Construction method of neurolyme disease model based on brain organoid

Abstract

The invention relates to the technical field of biological model construction, and discloses a construction method of a neurolyme disease model based on cerebrum orgonoids, which comprises the following steps: step 1, establishment and culture of the cerebrum orgonoids: inducing pluripotent stem cells (iPSCs) to be subjected to suspension culture to form embryoid bodies (EBs), inducing neuroepithelium, performing matrigel embedding to enable the neuroepithelium to be differentiated, and obtaining the cerebrum orgonoids; finally, culturing by using an orbit determination shaking table to make the brain organoid mature; and step 2, establishment of a neurolyme disease brain organ model: a pathogen Bb (Borreria burgdorferi) related to the neurolyme disease is introduced into the brain organ, a disease infection process is simulated, and the infection dose of the Bb is 1.0 * 10 < 5 > / each organ. According to the method, the whole maturation time of the brain organoid containing the microglial cells is successfully shortened by optimizing the culture conditions of the organoid, the structure and the function of the central nervous system can be more accurately simulated, and a more reliable experimental platform is provided for subsequently constructing a disease model and mining a key regulation mechanism.

Description

Technical Field

[0001] The present invention relates to the technical field of biological model construction, and specifically relates to a method for constructing a neuroborreliosis model based on brain organoids. Background Art

[0002] Lyme disease (LD) is a multi-stage, multi-system global tick-borne zoonosis caused by some genotypes of Borrelia burgdorferi sensu lato (Bbsl). Among the 22 different named Bbsl genotypes, 10 have been found in humans. Globally, most human infections are caused by Borrelia burgdorferi afzelii (B. afzelii), Borrelia burgdorferi garinii (B. garinii), and Borrelia burgdorferi sensu stricto (Bbss). Although different Bbsl genotypes differ in pathogenic mechanisms and tissue tropisms, Bbss (hereinafter referred to as Bb) remains a key object for studying Lyme disease, especially neuroborreliosis, due to its high infection rate and wide distribution globally. Lyme disease is a multi-stage, multi-system disease with diverse clinical manifestations. It can affect the skin, heart, joints, and nervous system. When Bbsl spreads to the nervous system, causing central and peripheral nerve damage, it is called Lyme neuroborreliosis (LNB), accounting for 10-15% of global Lyme disease cases. Its pathogenesis has not been fully elucidated, and the clinical manifestations can include inflammation of the nervous system, such as symptoms like headache, facial paralysis, memory loss, depression, cognitive impairment, and motor function impairment.

[0003] For neuroborreliosis, the current guideline-recommended approach is antibiotic treatment for 14 - 21 days. Although antibiotic treatment is usually effective, a proportion of patients develop persistent symptoms after antibiotic treatment, known as Post-treatment Lyme disease syndrome (PTLDS), which accounts for 10 - 20% of LNB. In addition, there is a well-recognized phenomenon that even after completion of antibiotic treatment, some Lyme disease patients develop persistent late chronic neuropsychiatric symptoms. The etiology and pathophysiological mechanisms of these persistent symptoms are still unclear. Regarding the causes of these post-treatment symptoms, various theories have been proposed, including persistent infection, autoimmunity, inflammatory processes, and damage to Central nervous system (CNS) tissues, and these conditions may coexist. Moreover, studies have shown that a low bacterial load in the CNS can trigger LNB, and the amplification mechanism of this disease process is also unknown. LNB imposes a significant burden on the public health system and has a considerable economic impact. For example, in the United States, the direct diagnosis and treatment costs of Lyme disease reach up to $1.3 billion annually, not including productivity losses and research costs. Neuroborreliosis is the most severe form of Lyme disease and accounts for the highest proportion of economic losses, especially the productivity losses due to the disabilities it causes. Therefore, studying the pathogenesis of neuroborreliosis and the causes of post-treatment persistent symptoms has great socio-economic significance.

[0004] Since 2005, the team has been dedicated to the research of Lyme disease. Since 2017, the team has started to conduct in-depth research on neuroborreliosis. The lack of a suitable research model has always been a bottleneck for the team in the research of neuroborreliosis. Due to ethical and technical limitations, the access to original human brain tissues is very limited, making it extremely difficult to directly study the impact of Bbsl on the human brain. Although the research on many infectious diseases relies on autopsy samples, in vitro human disease models are particularly important for in-depth exploration of the interaction between pathogens and hosts. Traditionally, researchers mainly use immortalized cell lines, primary human cells, monkey brain explants or animal models to study the effects of Bbsl infection. However, these traditional models have many deficiencies. Most immortalized cell lines are derived from cancer cells and cannot reflect the biological characteristics of normal cells. Primary human cells cultured in monolayers lack the three-dimensional structure of tissues, the extracellular matrix (ECM), and the interaction with other cell types (such as immune cells and neurons), resulting in their inability to fully represent the complexity of the human brain. These drawbacks limit our understanding of the role of Bb in different neuronal cell types. Although explants can partially overcome these problems and provide research conditions closer to the in vivo environment, they still have problems such as limited replicative ability, rapid senescence (i.e., reaching the Hayflick limit), and gradual loss of genetic heterogeneity. In addition, the dependence of explants on a large amount of fresh tissue limits their application in the research of a wide range of human diseases.

[0005] Due to this difference, certain Bbsl genotypes cannot effectively infect the central nervous system of mice. For example, after inoculating C3H mice with N40 subcutaneously in the chest and back, the research team did not find any signs of peripheral and central nervous system infection in the mice. More importantly, although we have in-depth knowledge of the immune system of mice, as natural reservoirs of Bbsl, rodents often do not show disease symptoms in vivo, while human infection with the same pathogen can cause Lyme disease. This difference in natural hosts hinders the possibility of using mouse models to analyze the immune response between pathogens and hosts after Bb infection. Therefore, there is an urgent need to find a reliable alternative model to establish a neuroborreliosis model and conduct in-depth research on its pathogenesis. This will help overcome the limitations of existing models and promote the progress of neuroborreliosis research. Summary of the Invention

[0006] The present invention aims to provide a method for constructing a neuroborreliosis model based on brain organoids to solve the problem in the prior art that traditional neuroborreliosis models are difficult to simulate the complexity of the human brain, thereby making it difficult to well simulate the pathophysiological manifestations of human neuroborreliosis.

[0007] To achieve the above object, the present invention adopts the following technical solution: A method for constructing a neuroborreliosis model of brain organoids, comprising the following steps:

[0008] Step 1. Establishment and culture of brain organoids: Induced pluripotent stem cells iPSCs are cultured in suspension to form embryoid bodies EBs and induce neuroepithelium. The neuroepithelium is differentiated by embedding in Matrigel, and finally the brain organoids are matured by culturing on an orbital shaker.

[0009] Step 2. Establishment of a neuroborreliosis brain organoid model: The pathogen Bb (Borrelia burgdorferi) related to neuroborreliosis is introduced into the brain organoids to simulate the disease infection process. The infection dose of Bb is 1.0×10 5 cells / organoid.

[0010] Preferably, as an improvement, in Step 1, the formation of EBs includes the following steps:

[0011] (1) Preparation of AggreWell TM 800

[0012] Add an anti-adhesion rinsing solution to AggreWell TM 800. After centrifugation to remove air bubbles and the anti-adhesion rinsing solution, add pre-warmed culture medium.

[0013] (2) Preparation of single-cell suspension

[0014] Label and remove differentiated cells, remove the culture medium, and rinse the culture wells with sterile PBS; after removing the rinsing solution, add ACCUTASE TM , and after incubation, remove ACCUTASE TM ; Add pre-warmed seeding medium to form a single-cell suspension. After centrifugation and removal of the supernatant, resuspend the cells in EB seeding medium. After trypan blue staining, count the number of live cells under a microscope using a Neubauer counting chamber, and adjust the cell concentration to 9.0×10 5 cells / ml.

[0015] (3) Formation of EBs

[0016] Add the cell suspension to AggreWell TM 800, 2 ml / well. After centrifugation, culture under the following conditions: 37 °C, 5% CO2, 95% humidity; Transfer the formed EBs to a U-bottom ultra-low adhesion 96-well plate and continue culturing with EBs formation medium until the diameter of the EBs reaches 500 - 600 μm.

[0017] Preferably, as an improvement, in step one, neural epithelium induction includes the following steps: Transfer the EBs into a 24-well ultra-low attachment culture dish, 1-2 per well, add pre-warmed induction medium and culture. The culture conditions are: culture at 37 °C, 5% CO2, and 95% humidity for 4 days.

[0018] Preferably, as an improvement, in step one, the method for expanding neural epithelium includes the following steps:

[0019] (1) Matrigel embedding

[0020] Remove the excess medium from the induced EBs, add 15 μL per for embedding;

[0021] (2) Neural epithelium culture

[0022] Rinse the embedded EBs with expansion medium and transfer them to a 6-well plate, culture at 37 °C, 5% CO2, and 95% humidity for 3 days; When epithelial buds appear on the surface of the EBs, it is the expanded neural epithelium.

[0023] Preferably, as an improvement, in step one, the method for culturing brain organoids to maturity is: Place the embedded organoids on an orbital shaker. The culture conditions are a shaker speed of 73 rpm, 37 °C, 5% CO2, and 95% humidity. During the culture period, change the medium every other day, and each time add pre-warmed maturation medium.

[0024] Preferably, as an improvement, in step two, the infection time of Bb is 96 h, and the medium volume is 2.5 ml per well.

[0025] Preferably, as an improvement, before suspension culture of iPSCs, plating and maintenance culture are carried out.

[0026] Preferably, the method for plating iPSCs is: Take P26 iPSCs, disinfect the cell cryopreservation tube with 70% alcohol, quickly thaw the cells with sterile warm water, disinfect the cell cryopreservation tube again with alcohol, transfer the cells to a centrifuge tube, add pre-warmed medium, after centrifugation, resuspend the cells in complete medium containing 10 μM Y27632, adjust the cell colony concentration to 50 - 80 cell colonies per well, and immediately add complete medium containing.

[0027] Preferably, the method for maintaining the culture of iPSCs is: After 24 hours, change to normal complete medium equilibrated to room temperature, change the medium every other day until the cells reach 80% confluence, and make the cell colony differentiation rate not exceed 20%; Mark the differentiated parts and remove the differentiated parts to prepare for cell passage.

[0028] Preferably, as an improvement, the EBs formation medium is: STEMdiff for the first 48 hours TM CerebralOrganoid EBs formation medium, and after 48 hours, it is the above-mentioned EBs formation medium without bFGF; the neural induction medium is: STEMdiff TM Cerebral Organoid neural induction medium; the neuroepithelial expansion medium is: STEMdiff TM Cerebral Organoid neuroepithelial induction medium; the maturation medium is: STEMdiff TM CerebralOrganoid maturation medium.

[0029] The principle and advantages of this solution are as follows: In this technical solution, the inventor comprehensively optimized the existing model construction method for the ethical and simulation problems existing in the existing neuroborreliosis model: in the cultivation stage of cerebral organoids, the number of starting hiPSCs was reduced (the number of starting cells directly determines the size and germ layer distribution of EBs. After optimization to 9.0×10 5 cells / mL, the size of EBs is moderate, which is conducive to the balanced development of ectoderm and mesoderm cells), the formation time of embryoid bodies (EBs) was prolonged (more sufficient time was given to the neural induction stage to promote the formation of neuroepithelial structures and improve the subsequent stratification and differentiation quality), and the embedding time was prolonged after neural induction. In the EBs formation stage, the AggreWell800 microwell culture dish can be used to form small EBs of uniform size. After 48 hours, the EBs are transferred to a 96-well U-bottom ultra-low adhesion culture dish to further prolong the culture time. The results show that the diameter of well-developed EBs increases significantly, up to 400 - 500 μm. After neuroepithelial induction in a 24-well ultra-low adhesion culture dish, we observed that the EBs further increased in size and the edges became more transparent, indicating successful neuroepithelial induction. The induced EBs were embedded on the 13th day and dense epithelial bud formation was observed 4 days later. Compared with the organoids 4 days after embedding, the organoids 7 days after embedding showed more significant neural differentiation characteristics, with significantly enlarged neuroepithelial buds, more orderly cell arrangement, and smoother edges, indicating positive progress in the neural induction process. Compared with the original protocol, the organoids at this stage are larger in volume, and the formed epithelial buds are relatively larger in volume. Research shows that ectodermal cells are usually located on the surface of EBs, while mesodermal and endodermal cells are located inside the EBs. Reducing the number of starting cells to form smaller EBs can increase the surface area of ectodermal cells, which is helpful for the development of ectodermal cells. At the same time, the other germ layer cells inside also spontaneously divide and increase in number.

[0030] Subsequently, the inventors conducted a series of evaluations on the organoids at various stages of maturity. The stereomicroscope showed that the organoids cultured for 48 days were similar in morphology and size to those cultured for 78 days, but lacked the typical choroidal pigmentation and black floating substances of the 78-day-old organoids. In addition, the results of HE staining and immunofluorescence staining showed that at 78 days of culture, the cell differentiation and tissue structure complexity of the organoids increased significantly, especially in the radial arrangement of the cortical plate, which is typical of the mature cerebral cortex. The radial structure may represent the path of neuronal migration, a process crucial for the formation of the hierarchical structure of the cerebral cortex. Magnified observation showed that the boundaries of the ventricle-like structures in the 78-day-old organoids were clearer compared to those in the 47-day-old organoids. Staining with the neuronal-specific marker TUBB3 for the cells surrounding the ventricle-like structures showed mature tissue stratification, indicating that these stratifications are composed of multiple different types of neurons and are one of the important markers of organoid maturity. This indicates that our organoid model has a high degree of accuracy in simulating brain development and verifies the effectiveness of the optimized protocol in simulating the complexity similar to human brain development.

[0031] The results of transcriptomic comparison showed that the maturity of the organoids induced by the fine-tuned protocol had reached a level comparable to that of the cerebral cortex of infants aged 1 to 4 months after birth. In the prior art, Luo et al. (2016) found through DNA methylation sequencing that the organoids at 40 to 60 days showed epigenetic characteristics of the fetal brain at 12-16 weeks of gestation. Trevino et al. (2020) used Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and found that the organoids at 40 to 80 days of age were similar to the human fetal brain at 8-10 weeks of gestation, and to achieve cortical similarity at the postnatal stage, the organoids needed to be cultured for 350 days. Tanaka Y et al. concluded by comparing the single-cell sequencing data of existing organoids that the organoids generated by either the guided or unguided protocols were similar to the fetal cortex in the mid-late pregnancy stage when cultured for 40-80 days. These results indicate that our improved protocol significantly improves the maturity of the organoids and shortens the time for organoid maturation.

[0032] Finally, in this technical solution, immunofluorescence staining was used to specifically label astrocytes and microglia which are closely related to our research. Co-staining of neurons and astrocytes showed that astrocytes with fibrous branches surrounded the orderly arranged neurons. The results showed that in 5 randomly selected sections from the 5 submitted organoids, all organoids exhibited GFAP-positive cells showing fibrous protrusions and branched structures, and these cells surrounded the mature nerves labeled by TUBB3, indicating the differentiation and maturation of astrocytes in the organoids. The merged images further revealed the complex interactions between astrocytes and neurons. Statistical analysis showed that in the sections of all 5 organoids, the distribution of astrocytes was consistent, and in each organoid, GFAP-positive cells showed typical branched structures, indicating the degree of maturation of these cells in the organoids. Astrocytes play a crucial role in the nervous system, mainly by supporting the morphology of neurons, providing mechanical support and maintaining neuronal stability. In addition, they also ensure the health of the neural environment by removing extracellular waste and dead nerve cells. Our optimized protocol successfully reproduced this important cell type, laying the foundation for the subsequent construction of a brain organoid model of neuroborreliosis.

[0033] In the immunofluorescence staining of microglia, the IBA-1 positive cells in the images of the test results showed the typical morphology of small and round non-activated microglia, indicating that our culture protocol successfully obtained this innate immune cell of the central nervous system. By performing immunofluorescence staining on the 5 submitted organoids, we randomly selected 5 sections from each organoid for observation and found that all organoids showed IBA-1 positive microglia. This result indicates that through our culture protocol, a sufficient number of microglia can be successfully obtained in brain organoids. Compared with the situation of the Lancaster team, where the formation time of EBs was shortened to increase the number of ectodermal cells, resulting in insufficient gliogenesis, Ormel et al. promoted the natural generation of microglia by reducing the heparin content. However, our research results show that by extending the formation time of EBs and without reducing the heparin concentration, more mesodermal cells can be obtained, thus naturally generating more microglia in the subsequent induction stage.

[0034] During the technology R & D period, the inventor team tried to cultivate according to the traditional Ormel scheme: in terms of the initial cell density, a cell concentration of more than 1.5×106 cells / mL was used, which resulted in too large EBs volume, easy hypoxia necrosis in the center, irregular morphology of some organoids, and poor neural epithelium induction effect. When trying 6 days for the EBs formation time, it led to a small number of microglial cells, mostly scattered distribution, and low maturity, without showing a typical IBA-1 positive morphology. In terms of the embedding treatment, embedding on the 11th day made many organoids fail to complete the expansion of neural epithelial tissue, resulting in structural disorder and low efficiency of epithelial bud formation after embedding, affecting subsequent differentiation, and none of them could achieve the expected effect of this scheme. In addition, in actual operation, the infected organoid tissue was significantly more fragile and prone to fragmentation during the embedding process, especially easy to cause damage to tissue layers during section preparation. Based on this, the embedding solution of this scheme was changed to a gelatin-sucrose mixture to provide a more gentle support environment. The pre-cooled gelatin bottom layer solidified in advance, and then the tissue was carefully embedded on the upper layer to reduce floating and shear force. The transfer steps were reduced, and the solidification was completed within a small volume operation to ensure the tissue remained intact. The quick-freezing time and temperature gradient were strictly controlled during the freezing process to prevent the damage of ice crystals to the structure. These optimization measures significantly improved the section integrity of the infected organoids, enabling smooth immunostaining with high image quality and good repeatability.

[0035] The beneficial effects of this technical solution are as follows:

[0036] 1. Short organoid maturation time: In the traditional method, it usually takes more than 52 days to construct brain organoids containing functional microglial cells, and nearly one year to reach the cortical maturity after birth. Through systematic optimization, this scheme significantly shortens the maturation time while retaining the multi-lineage cell structure.

[0037] 2. High microglial cell generation efficiency: In the existing schemes, it is difficult for microglial cells to naturally generate in organoids or the quantity is limited. Although the Ormel scheme can induce microglial cells, it takes a long time and the maturity is inconsistent. In this study, by extending the EBs formation time while maintaining the heparin concentration, the generation of mesoderm cells is enhanced, and the number and consistency of microglial cells are increased.

[0038] 3. Good coexistence stability of glial cells and neurons: In the traditional scheme, the rapid differentiation of neurons may inhibit the development of glial cells. This scheme coordinates the proportion of cells in different germ layers by controlling the initial cell quantity, extending the induction time before embedding, etc., to obtain organoids with clearer structure and more complete cell types.

[0039] In summary, by optimizing the conditions for organoid culture, the present technical solution has successfully shortened the overall maturation time of brain organoids containing microglia. This method has laid a solid foundation for our research, enabling us to more accurately simulate the structure and function of the central nervous system and providing a more reliable experimental platform for subsequent construction of disease models and exploration of key regulatory mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a G-banded karyotype analysis chart of hiPSC in the embodiment of the present invention.

[0041] Figure 2 It is an alkaline phosphatase staining chart of hiPSCs in the embodiment of the present invention.

[0042] Figure 3 It is a qRT-PCR result chart of stem cell markers and maintenance genes SOX2, OCT4, NANOG, and C-MYC of hiPSCs.

[0043] Figure 4 It is a verification chart of the endoderm differentiation ability of hiPSCs.

[0044] Figure 5 It is a verification chart of the mesoderm differentiation ability of hiPSCs.

[0045] Figure 6 It is a verification chart of the ectoderm differentiation ability of hiPSCs.

[0046] Figure 7 It is a chart of cell aggregation to form embryoid bodies (EBs) in the embodiment of the present invention.

[0047] Figure 8 It is a chart of the morphological changes of embryoid bodies during neural induction in the embodiment of the present invention.

[0048] Figure 9 It is a chart of the morphological characteristics of brain organoids during neural differentiation.

[0049] Figure 10 It is a morphological chart of mature brain organoids in the embodiment of the present invention.

[0050] Figure 11 It is a representative HE staining image of sections of mature brain organoids at different time points.

[0051] Figure 12 It is a PCA chart of the maturity of brain organoids.

[0052] Figure 13 It is an immunofluorescence staining chart of neuron markers of brain organoids in the embodiment of the present invention.

[0053] Figure 14Immunofluorescence staining images of GFAP, a specific marker for astrocytes, and TUBB3, a neuronal marker.

[0054] Figure 15 Immunofluorescence staining image of IBA-1, a specific marker for microglia.

[0055] Figure 16 Comparison graph of cytokines at each time point after stimulating brain organoids with LPS / LTA.

[0056] Figure 17 Representative hematoxylin-eosin staining image of brain organoid sections stimulated with LPS / LTA in the examples of the present invention.

[0057] Figure 18 qPCR result graph of inflammatory marker genes and homeostasis maintenance genes of astrocytes in the examples of the present invention.

[0058] Figure 19 Representative immunofluorescence staining images of GFAP, a specific marker for astrocytes, and TUBB3, a neuronal marker, after stimulation with LPS / LTA.

[0059] Figure 20 Western Blot result graph of GFAP, a specific marker for astrocytes, after stimulation with LPS / LTA.

[0060] Figure 21 qPCR result graph of inflammatory marker genes and homeostasis maintenance genes of microglia.

[0061] Figure 22 Representative immunofluorescence staining images of IBA-1, a specific marker for microglia, and the inflammatory marker CD68 after stimulation with LPS / LTA.

[0062] Figure 23 Western Blot result graph of IBA-1 / CD68 of microglia after stimulation with LPS / LTA.

[0063] Figure 24 Graph showing the effects of different Bb infection doses and time points on the survival status of brain organoids.

[0064] Figure 25 qPCR result graph of the dynamic proliferation of Bb infection in brain organoids.

[0065] Figure 26 Comparison graph of cytokines at each time point after Bb infection of brain organoids.

[0066] Figure 27 Hematoxylin-eosin staining image of brain organoids infected with Bb in the examples of the present invention.

[0067] Figure 28 RT-qPCR results of inflammatory marker genes and homeostasis maintenance genes in astrocytes after Bb infection

[0068] Figure 29 Representative immunofluorescence staining images of GFAP, a specific marker of astrocytes, and TUBB3, a neuronal marker, after Bb infection

[0069] Figure 29 Western Blot results of GFAP, a specific marker of astrocytes, after Bb infection

[0070] Figure 30 RT-qPCR results of inflammatory marker genes and homeostasis maintenance genes in microglia

[0071] Figure 31 Representative immunofluorescence staining images of IBA-1, a specific marker of microglia, and CD68, an inflammatory marker, after Bb infection

[0072] Figure 32 Western Blot results of IBA-1 / CD68 in microglia of brain organoids after Bb infection

[0073] Figure 33 Representative co-staining images of Bb markers and other cell markers in brain organoids after Bb infection

[0074] Figure 34 Apoptosis in brain organoids after Bb infection

[0075] Figure 35 Effect of CTRX treatment on the survival status of Bb-infected organoids

[0076] Figure 36 qPCR results of the proliferation dynamics of Bb in infected brain organoids after CTRX treatment

[0077] Figure 37 Hematoxylin-eosin staining of sections of infected brain organoids after CTRX treatment

[0078] Figure 38 Bar graphs of cytokines at different time points in Bb-infected brain organoids after CTRX treatment

[0079] Figure 39 RT-qPCR results of inflammatory marker genes and homeostasis maintenance genes in astrocytes of organoids after CTRX treatment

[0080] Figure 40RT-qPCR results of inflammatory marker genes and homeostasis maintenance genes of organoid microglia after CTRX treatment.

[0081] Figure 41 Western Blot results of the neuronal marker TUBB3 in infected organoids after CTRX treatment.

[0082] Figure 42 Light microscopy results after infection with Bb at different initial cell concentrations. Detailed implementation

[0083] The following is a further detailed description through specific implementation manners, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following implementation manners are conventional means well-known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0084] Example

[0085] A method for constructing a neuroborreliosis model based on brain organoids, comprising the following steps:

[0086] Step 1. Establishment of brain organoids

[0087] 1. Comprehensive identification of the maintenance culture, genetic stability and differentiation potential of iPSCs

[0088] ① Seeding of iPSCs (human induced pluripotent stem cells, SCI3-049): Take 1 tube of P26 iPSCs, disinfect the cell cryopreservation tube with 70% alcohol, quickly thaw the cells in warm sterile water at 37°C, disinfect the cell cryopreservation tube with alcohol again, transfer the cells to a 15 ml centrifuge tube with a 2 ml pipette, add 5 - 7 ml of pre-warmed medium, and centrifuge at 300×g for 5 min at room temperature. Resuspend the cells in 1 ml of pre-warmed complete medium containing 10 μM Y27632, and adjust the cell colony concentration. Add 0.5 ml of cell suspension to each well, and ensure that there are about 50 - 80 cell colonies per well. Immediately add the complete medium containing, 2 ml / well.

[0089] ② Detection of the karyotype integrity of iPSCs: (G-banding, this experiment should be carried out before the induction of brain organoids and every 5 - 10 passages of iPSCs, and repeated 3 times each time.)

[0090] A. Cell cycle block: Observe the cells at the metaphase of mitosis under a microscope (passage 3 times, day 5 after seeding, 80% confluence), add colchicine working solution to a final concentration of 0.1 μg / ml, and continue culturing for 16 - 24 hours.

[0091] B. Cell collection: Remove the cell culture medium, rinse twice with PBS, and add ACCUTASE TM , 10 ml / 75 cm 2 , and incubate at 37 °C for 5 - 10 min. Gently pipette with a 2-ml pipette to obtain single cells. Centrifuge at 300 × g for 5 min at room temperature, discard the supernatant, and collect the precipitate.

[0092] C. Hypotonic treatment: Resuspend the cell pellet in an equal volume of 0.075 M hypotonic solution as before centrifugation and incubate for 10 - 20 min. The specific time should be based on the slight swelling but not rupture of the cells under the microscope. Centrifuge at 300 × g for 5 min at room temperature, discard the supernatant, and collect the precipitate.

[0093] D. Preparation of cell observation slides: Drop 20 - 50 μL of the cell suspension onto a clean glass slide, and use another clean slide to make a thin smear to form a uniform cell thin film. Air dry at room temperature.

[0094] E. Fixing chromosome structure: Place it in an oven preheated to 72 - 75 °C and bake for 3 hours.

[0095] F. Cell membrane treatment: Drop 0.025% trypsin solution preheated to 37 °C onto the specimen slide for cell treatment. The dosage should be sufficient to completely cover the specimen, and the time should be based on the slight swelling of the cells observed under the microscope. Gently rinse the glass slide 3 times with 1×PBS preheated to 37 °C to fully remove the trypsin.

[0096] G. Chromosome band enhancement: At room temperature, drop a chromosome band enhancer sufficient to cover the entire cell area onto the specimen slide until the cell chromosomes become clearer and the separation between chromosomes becomes more obvious under the microscope. The time ranges from a few minutes to more than ten minutes. Gently rinse the glass slide 3 times with 1×PBS to fully remove the chromosome band enhancer. The results are as Figure 1 shown.

[0097] ③ Detection of iPSCs stem cell pluripotency maintenance:

[0098] A. Detection of iPSCs pluripotent proteins (alkaline phosphatase staining): According to the reagent instructions, for the cultured cells, after removing the culture medium, wash 3 - 4 times with 1×PBS, drain the excess water, cover all the cells with ALP fixative, incubate at 37 °C for 15 min, gently wash 3 - 4 times with distilled water, and then counterstain with nuclear fast red solution for 3 min and detect the results under the microscope.

[0099] As Figure 2 shown, the cultured cell population remains undifferentiated and shows strong AP activity. Among them, Figure 2A shows the overall view of hiPSCs cell colonies under a low-magnification microscope, showing the overall cell morphology and staining pattern, where alkaline phosphatase is blue and the cell nucleus is red. Figure 2 B is a magnified detail view, showing the dense cell structure and staining intensity (scale bar: 200 μm).

[0100] Combined with the qRT-PCR results of hiPSCs stemness markers and maintenance genes ( Figure 3 ), through qRT-PCR analysis, it was determined that hiPSCs maintained the expression of key stem cell marker genes during the culture process. In particular, the mRNA levels of OCT4, SOX2, and NANOG were continuously expressed, conforming to the characteristics of undifferentiated cells. In addition, although c-MYC is not a unique stem cell marker, its expression is related to the maintenance of cell pluripotency, and its expression was also confirmed in this detection. These results provide solid molecular biological evidence for the next application of iPSCs in disease model and regenerative medicine research. It was confirmed that the cultured iPSCs have good stem cell characteristics, providing ideal cell materials for subsequent experiments. Note: The control cells (neonatal foreskin fibroblasts) did not show the expression of these genes, so they are not shown in the figure (** indicates p < 0.01).

[0101] ④ Detection of the three-lineage differentiation ability of iPSCs: This experiment should be carried out before each downstream experiment of cell culture and repeated 3 times.

[0102] Detect according to the instructions. Briefly, the steps are as follows:

[0103] A. Preparation of reagents and consumables: One hour before plating, prepare a cell culture plate coated with Matrigel and STEMdiffTM Trilineage medium, and equilibrate at room temperature.

[0104] B. Plating: Treat the cultured cells into single cells according to the requirements of passage and adjust the cell concentration. The plating density of the endoderm is 4×105 cells / well, the plating density of the mesoderm is 1×105 cells / well, and the plating density of the ectoderm is 4×105 cells / well. Inoculate the above cell concentrations into a 24-well plate, with 3 replicates for each germ layer.

[0105] C. Culture: Add 0.5 mL / well of single-cell medium. Place the culture plate in an incubator at 37 °C, 5% CO2. Quickly and briefly shake the culture plate back and forth to evenly distribute the cells. Do not move the culture plate within 24 hours.

[0106] D. Induction: Add the induction reagents for the three germ layers to the culture wells of the three planned germ layers respectively. Induce the mesoderm and endoderm until the 5th day, while induce the ectoderm until the 7th day. Fix the cell slides with 4% paraformaldehyde.

[0107] E. Evaluation of differentiation: Immunofluorescence staining was performed using markers of each germ layer. PAX6 for the ectoderm, Brachyury (T) for the mesoderm, and SOX17 for the endoderm.

[0108] The identification results of the tri-lineage differentiation ability of hiPSCs are as Figure 4 , 5 , and Figure 6 show that through cell differentiation experiments and immunofluorescence staining verification, our hiPSCs demonstrated the ability to differentiate into three major cell lineages: endoderm, mesoderm, and ectoderm.

[0109] Among them, Figure 4 shows the ability of hiPSCs to differentiate into the endoderm. The upper row shows immunofluorescence staining (red) of the endoderm-specific marker protein SOX17, co-stained with the DAPI-labeled cell nuclei (blue). The merged image (the third panel, upper row) shows the co-localization of SOX17 and the cell nuclei. The lower row is the control group, in which no SOX17 positive signal was detected. Scale bar: 50 μm. (Note: Endo represents endoderm, Ctrl represents the control group)

[0110] Figure 5 shows the ability of hiPSCs to differentiate into the mesoderm. The upper row shows immunofluorescence staining (yellow) of the mesoderm-specific marker protein Brachyury, co-stained with the DAPI-labeled cell nuclei (blue). The merged image (the third panel, upper row) shows the co-localization of Brachyury and the cell nuclei. The lower row is the control group, in which no Brachyury positive signal was detected. Scale bar: 50 μm. (Note: Meso represents mesoderm, Ctrl represents the control group)

[0111] Figure 6 shows the ability of hiPSCs to differentiate into the ectoderm. The upper row shows immunofluorescence staining (green) of the ectoderm-specific marker protein PAX6, co-stained with the DAPI-labeled cell nuclei (blue). The merged image (the third panel, upper row) shows the co-localization of PAX6 and the cell nuclei. The lower row is the control group, in which no PAX6 positive signal was detected. Scale bar: 50 μm. (Note: Ecto represents ectoderm, Ctrl represents the control group)

[0112] The above results indicate that hiPSCs possess the pluripotent differentiation potential for downstream experiments, laying a solid foundation for subsequent application research.

[0113] Step 2. Cultivation of cerebral organoids

[0114] 1. Formation of EBs (0–9 days)

[0115] (1) AggreWell TM Preparation of 800

[0116] Add 500 μL / well of anti-adhesion rinsing solution and centrifuge at 1300×g for 5 min. Observe the plate under the microscope to ensure that there are no air bubbles in the microwells. If there are still foams, centrifuge again under the same conditions and observe until the air bubbles are completely removed. After removing the anti-adhesion rinsing solution, add 2 mL / well of pre-warmed culture medium.

[0117] (2) Single-cell suspension preparation

[0118] Label and remove the differentiated cells as before, remove the culture medium, and rinse the culture wells with 1 mL of sterile PBS. After removing the rinsing solution, add ACCUTASE TM 1 mL per well, incubate at 37 °C for 5 - 10 min, and then remove ACCUTASE TM . Add 1 mL of pre-warmed seeding medium and gently pipette with a pipette to form a single-cell suspension. Centrifuge at 300×g for 5 min, remove the supernatant, resuspend the cells in 1 mL of EB seeding medium, stain with trypan blue, and count the number of live cells under the microscope using a Neubauer counting chamber. Adjust the cell concentration to 9.0×10 5 cells / mL.

[0119] (3) Formation of EBs

[0120] Add the cell suspension to AggreWell TM 800, 2 mL / well, centrifuge at 100×g for 3 min, observe under the microscope that the cells are evenly distributed in the microwells, and culture at 37 °C in an environment of 5% CO2 and 95% humidity. After 24 h, EBs with round and smooth boundaries and a diameter of 100 - 200 μm will be observed to form. At 48 h after seeding, transfer the formed EBs to a U-bottom ultra-low adhesion 96-well plate and continue to culture with EBs formation medium, 100 μL / well, until the 9th day when the diameter of the EBs reaches 500 - 600 μm (see Figure 7 ).

[0121] 2. Neural epithelium induction (9 - 13 days)

[0122] (1) Preparation of induction medium

[0123] Take an appropriate amount of induction medium and restore it to room temperature.

[0124] (2) Induction

[0125] Use an ultra-low adhesion pipette tip with the tip cut off (opening diameter about 2 mm) to transfer the EBs into a 24-well ultra-low adhesion culture dish, 1 - 2 per well, and add 500 μL / well of pre-warmed induction medium. Culture in an environment of 37 °C, 5% CO2, and 95% humidity for 4 days, and the edges of the EBs will become more transparent (see Figure 8 ).

[0126] 3. Neural epithelium expansion (13 - 15 days)

[0127] (1) Matrigel embedding

[0128] Pre - cool all supplies in a - 20°C refrigerator and thaw them on ice simultaneously hESC - Qualified Matrix (354277) for 1 - 2 hours. Place the sealing film on the 200 μL pipette tip box and press it with gloved fingers to make embedding pits. Transfer the induced EBs into the pits, remove the excess culture medium, and immediately add 15 μL per pit Use the pipette tip to adjust the EBs to the center, and place the embedded EBs in an incubator at 27°C for 30 min to promote solidification

[0129] (2) Neural epithelium culture

[0130] Rinse the embedded EBs from the sealing film into a 6 - well plate with expansion medium and culture at 37°C, 5% CO2, and 95% humidity for 3 days without changing the medium. Epithelial buds can be seen on the surface of the EBs, which are the expanded neural epithelium (see Figure 9 ).

[0131] 4. Cerebral organoid maturation (15 - 78 days)

[0132] (1) Maturation culture

[0133] Place the embedded organoids on an orbital shaker at 73 rpm, 37°C, 5% CO2, and 95% humidity. Change the medium every other day, adding pre - warmed maturation medium each time until day 78. During this period, observe the development status of the organoids under a microscope and replenish the medium in a timely manner (see Figure 10 ). A. At day 47, the shape and size similar to those at day 78 can be seen under a stereomicroscope. B. At day 78, the long - term matured cerebral organoids show clear retinal epithelial cell characteristics (black arrows). C. Several organoid structures floating in the medium can be seen with the naked eye (white arrows). Scale bar: 1 mm (A, B); 2 mm (C).

[0134] (2) Morphological evaluation of cerebral organoids includes, in addition to the above - mentioned observation under a stereomicroscope( Figure 10 ), HE staining; maturity assessment; cell labeling and differentiation characterization of cerebral organoids

[0135] Note: The medium for EBs formation is STEMdiff TM Cerebral Organoid EBs formation medium for the first 48 hours, and the above - mentioned EBs formation medium without bFGF after 48 hours; the neural induction medium is STEMdiff TMCerebral Organoid neural induction medium; neural epithelial expansion medium: STEMdiff TM Cerebral Organoid neural epithelial induction medium; maturation medium: STEMdiff TM Cerebral Organoid maturation medium. In the examples of the present invention, the composition of the medium was not changed. It was mainly the reduction in the number of starting cells, the extension of the EBs formation time, and the extension of the time for embedding the induced EBs into Matrigel.

[0136] Step 3: Establishment of the pathological model of neuroborreliosis

[0137] MC-hCOs were stimulated with lipopolysaccharide (LPS) of Gram-negative bacteria and lipoteichoic acid (LTA) of Gram-positive bacteria to evaluate their immune responses and pathological changes under the action of bacterial pathogenic substances. According to laboratory experience and actual situations, 3 infection doses were determined as the starting points of infection (1.0×10 5 cells / organoid of each type, 1.5×10 5 cells / organoid of each type, 2×10 5 cells / organoid of each type). The selection of these doses was aimed at evaluating the inflammatory responses of organoids at different infection levels and their effects on cytokine secretion to determine the optimal infection dose, so as to simulate the pathological characteristics of neuroborreliosis and provide reliable model support for in-depth study of the disease mechanism and potential treatment methods.

[0138] 1. Bb culture

[0139] Take 1 vial of frozen Bb bacteria, quickly thaw it in warm water at 37°C, add 5 - 7 ml of pre-warmed BSK-II, and culture it in an incubator at 34°C, 5% CO2, and 95% humidity for 3 days. When there are 60 - 120 Bb per high-power microscopic field, transfer the above culture solution into a 50 ml centrifuge tube, add fresh BSK-II to 45 ml, and culture it for another 7 days under the same culture conditions. Count the bacteria with a Neubauer counting chamber under a high-power microscope and observe their viability to ensure that the bacteria are in the late logarithmic phase.

[0140] 2. Determination of Bb infection dose

[0141] Through the lactate dehydrogenase (LDH) release experiment and the analysis of the organoid survival curve, it was determined that when each organoid was infected with 1.0×105 Bb (treatment time 96 h, medium volume 2.5 ml / well), this infection dose showed good effectiveness and applicability, providing a reliable infection dose basis for the subsequent construction of the neuroborreliosis model.

[0142] 3. Comprehensive evaluation of the neuroborreliosis brain organoid model

[0143] The evaluation includes: the infection efficiency of Bb and its proliferation dynamics in brain organoids, the cytokine secretion dynamics (cytokine suspension array), histopathological changes (HE staining), pathological changes at the molecular level (qPCR of inflammatory markers, immunofluorescence staining and WB), the localization of Bb in brain organoids and its interaction with other cells, the response to antibiotic treatment and the evaluation of the model reliability.

[0144] Experimental examples

[0145] I. Morphological evaluation of brain organoids

[0146] (1) HE staining of brain organoid sections

[0147] To evaluate whether the internal structure of brain organoids has reached the same level of complexity as reported, we observed the HE staining of organoid sections at different time points (according to the operating steps in the HE staining kit instructions of Solarbio Life Science Co., Ltd.). The results are as Figure 11 shown. On the 47th day of culture ( Figure 11 A), the organoids showed initial organizational structures. The cells formed obvious clusters in a rose petal-like arrangement, and ventricular-like cavities appeared in the center of these clusters, indicating that nerve cells had begun to differentiate along specific developmental paths, and the formation of the ventricular-like structure predicted the gradual establishment of the spatial complexity of the organoids. By the 78th day of culture ( Figure 11 B), the maturity of the organoids had been significantly improved. Five randomly selected sections from each organoid showed consistent structural features, including obvious ventricular-like structures and mature nerve cell layer distributions. The nerve cells surrounding these ventricular-like structures showed mature tissue stratification, further verifying that the organoids mimicked the complexity similar to human brain development. Scale bar: 200 μm (upper panels of A and B); 50 μm (lower panels of A and B).

[0148] II. Evaluation of brain organoid maturity

[0149] The specific method steps are as follows:

[0150] ① RNA extraction

[0151] For brain organoid samples, compared with other samples, 1 mL of Trizol needs to be added after grinding in liquid nitrogen, and an additional 10 min at room temperature is required to improve the RNA extraction efficiency. The remaining steps are the same as the total RNA extraction of the existing technology, and 3 biological replicates are set for each experimental group.

[0152] ② Submission of RNA for inspection (entrusted to Guangzhou Gene Denovo Biotechnology Co., Ltd.)

[0153] ③ Comparison of transcriptome data

[0154] ④ Data Download and Processing

[0155] The GSE168408 dataset was downloaded from the Gene Expression Omnibus (GEO) database of NCBI. This dataset includes 27 single-cell RNA sequencing (snRNA-seq) samples, which cover various developmental stages of the human prefrontal cortex from fetal (22 weeks of gestation) to adulthood.

[0156] To conduct in-depth analysis of the transcriptome data regarding the degree of brain development and maturity in the model, we cited the transcriptome data related to human brain development provided by the PsychENCODE database. Data in two formats, 'Gene expression in counts' and 'Gene expression in RPKM', were downloaded. (These data provide information on gene expression levels at various developmental stages of the brain from fetal to adult, providing a valuable benchmark for our research.

[0157] ⑤ Data Transformation and Normalization

[0158] (1) To compare single-cell data with transcriptome data, we used the pseudobulk method of the muscat software package in R to transform the data. A key assumption of this method is that aggregating single-cell data of the same type or state can effectively represent the overall characteristics of the cell population.

[0159] ⑥ The reads per kilobase per million mapped reads (RPKM) value of each transcript was calculated using the TxDb.Hsapiens.UCSC.hg19.knownGene library, and the data was normalized to obtain the log2 value after RPKM normalization.

[0160] ⑦ Data Comparison

[0161] We compared these normalized data with the gene data related to human brain development provided by the latest version of the PsychENCODE database. Through this comparison, we identified the genes related to human brain development in the GSE168408 dataset.

[0162] Intersection analysis was performed on these genes in the dataset and the transcriptome data of brain organoids cultured for 78 days. In this way, we identified co-expressed genes.

[0163] ⑧ Principal Component Analysis (PCA)

[0164] Principal component analysis (PCA) was performed using the Prcomp package in R, which included z-score normalization of the gene expression data.

[0165] ⑨Data visualization

[0166] Preliminary PCA plots were generated in R and then further visually adjusted in Photoshop to optimize the presentation of the charts.

[0167] The transcriptome data of brain organoids at 47 days and 78 days were compared with the identified 14,879 common developmentally regulated genes, and principal component analysis (PCA) was performed on the results. The analysis results showed that among the 6 comparison samples, 3 brain organoids at 78 days exhibited a maturity comparable to that of the cerebral cortex at 1 to 4 months ( Figure 12 as shown by the green circles in the middle). Although there were certain differences in maturity among the samples, these differences were within the expected acceptable range. In contrast, the maturity of the brain organoids at 47 days varied greatly ( Figure 12 as shown by the gray circles in the middle). These results provided important biological basis and time point reference for the subsequent establishment of the infection model. Based on these analyses, we decided to select the brain organoids cultured for 78 days for the subsequent experiments to ensure the stability of the model.

[0168] III. Cell labeling and differentiation characterization of brain organoids

[0169] To evaluate whether the cultured organoids had cells closely related to the experimental purpose and their maturity, immunofluorescence staining was performed on neurons, astrocytes, and microglia.

[0170] ①Staining of the mature neuron-specific marker TUBB3

[0171] To preliminarily evaluate the neuronal network development and basic structural maturity of brain organoids, we first performed TUBB3 staining to reveal the overall location of the distribution of neurons. Microtubule-associated protein beta 3 (TUBB3), which is highly expressed in the microtubules of mature neurons, is commonly used as a neuron-specific marker. Figure 13The significant structural stratification of the organoids on the 78th day is shown. The TUBB3 staining reveals the layered arrangement of nerve cells, and these layered structures foreshadow the characteristics of the mature cortex, such as the formation of the cortical plate and the distinct ventriculoid structure. This structural feature was observed consistently in 5 randomly selected sections stained from the 5 submitted organoids, indicating that all the organoids exhibited this structural feature, reflecting their maturity and high mimicry of nervous system development. TUBB3 (green) stains neurons, and DAPI (blue) stains cell nuclei. On the 78th day, the left image shows the TUBB3 staining, and the right merged image shows more distinct neuronal layered structures (see brackets i, ii) and more mature ventriculoid structures (white dotted lines). Scale bar: 20 μm.

[0172] ② Immunostaining for the astrocyte-specific marker GFAP and the neuronal marker TUBB3

[0173] To investigate whether the brain organoids we cultured could mimic the interaction between astrocytes and neurons, we performed double immunostaining for astrocytes and neurons. We used the astrocyte-specific marker, glial fibrillary acidic protein (GFAP), and the neuronal marker TUBB3. Through this double staining, we aimed to evaluate the distribution and maturity of astrocytes. The results showed that in 5 randomly selected sections from the 5 submitted organoids, all the organoids exhibited GFAP-positive cells showing fibrous protrusions and branched structures, and these cells surrounded the mature nerves labeled by TUBB3, indicating the differentiation and maturity of astrocytes in the organoids. The merged image further revealed the complex interaction between astrocytes and neurons. This interaction indicates that the cultured brain organoids are mimicking the development of the mature brain in terms of organizational structure and functional complexity ( Figure 14 ). In the figure, GFAP (red) stains astrocytes, TUBB3 (green) stains neurons, and DAPI (blue) stains cell nuclei. The merged image shows that the TUBB3-positive cells are arranged in layers and surrounded by GFAP-positive fibrous and branched cells, revealing the complex interaction between astrocytes and neurons (the third panel). Scale bar: 20 μm.

[0174] ③ Immunostaining for the microglia-specific marker IBA-1

[0175] To evaluate whether our brain organoid model can mimic key cells in the immune response, we performed immunofluorescent staining of the microglia-specific marker, ionized calcium-binding adaptor molecule 1 (IBA-1), on serial sections. Microglia are the main immune cells in the normal brain, responsible for maintaining the stability of the neural environment and responding to inflammation, while interacting closely with neurons and other glial cells. As shown in the figure, the presence of IBA-1 positive cells in the brain organoids cultured for 78 days indicates that our organoid model indeed contains cell types capable of mimicking the immune response. Specifically, out of 100 randomly selected sections from 5 submitted organoids for staining, IBA-1 positive cells were found in 20 sections of 4 organoids, further supporting the potential of our organoid model to mimic the immune response( Figure 15 ). In the figure, IBA-1 (green) labels microglia, and DAPI (blue) labels the cell nuclei. The merged image (second panel) shows the presence of IBA-1 positive cells in the model, with their small and round morphology. Scale bar: 20 μm.

[0176] IV. LPS / LTA Stimulation Experiment

[0177] Three doses of 0.01 μg / mL, 0.05 μg / mL, and 0.1 μg / mL were set for LPS (lipopolysaccharide from Gram-negative bacteria); three doses of 1 μg / mL, 5 μg / mL, and 10 μg / mL were set for LTA (lipoteichoic acid from Gram-positive bacteria);

[0178] Table 1 Grouping Scheme for MC-hCOs Survival Experiment after LPS / LTA Stimulation

[0179]

[0180] The main evaluation indicators include:

[0181] ① Survival status of organoids: Analyze the survival rate of organoids under different treatment conditions and its time dynamics through the LDH release experiment;

[0182] The results are as Figure 16As shown, LPS / LTA stimulation significantly increased the expression of inflammatory factors in the organoids. As shown in the figure, a large number of cytokines were significantly upregulated at the early time points under LPS / LTA stimulation, and the number of elevated cytokines gradually decreased over time. Among them, IL-1β and IL-1ra were continuously and significantly upregulated at all time points, suggesting that they played a key role in the inflammatory response of brain organoids induced by LPS / LTA. The ELISA test results were consistent with those of the suspension chip. Among them, A. The Venn diagram shows the overlap and differences of cytokines significantly elevated at each time point after LPS stimulation screened by R software analysis. B. The Venn diagram shows the overlap and differences of cytokines significantly elevated at each time point after LTA stimulation screened by R software analysis. C. The bar chart shows the cytokines that continued to increase after ELISA verification of LPS / LTA stimulation. (Note: The cytokines in the Venn diagram satisfy Log2 FC>2, P<0.05; *P<0.05, **P<0.01)

[0183] According to the results of the cytokine chip, we adjusted the time points of the later experiments and selected the time points when the cytokine secretion was relatively high after 48 hours for section staining of the organoids. The selection of this time point was aimed at capturing the most typical pathological changes as the control baseline.

[0184] The experimental results are as Figure 17 shown. In the sections of 5 organoids submitted for inspection, 5 randomly selected sections from each organoid were stained with HE, and after LPS / LTA stimulation, characteristic cell death morphologies such as typical volume reduction, nuclear condensation, and nuclear fragmentation were observed in the brain organoids compared with the control group (red arrows in the lower row, LPS and LTA groups). Scale bar: 200 μm in the upper row, 50 μm in the lower row. This result indicates that under the action of pathogenic stimulants, the organoids underwent a significant cell death process.

[0185] ② Dynamics of cytokine secretion: Use a cytokine suspension chip to detect the secretion levels of key inflammatory factors and their changing trends over time;

[0186] The results are as Figure 18 shown: In the organoids after LPS / LTA stimulation, the expression of genes related to astrocyte inflammation and homeostasis maintenance was significantly upregulated compared with the control group. Among them, A. After LPS stimulation, the expression of astrocyte inflammation marker genes and homeostasis maintenance genes was significantly increased. B. After LTA stimulation, the expression of the above genes was also significantly increased. (Note: *P<0.05, **P<0.01)

[0187] ③ Histopathological changes: The cell morphology and tissue structure of the organoids were observed by HE staining; immunofluorescence staining showed that in the infected brain organoids, astrocytes exhibited morphological features of increased cell volume and more pseudopodia ( Figure 19 ). GFAP (red) labels astrocytes, TUBB3 (green) labels neurons, and DAPI (blue) labels cell nuclei. The merged images showed that compared with the control group, LPS / LTA stimulation caused obvious morphological changes in astrocytes (significantly increased cell volume and formed more pseudopodia). Unactivated astrocytes (yellow arrows), activated astrocytes (white arrows). Scale bar: 20 μm.

[0188] ④ Pathological changes at the molecular level: Quantitative real-time PCR (qPCR) was used to quantitatively analyze the expression levels of inflammatory marker genes, immunofluorescence staining was combined to localize inflammation-related proteins, and Western blot (WB) was further used to quantitatively analyze their expression levels.

[0189] The results are as Figure 20 shown: The WB experiment showed an increase in the expression of GFAP protein in the brain organoids after stimulation. A. Expression of GFAP protein in brain organoids. B. Statistical analysis of GFAP expression levels. Control group (gray), LPS group (pink), LTA group (green). Compared with the control group, the expression of GFAP was significantly upregulated in the LPS group and LTA group. (Note: *P < 0.05, **P < 0.01).

[0190] The qPCR experiment showed that in the organoids after LPS / LTA stimulation, the expression of microglia-related inflammatory genes and homeostasis maintenance genes was significantly increased compared with the control group ( Figure 21 ). The merged images showed that compared with the control group, LPS stimulation significantly caused an increase in microglia cell volume and aggregation, while after LTA stimulation, it mainly showed an increase in the volume of scattered microglia ( Figure 22 , activation status of microglia in brain organoids after LPS / LTA stimulation. IBA-1 (red) labels microglia, CD68 (green) labels activated microglia, and DAPI (blue) labels cell nuclei. The merged images showed that compared with the control group, LPS stimulation significantly caused an increase in microglia cell volume and aggregation, while after LTA stimulation, it mainly showed an increase in the volume of scattered microglia. Unactivated microglia (white arrows in the first row), activated microglia (white arrows in the second and third rows). Scale bar: 20 μm.). In 5 organoids submitted for inspection, 20 randomly selected sections from each organoid were subjected to immunofluorescence staining, and the results showed that each stimulated organoid exhibited activation and aggregation of microglia. Western Blot analysis further confirmed the upregulation of the microglia markers IBA-1 / CD68Figure 23 , A. Expression of IBA-1 and CD68 proteins in brain organoids. B. Statistical analysis of IBA-1 and CD68 expression levels. Control group (gray), LPS group (pink), LTA group (green). Compared with the control group, the expression of IBA-1 and CD68 was significantly upregulated in the LPS group and the LTA group. (Note: *P<0.05, **P<0.01).). Combining these results, it indicates that LPS / LTA stimulation significantly activates microglia in organoids.

[0191] V. Comprehensive evaluation of the brain organoid model of neuroborreliosis

[0192] A. Evaluation of Bb infection efficiency and proliferation dynamics in MC-hCOs using qPCR: The aim was to evaluate the infection efficiency of Bb on brain organoids and its proliferation within the organoids over time. For this purpose, we designed the following experimental protocol: The infection dose determined in the previous step was used to infect 10 organoids, and the same number of control groups were set. The absolute copy number of Bb in the infected organoids was detected by quantitative PCR (qPCR) to achieve the purpose of evaluating the infection efficiency and proliferation dynamics. Considering the accuracy and reliability of the detection, the flagellin gene B (abbreviated as flaB), which is stably expressed by Bb during the infection process, was selected as the target gene for qPCR analysis, so as to effectively evaluate its proliferation dynamics and infection efficiency in the environment of infected brain organoids.

[0193] B. Inflammatory evaluation of the MC-hCOs simulation model of neuroborreliosis: Cytokine suspension array, histopathological changes (HE staining), expression of inflammatory markers and homeostasis genes in astrocytes and microglia, immunofluorescence staining of inflammatory markers in astrocytes and microglia, WB detection of inflammatory markers in astrocytes and microglia, localization of Bb in brain organoids and its interaction with other cells.

[0194] The results are shown in Figures 24 - 31, and the results indicate that:

[0195] ① When the organoids were infected with 1.0×105 Bb, this infection dose showed good effectiveness and applicability ( Figure 24 ).

[0196] ② The qPCR results showed that in all the detected organoids (10 / 10), infection occurred, and the infection efficiency reached 100%. At the early stage of co-culture (4 hours), Bb could invade the organoids, and the initial bacterial load was approximately 125 per organoid. After 48 hours, the bacterial load increased to approximately 250 per organoid ( Figure 25 ).

[0197] ③Bb infection induced changes in the expression of multiple inflammatory factors in brain organoids. As shown in the figure, at the initial stage of infection, only a small number of cytokines were significantly upregulated. As the infection time prolonged, the expression levels of more cytokines gradually increased. Among them, IL-6, IL-8, and SCGF-β (stem cell growth factor-β) were continuously and significantly upregulated at all time points, indicating that they played a key role in the organoid inflammatory response induced by Bb. In addition, CXCL10, one of the cytokines significantly elevated in the cerebrospinal fluid of patients with neuroborreliosis, was detected at 4 hours post-infection and was significantly upregulated at subsequent time points ( Figure 26 , A. Venn diagram shows the overlap and differences among the cytokines significantly upregulated at each time point of Bb infection after screening by R software analysis. B. Bar chart shows the cytokines that were continuously and significantly upregulated after infection verified by ELISA. C. Bar chart shows the cytokines that were continuously and significantly upregulated after 12 hours verified by ELISA.).

[0198] ④ Organoid section staining was performed 48 hours later to capture the high-level cytokine secretion induced by Bb infection and the typical pathological changes consistent with it. In the sections of 5 organoids submitted for examination, after HE staining of 5 randomly selected sections from each organoid, cell death was significantly observed in the organoid sections after Bb infection. This significant increase in cell death indicates that live Bb had a significant cytotoxic effect on brain organoids during the infection process ( Figure 27 , compared with the control group, cells in the Bb infection group showed significant morphological changes, mainly including characteristics such as cell volume reduction and nuclear condensation, and these changes suggested that Bb infection induced cell death (bottom row, indicated by the red arrow). Scale bar: 200 μm in the top row, 50 μm in the bottom row.).

[0199] ⑤ The results of qPCR experiments showed that the expression of genes related to astrocyte inflammation and homeostasis maintenance in organoids after Bb infection was significantly upregulated compared with the control group ( Figure 28 ). In the 5 organoids submitted for examination, the same pathological changes were shown in 5 randomly selected sections, indicating that this morphological feature consistently existed in all organoids. Western Blot analysis further verified the increased expression of the astrocyte inflammation marker GFAP ( Figure 29 , 30 ). Among them, Figure 29Shows the distribution and status of neurons and astrocytes in brain organoids after Bb infection. GFAP (red) labels astrocytes, TUBB3 (green) labels neurons, and DAPI (blue) labels cell nuclei. The merged image shows that compared with the control group, Bb infection significantly increased the volume of astrocytes and promoted the formation of more pseudopodia. Unactivated astrocytes (upper row, white arrow), activated astrocytes (lower row, white arrow). Scale bar: 20 μm. Figure 30 A. Expression of GFAP protein in brain organoids. B. Statistical analysis of GFAP expression levels. Control group (gray), Bb group (pink). Compared with the control group, the expression of GFAP in the Bb group was significantly upregulated. (Note: *P<0.05, **P<0.01).

[0200] ⑥ qPCR experiments showed that in organoids after Bb infection, the expression of microglia-related inflammatory genes and homeostasis-maintaining genes was significantly increased compared with the control group ( Figure 31 ). Immunofluorescence staining results showed that microglia in infected organoids presented morphological changes of increased volume, and activated glial cells could be seen aggregating into clusters ( Figure 32 ). Western Blot analysis further confirmed the upregulation of the microglia markers IBA-1 / CD68 ( Figure 33 ). Collectively, these results indicate that Bb infection significantly activates microglia in organoids.

[0201] ⑦ Through immunofluorescence co-staining, we observed the interaction between Bb and neurons and astrocytes in brain organoids. Figure 34 Shows that there is obvious overlap between the fluorescence signal of Bb and the signals of neurons and astrocytes (upper row: Bb (white), astrocytes (red), neurons (green), cell nuclei (blue). The merged image shows that the fluorescence signal of Bb overlaps with that of astrocytes (white arrow). Lower row: The fluorescence signal of Bb overlaps with that of neurons (white arrow), and the surrounding astrocyte signals increase, while the fluorescence signal of neurons weakens, showing a scattered arrangement). Specifically, among the 5 organoids submitted for inspection, we randomly selected 30 sections for co-staining of Bb with neurons and astrocytes. The statistical results showed that in each organoid, there were 5 sections with overlapping fluorescence signals of Bb with neurons or astrocytes. Among all the sections, a total of 100 overlapping signals were found, of which 79 overlapped with neurons and the remaining 21 overlapped with astrocytes.

[0202] These results indicate that the overlapping signal of Bb with neurons is significantly higher than that with astrocytes, suggesting that Bb may directly act on neurons, affect their functional microenvironment, and thus trigger neuronal damage and disorders in the neural network structure. In the interaction with astrocytes, the enhanced signal of astrocytes around Bb indicates that these glial cells may be activated during infection and play a role in the local inflammatory response. These activated astrocytes may help clear Bb or cell debris after neuronal death.

[0203] ⑧Bb infection leads to neuronal damage in brain organoids, such as Figure 35 shown, Bb infection results in an increase in apoptotic cells in brain organoids.

[0204] VI. Treatment Tests

[0205] (1) Antibiotic selection: To evaluate the effect of treatment intervention on the pathological changes caused by Bb infection in brain organoids, explore the role of antibiotics on Bb invading organoids after treatment, and further analyze the potential significance of these results in persistent Lyme disease, we designed the following experimental protocol.

[0206] (2) Toxicity assessment of ceftriaxone sodium on the brain organoid model

[0207] To further evaluate the potential toxicity of ceftriaxone sodium in the brain organoid model, this study referred to the data of Agger et al., who pointed out that for the Borrelia burgdorferi B31 strain, the minimum inhibitory concentration (MIC) of ceftriaxone sodium is 0.02 μg / mL, and the minimum bactericidal concentration (MBLC) is 0.06 μg / mL. Based on this, we set up three experimental groups: low-dose group: 0.01 μg / mL (lower than MIC), medium-dose group: 0.02 μg / mL (equal to MIC), high-dose group: 0.12 μg / mL (twice the MBLC). The treatment time was extended to 96 hours to more fully explore the long-term effects at different doses. After the experiment, the potential toxicity of ceftriaxone sodium was evaluated by the LDH release experiment.

[0208] Table 2 Grouping scheme for the potential toxicity assessment of ceftriaxone sodium on brain organoids

[0209]

[0210] (3) Ceftriaxone sodium treatment protocol

[0211] Based on the previous toxicity assessment experiment of ceftriaxone sodium in the brain organoid model, we determined that 0.01 μg / mL - 0.12 μg / mL is safe for brain organoids, without causing obvious cell death or damage to the 3D structure of brain organoids. Considering the limited resources and following the principle of the minimum dose of antibiotic treatment in clinical simulation, in this experiment, a dose higher than the medium dose, i.e., 2 times the MIC (0.04 μg / mL), was selected to treat the Bb-infected organoids. Previous comprehensive evaluations showed that Bb can invade the brain organoid parenchyma 4 hours after infection, but does not multiply significantly within 48 hours. At the same time, literature reports indicate that it takes 72 hours for ceftriaxone to completely kill Bb in vitro. Combining these pieces of information, we decided to add Bb and ceftriaxone to the organoids simultaneously at the early stage of establishing the infection model and treat them for 96 hours to better simulate the situation of early clinical intervention.

[0212] Table 3 Grouping scheme for ceftriaxone treatment in infected brain organoids

[0213]

[0214] (4) Experimental content: Comprehensive evaluation of pathological changes in the model after ceftriaxone sodium treatment

[0215] ① Effect of treatment on the survival rate of the model - Survival curve: To evaluate the effect of antibiotic treatment on the survival status of MC-hCOs after infection, an untreated PBS control group and an antibiotic treatment group were set up in the experiment to visually show the effect of antibiotic treatment and time on the survival status of MC-hCOs.

[0216] Table 4 Grouping scheme for the survival curve of MC-hCOs after ceftriaxone treatment

[0217]

[0218] The survival curve is as Figure 36 shown, the results of the LDH release experiment of Bb-infected organoids under the selected CTRX dose treatment. The results show that antibiotic treatment significantly increased the survival rate of infected organoids.

[0219] ② Quantitative analysis of the effect of treatment on the dynamic changes of Bb invading MC-hCOs: By qPCR method, quantitative analysis of the dynamic changes of Bb load in samples at each sampling time point after treatment was carried out. At the end point of treatment at 96 hours, it was observed that all Bb in the supernatant lost their motility, indicating that Bb had completely died. Subsequently, the treated organoids were minced and placed in BSKII medium for Bb culture to comprehensively evaluate the effect of CTRX treatment on Bb invading the organoids. The specific steps of qPCR are the same as above.

[0220] The results are asFigure 37 As shown, CTRX treatment significantly reduced the Bb load in Bb-infected brain organoids.

[0221] ③ Effect of treatment on morphological changes (HE staining of sections), and the results are as Figure 38 shown. Dead cells still existed in the brain organoids after CTRX treatment. This indicates that although the treatment effectively controlled the progression of Bb infection, the damaged cells in the organoids were not completely cleared.

[0222] ④ Effect of treatment on cytokine secretion in infected MC-hCOs: The secretion of these cytokines in the supernatant after treatment was detected by ELISA, and the operation was strictly carried out according to the instructions of the Dayou kit to evaluate the effect of treatment on their secretion levels.

[0223] The results are as Figure 39 shown. After CTRX treatment, the secretion levels of IL-6, IL-8, SCGF-β, and CXCL10 in the brain organoids were significantly reduced. However, compared with the control group, the secretion levels of IL-6, IL-8, and SCGF-β were still significantly higher than those of the control group at all time points, and the secretion amount of CXCL10 at 12 hours was also significantly different from that of the control group.

[0224] ⑤ Effect of treatment on the activation of glial cells in infected MC-hCOs: The qPCR detection method for inflammation-related genes and homeostasis genes and the WB procedure for inflammation markers were the same as above.

[0225] As Figure 40 shown, CTRX treatment significantly reduced the expression of inflammation-related genes in astrocytes.

[0226] ⑥ Effect of treatment on neuron death in infected MC-hCOs: The effect of CTRX treatment on infected organoids on neurons was detected by WB experiment.

[0227] As Figure 41 shown, compared with the control group, in the infected brain organoids of the CTRX treatment group, the expression of inflammation marker genes in microglia was significantly reduced, while the expression of homeostasis maintenance genes was significantly increased. However, the expression of inflammation genes in some microglia still showed significant differences. (Note: *P<0.05, **P<0.01)

[0228] As Figure 42 shown, the results of Western Blot experiment showed that although CTRX treatment significantly controlled the infection process of Bb, the protein expression of neuron markers in the infected organoids was still significantly lower than that of the control group. This result indicates that although Bb was effectively killed, the Bb that had invaded the organoids still caused neuron damage.

[0229] AsFigure 43 As shown, it is the influence of different initial cell concentrations on the infection tolerance of brain organoids. It can be seen from the figure that after the formation of mature organoids, the organoids with fewer initial cells show stronger tolerance to infection compared to those with more initial cells.

[0230] In summary, this study successfully constructed an MC-hCOs model that highly mimics the key pathological changes of neuroborreliosis, providing a feasible alternative for the study of neuroborreliosis. This model can effectively reproduce important pathological features such as the immune response, glial cell activation, and neuronal damage in the central nervous system of neuroborreliosis, and has high biological relevance. Although the current model lacks the participation of peripheral immune cells, which limits its application in simulating the systemic immune response, this model avoids the complex ethical issues of the rhesus monkey model and shows great potential as a research platform for neuroborreliosis. Future research can further optimize this model by introducing peripheral immune cells to enhance its role in simulating the complete immune response and pathological process, thereby providing a more valuable tool for the mechanism research and clinical application of neuroborreliosis. Through the treatment tests and drug screening conducted with this model, the efficacy and safety of drugs can be evaluated more effectively, accelerating the research and treatment progress of neuroborreliosis.

[0231] The above are only the embodiments of the present invention, and common general knowledge such as specific technical solutions and / or characteristics known in the solution are not described in detail here. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can still be made, and these should also be regarded as the protection scope of the present invention, which will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a neurological Lyme disease model based on brain organoids, characterized in that: The steps include: Step 1: Establishment and culture of brain organoids: iPSCs are cultured in suspension to form embryoid bodies (EBs) and induce neural epithelium, which are then embedded in matrix gel to differentiate into neural epithelium. Finally, the brain organoids are cultured in an orbital shaker to mature. Step 2: Establishment of a brain organoid model for neurological Lyme disease: Introduce the pathogen Bb associated with neurological Lyme disease into the brain organoids to simulate the disease infection process. The infection dose of Bb is 1.0×10 5 per organoid.

2. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 1, characterized in that: In step 1, the formation of EBs includes the following steps: (1) AggreWell TM 800 Preparation AggreWell TM Add anti-adhesion flushing solution to 800, centrifuge to remove bubbles and anti-adhesion flushing solution, and then add preheated culture medium; (2) Preparation of single cell suspension Mark and remove differentiated cells, remove the culture medium, and rinse the culture wells with sterile PBS; After removing the rinse solution, add ACCUTASE TM After incubation, remove ACCUTASE TM ; Add pre-warmed inoculation medium to form a single cell suspension, centrifuge and remove the supernatant, then resuspend the cells in EB inoculation medium, stain with trypan blue, and count the number of live cells under a Neubauer counting microscope, and adjust the cell concentration to 9.0×10 5 Pieces / ml; (3) Formation of EBs Add the cell suspension to AggreWell TM 800, 2 ml / well, culture after centrifugation, the culture conditions are: 37°C, 5% CO2, 95% humidity; the formed EBs were transferred to a U-bottom ultra-low viscosity 96-well plate, and the EBs formation culture medium was continued to be cultured until the EBs diameter reached 500-600μm.

3. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 2, characterized in that: In step 1, neuroepithelial induction includes the following steps: EBs are transferred into a 24-well ultra-low-adhesion culture dish, 1-2 per well, and pre-warmed induction medium is added for culture. The culture conditions are: 37°C, 5% CO2, and 95% humidity for 4 days.

4. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 3, characterized in that: In step 1, the method of neuroepithelial expansion includes the following steps: (1) Matrigel embedding Remove excess culture medium from induced EBs and add 15 μL / each Carry out embedding; (2) Neuroepithelial culture The embedded EBs were rinsed with expansion medium into a 6-well plate and cultured at 37°C, 5% CO2, 95% humidity for 3 days; epithelial buds appeared on the surface of the EBs, which were the expanded neuroepithelium.

5. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 4, characterized in that: In step 1, the method for maturation culture of brain organoids is as follows: the embedded organoids are placed on an orbital shaker, and the culture conditions are a shaker speed of 73 rpm, 37°C, 5% CO2, and 95% humidity. The medium is changed every other day during the culture period, and pre-warmed maturation culture medium is added each time.

6. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 5, characterized in that: In step 2, the infection time of Bb was 96 h, and the culture medium volume was 2.5 ml / well.

7. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 6, characterized in that: In step 1, iPSCs are plated and maintained in culture before suspension culture.

8. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 7, characterized in that: The method for plating iPSCs is as follows: take P26 iPSCs, disinfect the cell cryopreservation tubes with 70% alcohol, thaw the cells quickly with sterile warm water, disinfect the cell cryopreservation tubes with alcohol again, transfer the cells to centrifuge tubes, add pre-warmed culture medium, resuspend the cells in complete culture medium containing 10uM Y27632 after centrifugation, adjust the cell colony concentration to 50-80 cell colonies per well, and immediately add complete culture medium containing.

9. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 8, characterized in that: The method for maintaining iPSCs culture is: after 24 hours, change to ordinary complete culture medium equilibrated to room temperature, change the medium every other day until the cells reach 80% fusion and the cell colony differentiation rate does not exceed 20%; mark the differentiated part and remove the differentiated part to prepare for cell passaging.

10. The method for constructing a neurological Lyme disease model based on brain organoids according to claim 9, characterized in that: EBs formation medium: STEMdiff for the first 48 hours TM Cerebral Organoid EBs formation medium, 48 hours later the above EBs formation medium without bFGF; neural induction medium: STEMdiff TM Cerebral Organoid Neural Induction Medium; Neuroepithelial Expansion Medium: STEMdiff TM Cerebral Organoid Neuroepithelial Induction Medium; Maturation Medium: STEMdiff TM Cerebral Organoid Maturation Medium.

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