Preparation method and application of three-dimensional spleen cell spheroids

Abstract

The present application belongs to the field of cell culture and tissue engineering, and particularly relates to a preparation method and application of three-dimensional spleen cell spheroids. The preparation method of the three-dimensional spleen cell spheroids comprises ultra-low adhesion agarose well plate preparation, primary spleen cell separation, self-assembly into spheroids without matrix glue, and microtissue steady-state maintenance culture. The preparation method has extremely low raw material cost, excellent experimental repeatability, and no exogenous matrix background interference, and is suitable for high-throughput screening of active pharmaceutical ingredients, analysis of immune antioxidant molecular mechanisms, and in-vitro drug immunotoxicity evaluation.

Description

Technical Field

[0001] This invention belongs to the field of cell culture and tissue engineering, specifically relating to a method for preparing three-dimensional spleen cell spheres and their application. Background Technology

[0002] The spleen is the largest peripheral immune organ in the body, containing a complete subset of immune cells, including T lymphocytes, B lymphocytes, macrophages, and dendritic cells. It plays a central role in the body's immune response, inflammation regulation, and oxidative stress defense. Therefore, primary mixed cells derived from the spleen have long been an important in vitro model for evaluating the immunomodulatory, antioxidant, and anti-inflammatory activities of drugs.

[0003] Currently, traditional spleen cell research commonly employs two-dimensional plate adherent culture. However, in two-dimensional culture systems, cells are discretely distributed, lacking three-dimensional intercellular contact and a paracrine immune microenvironment. This makes it difficult to realistically simulate the three-dimensional microenvironment of spleen immune cells in vivo, resulting in weak oxidative stress pathway responses, low inflammatory response capacity, and poor correlation with actual in vivo conditions. This severely restricts the accurate evaluation of drug activity based on spleen cells.

[0004] To overcome the inherent limitations of two-dimensional culture, researchers have attempted to construct three-dimensional spleen cell spheroid models to more realistically simulate the immune microenvironment of the spleen in vivo. However, current three-dimensional spleen cell construction techniques heavily rely on commercially available imported ultra-low adsorption plates or matrix gel coating systems. Regarding commercially available ultra-low adsorption culture plates, these products are expensive, significantly increasing experimental costs for large-scale cell spheroid culture; furthermore, the coating varies greatly between different production batches, making them unsuitable for large-scale drug screening. As for matrix gel coating systems, animal-derived basement membrane matrices, represented by Matrigel, are currently the most commonly used scaffold materials in three-dimensional cell culture and organoid construction. However, these natural matrices contain various unknown cytokines and exhibit significant batch-to-batch variability, severely interfering with the precise mechanism evaluation of active ingredients in traditional Chinese medicine, resulting in high background noise and poor data reproducibility, posing serious challenges to experimental reproducibility and result reliability. Simultaneously, the high cost of matrix gels themselves further increases the economic burden of experiments. Chinese patent application CN110106136A discloses the preparation and application of an ultra-low adhesion coating solution. The solution is prepared by mixing agarose and dimethyl sulfoxide (DMSO), achieving a stable and non-toxic ultra-low adhesion effect through precise control of concentration and temperature. However, this method essentially still involves physical / chemical modification of the culture plate surface, only addressing the issue of preventing cell adhesion, and DMSO has potential toxicity to cells. Most existing three-dimensional spleen culture protocols employ techniques such as encapsulating spleen cells in a semi-solid basement membrane matrix or embedding them in matrix gel followed by the addition of exogenous cytokines to induce three-dimensional structure formation. These methods all rely on exogenous biological matrices or artificial scaffold materials as support for spheroidization, rather than being a purely self-assembly system driven by the cells' natural adhesion properties. Chinese patent application CN122484042A discloses a method based on GelMA... A paper fiber scaffold-based co-culture system for tumor organoids and immune cells utilizes GelMA, which has a bilayer heterogeneous structure. A paper-fiber composite scaffold, with a GelMA layer to simulate the vascular barrier and a paper fiber layer to support deep infiltration of immune cells, was successfully constructed in vitro using a time-controlled co-culture strategy. This involved allowing tumor organoids to self-assemble before introducing immune cells, ultimately resulting in a mature tumor immune microenvironment system. However, this method is cumbersome in its preparation process and the components are unrelated to the spleen's natural microenvironment, failing to realistically simulate the spontaneous aggregation of spleen immune cells in vivo. This also limits the model's application in exploring cell self-organization behavior and related signaling pathway regulatory mechanisms.

[0005] Therefore, there is an urgent need to develop a low-cost method for preparing three-dimensional spleen cell sphere microtissues that is completely free of matrix gel and exogenous scaffolds and based on the natural self-assembly characteristics of cells, in order to meet the urgent technical needs in the fields of traditional Chinese medicine immunomodulation, anti-oxidation and Nrf2 pathway mechanism research. Summary of the Invention

[0006] To address one of the aforementioned technical problems in the prior art, in a first aspect, the present invention provides a method for preparing three-dimensional spleen cell spheres, comprising the following steps: (1) Preparation of ultra-low adhesion agarose well plates: Cell culture grade low melting point agarose is dissolved in serum-free culture medium, heated to dissolve and sterilized, added to cell culture container, allowed to stand and solidify to form ultra-low adhesion gel bottom layer; after ultraviolet sterilization, it is soaked and rinsed with culture medium containing 10% fetal bovine serum to balance osmotic pressure and obtain ultra-low adhesion agarose well plates. (2) Isolation of primary spleen cells: mouse spleen tissue was taken and cut into pieces, and then digested gently with digestive juice. After filtration, centrifugation and erythrocyte lysis, primary spleen cells were obtained. (3) Matrix-free self-assembly into spheres: The primary spleen cells obtained in step (2) were resuspended in complete culture medium containing 10% fetal bovine serum and seeded into the ultra-low adhesion agarose wells prepared in step (1). After static culture, the cells spontaneously aggregated to form three-dimensional spleen cell spheres. (4) Micro-tissue homeostasis maintenance culture: After the three-dimensional spleen cell spheres are formed, half of the medium is changed regularly to maintain the culture and obtain three-dimensional spleen cell sphere micro-tissue with uniform structure and stable immune function.

[0007] In some embodiments, in step (1), the mass-volume ratio of the low-melting-point agarose is 1.0% to 2.0%, preferably 1.5%.

[0008] In some embodiments, in step (1), the culture medium comprises a basal culture medium, an antimicrobial agent, and a culture medium supplement.

[0009] In some embodiments, the basal culture medium is selected from RPMI-1640, DMEM, DMEM / F12 or AdvancedDMEM / F12 medium, preferably RPMI-1640.

[0010] In some embodiments, in step (1), the soaking and rinsing time of the culture medium is 40-80 min, preferably 60 min, to displace the internal osmotic pressure of the gel and avoid osmotic pressure damage to the primary cells.

[0011] In some embodiments, in step (2), the digestive fluid contains one or more of the following: collagenase, calcium chloride, zinc chloride, DNase, neutral protease, fetal bovine serum, Liberase enzyme, and antibiotics.

[0012] In some embodiments, the digestive solution is a mixed digestive solution of collagenase IV and DNase I prepared with calcium magnesium HBSS.

[0013] In some embodiments, the concentration of collagenase IV in the digestive fluid is 50-200 U / mL, and the concentration of DNase I is 10-40 U / mL. Preferably, the concentration of collagenase IV is 75-125 U / mL, and the concentration of DNase I is 15-25 U / mL. More preferably, the concentration of collagenase IV is 100 U / mL, and the concentration of DNase I is 20 U / mL.

[0014] In some embodiments, in step (2), the mild digestion conditions are digestion at 37°C for 10 to 30 minutes, preferably 20 minutes.

[0015] In some embodiments, in step (2), the red blood cell lysis is performed using ammonium chloride-potassium (ACK) buffer as the lysis buffer, and the lysis time is 2 to 8 minutes, preferably 3 minutes.

[0016] In some embodiments, in step (3), the primary spleen cells are seeded at a density of 5 × 10⁶ cells per well. 4 ~2×10 5 Cells, preferably 1×10 per well 5 Each cell.

[0017] In some implementations, in step (4), after culturing for 48 h, the cells spontaneously assemble to form dense spherical micro-tissues, and half of the medium is replaced every 48 h. The cell culture period of 2 to 5 days is the functional stability window of the model.

[0018] In some embodiments, the method for preparing the three-dimensional spleen cell spheroid microtissue includes the following steps: (1) Preparation of ultra-low adhesion agarose well plates: Take cell culture grade low melting point agarose and prepare a 1.5% agarose solution with serum-free RPMI 1640 medium as solvent. Autoclave at 115 ℃ for 30 min and keep warm at 50 ℃. Add 50 μL of agarose solution to each well of a sterile U-shaped bottom 96-well plate, let it solidify at room temperature, sterilize with ultraviolet light, and then soak and wash with RPMI 1640 complete medium containing 10% fetal bovine serum to balance the osmotic pressure to obtain ultra-low adhesion culture well plates. (2) Isolation of primary mouse spleen cells: The mouse spleen was aseptically removed and cut into pieces. The digestive solution containing collagenase IV and DNase I was prepared using calcium magnesium HBSS buffer for gentle enzymatic digestion. After filtration, red blood cells were lysed with ammonium chloride-potassium (ACK) buffer, centrifuged and washed to obtain highly active primary mixed spleen immune cells. (3) Matrix-free self-assembly into spheres: The purified spleen cells were resuspended in RPMI 1640 complete medium containing 10% fetal bovine serum, and the cell suspension concentration was adjusted to 1×10⁻⁶. 6Cells / mL, seeded with 100 μL of cell suspension per well, i.e., 1 × 10⁶ cells / mL per well. 5 One spleen cell was placed in a cell culture incubator for static culture. (4) Micro-tissue homeostasis maintenance culture: After 48 hours of culture, the cells spontaneously assemble to form dense spherical micro-tissues. Half of the medium is replaced every 48 hours using a wide-mouth pipette tip. No matrix gel or exogenous scaffold material is added throughout the process. Three-dimensional spleen cell spherical micro-tissues with uniform structure and stable immune function are obtained during the culture period.

[0019] In a second aspect, the present invention provides a three-dimensional spleen cell sphere microstructure obtained by the preparation method of the three-dimensional spleen cell spheres described in the first aspect.

[0020] Thirdly, the present invention provides the application of the three-dimensional spleen cell spheroid microtissue described in the second aspect in drug antioxidant activity screening, drug immunomodulatory activity evaluation, drug anti-inflammatory activity screening, and the mechanism of action of the spleen immune microenvironment in vitro.

[0021] Its application in the establishment of oxidative stress models and drug screening, including screening for drugs with antioxidant activity, anti-inflammatory activity, and immunotherapy drugs. In some implementations, the application includes the construction of an oxidative stress model.

[0022] In some implementations, the application includes Nrf2 pathway detection.

[0023] In some implementations, the application includes NF-κB pathway detection.

[0024] This invention utilizes cell culture-grade low-melting-point agarose combined with serum-free RPMI 1640 medium to prepare an inert, ultra-low adhesion substrate, which is then used to modify a conventional U-shaped 96-well plate. No matrix gel, artificial scaffolds, or exogenous extracellular matrix are added throughout the process. The model achieves spontaneous self-assembly into spheroids by relying on the natural intercellular adhesion properties of primary mixed mouse spleen cells. This invention overcomes the technical bottlenecks of existing three-dimensional spleen cell spheroid models, which rely on imported commercially available ultra-low adsorption culture plates, have high matrix gel costs, suffer from complex matrix compositions and large batch-to-batch variations, and are susceptible to interference from exogenous biological matrices in the screening of traditional Chinese medicine efficacy and pathway mechanism detection. By optimizing the agarose gel formulation, cell seeding density, and intermittent half-volume medium replacement culture protocol, it is possible to obtain batches of three-dimensional spleen cell spheroids with uniform size, dense and stable structure, and complete preservation of the native spleen's immune microenvironment. The functional stability window of this model is 2-5 days after culture, and it can be used for constructing oxidative stress injury models, evaluating the activity of drugs related to the Nrf2 antioxidant pathway and the NF-κB inflammatory pathway. This preparation method has extremely low raw material costs, excellent experimental repeatability, and no background interference from exogenous matrix. It is suitable for high-throughput screening of active ingredients in traditional Chinese medicine, analysis of the molecular mechanism of immune antioxidation, and in vitro drug immunotoxicity assessment.

[0025] Compared with the prior art, the present invention has the following beneficial technical effects: (1) Completely free of matrix gel and exogenous scaffold: pure cell self-assembly, no interference from exogenous biological matrix, clean background, extremely suitable for the precise study of the antioxidant and anti-inflammatory mechanisms of traditional Chinese medicine compound and natural product, solving the industry pain point of matrix gel interference in drug efficacy evaluation; (2) Low cost can replace imported consumables: the effect of self-made agarose coated plates is equivalent to that of commercial ultra-low adsorption plates, and the cost is reduced by more than 90%, making them suitable for high-throughput drug screening; (3) Preservation of the native immune microenvironment: without cell sorting or modification of cell ratio, the spleen’s natural immune cell interaction system is completely preserved, and the in vivo correlation is far superior to that of a single cell line model; (4) The model has high stability and good reproducibility: the spheres are uniform in size, without scattered cells or excessive fusion, and the function is stable for 2-5 days. The data on Nrf2 oxidative stress and inflammatory factors show little fluctuation. (5) Simple operation and batch preparation: The process is simple, the error tolerance is high, and it can be standardized and replicated, making it suitable for routine pharmacological evaluation in the laboratory and the transformation of patented technologies. Attached Figure Description

[0026] Figure 1 HE staining results show the three-dimensional morphology of spleen cell spheroids.

[0027] Figure 2 The results show the expression of HO-1 and NQO1 proteins downstream of the Nrf2 pathway under tBHQ drug stimulation.

[0028] Figure 3 The results of ROS fluorescence intensity in the H2O2-induced oxidative stress model are shown.

[0029] Figure 4 The study showed the secretion levels of cytokines TNF-α and IL-6 in the LPS inflammation model and the effects of drug intervention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0031] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0032] In the description of the invention, the numerical values ​​of time, temperature, ratio, and mass involved can be based on actual measurements, standard equipment parameters, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0033] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0034] The terms “about” or “around” as used herein are as understood by one of ordinary skill in the art and vary within a range depending on the context in which they are used. If one of ordinary skill in the art is not familiar with the use of the term in the context in which it is used, “about” or “around” will mean a particular value plus or minus 10%, such as 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0035] Unless otherwise specified, when the content, time, or other values ​​or parameters used herein are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, they should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1~5” is disclosed, the described range should be interpreted as including ranges “1~4”, “1~3”, “1~2”, “1~2 and 4~5”, “1~3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range includes its endpoints and all integers and fractions within that range.

[0036] The terms “comprising” and / or “including” as used herein specify the presence of the stated features, integers, steps, operations, units and / or components, without excluding the presence or addition of one or more other features, integers, steps, operations, units, components and / or combinations thereof.

[0037] The cell spheroid culture used in this article is a widely used three-dimensional multicellular culture model. Its principle lies in the fact that when cell adhesion to the culture substrate is disrupted, cells tend to aggregate and form three-dimensional cell spheroids. This three-dimensional structure promotes spatial exchange of substances and signals between cells, and can, to some extent, replicate the in vivo survival state of cells, exhibiting stronger physiological relevance than two-dimensional culture. The "spleen cell spheroids" used in this article refer to aggregates of 1000 or more single cells forming three-dimensional spherical structures. Because they can more accurately replicate the structure and physical properties of the three-dimensional tissue surrounding cells in the microenvironment, they are effectively used in drug screening, treatment, and related research fields.

[0038] The term "culture medium" as used in this article refers to a medium containing the components required for cell culture. Culture media can contain basal medium and various additives. Culture media can be prepared by combining basal medium with additives. Culture media can further contain any one or both of extracellular matrix and substances that aid cell proliferation.

[0039] The term "basal medium" as used herein can refer to any known cell culture medium, such as Durbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle Basal Medium (BME), RPMI-1640, KnockOut DMEM, KnockOut DMEM / F12, neurobasal, or DMEM / F12. In one embodiment, the basal medium is RPMI-1640. In one embodiment, the basal medium is DMEM / F12. In one embodiment, the basal medium comprises combinations of the above-described exemplary basal media in various proportions.

[0040] The term "collagenase" as used herein refers to collagenase, which hydrolyzes proline in the intercellular matrix, thereby dissociating cells. Collagenase has a strong digestive effect on collagen, acting primarily on the intercellular matrix with minimal damage to cells, making it suitable for digesting and separating fibrous tissue and harder cancerous tissue. Calcium and magnesium ions and serum do not affect the activity or digestive effect of collagenase. In some embodiments, the collagenase is selected from collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V, or any combination thereof.

[0041] The term "matrix or extracellular matrix" as used in this article refers to a three-dimensional matrix that can polymerize at room temperature to form a biologically active matrix that mimics the structure, composition, physical properties, and function of the in vivo cell basement membrane, thus facilitating cell culture and differentiation in vitro. Extracellular matrices are commercially available, such as extracellular matrix proteins (Invitrogen) or basement membrane preparations derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex (registered trademark) Basement Membrane Extract (Trevigen) or Matrigel (registered trademark) (Corning)). Extracellular matrices can be synthetic (e.g., ProNectin (Sigma Z378666)). Extracellular matrices can be one or a mixture of two or more components.

[0042] The "Nrf2 (nuclear factor E2-related factor 2)" used in this article is a transcription factor that plays a crucial role in maintaining cellular oxidative stress homeostasis. It mediates the cell's protective response to oxidative stress by regulating the expression of antioxidant response element-dependent genes. Oxidative stress can release large amounts of reactive oxygen species (ROS), inducing intracellular lipid peroxidation, decreased enzyme activity, and changes in protein structure, leading to oxidative damage to cells and tissues. The Nrf2 pathway is one of the most important antioxidant pathways and can regulate the expression of endogenous antioxidant enzymes. Under normal physiological conditions, Nrf2 binds to Kelch-like ECH-associated protein 1 (Keap 1) in the cytoplasm and is degraded through ubiquitination, maintaining Nrf2 levels at physiologically necessary levels. When the body is subjected to oxidative stress, ROS can modify the cysteine ​​residues of Keap 1, causing dissociation from Nrf2. Nrf2 is released through deubiquitination and enters the nucleus, where it binds to antioxidant response elements (AREs), thereby upregulating the expression of antioxidant enzymes and phase II detoxification enzymes to scavenge excess free radicals, such as heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Studies have found that activating the Nrf2-ARE signaling pathway can promote the transcription of cellular protective genes, reduce damage from oxidative stress, and play a protective role in the body. In the three-dimensional spleen cell spheroid model of the present invention, the activation effect of the drug on the Nrf2 pathway can be evaluated by detecting Nrf2 nuclear translocation, mRNA and protein expression levels of Nrf2 downstream target genes HO-1 and NQO1.

[0043] The term "NF-κB" (nuclear factor-kappa B, activated B cell nuclear factor-kappa light chain enhancer, NFkB, NFκB, or NFKB) used in this article refers to a family of transcription factor proteins widely distributed in animal cells. These proteins dimerize to form transcription factors, regulate gene expression, and participate in various biological processes. Inhibiting NF-κB can alleviate inflammatory responses and is used to treat inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. The NFkB pathway is a key cellular signaling pathway involved in immunity, inflammation, and acute-phase responses, but it also participates in controlling cell survival, proliferation, and apoptosis. NFkB transcription factors can be activated by various stimuli, such as reactive oxygen species, pathogens, cytokines, and the activation of T cell and B cell receptors in immune cells. After activation, NFkB inhibitors are phosphorylated and undergo ubiquitous protein-dependent degradation by the proteasome, and the NFkB dimer translocates to the cell nucleus, where it acts as a transcription factor.

[0044] This invention relates to a method for preparing three-dimensional spleen cell sphere microtissues through matrix gel-free self-assembly, comprising four core steps: self-made ultra-low adhesion gel substrate, gentle dissociation of primary spleen cells, scaffold-free self-assembly into spheres, and rhythmic steady-state culture.

[0045] 1) Homemade culture medium-grade agarose ultra-low adhesion substrate: Using a 1.5% cell culture-grade low-melting-point agarose + serum-free RPMI1640 system, a completely inert, cell adhesion-free bottom coating is formed after solidification, replacing imported commercial ultra-low adsorption plates and completely eliminating batch-to-batch coating variations.

[0046] 2) Mild dissociation of native spleen immune cells: It uses calcium- and magnesium-containing HBSS to activate collagenase, combined with DNase I to eliminate DNA adhesion of dead cells, preserving the proportion of natural mixed immune cell subsets in the spleen, without cell sorting or disrupting the native immune microenvironment.

[0047] 3) Spheres are formed purely by self-assembly without any matrix adhesive: Utilizing the high spontaneous adhesion properties of spleen cells, they settle under gravity on an inert, non-adhesive substrate and autonomously bind with each other. Without the assistance of any matrix gel, scaffold, or additives, they spontaneously form dense, regular three-dimensional spherical micro-tissues within 48 hours.

[0048] 4) Low-disturbance steady-state maintenance culture: A 48-hour half-volume gentle medium replacement system was established, with complete culture in 10% serum throughout the process, to maintain the activity of immune cells and the stability of the Nrf2 and NF-κB pathways. The model's functional window was stable, and it could be used for batch drug administration and screening.

[0049] This invention achieves the following: (1) using culture medium to dissolve agarose to prepare an ultra-low adhesion bottom layer, effectively avoiding damage to primary immune cells caused by osmotic pressure imbalance of pure hydrogel, and is suitable for three-dimensional culture of highly active primary cells; (2) without sorting immune cells, using a complete native mixed spleen cell pure self-assembly system, completely preserving the natural multicellular immune microenvironment of the spleen, with a biomimetic degree far higher than that of a single immune cell spheroid model; (3) establishing a pure cell self-assembly spheroidization process without matrix gel or exogenous scaffold assistance, solving the three core defects of existing technologies in one go: matrix components interfering with drug efficacy evaluation, high cost of imported consumables, and large differences in results between different experimental batches, and possessing outstanding creativity and industrial practical value.

[0050] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0051] Example For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0052] Example 1 This embodiment provides a method for preparing matrix-free three-dimensional spleen cell spheres, including the following steps: 1) Preparation of ultra-low adhesion agarose well plates Weigh 1.5 g of cell culture-grade low-melting-point agarose, add 100 mL of serum-free RPMI 1640 medium, microwave until completely dissolved, autoclave at 115 °C for 30 min, and cool to 50 °C and incubate. Add 50 μL of agarose solution to each well of a sterile 96-well U-bottom plate, let stand at room temperature for 30 min until completely solidified, and sterilize under UV light in a clean bench for 30 min. Rinse each well with 100 μL of 10% FBS RPMI 1640 complete medium for 60 min, and discard the rinse solution.

[0053] 2) Isolation of primary spleen cells Spleens were aseptically harvested from mice, mechanically minced, and digested with collagenase IV (100 U / mL) + DNase I (20 U / mL) prepared with calcium and magnesium HBSS. The mixture was then gently digested at 37 °C for 20 min. The cells were filtered through a 70 μm filter, centrifuged, and lysed with ammonium chloride-potassium (ACK) buffer for 3 min. Washing was terminated with ice-cold culture medium to obtain purified viable spleen cells.

[0054] 3) The boards are self-assembled into a ball. Spleen cells were resuspended in 10% FBS RPMI 1640 complete medium and the cell concentration was adjusted to 1×10⁻⁶. 6 cells / mL, seed 100 μL of cell suspension per well, i.e., 1×10 cells / mL per well. 5 Cells were placed in a 37°C, 5% CO2 incubator for static culture.

[0055] 4) Steady-state cultivation and maintenance After 48 hours of culture, the cells completely self-assembled to form dense and regular three-dimensional spleen cell spheres. Figure 1 HE-stained centrifuged smear of 3D cultured primary spleen cells. Figure 1 It can be seen that the cells inside the sphere are tightly arranged and there is no central necrotic area.

[0056] The medium was replaced halfway every 48 hours using a wide-mouth pipette tip, without disturbing the spheroids. After 2-5 days of culture, the cells could be used for oxidative stress model construction, Nrf2 pathway detection, and anti-inflammatory drug screening. In this example, the three-dimensional spleen cell spheroids prepared achieved a spheroidization rate of over 85%, with uniform spheroid diameter (250-300 μm, CV value <10%), and cell viability >95% as determined by trypan blue staining.

[0057] Example 2 This embodiment provides a method for preparing matrix-free three-dimensional spleen cell spheres, including the following steps: 1) Preparation of ultra-low adhesion agarose well plates Weigh 1.0 g of cell culture-grade low-melting-point agarose and add it to 100 mL of serum-free RPMI 1640 medium. Dissolve completely in microwave, autoclave at 115 °C for 30 min, and cool to 50 °C and incubate. Add 50 μL of the agarose solution to each well of a sterile 96-well U-bottom plate and let it stand at room temperature for 60 min to allow it to solidify completely. Sterilize under UV light in a laminar flow hood for 30 min. Rinse each well with 100 μL of RPMI 1640 complete medium containing 10% fetal bovine serum for 80 min, and discard the rinse solution.

[0058] 2) Isolation of primary spleen cells Spleens were aseptically harvested from mice, mechanically minced, and digested with collagenase IV (75 U / mL) + DNase I (15 U / mL) containing calcium and magnesium HBSS. The mixture was then gently digested at 37 °C for 30 min. The cells were filtered through a 70 μm filter, centrifuged, and subjected to ACK cytokinesis for 8 min. Washing was stopped with ice-cold culture medium to obtain purified viable spleen cells.

[0059] 3) The boards are self-assembled into a ball. Spleen cells were resuspended in 10% FBS RPMI 1640 complete medium and the cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 5cells / mL, seed 100 μL of cell suspension per well, i.e., 5 × 10⁶ cells / mL per well. 4 Cells were placed in a 37°C, 5% CO2 incubator for static culture.

[0060] 4) Steady-state cultivation and maintenance After 48 hours of culture, the cells completely self-assembled to form dense and well-organized three-dimensional spleen cell spheres. HE staining results showed that the intercellular spaces within the spheres were slightly larger compared to Example 1.

[0061] The medium was replaced halfway every 48 hours using a wide-mouth pipette tip without disturbing the spheroids. After culturing for 2-5 days, the cells could be used for oxidative stress model construction, Nrf2 pathway detection, and anti-inflammatory drug screening. The three-dimensional spleen cell spheroids prepared in this example achieved a spheroidization rate of over 70%, with uniform spheroid diameter (200-260 μm, CV value <12%) and cell viability >90%.

[0062] Example 3 This embodiment provides a method for preparing matrix-free three-dimensional spleen cell spheres, including the following steps: 1) Preparation of ultra-low adhesion agarose well plates Weigh 2.0 g of cell culture-grade low-melting-point agarose and add it to 100 mL of serum-free RPMI 1640 medium. Dissolve completely in microwave, autoclave at 115 °C for 30 min, and cool to 50 °C for constant temperature. Add 50 μL of the agarose solution to each sterile 96-well U-bottom plate and let it stand at room temperature for 20 min to allow it to solidify completely. Then sterilize under UV light in a laminar flow hood for 30 min. Rinse each well with 100 μL of RPMI 1640 complete medium containing 10% fetal bovine serum for 40 min, and discard the rinse solution.

[0063] 2) Isolation of primary spleen cells Spleens were aseptically harvested from mice, mechanically minced, and digested with collagenase IV (125 U / mL) + DNase I (25 U / mL) prepared with calcium and magnesium HBSS. The mixture was then gently digested at 37 °C for 15 min. The cells were filtered through a 70 μm filter, centrifuged, and subjected to ACK cytokinesis for 2 min. Washing was stopped with ice-cold culture medium to obtain purified viable spleen cells.

[0064] 3) The boards are self-assembled into a ball. Spleen cells were resuspended in 10% FBS RPMI 1640 complete medium and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6 cells / mL, seed 100 μL of cell suspension per well, i.e., 2 × 10⁶ cells / mL per well. 5 Cells were placed in a 37°C, 5% CO2 incubator for static culture.

[0065] 4) Steady-state cultivation and maintenance After 48 hours of culture, the cells completely self-assembled to form dense and well-organized three-dimensional spleen cell spheres. HE staining results showed that the cells inside the spheres were arranged slightly more tightly compared to Example 1.

[0066] The medium was replaced halfway every 48 hours using a wide-mouth pipette tip without disturbing the spheroids. After 2-5 days of culture, the cells can be used for oxidative stress model construction, Nrf2 pathway detection, and anti-inflammatory drug screening. The three-dimensional spleen cell spheroids prepared in this example achieved a spheroidization rate of over 80%, with uniform spheroid diameter (350-400 μm, CV value <10%) and cell viability >85%.

[0067] Comparative Example 1 The only difference from Example 1 is that in step 1), 0.5 g of cell culture grade low melting point agarose was weighed and added to 100 mL of serum-free RPMI 1640 medium in the preparation of ultra-low adhesion agarose well plates.

[0068] Comparative Example 2 The only difference from Example 1 is that in step 1) the preparation of ultra-low adhesion agarose well plates, 1.5 g of cell culture grade low melting point agarose is weighed and added to 100 mL of RPMI 1640 medium containing 10% FBS.

[0069] Comparative Example 3 The only difference from Example 1 is that step 1) uses a commercially available ultra-low adsorption 96-well U-type substrate (Beyotime, FULA961-1pc).

[0070] Comparative Example 4 The only difference from Example 1 is that step 1) uses a commercially available Matrigel-coated 96-hole U-shaped base plate (Corning, 356259).

[0071] Example 4: Model Performance Verification Three-dimensional spleen cell spheres prepared by the method in Examples 1-3 of this invention: 1) Spheroid formation rate > 70%, spheroids are uniform in size and have no adherent cells; 2) Compared to 2D culture, the sensitivity of inflammatory stress response was significantly improved; 3) No matrix gel interference throughout the process, resulting in low background and strong repeatability in the screening of the efficacy of traditional Chinese medicine extracts; 4) The model can maintain its function stably for 5 days, meeting the complete pharmacological experimental cycle of drug pretreatment, modeling, and detection.

[0072] This embodiment specifically verifies the three-dimensional spleen cell spheres prepared by the methods in Examples 1-3 and Comparative Examples 1-4. The verification of the three-dimensional spleen cell spheres prepared by each method was repeated at least 3 times.

[0073] 1) Evaluation of pellet formation rate and morphology Observation under an inverted microscope showed that the three-dimensional spleen cell spheres prepared by the methods in Examples 1-3 exhibited significant cell aggregation after 24 hours of culture, and formed dense and regular three-dimensional spheres after 48 hours, with a spheroidization rate >70%, a sphere diameter of 200-400 μm, and uniform size (CV value <15%). Figure 1 As shown, HE staining revealed that the cells inside the sphere were tightly packed, with no obvious central necrotic area.

[0074] However, the three-dimensional spleen cell spheres prepared by the method in Comparative Example 1 had insufficient gel strength due to the low agarose concentration, resulting in gel rupture at the bottom of some wells. After 48 hours of culture, approximately 35% of the wells showed cell adhesion growth, with a sphere formation rate of only 40%–50%, and significant batch-to-batch variations. The three-dimensional spleen cell spheres prepared by the method in Comparative Example 2 (using RPMI 1640 with 10% FBS to prepare the agarose substrate) exhibited altered ultra-low adhesion characteristics due to the adsorption of protein and lipid components from the FBS onto the agarose surface. After 24 hours of culture, most cells remained in an adherent, extended state, with only a few wells forming cell spheres, resulting in a sphere formation rate of less than 30%. Furthermore, the spheres were irregular in shape, and some showed outward migration and growth at the edges. The three-dimensional spleen cell spheres prepared by the method in Comparative Example 3 (commercial ultra-low adsorption plate) achieved a sphere formation rate of approximately 86% after 48 hours of culture, showing no significant difference from the three-dimensional spleen cell spheres prepared by the methods in Examples 1–3. However, the CV value of the sphere formation rate among the three batches was 18.5% (>15%). Similar batch-to-batch differences were observed in the three-dimensional spleen cell spheres prepared by the method in Comparative Example 4 (commercial Matrigel coated plates), with some batches having thicker gels that caused the spheres to become embedded in the matrix.

[0075] 2) Cell viability evaluation Calcein-AM / PI staining results showed that the cell viability was >95% on day 1, >90% on day 3, and remained above 85% on day 5, indicating that the spleen cell spheres prepared by the methods in Examples 1-3 can stably maintain function in vitro for at least 5 days.

[0076] 3) Drug screening suitability evaluation Three-dimensional spleen cells were treated with tert-butylhydroquinone (tBHQ, 10 μmol / L) for 24 h to detect the expression levels of downstream target genes of Nrf2. Figure 2 As shown, compared with the control group, the protein expression levels of HO-1 and NQO1 in the treatment group were significantly increased ( P <0.01). This indicates that the spleen cell spheres prepared by this method are sensitive to drug stimulation, have good data repeatability, and low background noise, making them suitable for precise mechanism evaluation of active ingredients in traditional Chinese medicine (the figure shows the detection results of matrix-free three-dimensional spleen cell spheres in Example 1).

[0077] However, Comparative Example 1 could not be used for drug stimulation experiments due to insufficient cell spheroid formation. In Comparative Example 2, due to the majority of cells adhering to the plate, there was no significant difference in HO-1 and NQO1 levels after tBHQ stimulation compared to the control group (P>0.05), and the data fluctuated greatly. Comparative Example 3 (commercial ultra-low adsorption plate) showed comparable fold induction of HO-1 and NQO1 and experimental repeatability to Examples 1-3 after tBHQ stimulation (P>0.05), but its cost per well was approximately 8-10 times that of the self-made substrate of this invention, and the difference in spheroid formation rate between batches led to an imbalance in experimental sample size. In Comparative Example 4 (Matrigel coated plate), the basal expression level of HO-1 in the unstimulated state was 3.2 times that of the unstimulated group in Example 1 (Matrigel coated plate). P <0.01), indicating that the growth factors and cytokines contained in Matrigel itself have significantly activated the downstream signaling of the Nrf2 pathway, resulting in severe background interference; the HO-1 induction fold after tBHQ stimulation was only 1.8-fold (results not shown), the dynamic response range was compressed, and the data reliability was poor.

[0078] The results show that the matrix-free self-assembly method of the present invention for constructing three-dimensional spleen cell spheroid microtissue achieves low-cost, highly stable, and highly in vivo correlated immune microtissue model construction. When used for drug screening, it can effectively avoid the interference of exogenous biological matrix on pathway detection and significantly improve the reliability of accurate evaluation of active ingredients of traditional Chinese medicine.

[0079] 4) Establishment of a three-dimensional model of oxidative stress in spleen cells The three-dimensional spleen cell spheres prepared in Example 1 were used. The original culture supernatant was discarded and replaced with serum-free RPMI1640 medium containing 200 μmol / L hydrogen peroxide (H2O2). After 12 h incubation at 37°C and 5% CO2, samples were collected for intracellular reactive oxygen species (ROS) level detection. Serum-free RPMI1640 medium without H2O2 was used as a blank control.

[0080] The ROS level in three-dimensional splenocyte spheroids was detected using the DCFH-DA fluorescent probe method. Three-dimensional splenocyte spheroids treated with H2O2 were collected, washed twice with serum-free RPMI 1640 medium, and then incubated with DCFH-DA probe working solution at a final concentration of 10 μmol / L for 30 min in the dark. After incubation, the cells were washed three times with serum-free medium, and the fluorescence intensity was detected using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0081] The results are as follows Figure 3The results showed that, compared with the blank control group, the ROS level in the H2O2-treated group exhibited a significant upward trend. These results indicate that H2O2 treatment can induce oxidative stress in three-dimensional spleen cell spheres. The matrix-free self-assembly of three-dimensional spleen cell sphere microtissues constructed in this invention can be used to construct oxidative stress injury models.

[0082] 5) Three-dimensional spleen cell spheres for screening anti-inflammatory drugs Three-dimensional spleen cell spheres prepared in Example 1 were treated with 1 μg / mL lipopolysaccharide (LPS) for 12 h to induce an inflammatory response. After establishing the inflammatory model, a model control group, a positive control group (dexamethasone 1 μmol / L), and groups treated with different concentrations of the tested traditional Chinese medicine extracts were set up. Samples were collected after 12 h of treatment. The secretion levels of various inflammatory cytokines were detected by ELISA. The results are as follows: Figure 4 The results showed that the secretion of TNF-α and IL-6 in the three-dimensional spleen cell spheres was significantly increased compared with the blank group after LPS stimulation (P<0.01); the secretion of TNF-α and IL-6 in the positive control group was significantly decreased compared with the model control group (P<0.01); the Chinese herbal extracts tested inhibited the secretion of the above inflammatory factors in a dose-dependent manner (P<0.01). These results indicate that the three-dimensional spleen cell spheres prepared in this invention can be effectively used for in vitro activity screening of anti-inflammatory drugs.

[0083] This invention provides a method for preparing and applying matrix-free, self-assembled three-dimensional spleen cell spheroid microtissues. It overcomes the technical bottlenecks of existing three-dimensional spleen cell spheroid models, which rely on imported commercially available ultra-low adsorption culture plates, have high matrix gel costs, suffer from complex matrix composition and large batch-to-batch variations, and are susceptible to interference from exogenous biological matrices in the screening of traditional Chinese medicine efficacy and pathway mechanism detection. By optimizing the agarose gel formulation, cell seeding density, and intermittent half-volume medium replacement culture protocol, it is possible to obtain batches of three-dimensional spleen cell spheroid microtissues that are uniform in size, structurally dense and stable, and completely preserve the native spleen's immune microenvironment. The functional stability window of this model is 2-5 days after culture, and it can be used for constructing oxidative stress injury models, evaluating the activity of drugs related to the Nrf2 antioxidant pathway and the NF-κB inflammatory pathway. This preparation method has extremely low raw material costs, excellent experimental reproducibility, and no exogenous matrix background interference, making it suitable for high-throughput screening of active ingredients in traditional Chinese medicine, analysis of immune antioxidant molecular mechanisms, and in vitro drug immunotoxicity assessment.

[0084] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing three-dimensional spleen cell spheres, characterized in that, Includes the following steps: (1) Preparation of ultra-low adhesion agarose well plates: Cell culture grade low melting point agarose is dissolved in serum-free culture medium, heated to dissolve and sterilized, added to cell culture container, allowed to stand and solidify to form ultra-low adhesion gel bottom layer; after ultraviolet sterilization, it is soaked and rinsed with culture medium containing 10% fetal bovine serum to balance osmotic pressure and obtain ultra-low adhesion agarose well plates. (2) Isolation of primary spleen cells: mouse spleen tissue was taken and cut into pieces, and then digested gently with digestive juice. After filtration, centrifugation and erythrocyte lysis, primary spleen cells were obtained. (3) Matrix-free self-assembly into spheres: The primary spleen cells obtained in step (2) were resuspended in complete culture medium containing 10% fetal bovine serum and seeded into the ultra-low adhesion agarose wells prepared in step (1). After static culture, the cells spontaneously aggregated to form three-dimensional spleen cell spheres. (4) Micro-tissue homeostasis maintenance culture: After the three-dimensional spleen cell spheres are formed, half of the medium is changed regularly to maintain the culture and obtain three-dimensional spleen cell sphere micro-tissue with uniform structure and stable immune function.

2. The method for preparing three-dimensional spleen cell spheres according to claim 1, characterized in that, In step (1), the mass-volume ratio of the low-melting-point agarose is 1.0% to 2.0%, preferably 1.5%.

3. The method for preparing three-dimensional spleen cell spheres according to claim 1, characterized in that, In step (1), the culture medium includes a basal culture medium, an antimicrobial agent, and a culture medium supplement, wherein the basal culture medium is selected from RPMI-1640, DMEM, DMEM / F12 or Advanced DMEM / F12 culture medium, preferably RPMI-1640.

4. The method for preparing three-dimensional spleen cell spheres according to claim 1, characterized in that, In step (1), the soaking and rinsing time of the culture medium is 40-80 min, preferably 60 min.

5. The method for preparing three-dimensional spleen cell spheres according to claim 1, characterized in that, In step (2), the digestive fluid contains one or more of the following: collagenase, calcium chloride, zinc chloride, DNase, neutral protease, fetal bovine serum, Liberase enzyme, and antibiotics; The mild digestion conditions are digestion at 37°C for 10-30 min, preferably 20 min; the erythrocyte lysis uses ammonium chloride-potassium (ACK) buffer as the lysis buffer and the lysis time is 2-8 min.

6. The method for preparing three-dimensional spleen cell spheres according to claim 5, characterized in that, The digestive solution is a mixed digestive solution of collagenase IV and DNase I prepared with calcium and magnesium HBSS, wherein the concentration of collagenase IV is 50-200 U / mL, the concentration of DNase I is 10-40 U / mL, preferably the concentration of collagenase IV is 75-125 U / mL, the concentration of DNase I is 15-25 U / mL, and more preferably the concentration of collagenase IV is 100 U / mL and the concentration of DNase I is 20 U / mL.

7. The method for preparing three-dimensional spleen cell spheres according to claim 1, characterized in that, In step (3), the primary spleen cells are seeded at a density of 5 × 10⁶ cells per well. 4 ~2×10 5 Cells, preferably 1×10 per well 5 Each cell.

8. The method for preparing three-dimensional spleen cell spheres according to claim 1, characterized in that, In step (4), after culturing for 48 h, the cells spontaneously assemble to form dense spherical micro-tissues. Half of the medium is replaced every 48 h. The cell culture period of 2-5 days is the window period for the model to stabilize.

9. A three-dimensional spleen cell sphere microstructure obtained by the preparation method of the three-dimensional spleen cell sphere according to any one of claims 1 to 8.

10. The application of the three-dimensional spleen cell spheroid microtissue as described in claim 9 in the establishment of an in vitro spleen immune microenvironment model and in drug screening, wherein drug screening includes screening for antioxidant active drugs, screening for anti-inflammatory active drugs, and screening for immunomodulatory drugs.

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