Target cell lentivirus infection rate detection method based on dichotomy
By employing a cell-dichotomy method, the accuracy and cost issues of existing lentivirus infection rate detection methods are resolved. This provides an efficient and low-cost method for infection rate determination that does not require exogenous markers or equipment, and is applicable to the detection of infection rates in various target cells.
Patent Information
- Application Number
- CN202511481412.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-16
- Publication Date
- 2026-01-30
AI Technical Summary
Existing methods for detecting lentivirus infection rates suffer from problems such as high subjectivity, high equipment dependence, high cost, and inaccurate results. In particular, they are difficult to accurately measure infection rates when infection is difficult in primary cells and T cells.
The cell dichotomy method was used, in which target cells were infected and prepared into single-cell suspensions and divided into two parallel samples. One sample was treated with antibiotics and the other was used as a control group for cell counting. The infection rate was calculated by combining cell counts at three key time points.
It achieves accurate detection without the need for external labeling and special equipment, reduces experimental costs, is applicable to a variety of experimental scenarios, and improves the accuracy and reliability of detection results.
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Figure CN121428059A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for quantitatively detecting infection rate of lentivirus on target cells. BACKGROUND
[0002] Lentivirus (LV) belongs to the family of retroviruses and the genus of lentivirus, and has unique advantages in the field of gene editing. It can accommodate up to 10 kb of target fragments for efficient infection in various cell division and non-division stages, and stably integrate the carried exogenous DNA fragments into the genome of target cells for sustained expression. Among them, human immunodeficiency virus type 1 (HIV-1) is the most studied and widely used lentivirus. Its safety in use has been improved by biological means to delete its virulence-related genes, split auxiliary genes, introduce self-inactivation design and increase safety switches. At present, lentivirus vectors have been deeply involved in basic biological research and clinical treatment, and are an indispensable important tool in gene editing tool delivery and stable cell line construction and other application scenarios. At the same time, through optimization of vector design and targeting strategy, there is still potential to further expand its application range.
[0003] After the preparation of lentivirus, obtaining the infection rate data of the lentivirus preparation of this batch on target cells is the premise of realizing accurate controllability and good reproducibility in subsequent experiments. Some researchers will measure the titer of lentivirus after packaging, but due to the difficulty of infection of primary cells and T cells, it is still necessary to further test the actual infection rate of target cells. For example, when constructing a primary stable cell line with the help of lentivirus, too low infection rate will cause waste of cells, and too high concentration of lentivirus will cause certain toxicity to target cells, affecting cell viability. The infection rate determination result can help the experimenter to choose between the two. In some special scenarios, such as CRISPR screening, which requires one sgRNA in the library to enter one target cell, and in order to save the amount of cells, it is best to limit the infection rate to the range of 20%-50%, at this time the determination of target infection rate is essential.
[0004] The existing methods for determining the infection rate of lentivirus target cells mainly include fluorescence labeling combined with microscope counting and flow cytometry analysis, qPCR detection, antibiotic screening combined with cell counting, etc. The method based on fluorescence labeling is highly subjective, and the experimental operation of flow cytometry is relatively complex, and it is highly dependent on experimental equipment. Primary cells are also not suitable for this method due to the interference of spontaneous fluorescence. In addition, both methods require a reporter gene, and the application scenario is limited. The qPCR detection method for detecting the genomic copy number of lentivirus integration requires cell genome extraction and qPCR detection operations. The experimental results are easily affected by the efficiency of the kit, and it is difficult to distinguish between free viruses and integrated viruses, which often overestimates the infection rate of lentivirus. Commercial kits are mostly based on the qPCR detection method, which has the same problems as above, and increases the additional experimental cost. The traditional antibiotic screening combined with cell counting method for determining the infection rate of lentivirus uses the number of cells in a single sample as the denominator for infection rate calculation. Due to the different infection amounts of different lentiviruses, the cell viability is different, so the number and state of cells in each group are different. The single total cell number cannot reflect the true situation of each sample, which will cause deviation in the calculation of the infection rate. SUMMARY
[0005] In order to solve the problems of the prior art, the present application provides a method for accurately detecting the infection rate of lentivirus based on cell bisection, which does not require specific markers and has a wide range of application scenarios, and can reflect the true infection ability of lentivirus in target cells.
[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows: A method for quantitatively detecting the infection rate of lentivirus on target cells based on cell bisection, comprising: Infecting target cells with a lentivirus-containing sample to be tested; preparing target cells integrated with lentivirus into a single cell suspension, dividing it into two parallel samples with the same number and state and inoculating to obtain antibiotic-treated cells and cells without antibiotic treatment; Counting the positive infection cells Ne after adding antibiotics to the drug screening group; at three time points when performing bisection operation, when antibiotics start screening, and after antibiotics screening is completed, counting the control group without adding drugs can obtain total cell numbers No.2, No.1 and No.3; based on the total cell number, the infection rate of lentivirus on target cells is calculated.
[0007] In some embodiments, the infection of target cells with a lentivirus-containing sample to be tested is to add lentivirus to the target cell culture system using a 1 / 2 small volume infection method; when lentivirus infects target cells, replace 1 / 2 of the culture volume with fresh culture medium, and supplement the other half of the culture system after lentivirus infection for a period of time.
[0008] In some embodiments, the polystyrene sulfonate is added to promote the lentivirus transduction in the step of infecting the target cells with the lentivirus-containing sample to be tested.
[0009] In some embodiments, the other half of the culture medium is supplemented after 4 hours of lentivirus infection in the step of infecting the target cells with the lentivirus-containing sample to be tested.
[0010] In some embodiments, the bisecting operation is performed at 24 hours of lentivirus infection in the step of infecting the target cells with the lentivirus-containing sample to be tested.
[0011] In some embodiments, after the sample is prepared into a single-cell suspension and is equally divided, it is inoculated into a culture container with the same or larger volume as the original one.
[0012] In some embodiments, the calculation formula for obtaining the infection rate of the lentivirus to the target cells based on the total number of cells is: Infection rate.1 = Ne / No.1 x 100% Infection rate.2 = Ne / No.2 x 100% Infection rate.3 = Ne / No.3 x 100%.
[0013] In some embodiments, after obtaining the No.2 data of the control group, the cells do not need to be inoculated into a new culture container; after performing the cell bisecting operation, the control group is added with normal complete culture medium for continuous culture until the drug screening group starts to add drugs; when the drug screening group replaces the complete culture medium containing antibiotics, the control group is replaced with normal complete culture medium for continuous culture until the end of the antibiotic screening. When calculating the infection rate, Ne and No are uniformly used as the number of living cells or the total number of cells.
[0014] In some embodiments, the lentivirus titer is known, and different MOI gradients are set when the lentivirus is infected. When the lentivirus titer is unknown, the lentivirus concentration multiple should be considered, and different lentivirus volume gradients are set.
[0015] In some embodiments, human umbilical cord-derived mesenchymal stem cells are used as the target cells.
[0016] Compared with the prior art, the application has the following beneficial effects: In view of the deficiencies of the existing lentivirus infection rate detection methods, the present application is based on a cell bisection method to accurately determine the lentivirus infection rate, which can ensure that the number and state of the drug screening group and the control group cells are completely the same, does not require any reporter gene and exogenous marker, does not depend on kits and special instruments and equipment, has strong operability, low detection cost, and is widely applicable to various experimental scenarios. In addition, the present application also compares the infection rate calculation results of different experimental nodes for sampling, and selects the optimal scheme to ensure the accuracy of the detection results.
[0017] Further, the present application ensures that the initial number and state of the drug screening group and the control group cells are completely the same through the cell bisection method, and accurately determines the actual infection rate of the lentivirus to different target cells on this basis. At the same time, according to practical experience, the calculation method of infection rate.1 is the most accurate, but the optimal value is not unique under different target cell types and states.
[0018] Further, the present application has a simple operation method, which greatly reduces the experimental cost. Without any additional markers, it is not limited by experimental equipment such as flow cytometry, and does not require genome extraction or qPCR experimental operations.
[0019] Further, the present application has a wide range of applications and is applicable to various experimental scenarios and various target cells. After obtaining the lentivirus suspension, this method can be used to determine the infection rate of lentivirus to target cells under different volumes. General determination methods can also be used to preliminarily obtain lentivirus titer data, and then the present application can be used to verify the actual infection rate of cells under different MOIs, especially in cases where there are specific requirements for the infection rate. In addition, for some primary cells or T cells that are difficult to infect, the lentivirus titer data determined by non-target cells are usually quite different from the actual infection rate, and the detection of the infection rate is essential. BRIEF DESCRIPTION OF DRAWINGS
[0020] The accompanying drawings are used to illustrate the principles and operation of the present application, and facilitate a further understanding of the present application, and constitute a part of the specification. The illustrative embodiments and descriptions of the present application are used to explain the present application, and do not constitute an improper limitation on the present application.
[0021] Figure 1 A schematic diagram of the principle of cell bisection method for determining the infection rate of lentivirus to target cells; Figure 2 An experimental operation flowchart for quantitatively detecting the infection rate of lentivirus to target cells; Figure 3 Cell density of hMSCs in different samples when calculating infection rate.1 Figure 1 ; Figure 4 Cell density of hMSCs in different samples when calculating infection rate.1 Figure 2 . DETAILED DESCRIPTION
[0022] In order to more clearly show the technical solutions and advantageous features of the present application, the present application is described in detail below in combination with the drawings and examples. It should be noted that the examples shown are only one application demonstration of the present application and cannot be understood as limiting the embodiments and protection scope of the present application. Unless otherwise specified, the scientific and technical terms used herein have the same meaning as understood by the general skilled person in the same field as the present application; the reagents and materials and instruments used in the present application, such as those not mentioned by the manufacturer, are conventional goods, which are prepared and used in a conventional manner or in a manner recommended by the product instructions; the detection methods used in the present application, such as those not specifically mentioned, are conventional methods.
[0023] It should be noted that after reading the content of the present application, the experimenters can make modifications to the present application, but equivalent embodiments that do not deviate from the basic principles of the present application are within the protection scope of the present application.
[0024] The present application relates to a method for quantitatively detecting the infection rate of lentivirus on target cells, in particular to a precise determination technology for specific infection rate of lentivirus on target cells by cell bisection method. It is suitable for virus production process optimization, gene therapy, cell function research and other fields, especially for lentivirus infection efficiency evaluation of cells such as primary cells which are difficult to infect.
[0025] The present application takes human umbilical cord-derived mesenchymal stem cells (hMSCs) as the target cell model. After lentivirus infection, cell bisection method is used to obtain two groups of parallel samples with the same number and state of cells, which are defined as drug screening group (antibiotic treated cells) and control group (no antibiotic treated cells) respectively. The drug screening group performs antibiotic screening to obtain the number of positive infected cells, and the control group does not add antibiotics to count the total number of cells when the drug screening group is added. The actual cell infection rate is calculated according to the number of positive infected cells and the total number of cells.
[0026] Further, the method for quantitatively detecting the infection rate of lentivirus on target cells based on cell bisection method comprises the following steps: (1) Cell bisection method operation The target cells are infected with the sample to be tested containing recombinant lentivirus. The target cells integrated with lentivirus are prepared into a single cell suspension, which is divided into two parallel samples with the same number and state and inoculated into new culture containers to obtain antibiotic treated cells (drug screening group) and no antibiotic treated cells (control group).
[0027] Specifically, a small volume infection method is used, and only half of the cell culture volume of medium is added when lentivirus infects target cells. This operation can increase the contact opportunity between lentivirus and target cells and improve the infection efficiency. The other half of the culture system is then supplemented.
[0028] After lentivirus gradient infection of the target cells, all cell samples are prepared into single cell suspensions, and then each sample is divided into two parts and reseeded into two culture containers to obtain a drug screening group and a control group, so that the initial cell number and state of the two parallel samples are completely the same. After the exogenous fragment in the lentivirus is fully integrated into the genome of the target cells, the corresponding antibiotic is added to the drug screening group according to the resistance gene carried by the target plasmid, and the positive infection cells are screened for a sufficient time. The control group does not add the antibiotic to obtain the total cell number.
[0029] (2) Cell counting and infection rate calculation After the drug screening group is added with the antibiotic for screening, the positive infection cells Ne are counted. At three time points of performing the bisection operation, starting the antibiotic screening, and completing the antibiotic screening, the control group without the drug is counted to obtain the total cell number No.2, No.1 and No.3. The infection rate of the lentivirus to the target cells is calculated.
[0030] The calculation formula is: Infection rate.1 = Ne / No.1 x 100% Infection rate.2 = Ne / No.2 x 100% Infection rate.3 = Ne / No.3 x 100%.
[0031] By performing the cell bisection operation after the lentivirus infects the target cells, the initial cell number and cell state of the antibiotic-treated cells (the drug screening group) and the cells without the antibiotic treatment (the control group) are completely the same. Meanwhile, the time node of sampling the control group is optimized, the control group cell number at the time when the screening group sample starts to be added with the drug is selected as the denominator for the infection rate calculation, and the accuracy of the experimental results is further improved. The application discloses a method for simply and efficiently determining the actual infection rate of lentivirus to target cells without the aid of exogenous markers and other instruments and equipment, which has a wide application range, strong operability, and low cost.
[0032] The specific steps are as follows: After the antibiotic screening is completed, the positive infection cell number of the drug screening group is counted and recorded as Ne. At the time when the antibiotic starts to be screened, the cell sample in the control group is prepared into a single cell suspension, and the total cell number at the time when the antibiotic starts to be screened is counted and recorded as No.1. Finally, the actual target cell infection rate of the lentivirus under specific conditions is calculated as follows: Infection rate.1 = Ne / No.1 x 100% The present application research found that when performing the dichotomous operation, half of the cells are inoculated into a new culture container as a drug screening group, and the other half of the cells are directly counted as the total cell number of the control group, recorded as No. 2. When No. 2 is used as the total cell number for infection rate calculation, the cell infection rate will be overestimated. In fact, during the process of lentivirus infection of target cells, both the positive cells successfully infected with lentivirus and the negative cells not infected with lentivirus have a certain proliferation phenomenon. Using the calculation method of infection rate.2=Ne / No.2x100%, the proliferation of total cells during lentivirus infection is ignored, resulting in an overestimated infection rate.
[0033] In addition, the present application research also found that the control group counted again after the completion of the drug screening group screening will also cause deviation in the calculation results of the infection rate. After performing the dichotomous operation to obtain the drug screening group and the control group samples, the drug screening group is added with antibiotics for pressure screening, and the control group is not added with antibiotics and kept in normal culture state until the end of the screening. After the screening is completed, the drug screening group sample is counted to obtain the number of positive infected cells Ne, and at this time the control group sample is counted to obtain the total cell number No. 3. During the antibiotic screening process, the control group cells still have a certain proliferation phenomenon due to the absence of survival pressure. Using the calculation method of infection rate.3=Ne / No.3x100%, the excessive total cell proliferation condition is contained, thereby underestimating the actual cell infection rate. In fact, for cells with slow proliferation rate or poor state after virus infection, the proliferation phenomenon is not obvious, and in some cases, infection rate.3 and infection rate.2 have similar reference value.
[0034] In some embodiments, the quantified cells are inoculated into the culture container by counting. The cells are counted before lentivirus infection. By reducing the amount of lentivirus solution added through operations such as lentivirus concentration, the non-culture system volume introduced by the addition of lentivirus can be reduced while increasing the concentration of lentivirus, and the virus infection efficiency can be improved by 1 / 2 small volume infection method.
[0035] In some embodiments, the lentivirus infection efficiency is improved by adding polybrene.
[0036] In some embodiments, the time for supplementing the other half of the culture system by 1 / 2 small volume infection method is usually 4 hours after lentivirus infection. Given the lentivirus titer, different MOI gradients are set during lentivirus infection.
[0037] In some embodiments, the lentivirus titer is unknown, and different lentivirus volume gradients should be set considering the lentivirus concentration multiple. After performing the dichotomous operation, the drug screening group and the control group cells are inoculated into culture containers with the same or larger volume than before digestion.
[0038] In some embodiments, cell counting is performed using a fully automated cell counter to standardize the counting. The target cells are the cells of interest for the subsequent experiments, including primary cells that are difficult to infect.
[0039] More specifically, in step one, 1 / 2 volume infection is used to add lentivirus into the target cell culture system. When lentivirus infects the target cells, 1 / 2 volume of fresh medium is replaced, and the other half of the culture system is supplemented after a period of lentivirus infection to increase the transduction efficiency of lentivirus. Polybrene is added to promote lentivirus transduction. Generally, the other half of the medium is supplemented after 4 hours of lentivirus infection. Generally, the split operation is performed at 24 hours of lentivirus infection. After the sample is prepared into a single cell suspension and divided, it is inoculated into a culture container with the same or larger volume as the original.
[0040] In step two, after the control group obtains No. 2 data, it is not necessary to inoculate the cells into a new culture container. To obtain No. 1 data, after performing the cell split operation, the control group is added with normal complete medium for continuous culture until the drug screening group starts to add drugs. To obtain No. 3 data, when the drug screening group replaces the complete medium containing antibiotics, the control group is replaced with normal complete medium for continuous culture until the end of antibiotic screening. Due to the differences in characteristics of target cells, the best value of the three infection rate calculation results shown in step two is not fixed or unique. Cell counting is standardized using a cell counter. When calculating the infection rate, Ne and No use the number of viable cells or the number of total cells. Human umbilical cord-derived mesenchymal stem cells are used as a target cell model.
[0041] Example 1: Accurate determination of lentivirus infection rate of hMSCs as target cells by cell split method The information of reagents, consumables and instruments involved in the examples is shown in Table 1.
[0042] Table 1. Vectors, reagents, consumables and instruments
[0043] The time flow of the experimental operation is shown in Figure 2 , which completely corresponds to the time description in the following examples.
[0044] (1) Cell split operation: Day 0, plating: cell counting, hMSCs were inoculated into a 12-well plate at a density of 1 x 10 4 cells / cm 2 , so that the confluence was about 30% after adhesion; Note: The plating density can be adjusted according to the growth status of the cells, and the standard is to grow a monolayer in 48-72 hours.
[0045] Day 1, Lentivirus infection: a. After the cells adhered to the bottom of the plate, the cells in one well were counted after trypsin digestion, as shown in Figure 1 The initial cell culture plate, recorded as Nori = 2.70 x 10 4 cells. The old culture medium was discarded from the remaining sample wells, and 1 / 2 of the culture volume of fresh DMEM complete medium (DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin) was added, i.e. 0.5 mL per well, and polybrene was added to a final concentration of 8 µg / mL to promote lentivirus infection.
[0046] b. In the remaining sample wells, lentivirus packaged with the plasmid pKLV2-U6gRNA5-PGKpuro2ABFP (used in the following examples) was added according to the gradient in Table 2; Table 2. Lentivirus infection titer
[0047] Note: If the lentivirus titer is not determined, the data in Table 2 "virus volume" can be used for gradient setting. Similarly, if the lentivirus titer is known, the data in Table 2 "MOI" can be used for gradient setting. In this example, the lentivirus titer is known, so the corresponding infection gradient is set according to the MOI, and the "virus volume" is calculated from the lentivirus titer and the number of cells. The gradient can be adjusted according to the experimental purpose.
[0048] c. After 4 hours of lentivirus infection, the remaining 1 / 2 culture volume of DMEM complete medium was supplemented, and polybrene was added to a final concentration of 8 µg / mL; Day 2, Cell split: a. After 24 hours of lentivirus infection, the old culture medium in the sample wells was discarded, the sample wells were washed 1-2 times with PBS, and trypsin was used to prepare a single cell suspension. After centrifugation at 1000 rpm for 5 minutes at room temperature, cell pellets were obtained from different sample wells; b. Each cell pellet was resuspended in fresh DMEM complete medium, and each sample was divided into two equal parts and inoculated into two corresponding wells of a new 12-well plate, one well was recorded as the drug screening group, and the other well was the control group, as shown in Figure 1 The medium was supplemented to 1 mL, and the culture was continued at 37°C, 5% CO2; (2) Cell counting and infection rate calculation: Day 4, puromycin screening: After 72 hours of lentivirus infection, the following operations were performed for each cell sample under each infection condition: a. The drug screening group was replaced with new DMEM complete medium and 1.2 pg / mL puromycin was added for pressure screening; b. The control group used trypsin digestion to obtain No. 1 data. The experimental operation is shown in Figure 1 , and the results are shown in Table 3.
[0049] Day 7: After 72 hours of puromycin screening, the sample wells of the drug screening group were trypsinized and counted, and the experimental operation is shown in Figure 1 , to obtain Ne data. The results are shown in Table 3.
[0050] According to the above formula, the actual infection rate of the batch of lentivirus on hMSCs.1 was calculated, and the results are shown in Table 3: Table 3. Counting results and infection rate data of hMSCs infection rate.1
[0051] During the experiment, the density distribution of cells in different samples when counting is shown in Figure 3 and Figure 4 . The observed proportion of positive cells is basically the same as the calculated results of infection rate.1.
[0052] Example 2: Determination of lentivirus infection rate.2 on hMSCs as target cells based on cell dichotomy This example provides a method for determining the lentivirus infection rate.2 of target cells based on cell dichotomy. The same as the experimental operation of Example 1, this time the No. 2 data obtained by counting participates in the infection rate calculation.
[0053] (1) Cell dichotomy operation: hMSCs plating and lentivirus infection were performed according to the same standard as above. After 24 hours of lentivirus infection, the cells in each sample well were trypsinized and counted, and half of the cells in each sample were re-plated to obtain the drug screening group; 1 / 2 of the number of cells at this time was taken as the No. 2 value of the control group, and the counting node is shown in Figure 2 . The results are shown in Table 4.
[0054] (2) Cell counting and infection rate calculation: After 72 hours of lentivirus infection, the drug screening group was replaced with new DMEM complete medium and 1.2 pg / mL puromycin was added for pressure screening; After 72 hours of puromycin screening, the sample wells of the drug screening group were trypsinized and counted, and the experimental operation is shown in , to obtain Ne data. The results are shown in Table 4.
[0055] According to the above formula, the infection rate.2 of the batch of lentivirus on hMSCs was calculated, and the results are shown in Table 4. It can be seen that the results of infection rate.2 are significantly higher than infection rate.1.
[0056] Table 4. Counting results and infection rate data of hMSCs
[0057] Example 3: Determination of lentivirus infection rate.3 with hMSCs as target cells based on cell dichotomy This example provides a method for determining the lentivirus infection rate.3 of target cells based on cell dichotomy. The same experimental operation as in Example 1 is used, and the No. 3 data is obtained by counting. The infection rate is calculated.
[0058] (1) Cell dichotomy operation: hMSCs are plated and infected with lentivirus according to the same standards as above. The drug screening group and the control group are obtained by cell dichotomy. After 72 hours of lentivirus infection, the drug screening group is replaced with new DMEM complete medium and 1.2 μg / mL puromycin is added for pressure screening; the control group is replaced with new DMEM complete medium for continuous culture.
[0059] (2) Cell counting and infection rate calculation: After 72 hours of puromycin screening, the drug screening group and the control group are counted respectively to obtain the Ne data and the No. 3 data. The counting nodes are as shown in Figure 2 , and the data processing and infection rate calculation are performed according to the same method as above.
[0060] Based on the analysis of the above examples, it can be concluded that in the scheme of the present application, the key characteristic of lentivirus is that after infecting target cells, it will integrate its genome into the target cell genome. Only the positive cells that have integrated lentivirus can survive in an antibiotic environment, and the negative cells that have not integrated will be killed. The core principle of this method is to accurately quantify the proportion of positive cells in total cells, construct a strict control through cell dichotomy, and then monitor the cell growth dynamics through three counting nodes to finally eliminate the interference of cell proliferation or death on the counting results during the culture process.
[0061] First, infect the target cells with the sample to be tested containing lentivirus. After infection, if the lentivirus successfully integrates into the target cell genome, the target cells will express the antibiotic resistance gene carried by the lentivirus (which is the molecular basis for subsequent screening of positive cells); and the negative cells that have not integrated the lentivirus, due to the lack of resistance genes, cannot tolerate the subsequent antibiotic treatment.
[0062] After the infection is completed, all target cells are prepared into a single cell suspension (to ensure that the cells are completely dispersed to avoid the influence of cell clumps on the accuracy of counting). Then perform the dichotomy operation: evenly divide the single cell suspension into two parallel samples with identical quantity and state.
[0063] The key logic here is homogeneity - the two samples are derived from the same batch of infected cells, and theoretically they contain the same proportion of positive cells, without sample heterogeneity differences. One of the samples is used as a drug screening group (experimental group) to screen and count positive cells; the other is used as a control group (blank group) without adding antibiotics, only for monitoring the natural growth dynamics of cells during culture.
[0064] The two samples obtained by dichotomy will be treated in the same culture environment (only the drug screening group adds antibiotics), and cell counting will be performed on the control group at three key time points (the drug screening group only counts viable cells after the screening ends), and the three nodes have different focuses: The first node: when the dichotomy operation is performed (i.e., when the single-cell suspension is divided into two parts). At this time, the control group is counted to obtain the total cell number No.2.
[0065] The second node: when the antibiotic screening starts (i.e., when the antibiotic is added to the drug screening group). At this time, the control group is counted again to obtain the total cell number No.1. This value is the starting benchmark for cell growth dynamics - it records the total cell amount of the two samples (due to homogeneity, the total cell number of the drug screening group is consistent with the control group No.1 at this time) at the start of drug screening, providing starting data for subsequent calculation of cell proliferation efficiency.
[0066] The third node: when the antibiotic screening is completed (i.e., when the negative cells in the drug screening group have been completely killed, and only positive cells survive). At this time, both samples are counted simultaneously: the drug screening group is counted to obtain the number of surviving positive cells Ne; the control group is counted to obtain the total cell number No.3.
[0067] Traditional lentivirus infection rate detection methods (such as fluorescence labeling counting, direct drug screening counting) often have problems such as large sample heterogeneity, cell growth interference with counting results, and poor repeatability. This method precisely solves these pain points through dichotomy and three counting nodes, with the following specific advantages: 1. Cell dichotomy: eliminate sample heterogeneity and reduce systematic error Traditional methods generally divide the control group and experimental group when the cells are plated, and virus infection is performed separately, resulting in differences in cell state.
[0068] The dichotomy of this method ensures that the two samples are derived from the same batch of single-cell suspension, with consistent quantity and state, eliminating errors caused by sample heterogeneity from the source. This provides a reliable basis for subsequent proliferation correction, significantly reducing systematic error.
[0069] 2. Three counting nodes: dynamic correction of cell growth, avoiding static counting bias Traditional methods mostly use static counting: for example, count the total cell number N0 at the time of infection, count the positive cell number Ne after drug screening, and directly calculate the infection rate as Ne / N0. However, this method ignores the cell proliferation during drug screening - Ne is the number of positive cells after proliferation, N0 is the initial number at the time of infection, and the direct ratio will seriously overestimate the infection rate.
[0070] 3. Antibiotic screening + dichotomous control: dual protection of specificity and accuracy Compared with fluorescence labeling counting (which may cause false negatives or false positives due to weak fluorescence intensity or cell autofluorescence), antibiotic screening only allows positive cells with integrated lentivirus to survive, which can specifically enrich positive cells and reduce misjudgment.
[0071] The internal control constructed by dichotomous method avoids external interference such as differences in cell state and operation errors of different batches. For example, if a batch of cells grows abnormally, the problem can be found and invalid data can be excluded in time, which is more reliable than single-sample drug screening counting (without control, it is impossible to judge whether the cell growth is normal).
[0072] 4. High repeatability of operation: standardized process reduces human error The key steps of this method (single cell suspension preparation, dichotomous division, and time control of three counting nodes) can be completely standardized. This process standardization + real-time quality control design reduces the result fluctuation caused by human operation (such as cell counting deviation and uneven sample distribution), so that the experimental results of different experimenters and different batches are more comparable.
[0073] The above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the specific embodiments of the present application can still be modified or replaced, and any modification or replacement without departing from the spirit and scope of the present application should be covered within the protection scope of the claims of the present application.
Claims
1. A method for quantitatively detecting the infection rate of lentivirus on target cells based on cell bisection, characterized in that, The method comprises the following steps: Infecting target cells with a sample containing lentivirus; Preparation of a single-cell suspension of target cells integrated with lentivirus, division into two parallel samples with the same amount and state, and inoculation to obtain antibiotic-treated cells and antibiotic-free treated cells; Counting the positive infected cells Ne after adding antibiotic screening to the drug screening group, and counting the total cell number No.2, No.1 and No.3 at three time points of performing the dichotomy operation, starting antibiotic screening and completing antibiotic screening; and calculating the lentivirus infection rate of target cells based on the total cell number.
2. The method for quantitatively detecting the infection rate of lentivirus on target cells based on cell dichotomy according to claim 1, characterized in that, The sample containing lentivirus is added to the target cell culture system by using 1 / 2 small volume infection method; fresh culture medium is replaced when the target cells are infected with lentivirus, and the other half of the culture system is supplemented after a period of lentivirus infection.
3. The method for quantitatively detecting the infection rate of lentivirus on target cells based on cell binary method according to claim 1 or 2, characterized in that, In the step of infecting target cells with a sample containing lentivirus, polybrene is also added to promote lentivirus transduction.
4. The method for quantitatively detecting the infection rate of lentivirus on target cells based on cell dichotomy according to claim 1 or 2, characterized in that, In the step of infecting target cells with a sample containing lentivirus, the other half of the culture medium is supplemented after 4 hours of lentivirus infection.
5. The method for quantitatively detecting the infection rate of lentivirus on target cells based on cell dichotomy according to claim 1 or 2, characterized in that, In the step of infecting target cells with a sample containing lentivirus, the dichotomy operation is performed at 24 hours of lentivirus infection.
6. The method for quantitatively detecting infection rate of lentivirus on target cells based on cell dichotomy according to claim 1, characterized in that, After the sample is prepared into a single-cell suspension and divided, it is inoculated into a culture container with the same or larger volume as the original one.
7. The method for quantitatively detecting infection rate of lentivirus on target cells based on cell dichotomy according to claim 1, characterized in that, In the step of calculating the lentivirus infection rate of target cells based on the total cell number, the calculation formula is: Infection rate.1=Ne / No.1×100% Infection rate.2=Ne / No.2×100% Infection rate.3=Ne / No.3×100%.
8. The method for quantitatively detecting infection rate of lentivirus on target cells based on cell dichotomy according to claim 1, characterized in that, After obtaining No.2 data of the control group, the cells do not need to be inoculated into a new culture container; after performing the cell dichotomy operation, the control group is added with normal complete culture medium for continuous culture until the drug screening group starts to add drugs; when the drug screening group is replaced with complete culture medium containing antibiotics, the control group is replaced with normal complete culture medium for continuous culture until the end of antibiotic screening; When calculating the infection rate, Ne and No use the number of living cells or the total number of cells.
9. The method for quantitatively detecting infection rate of lentivirus on target cells based on cell dichotomy according to claim 1, characterized in that, Given the lentivirus titer, set different MOI gradients when the lentivirus is infected; If the lentivirus titer is unknown, the lentivirus concentration multiple should be considered, and different lentivirus volume gradients should be set.
10. The method for quantitatively detecting the infection rate of lentivirus on target cells based on cell binary method according to any one of claims 1-9, characterized in that, Human umbilical cord-derived mesenchymal stem cells are used as target cells.