A method for detecting the activity of islet cells derived from human pluripotent stem cells

Abstract

The present invention discloses a method for detecting the activity of islet cells derived from human pluripotent stem cells: the islet cell spheres to be detected are digested to obtain a single-cell suspension; 0.1-5% HSA by volume is added to the PBS buffer as a cell protection solution, the cell protection solution is added to the single-cell suspension, and then PI staining solution is added for staining; the stained sample is subjected to flow cytometry to obtain the proportion data of live cells. The present invention uses a low-concentration PI staining solution for staining to improve the staining effect. The stained cells are sorted by flow cytometry. HSA is used as a cell protection solution to protect the islet cells during the flow cytometry detection, reduce the sample preparation time and the damage to islet cells caused by flow cytometry detection, maintain cell activity, can be standardized and normalized, avoid human judgment errors, objectively and accurately detect the islet viability, has high repeatability, and has a low detection cost, and is expected to be applied to the determination of viability before clinical islet transplantation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for detecting the activity of islet cells derived from human pluripotent stem cells. Background Art

[0002] Diabetes is a chronic disease with a relatively high incidence rate, which is caused by the lack, dysfunction or insufficiency of pancreatic islet β cells (cells that release insulin and control glucose metabolism). Patients with type 1 diabetes (T1DM) and advanced type 2 diabetes (T2DM) need lifelong exogenous insulin therapy. However, the traditional therapy of infusing insulin to relieve diabetes may lead to an increase in the patient's insulin dependence and cannot cure diabetes.

[0003] Islet cell transplantation can be used for the treatment of diabetes, but the donor sources of islet cells are very few, and there are problems such as donor matching and immune rejection. Human pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells (iPSCs), differentiating into islet cells in vitro has been a research hotspot in the past decade. Islet cells differentiated in vitro can obtain sufficient β cells for cell transplantation in diabetic patients. Cells derived from human pluripotent stem cells can solve the limitations of using human cadaveric islets as donors and can be used as a model system to understand the pathogenic mechanisms leading to various forms of diabetes. However, the number, activity and good function of islets before transplantation are important factors for the success of transplantation.

[0004] The currently accepted and commonly used method for evaluating islet activity is to use the cell-permeable esterase substrate fluorescein diacetate (FDA) and the viability probe propidium iodide (PI) that penetrates unhealthy / dead cells to determine the percentage of live cells in the islet preparation. However, there is no definite measurement standard for judging the islet viability after FDA / PI staining. It mainly relies on the subjective judgment of preclinical physicians and gives an estimated value of islet activity. There is no quantitative data directly indicating the activity of islets, which will lead to a great subjectivity in the judgment results of islet viability and also a large error.

[0005] Therefore, it is necessary to develop a detection method that can standardize and quantify the islet cell viability, provide accurate data reference for clinical applications, and facilitate product quality control and verification. Summary of the Invention

[0006] The object of the present invention is to provide a method for detecting the activity of islet cells derived from human pluripotent stem cells, which can be standardized in operation, is not affected by human subjective judgment, can accurately quantify, and the data is more accurate.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A method for detecting the activity of islet cells derived from human pluripotent stem cells, the method comprising the following steps:

[0009] (1) Digest the islet cell spheres to be detected to obtain a single-cell suspension.

[0010] The islet cell spheres to be detected are human pluripotent stem cell-derived islet cell spheres, which are obtained by chemically small molecule-induced culture of human pluripotent stem cells.

[0011] (2) Add HSA with a volume fraction of 0.1-5% to the PBS buffer as a cell protection solution. Add the cell protection solution to the single-cell suspension, and then add the PI staining solution for staining.

[0012] (3) Perform flow cytometry on the stained sample to obtain the proportion data of live cells.

[0013] In the step (2), the PBS buffer is a commercially available 1X PBS buffer.

[0014] In the step (2), the volume ratio of the single-cell suspension to the cell protection solution is 200:80-120, the concentration of the PI staining solution is 0.02-0.04 mg / mL, and the volume ratio of the single-cell suspension to the PI staining solution is 200:20-40.

[0015] Preferably, the volume fraction of HSA in the cell protection solution is 1.5-5%, and more preferably 5%.

[0016] Furthermore, preferably in the step (2), 100 μL of the cell protection solution is added to every 200 μL of the single-cell suspension, and 30 μL of the PI staining solution with a concentration of 0.025 mg / mL is added.

[0017] The PI staining solution is prepared by dissolving PI in the PBS buffer.

[0018] The staining time is preferably 10-20 min in the dark at room temperature.

[0019] The present invention discovers that directly detecting the single-cell suspension on the machine after terminating digestion will cause the cells to easily die and the viability rate to be low. The applicant screens and discovers that HSA (human serum albumin) has a protective effect on cells. Adding HSA can maintain cell activity and improve the viability rate of islet cells.

[0020] In the step (1), it is preferred to use Accutase enzyme for digestion. The digestion is generally carried out at 37 °C for 15-25 min, preferably 20 min.

[0021] Specifically, preferably the step (1) is carried out according to the following steps:

[0022] Add 20 μL of the islet cell spheres to be detected to 1 mL of Accutase enzyme, and digest at 37 °C for 15-25 min, preferably digest for 20 min, to obtain a single-cell suspension.

[0023] If the digestion time is too short, the pancreatic islet cells are not completely digested, and the results of flow cytometry detection are inaccurate, and the dye cannot stain all cells. If the digestion time is too long, it will cause cell death and also lead to inaccurate results.

[0024] The beneficial effects of the present invention are as follows:

[0025] Staining is performed using a PI staining solution with a low concentration to improve the staining effect. The stained cells are sorted by flow cytometry. Among them, HSA is used as a cell protection solution to protect the pancreatic islet cells during the flow detection process, reduce the damage to the pancreatic islet cells during the flow detection, maintain cell viability, and can also protect the cells during a relatively long sample preparation time. The obtained data is accurate and reliable, and can be standardized and normalized for operation, avoiding human judgment errors, objectively and accurately detecting the viability of pancreatic islets, with high repeatability. The PI staining is close to the existing PI / FDA staining method and is easily recognized clinically. It is expected to be applied to the determination of viability before clinical pancreatic islet transplantation, for quality control of clinical products, and can also be used for various screening experiment verifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a graph of the flow cytometry detection results of the viability of pancreatic islet cells after different digestion times and PI staining in Example 2.

[0027] Figure 2 It is a graph of the flow cytometry detection results of the viability of pancreatic islet cells after different digestion times and PI staining in Example 3.

[0028] Figure 3 It is a graph of the flow cytometry detection results of the viability of pancreatic islet cells after PI staining under the protection of different concentrations of HSA in Example 4.

[0029] Figure 4 It is a graph of the staining results using FDA / PI in the comparative example.

[0030] Figure 5 It is a graph of the flow cytometry detection results of the viability of pancreatic islet cells in Example 5.

[0031] Figure 6 It is a graph of the flow cytometry detection results of the viability of pancreatic islet cells at different placement times in Example 6. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The following takes specific examples to further illustrate the technical solutions of the present invention, but the protection scope of the present invention is not limited thereto.

[0033] Example 1

[0034] Prepare reagents:

[0035] Step 1: 100x PI stock solution: Weigh 0.5 mg of PI and dissolve it in 1 mL of PBS. After mixing, a stock solution of 0.5 mg / mL is obtained. Store it in the medical refrigerator (4°C) in the flow cytometry room in the dark after use, and the shelf life is one month.

[0036] Step 2: 5x PI working solution: Take an appropriate amount of 100x PI stock solution and dilute it 20-fold with PBS to obtain a 5x PI working solution (PI concentration 0.025 mg / mL).

[0037] Step 3: Preparation of cell protection solution: Add 5% HSA (by volume) to the PBS buffer to prepare the cell protection solution.

[0038] Step 4: Aliquot Accutase according to the usage amount and place it in a 37°C water bath for sufficient preheating for at least 15 min.

[0039] Example 2

[0040] The cell spheres to be detected are islet cell spheres derived from human pluripotent stem cells after induction culture.

[0041] Step 1 Sampling: Place the cell spheres to be detected in a biosafety cabinet. Insert a low-adsorption pipette tip into a 20 μL pipette, and aspirate 20 μL of full-volume cell spheres into a clean 1.5 mL EP tube. During the aspiration of cell spheres, avoid aspirating cell supernatant into the pipette tip. If necessary, repeat this step until the above requirements are met.

[0042] Step 2 Digestion: Add 1 mL of Accutase enzyme to the 1.5 mL EP tube and incubate at 37°C. Take 3 groups of EP tubes and digest them for 5 min, 10 min, and 15 min respectively. During digestion, invert the EP tube several times every 5 min to ensure complete digestion. The digested single cells are stained and counted using a NC cell counter.

[0043] Step 3 Sieving: Pass the digested cell suspension through a 40 μm cell sieve.

[0044] Step 4 Staining: Take 200 μL of the cell suspension after sieving in three groups and operate according to the following conditions:

[0045] ① Add 100 μL of cell protection solution (PBS buffer) to 200 μL of cell suspension, and then add 3 μL of 100X PI stock solution;

[0046] ② Add 100 μL of cell protection solution (PBS buffer) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution;

[0047] ③ Add 200 μL of cell suspension to 100 μL of cell protection solution (PBS buffer with 0.1% FBS by volume), and then add 30 μL of 5X PI working solution;

[0048] Stain at room temperature in the dark for 10 min.

[0049] Step 5 Detection: After staining, transfer the sample in the tube to a flow cytometry tube, and then perform flow cytometry detection on the machine. The obtained results are as Figure 1 shown in and Table 1. When detecting and analyzing data, select channels 2 and 4, and the proportion of live cells is shown as Q4.

[0050] Table 1

[0051]

[0052]

[0053] Table 1 and Figure 1 The results show that:

[0054] 1. When the digestion time is 5 min, about half of the cell spheres are not completely digested, and the number of pipetting and mixing times is relatively large, which may be the reason for the low viability counted by NC.

[0055] 2. When the digestion time is 10 min, a small amount of cell spheres are not completely digested, and multiple pipetting and mixing are also required.

[0056] 3. When the digestion time is 15 min, the cell spheres are completely digested.

[0057] 4. Adding 0.1% FBS to the cell protection solution has almost no effect on the viability stained by PI; there is no significant difference between the low-concentration PI staining solutions ② and ③ and the control group ①.

[0058] 5. There is a large difference between the viability results of the NC counter and the PI viability results. Since the staining and counting methods used by the NC counter are different from the FDA / PI staining method used for the conventional judgment of islet viability, and therefore only the NC counter is used as a reference, but the method of using the NC counter to detect the islet viability is not considered in the present invention.

[0059] Example 3

[0060] Step 1 Sampling: Place the cell spheres to be detected in the biosafety cabinet. Insert a low-adsorption pipette tip into a 20 μL pipette, and aspirate 20 μL of full-volume cell spheres into a clean 1.5 mL EP tube. Avoid aspirating cell supernatant into the pipette tip during the aspiration of cell spheres. If necessary, repeat this step until the above requirements are met.

[0061] Step 2 Digestion: Add 1 mL of Accutase enzyme to a 1.5 mL EP tube and incubate at 37°C. Take 3 sets of EP tubes and digest for 15 min, 20 min, and 25 min respectively. During digestion, invert the EP tube several times every 5 min to ensure complete digestion.

[0062] Step 3 Sieving: Pass the digested cell suspension through a 40 μm cell sieve.

[0063] Step 4 Staining: Take 200 μL of the cell suspension after sieving in three groups and operate according to the following conditions:

[0064] ① Add 100 μL of cell protection solution (PBS buffer) to 200 μL of cell suspension, and then add 3 μL of 100X PI stock solution;

[0065] ② Add 100 μL of cell protection solution (PBS buffer) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution;

[0066] ③ Add 100 μL of cell protection solution (PBS buffer with 5% HSA by volume) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution;

[0067] Stain at room temperature in the dark for 10 min.

[0068] Step 5 Detection: After staining, transfer the sample in the tube to a flow cytometry tube, and then perform flow cytometry detection on the machine. The obtained results are as Figure 2 shown in and Table 2. When detecting and analyzing data, select channels 2 and 4, and the proportion of live cells is shown as Q4.

[0069] Table 2

[0070]

[0071] Table 2 and Figure 2 The results show that:

[0072] 1. The viability of ③ is higher than that of ① and ②. It can be seen that adding HSA to the cell protection solution will increase the cell viability; while the protection effect of FBS in Example 2 before was poor. It is speculated that albumin in HSA plays a key role, and the albumin content in FBS is very low, so the ability to maintain cell activity is poor.

[0073] 2. The viability of ② and ③ is higher than that of the control group ①. It can be seen that reducing the working concentration of the PI staining solution can increase the cell viability. The final concentration of PI in ② and ③ (50X) is half of that in the control group ① (100X), but the staining effect is better and the cell viability detection result is better. This may be because although the final concentration decreases, the working solution concentration of PI decreases, the volume of the PI working solution increases, and the increase in the volume of the staining solution helps the PI to fully contact the cells and improve the staining effect.

[0074] 3. From the perspective of digestion time, digesting for 20 minutes can not only ensure complete digestion of the cell spheres, but also has no effect on the viability of the cell spheres themselves.

[0075] Example 4

[0076] Step 1 Sampling: Place the cell spheres to be detected in the biosafety cabinet. Insert a low-adsorption pipette tip into a 20 μL pipette, and aspirate 20 μL of full-volume cell spheres into a clean 1.5 mL EP tube. Avoid aspirating cell supernatant into the pipette tip during the aspiration of cell spheres. Repeat this step if necessary until the above requirements are met.

[0077] Step 2 Digestion: Add 1 mL of Accutase enzyme to the 1.5 mL EP tube, and incubate at 37 °C. Take 3 groups of EP tubes and digest for 20 minutes. During digestion, invert the EP tubes several times every 5 minutes to ensure complete digestion.

[0078] Step 3 Sieving: Pass the digested cell suspension through a 40 μm cell sieve.

[0079] Step 4 Staining: Take 200 μL of the sieved cell suspension and operate according to the following conditions:

[0080] ① Add 100 μL of cell protection solution (PBS buffer with 0.1% HSA by volume) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution;

[0081] ② Add 100 μL of cell protection solution (PBS buffer with 5% HSA by volume) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution;

[0082] ③ Add 100 μL of cell protection solution (PBS buffer with 10% HSA by volume) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution;

[0083] ④ Add 100 μL of cell protection solution (PBS buffer with 25% HSA by volume) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution;

[0084] Stain at room temperature in the dark for 10 minutes.

[0085] Step 5 Detection: After the staining is completed, transfer the sample in the tube to a flow cytometry tube, and then perform flow cytometry detection on the machine. The obtained results are as Figure 3 shown in and Table 3. When detecting and analyzing data, select channels 2 and 4, and the proportion of live cells is shown in Q4.

[0086] Table 3

[0087] Condition HSA concentration in the protective solution PI viability ① 0.1％ 91.3％ ② 5％ 94.3％ ③ 10％ 93.1％ ④ 25％ 93.2％

[0088] The results of Table 3 and Figure 3 show that the concentration of HSA in the protective solution is too low, the protective effect on cells is poor, the PI viability rate is low, and too high a concentration of HSA will also affect the PI viability rate. The more preferred concentration range of HSA is 1.5 - 5%, and the most preferred is 5%.

[0089] It should be noted that the islet cell spheres used in Examples 2, 3, and 4 of the present invention are obtained by induced culture from different batches of human pluripotent stem cell sources, and there are differences in the viability rates between different batches. Therefore, the viability rate data between different examples cannot be compared.

[0090] Comparative Example

[0091] Directly take samples of the cells to be detected for FDA / PI staining. The specific steps are as follows:

[0092] 1. Prepare the PI working solution:

[0093] 1) Weigh 5 mg of PI and dissolve it in 10 mL of PBS to obtain a stock solution of 0.5 mg / mL. After aliquoting, store it in the dark at 4°C.

[0094] 2) Dissolve 50 μL of the PI stock solution in 450 μL of PBS to dilute it into a working solution of 0.05 mg / mL.

[0095] 2. Prepare the FDA working solution:

[0096] 1) Take 100 mg of FDA and dissolve it in 20 mL of acetone to obtain a stock solution of 5 mg / mL. After aliquoting, store it in the dark at -20°C.

[0097] 2) Take 10 μL of the FDA stock solution and dissolve it in 990 μL of PBS to dilute it into a working solution of 0.05 mg / mL, and use it up within 30 minutes.

[0098] (Note: Ensure the full dissolution of the dye when preparing the dye, otherwise blocky impurities may appear during staining)

[0099] 3. Sampling and staining

[0100] 1) Take an appropriate amount of islet cell spheres from human pluripotent stem cell sources and transfer them to one well of a low-attachment six-well plate;

[0101] 2) Preparation of staining solution: First, add 200 μL of PBS to a 1.5 mL EP tube, and then sequentially add 0.2 μL of FDA and 2 μL of PI working solution, and mix well.

[0102] 3) Staining: Add the prepared staining solution to the well plate containing cell spheres and incubate in the dark for 10 min.

[0103] 4. Observe by taking pictures with a fluorescence microscope.

[0104] The results are as Figure 4 shown. Figure 4 In the figure, the upper and middle pictures are fluorescence photos of islet cell spheres under a 4-fold microscope, and the lower picture is a fluorescence photo under a 10-fold microscope.

[0105] Among them, the excitation light intensity of the upper and lower pictures is 100, and the excitation light intensity of the middle picture is 500.

[0106] Figure 4 As can be seen from the upper and lower pictures of Figure 4 In the middle picture of

[0107] From Figure 4 it can be seen that this FDA / PI staining method can only roughly estimate the viability of islet cells, and accurate data cannot be obtained, and the estimation error is also relatively large.

[0108] Example 5

[0109] Step 1 Sampling: Place the cell spheres to be detected in a biosafety cabinet. Insert a low-adsorption pipette tip into a 20 μL pipette, and aspirate 20 μL of full-volume cell spheres into a clean 1.5 mL EP tube. Avoid aspirating cell supernatant into the pipette tip during the aspiration of cell spheres. If necessary, repeat this step until the above requirements are met.

[0110] Step 2 Digestion: Add 1 mL of Accutase enzyme to a 1.5 mL EP tube, and incubate at 37 °C. Take 3 groups of EP tubes and digest for 20 min. During digestion, the EP tubes need to be inverted up and down several times every 5 min to ensure complete digestion.

[0111] Step 3 Sieving: Pass the digested cell suspension through a 40 μm cell sieve.

[0112] Step 4 Staining: Add 100 μL of cell protection solution (PBS buffer containing 5% HSA by volume) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution; stain at room temperature in the dark for 10 min.

[0113] Step 5 Detection: After the staining is completed, transfer the sample in the tube to a flow cytometry tube, and then perform flow cytometry detection on the machine. The obtained results are as follows Figure 5 shown. When detecting and analyzing the data, select channels 2 and 4, and the proportion of live cells is shown in Q4.

[0114] Example 6

[0115] Step 1 Sampling: Place the cell spheres to be detected in a biosafety cabinet. Insert a low-adsorption pipette tip into a 20 μL pipette, and aspirate 20 μL of full-volume cell spheres into a clean 1.5 mL EP tube. Avoid aspirating the cell supernatant into the pipette tip during the aspiration of cell spheres. If necessary, repeat this step until the above requirements are met.

[0116] Step 2 Digestion: Add 1 mL of Accutase enzyme to the 1.5 mL EP tube, and incubate at 37 °C. Take 3 groups of EP tubes and digest for 20 min. During the digestion, invert the EP tube several times every 5 min to ensure complete digestion.

[0117] Step 3 Sieving: Pass the digested cell suspension through a 40 μm cell sieve.

[0118] Step 4 Staining: Take 200 μL of the cell suspension after sieving and operate according to the following conditions:

[0119] ① Add 100 μL of PBS buffer to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution; prepare three groups;

[0120] ② Add 100 μL of cell protection solution (PBS buffer with 5% HSA by volume) to 200 μL of cell suspension, and then add 30 μL of 5X PI working solution; prepare three groups;

[0121] Stain at room temperature in the dark for 10 min.

[0122] Step 5 Detection:

[0123] After staining, the three groups of samples under condition ① are immediately detected on the machine, detected on the machine after being placed for 30 minutes, detected on the machine after being placed for 1 hour, and detected on the machine after being placed for 2 hours.

[0124] The three groups of samples under condition ② are immediately detected on the machine, detected on the machine after being placed for 30 minutes, detected on the machine after being placed for 1 hour, and detected on the machine after being placed for 2 hours.

[0125] The obtained results are shown in Table 4 and Figure 6 as follows:

[0126] Table 4

[0127]

[0128] The results in Table 4 show that after adding the protective solution in Condition ②, the longer the placement time, the cell viability did not continue to decline, but remained above 90%. In Condition ①, only PBS buffer was added. After a long placement time, the cell viability continued to decline, and there were significant differences in the PI detection results.

[0129] In actual clinical applications, during large-scale detections, the sample preparation time is long, and there may be differences in the time when samples of the same batch are loaded onto the machine. The cell protective solution can protect cell viability for a long time without being affected by time, and the detection results are in line with the actual situation of the product. Without adding the cell protective solution, the cell viability is affected by the sample preparation time, and there are large errors between the detection results and the actual samples.

[0130] The viability detection of the invention has high repeatability and high accuracy of detection data.

Claims

1. A method for detecting the activity of islet cells derived from human pluripotent stem cells, characterized in that the method comprises the following steps: (1) The islet cell spheres to be detected are digested with Accutase enzyme to obtain a single-cell suspension; the islet cell spheres to be detected are islet cell spheres derived from human pluripotent stem cells, which are obtained by inducing and culturing human pluripotent stem cells with small chemical molecules; (2) Add HSA with a volume fraction of 0.1-5% to the PBS buffer as a cell protection solution. Add the cell protection solution to the single-cell suspension, and then add PI staining solution for staining; the volume ratio of the single-cell suspension to the cell protection solution is 200:80-120; the concentration of the PI staining solution is 0.02-0.04 mg / mL, and the volume ratio of the single-cell suspension to the PI staining solution is 200:20-40; (3) The stained sample is subjected to flow cytometry to obtain the proportion data of living cells.

2. The method according to claim 1, characterized in that in the step (2), the volume fraction of HSA in the cell protection solution is 5%.

3. The method according to claim 1, characterized in that in the step (2), 100 μL of the cell protection solution is added to every 200 μL of the single-cell suspension, and 30 μL of the PI staining solution with a concentration of 0.025 mg / mL is added, and the volume fraction of HSA in the cell protection solution is 5%.

4. The method according to claim 1, characterized in that in the step (2), the staining is carried out in the dark at room temperature for 10-20 min.

5. The method according to claim 1, characterized in that in the step (1), the digestion is carried out at 37 °C for 15-25 min.

6. The method according to claim 1, characterized in that the step (1) is carried out according to the following steps: Add 20 μL of the islet cell spheres to be detected to 1 mL of Accutase enzyme, and digest at 37 °C for 20 min to obtain a single-cell suspension.

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