Want to make creations as awesome as this one?

No description

Transcript

Organofluidics

Organoid : a 3D multicellular in vitro tissue constructRobust advancement in in vitro platformsIntrinsic hypoxia jeopardizes the culture sustainability

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

AIMS

CONTEXT

Long-term culture

Reduce hypoxia and necrotic core

Fund microchip construction

EXPERIMENTAL APPROACH

EXPECTEDOUTCOME

BUDGET & TIMING

Most expensive part : microchip construction

By company ~ 1,500€With contributors

Sup'Biotech Interactive Scientific Poster ©

ABSTRACT

COLLAB.

REFS.

Increase the nutrient and oxygen supply

Adaptation of the Spin∞ chip

Organofluidic microchip

Macrofluidic systemLong-term cultureMini spinning bioreactorPerfusion channels to provide nutrients and oxygen

Overview (by AutoCAD)

Microfluidic systemMicropillars: Interface of the cult ure chamber and perfusion channelPerfusion channels to provide nutrients and oxygenLong-term cultureSupported by microfluidics expert

CONTEXT

1. Definition of an organoid

3D multicellular in vitro tissue constructAble to mimic its corresponding in vivo organGive scientists a detailed view of the formation and growth of the organsNew insights on human development and models of diseaseIn vitro models in the long term

2

3

1

Organofluidics

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

2. What are the limits ?

Proper organ microenvironment is complex and difficult to reproduceCurrent technologies do not allow uniform organoid productionFormation of a necrotic core within the organoidDifficulty to obtain mature organoids in vitro

Representation of the challenges (via BioRender)

CONTEXT

2

3

1

Organofluidics

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

3. Several methods to overcome hypoxia core issue

Air–liquid interface method [1][2]:Cells are cultured on a thin, microporous membraneEnhance the uptake of oxygen by the organoidMicrofluidic approach [3]:Inject culture medium into a microfluidic chipImproved interaction between organoid and the culture mediumMini bioreactors [4]:Using motorized helixIncreased flow of nutrients and an oxygenation of the medium

[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.

Representation of the existing method (via BioRender)

CONTEXT

3

1

2

4. The Organofluidics project

Use microfluidics to optimize organoid microenvironnementInduce the influx of nutrients into the organoid & increase its nutrient absorptionCreating microfluidic chip prototypesInvestigate the effectiveness of spheroids on microfluidic systems

Organofluidics

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

Organofluidics' logo

AIMS

Use a microfluidic chip to reduce the hypoxia coreForm spheroids by using the hanging drop method with tumor cellsInterest to use a microfluidic systemProvide a constant flow rate of nutrient and gas exchangeAllowing a long-term culture in 3D

Organofluidics

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

1

Macrofluidic system (via BioRender)

Microfluidic system

Reproduce the Spin ∞ designA cover adapted for a 12 well plateUse of motorized helixInterest to use a macrofluidifc systemTo induce an increased nutrient flowProvide a medium oxygenation

Macrofluidic system

Microfluidic system (via BioRender)

EXPERIMENTAL APPROACH

1

Organofluidics

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

Adaptation of the Spin∞ chip

[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/

Side view (by AutoCAD)

Overview (by AutoCAD)

Design inspired by Romero-Morales et al. (2019)Design will be performed on AutoCADMini spinning bioreactorAdapted to commercialised well-plateWill be printed with a 3D printer (Neva from DAGOMA)

Macrofluidic: The Spin∞ chip

Overview of the Spin∞ chip [4]

Bottom view of the Spin∞ chip [4]

Modification of the helix

Modification of the shape of the propeler

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

Picture of the impeller [5]

EXPERIMENTAL APPROACH

2

Organofluidics

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

Microfluidic chip

[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.

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

Schematic diagram of the microfluidic chip of Organofluidic [7]

EXPERIMENTAL APPROACH

3

Organofluidics

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

[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

Process of manufacturing of the microfluidic chip

Design the maskCreate the mold by photolitographyCreate the chip from the moldExperimental approach

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

EXPERIMENTAL APPROACH

4

Organofluidics

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

BUDGET & TIMING

GANTT CHART

Organofluidics

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

2

1

3

BUDGET & TIMING

OVERALL BUDGET

Cell culture: consumablesPrinting in 3D: 3D printer + PLAIn the Microchip design: license of the CAO software AutoCAD is expensive

1

2

3

Organofluidics

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

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

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

1

3

2

BUDGET & TIMING

Working with potential collaborators: cost effective strategy for microchip fabrication

Organofluidics

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

Fabrication of the chip in one step

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

Fabrication of the chip in several steps

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

EXPECTED OUTCOME

1. Overall budget

Cell culture: equipment available at Sup'Biotech, 200€/week of Fil rouge will be dedicated on this part1 free year of AutoCAD with a student licenseA 3D printer (NEVA from DAGOMA) is available at the SBIP workshop

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

2. Microchip (microfluidic)

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

Organofluidics

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

4. Perspectives

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

3. Mini bioreactor (macrofluidic)

We want to reproduce the spin ∞ designAll the helix will be optimised and printed in 3DDifferent model of helix can be designed and comparedThe need for a collaboration is much less decisive than for the microfluidics

BIBLIOGRAPHY

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, 48–532. Rossi, G.; Manfrin, A.; Lutolf, M.P. Progress and potential in organoid research. Nat. Rev. Genet. 2018, 19, 671–6873. 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, 16774. 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.e000845.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/app60200407.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

Organofluidics

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

ABSTRACT

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.

Organofluidics

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

COLLABORATIONS & ACKNOWLEDGEMENTS

Organofluidics

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

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.

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.