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CA-3078426-C - MICROPHYSIOLOGICAL ORGANOID CULTURE

CA3078426CCA 3078426 CCA3078426 CCA 3078426CCA-3078426-C

Abstract

The present invention is based on the field of cultivating biological cells and tissues having an organ-like function on a micro-physiological scale and relates to a method for the microphysiological co-cultivation of 3D organoid tissue and at least one 2D cell layer.

Inventors

  • Peter Loskill
  • Christopher Probst
  • Stefan Liebau
  • Kevin Achberger
  • Jasmin Haderspeck

Assignees

  • FRAUNHOFER-GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG E.V.
  • EBERHARD KARLS UNIVERSITAT TUBINGEN

Dates

Publication Date
20260505
Application Date
20181001
Priority Date
20171005

Claims (17)

  1. CLAIMS 1. A method for the microphysiological co-cultivation of organoid tissue (10) in a bioreactor vessel (30) with a semi-permeable membrane (33) on the bottom, comprising the steps: (a) (b) seeding cells of at least one first cell type onto the membrane (33), cultivating these seeded cells to form at least one 2D cell layer (20) supported on this membrane (33) and (c) introducing into the bioreactor vessel (30) on the supported 2D cell layer (20) - an organoid (10) containing cells of at least two further cell types which are arranged in a defined 3D structure relative to each other, and - a hydrogel (15) with the proviso that the organoid (10) in the bioreactor vessel (30) is spaced apart from the supported 2D cell layer (20) by the introduced hydrogel (15).
  2. 2. The method according to claim 1, wherein in step (c) the further proviso applies that the organoid (10) in the bioreactor vessel (30) is also spaced from the walls (38) of the bioreactor vessel (30) by way of the hydrogel (15).
  3. 3. The method of claim 1 or 2, wherein in step (c) the organoid (10) is introduced into the bioreactor vessel (30) together with the hydrogel (15).
  4. 4. (c1) The method of claim 1 or 2, wherein step (c) contains the substeps of: introducing some of the hydrogel to form a defined spacer layer (18) relative to the 2D cell layer (20) and optionally relative to the walls (38) of the bioreactor vessel (30), and then (c2) introducing the organoid (10) on the hydrogel spacer layer (18) formed. 15
  5. 5. The method according to any one of claims 1 to 4, wherein in the cultivation of the 2D cell layer (20) on the bottom semipermeable membrane (33) at the basal pole (24) thereof is perfused separately from the apical pole (22) thereof.
  6. 6. An in vitro tissue culture in a bioreactor vessel (30) with a semipermeable membrane (33) on the bottom, containing: - a 2D cell layer (20) containing at least a first cell type on the semipermeable membrane (33), - an organoid (10) containing cells of at least two further cell types which are arranged in a defined 3D structure relative to each other, and - a hydrogel (15) in which the organoid (10) is embedded in the bioreactor vessel (30) and which is spaced apart from the bottom 2D cell layer (20) by a defined distance.
  7. 7. The in vitro tissue culture according to claim 6, wherein the defined distance of the organoid (10) to the 2D cell layer (20) is 1 to 100 µm.
  8. 8. The in vitro tissue culture according to claim 6, wherein the defined distance of the organoid (10) to the 2D cell layer (20) is 2 to 20 µm.
  9. 9. The in vitro tissue culture according to any one of claims 6 to 8, wherein the organoid (10) is embedded in the hydrogel (15) so that said organoid is also spaced from the walls (38) of the bioreactor vessel (30).
  10. 10. The in vitro tissue culture according to one of claims 6 to 9, wherein the cell type of the 2D cell layer (20, 25) is selected from the group consisting of: - epithelial cells, - epithelial-like cells, - endothelial cells, - stromal cells containing fibrocytes and/or fibroblasts, and - muscle cells containing myoblasts, myocytes and/or muscle fibers. 16
  11. 11. The in vitro tissue culture according to one of claims 6 to 10, wherein a first 2D cell layer (20) is disposed on top of the membrane (33) facing the organoid (10).
  12. 12. The in vitro tissue culture according to claim 11, wherein a further 2D cell layer (25) is arranged on the bottom of the membrane (33) facing away from the organoid (10).
  13. 13. The in vitro tissue culture according to claim 11, wherein the first 2D cell layer (20) on the top of the membrane (33) facing the organoid (10) contains or consists of epithelial cells.
  14. 14. The in vitro tissue culture according to claim 12, wherein the first 2D cell layer (20) on the top of the membrane (33) facing the organoid (10) contains or consists of epithelial cells.
  15. 15. The in vitro tissue culture according to claim 14, wherein the further 2D cell layer (25) on the bottom of the membrane (33) facing away from the organoid (10) contains or consists of endothelial cells.
  16. 16. The in vitro tissue culture according to one of claims 6 to 15, wherein the organoid (10) is selected from the group of self-organizing multi-cell type tissues or multi-cell type tissues with defined 3D structures which can be produced by cell pressure, consisting of: retinal organoids, brain organoids, pancreatic organoids, and intestinal organoids.
  17. 17. The in vitro tissue culture according to one of claims 6 to 16, wherein the organoid (10) is a retina organoid which contains at least photoreceptor cells and cells of at least one other cell type of the neural vertebrate retina, and wherein the 2D cell layer (20) is a confluent monolayer of retinal pigment epithelial cells. 17

Description

Microphysiological organoid culture DESCRIPTION The present invention is in the field of the cultivation of biological cells and tissues with organ-like function on a microphysiological scale and provides a method for the microphysiological co-cultivation of 3D organoid tissue and at least one 2D cell 5 layer. Cell and stem cell-based in vitro models are being developed which, on the other hand, can replace ethically problematic and cost-intensive animal models in the research of genetic or idiopathic diseases of the human body and in the development of prophylactic and therapeutic agents. 10 Microphysiological (MPS) or so-called "Organ-on-a-Chip" (OoaC) systems enable the cultivation of endogenous cells such as cell lines, primary cells, cells of an embryonic origin or induced pluripotent stem cells (iPSZ) under physiological conditions in order to reconstruct specific tissues such as lungs, heart, intestines and kidneys. Complex stem cell-based organ systems from several cell types have been 15 developed, so-called organoids. These arise largely independently during in vitro differentiation and self-organizingly under the influence of fewer external signaling molecules. Examples of this are pancreatic, intestinal, brain or retinal organoids. To a certain extent, they are able to simulate physiological relationships, since they combine to form complex cell assemblies in a self-organizing manner. For example, 20 patient-specific in vitro organoid systems can be made available from a patient's own stem cells (individualized iPS cells), in particular those useful for the development of 2 individualized therapy, screening for drug effects and drug safety, or for researching the basics of diseases and physiological relationships in the organ systems. The disadvantage is that there is currently no vascular supply in the cultivation of such organoids and in particular there is no guarantee of development of the 5 organoids beyond a certain level of embryonic maturity, nor can interaction with cell types not contained in the organoid or when such cell types are in unphysiological cell orientations. Thus, in known cultivation processes, deficient supply, cell death and unphysiological conditions presumably occur due to a lack of supply and interaction, which complicates the usefulness of the findings found in vitro. 10 Particularly promising organoid systems include so-called retina organoids which are intended to make the complex interactions in the multilayered retina simulable. Retina organoids can be obtained from patient-specific iPS cells in particular, and include all cell types of the neural retina: Photoreceptors, retinal neurons and glial cells, in a complex interplay similar to an embryonic situation. The prophylaxis and 15 therapy of common and severe diseases of the retina, such as age-related macular degeneration (AMO) or retinitis pigmentosa (RP), which are the main causes of blindness in humans, are an important motivation for the development of the most physiological retinal organ systems possible. However, to model a retina that is as physiological as possible, the current retina organoid systems lack a) intercellular 20 interaction of the photoreceptors ( especially the outer segments thereof) with retinal pigment epithelial cells (RPE), b) integration of subpigment epithelial endothelial cells and vessels of the choroidea and c) a physiological extracellular matrix (EZM), especially in the interaction area of the RPE, i.e. the so-called interphotoreceptor matrix. 25 The present invention was based on the technical problem of providing methods and agents for improved cultivation of organ-like organ systems for the research and 3 development of prophylactic or therapeutic agents from patient-specific cells in particular, primarily iPS cells, which have the disadvantages mentioned of known organoid cultures, in particular incomplete maturation, cell death and overcoming a lack of cell interaction. 5 The technical problem is completely solved by a method for the co-cultivation of organoid tissue in a bioreactor vessel with a semi-permeable membrane on the bottom, in particular on a micro-physiological scale. The process contains at least the following steps: Step (a): seeding cells of at least one first cell type in the bioreactor vessel onto the membrane, step (b ): culturing these seeded cells so that a 2D cell 10 layer, in particular a confluent, supported 2D cell layer, forms on the membrane, and in particular immediately thereafter step (c): introducing the organoid, which contains cells of at least two further cell types which are arranged in a defined 3D structure relative to one another, and hydrogel into the bioreactor vessel (30) and onto the supported 2D cell layer (20), specifically with the proviso that the organoid 15 in the bioreactor vessel into which it is introduced is kept at a defined distance from the supported 2D cell layer by the hydrogel which has also been introduced previou