Method for labeling APC and t cells on basis of lip3000 liposome transfection
By using the Lipofectamine 3000 liposome transfection method, oligonucleotide fragment tag sequences are delivered into cells, solving the problems of cross-contamination and low labeling efficiency in existing technologies. This enables high-throughput, low-cost single-cell sample labeling, supporting APC and T cell labeling and tumor neoantigen screening in TCR-T cell therapy.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- SHENZHEN HUADA GENE INST
- Filing Date
- 2025-01-26
- Publication Date
- 2026-07-30
AI Technical Summary
Existing cell labeling methods are prone to cross-contamination, have low labeling efficiency, limited application of endogenous tags, and are costly. Furthermore, existing technologies struggle to achieve high-throughput, low-cost multiplexing of single-cell samples.
Using Lipofectamine 3000 liposomes as a carrier, oligonucleotide fragment tag sequences were delivered into cells. Cell labeling was performed using droplet microfluidic technology, achieving efficient and low-cost single-cell sample labeling while avoiding tag detachment and cross-contamination.
It achieves efficient and low-cost single-cell sample labeling, significantly improves the high-throughput of cell pairing analysis, reduces cross-contamination rate, is suitable for APC and T cell labeling in TCR-T cell therapy, and supports efficient screening of TCR and tumor neoantigens.
Smart Images
Figure PCTCN2025075136-FTAPPB-I100001 
Figure PCTCN2025075136-FTAPPB-I100002 
Figure PCTCN2025075136-FTAPPB-I100003
Abstract
Description
A method for transfecting and labeling APCs and T cells based on LIP3000 liposomes Technical Field
[0001] This invention belongs to the field of biotechnology and relates to a new method for transfecting labeled APCs and T cells based on LIP3000 liposomes (adjuvant TCRT therapy). Background Technology
[0002] With the continuous development of gene sequencing and bioinformatics analysis tools, single-cell sequencing technology is gaining increasing importance in biomedical research because population analysis cannot resolve differences in expression between cells. Single-cell sequencing technology helps researchers understand various biological functions at the single-cell level and reveals complex cell interaction mechanisms. In recent years, research in tumor immunotherapy has made rapid progress, with adoptive cell transfer therapy (ACT) showing promising clinical efficacy. Adoptive T-cell transfer therapy screens T cells that specifically recognize tumor antigens, undergoes long-term in vitro clonal culture, and then reinfuses them into the patient to mediate durable cancer regression.
[0003] Chimeric antigen receptor-modified T cells (CAR-T) and T cell receptor-T cell (TCR-T) are two of the most promising treatment technologies in adoptive cell transfer therapy.
[0004] Chimeric T-cell therapy (TCR-T) is one of the most promising technologies for targeted cancer therapy. It involves modifying patient-derived T cells to express receptors on their surface that recognize tumor antigens, thereby precisely killing tumor cells. Because T cells are heterogeneous, different T cells respond differently to tumor cells. Therefore, cell samples need to be labeled to allow for screening and analysis of T cell antigen specificity and other characteristics at the single-cell level.
[0005] In T-cell receptor T-cell therapy, HLA (human leukocyte antigen, an expression product of the human major histocompatibility complex (MHC)) on antigen-presenting cells (APCs) presents tumor antigen peptide fragments on the surface of APCs. Genetically modified TCRs can recognize these tumor peptide fragments on the surface of APCs with high affinity and specificity, thereby activating the T cells' ability to kill tumor cells. Because T cells are heterogeneous, different T cells respond differently to tumor cells. Therefore, it is necessary to label T cell and APC samples to enable screening and analysis of T cell antigen specificity and other characteristics at the single-cell level.
[0006] In TCR-T therapy applications, the major histocompatibility complex (MHC)-antigen peptide tetramer (MHC tetramer) technology has been developed. This technology consists of a complex of four MHC-antigen peptide monomers and a fluorescent dye, based on streptavidin, which crosslinks the four MHC monomers to form the MHC tetramer. The MHC tetramer can recognize and bind to the TCR on the surface of T cells. However, this technology is currently mainly implemented using well plates, and the throughput is limited by the well plates, resulting in low efficiency. Droplet microfluidics can generate high-throughput, independent droplet environments to act as bioreactors, and has shown significant advantages in TCR and tumor neoantigen screening. However, when using droplet microfluidics to pair TCR-T cells with APC cells, efficient labeling of cell samples is required to distinguish T cells carrying different TCRs or APC cells expressing different antigen fragments.
[0007] Currently, representative single-cell sequencing platforms such as Drop-Seq, inDrop, and 10X Genomics Chromium provide high-throughput single-cell information. However, due to the high cost of single-cell sequencing sample preparation, the large amount of manpower required, the ambiguity in single-cell identification, and batch effects, the application scope of single-cell sequencing technology remains limited.
[0008] Current methods for cell labeling utilize genetic barcoding, where each patient's single nucleotide polymorphism (SNP) serves as a sample tag sequence to identify each cell, enabling simultaneous pooling and sequencing of multiple samples. While this method partially addresses the multiplexing problem, its applicability is limited to samples with genetic differences.
[0009] Because cells highly express certain specific proteins on their cell membrane surfaces, some research teams have designed antibodies to link sample tag sequences, allowing these tags to bind to cells. This involves using antibodies linked to oligonucleotide chains as tags. This method can simultaneously detect gene expression levels and the expression levels of membrane-specific proteins. However, this method suffers from the risk of the membrane tags detaching and attaching to other cells, causing cross-contamination. Furthermore, some cells may not express certain membrane proteins, leading to tagging failure.
[0010] Some teams have also used lentivirus technology to transduce barcode oligonucleotides into cells, achieving long-term stable expression of barcodes in living cells. However, this method is time-consuming and costly.
[0011] One team proposed a barcoding strategy for single-cell sequencing samples based on concanavalin A. This strategy involves attaching concanavalin A or its analogues, linked to a sample tag sequence, to the cell membrane or nuclear membrane, allowing different cell samples to carry different tag sequences. This strategy is simple to operate and low in cost. However, the binding of concanavalin A to cell membrane glycoproteins is non-specific and not tight, leading to easy tag detachment and cross-contamination.
[0012] Therefore, single-cell sequencing analysis requires a low-cost and high-efficiency method to label APC and T cell samples under different experimental conditions in order to achieve sample reuse.
[0013] Invention Overview
[0014] To address the problems of cross-contamination, low labeling efficiency, and limited application of endogenous tags in existing cell labeling methods, this invention utilizes Lipo3000 transfection to deliver exogenous tag sequences (oligonucleotide chains) into cells via endocytosis, thereby labeling cell samples under different experimental conditions. This method ensures the tag sequence enters the cell without detachment and contamination of other cells. Furthermore, this invention employs droplet microfluidics to pair labeled APC cells with TCR-T cells, significantly improving the high-throughput of pairing analysis and enabling efficient screening of TCR and tumor neoantigens. Moreover, this method requires only a single RNA sequencing run to complete the sequencing analysis of all single-cell samples, offering low cost and high labeling success rate.
[0015] The key point of this invention lies in developing a highly efficient, low-cost, non-specific, and low-cross-contamination method for cell labeling. This invention enables the development of kits based on Lipo 3000 liposome transfection specifically labeled APCs and T cells. It can be applied to distinguish samples of different single cells through nuclear labeling, ensuring the label does not detach. This effectively solves the cross-contamination problem caused by other multi-path labeling methods currently on the market, and shows great promise.
[0016] In a first aspect, the present invention provides a method for labeling multiple cells, which delivers tag sequences of different cells into the corresponding cells to achieve labeling of multiple cells.
[0017] In the method described above, the carriers used for delivering the tag sequences of the different cells are liposomes, reagents required for electroporation, or reagents required for calcium phosphate transfection.
[0018] The method described above uses liposomes as a carrier to deliver tag sequences of different cells into the corresponding cells, thereby achieving the labeling of multiple cells.
[0019] In the method described above, the tag sequence for each type of cell is different.
[0020] And / or, the tag sequence of the cell is an oligonucleotide fragment.
[0021] The method described above, the method for labeling multiple cells, further includes the following steps:
[0022] 1) Separate labeled single cells using microfluidic technology;
[0023] 2) Capture the tag sequence for each single cell;
[0024] 3) Construct sequencing libraries and detect tag sequences.
[0025] In the method described above, the liposome is Lipofectamine 3000.
[0026] In the method described above, the step of using liposomes as a carrier to deliver the tag sequences of different cells into the corresponding cells is to use Lipofectamine 3000 to transfect the tag sequences of the different cells into the corresponding cells.
[0027] In the method described above, the cells are TCR-T cells.
[0028] In the method described above, the cells are APC cells or TCR-T cells.
[0029] In a second aspect, the present invention provides the application of the method described in the first aspect in single-cell sequencing;
[0030] Alternatively, the present invention provides the application of the method described in the first aspect in single-cell nuclear labeling;
[0031] Alternatively, the present invention provides the application of the method described in the first aspect in cell labeling in TCR-T;
[0032] Alternatively, the present invention provides the application of the method described in the first aspect in the screening of TCRs or tumor neoantigens.
[0033] Thirdly, the present invention provides a method for labeling the nuclear cells of a single cell, comprising the steps of the method described in the first aspect.
[0034] Fourthly, the present invention provides a method for labeling cells in TCR-T cells, which is a step of the method described in the first aspect for labeling cells in TCR-T cells.
[0035] Fifthly, the present invention provides a method for screening TCRs or tumor neoantigens, comprising the following steps: labeling TCR-T cells using the steps described in the first aspect, and then performing library construction and sequencing to achieve screening of TCRs or tumor neoantigens.
[0036] Sixthly, the present invention provides a single-cell sequencing method, comprising the following steps:
[0037] 1) Labeling cells using the steps of the method described in the first aspect,
[0038] 2) The labeled cells obtained in step 1) are used for library construction and sequencing to achieve single-cell sequencing.
[0039] In a seventh aspect, the present invention provides a kit for labeling various cell types, comprising the following:
[0040] 1) The liposomes described in the first aspect;
[0041] 2) The tag sequence described in the first aspect.
[0042] The present invention also includes the following forms: In the process of drug development, in order to capture the different responses of highly heterogeneous samples such as tumor cells to drugs, it is necessary to obtain single-cell gene expression profiles. In this context, a strategy for multi-channel single-cell transcriptome sequencing was designed. This strategy uses Lipofectamine 3000 to transiently transfect cells with short barcode oligonucleotides, labeling different cell samples such as HEK293T and NIH 3T3, which is a variation of the present invention. Other transfection methods for oligonucleotide tag sequences, such as electroporation and calcium phosphate transfection, can replace the Lipofectamine 3000 transfection method. Attached Figure Description
[0043] Figures 1 to 8 show the data from the machine. Embodiments of the present invention
[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0046] Example 1: Establishment of a method for transfecting labeled APCs and T cells using LIP3000 liposomes.
[0047] 1. Cell passage and plating:
[0048] The day before the experiment, APC cells and T cells were passaged and plated in six-well plates, with two wells for each type of cell (one as a negative control, without cell tagging during transfection, and the other as the experimental group). Each well contained 500,000 cells to ensure that the cell density was at least 80% by the time of transfection the next day.
[0049] 2. Lipofectamine TM 3000 transfections and cultures
[0050] The next day, after the cell density reached 80%, Lipofectamine was used. TM 3000 different cell tags were transfected into the cells, centrifuged for 1 hour, and then incubated at 37 degrees Celsius in a 5% carbon dioxide incubator for 4 hours.
[0051] 3. Database construction
[0052] After culturing for 4 hours, the cells were removed and used to construct a library using the BGI Genomics High-Throughput Single-Cell RNA Library Preparation Kit to obtain a single-cell RNA library.
[0053] 4. Single-cell sequencing, data processing and splitting
[0054] The single-cell RNA library was sequenced, and the resulting sequencing data was V2.
[0055] The V2 data was first filtered to identify those containing only two types of tag information. Then, by comparing the negative control data of APC cells and T cells with the experimental group data, it was analyzed whether APC cells and T cells contained transfected cell tags (with known sequences). Finally, it was examined whether Lipofectamine could be used to achieve the desired results. TM 3000 successfully transfected the cell tag into the cell.
[0056] Example 2: Transfection of labeled T2 cells and Jurkat cells using LIP3000 liposomes
[0057] I. The experimental materials used in this embodiment are as follows:
[0058] 1. 5E5 T2 cells
[0059] 2.5E5 Jurkat cells
[0060] 3. Lipofectamine TM 3000 (Brand: Invitrogen)
[0061] 4. BGI Genomics High-Throughput Single-Cell RNA Library Preparation Kit
[0062] II. Experimental Steps in this Embodiment
[0063] 1. Cell plating:
[0064] One day in advance, seed T2 cells and Jurkat cells in 6-well plates with 5E5 cells per well, two wells for each cell type, and one well as a negative control (containing only Lipofectamine). TM 3000, P3000 and Opti-MEM TM Culture medium (without cell labels), one well for the experimental group (using Lipofectamine). TM 3000 transfects cell tags into the cells; Lipofectamine TM 3000, P3000 and Opti-MEM TM (All culture media added).
[0065] Table 1 shows the cell locations in the well plate.
[0066] 2. Lipofectamine TM 3000 transfections
[0067] Lipofectamine was used on the second day. TM 3000 (Brand: Invitrogen): Refer to Lipofectamine TM According to the 3000 instruction manual, the following reagents should be prepared first:
[0068] Tube A: Since the reagent from tube A is required for all four wells, the amount needed for all four wells has been prepared directly.
[0069] Table 2 shows the reagents in tube A.
[0070] Table 3 shows the reagents in tube B.
[0071] Table 4 lists the reagents in tube C.
[0072] Table 5 shows the reagents in tube D (negative control reagents).
[0073] After preparing the above reagents, begin preparing the reagents for tubes D, E, and F.
[0074] E tube: Take 250 μl from each of tubes A and D, mix well by pipetting, and incubate at room temperature for 15 minutes;
[0075] F tube: Take 125ul from each of tubes A and B, mix well by pipetting, and incubate at room temperature for 15 minutes;
[0076] G tube: Take 125ul from each of tubes A and C, mix well by pipetting, and incubate at room temperature for 15 minutes;
[0077] After incubation at room temperature, add 250 μL of reagent from tube E to each of the two wells of the T2 cell negative control and the Jurkat cell negative control in the six-well plate; add 250 μL of reagent from tube F to the well of the T2 cell experimental group and 250 μL of reagent from tube G to the well of the Jurkat cell experimental group (after adding the reagents, thoroughly mix the cells by pipetting).
[0078] After mixing by pipetting, seal the six-well plate with sealing film and centrifuge it in a centrifuge (centrifugation conditions: speed: 300G, time: 1 hour). After centrifugation, incubate the cells in a 37°C, 0.5% CO2 incubator for 4 hours to obtain transfected cells.
[0079] 3. Database construction
[0080] The transfected cells obtained in step 2 above were taken out and used to construct libraries using the BGI Genomics High-Throughput Single-Cell RNA Library Preparation Kit to obtain RNA libraries for different cells.
[0081] 4. Sequencing and Data Processing
[0082] RNA libraries of different cells constructed in the above 3 were sequenced to obtain V2 data of sequencing results for cells in different wells (Figures 1-8).
[0083] The V2 data mentioned above first filters out data containing only two types of tag information. Then, by comparing the negative control data of T2 cells and Jurkat cells with the experimental group data, it is analyzed whether the T2 experimental group and Jurkat experimental group contain transfected cell tags (with known sequences), thus realizing the use of Lipofectamine. TM 3000 successfully transfected the cell tag into the cell. Industrial applicability
[0084] Currently, methods for labeling cells include endogenous genetic barcoding, antibody labeling, concanavalin A labeling, or using lentiviral technology to transduce barcoded oligonucleotides into cells. However, endogenous genetic barcoding is only applicable to samples with genetic differences. Antibody labeling involves the easy detachment of membrane tags that attach to other cells, causing cross-contamination. Furthermore, some cells may not express certain membrane proteins, leading to labeling failure. The binding of concanavalin A to cell membrane surface glycoproteins is non-specific and not tight, causing the tag sequence to easily detach and resulting in cross-contamination. Lentiviral transduction methods are time-consuming and costly.
[0085] The method of this invention has a transfection efficiency of up to 99%, a low cross-contamination rate during data splitting, and is non-specific, applicable to any cell type. Furthermore, Lipofectamine... TM The 3000 reagent kit is also relatively inexpensive, with one kit costing approximately 4000 yuan. Therefore, this invention has the advantages of high efficiency, low cross-contamination rate, and low cost due to lack of specificity.
Claims
1. A method for labeling multiple cells, wherein different cell tag sequences are delivered into the corresponding cells to achieve labeling of multiple cells.
2. The method according to claim 1, characterized in that: The delivery of the tag sequences for different cells uses liposomes, reagents for electroporation, or reagents for calcium phosphate transfection as carriers.
3. The method according to claim 1 or 2, characterized in that: The method involves using liposomes as carriers to deliver tag sequences of different cells into the corresponding cells, thereby achieving the labeling of multiple cell types.
4. The method according to any one of claims 1-3, characterized in that: The tag sequence is different for each type of cell. And / or, the tag sequence of the cell is an oligonucleotide fragment.
5. The method according to claim 3 or 4, characterized in that: The method for labeling multiple cells further includes the following steps: 1) Separate labeled single cells using microfluidic technology; 2) Capture the tag sequence for each single cell; 3) Construct sequencing libraries and detect tag sequences.
6. The method according to any one of claims 3-5, characterized in that: The use of liposomes as a carrier to deliver the tag sequences of different cells into the corresponding cells refers to the use of Lipofectamine 3000 to transfect the tag sequences of the different cells into the corresponding cells.
7. The method according to any one of claims 1-6, characterized in that: The cells mentioned are cells from TCR-T.
8. The method according to claim 7, characterized in that: The cells are APC cells or TCR-T cells.
9. The application of the method according to any one of claims 1-8 in single-cell sequencing; Or the application of the method described in any of claims 1-8 in single-cell nuclear labeling; Or the application of the method described in any of claims 1-8 in cell labeling in TCR-T; Or its application in the screening of TCR or tumor neoantigens using any of the methods described in claims 1-8.
10. A method for labeling the nuclear cells of a single cell, comprising the steps of any one of the methods described in claims 1-8.
11. A method for labeling cells in TCR-T cells, comprising labeling TCR-T cells using the steps of any one of claims 1-8.
12. A method for screening TCRs or tumor neoantigens, comprising the following steps: labeling TCR-T cells using the steps of any one of the methods described in claims 1-8, and then performing library construction and sequencing to achieve screening of TCRs or tumor neoantigens.
13. A single-cell sequencing method, comprising the following steps: 1) Labeling cells using the steps of any one of claims 1-8, 2) The labeled cells obtained in step 1) are used for library construction and sequencing to achieve single-cell sequencing.
14. A kit for labeling multiple cell types, comprising the following: 1) The liposomes according to any one of claims 2-8; 2) The tag sequence of any one of claims 2-8.