Expansion method of single cell, single cell nucleus or single subcellular structure and application thereof

Abstract

The invention discloses a single cell, single cell nucleus or single subcellular structure expansion method and application thereof. The expansion method comprises the following steps: dividing raw materials for forming the hydrogel into at least two groups, and respectively introducing the raw materials into a single cell, a single cell nucleus or a single subcellular structure; a step of forming a hydrogel by polymerization; and putting the single cell, single cell nucleus or single subcellular structure with the hydrogel inside into a hypotonic solution to expand. According to the invention, a hydrogel forming region can be controlled in a single cell, single cell nucleus or single subcellular structure, high monodispersity is still maintained after expansion, and a flowable single cell, single cell nucleus or single subcellular structure suspension can be formed; the method can be directly applied to multiple single-cell omics detection platforms such as a microfluidic liquid drop platform, a microwell plate sorting platform and an FACS sorting platform, multiple single-cell omics processes are carried out, the capture efficiency of molecules such as DNA / RNA / protein is improved, and the omics data quality is improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a method for expanding single-cell, single-nucleus, or single-subcellular structures and its application. Background Technology

[0002] Single-cell omics (including single-cell transcriptomics, single-cell genomics, and single-cell proteomics) is an important tool for elucidating cellular heterogeneity and complex biological systems. However, in existing single-cell omics experiments, signal capture efficiency is often limited by the following factors: for platforms that use cell lysis in droplets for molecular capture (such as 10X Genomics), incomplete cell lysis (especially of bacteria) leads to insufficient RNA / DNA release; for platforms that use in-situ reactions in fixed cells for molecular capture (such as smRandom-seq), the diffusion distance of captured proteins or nucleic acids is limited, making it difficult for them to enter the capture system.

[0003] Cell swelling is beneficial for solving the above problems. However, traditional swelling techniques (such as Expansion Microscopy for microscopic imaging) are mainly used to achieve proportional magnification of tissue sections, adherent cells, cell clusters obtained by centrifugation, or whole organoids in gels to improve microscopic resolution. This technique has good effect on overall sample swelling, but it has obvious limitations when applied at the single cell scale. This is because existing methods usually use single-step cross-linking or exogenously initiated polymerization systems, so that the polymerization reaction occurs simultaneously inside and outside the cell, making it difficult to achieve spatial control of the gel formation area. What is obtained is a "cell-hydrogel complex", and the extracellular space is also surrounded by a large amount of gel. This structure is prone to the following problems: (1) the extracellular hydrogel restricts the free expansion of individual cells, resulting in uneven swelling ratio; (2) the mechanical coupling between the cell and the external gel will produce a pulling effect, causing cell structure deformation; (3) the extracellular gel is difficult to remove, affecting subsequent single-cell operations such as fluorescent labeling, imaging, or molecular analysis. Furthermore, traditional chemical or thermal initiation systems have strong diffusivity and low polymerization efficiency within the cellular microenvironment, resulting in a sparse gel network and insufficient mechanical support within the cell, which leads to structural collapse or morphological distortion during the expansion process.

[0004] While some literature has attempted to improve permeability by adjusting the type of cross-linking agent or adding surfactants, it is still impossible to achieve specific control of intracellular gelation, and it is difficult to avoid the formation of extracellular gel. For experimental systems that require subsequent single-cell RNA extraction, spatial omics sequencing, or high-resolution imaging, this external gel residue will significantly reduce experimental compatibility and molecular recovery efficiency.

[0005] Therefore, how to achieve hydrogel polymerization limited to the cell interior at the single-cell scale while ensuring the structural integrity, expansion uniformity, and compatibility with downstream analyses of single cells has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a method for expanding single-cell, single-nucleus, or single-subcellular structures and its applications.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0008] This invention first provides a method for expanding a single cell, a single cell nucleus, or a single subcellular structure, the expansion method comprising:

[0009] The steps involve dividing the raw materials used to form the hydrogel into at least two groups and introducing them into single-cell, single-nucleus, or single-subcellular structures, respectively.

[0010] The steps involved in polymerization to form a hydrogel;

[0011] The step of placing a single cell, single nucleus, or single subcellular structure containing a hydrogel inside into a hypotonic solution to swell.

[0012] This invention divides the raw materials used to form hydrogels into at least two groups, and then introduces these at least two groups of raw materials into single cells, single cell nuclei, or single subcellular structures respectively. At the end of the introduction process, the single cells, single cell nuclei, or single subcellular structures contain all the raw materials for forming hydrogels, while the outside of the cells, cell nuclei, or subcellular structures contains only the last group of introduced raw materials. When polymerizing to form a hydrogel, hydrogels can be formed inside the single cells, single cell nuclei, or single subcellular structures, but hydrogels will not be formed outside the cells, cell nuclei, or subcellular structures. This approach not only allows for the control of hydrogel formation within single-cell, single-nucleus, or single-subcellular structures, but also ensures that these structures maintain high monodispersity, enabling the formation of flowable suspensions. These suspensions can be directly applied to various single-cell omics detection platforms, such as microfluidic droplet platforms, microplate sorting platforms, and FACS sorting platforms. They can then be integrated into various omics processes, including transcriptomics, genomics, and proteomics, utilizing single-cell, single-nucleus, or single-subcellular structures. This helps improve the capture efficiency of DNA, RNA, and proteins, thereby enhancing the quality of omics data.

[0013] The raw materials used to form hydrogels can be divided into at least two major groups: raw materials used to form the hydrogel skeleton and hydrogel monomers (including monomers, crosslinking agents, free radical initiators, etc.). Each major group can be further subdivided into multiple subgroups, which can be specifically set up by those skilled in the art as needed.

[0014] To facilitate the explanation of the technical concept of the present invention, the present invention provides one expansion method as an example, which includes the following steps:

[0015] (1) Introducing polymerizable anchoring agents into pre-fixed single-cell, single-nucleus, or single-subcellular structures;

[0016] The polymerizable anchoring agent has polymerizable groups for participating in free radical polymerization reactions, and a first anchoring functional group for anchoring macromolecules within cells, cell nuclei, or subcellular structures.

[0017] (2) Introduce photosensitive initiation anchoring agents into single cells, single cell nuclei, or single subcellular structures;

[0018] The photosensitive initiating anchoring agent has a photosensitive initiating group and a second anchoring functional group for anchoring macromolecules in cells, cell nuclei or subcellular structures;

[0019] (3) Introducing hydrogel monomers into single-cell, single-nucleus, or single-subcellular structures;

[0020] (4) Photo-induced treatment of single cells, single nuclei or single subcellular structures treated in steps (1), (2) and (3) to induce hydrogel polymerization;

[0021] (5) Place a single cell, single cell nucleus or single subcellular structure with hydrogel inside into a hypotonic solution to swell.

[0022] In step (1), the polymerizable anchoring agent introduced can not only polymerize to form a hydrogel framework through its polymerizable groups, but also anchor the hydrogel framework to macromolecules in cells, nuclei, or subcellular structures through the first anchoring group; the photosensitive initiating anchoring agent introduced in step (2) can anchor the photosensitive initiating group to macromolecules with the second anchoring functional group, adjacent to the hydrogel framework, and promote the formation of a three-dimensional hydrogel network on the hydrogel framework; the hydrogel monomer introduced in step (3) can form a three-dimensional hydrogel network around the hydrogel framework; there is no necessary sequential reaction dependence between steps (1), (2), and (3), so they can be carried out in any order; however, photo-induced treatment is only carried out after steps (1), (2), and (3) are completed, so as to avoid hydrogel polymerization outside cells, nuclei, or subcellular structures, and make the hydrogel only form inside cells, nuclei, or subcellular structures.

[0023] Meanwhile, to avoid the formation of hydrogels on the outside of single cells, single cell nuclei or single subcellular structures, at least after the first two steps of steps (1), (2) and (3), the single cells, cell nuclei or subcellular structures are resuspended in buffer solution before proceeding to the next step, so as to avoid the formation of hydrogels due to the encounter of polymerizable anchoring agents, photosensitive initiating anchoring agents and hydrogel monomers on the outside of cells, cell nuclei or subcellular structures.

[0024] In the expansion method of the present invention, the macromolecule includes at least one of polypeptides and nucleotide chains;

[0025] The nucleus or subcellular structure is a nucleus or subcellular structure located inside the cell, or the nucleus or subcellular structure is a nucleus or subcellular structure independent of the cell.

[0026] The subcellular structures include the endoplasmic reticulum, mitochondria, Golgi apparatus, ribosomes, chloroplasts, vacuoles, and centrosomes.

[0027] This invention does not require that the cell nucleus or subcellular structure be located outside or inside the cell. When it is outside the cell, general macromolecules can be used to anchor the cell nucleus or subcellular structure. When it is inside the cell, in order to achieve precise positioning, a first anchoring functional group that can specifically anchor a specific macromolecule within the cell nucleus or subcellular structure is required.

[0028] Preferably, in the expansion method of the present invention, the first anchoring functional group and the second anchoring functional group achieve anchoring of the macromolecule through covalent or non-covalent interactions with the macromolecule.

[0029] As a further preferred embodiment, the first anchoring functional group and the second anchoring functional group are independently selected from at least one of the following: functional groups targeting the amino group of a polypeptide, functional groups targeting the thiol group of a polypeptide, functional groups targeting the carboxyl group of a polypeptide, functional groups targeting the hydroxyl or phosphate group of a nucleotide, general functional groups that participate in macromolecular crosslinking or labeling, and functional groups that are coupled to macromolecules through crosslinking bridging agents.

[0030] Wherein, the functional group of the amino group of the targeted polypeptide is selected from at least one of carboxyl group, N-hydroxysuccinimide ester, isocyanate group, aldehyde group, ketone group, epoxy group, haloalkyl group, and thioester group;

[0031] The functional group of the targeted peptide thiol group is selected from at least one of maleimide group, epoxy group, haloalkyl group, disulfide bond, and thiol group;

[0032] The functional group of the carboxyl group of the targeted polypeptide is selected from at least one of amino, aromatic amine, hydroxyl, hydrazine, hydrazinyl, hydrazone, and oxime groups;

[0033] The functional group targeting the hydroxyl or phosphate group of the nucleotide is selected from at least one of haloalkyl, phosphate, borate, and borate groups;

[0034] The general functional group that participates in macromolecular crosslinking or labeling is selected from at least one of azide, alkynyl, biotinyl, urea, and carbamate groups;

[0035] The functional groups that are coupled to the macromolecules by the cross-linking bridging agent are usually not directly coupled to the biological macromolecules. They need to be coupled to the biological macromolecules with the help of the cross-linking bridging agent. At least one of the following groups can be selected: quaternary ammonium salt group, thioether group, phosphono group, phosphonic acid group, imidazole group, pyridinyl group, thiazolyl group, indole group, and thiourea group.

[0036] Obviously, when other types of first or second anchoring functional groups (i.e. functional groups that do not necessarily need to be coupled to macromolecules through cross-linking bridging agents) are selected, cross-linking bridging agents can also be used to promote the hydrogel skeleton to be anchored to macromolecules more quickly and stably.

[0037] Preferably, in the expansion method of the present invention, the polymerizable group is an unsaturated group with free radical polymerization reactivity or photoinduced polymerization reactivity; the unsaturated group includes, but is not limited to: double bond unsaturated hydrocarbon groups (such as vinyl, allyl, styrene, norbornene, cycloalkenyl, acryloyl, methacryloyl, etc.), unsaturated ester groups (such as vinyl ester, acrylate, methacrylate, fumarate, maleate, etc.), and strongly electrophilic unsaturated groups (such as maleimide, vinyl ether, vinyl sulfone, etc.).

[0038] Preferably, in the expansion method of the present invention, the photoinitiating group is a photoinitiating group that generates free radicals or active intermediates under light irradiation; the photoinitiating group includes, but is not limited to: photoinitiating cleavage type groups (such as onium salt photoinitiating groups such as diaryliodonium salt group, triarylthiodonium salt group, etc., azide group), photoinduced hydrogen abstraction type groups (such as carbonyl photoinduced hydrogen abstraction groups such as anthraquinone group, benzophenone group, thioxanthone group, benzoyl group, acetophenone group, etc., benzofuranone group, benzothiazolyl group), and photoinduced isomerization or cyclization type groups (such as azo group, coumarin group, flavonoid photoinitiating group, etc.).

[0039] Preferably, in the expansion method of the present invention, a crosslinking bridging agent is introduced simultaneously with the introduction of a polymerizable anchoring agent or a photosensitive initiating anchoring agent to promote crosslinking between the first anchoring functional group or the second anchoring functional group and the target group.

[0040] This invention does not have special requirements for the type of crosslinking bridging agent, as long as it can achieve bridging crosslinking between amines, amines-carboxyl groups, amines-thiol groups, or hydroxyl groups. It can be selected from formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, succinaldehyde, glyoxal dimethyl acetal, glutaraldehyde derivatives, N-hydroxysuccinimide (NHS) and its active esters, NHS-ester crosslinking agents, carbodiimide crosslinking agents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), combinations of EDC and N-hydroxysuccinimide (NHS), diisocyanate compounds, maleimide bifunctional crosslinking agents, epoxy crosslinking agents, disulfide bond crosslinking agents, photosensitive crosslinking agents, etc.

[0041] Preferably, in the expansion method of the present invention, the polymerizable anchoring agent is selected from N-(3-aminopropyl)methacrylamide hydrochloride, N-(3-aminopropyl)acrylamide, and N-(2-aminoethyl)methacrylamide;

[0042] The photosensitive initiating anchoring agent is selected from at least one of the following: 4-benzoylaniline, 4,4'-diaminobenzophenone, 2-aminothioxanthone, 2-aminoanthraquinone, 4-aminoacetophenone, and 4-benzoylbenzoic acid-N-succinimide ester;

[0043] Preferably, in step (2) of the expansion method of the present invention, an ultraviolet photoinitiator is introduced at the same time as the photosensitive initiation anchoring agent is introduced into the single cell to improve the initiation efficiency.

[0044] Preferably, in the expansion method of the present invention, step (1) is implemented by: resuspending the pre-fixed single cell, nucleus or subcellular structure in a solution containing a polymerizable anchoring agent, and incubating at 4°C to room temperature for 1-14 h (incubating at room temperature for 1-3 h, or incubating at 4°C for 8-14 h) to introduce the polymerizable anchoring agent;

[0045] Step (2) is implemented by resuspending the single cell, nucleus or subcellular structure treated in step (1) in a solution containing a photosensitive initiation anchoring agent and incubating it at 4°C to room temperature for 1-14 h (1-3 h at room temperature or 8-14 h at 4°C) under light-protected conditions to introduce the photosensitive initiation anchoring agent.

[0046] Step (3) is implemented by resuspending a single cell, single cell nucleus or single subcellular structure in a hypertonic solution containing hydrogel monomers and incubating it at 4℃-room temperature for 1-14 h (1-3 h at room temperature or 8-14 h at 4℃) under light-protected conditions to introduce hydrogel monomers.

[0047] In this invention, "hypertonic solution" and "hypotonic solution" are relative concepts. "Hypertonic solution" refers to a solution with a higher osmotic pressure compared to the solution used in the subsequent expansion step; "hypotonic solution" refers to a solution with a lower osmotic pressure compared to the solution used in the polymerization step. As long as the above relative osmotic pressure relationship is satisfied, the technical effects of this invention can be achieved, and the absolute osmotic pressure of the solution is not limited.

[0048] As a further preferred embodiment, in the above-described expansion method, the hypertonic solution contains, by mass fraction:

[0049] The composition includes 10-25% amide monomer, 5-20% carboxylic acid monomer, 0.05-1% crosslinking agent, 0.05-1% free radical initiator, 0.01-0.5% anti-agglomeration agent, 0.1-3% hypertonic solute, and the balance being water.

[0050] The crosslinking agent is selected from at least one of N,N′-methylenebisacrylamide, divinyl sulfone, and polyethylene glycol diacrylate; the free radical initiator is selected from at least one of sodium persulfate, ammonium persulfate, and azobisisobutyronitrile; the anti-agglomeration agent is selected from nonionic surfactants such as polysorbate 20 (Tween-20), poloxamer 407, and octylphenoxy polyoxyethylene alcohol (Triton X-100); and the hypertonic solute is selected from at least one of sodium chloride, potassium chloride, sucrose, and mannitol.

[0051] The role of anti-agglomeration agents is to further prevent adhesion between single cells, single cell nuclei, or single subcellular structures during the expansion process. Hypertonic solutes are used to temporarily create a hypertonic environment during the introduction of hydrogel monomers to improve the efficiency and uniformity of hydrogel monomers penetrating into cells, cell nuclei, or subcellular structures, and to facilitate a more complete expansion effect in subsequent hypotonic solutions.

[0052] As a further preferred embodiment, in step (4) of the above-mentioned expansion method, the photo-induced treatment includes: ultraviolet light irradiation for 3-5 minutes.

[0053] As a further preferred option, in step (5), the proteins inside a single cell, single cell nucleus, or single subcellular structure are first enzymatically digested, and then placed in a hypotonic solution to swell. Enzymatic digestion is not necessary; its purpose is to soften the internal structure of the cell, nucleus, or subcellular structure, promote the expansion of the hydrogel system, and facilitate the release and diffusion of nucleic acids. If transcriptomic or genomic analysis is to be performed subsequently, enzymatic digestion is usually performed; if proteomic analysis is to be performed subsequently, enzymatic digestion is not required or only a weak enzymatic digestion is performed to avoid large-scale protein degradation.

[0054] This invention does not have any special requirements for the type of protease used in enzymatic hydrolysis, as long as it can hydrolyze the protein under mild conditions without significantly damaging the hydrogel structure. At least one of proteinase K, trypsin, pepsin, chymotrypsin, papain, bromelain, streptomycin, thermophilic protease, subtilisin, and metalloproteinase can be selected.

[0055] The present invention also provides the application of the above-described expansion method in transcriptomic, genomic, proteomic, or spatial omics analysis of single-cell, single-nucleus, or single-subcellular structures.

[0056] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0057] 1. In this invention, the raw materials used to form a hydrogel are divided into at least two groups, and then these at least two groups of raw materials are introduced into a single cell, a single cell nucleus, or a single subcellular structure, respectively. At the end of the introduction process, the single cell, single cell nucleus, or single subcellular structure contains all the raw materials for forming a hydrogel, while the outside of the cell, cell nucleus, or subcellular structure contains only the last group of introduced raw materials. When the hydrogel is polymerized to form a hydrogel, a hydrogel can be formed inside the single cell, single cell nucleus, or single subcellular structure, while a hydrogel will not be formed outside the cell, cell nucleus, or subcellular structure. This not only allows the hydrogel formation area to be controlled within single cells, single nuclei, or single subcellular structures, but also ensures that the single cells, single nuclei, or single subcellular structures containing hydrogels maintain high monodispersity (over 90%). This enables the formation of flowable suspensions of single cells, single nuclei, or single subcellular structures, which can be directly applied to various single-cell omics detection platforms such as microfluidic droplet platforms, microplate sorting platforms, and FACS sorting platforms. These suspensions can then be used in various omics processes, including transcriptomics, genomics, and proteomics, based on single cells, single nuclei, or single subcellular structures. This helps improve the capture efficiency of molecules such as DNA, RNA, and proteins, and enhances the quality of omics data.

[0058] 2. This invention utilizes anchoring functional groups to anchor polymerizable groups and photoinitiating groups to macromolecules within cells, cell nuclei, or subcellular structures. On one hand, polymerizable groups polymerize in situ to form a hydrogel framework. After the hydrogel monomers infiltrate into cells, cell nuclei, or subcellular structures, the photoinitiating groups can induce the hydrogel monomers to polymerize on the hydrogel framework under light induction, forming a hydrogel network in situ, which can achieve a volume expansion of approximately 2-10 times.

[0059] 3. Depending on the subsequent analytical needs, enzymatic digestion can be selectively performed before expansion. For cases requiring transcriptome or genome analysis, enzymatic digestion followed by expansion makes the structure of cells, nuclei, or subcellular structures more porous, facilitating cell lysis, RNA / DNA release and diffusion, thereby improving nucleic acid capture efficiency, increasing the number of UMIs, the number of detectable genes, and the sensitivity of mutation site detection. For cases requiring proteome analysis, enzymatic digestion can be omitted or only weakly performed to preserve intracellular protein structures and epitopes, allowing the expanded cells, nuclei, or subcellular structures to still be suitable for antibody labeling or proteomics analysis, thus improving the detectability of protein signals.

[0060] 4. The method of the present invention is applicable to eukaryotic cells, prokaryotic cells, pathogen-infected cells, fresh / fixed cells, isolated cell nuclei, FFPE-derived cell nuclei, as well as subcellular structures such as lysosomes, mitochondria, and chloroplasts, and can significantly expand the range of samples that can be analyzed by single-cell omics. Attached Figure Description

[0061] Figure 1 This is a schematic flowchart of a single-cell expansion method according to the present invention;

[0062] Figure 2 A morphological comparison of single human colonic epithelial cells Caco-2 before and after expansion under a fluorescence microscope;

[0063] Figure 3 Scanning electron microscope (SEM) images of single human colonic epithelial cells Caco-2 before and after expansion;

[0064] Figure 4 Comparative images of the morphology of single cells of human embryonic kidney cells 293T before and after expansion under a fluorescence microscope;

[0065] Figure 5 These are morphological comparison images of single mouse fibroblast 3T3-L1 cells before and after swelling under a fluorescence microscope.

[0066] Figure 6A comparison of cDNA capture qPCR results for single-cell transcriptome sequencing technology in human and mouse cell mixed samples before and after expansion.

[0067] Figure 7 Nucleic acid gel electrophoresis images of cDNA products used in single-cell transcriptome sequencing technology before and after expansion of a mixed sample of human and mouse cells;

[0068] Figure 8 A comparison of the number of UMIs and the number of genes detected obtained by single-cell transcriptome sequencing technology before and after expansion of human and mouse cell mixed samples;

[0069] Figure 9 These are morphological comparison images of individual cell nuclei of mouse muscle cells before and after expansion, taken under a fluorescence microscope. Detailed Implementation

[0070] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0071] Example 1

[0072] This embodiment describes a method for expanding a single cell (flow diagram shown below). Figure 1 (As shown), including the following steps:

[0073] (1) Introducing polymerizable anchoring agents into pre-fixed single cells;

[0074] Specifically, human colonic epithelial cells Caco-2 were first selected as model cells and seeded in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixed antibiotic solution (100X penicillin-streptomycin solution, NO.E607011, Sangon Biotech (Shanghai) Co., Ltd.). The cells were then cultured in a constant temperature incubator at 37 ℃ and 5% CO2. After the cells reached the logarithmic growth phase, two aliquots of approximately 1×10⁻⁶ cells were... 6 Caco-2 cells were collected by centrifugation and resuspended in calcium- and magnesium-free PBS solution; paraformaldehyde (PFA) solution was added to a final concentration of 4%, and the cells were fixed at room temperature for 10 min; after fixation, the cells were washed twice with 1×PBS; RNase inhibitor (MurineRNase inhibitor (40 U / μL), NO. 10603ES05, ​​Yisheng Biotechnology (Shanghai) Co., Ltd.) was added to a final concentration of 1 U / μL, and the cells were temporarily stored at 4℃.

[0075] One of the washed cell samples was used as a control group, and the other washed cell sample was resuspended in a solution containing 10% N-(3-aminopropyl)methacrylamide hydrochloride (APMA), while an equal volume of 4% (i.e., 4 g / 100 mL) paraformaldehyde solution was added, so that the cells were in an environment where APMA and free formaldehyde coexisted, and incubated at 4 ℃ for 12 h.

[0076] (2) Introduce a photosensitive initiation anchor into Caco-2 cells treated in step (1);

[0077] Specifically, the Caco-2 cells treated in step (1) were washed twice with 1× PBS buffer containing 0.05% Tween-20, resuspended in 1.0 mg / mL 4-benzoylbenzamide solution, and incubated at room temperature for 1 h in the dark.

[0078] The photoinitiator and anchor used in this embodiment, 4-benzoylbenzoic acid-N-succinimide ester (CAS No.: 91990-88-4; hereinafter referred to as BBS-NHS), is a benzophenone-based photoinitiator and anchor. Its molecular structure contains a benzophenone photoinitiator group and an activated ester functional group that can anchor to intracellular macromolecules.

[0079] (3) Introduce hydrogel monomers into the Caco-2 cells treated in step (2);

[0080] Specifically, 100 μL of 40% acrylamide stock solution, 400 μL of 20% sodium acrylate stock solution, 50 μL of 2% N,N′-methylenebisacrylamide (Bis) stock solution, 100 μL of 10% ammonium persulfate (APS) stock solution, 100 μL of 1 mol / L NaCl stock solution, and 5 μL of 1% Triton X-100 stock solution were mixed to obtain 1 mL of a hypertonic solution containing hydrogel monomers.

[0081] Then, the Caco-2 cells treated in step (2) were washed twice with 1× PBS buffer containing 0.05% Tween-20, resuspended in the hypertonic solution containing the hydrogel monomer, and incubated at room temperature for 1 h in the dark.

[0082] (4) The Caco-2 cells treated in steps (1), (2) and (3) were subjected to photo-induced treatment to induce hydrogel polymerization;

[0083] Specifically, Caco-2 cells treated in steps (1), (2) and (3) are exposed to ultraviolet light for 3 minutes to stimulate the photosensitive groups on BBS-NH in the cells, which triggers the polymerization of monomers such as acrylamide / sodium acrylate / Bis mainly in the cell interior to form a hydrogel network and form a small number of attached structures on the cell surface (no continuous hydrogel structure is formed in the extracellular environment), thereby achieving in situ embedding of single cells.

[0084] (5) Place a single cell with hydrogel inside into a hypotonic solution to swell;

[0085] Specifically, the cells treated in step (4) were first washed three times with 1×PBS buffer containing 0.05% Tween-20 to remove residual hydrogel monomers from the extracellular environment. Then, the cells were resuspended in 0.1 g / mL proteinase K solution and treated at 50 °C for 10 min to remove most of the intracellular proteins and soften the structure. Subsequently, the cells were resuspended in 1×PBS buffer (i.e., hypotonic solution) for 30-60 min. This buffer has a lower osmotic pressure than the aforementioned hypertonic solution, causing the hydrogel formed inside the cells to absorb water and swell, thereby achieving the expansion of single cells, single nuclei, or single subcellular structures. Finally, the cells were washed three times with 1×PBS buffer containing 0.05% Tween-20.

[0086] The monodisperse expanded Caco-2 cells obtained above and the control group Caco-2 cells that were fixed and washed were used to fluoresce the cell nuclei with 4',6-diamidino-2-phenylindole (DAPI) and observed under white light and fluorescence microscopes. Figure 2 The observation results showed that after expansion, the Caco-2 cells had intact structure and no obvious lysis, and the cell diameter increased by about 2 to 3 times compared with the control group.

[0087] The monodisperse expanded Caco-2 cells obtained above and the control group Caco-2 cells that were fixed and washed were taken, and the cells were fixed, dehydrated, and critically dried before being observed by scanning electron microscopy. Figure 3 The results showed that the internal structure of cells in the swelling group was significantly looser than that in the control group, the overall volume was larger, and the cell morphology was well maintained.

[0088] Example 2

[0089] This embodiment describes a method for expanding a single cell, which is basically the same as that in Embodiment 1, except that:

[0090] 1. Human embryonic kidney cells 293T were used as model cells;

[0091] 2. The polymerizable anchoring agent used in step (1) is N-(2-aminoethyl)methacrylamide, and its introduction method, fixation conditions and incubation method are the same as those in Example 1;

[0092] 3. The photosensitive initiation anchoring agent used in step (2) is 2-aminothioxanthone. The 293T cells treated in step (1) are resuspended in a solution containing 0.5 mg / mL of 2-aminothioxanthone and incubated at room temperature for 2 h under light-protected conditions.

[0093] 4. In step (4), ultraviolet light is used to irradiate for 4 min to induce hydrogel polymerization.

[0094] The resulting monodisperse expanded 293T cells and control cells that were fixed and washed were stained with 4',6-diamidino-2-phenylindole (DAPI) for their nuclei and observed under white light and fluorescence microscopes.

[0095] Figure 4 The results showed that the expanded 293T cells had intact structure and no obvious lysis. The cell diameter was about 2 to 3 times larger than that of the control group, and the volume was significantly expanded.

[0096] Example 3

[0097] This embodiment describes a method for expanding a single cell, which is basically the same as that in Embodiment 1, except that:

[0098] 1. Mouse fibroblast 3T3-L1 cells were used as the model cells;

[0099] 2. The polymerizable anchoring agent used in step (1) is N-(3-aminopropyl)acrylamide, which is introduced by incubation at 4 ℃ for 10 h;

[0100] 3. In step (3), the hypertonic solute in the hypertonic solution is sucrose, and the rest of the composition is the same as in Example 1.

[0101] The obtained monodisperse expanded 3T3-L1 cells and control cells that were fixed and washed were stained with DAPI and observed under white light and fluorescence microscopes.

[0102] Figure 5 The results showed that the expanded 3T3-L1 cells had intact structure and no obvious lysis. The cell diameter was about 2 to 3 times larger than that of the control group, and the volume was significantly expanded.

[0103] Example 4

[0104] This embodiment describes a method for expanding a single cell, which is basically the same as that in Embodiment 1, except that: human embryonic kidney cells 293T and mouse fibroblasts 3T3-L1 are mixed in a 1:1 ratio to obtain a human-mouse cell hybrid sample; this human-mouse cell hybrid sample is used as the model cell.

[0105] The obtained monodisperse expanded cells and the control group cells that were only fixed and washed were respectively applied to single-cell transcription library construction and high-throughput sequencing based on the microfluidic droplet platform. The overall process was carried out in accordance with the method described in Example 3 of Chinese Invention Patent Application No. 202310156589.3, and will not be repeated in this example.

[0106] Figure 6 The qPCR results showed that the Ct value of the expanded group samples decreased by about 3 cycles compared with the control group, which suggests that the cDNA capture amount increased by about 8 times.

[0107] Figure 7 The results of nucleic acid gel electrophoresis showed that the cDNA bands obtained from the swollen group samples were distributed in the range of about 150 to 400 bp. The overall bands were slightly larger than those in the control group and were more diffuse, with no obvious single concentrated main band, suggesting that the captured transcript lineage was more abundant.

[0108] The amplified cDNA underwent end repair and A-tailing using the TA cloning adapter library construction method, and sequencing adapters were ligated using a library construction kit to obtain sequencing libraries. High-throughput sequencing was performed using the Illumina sequencing platform. The sequencing data were preprocessed, and unique molecular identifiers (UMIs) and cell-specific barcodes were extracted from Read1. Read2 was then aligned using STARsolo to generate a gene expression matrix.

[0109] Figure 8 The results showed that higher UMI numbers and gene numbers were detected in cells after the swelling treatment.

[0110] In summary, the experimental results show that, compared with the unexpanded control samples, the cells expanded by the expansion method of this invention exhibit higher nucleic acid capture efficiency and richer molecular detection depth in single-cell transcriptomics experiments.

[0111] Example 5

[0112] This embodiment provides a method for expanding intracellular cell nuclei. This method is essentially the same as the single-cell expansion method described in Embodiment 1, except that: cell nuclei isolated from mouse muscle tissue are used as the expansion target, and the isolated cell nuclei are obtained through a gentle lysis method before expansion. Specifically, it includes:

[0113] (1) Introducing a polymerizable anchoring agent into a pre-fixed single-cell nucleus;

[0114] Specifically, fresh or pre-preserved mouse muscle tissue was first taken and washed in pre-chilled 1×PBS buffer to remove blood and impurities. The muscle tissue was then minced and resuspended in lysis buffer containing 0.1% Triton X-100. The tissue was gently homogenized on ice and a disposable homogenizer was used for gentle homogenization to promote cell membrane lysis and release of the nucleus. After lysis, unlyzed tissue fragments were removed by low-speed centrifugation, and the nucleus pellet was collected by further low-speed or medium-speed centrifugation. The obtained nuclei were gently washed twice with 1×PBS buffer to obtain structurally intact isolated nuclei.

[0115] The resulting cell nuclei were then resuspended in a solution containing 10 wt% N-(2-aminoethyl)methacrylamide, and a small amount of paraformaldehyde was added as a crosslinking bridging agent (the final concentration of paraformaldehyde in the reaction system was 1 wt%) to promote the anchoring reaction. The mixture was then incubated at 4°C for 8 h.

[0116] (2) Introducing a photosensitive initiation anchor into a single cell nucleus;

[0117] Specifically, the cell nuclei treated in step (1) were washed with 1× PBS buffer and then resuspended in a solution containing 1.0 mg / mL 2-aminothioxanthone and incubated at room temperature for 1 h under light-protected conditions.

[0118] (3) Introducing hydrogel monomers into single cell nuclei;

[0119] Specifically, the cell nuclei treated in step (2) were resuspended in a hypertonic solution containing hydrogel monomers. The hypertonic solution consisted of: 20 wt% acrylamide, 15 wt% sodium acrylate, 0.10 wt% N,N'-methylenebisacrylamide, 0.10 wt% ammonium persulfate, 0.5 mol / L sodium chloride, 0.05 wt% Triton X-100, with the remainder being deionized water. The solution was incubated for 1 h in the dark.

[0120] (4) Photo-induced treatment of the single cell nuclei treated in step (3) to induce hydrogel polymerization;

[0121] Specifically, the sample was exposed to ultraviolet light for 5 minutes to excite the photosensitive initiating groups anchored in the cell nucleus, thereby inducing the polymerization reaction of hydrogel monomers in the cell nucleus and its adjacent surface areas to form a hydrogel network.

[0122] (5) The single cell nucleus with hydrogel inside is placed in a hypotonic solution and swells;

[0123] Specifically, the cells were first washed with 1×PBS buffer to remove residual monomers. Then, the nuclei were resuspended in a solution containing 0.1 g / mL trypsin and treated at 45°C for 10 min to remove some nuclear proteins and soften the nuclear structure. Subsequently, the treated nuclei were placed in 1×PBS buffer for 30 min to allow the hydrogel formed inside the nucleus to absorb water and swell, thereby achieving the expansion of the nucleus.

[0124] The swollen cell nuclei and the unswollen control group cell nuclei were observed under a microscope.

[0125] Figure 9 The results showed that the expanded mouse muscle cell nuclei had intact structures and good morphology, and the nucleus diameter was about 2 times larger than that of the control group, indicating that the method of the present invention can achieve the expansion of cell nuclei.

Claims

1. A method for expanding a single cell, a single cell nucleus, or a single subcellular structure, characterized in that, include: The steps involve dividing the raw materials used to form the hydrogel into at least two groups and introducing them into single-cell, single-nucleus, or single-subcellular structures, respectively. The steps involved in polymerization to form a hydrogel; The step of placing a single cell, single nucleus, or single subcellular structure containing a hydrogel inside into a hypotonic solution to swell.

2. The expansion method as described in claim 1, characterized in that, Includes the following steps: (1) Introducing polymerizable anchoring agents into pre-fixed single-cell, single-nucleus, or single-subcellular structures; The polymerizable anchoring agent has polymerizable groups for participating in free radical polymerization reactions, and a first anchoring functional group for anchoring macromolecules within cells, cell nuclei, or subcellular structures. (2) Introduce photosensitive initiation anchoring agents into single cells, single cell nuclei, or single subcellular structures; The photosensitive initiating anchoring agent has a photosensitive initiating group and a second anchoring functional group for anchoring macromolecules in cells, cell nuclei or subcellular structures; (3) Introducing hydrogel monomers into single-cell, single-nucleus, or single-subcellular structures; (4) Photo-induced treatment of single cells, single nuclei or single subcellular structures treated in steps (1), (2) and (3) to induce hydrogel polymerization; (5) Place a single cell, single cell nucleus or single subcellular structure with hydrogel inside into a hypotonic solution to swell.

3. The expansion method as described in claim 2, characterized in that, The macromolecules mentioned include at least one of polypeptides and nucleotide chains; The nucleus or subcellular structure is a nucleus or subcellular structure located inside the cell, or the nucleus or subcellular structure is a nucleus or subcellular structure independent of the cell. The subcellular structures include the endoplasmic reticulum, mitochondria, Golgi apparatus, ribosomes, chloroplasts, vacuoles, and centrosomes.

4. The expansion method as described in claim 2, characterized in that, The first anchoring functional group and the second anchoring functional group achieve anchoring of the macromolecule through covalent or non-covalent interactions with the macromolecule; The first anchoring functional group and the second anchoring functional group are independently selected from at least one of the following: functional groups that target the amino group of a polypeptide, functional groups that target the thiol group of a polypeptide, functional groups that target the carboxyl group of a polypeptide, functional groups that target the hydroxyl or phosphate group of a nucleotide, general functional groups that participate in macromolecular crosslinking or labeling, and functional groups that are coupled to macromolecules through crosslinking bridging agents. The polymerizable group is an unsaturated group that has free radical polymerization reactivity or photoinduced polymerization reactivity; The photosensitive initiating group is a photosensitive group that generates free radicals or active intermediates under light irradiation conditions.

5. The expansion method as described in claim 4, characterized in that, A crosslinking bridging agent is introduced simultaneously with a polymerizable anchoring agent or a photosensitive initiating anchoring agent to promote crosslinking between the first anchoring functional group or the second anchoring functional group and the target group; The polymerizable anchoring agent is selected from N-(3-aminopropyl)methacrylamide hydrochloride, N-(3-aminopropyl)acrylamide, and N-(2-aminoethyl)methacrylamide; The photosensitive initiating anchoring agent is selected from at least one of the following: 4-benzoylaniline, 4,4'-diaminobenzophenone, 2-aminothioxanthone, 2-aminoanthraquinone, 4-aminoacetophenone, and 4-benzoylbenzoic acid-N-succinimide ester.

6. The expansion method as described in claim 4, characterized in that, In step (2), a UV photoinitiator is introduced into the single cell at the same time as the photoinitiator is introduced to improve the initiation efficiency.

7. The expansion method as described in claim 1, characterized in that, In step (1), the pre-fixed single cells, nuclei or subcellular structures are resuspended in a solution containing a polymerizable anchoring agent and incubated at 4°C to room temperature for 1-14 h to introduce the polymerizable anchoring agent; In step (2), single cells, nuclei, or subcellular structures are resuspended in a solution containing a photosensitive initiation anchoring agent and incubated at 4°C to room temperature for 1-14 h under light-protected conditions to introduce the photosensitive initiation anchoring agent. In step (3), single cells, nuclei or subcellular structures are resuspended in a hypertonic solution containing hydrogel monomers and incubated at 4°C to room temperature for 1-14 h under light-protected conditions to introduce hydrogel monomers. There is no necessary sequential dependence between steps (1), (2) and (3), so they can be performed in any order. However, at least after the first two steps are completed, the single cell, nucleus or subcellular structure should be resuspended in buffer solution before proceeding to the next step.

8. The expansion method as described in claim 7, characterized in that, The hypertonic solution contains, by mass fraction: The composition consists of 5-30% amide monomer, 1-20% carboxylic acid monomer, 0.01-2% crosslinking agent, 0.01-2% free radical initiator, 0.01-1% anti-agglomeration agent, 0.1-5% hypertonic solute, and the balance being water. The crosslinking agent is selected from at least one of N,N′-methylenebisacrylamide, divinyl sulfone, and polyethylene glycol diacrylate; the free radical initiator is selected from at least one of sodium persulfate, ammonium persulfate, and azobisisobutyronitrile; the anti-agglomeration agent is selected from nonionic surfactants; and the hypertonic solute is selected from at least one of sodium chloride, potassium chloride, sucrose, and mannitol.

9. The expansion method as described in claim 7, characterized in that, In step (5), the proteins inside a single cell, a single cell nucleus, or a single subcellular structure are first enzymatically digested, and then placed in a hypotonic solution to swell.

10. The application of the expansion method as described in any one of claims 1-9 in transcriptomic, genomic, proteomic, or spatial omics analysis of single-cell, single-nucleus, or single-subcellular structures.

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