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US-12618035-B2 - Microfluidic device

US12618035B2US 12618035 B2US12618035 B2US 12618035B2US-12618035-B2

Abstract

The present invention relates to a microfluidic device ( 1 ) for cultivating cells, in particular for generating brain organoids, comprising at least two fluid channels ( 2 ) positioned essentially opposite to each other and a main chamber ( 3 ) located between the fluid channels ( 2 ), wherein the main chamber ( 3 ) comprises at least one preferably sealable access opening, and each of the at least two fluid channels ( 2 ) is fluidly connected to the main chamber ( 3 ) at at least one point of contact ( 4 ), wherein a slotted structure ( 5 ) is provided at each point of contact ( 4 ) separating the main chamber ( 3 ) from the respective fluid channel ( 2 ), wherein the slotted structure ( 5 ) is permeable to a liquid.

Inventors

  • Sarah SPITZ
  • Peter Ertl
  • Jens Schwamborn
  • Silvia BOLOGNIN

Assignees

  • TECHNISCHE UNIVERSITÄT WIEN

Dates

Publication Date
20260505
Application Date
20210505
Priority Date
20200508

Claims (17)

  1. 1 . A microfluidic device ( 1 ) for cultivating cells, comprising at least two fluid channels ( 2 ) positioned essentially opposite to each other and a main chamber ( 3 ) located between the fluid channels ( 2 ), wherein the main chamber ( 3 ) comprises at least one access opening, and each of the at least two fluid channels ( 2 ) is fluidly connected to the main chamber ( 3 ) at at least one point of contact ( 4 ), wherein a slotted structure ( 5 ) is provided at each point of contact ( 4 ) separating the main chamber ( 3 ) from the respective fluid channel ( 2 ), wherein the slotted structure ( 5 ) is permeable to a liquid, wherein the main chamber ( 3 ) comprises an inner top surface ( 6 ), an inner bottom surface ( 7 ) situated opposite to the inner top surface ( 6 ), and an array of pillars ( 8 ) extending from the inner top surface ( 6 ) into the cross section of the main chamber ( 3 ) in direction of the inner bottom surface ( 7 ), wherein the inner bottom surface ( 7 ) of the main chamber ( 3 ) comprises at least one recess ( 9 ).
  2. 2 . The microfluidic device ( 1 ) according to claim 1 , wherein the contact points ( 4 ) are formed by elongated contact areas located on opposite sides of the main chamber ( 3 ).
  3. 3 . The microfluidic device ( 1 ) according to claim 2 , wherein the distance between the elongated contact areas is smaller than a length of the main chamber ( 3 ) of the microfluidic device ( 1 ).
  4. 4 . The microfluidic device ( 1 ) according to claim 1 , wherein the at least one recess ( 9 ) has a substantial cylindrical or substantial hemispherical shape.
  5. 5 . The microfluidic device ( 1 ) according to claim 1 , wherein each of the at least two fluid channels ( 2 ) is fluidly connected to a respective fluid channel inlet ( 2 a ) and a respective fluid channel outlet ( 2 b ).
  6. 6 . The microfluidic device ( 1 ) according to claim 5 , wherein the fluid channel inlet ( 2 a ) and the fluid channel outlet ( 2 b ) are fluidly connected to medium reservoirs ( 2 c , 2 d , 2 e , 2 f ).
  7. 7 . The microfluidic device ( 1 ) according to claim 6 , wherein the medium reservoirs ( 2 c , 2 f ) fluidly connected to the fluid channel inlet ( 2 a ) have a larger volume than the medium reservoirs ( 2 d , 2 e ) fluidly connected to the fluid channel outlet ( 2 b ).
  8. 8 . The microfluidic device ( 1 ) according to claim 6 , wherein the medium reservoirs ( 2 c , 2 f ) fluidly connected to the fluid channel inlet ( 2 a ) have a smaller volume than the medium reservoirs ( 2 d , 2 e ) fluidly connected to the fluid channel outlet ( 2 b ).
  9. 9 . The microfluidic device ( 1 ) according to claim 6 , wherein the medium reservoirs ( 2 c , 2 f ) fluidly connected to one fluid channel ( 2 ) of the at least two fluid channels have a larger volume than the medium reservoirs ( 2 d , 2 e ) fluidly connected to another fluid channel ( 2 ) of the at least two fluid channels.
  10. 10 . The microfluidic device ( 1 ) according to claim 1 , wherein the slotted structure ( 5 ) comprises an electrically conductive material to form an electrode.
  11. 11 . The microfluidic device ( 1 ) according to claim 1 , wherein the slots of the slotted structure ( 5 ) are spaced apart from each other by at least 50 μm.
  12. 12 . The microfluidic device ( 1 ) according to claim 1 , wherein an inner surface of the main chamber, an inner surface of the at least two fluid channels ( 2 ), and/or the slotted structure ( 5 ) is covered by an antifouling layer.
  13. 13 . The microfluidic device ( 1 ) according to claim 1 , wherein the array of pillars ( 8 ) extending from the inner top surface ( 6 ) into the cross section of the main chamber ( 3 ) in direction of the inner bottom surface ( 7 ) comprises at least some pillars connecting the inner top surface ( 6 ) and the inner bottom surface ( 7 ).
  14. 14 . The microfluidic device ( 1 ) according to claim 1 , wherein the access opening is sealable.
  15. 15 . The microfluidic device ( 1 ) according to claim 11 , wherein the slots of the slotted structure ( 5 ) are spaced apart from each other by at least 80 μm.
  16. 16 . The microfluidic device ( 1 ) according to claim 11 , wherein the slots of the slotted structure ( 5 ) are spaced apart from each other by at least 100 μm.
  17. 17 . The microfluidic device ( 1 ) according to claim 11 , wherein the slots of the slotted structure ( 5 ) are spaced apart from each other by at least 120 μm.

Description

TECHNICAL FIELD The present invention relates to the field of microfluidic test devices. BACKGROUND ART With yet unknown aetiology Parkinson disease (PD) constitutes the second most common neurodegenerative disease worldwide. Characterized by a loss of opaminergic neurons within the substantia nigra of the human midbrain, Parkinson disease results in a variety of symptoms including rigidity, akinesia as well as tremor. In addition, the disease is linked to a broad spectrum of non-motor symptoms such as disorders of the mood, cognitive dysfunction and hallucinosis. While several aspects including genetic and environmental factors have been identified to play a role in Parkinson disease the underlying causes still remain unknown. This can at least in part be explained by the inability of animal models to adequately mimic important aspects of the human brain including histomorphology, spatiotemporal self-organization and neurodevelopment as well as the multifactorial nature of PD, all of which have called for sophisticated in vitro models capable of emulating and thus studying the disease and testing potential drug candidates. With the emergence of induced pluripotent stem cell (iPSC) technology, it has now become possible to generate complex biological structures in vitro, including that of the human brain and midbrain on a personalized level. While iPSC derived human midbrain organoids were shown to display spatial organizations including cell-cell interactions as well as characteristic functions such as neuronal activity that mimic that of the human brain, they still fail to take mechanical stresses such as fluid flow into account. Interstitial fluid flow not only plays an important role in the delivery of nutrients and removal of metabolic waste but it implicates non-synaptic cell-cell communication, ionic homeostasis, cell migration as well as immune function. Furthermore, it is involved in drug delivery, distribution and clearance and thus of considerable importance when employing hMOs in the context of drug screening applications. The production of organoids, in particular of brain or midbrain organoids, in microwell and cell culture plate formats are well-established methods. However, the formation of with such methods displays reproducability issues and the organoids formed show typically unphysiologic necortic cores. Hence, conventional strategies in induced pluripotent stem cell technology (iPSC) organoid technology are based on microwell and cell culture plate formats, lacking active nutrient and oxygen supply as well as the removal of degradation products at the same time resulting in inferior physiologic properties. WO 2017/035119 A1 discloses a microfluidic system for modelling the blood brain barrier. US 2014/0057311 A1 discloses a microfluidic device comprising one or more fluid channels and respective fluid channel inlets and fluid channel outlets. US 2011/0256574 A1 discloses a microfluidic continuous flow device with different compartments. WO 2009/089189 A2 discloses a microfluidic cell culture system with a medium inlet reservoir fluidically connected to a plurality of microfluidic medium channels. MARIANA S. VIEIRA ET AL: “Neural stem cell differentiation into mature neurons: Mechanisms of regulation and biotechnological applications”, BIOTECHNOLOGY ADVANVCES., vol. 36, no. 7, 1 Nov. 2018 (2018-11-01), pages 1946-1970, XP055705717, ISSN: 0734-9750 discloses biological background information about stem cell differentiation. There is currently no in vitro system available which allows producing brain organoids to be used in tests which simulate an in vivo environment. Hence, it is an object of the present invention to provide method and means to overcome the drawbacks of current systems known in the art. SUMMARY OF THE INVENTION In order to overcome the drawbacks of the methods and devices known in the art for producing organoids, in particular brain organoids, the present invention provides a microfluidic device for cultivating cells, in particular for generating brain organoids, comprising at least two fluid channels positioned essentially opposite to each other and a main chamber located between the fluid channels, wherein the main chamber comprises at least one preferably sealable access opening, and each of the at least two fluid channels is fluidly connected to the main chamber at at least one point of contact, wherein a slotted structure is provided at each point of contact separating the main chamber from the respective fluid channel, wherein the slotted structure is permeable to a liquid. The main chamber comprises an inner top surface, an inner bottom surface situated opposite to the inner top surface, and an array of pillars extending from the inner top surface into the cross section of the main chamber in direction of the inner bottom surface. It turned surprisingly out that the microfluidic device of the present invention allows longterm cultivation of mammalian cells to produ