Maturation and industrial scale production of brain organoids

Abstract

The invention relates to brain organoids whose functional maturation and production can be completed on an industrial scale in the RCCS-microgravity bioreactors and their use as an in vitro three-dimensional brain tissue model.

Description

[0001] MATURATION AND INDUSTRIAL SCALE PRODUCTION OF BRAIN

[0002] ORGANOIDS

[0003] Technical Field of the Invention

[0004] The invention relates to brain organoids, whose functional-molecular maturation is completed on an industrial scale in RCCS (Rotary Cell Culture System)-microgravity bioreactors with bioengineering-tissue engineering techniques, and their use in the pharmaceutical industry as an in vitro three-dimensional (3D) brain tissue model that can be used with high output and safety for preclinical drug screening tests.

[0005] State of the Art of the Invention (Prior Art)

[0006] Although two-dimensional (2D) cell culture systems realized on typical tissue culture plates, where roughly dose-dependent trials are performed for in vitro imaging and drug screening administrations, are used extensively in basic studies due to their low cost and high control, they cannot adequately mimic the healthy living phenotype because of the disadvantages associated with the inability to reach the complexity of in vivo cell and tissue organization, tissue-specific architectural, mechanical and biochemical cues, and loss of cell-cell and cell- extracellular matrix (ECM) interactions. Three-dimensional (3D) cell culture technology, which provides more suitable conditions for in vivo tissue physiology, anatomy and structure, manipulates genetic and microenvironment factors independently by imitating ECM in normal tissue, thus reducing the use of experimental animals, is rapidly evolving with microfabrication techniques, bioengineering and tissue engineering approaches following recent advances in cell biology discovery. 3D cell culture techniques, which have their own advantages and disadvantages, can be examined in general with (i) tissue scaffolds that can be produced in a hydrogel, film, sphere, porous or nanofiber form, (ii) bioprinting products, (iii) organ-on-a-chip systems produced by microfluidic techniques, (iv) spheroids that can be produced in low adhesion plate, hanging drop plate, spinner bioreactors and micro-nano patterned surfaces, and also (v) organoids derived from tissue or stem cells.

[0007] Primarily in brain organoid formation process, in vitro induced pluripotent stem cells (iPSCs) develop into aggregates termed “Embryoid Bodies (EBs)”.EBs differentiate into 3 germ layers including endoderm, ectoderm, and mesoderm. Then, after the transfer of EBs to the minimal environment, the ectoderm (outermost layer) acquires more neuronal features and then differentiates into the neuroepithelium, which is the source of neural progenitors. One of the key points here is to place the suspended 3D post-EB aggregates on a specialized tissue scaffold, such as the matrigel matrix that mimics ECM components, and to promote the formation of apicobasally polarized neuroepithelial buds by supporting the self-organization of the neuroepithelium. The second critical point is to provide dynamic agitation of brain organoids, which start to be produced under static conditions, as floating cultures, using devices such as conventional spinner bioreactor or orbital shaker after this stage. These conditions promote the delivery of oxygen and nutrients to the tissue while promoting neuronal self-organization. It was noted that this promotes the formation of larger brain organoids with well-defined fluid-filled cavites resembling ventricles.

[0008] In addition to its wide range of uses and advantages such as mimics specific brain regions (cerebral cortex, cerebellum, midbrain, forebrain, hypothalamus, hippocampus, and even the medial ganglionic region, a critical ventral brain area that produces cortical interneurons) with the addition of different situmulation factors, containing different cell types, performing specific functions and providing long-term testing with unchanged phenotype and karyotype, there are some problems and difficulties of brain organoid formation protocols with existing dynamic systems where similar biomechanical forces cannot be fully achieved in vivo-like functionality. These challenges are listed as the lack of natural microenvironment of stromal, muscle, blood vessel endothelium and immune cells such as microglia, the necessity of a long-term and strenuous culture process for full neuronal-structural development, the insufficiency of electrophysiological functionality, the size problems due to the presence of dead zones caused by the inadequacy of nutrient-oxygen transition to the inner layers (they can be produced in a maximum size of 4-5 mm), bath-to-batch variability due to very limited controllability on biochemical and biophysical factors in 3D dynamic culture, low reproducibility, harvestability, standardization and industrial scale-up rate. Even so, they can be solved by bioengineering approaches that require the use of different biomaterials and bioreactor types.

[0009] In the whole brain organoid formation process, conventional agitation systems such as spinner flask or orbital shaker are still more commonly used today due to their easier access, for the maturation stage after the generation stage, which is carried out in static culture for approximately 15 days. However, these systems still have shortcomings, such as the lack of a natural microenvironment of stromal cells, muscle, blood vessel endothelium and immune cells such as microglia, the necessity of a long-term and strenuous culture process for full neuronal -structural development, the insufficiency of electrophysiological functionality, the size problems due to the presence of dead zones caused by the inadequacy of nutrient-oxygen transition to the inner layers (they can be produced in maximum size of 4-5 mm), variations on batch-to-batch and inter-batch production due to very limited controllability on biochemical and biophysical factors in 3D dynamic culture, low harvestability, standardization and industrial scale-up rate, due to the inability to fully provide in vivo similar biomechanical forces.

[0010] Miniaturized spinning bioreactors have been developed on a platform with a design that will improve the dynamic system for maturation of various region-specific brain organoids, reduce inter-batch variability and consider scale-up process, for use in modeling various diseases and high throughput drug screening. In addition, effective bioreactor systems named as Vertical- Wheel bioreactors (VWBRs) have been developed, in which whole organoid formation is carried out after cell inoculatin on a single platform, which reduces the risk of contamination, without requiring ECM matrix embedding stage where the controlled flow can be achieved. However, this type of small-scale bioreactor systems are still not well characterized, uncontrolled and perfused are not compatible and dead space-aggregation formation can be seen in the agitator sub-zones. In addition, there have been deficiencies in terms of full functional-structural maturation, effective neuronal development, ideally enough shear stress, survival, and organoid quality.

[0011] In order to provide better neuronal development in brain organoids, to show higher structural- functional maturation, to show various brain layers, to provide better electrophysiological functionality, to maintain longer-term viability by oxygen-nutrient transition in the inner regions, and to ensure reproducible, standardized and reliable production by reducing batch- to-batch variability, there is also an innovative microfluidic platform system for organoid maturation operated on rocker shaker, which is stated to support in vzvo-like cerebrospinal and interstitial bi-directional controlled dynamic fluid flow conditions in the brain. For similar purposes, there is a study of brain organoid maturation performed on milifluidic platforms with millivolume wells connected by tubing and parallel-series connections. And, one-stop microfluidic and microcapillary systems, in which all generation and maturation processes, including EB formation, are carried out on a single platform, have also been studied. But the fabrication, sterilization, optimization and microcontrol of these platforms, and also validation of the ideal culture are both time-consuming and strenuous. In addition, these systems still have unresolved problems, such as scale-up, harvestability and ease of use, which are the most important criteria for their use by pharmaceutical companies that perform high throughput drug screening on an industrial scale.

[0012] Brief Summary and Objectives of the Invention

[0013] The invention relates to the realization of the ideal maturation process of 3D brain organoids with high harvestibility rates on an industrial scale in RCCS-microgravity bioreactors developed by NASA-Synthecon Inc.®.

[0014] Within the scope of the invention, in addition to conventional methods, RCCS-microgravity bioreactors that provide controlled dynamic flow conditions by mimicking laminar fluid flow in the brain will be able to support the production of brain organoids with ideal cerebral ventricular spaces-different layers and cellular content, which can survive in larger sizes and for a long time by supporting growth factor diffusion and metabolite exchange, whose molecular-functional-electrophysiological-structural maturation has been completed, which are highly harvestable, up-scalable, reproducible, fully standardized and reduced batch-to- batch variability. With this invention, it will be possible to produce brain organoid models that can be used with high safety and output in preclinical potential drug screening tests by industrial pharmaceutical companies. In the RCCS-microgravity bioreactor, more efficient mass transfer is realized between the organoid surface and its center with the laminar flow regime and low shear stress that occurs under controlled dynamic conditions, nutrient and metabolic waste gradients that more closely mimic the natural growth environment are formed and different cell phenotypes are correctly directed during differentiation. In addition, cells are induced mechanotransductively by providing idealy enough shear stress with the laminar fluid flow. This provides in vzvo-like structural development and transcriptional outputs.

[0015] Thus, simultaneously, many potential drug molecules will be able to be screened at different concentrations, in repeated tests, on functionally very similar brain organoids. The discovery of new drug molecules and the possible effects of therapeutics will be able to be examined in a multifaceted way.

[0016] These brain organoids, whose maturation has been completed in the RCCS system, will be able to be used as a useful in vitro model in preclinical trials for the entire healthcare- pharmaceutical industry working on neurodegenerative, neurodevelopmental and neurotoxic diseases.

[0017] In addition to whole brain organoid maturation, it will also be able to be used for the maturation of region-specific brain organoids, with various inhibitor-activator factors preferably added to the culture medium content at the generation stage.

[0018] In addition to the formation of brain organoids whose maturation will be completed in the RCCS system after the organoid generation stage in static culture, brain organoid formation in continuous culture will be possible with culture medium changes to be made only at certain times as a one-stop process, directly after s eeding of iPSCs in the form of single cells to the bioreactor system, without changing the culture plate, without depending on ECM matrix embedding processes, and by reducing hand manipulation and contamination risk.

[0019] The RCCS -microgravity bioreactor culture, validated for the maturation of 3D brain organoids, will be able to be used not only for the maturation of brain organoids, but also for other iPSC-induced organoid maturation such as kidney, intestine, lung and heart, and the target model and patient group will be able to be increased.

[0020] Definitions of Drawings Illustrating the Invention

[0021] The figures and related explanations required for a better understanding of the subject of the invention are as follows.

[0022] Figure 1: A) Brain organoid generation and maturation process in the RCCS-STLV (Rotary Cell Culture System- Slow Turning Lateral Vessel) bioreactor, B) macroscopic-microscopic- H&E staining images, C) RCCS system COMSOL simulation outputs, D) time-dependent organoid size analysis, E) CD45+ / CDl lb+ double positive microglia cell sorting from RCCS organoids, F) TUNEL / Immunofluorescence staining images, G) qRT-PCR analysis heat map results (original study data).

[0023] Detailed Description of the Invention

[0024] Rotary Cell Culture System (RCCS)

[0025] The main equipment of the RCCS-microgravity bioreactor developed by NASA-Synthecon Inc.® are horizontally rotating culture plates (STLV-Slow Turning Lateral Vessel) full-filled with growth medium and equipped with a membrane that allows proper gas exchange. In the RCCS bioreactor system, cells / tissues are cultured in a high aspect ratio cylindrical vessel completely filled with medium. The vessel rotates horizontally, causing the medium inside to rotate as well. If it is operating within a certain speed range, after a while the medium will rotate at the same speed as the vessel itself, unlike in other bioreactor systems where the liquid moves according to the culture plate walls. This minimizes the effect of earth's gravity on the particles within the culture plate, resulting in an effective gravitational force of IO-2x g. Furthermore, the particles in the vessel have a nearly zero terminal velocity and therefore move with the medium on the axis of rotation, with limited motion alongside other axes, a cell returns to approximately the same position with each full culture plate rotation. Therefore, cells can be cultured in suspension in a laminar regime with minimal shear stress (<5x l0-2Pa), and shear stress can be further reduced due to the absence of headspace in the culture plate and thus no air bubbles. Achieving both of these effects requires the RCCS to rotate within a limited speed range, and a low rotational speed causes cells to settle because they cannot overcome the earth's gravitational force, while high rotational speed results in a dominant centrifugal force that will push the cells against the outer wall. For this reason, ideal speed optimization is important.

[0026] The rotational speed can be adjusted via an external motor to maintain the continuous falling motion of the samples, similar to the microgravity environment.

[0027] Thus, unlike conventional dynamic systems that exhibit inhomogeneous shear stress, a controlled laminar flow state with reduced shear stress can be created within the RCCS bioreactor, long-term precise control of many parameters such as increased oxygen and nutrient delivery, natural flow conditions, reduction of cell damage with non-damaging shear stress and cell-cell / cell-environment interactions can be achieved with increased mass transfer, and it allows the generation and continuity of homogeneous organoids that can mimic the relevant organ architecture by minimizing the variations that may occur between productions.

[0028] In this context, brain organoid maturation carried out in the RCCS bioreactor, as included in the invention, supports the production of brain organoids with ideal cerebral ventricular spaces-different layers and cellular content, which can survive in larger sizes and for a long time by supporting growth factor diffusion and metabolite exchange, whose molecular- functional -electrophysiological -structural maturation has been completed, which are highly harvestable, up-scalable, reproducible, fully standardized and reduced batch-to-batch variability.

[0029] RCCS-microgravity bioreactors provide controlled dynamic flow conditions by mimicking laminar fluid flow in the brain. It allows high harvestability and reproducibility of the maturated brain (cerebral) organoids by exhibiting homogeneous hemodynamic forces that facilitate long-term precise control of cell-cell interactions. It supports increased mass transfer and reduced cell damage with reduced shear stress. With these advantages, the RCCS system, compared to other systems, can support the production of brain organoids with ideal cerebral ventricular spaces-different layers and cellular content, which can survive in larger sizes and for a long time by supporting growth factor diffusion and metabolite exchange, whose molecular-functional-electrophysiological-structural maturation has been completed, which are highly harvestable, up-scalable, reproducible, fully standardized and reduced batch-to- batch variability.

[0030] In the prototype study carried out in the RCCS-STLVs system with a working volume of 55 mL, 100 brain organoids subjected to molecular-functional-structural maturation could be harvested using IL cerebral organoid differentiation medium at the end of 120 days with a 95% harvestability rate. When scaled up to a working volume of 500 mL in one batch, it is predicted that 1000 organoids can be harvested without any batch-to-batch and inter-batch variation.

[0031] Brain organoid generation step in the static phase (Figure 1A)

[0032] Lancaster et al., 2013 technique was used for early-stage brain organoid generation in static culture lasting approximately 15 days. In this respect,

[0033] Embryoid body (EB) formation and germ layer differentiation: Until differentiation is initiated, the human-derived iPSCs were cultured in matrigel-coated 6-well plates in mTESRl medium. For single cell suspension, after iPSC colonies were washed with PBS without Ca+2and Mg+2, they were incubated with 1 ml of “gentle cell dissociation reagent” for 6 minutes, or with 0.5 mM EDTA for 4 minutes at 37°C, pipetted homogeneously by adding 3 ml of DMEM F12, and the cells were collected in the falcon tube by allowing them to rise from the surface. After centrifugation at 500 rpm for 5 minutes, the pellet was homogenized in 2 ml of hESC medium [For 100 mL preparation: 80 ml DMEM F12, 20 mL KOSR, 3 ml FBS, 1 mL Glutamax, 1 mL NEAA, 0.7 pL BME, and finally 100 p Tocris (50 pM from 50 mM stock) and 4 plL bFGF (4 ng / mL from 100 pg / mL stock) were mixed] and cell count was performed in a hemocytometer. Then, iPSCs prepared as a single-cell suspension were seeded into U- bottom 96-well ultra-low adhesion plates (U-bottom 96-well ULA plate) in hESC medium at 9000 cells / well / 150 pL. Without damaging the EBs formed in the plate wells, the hESC medium was changed by half every other day and observed microscopically for about 5-6 days until they started to brighten and their diameters became greater than 350-400 gm (they are sharply bounded smooth structures).

[0034] Induction of neuroepithelial structure: When the EBs have smooth borders and reach a size of approximately 500-600 pm, they were carefully transferred into 24-well ultra-low adhesion plates in an intact state with 200 pL truncated pipette tips (maximum 4 EBs were transferred into each well to avoid aggregate formation), they were fed with 500 pL of neural induction medium (DMEM-F12 nutrient medium containing 1% N2 supplement, 1% Glutamax, 1% MEM-NEAA, 1% Penicillin / Streptomycin and 1 pg / mL Heparin). EBs were monitored for approximately 4-5 days with a medium change every other day until they formed externally brighter structures indicating neuroectodermal differentiation (radially organized, optically translucent neuroectoderm structures are present).

[0035] Transfer of neuroepithelial tissue into matrigel drops: In order to form 4x4 matrigel drops, parafilms cut in sizes to fit in a 60 mm petri dish and a few pairs of gloves were subjected to UV sterilization in the cabinet, and microconcave wells were created by pressing 200 pL pipette tip box pores by hand on the parafilm surface. Then, a neuroepithelial tissue was carefully transferred into each microconcave structure with a 1000 pL truncated pipette tip, excess liquid was drawn and 25 pL of matrigel liquefied for 1-2 hours at +4 °C was added onto them. A 10 pL pipette tip was quickly used to position the tissues in the center of the matrigel drops and to remove air bubbles. After waiting for about half an hour at 37 °C for the matrigel drops to polymerize, the solidified drops were transferred into 6-well suspended culture plates or 60 mm petri dishes by flowing with cerebral organoid differentiation medium that does not contain vitamin A [For 100 mL preparation: 50 ml DMEM-F12, 50 mL Neurobasal medium, 0.5 mL N2 supplement, 25 pL Insulin, 1 mL Glutamax supplement, 0.5 ml MEM-NEAA, 1 mL penicillin-streptomycin, 35 pL 2-Mercaptoethanol (prepared at 1 : 100 dilution in DMEM-F12) and 1 mL B27 supplement w / o vit A], The embedded tissues were cultured for 3-4 days until they began to form many more enlarged neuroepithelial buds containing fluid-filled spaces, with a medium change every other day. The maturation of cerebral organoid structures, which began to develop with this process, was maintained in a cerebral organoid differentiation medium containing vitamin A in dynamic culture as well as in static culture (there are fluid-filled cystic structures without well-developed neuroepithelium). It has been observed that the cerebral organoids developed up to this stage are microscopically and macroscopically similar to the literature. Brain organoid maturation in the RCCS-Microgravity bioreactor (Figures 1B,1C)

[0036] About 100 cerebral organoids developed at an early stage were added to a 55 mL RCCS- STLVs vessel for a dynamic microgravity-guided maturation stage. The entire volume of the culture plate was adjusted to be completely filled with cerebral organoid differentiation medium containing vitamin A, and all air bubbles were removed using syringes. STLVs were run at an initial rpm of 10 RPM (slightly increased as the organoids grew to avoid precipitation) in the RCCS-1 system in an incubator at 37°C and 5% CO2. The speed was increased by 1 RPM as needed to prevent precipitation of the organoids, the maturation medium (around3 / 4 volume) was changed once a week, air bubbles were removed as needed during the culture period, and the culture was maintained for 120 days. In the preferred applications of the invention, the medium can be changed in the range of 70%-90%. When it is less, the nutrient becomes insufficient, a renewal is required during the week and this causes additional costs. More is not recommended, and growth factors-cytokines in the old medium remaining in small amounts have a positive effect on the culture. Computational Fluid Dynamics (CFD) simulations were performed to determine the shear stress of RCCS under laminar flow conditions using COMSOL Multiphysics.

[0037] Characterization of brain organoids (Figures ID, IE, IF, 1G)

[0038] Samples of the matured brain organoids were taken on certain days (30, 60 120), macroscopic-microscopic-Hematoxylin&Eosin staining images were obtained, harvestability, reproducibility and organoid size analysis were made, and their cellular components, regionspecific layers, molecular-functional maturation and survivability were characterized by immunohistology, qRT-PCR, FACS, TUNEL and apoptosis tests in terms of MAP2, NEUN, CD 11b, GFAP, SI 00b, CD31, MBP, OLIG2, SOX1, SOX2, PAX6, TUJ1, NCAD, FOXG1, TBR1, TBR2, PROXI, CTIP2, SATB2, PSD95, NESTIN, KROX20, TTR, VGLUT1 and Ki67 genes-proteins.

[0039] In the flow simulation, a very low maximum shear stress of approximately 2.32xl0'4Pa was calculated with laminar flow at the tried 15 rpm flow rate. It was determined that the brain organoids matured with a 95% harvestability had well-distinguished flat-round shapes and the ventricular structures were quite homogeneous and high in size, and at the end of the 120th day, harvest was carried out with an average size of 2.87 mm and with a maximum size of 3.60 mm, resulting in a very good yield. At the end of the TUNEL test, organoids rich in maturation markers with cellular components and different layers were producedwith almost no apoptotic region found inside the organoid.

[0040] In the prototype study carried out in an RCCS-STLV bioreactor with a working volume of 55 mL, 100 iPSC-derived brain organoids which have reached maturity in terms of molecular- functional -structural aspects could be harvested using IL cerebral organoid differentiation medium after 120 days with a 95% harvestability. Characterization of organoid maturation was supported by flow modeling, immunofluorescence, qRT-PCR, TUNEL, apoptosis and western blot analyses. When scaled up to a working volume of 500 mL in one batch, it is predicted that 1000 organoids can be harvested without any batch-to-batch or inter-batch variation.

[0041] Thus, simultaneously, many potential drug molecules will be able to be screened at different concentrations, in repeated tests, on functionally very similar brain organoids. The discovery of new drug molecules and the possible effects of therapeutics will be able to be examined in a multifaceted way.

[0042] These brain organoids, whose maturation has been completed in the RCCS system, will be able to be used as a useful in vitro model in preclinical trials for the entire healthcarepharmaceutical industry working on neurodegenerative, neurodevelopmental and neurotoxic diseases.

[0043] In addition to whole brain organoid maturation, it will also be able to be used for the maturation of region-specific brain organoids, with various inhibitor-activator factors preferably added to the culture medium content at the generation stage.

Claims

CLAIMS1. A brain organoid maturation method, characterized in that it comprises inhibitor-free maturation of early-stage brain organoids developed from human-derived induced pluripotent stem cells (iPSC) in static culture, in a rotary cell culture system (RCCS- STLV bioreactor).

2. A maturation method according to Claim 1, characterized in that it comprises the following process steps: i. carrying out the simulation of shear stress and flow regime in culture for the organoid maturation process, ii. adding the early-stage generated brain organoid to the RCCS-STLV culture vessel, iii. adjusting the entire volume of the culture plate to be completely filled with cerebral organoid differentiation medium containing vitamin A and eliminating air bubbles, iv. running STLVs on the RCCS system at an initial RPM of 10 RPM and increasing to 15 RPM as the organoids grow, v. changing the culture medium and removing air bubbles as needed during the culture period, vi. maintaining the culture for 120 days.

3. A maturation method according to Claim 2, characterized in that the brain organoid added to the culture vessel in step ii is generated at an early-stage, approximately on the 15th day of static culture.

4. A maturation method according to Claim 2, characterized in that STLVs are run in an incubator at 37°C and 5% CO2 in step iv.

5. A maturation method according to Claim 2, characterized in that the culture medium is changed in the range of 70%-90% by volume and once a week in step v.

6. A maturation method according to Claim 2, characterized in that it comprises the step of increasing the current RPM by 1 unit as a process of optimizing the ideal rotational speed to prevent the precipitation of organoids between steps iv and vi and to provide laminar flow regime with controlled shear stress on the organoids.

7. A maturation method according to Claim 2, characterized in that it comprises the process steps of characterizing the produced brain organoids by immunohistology, qRT-PCR, FACS and TUNEL, apoptosis test analyses.A mature brain organoid produced by the method according to Claims 1-7. A mature brain organoid according to Claim 8 for use as an in vitro three-dimensional brain model. A mature brain organoid according to Claim 8 for use in high throughput drug screening tests for neurological activity, neurotoxicity, neurodegenerative and neurodevelopmental diseases and / or neuroinflammation.

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