A method for improving functional detection of pancreatic islet differentiated cells by optimizing a flow-based sample preparation process

Abstract

This invention discloses an optimized flow cytometry sample preparation method to improve the accuracy of functional detection of pancreatic islet differentiated cells. The method significantly improves single-cell viability (>90%), ensuring that the flow cytometry results of pancreatic β cells (C-peptide / NKX6.1 double positive), pancreatic α cells (GCG single positive), and pancreatic progenitor cells (PDX1 / NKX6.1 double positive) accurately reflect differentiation efficiency. This is highly consistent with GSIS functional detection and in vivo transplantation results, thereby reducing production errors and batch waste, and is applicable to quality control at all stages of islet differentiation.

Description

Technical Field

[0001] This invention relates to the field of differentiated stem cell function detection, and more specifically to a method for improving the functional detection of pancreatic islet differentiated cells by optimizing the flow cytometry sample preparation process. Background Technology

[0002] Pancreatic islet cells, especially β cells, are crucial for blood sugar control. For some diabetic patients, particularly those with type 1 diabetes, islet transplantation is an effective treatment. However, islet donors are very limited. Obtaining islet organoids for transplantation through stem cell differentiation is a promising alternative.

[0003] The differentiation of pancreatic β cells from embryonic stem cells or induced pluripotent stem cells (iPSCs) involves multiple stages of functional testing, and the results of each stage are crucial to the final outcome of that batch of differentiated cells. By using a combination of compounds in a specific sequence to simulate the development of pancreatic β cells, embryonic stem cells are differentiated sequentially into several stages: definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitors (PP cells), endocrine progenitors, and mature islet organoids. Functional testing of key differentiation stages, such as definitive endoderm, pancreatic progenitors (PP cells), and mature islet organoids, typically uses flow cytometry to determine the differentiation trend, making the flow cytometry sample preparation process crucial at each testing stage. Summary of the Invention

[0004] This invention is the first to discover that the flow cytometry sample preparation process affects the functional detection of differentiated pancreatic islet cells. Traditional flow cytometry sample preparation (e.g., static digestion, forced cell agitation, and failure to terminate digestion promptly) suffers from drawbacks such as low cell viability, uneven dissociation, distorted detection results, and batch judgment errors. This invention, through an integrated sample preparation scheme that optimizes washing, gentle dynamic digestion, timely termination of digestion, and standardized fixation and resuspension, significantly improves single-cell viability (>90%). This ensures that the flow cytometry results for pancreatic β cells (C-peptide / NKX6.1 double-positive), pancreatic α cells (GCG single-positive), and pancreatic progenitor cells (PDX1 / NKX6.1 double-positive) accurately reflect differentiation efficiency, showing high consistency with GSIS functional detection and in vivo transplantation results. This invention reduces production errors and batch waste, and is applicable to quality control at all stages of islet differentiation in adherent and 3D cell spheroids, providing stable and reliable detection support for large-scale production and clinical transplantation of stem cell islets.

[0005] Technical issues This invention aims to solve the problems of low cell viability, uneven dissociation, detection distortion, and batch misjudgment caused by existing flow cytometry sample preparation of pancreatic islet differentiated cells.

[0006] Technical solution This invention provides a method for optimizing flow cytometry sample preparation to improve the accuracy of functional detection of pancreatic islet differentiated cells, characterized by comprising the following steps: (1) Sampling and washing: Take pancreatic islet differentiated adherent cells or 3D cell spheres differentiated from pluripotent stem cells and wash them with cell washing solution; (2) Dynamic digestion: Add digestive fluid and digest under dynamic conditions on a shaker or biological stirrer; (3) Termination of digestion: Add the cell washing solution to terminate digestion, centrifuge and discard the supernatant; (4) Fixation, resuspension and flow cytometry: After fixing the cells, the cells are resuspended to prepare a single-cell suspension for flow cytometry detection.

[0007] In one specific embodiment, the pluripotent stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0008] In one specific embodiment, the islet differentiation cells are in a differentiation stage selected from the following: endoderm stage, pancreatic precursor stage, endocrine precursor cell stage, and islet organoid stage.

[0009] In one specific embodiment, the cell washing solution in step (1) comprises a basal culture medium and 1% 20% (v / v) serum or a serum substitute, wherein the basal culture medium is selected from one or more of DPBS, DMEM, DMEM / F12, RPMI 1640, MCDB 131, and mTeSR1, and / or the serum or serum substitute is selected from one or more of FBS, BSA, HAS (Human Serum Albumin), and KOSR (KnockOut Serum Replacement). These culture media are well known in the art and commercially available, for example, from Gibco, MCE, STEMCELL Technologies, or Thermo Fisher Scientific.

[0010] In one specific embodiment, in step (1): (i) the cells are washed 13 (1, 2, or 3) times with the cell washing solution; and / or (ii) the sample size of the adherent cells is 2 × 10⁻⁶. 5 2×10 6The number of samples, or the number of 3D cell spheres, is 20,010,000.

[0011] In one specific embodiment, the digestive fluid in step (2) is selected from one or more of ReleSR, Accutase, TrypLE, CTSTrypLE, and Trypsin. These digestive fluids are well known in the art and are commercially available, for example, from Gibco, MCE, STEMCELL Technologies, or Thermo Fisher Scientific.

[0012] In one specific embodiment, the conditions for dynamic digestion in step (2) are: shaker speed 160 rpm, biological stirrer speed 130 rpm, and time 2-20 min (or between 1, 2, 3, 4, 5, 8, and 10 min).

[0013] In one specific embodiment, the centrifugation conditions in step (3) are 100-1000 g and centrifugation time is 110 min (or between 1, 2, 3, 4, 5, 8, and 10 min).

[0014] In one specific embodiment, the biomarkers detected by flow cytometry in step (4) include: pancreatic β cells: C-peptide / NKX6.1 double positive; pancreatic α cells: GCG (glucagon) single positive; pancreatic progenitor cells: PDX1 / NKX6.1 double positive.

[0015] In one specific embodiment, the fixation in step (4) is performed using paraformaldehyde at 4°C for 15-60 min.

[0016] In one specific embodiment, the cell viability after sample preparation is ≥90%; and / or the pancreatic β-cell detection efficiency is 40%-70%, the pancreatic progenitor cell double positivity rate is 60%-80%, and the detection results are consistent with the blood glucose lowering effect of GSIS and transplantation.

[0017] Technical effect 1. Cell viability was significantly improved, with a viability rate >90% after sample preparation; 2. Flow cytometry results accurately reflect differentiation efficiency, with pancreatic β-cells accounting for 40%–70% and α-cells for 10%–20%, consistent with GSIS function and in vivo transplantation glucose-lowering effect; 3. Avoid misjudging high-quality cell batches, reduce production costs, and support large-scale production; 4. Suitable for adherent cells and 3D cell spheres, covering the entire process from endoderm, pancreatic precursors, endocrine precursors to mature islets. Attached Figure Description

[0018] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein: Figure 1 Flow cytometry analysis of pancreatic β cells from pluripotent stem cells differentiated into pancreatic islet organoids. Islet cells were identified by double-positive expression of C-peptide and NKX6.1, as shown in the figure above: Group A had 44.2% pancreatic β cell content, Group B had 67.0%, Group C had 53.9%, and Group D had 58.1%.

[0019] Figure 2 This study describes the flow cytometry analysis of pancreatic α cells from pluripotent stem cells differentiated into pancreatic islet organoids. Islet cells were identified by single-positive GCG (glucagon) expression, specifically Q1+Q2 in the flow cytometry plot. In the above figure, group A had 10.13% pancreatic α cell content, group B had 9.25%, group C had 11.16%, and group D had 12.16%. Group E served as the control group.

[0020] Figure 3 This study assesses insulin secretion function in pancreatic islet organoids differentiated from pluripotent stem cells using glucose-stimulated insulin release (GSIS). A high glucose level > a low glucose level indicates that the islet cells in groups A, B, C, and D above are functional and capable of responding to insulin secretion in response to both high and low glucose concentrations.

[0021] Figure 4 To investigate the transplantation efficacy of pluripotent stem cell-differentiated pancreatic islet organoids, the differentiated islet organoids were transplanted into diabetic mice. (A) Before transplantation, STZ (streptozotocin) treatment was administered to induce type 1 diabetes in SCID / Beige mice, resulting in elevated blood glucose levels. On day 10, after transplantation of the pluripotent stem cell-differentiated islets, diabetes reversal was achieved within 3 months, with blood glucose levels returning to normal (<11.1 mmol / L). After the experiment, the grafts were removed, and the mice exhibited a return to diabetic symptoms. (B) Three weeks post-transplantation, an intraperitoneal glucose tolerance test (IVGTT) was performed. Successfully transplanted mice responded to intraperitoneal glucose stimulation and achieved blood glucose recovery within 2 hours, while non-transplanted diabetic mice (CTRL1 and CTRL2) failed to achieve a blood glucose decrease within 2 hours, exhibiting diabetic symptoms. (C) Human C-peptide (approximately 200-800 pM) was detectable in mouse serum after transplantation. In contrast, human C-peptide was undetectable in mouse serum before transplantation and after graft removal.

[0022] Figure 5This study describes a flow cytometry assay for pancreatic β-cell content in pluripotent stem cell-derived pancreatic organoids. Samples of pancreatic organoids from the same batch of pluripotent stem cells were prepared using this non-patented method, and the β-cell content was analyzed by flow cytometry. The results in the graph above show that group A had a β-cell content of 0.66%, group B 0.45%, group C 0.23%, group D 0.28%, group E 0.036%, and group F 0.04%. This assay fails to accurately reflect the β-cell content and does not correlate with pancreatic function (GSIS and in vivo transplantation efficacy). Group G serves as the control group.

[0023] Figure 6 This study describes a flow cytometry assay for detecting pancreatic α cells in pluripotent stem cell-derived islet organoids using a non-patented method. Islet cells are identified by single-positive GCG (glucagon) expression, represented by Q1+Q2 in the flow cytometry plot. Samples from the same batch of pluripotent stem cell-derived islet organoids were prepared using this non-patented method, and the α cell content was analyzed by flow cytometry. In the above figure, the α cell content was 6.66% in group A, 4.83% in group B, 2.69% in group C, 15.84% in group D, 6.10% in group E, and 6.17% in group F. The flow cytometry plots show almost no GCG-positive cell populations, failing to accurately reflect the differentiation ratio of islet α cells. Group G serves as the control group.

[0024] Figure 7 This invention describes the flow cytometry detection of pancreatic progenitor cells differentiated from pluripotent stem cells using the method of this invention. Pancreatic progenitor cells are identified by double-positive expression of PDX1 and NKX6.1, as shown in part Q2 of the flow cytometry plot. After sampling pancreatic progenitor cells differentiated from pluripotent stem cells of the same batch, the flow cytometry samples prepared using the patented method were analyzed to determine the pancreatic progenitor cell content. As shown in the above figure, group A had a content of 66.1%, group B had a content of 84.8%, and group C had a content of 70.9%. The double-positive expression cell population of pancreatic progenitor cells was higher than 60%, which is higher than... Figure 8 The results of detection of pancreatic progenitor cells digested using a non-patented method were compared. Group D served as the control group.

[0025] Figure 8This study describes a flow cytometry assay for detecting pancreatic progenitor cells differentiated from pluripotent stem cells using a non-patented method. Pancreatic progenitor cells are characterized by double-positive expression of PDX1 and NKX6.1, as shown in portion Q2 of the flow cytometry plot. Samples of pancreatic progenitor cells differentiated from pluripotent stem cells from the same batch were prepared using this non-patented method, and the pancreatic progenitor cell content was analyzed by flow cytometry. Group A had a content of only 48.8%, Group B only 46.9%, and Group C only 10.4%. Group D served as the control group. The results showed significant numerical fluctuations in the flow cytometry results, far less than the 60-80% double-positive differentiation results. This failed to reflect the differentiation efficiency of pancreatic progenitor cells, potentially interfering with production release and hindering accurate detection of pancreatic progenitor cells. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise indicated, the terms used herein have their general technical meanings as understood by those skilled in the art.

[0028] In this invention, the singular articles “a” and “the” cover a plurality of indicators unless the context clearly indicates otherwise. All references cited herein are incorporated herein by reference in their entirety.

[0029] The use of "comprising" or "including" in this invention is an open-ended description that includes all specified components or steps described, as well as other specified components or steps that do not materially affect the description.

[0030] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0031] The "flow cytometry sample preparation" described in this invention refers to the process of obtaining a single-cell suspension for flow cytometry detection. Flow cytometry typically includes steps such as single-cell suspension preparation, cell counting and viability detection, cell fixation and permeabilization, antibody staining, washing, and resuspending. This invention focuses on optimizing the single-cell suspension preparation steps in a flow cytometry method for detecting the functionality of pancreatic islet differentiated cells.

[0032] Currently, methods for determining whether stem cell differentiation has reached the expected differentiation stage are very limited. Flow cytometry detection of functional markers at each stage of differentiation has become an important and crucial indicator for assessing the differentiation process. However, the flow cytometry sample preparation process affects the functional detection of pancreatic islet differentiation cells. Different sample preparation methods result in different test results, influencing whether a batch of cells is transplanted or discarded during differentiation. This invention improves the accuracy of functional detection at the differentiation stage by optimizing the flow cytometry sample preparation method, reducing production costs caused by detection errors due to sample preparation methods, and providing more accurate and effective data support for future large-scale production.

[0033] This invention relates to a method for improving the functional detection of pancreatic islet differentiated cells by optimizing the flow cytometry sample preparation process. It plays a role in detecting the differentiation efficiency of human pluripotent stem cells (iPSCs / ESCs) at various stages of differentiation into pancreatic islet organoids (including the stages of endoderm, pancreatic progenitor cells, endocrine progenitor cells, and pancreatic islet organoids), providing data support for large-scale production. Compared to conventional (non-patented) methods for digesting pancreatic islet organoids to prepare single-cell samples for flow cytometry detection, this invention can improve cell viability during the digestion and sample preparation process, accurately detect the differentiation efficiency of islet cells, reduce cell breakage or death caused by the dissociation of cell spheroids or adherent cells during sample preparation, reduce errors in differentiation efficiency detection, and improve the accuracy of flow cytometry detection. This increases production batch stability, reduces the production cost of pancreatic differentiated cells, provides accurate and effective data support for large-scale production, and brings good expectations for the in vivo transplantation effect of differentiated mature islet cells. It effectively reduces the risk of diabetes mellitus' inability to regulate blood sugar stability, thereby reducing diabetic complications. It is of great significance for improving the quality of life of the injured and sick, reducing the burden on society and families, and promoting the long-term harmonious and healthy development of Chinese society.

[0034] The present invention is further illustrated in the following embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents, materials, etc., used in the embodiments are commercially available. Unless otherwise specified, the methods used in the embodiments of this application are conventional methods in the prior art.

[0035] The pancreatic islet cells (including adherent cells and 3D cell spheres) differentiated from pluripotent stem cells (iPSCs) used in the embodiments can be prepared with reference to published methods, particularly Chinese Patent Application No. 2025111697686 entitled "Method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells" and Chinese Patent Application No. 2023113510622 entitled "A pancreatic endocrine cell and a method for its preparation".

[0036] Example 1: Flow cytometry sample preparation, detection method, and transplantation efficacy verification of pancreatic islet cell differentiation from pluripotent stem cells. Flow cytometry sampling and sample preparation steps: (1) For adherent cells, each flow cytometry sample should contain an estimated 2E6 cells. Remove the adherent cells from the incubator and aspirate the culture medium using a washing pump. Wash three times with DPBS (gibco) + 2% BSA (Thermo Fisher Scientific). Discard the washing solution after washing.

[0037] Digest the cells in 1 mL TrypLE (Thermo Fisher Scientific) on a shaker (20 rpm) or a biological stirrer (Huakan Biotechnology 5 mL stirred culture plate) (20 rpm) for 10 min. Then, add 3 mL of cell washing buffer to terminate the digestion. Centrifuge at 200 g for 2 min, and discard the supernatant. Add 1 mL of paraformaldehyde to the cell pellet and fix at 4°C for 30 min. Centrifuge at 1000 g for 3 min, and discard the supernatant. Finally, resuspend the cells in 1 mL of cell washing buffer and count the single cells. Store the sample at 4°C for flow cytometry detection of C-peptide / GCG (C-peptide and glucagon, glucagon-positive expression represents pancreatic α-cell population) and C-peptide / NKX6.1 (C-peptide and NKX6.1 double-positive expression represents pancreatic β-cell population).

[0038] (2) For cell spheres cultured in 3D, approximately 1000 cell spheres are required for each flow cytometry sample. The cell spheres are centrifuged at 100 g for 3 min along with the culture medium, allowing them to settle to the bottom of the centrifuge tube. The supernatant is then discarded. The cells are washed three times with DPBS (gibco) + 2% BSA (ThermoFisher Scientific) and centrifuged at 200 g for 3 min. The supernatant is discarded after centrifugation.

[0039] Digest the cells in 1 mL of TrypLE (Thermo Fisher Scientific) on a shaker (30 rpm) or a biological stirrer (Huakan Biotechnology 5 mL stirred culture plate) for 10 min. Then, add 3 mL of cell washing buffer to terminate the digestion. Centrifuge at 200 g for 3 min, discarding the supernatant. Add 1 mL of paraformaldehyde to the cell pellet and fix at 4°C for 30 min. Centrifuge at 1000 g for 3 min, discarding the supernatant. Finally, resuspend the cells in 1 mL of cell washing buffer and count the single cells. Store the sample at 4°C for flow cytometry analysis of C-peptide / GCG and C-peptide / NKX6.1.

[0040] The following are the results of four independent samples digested with pancreatic islet spheres using the above method. The sample volume was 10 μL, and the cells were counted using a CountStar Rigel S2. The results (see Table 1 below) show that the viability was higher than 90%, and the results were consistent.

[0041] Table 1 Flow cytometry results (Beckman CytoFLEX flow cytometer), see [link / reference]. Figure 1 and Figure 2 Four independent samples of pancreatic islet cell spheres digested according to the above-mentioned method were labeled as groups A, B, C, and D, with group E serving as a negative control. Figure 1 These are the results of pancreatic islet beta cell detection in groups ABCD. Figure 2 These are the results of pancreatic islet alpha cell detection in groups ABCD.

[0042] The antibodies involved in flow cytometry detection are shown in Table 2 below: Table 2 Note: NKX6.1 uses two different antibodies. The NKX6.1 used for detecting the PP phase and the NKX6.1 used for detecting the beta phase are different antibodies, so as to distinguish the colors.

[0043] In addition, insulin secretion function of pancreatic islet organoids differentiated from pluripotent stem cells was tested, specifically glucose-stimulated insulin release (GSIS), according to two published patent applications: Chinese Patent Application No. 2025111697686 entitled "Method for Increasing the Yield and Improving the Stability of Pluripotent Stem Cell Differentiation into Pancreatic Islet Organoids" and Chinese Patent Application No. 2023113510622 entitled "A Pancreatic Endocrine Cell and its Preparation Method". Four independent samples of pancreatic islet spheroids undergoing GSIS testing according to the above-mentioned methods were labeled as groups A, B, C, and D. The results are as follows: Figure 3 As shown.

[0044] In addition, the transplantation efficacy of pluripotent stem cell-differentiated islet organoids was verified. The grafts were combined samples from four independent tests of islet cell spheres performed using the aforementioned method. Because of the good consistency in quality testing, the combined samples were used for in vivo efficacy testing of islet transplantation in diabetic mice. The results are as follows. Figure 4 As shown.

[0045] In summary, the above experimental results demonstrate that, in the flow cytometry detection method of this invention, the differentiation efficiency of pancreatic β cells (C-peptide / NKX6.1 double positive) is generally between 40% and 70%; the differentiation efficiency of pancreatic α cells (GCG single positive) is generally between 10% and 20%; such islets are functional and have a hypoglycemic effect after transplantation.

[0046] According to the method of this invention, the detection results after flow cytometry digestion accurately reflect the islet differentiation effect, consistent with the GSIS functional detection results (high glucose > low glucose), proving that islet cell differentiation is functional. After flow cytometry single-cell sample preparation, the cell viability is generally greater than 90%, indicating good sample preparation quality.

[0047] Comparative Example 1: Conventional (non-patented method) digestion of pancreatic islets The same 3D pancreatic islet cell spheres differentiated from pluripotent stem cells as in Example 1 were used.

[0048] Control A: Digest cells with 1 mL TrypLE for 30 min, incubate in an incubator, then remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Do not add cell washing buffer to stop digestion. Centrifuge at 1000g for 3 min, and discard the supernatant. Add 1 mL paraformaldehyde to the cell pellet, fix at 4℃ for 60 min, then centrifuge at 1000g for 3 min, and discard the supernatant. Finally, resuspend the cell pellet in 1 mL cell washing buffer and count single cells. Store the sample at 4℃ for flow cytometry detection of C-peptide / GCG and C-peptide / NKX6.1.

[0049] Control B: Digest cells with 1 mL TrypLE for 30 min, incubate in an incubator, then remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Add 3 mL of cell washing buffer to stop digestion, centrifuge at 1000 g for 3 min, and discard the supernatant. Add 1 mL of paraformaldehyde to the cell pellet, fix at 4 °C for 60 min, centrifuge at 1000 g for 3 min, and discard the supernatant. Finally, resuspend the cell pellet in 1 mL of cell washing buffer and count single cells. Store the sample at 4 °C for flow cytometry detection of C-peptide / GCG and C-peptide / NKX6.1.

[0050] Control C: Digest cells with 1 mL Accutase for 30 min, incubate in an incubator, then remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Do not add cell washing buffer to stop digestion. Centrifuge at 1000 g for 3 min, and discard the supernatant. Add 1 mL paraformaldehyde to the cell pellet, fix at 4 °C for 60 min, then centrifuge at 1000 g for 3 min, and discard the supernatant. Finally, resuspend the cell pellet in 1 mL cell washing buffer and count single cells. Store the sample at 4 °C for flow cytometry detection of C-peptide / GCG and C-peptide / NKX6.1.

[0051] Control D: Digest cells with 1 mL Accutase for 30 min, incubate in an incubator, then remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Add 3 mL of cell washing buffer to stop digestion, centrifuge at 1000 g for 3 min, and discard the supernatant. Add 1 mL of paraformaldehyde to the cell pellet, fix at 4 °C for 60 min, centrifuge at 1000 g for 3 min, and discard the supernatant. Finally, resuspend the cell pellet in 1 mL of cell washing buffer and count single cells. Store the sample at 4 °C for flow cytometry detection of C-peptide / GCG and C-peptide / NKX6.1.

[0052] Control E: Digest cells with 1 mL TrypLE for 20 min, stirring with a stirrer. After digestion, remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Do not add cell washing buffer to stop digestion. Centrifuge at 1000g for 3 min, and discard the supernatant. Add 1 mL paraformaldehyde to the cell pellet, fix at 4℃ for 60 min, and centrifuge at 1000g for 3 min, discarding the supernatant. Finally, resuspend the cell pellet in 1 mL cell washing buffer and count single cells. Store the sample at 4℃ for flow cytometry detection of C-peptide / GCG and C-peptide / NKX6.1.

[0053] Control F: Digest with 1 mL Accutase for 20 min, stirring with a stirrer. After digestion, remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Add 3 mL of cell washing buffer to stop digestion, and centrifuge at 1000 g for 3 min. Discard the supernatant after centrifugation. Add 1 mL of paraformaldehyde to the cell pellet, fix at 4 °C for 60 min, and centrifuge at 1000 g for 3 min. Discard the supernatant after centrifugation. Finally, resuspend the cell pellet in 1 mL of cell washing buffer and count single cells. Store the sample at 4 °C for flow cytometry detection of C-peptide / GCG and C-peptide / NKX6.1.

[0054] Table 3 below shows the results of digested islet detection, with a sample volume of 10 μL, analyzed using a cell counter (CountStar Rigel S2): Table 3 Flow cytometry results (Beckman CytoFLEX flow cytometer, antibody detected as above), see [link / reference]. Figure 5 and Figure 6 The pancreatic islet cell spheres digested using the above-mentioned non-inventive method were respectively associated with groups A, B, C, D, E, and F, with group G serving as a negative control.

[0055] The results show that treating pancreatic islet cells using conventional (non-patented) cell preparation methods results in low cell viability (fluctuating around 60%, far lower than the digestion method of this invention), and the flow cytometry results after sample preparation fail to accurately reflect islet differentiation. The main reasons for this are: 1. In the control groups ABCD, the pancreatic islet cells were not digested with simultaneous digestion and stirring. Being left stagnant in the incubator prevented uniform digestion by the digestive solution (whether the weak digestive solution Accutase or the strong digestive solution TrypLE). Even extending the digestion time to 30 minutes resulted in incomplete digestion of central islet cells, and the dispersal of cells by pipetting caused significant damage to islet cells, ultimately distorting the flow cytometry results. 2. In the control groups EF, the cell stop solution effectively terminated the enzyme's action. Although flow cytometry offers methods to directly fix the target without a stop solution to accelerate detection, the experimental results show that without adding a stop solution after sample preparation, cells die rapidly, resulting in a rapid decrease in viability and unsatisfactory flow cytometry results. Even using paraformaldehyde to fix cells resulted in ineffective flow cytometry detection. This leads to errors in the assessment of islet cell differentiation efficiency. The differentiation efficiency of pancreatic β cells (C-peptide / NKX6.1 double positive) is generally around 10%; the differentiation efficiency of pancreatic α cells (GCG single positive) generally fluctuates below 20%, resulting in large fluctuations in the test data and failing to show the functional characteristics of the islets (such as GSIS>1, indicating a hypoglycemic effect when transplanted in vivo).

[0056] Example 2: Flow cytometry sample preparation and detection method for pancreatic progenitor cell differentiation from pluripotent stem cells. For pancreatic progenitor cell spheroids differentiated from pluripotent stem cells in 3D culture, approximately 1000 cell spheroids are collected for each flow cytometry sample. The spheroids are centrifuged at 200 g for 3 minutes along with the culture medium, allowing them to settle to the bottom of the centrifuge tube. The supernatant is then discarded. The cells are washed three times with DPBS + 2% BSA, followed by centrifugation at 200 g for 3 minutes. The supernatant is discarded after centrifugation.

[0057] Using 1 mL of TrypLE, place it in an incubator and shake at 30 rpm or a biological stirrer at 30 rpm (Huakan Biotechnology 5 mL stirred culture plate) for 10 min to digest. Then add 3 mL of cell washing buffer to stop digestion. Centrifuge at 200 g for 3 min, and discard the supernatant after centrifugation. Add 1 mL of paraformaldehyde to the cell pellet, fix at 4℃ for 30 min, and centrifuge at 1000 g for 3 min, and discard the supernatant after centrifugation. Finally, resuspend the cell pellet in 1 mL of cell washing buffer and count single cells. Store the sample at 4℃ for flow cytometry detection of PDX1 / NKX6.1. The detection results are as follows: Figure 7 As shown, groups A, B, and C are pancreatic progenitor cell spheres from three independent pluripotent stem cell differentiations, while group D is a negative control.

[0058] Comparative Example 2: Detection of pancreatic progenitor cells by conventional (non-patented method) digestion The same 3D cell spheres as in Example 2 were used, and the detection method was the same as described above.

[0059] Control A: Digest cells with 1 mL TrypLE for 10 min, incubate in an incubator, then remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Add 3 mL of cell washing buffer to stop digestion, centrifuge at 1000 g for 3 min, and discard the supernatant. Add 1 mL of paraformaldehyde to the cell pellet, fix at 4 °C for 60 min, centrifuge at 1000 g for 3 min, and discard the supernatant. Finally, resuspend the cell pellet in 1 mL of cell washing buffer and count single cells. Store the sample at 4 °C for flow cytometry detection of PDX1 / NKX6.1.

[0060] Control B: Digest cells with 1 mL TrypLE for 20 min, incubate in an incubator, then remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Add 3 mL of cell washing buffer to stop digestion, centrifuge at 1000 g for 3 min, and discard the supernatant. Add 1 mL of paraformaldehyde to the cell pellet, fix at 4 °C for 60 min, centrifuge at 1000 g for 3 min, and discard the supernatant. Finally, resuspend the cell pellet in 1 mL of cell washing buffer and count single cells. Store the sample at 4 °C for flow cytometry detection of PDX1 / NKX6.1.

[0061] Control C: Digest cells with 1 mL Accutase for 10 min, incubate in an incubator, then remove from the incubator, mix cells by pipetting, and disperse any undigested cells. Add 3 mL of cell washing buffer to stop digestion, centrifuge at 1000 g for 3 min, and discard the supernatant. Add 1 mL of paraformaldehyde to the cell pellet, fix at 4 °C for 60 min, centrifuge at 1000 g for 3 min, and discard the supernatant. Finally, resuspend the cell pellet in 1 mL of cell washing buffer and count single cells. Store the sample at 4 °C for flow cytometry detection of PDX1 / NKX6.1. Detection results are as follows: Figure 8 As shown, groups A, B, and C are samples of pancreatic progenitor cell spheres digested by the non-inventive method described above, corresponding to groups A, B, and C respectively, while group D is a negative control.

[0062] The experimental results show that treating pancreatic islet cells with conventional (non-patented) cell preparation methods results in low cell viability (fluctuating by about 60%). The detection results after flow cytometry digestion also fail to accurately reflect the islet differentiation effect, causing errors in the detection and judgment of islet cell differentiation efficiency, resulting in large fluctuations in the detection data, and failing to show the functional characteristics of the islets (such as GSIS>1, indicating a hypoglycemic effect when transplanted in vivo).

[0063] This invention was used to prepare single-cell samples of pancreatic islet organoids differentiated from pluripotent stem cells, and flow cytometry analysis was performed. The results after flow cytometry digestion accurately reflected the islet differentiation effect, consistent with the GSIS functional assay results, demonstrating that islet cell differentiation is functional. After flow cytometry preparation of single-cell samples, cell viability was improved, and the sample preparation quality was good. In flow cytometry analysis, the differentiation efficiency of pancreatic β cells (C-peptide / NKX6.1 double positive) was detected more accurately.

[0064] In the example of flow cytometry sample preparation for pancreatic progenitor cells, the conventional (non-patented) cell preparation method for pancreatic progenitor cells will result in cell loss or damage, leading to a lower positive result (10-40%) in the final flow cytometry detection, which will cause confusion in the judgment of the differentiation quality and differentiation effect of pancreatic progenitor cells.

[0065] According to the method of the present invention, the detection results after flow cytometry digestion can accurately reflect the differentiation effect of pancreatic progenitor cells (between 60-80%), and the differentiation ratio of pancreatic progenitor cells with double positive PDX1 / NKX6.1 is significantly improved.

[0066] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0067] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. Furthermore, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, and should also be regarded as the content disclosed by the present invention.

Claims

1. A method for optimizing flow cytometry sample preparation to improve the accuracy of functional detection of pancreatic islet differentiated cells, characterized in that, Includes the following steps: (1) Sampling and washing: Take pancreatic islet differentiated adherent cells or 3D cell spheres differentiated from pluripotent stem cells and wash them with cell washing solution; (2) Dynamic digestion: Add digestive fluid and digest under dynamic conditions on a shaker or biological stirrer; (3) Termination of digestion: Add the cell washing solution to terminate digestion, centrifuge and discard the supernatant; (4) Fixation, resuspension and flow cytometry: After fixing the cells, the cells are resuspended to prepare a single-cell suspension for flow cytometry detection.

2. The method of claim 1, wherein the pluripotent stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

3. The method according to claim 1, wherein the islet differentiation cells are in a differentiation stage selected from the following: endoderm stage, pancreatic precursor stage, endocrine precursor cell stage, and islet organoid stage.

4. The method according to claim 1, wherein the cell washing solution in step (1) comprises a basal culture medium and 1% 20% (v / v) serum or a serum substitute, wherein the basal culture medium is selected from one or more of DPBS, DMEM, DMEM / F12, RPMI1640, MCDB131 and mTeSR1, and / or the serum or serum substitute is selected from one or more of FBS, BSA, HSA, and KOSR.

5. The method according to claim 1, wherein in step (1): (i) Wash 13 times with the cell washing solution; and / or (ii) The sample size of the adherent cells is 2 × 10⁻⁶. 5 2×10 6 The number of samples, or the number of 3D cell spheres, is 20,010,000.

6. The method according to claim 1, wherein the digestive fluid in step (2) is selected from one or more of ReleSR, Accutase, TrypLE, CTSTrypLE and Trypsin.

7. The method according to claim 1, wherein the conditions for dynamic digestion in step (2) are: shaking speed of 160 rpm, biological stirrer speed of 130 rpm, and time of 2-20 min.

8. The method according to claim 1, wherein the centrifugation conditions in step (3) are 100-1000 g and centrifugation time is 110 min.

9. The method according to claim 1, wherein the biomarker detected by flow cytometry in step (4) comprises: Pancreatic β cells: C-peptide / NKX6.1 double positive; Pancreatic α cells: GCG single positive; Pancreatic progenitor cells: PDX1 / NKX6.1 double positive.

10. The method according to any one of claims 1 to 9, wherein the fixation in step (4) is performed by paraformaldehyde fixation at 4°C for 15 to 60 min; wherein the cell viability after sample preparation is ≥90%; and / or the pancreatic β-cell detection efficiency is 40% to 70%, the pancreatic progenitor cell double positivity rate is 60% to 80%, and the detection results are consistent with the hypoglycemic effect of GSIS and transplantation.

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