A highly efficient and gentle method for dissociating mammalian blastocyst single cells
By using a composite dissociation solution and simplifying the operation process, the problems of low dissociation efficiency, poor cell viability, and complex operation in single-cell dissociation technology have been solved, achieving efficient and gentle blastocyst single-cell dissociation, which is suitable for high-throughput experiments.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE SECOND AFFILIATED HOSPITAL OF NANJING MEDICAL UNIV
- Filing Date
- 2026-01-19
- Publication Date
- 2026-05-29
AI Technical Summary
Existing single-cell dissociation technologies suffer from low dissociation efficiency, poor cell viability, complex operation, and cell morphology damage, making it difficult to meet high-throughput requirements.
A composite dissociation solution containing EDTA, TrypLE™ Express enzyme, ACCUTASE cell digestion solution, and KSOM embryo culture medium is used. Combined with calcium- and magnesium-free benchtop solution to remove the zona pellucida and pipetting with an oropharynx, the blastocysts are incubated at 35-37°C, simplifying the process to two steps.
It significantly improves single-cell dissociation efficiency and cell viability, shortens operation time, ensures cell morphology integrity, and is suitable for high-throughput experiments.
Smart Images
Figure CN122104560A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of developmental biology technology, specifically relating to a highly efficient and gentle method for the dissociation of single-cell mammalian blastocysts. Background Technology
[0002] Single-cell dissociation technology is crucial in biological research, especially when dealing with special samples such as blastocysts, which present significant challenges. Blastocysts consist of a stable three-dimensional structure formed by the dense adhesion of the trophoblast (TE) and inner cell mass (ICM) through tight junction proteins (such as E-cadherin). The outer zona pellucida (ZP), acting as a glycoprotein shell, forms a physical barrier, hindering enzyme penetration. Simultaneously, the internal pressure generated by the blastocyst cavity fluid further strengthens intercellular connections, making conventional trypsin / collagenase systems difficult to effectively dissociate cells. Existing methods, such as commercially available kits, require multiple steps (acid treatment of the zona pellucida → mechanical dissection → combined enzymatic digestion), rely on microscopic instruments (such as laser cutting or puncture), take more than 90 minutes, and generally result in cell viability below 70%. While micropipette methods, laser-captured microdissection, or microfluidic techniques can be used for single-cell isolation, they suffer from operational complexity, expensive equipment, or low cell yield, making it difficult to meet high-throughput requirements. In summary, existing single-cell dissociation techniques have the following technical problems: (1) Low dissociation efficiency: Conventional trypsin / collagenase systems are difficult to effectively dissociate blastocysts, and existing commercial kits require multiple steps and take more than 90 minutes. (2) Poor cell viability: The cell viability of existing methods is generally below 70%. Although micropipette, laser capture microdissection, or microfluidic technology can be used for single-cell separation, the cell yield is low. (3) Complex operation: Existing methods rely on microscopic instruments (such as laser cutting or puncture), which are complex to operate and difficult to meet high-throughput requirements. (4) Damaged cell morphology: Traditional acidic solutions damage cells, resulting in incomplete cell morphology and affecting the accuracy of subsequent experiments. Therefore, developing an efficient and mild single-cell dissociation method for mammalian blastocysts is an urgent technical problem to be solved in this field. Summary of the Invention
[0003] The purpose of this invention is to address the technical problems raised in the background art by providing an efficient and gentle method for the dissociation of single-cell mammalian blastocysts.
[0004] The objective of this invention can be achieved through the following technical solutions:
[0005] In a first aspect, the present invention claims protection for a composite dissociation solution for single-cell dissociation of mammalian blastocysts, the composite dissociation solution containing the following components at final concentrations: EDTA 4-6 mM, TrypLE™ Express enzyme 8%-20% (v / v), ACCUTASE cell digestion solution 8%-20% (v / v), and KSOM embryo culture medium to 100%.
[0006] Preferably, the final concentrations of each component are: EDTA 4-6 mM, TrypLE™ Express enzyme 8%-12% (v / v), ACCUTASE cell digestion solution 8%-12% (v / v), and KSOM embryo culture medium to 100%. More preferably, the final concentrations of each component are: EDTA 5 mM, TrypLE™ Express enzyme 10% (v / v), ACCUTASE cell digestion solution 10% (v / v), and KSOM embryo culture medium to 100%.
[0007] As a more preferred technical solution, the composite dissociation solution further comprises collagenase type IV, neutral protease type II, and DNase; the final concentrations of collagenase type IV, neutral protease type II, and DNase in the composite dissociation solution are as follows: collagenase type IV 0.6-1.5 mM, neutral protease type II 0.6-1.5 U / mL, and DNase 30-70 ug / mL. Preferably, the concentrations are: collagenase type IV 0.8-1.2 mM, neutral protease type II 0.8-1.2 U / mL, and DNase 40-60 ug / mL. More preferably, the concentrations are: collagenase type IV 1 mM, neutral protease type II 1 U / mL, and DNase 50 ug / mL.
[0008] In a specific embodiment of the present invention, the composite dissociation solution can have two formulations:
[0009] Formula 1: EDTA 4-6 mM, TrypLE™ Express enzyme 8%-20% (v / v), ACCUTASE cell digestion solution 8%-20% (v / v), KSOM embryo culture medium to 100%.
[0010] Formula 2: EDTA 4-6 mM, TrypLE™ Express enzyme 8%-20% (v / v), ACCUTASE cell digestion solution 8%-20% (v / v), collagenase type IV 0.6-1.5 mM, neutral protease type II 0.6-1.5 U / mL, DNase 30-70 ug / mL, KSOM embryo culture medium to 100%. Preferably: EDTA 4-6 mM, TrypLE™ Express enzyme 8%-12% (v / v), ACCUTASE cell digestion solution 8%-12% (v / v), collagenase type IV 0.8-1.2 mM, neutral protease type II 0.8-1.2 U / mL, DNase 40-60 ug / mL, KSOM embryo culture medium to 100%. Secondly, this invention claims protection for a highly efficient and mild method for the dissociation of single-cell mammalian blastocysts, the method comprising the following steps:
[0011] (1) Removal of zona pellucida: The blastocyst is placed in a calcium- and magnesium-free terephthalic solution and the zona pellucida is dissolved and completely removed by physical blowing.
[0012] (2) Enzymatic hydrolysis incubation: The blastocysts treated in step (1) are transferred to the composite dissociation solution of any one of claims 1-4 pre-equilibrated at 35℃-37℃ and incubated for 15-30 minutes, during which intermittent pipetting is performed until the blastocysts are completely dissociated into single cells;
[0013] (3) Washing: Use dPBS to wash the dissociated single cells to remove residual dissociation solution.
[0014] Furthermore, after step (1) and before step (2), there is an additional step: using an oral pipette with a diameter smaller than that of the blastocyst to blow on the blastocyst after removing the zona pellucida, so as to completely drain the blastocyst cavity fluid and loosen the blastocyst structure.
[0015] Furthermore, the physical blowing operation described in step (1) involves repeatedly blowing using a straw; the intermittent blowing described in step (2) involves blowing once every 5 minutes.
[0016] Thirdly, the present invention seeks protection for the use of the above-described composite dissociation solution in the preparation of reagents or kits for the dissociation of single cells from mammalian blastocysts.
[0017] Fourthly, the present invention claims protection for a reagent or kit for the dissociation of single cells from mammalian blastocysts, the reagent or kit containing the aforementioned composite dissociation solution.
[0018] Fifthly, the present invention claims protection for mammalian blastocyst single cells obtained by the above-described method, wherein the viability of the single cells is greater than 90%. The single cells are suitable for flow cytometry sorting, single-cell sequencing, or embryonic stem cell line establishment.
[0019] This invention provides a highly efficient and gentle two-step method for single-cell blastocyst dissociation. First, a calcium- and magnesium-free benchtop solution is used to dissolve the zona pellucida, avoiding cell damage caused by traditional acidic solutions (such as Tyrode's acid), removing the zona pellucida barrier within 5 minutes. Then, the blastocysts are placed in a specialized composite dissociation solution and incubated at 35-37°C for 15-20 minutes to gently lyse intercellular connections; intermittent pipetting is performed during this time until the blastocysts are completely dissociated into single cells. The entire process takes only 30 minutes and requires no centrifugation or micromanipulation. The dissociation solution environment maintains osmotic pressure balance, reduces apoptosis signal activation, and ensures intact cell morphology. This approach significantly reduces mechanical stress and chemical damage by simplifying and optimizing the dissociation system and operational procedures, making it suitable for subsequent high-precision analyses such as flow cytometry or single-cell sequencing.
[0020] In a specific embodiment of the present invention, the efficient and gentle mammalian blastocyst single-cell dissociation method specifically includes the following steps:
[0021] (1) Collect embryonic cells at the blastocyst stage, place a single blastocyst in a drop of calcium- and magnesium-free thallium solution, and repeatedly blow with an oral pipette until the zona pellucida is dissolved and removed. Then, observe with a stereomicroscope to ensure that the zona pellucida has been completely removed.
[0022] (2) Use a small-diameter pipette smaller than the diameter of the blastocyst to repeatedly blow on the embryo after removing the zona pellucida until the blastocyst cavity fluid in the blastocyst is completely drained, making the blastocyst structure loose.
[0023] (3) Transfer the treated blastocysts into droplets of a composite dissociation solution that has been balanced at 35-37℃. Use a pipette to repeatedly blow the blastocysts every 5 minutes. After incubation for 15 minutes, the blastocysts will be dispersed into single blastocyst cells.
[0024] (4) Use dPBS to wash the detached blastocyst single cells. Repeat the washing three times to remove residual lysis buffer and other impurities, ensuring the purity and activity of the single cell sample and providing high-quality samples for subsequent experiments.
[0025] The functions of each technical feature in the technical solution of this invention are as follows:
[0026] (1) Function of calcium- and magnesium-free metabolite solution: calcium- and magnesium-free metabolite solution can effectively dissolve the zona pellucida of the blastocyst, avoid damage to cells caused by traditional acidic solutions, and quickly remove the zona pellucida barrier, providing convenience for subsequent operations.
[0027] (2) Selection of small-diameter pipettes: Using small-diameter pipettes smaller than the diameter of the blastocyst allows for more precise operation, avoids unnecessary mechanical damage to the blastocyst cells, and ensures that the blastocyst cavity fluid is completely drained.
[0028] (3) 35-37℃ constant temperature incubation: 35-37℃ is the suitable temperature for cell physiological activities. Constant temperature incubation can ensure that the blastocyst lysis complex buffer works under the best conditions, gently lysing intercellular connections and reducing cell damage.
[0029] (4) Importance of dPBS washing: Repeated washing with dPBS can remove residual lysis buffer and other impurities, ensuring the purity and activity of single-cell samples, and providing high-quality samples for subsequent flow cytometry or single-cell sequencing experiments.
[0030] (5) Using the composite dissociation solution of the present invention, the dissociation solution environment can maintain osmotic pressure balance, reduce the activation of apoptosis signals, and ensure the integrity of cell morphology.
[0031] The beneficial effects of this invention are:
[0032] The core advantages of this invention lie in its high efficiency, high viability, and simplified operation. Compared with traditional methods: 1. Reduced steps and simplified operation: The dissociation process is simplified from 3-4 steps to two steps (zona pellucida dissolution + enzymatic incubation), reducing the total time from more than 90 minutes to less than 30 minutes, significantly improving dissociation efficiency; 2. Enhanced cell viability: Cell viability is increased from about 70% to more than 90% due to mild reagents and short treatment time, reducing membrane damage; 3. Guaranteed cell morphology integrity: Incubation at 37°C for 15-20 minutes gently lyses intercellular connections, reducing cell damage. The dissociation solution environment maintains osmotic pressure balance, reduces apoptosis signal activation, and ensures cell morphology integrity, making it suitable for subsequent high-precision analyses such as flow cytometry or single-cell sequencing; 4. Enhanced compatibility: Dissociated cells can be directly used in downstream experiments, such as ensuring accurate surface antigen recognition during flow cytometry due to membrane integrity, and avoiding gene expression bias caused by apoptosis in single-cell sequencing due to high viability. This protocol is particularly suitable for embryo fate studies, providing high-fidelity single-cell samples for chromosomal heterogeneity analysis of the trophoblast and inner cell mass of blastocysts. 5. Simple operation: No centrifugation or micromanipulation is required, reducing operational complexity and dependence on microscopic instruments, thus meeting high-throughput requirements. Single-cell dispersion can be achieved simply by gently blowing with a pipette, eliminating the need for complex mechanical dissection or laser cutting. Attached Figure Description
[0033] Figure 1 This is a flowchart of the blastocyst dissociation process of the present invention.
[0034] Figure 2 This is an image of a mouse blastocyst.
[0035] Figure 3 This is a diagram showing the results of routine pancreatic enzyme dissociation.
[0036] Figure 4 This is a graph showing the dissociation results of a commercially available reagent.
[0037] Figure 5 This is a diagram showing the dissociation results of the composite dissociation solution and blastocyst in this invention.
[0038] Figure 6 This is a diagram showing the dissociation results of the composite dissociation solution for two blastocysts in this invention.
[0039] Figure 7 Results of blastocyst dissociation cell viability; A: Commercial reagent, B: Composite dissociation solution one of the present invention, C: Composite dissociation solution two of the present invention.
[0040] Figure 8 This is a graph showing the detection of live cells by commercially available reagents (red: dead cells; green: live cells).
[0041] Figure 9This is a detection diagram of the two dissociation live cells using the special composite dissociation solution of this invention (red: dead cells; green: live cells). Detailed Implementation
[0042] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0043] I. Reagent Preparation:
[0044] TrypLE™ Express enzyme: a non-animal-derived recombinant trypsin substitute.
[0045] ACCUTASE: A cell digestion solution containing proteolytic enzymes and collagenases.
[0046] Table 1. Commodity reagents and their product numbers
[0047]
[0048] II. Reagent Preparation:
[0049] Table 2. Formulation of Special Composite Dissociation Solution
[0050]
[0051] Table 3. Formulation of Special Composite Dissociation Solution
[0052]
[0053] Table 4. Three-Formulation of Special Composite Dissociation Solution
[0054]
[0055] Table 5. Formulas for four types of special composite dissociation solutions
[0056]
[0057] Example 1: Blastocyst dissociation in C57B6J strain mice
[0058] I. Experimental Materials:
[0059] C57B6J strain mouse blastocysts, calcium- and magnesium-free benchtop solution, special composite dissociation solution, dPBS, pipettes (including small-diameter pipettes), stereomicroscope, and 37°C constant temperature incubation equipment.
[0060] II. Experimental Procedure:
[0061] (1) Removal of the zona pellucida: The overall dissociation process is as follows Figure 1 Collect embryonic cells at the blastocyst stage (such as...) Figure 2 (As shown). Place a single blastocyst in a drop of calcium- and magnesium-free tetrodotoxin and repeatedly pipette until the zona pellucida is dissolved and removed. Observe using a stereomicroscope to ensure that the zona pellucida has been completely removed.
[0062] (2) Blastocyst fluid drainage: Use a small-diameter pipette smaller than the diameter of the blastocyst to repeatedly blow on the embryo after removing the zona pellucida until the blastocyst fluid in the blastocyst is completely drained, making the blastocyst structure loose.
[0063] (3) Blastocyst dissociation: The treated blastocysts were transferred to droplets of two pre-equilibrated composite dissociation solutions (Table 2 and Table 3) at 37°C. Simultaneously, the blastocysts were transferred to trypsin dissociation drops (Yeasen, 40123ES60) and commercial dissociation reagents (Shanghai Xuanya Biotechnology, XY-D-1908) as controls. Dissociation in the trypsin droplets required approximately 1 hour. After dissociation, the cells still appeared as large clumps, indicating poor dissociation efficiency. Figure 3 After using a commercial kit, the cells were relatively loose, but not completely dissociated. Figure 4 In the compound dissociation solution, the embryos were repeatedly aspirated using a pipette every 5 minutes. After incubation for 30 minutes, the blastocysts were dispersed into single-cell blastocysts, resulting in good dissociation, but the dissociation time was relatively long. Figure 5 In the second compound dissociation solution, the embryos are repeatedly aspirated using a pipette every 5 minutes. After incubation for 15 minutes, the blastocysts will be dispersed into single blastocyst cells. This method offers the best dissociation effect, the shortest processing time, and the best results. Figure 6 Meanwhile, we used AOPI reagents to statistically analyze cell viability. The results showed that the single-cell viability of commercially available kits was inferior to that of our method. Figure 7 The specific fluorescence presentation of live cells after dissociation using commercial reagents and the proprietary composite dissociation solution of this invention is shown in the figure. Figure 8 and 9 As shown.
[0064] (4) Single-cell washing: Wash the dissociated blastocyst single cells with dPBS, repeating the washing three times to remove residual lysis buffer and other impurities. The washed single cells can then be used for subsequent experiments.
[0065] Example 2: Isolation of ICR strain mouse blastocysts
[0066] I. Experimental Materials:
[0067] ICR strain mouse blastocysts, calcium- and magnesium-free benchtop solution, special composite dissociation solution, dPBS, pipettes (including small-diameter pipettes), stereomicroscope, and 37°C constant temperature incubation equipment.
[0068] II. Experimental Procedure:
[0069] (1) Removal of zona pellucida: Collect embryonic cells at the blastocyst stage. Place a single blastocyst in a drop of calcium- and magnesium-free thallium solution and repeatedly pipette until the zona pellucida is dissolved and removed. Observe using a stereomicroscope to ensure that the zona pellucida has been completely removed.
[0070] (2) Blastocyst fluid drainage: Use a small-diameter pipette smaller than the diameter of the blastocyst to repeatedly blow on the embryo after removing the zona pellucida until the blastocyst fluid in the blastocyst is completely drained, making the blastocyst structure loose.
[0071] (3) Blastocyst dissociation: The treated blastocysts are transferred into droplets of a special composite dissociation solution II that has been equilibrated at 37°C. The blastocysts are repeatedly blown into the solution every 5 minutes using a pipette. After incubation for 15 minutes, the blastocysts will be dispersed into single blastocyst cells.
[0072] (4) Single-cell washing: Wash the dissociated blastocyst single cells with dPBS, repeating the washing three times to remove residual lysis buffer and other impurities. The washed single cells can then be used for subsequent experiments.
Claims
1. A composite dissociation solution for the dissociation of single cells from mammalian blastocysts, characterized in that, The composite dissociation solution contains the following components at final concentrations: EDTA 4-6 mM, TrypLE™ Express enzyme 8%-20%, ACCUTASE cell digestion solution 8%-20%, and the remainder is KSOM embryo culture medium.
2. The composite dissociation solution according to claim 1, characterized in that, The final concentrations of each component are: EDTA 4-6 mM, TrypLE™ Express enzyme 8%-12%, ACCUTASE cell digestion solution 8%-12%, and the remainder is KSOM embryo culture medium.
3. The composite dissociation solution according to claim 2, characterized in that, The final concentrations of each component were: EDTA 5 mM, TrypLE™ Express enzyme 10%, ACCUTASE cell digestion solution 10%, and the remainder was KSOM embryo culture medium.
4. The composite dissociation solution according to claim 1, 2, or 3, characterized in that, The composite dissociation solution also contains collagenase type IV, neutral protease type II, and DNase; the final concentrations of collagenase type IV, neutral protease type II, and DNase in the composite dissociation solution are as follows: collagenase type IV 0.6-1.5 mM, neutral protease type II 0.6-1.5 U / mL, and DNase 30-70 ug / mL.
5. A highly efficient and gentle method for the dissociation of single-cell mammalian blastocysts, characterized in that, The method includes the following steps: (1) Removal of zona pellucida: The blastocyst is placed in a calcium- and magnesium-free terephthalic solution and the zona pellucida is dissolved and completely removed by physical blowing. (2) Enzymatic hydrolysis incubation: The blastocysts treated in step (1) are transferred to the composite dissociation solution of any one of claims 1-4 pre-equilibrated at 35℃-37℃ and incubated for 15-30 minutes, during which intermittent pipetting is performed until the blastocysts are completely dissociated into single cells; (3) Washing: Use dPBS to wash the dissociated single cells to remove residual dissociation solution.
6. The method according to claim 5, characterized in that, After step (1) and before step (2), there is an additional step: using an oral pipette with a diameter smaller than that of the blastocyst to blow the blastocyst after removing the zona pellucida, so as to completely drain the blastocyst cavity fluid and loosen the blastocyst structure.
7. The method according to claim 5, characterized in that, The physical blowing operation described in step (1) is to repeatedly blow using a straw; the intermittent blowing described in step (2) is to blow once every 5 minutes.
8. Use of the composite dissociation solution according to any one of claims 1-4 in the preparation of reagents or kits for the dissociation of single cells from mammalian blastocysts.
9. A reagent or kit for the single-cell dissociation of mammalian blastocysts, characterized in that, The reagent or kit contains the composite dissociation solution as described in any one of claims 1-4.
10. A single mammalian blastocyst cell obtained by dissociation using the method described in any one of claims 5-7, characterized in that, The viability of the single cells is greater than 90%.