth & YATES Frank*

Organofluidics

• Use microfluidics to optimize organoid microenvironnement
• Induce the influx of nutrients into the organoid & increase its nutrient absorption
• Creating microfluidic chip prototypes
• Investigate the effectiveness of spheroids on microfluidic systems

4. The Organofluidics project

CONTEXT

Microfluidic system (via BioRender)

Macrofluidic system

• Reproduce the Spin ∞ design
• A cover adapted for a 12 well plate
• Use of motorized helix
• Interest to use a macrofluidifc system
• To induce an increased nutrient flow
• Provide a medium oxygenation

Microfluidic system

Macrofluidic system (via BioRender)

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

• Use a microfluidic chip to reduce the hypoxia core
• Form spheroids by using the hanging drop method with tumor cells
• Interest to use a microfluidic system
• Provide a constant flow rate of nutrient and gas exchange
• Allowing a long-term culture in 3D

AIMS

Picture of the impeller [5]

• Pitched blades angle set at 45° for a radial and axial flow
• Better overall mixing & higher oxygen mass transfer rate KLa

Modification of the shape of the propeler

Modification of the helix

Bottom view of the Spin∞ chip [4]

Overview of the Spin∞ chip [4]

Macrofluidic: The Spin∞ chip

• Design inspired by Romero-Morales et al. (2019)
• Design will be performed on AutoCAD
• Mini spinning bioreactor
• Adapted to commercialised well-plate
• Will be printed with a 3D printer (Neva from DAGOMA)

Overview (by AutoCAD)

Side view (by AutoCAD)

[4] Romero-Morales, A. I., O’Grady, B. J., Balotin, K. M., Bellan, L. M., Lippmann, E. S., & Gama, V. (2019). Spin∞: an updated miniaturized spinning bioreactor design for the generation of human cerebral organoids from pluripotent stem cells. HardwareX, 6, 20. https://doi.org/10.1016/j.ohx.2019.e00084[5] Mirro, R., & Voll, K. (2009). Which Impeller Is Right for Your Cell Line? - BioProcess InternationalBioProcess International. BioProcess International, 52–57. https://bioprocessintl.com/analytical/cell-line-development/which-impeller-is-right-for-your-cell-line-183538/

Adaptation of the Spin∞ chip

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

EXPERIMENTAL APPROACH

Schematic diagram of the microfluidic chip of Organofluidic [7]

• Design inspired by Yaqing et al. (2018)
• Pre-formed spheroids will be suspended into ice-cold Matrigel
• Chip will be incubated at 37°C
• A controlled flow rate using a KNF pump (Ref: N86KN. 18)

[6] Yaqing Wang, Li Wang, Yaqiong Guo, Yujuan Zhu, Jianhua Qin, “Engineering stem cell- derived organoids in a perfusable organ-on-a-ship system”, RSC Adv., 2018, 8, 1677.

Microfluidic chip

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

EXPERIMENTAL APPROACH

Schematic diagram of the process of manufacturing of the microfluidic chip [7]

1. Design the mask
2. Create the mold by photolitography
3. Create the chip from the mold
4. Experimental approach

Process of manufacturing of the microfluidic chip

[7] Ma, Y., Thiele, J., Abdelmohsen, L., Xu, J., & Huck, W. T. S. (2014). Biocompatible macro-initiators controlling radical retention in microfluidic on-chip photo-polymerization of water-in-oil emulsions. Chemical Communications, 50(1), 112–114. https://doi.org/10.1039/c3cc46733c

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

EXPERIMENTAL APPROACH

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

EXPERIMENTAL APPROACH

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

GANTT CHART

BUDGET & TIMING

Provisional budget of the Organofluidic's project (by Excel)

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

One of the most expensive parts of our project is in the microchip construction

Organofluidics

• Cell culture: consumables
• Printing in 3D: 3D printer + PLA
• In the Microchip design: license of the CAO software AutoCAD is expensive

OVERALL BUDGET

BUDGET & TIMING

• PDMS: 34€ (for 30mL)
• Negative photoresist : 178€
• Rent room, spin-coated, plasma oven by Institute of Pierre-Gilles de Gennes
• Alveole with PRIMO technique

Fabrication of the chip in several steps

Costs around 1,500€ (price given by 4D cell)

Fabrication of the chip in one step

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

Working with potential collaborators: cost effective strategy for microchip fabrication

BUDGET & TIMING

• We want to reproduce the spin ∞ design
• All the helix will be optimised and printed in 3D
• Different model of helix can be designed and compared
• The need for a collaboration is much less decisive than for the microfluidics

3. Mini bioreactor (macrofluidic)

• Our goal is to make the long term culture of organoids possible, for more than one year
• Possibility to mature organoids (notably in brain organoids which are only in pre-natal stages)
• Facilitate the culture of organoids

4. Perspectives

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

• No equipment available at Sup'Biotech to fabricate a mask and a microchip
• Specific materials are required for microfluidic
• Collaborations are crucial for the viability of this project: Dr. Gobaa & Mr. Opitz

2. Microchip (microfluidic)

Culture organoids for the long-term could lead to new models for the study of disease

• Cell culture: equipment available at Sup'Biotech, 200€/week of Fil rouge will be dedicated on this part
• 1 free year of AutoCAD with a student license
• A 3D printer (NEVA from DAGOMA) is available at the SBIP workshop

1. Overall budget

EXPECTED OUTCOME

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

1. Quadrato, G.; Nguyen, T.; Macosko, E.Z.; Sherwood, J.L.; Yang, S.M.; Berger, D.R.; Maria, N.; Scholvin, J.; Goldman, M.; Kinney, J.P.; et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 2017, 545, 48–53 2. Rossi, G.; Manfrin, A.; Lutolf, M.P. Progress and potential in organoid research. Nat. Rev. Genet. 2018, 19, 671–687 3. Yaqing Wang, Li Wang, Yaqiong Guo, Yujuan Zhu, Jianhua Qin, “Engineering stem cell- derived organoids in a perfusable organ-on-a-chip system”, RSC Adv., 2018, 8, 1677 4. Romero-Morales, A. I., O’Grady, B. J., Balotin, K. M., Bellan, L. M., Lippmann, E. S., & Gama, V. (2019). Spin∞: an updated miniaturized spinning bioreactor design for the generation of human cerebral organoids from pluripotent stem cells. HardwareX, 6, 20. https://doi.org/10.1016/j.ohx.2019.e00084 5. Mirro, R., & Voll, K. (2009). Which Impeller Is Right for Your Cell Line? - BioProcess InternationalBioProcess International. BioProcess International, 52–57. Ahmad Khalili, A., Ahmad, M., Takeuchi, M., Nakajima, M., Hasegawa, 6. Y., & Mohamed Zulkifli, R. (2016). A Microfluidic Device for Hydrodynamic Trapping and Manipulation Platform of a Single Biological Cell. Applied Sciences, 6(2), 40. https://doi.org/10.3390/app6020040 7. Ma, Y., Thiele, J., Abdelmohsen, L., Xu, J., & Huck, W. T. S. (2014). Biocompatible macro-initiators controlling radical retention in microfluidic on-chip photo-polymerization of water-in-oil emulsions. Chemical Communications, 50(1), 112–114. https://doi.org/10.1039/c3cc46733c

BIBLIOGRAPHY

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

An organoid is a three-dimensional (3D) aggregation of cells, obtained by differentiated stem cells. It is considered scientifically more important in mimicking the systemic and physiological conditions of in organs relative to two-dimensional cell culture. One of the biggest challenges in the area remains to increase the viability of the inner cells of this 3D cell culture, which tends toward necrosis because of a lack of nutrients and the oxygen uptake the core. Several approaches for vascularization are being established to solve the issue of hypoxia, such as using a controlled organ-on-a-chip system. Based on the literature, we design few microfluidic chips and one macrofluidic chip, presented in this Grant form application.

ABSTRACT

The authors ackowledge Dr. Yates for his precious advices and his support all along the project, and also Dr. Perdiz for providing the cells needed for the project.

The authors would like to give thank to Mr. Opitz (from Alveole) for his precious advices on the fabrication of microfluidic chip and PRIMO and to Dr. Gobaa (from Institut Pasteur) for his expertise on the microfluidic and biomaterials.

SAXENA Mohit, LIN Alison, PIGEARD Benjamin, TRAN-RAJAU Jaouen, TRY Élisabeth & YATES Frank*

Organofluidics

COLLABORATIONS & ACKNOWLEDGEMENTS