Stem cell viability and growth curve detection method based on full-field cell scanning analyzer

Abstract

The present invention relates to the field of cell biology detection technology, and in particular to a method for detecting stem cell viability and growth curve based on a full-view cell scanning analyzer. The method is based on a full-view cell scanner to detect the viability and growth curve of stem cells. Compared with traditional cell counting, the instrument scanning method can complete the counting of a large number of cells in a short time with high efficiency. At the same time, the image analysis method is adopted, the operation is standardized, and the results obtained by scanning are highly accurate and repeatable.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology detection, and in particular to a method for detecting stem cell viability and growth curve based on a full-view cell scanning analyzer. Background Art

[0002] Stem cells are a type of cell with the ability of self-renewal and multidirectional differentiation potential. The types of stem cells commonly used in drug development or clinical applications include pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), as well as adult stem cells such as mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs).

[0003] Due to the characteristics of self-renewal, multidirectional differentiation, and immune regulation, stem cells are widely used in drug screening, cell therapy, regenerative medicine, cosmetic surgery and other fields. Whether it is drug development or clinical application, the cell activity of stem cells is always a key quality attribute that needs to be closely monitored.

[0004] Cell activity detection is an important means to evaluate the survival status, proliferation ability, and metabolic activity of cells. Conventional evaluation indicators include cell morphology, cell survival rate, cell growth curve, cell cycle, etc. Among them, cell survival rate is an indicator for evaluating the proportion of healthy living cells in a cell population, and growth curve is a method for evaluating the growth and proliferation of cells during in vitro culture. It is often used in stem cells to calculate the population doubling time of cells. It is of great significance for evaluating the growth characteristics, differentiation potential, environmental impact, predicting the cell life cycle, and optimizing culture conditions of stem cells.

[0005] Traditional methods for measuring cell activity include conventional cell counting methods based on trypan blue staining or AO / PI staining, and colorimetric methods based on MMT, CCK-8, etc. For the determination of cell growth curves, it is necessary to collect cells inoculated at the same density at different culture times for digestion and counting. The operation is relatively cumbersome and there are large differences between replicate wells. Summary of the invention

[0006] The purpose of the present invention is to provide a method for detecting stem cell viability based on a full-field cell scanning analyzer, which has the advantages of being rapid, efficient, easy to operate, highly accurate and highly repeatable.

[0007] Another object of the present invention is to provide a method for detecting stem cell growth curve based on a full-field cell scanning analyzer, which has the advantages of being fast, efficient, easy to operate, highly accurate and highly repeatable.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A method for detecting stem cell viability based on a full-view cell scanning analyzer, the method comprising the following steps:

[0010] S1. Digest the stem cells to be tested with digestion solution, collect the cell pellet by centrifugation, resuspend the cells with D-PBS, and gently pipette until they become single cells or small cell clusters;

[0011] S2, mix the cell suspension obtained in S1 and AO / PI staining solution, and incubate at room temperature for 5-15 minutes to obtain a suspension;

[0012] AO / PI staining solution is a mixture of AO staining solution and PI staining solution, wherein the final concentrations of AO dye and PI dye are 10-20 μg / mL and 50-100 μg / mL, respectively;

[0013] S3. Take the suspension of S2 and add it to the cell culture plate, centrifuge for 5 minutes or let it stand for 10 minutes, and use a full-view cell scanning analyzer to observe and detect the cell viability.

[0014] Preferably, the stem cells to be tested include various types of stem cells or stem cells at different stages and their derivatives, including induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, hematopoietic stem cells and their derivatives; as well as seed cell banks, master cell banks, working cell banks, and final product cells.

[0015] Preferably, the preparation method of AO / PI staining solution is: dilute 1 mg / mL AO staining solution and 1 mg / mL PI staining solution to 20 μg / mL and 100 μg / mL respectively, and mix them in equal proportions to obtain AO / PI staining solution.

[0016] Preferably, the cell suspension and the AO / PI staining solution are mixed in equal volumes.

[0017] Preferably, in S3, the setting parameters of the full-field cell scanning analyzer are: AO (acridine orange) dye and PI (propidium iodide) dye are selected as the staining solution, "Green Illumination" is selected for acridine orange staining, and the "Exposure Time" is set to about 10000μs; "Red Illumination" is selected for propidium iodide staining, and the "Exposure Time" is set to about 6000μs; the "Intensity Threshold" of the green light channel is adjusted to 4, and the "Precision" is selected as "High"; the "Cell Diameter" is adjusted to 10; the "Intensity Threshold" of the red light channel is adjusted to 10, and the "Precision" is selected as "High"; and the "Cell Diameter" is adjusted to between 8-10.

[0018] A method for detecting stem cell growth curve based on a full-field cell scanning analyzer, the method comprising the following steps:

[0019] S1. Digest the stem cells to be tested with digestion solution, then resuspend them with culture medium and adjust the cell density to 1×10 4 -2×10 4 Pieces / mL;

[0020] S2: Inoculate the stem cells obtained in S1 into a cell culture plate and incubate at 37°C and 5% CO 2 The cells were cultured under the condition of 4% paraformaldehyde and 1% paraformaldehyde. The supernatant was discarded every 24 hours, and the cells were washed with D-PBS and replaced with fresh culture medium. The cells were scanned with an automatic cell scanner for a total of 5-8 days.

[0021] S3. According to different cell types and cell morphologies, select cell counting or cell density for analysis;

[0022] S4. Draw a growth curve based on the cell number or cell density obtained from the analysis at different time points, and calculate the population doubling time of the sample cells according to the Patterson formula.

[0023] Preferably, the stem cells to be tested include various types of stem cells or stem cells at different stages and their derivatives, including induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, hematopoietic stem cells and their derivatives; as well as seed cell banks, master cell banks, working cell banks, and final product cells.

[0024] Preferably, in S3, relatively regular morphological adherent cells or suspension cells including iPS cells are analyzed by cell counting, corresponding to the "Cell Counting" mode of the instrument; irregularly shaped cells including MSC cells cannot be directly circled and counted, and are analyzed by cell density, corresponding to the "Confluence" mode of the instrument.

[0025] Preferably, the Intensity Threshold is set to identify cells according to the pixel intensity of 4;

[0026] In the "Cell Counting" mode, set the Cell Diameter according to the expected cell diameter of 8-15. Set the Minimum Cell Area to 50 to exclude impurities that are too small until all cells are circled;

[0027] For the "Confluence" mode, set the Cell Diameter according to the expected cell diameter 8;

[0028] Select "High" for "Precision", set Min Cluster Size to 2000, and adjust related parameters according to the specific cell type until all cells are circled.

[0029] As an advanced cell analysis device, the Celigo full-field cell scanning analyzer can provide quantitative data analysis of whole-well imaging in multiple fields of view such as bright field and fluorescence. Based on the Celigo full-field cell scanner, cells can be directly scanned at different culture times, and the cell number and cell density corresponding to different culture times can be directly obtained through preset parameters. It has strong operability, good repeatability and can achieve non-destructive analysis of cells.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. This method is based on the detection of stem cell viability and growth curve using a full-field cell scanner. Compared with traditional cell counting, the instrument scanning method can complete the counting of a large number of cells in a short time with high efficiency. At the same time, the image analysis method is adopted, the operation is standardized, and the scanning results are highly accurate and reproducible.

[0032] 2. The traditional cell viability counting method is generally trypan blue staining, which is easily interfered by cell debris and impurities in the sample. AO / PI fluorescence staining can stain the DNA in the nuclei of dead and live cells respectively, effectively distinguish dead cells from debris, and reduce the influence of changes in cell refractive index on the staining effect. Cell viability detection based on full-field cell scanner can realize data collection of an entire cell culture plate in a short time, and can effectively count and analyze the viability of cell clusters that are not single cells.

[0033] 3. The traditional method of measuring cell growth curve involves multiple sets of repeated cell plating processes. When counting cells at different time points, the cells need to be digested and counted, which is complicated and has poor repeatability. This method is based on a full-view cell scanner, which can continuously count and collect images of the same cell culture plate at multiple time points. After the test, the cells are intact and can continue to be cultured. It can not only reduce the amount of cell inoculation, but also reduce the errors introduced by cell inoculation, digestion and other operations. By adjusting different analysis modes, cell types with different morphologies can be delineated and analyzed. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the fluorescence distribution and delineation diagram of AO / PI stained cells;

[0035] Figure 2 This is a focused picture of Day 3 iPSC cells;

[0036] Figure 3 This is a picture of Day3 iPSC cells after they were circled;

[0037] Figure 4 This is a focused picture of Day 3 MSC cells;

[0038] Figure 5 This is the picture after Day3 MSC cells were circled;

[0039] Figure 6 is the iPSC cell growth curve;

[0040] Figure 7 This is the MSC cell growth curve. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0043] The present invention is now further described in conjunction with specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial channels).

[0044] Experimental materials and equipment used in the examples of the present invention:

[0045] MSC cells were purchased from ATCC, catalog number: PCS-500-010; MSC culture medium was purchased from STEMCELL, catalog number: 05445;

[0046] iPSC cells were purchased from ATCC, catalog number: ACS-1007; iPSC culture medium was purchased from STEMCELL, catalog number: 85850;

[0047] D-PBS, Dulbecco's phosphate buffered saline, purchased from STEMCELL, catalog number: 37350;

[0048] AO dye and PI dye were purchased from Solebol with the product numbers: CA1143 and C0080, respectively;

[0049] Celigo fully automatic scanning analyzer, purchased from Nexcelom, model: 200-BFFL-S.

[0050] Example 1 Cell viability detection sample pretreatment and staining

[0051] Take cells with a confluence of 80%-90% and good growth status, digest them with digestion solution, collect the cell suspension, centrifuge at 1300 rpm for 5 minutes, discard the supernatant, and resuspend them with D-PBS to obtain a cell suspension.

[0052] Dilute AO staining solution (1 mg / mL) and PI staining solution (1 mg / mL) to 20 μg / mL and 100 μg / mL respectively, and mix them in equal proportions to form AO / PI staining solution. Take 20 μL of cell suspension and 20 μL of AO / PI staining solution, mix them evenly, incubate them at room temperature for 10 minutes, take 30 μL-40 μL of suspension and add it to a 96-well plate, let it stand for 10 minutes, and count the cells using Celigo full-view cell scanning analyzer.

[0053] Example 2 Cell viability detection method

[0054] Set up the Celigo full-field cell scanning analyzer and create a new scan file.

[0055] Scan interface settings: Select Celigo application: Expression Analysis (Target 1+2), set "Target1Channel" (AO), select "Green Illumination" for acridine orange staining. Set "Exposure Time" to about 10000μs; set "Target 2Channel" (PI), select "Red Illumination" for propidium iodide staining. Set "Exposure Time" to about 6000μs.

[0056] On the green / AO channel, click "Hardware autofocus" and click "Register Auto". Switch to the red / PI channel, also click "Register Auto" and in "Set Offset", set the optimal offset between +5-10μm. Select the well to be collected and click "Start Scan" to collect the image.

[0057] Analysis interface settings: Check "Well Mask", enter the volume of the cell suspension to be tested in "Sample Volume"; select "Fluorescence" in the Algorithm item of the green and red fluorescence channels; adjust the "IntensityThreshold" of the green light channel to 4, select "High" for "Precision"; adjust the "Cell Diameter" to 10; adjust the "Intensity Threshold" of the red light channel to 10, select "High" for "Precision"; adjust the "Cell Diameter" to between 8-10. Check "Separate Touching Objects" and "Background Correction" at the same time. Select the required wells and click "Start Analyze" to analyze.

[0058] After the analysis is completed, the fluorescence distribution of cells stained with AO and PI dyes and the fluorescence circumscription can be viewed through "Image Display" and "Graphic Overlay".

[0059] Data analysis: In the "Information" interface of each well, you can see the specific information of the selected well. "Total Concentration", "% Target 1 (only)", and "Target 1 (only) Concentration" are cell concentration, cell viability, and live cell concentration, respectively.

[0060] Example 3 Cell viability test results

[0061] Two different stem cell types, iPSC and MSC, were selected, and two different staining and cell counting methods were used to determine cell viability. In addition to the Celigo-based cell viability detection method shown in Examples 1 and 2, another method used a traditional trypan blue staining counting method, that is, by mixing with an equal volume of 0.2% trypan blue dye and dropping it onto a cell counting plate, and using a cell counter for detection. The AO / PI stained cell fluorescence distribution and demarcation diagram are shown in Figure 2. Figure 1 The detection results of the two staining methods for iPSC and MSC cells are shown in Tables 1-2.

[0062] Table 1 Detection results of iPSC cells by different staining methods

[0063]

[0064] Table 2 Detection results of different staining methods for MSC cells

[0065]

[0066]

[0067] Different loading volumes were tested to verify the robustness of the method under minor changes, and the results are shown in Table 3.

[0068] Table 3 iPSC AO / PI method durability test results

[0069]

[0070] According to the results of Tables 1 to 3, for all cell types, the cell counting results of the trypan blue staining method are slightly greater than those of the AO / PI fluorescent staining method, and the inter-group variation is large. In particular, for cell types that are difficult to digest as single cells, or are generally digested as small cell clumps for passage, such as iPSC, which are prone to differentiation, the conventional trypan blue staining method is easily interfered by factors such as the size of cell clumps and cell fragments in the sample, resulting in inaccurate cell viability measurement. The detection method described in the present invention combines Celigo instrument measurement with AO / PI staining, which can accurately stain live and dead cells, effectively distinguish dead cells from cell fragments, and better interpret cell size and cell particles, with better result consistency and accuracy.

[0071] Example 3 Growth curve detection method

[0072] Take stem cells with a confluence of 80%-90% and good growth status, digest them with digestion solution, collect the cell suspension, and 4 -2×10 4 The cells were inoculated at a density of 100 / mL in a well plate and incubated at 37°C and 5% CO 2 Every 24 hours, the supernatant was discarded, the suspended dead cells were washed with D-PBS, and fresh culture medium was replaced, and the cells were scanned using an automatic cell scanner.

[0073] Set up the Celigo full-field cell scanning analyzer and create a new scan file.

[0074] Scan interface settings: In the Application section, click the "Please Select" drop-down menu and select "CellCounting", "Direct Cell Counting" or "Confluence", "Confluence 1". For adherent cells or suspension cells with relatively regular morphology, such as iPS cells, select the former to analyze by cell counting. For cells with irregular shapes, such as MSC cells, which cannot be directly circled and counted, select the latter to analyze by cell density.

[0075] Click Live mode. Select a well in the cell layout in the well plate navigation area and click Apply to automatically expose under the bright field light source. Click Find Focus several times until the cell field of view appears, and click Focus Setup. To facilitate cell identification, select "Dark Focus" and then click Register Auto. After the device autofocus operation is completed, click Focus Setup again to close the dialog box. The focused cells are as follows: Figure 2 , Figure 4 shown.

[0076] Select Plate mode in the well plate location area on the right side of the interface and click Selection. Highlight all wells to be imaged. Click Start Scan to begin imaging.

[0077] Analysis interface settings: Click Selection in the well plate navigation area. Click the blank next to A1 to cancel all selected wells. Click the View button and click any green well in the navigation area to view the image. Set IntensityThreshold to 4 for pixel intensity to identify cells. In the "Cell Counting" mode, set CellDiameter to 8-15 according to the expected cell diameter. Set Minimum Cell Area to 50 to exclude impurities that are too small until all cells are circled, such as Figure 3 For the "Confluence" mode, set the Cell Diameter to 8 according to the expected cell diameter. Select "High" for "Precision" and set the Min Cluster Size to 2000. You can adjust the relevant parameters according to the specific cell type until all cells are circled, as shown in the figure below. Figure 5 shown.

[0078] Click on the Well Mask display. Check the Well Mask option. Reduce %Well Mask as appropriate to eliminate interference from well edge impurities on the analysis.

[0079] Select the desired wells for analysis and click “Start Analyze”.

[0080] After the analysis is completed, the cell number or cell density of the selected well can be obtained.

[0081] Example 4 iPSC cell growth curve detection results

[0082] iPSC cells were selected and 2×10 4 / mL was inoculated in a six-well plate. Day 1 detection started 24 hours later, and the detection lasted for 5 days. On the fifth day, the cell density reached more than 90%, which was beyond the instrument's recognition range. The specific information is shown in Table 4:

[0083] Table 4 iPSC cell growth curve results

[0084]

[0085]

[0086] The population doubling time of sample cells was calculated according to Patterson's formula:

[0087]

[0088] Where t is the termination time of cell culture (unit: h), t 0 Cell culture starting time (unit: h), N t N is the number of cells at the end of culture. 0 is the starting cell number for culture.

[0089] Growth curve Figure 6 As shown, the population doubling time is: PDT = 32.29h.

[0090] Example 5 MSC cell growth curve test results

[0091] Select MSC cells and add 1×10 4 The density of 100 μg / mL was inoculated in a six-well plate. After 24 hours, the Day 1 test was started for a total of six days, and the plateau phase was reached on the fifth day. The specific information is shown in Table 5.

[0092] Table 5 MSC cell growth curve results

[0093]

[0094] The population doubling time of sample cells was calculated according to Patterson's formula:

[0095]

[0096] Where t is the termination time of cell culture (unit: h), t 0 Cell culture starting time (unit: h), N t N is the cell number (cell density) at the end of culture. 0 is the starting cell number (cell density) of the culture.

[0097] Growth curve Figure 7 As shown, the population doubling time is: PDT = 30.96h.

[0098] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0099] The above is a detailed introduction to a method for detecting stem cell viability and growth curve based on a full-field cell scanning analyzer provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting stem cell viability based on a full-view cell scanning analyzer, characterized in that The method comprises the following steps: S1. Digest the stem cells to be tested with digestion solution, collect the cell pellet by centrifugation, resuspend the cells with D-PBS, and gently pipette until they become single cells or small cell clusters; S2, mix the cell suspension obtained in S1 and AO / PI staining solution, and incubate at room temperature for 5-15 minutes to obtain a suspension; AO / PI staining solution is a mixture of AO staining solution and PI staining solution, wherein the final concentrations of AO dye and PI dye are 10-20 μg / mL and 50-100 μg / mL, respectively; S3. Take the suspension of S2 and add it to the cell culture plate, centrifuge for 5 minutes or let it stand for 10 minutes, and use a full-view cell scanning analyzer to observe and detect the cell viability.

2. The method for detecting stem cell viability according to claim 1, characterized in that The preparation method of AO / PI staining solution is as follows: dilute 1 mg / mL AO staining solution and 1 mg / mL PI staining solution to 20 μg / mL and 100 μg / mL respectively, and mix them in equal proportions to obtain AO / PI staining solution.

3. The method for detecting stem cell viability according to claim 1, characterized in that: The cell suspension and AO / PI staining solution were mixed in equal volumes.

4. The method for detecting stem cell viability according to claim 1, characterized in that: In S3, the setting parameters of the full-view cell scanning analyzer are: Select AO (acridine orange) dye and PI (propidium iodide) dye as the staining solution, select "Green Illumination" for acridine orange staining, and set the "Exposure Time" to about 10000μs; select "Red Illumination" for propidium iodide staining, and set the "Exposure Time" to about 6000μs; adjust the "Intensity Threshold" of the green light channel to 4, select "High" for "Precision"; adjust the "Cell Diameter" to 10; adjust the "Intensity Threshold" of the red light channel to 10, select "High" for "Precision"; adjust the "Cell Diameter" to between 8-10.

5. A method for detecting stem cell growth curve based on a full-view cell scanning analyzer, characterized in that The method comprises the following steps: S1. Digest the stem cells to be tested with digestion solution, then resuspend them with culture medium and adjust the cell density to 1×10 4 -2×10 4 Pieces / mL; S2, inoculate the stem cells obtained in S1 into a cell culture plate and culture them at 37°C and 5% CO2; discard the supernatant every 24 hours, wash with D-PBS and replace with fresh culture medium, and scan with an automatic cell scanner for a total of 5-8 days; S3. According to different cell types and cell morphologies, select cell counting or cell density for analysis; S4. Draw a growth curve based on the cell number or cell density obtained from the analysis at different time points, and calculate the population doubling time of the sample cells according to the Patterson formula.

6. The detection method according to claim 1 or 5, characterized in that: The stem cells to be tested include various types of stem cells or stem cells at different stages and their derivative cells, including induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, hematopoietic stem cells and their derivative cells; as well as seed cell banks, master cell banks, working cell banks, and final product cells.

7. The detection method according to claim 5, characterized in that: In S3, adherent cells or suspension cells with relatively regular morphology, including iPS cells, are analyzed by cell counting, which corresponds to the instrument's "Cell Counting" mode. Irregularly shaped cells, including MSC cells, cannot be directly circled and counted, so they are analyzed by cell density, which corresponds to the instrument's "Confluence" mode.

8. The detection method according to claim 5, characterized in that: Identify cells according to the pixel intensity 4 by setting the Intensity Threshold; In the "Cell Counting" mode, set the Cell Diameter to 8-15 according to the expected cell diameter; set the Minimum Cell Area to 50 to exclude impurities that are too small until all cells are circled; For "Confluence" mode, set Cell Diameter according to the expected cell diameter 8; Select "High" for "Precision", set Min Cluster Size to 2000, and adjust related parameters according to the specific cell type until all cells are circled.

Images