Digestive fluids, methods and uses
By using digestive fluid containing hyaluronidase and collagenase to digest pancreatic islet organoids into single cells, the problem of difficult to maintain the secretion of cells in the long-term culture process is solved, observation and analysis of individual cells is achieved, and the activity and function of cells are maintained.
Patent Information
- Application Number
- CN202510086134.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art is difficult to maintain the continuous insulin secretion state of islet organoid cells during long-term culture, and it is difficult to observe the state of each cell.
The organoid is digested into single cells using a digestive fluid containing hyaluronidase and collagenase, so as to facilitate observation and analysis of single cells.
By digesting the organoid into single cells, the observation and analysis of each cell state is achieved, and the digested single cells still have adherent growth ability and do not affect cell activity and function.
Smart Images
Figure CN119875993A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of cell biology technology, and in particular to a digestive solution. The present invention also relates to a preparation method of the digestive solution, a use of the digestive solution, and a digestion method of an organoid. Background Art
[0002] Diabetes is a metabolic disease characterized by elevated blood sugar. When blood sugar levels rise, patients will develop a series of complications such as cardiovascular disease, eye lesions, diabetic nephropathy, nerve damage, diabetic foot, etc., which will cause a lot of pain to patients and also bring a heavy burden to their families.
[0003] Traditional treatments for diabetes cannot fundamentally cure diabetes. In 2000, Dr. Shapiro and others proposed a pancreatic islet transplantation method combined with an immunosuppressive regimen, which achieved an insulin-independent effect. Although pancreatic islet transplantation can fundamentally cure diabetes, it requires a large and stable source of donors. The extreme shortage of donors has prevented this method from becoming the main treatment for diabetes. How to fundamentally alleviate the symptoms of hyperglycemia in diabetics is still a hot topic in current research.
[0004] Pancreatic organoids are three-dimensional cultures that contain some key characteristics of pancreatic islets. Specifically, they use the self-renewal and multidirectional (or unidirectional) differentiation potential of stem cells (pluripotent stem cells or unipotent stem cells) or progenitor cells to reconstruct and develop into pancreatic islets in vitro, with structures and functions similar to natural pancreatic islets. However, how to keep pancreatic organoid cells in a state of continuous insulin secretion during long-term culture requires further observation.
[0005] Traditional methods often use enzyme-linked immunosorbent assay to detect the total secretion of insulin, but it is impossible to observe the status of each cell. Summary of the invention
[0006] In view of this, the present invention aims to provide a digestion solution suitable for digesting organoids into single cells, thereby facilitating single cell observation.
[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:
[0008] A digestive fluid comprises a solvent and a solute dispersed in the solvent; the solute comprises hyaluronidase and collagenase.
[0009] Furthermore, the concentration of the hyaluronidase in the digestive fluid is between 25 mg / ml and 50 mg / ml.
[0010] Furthermore, the concentration of the collagenase in the digestive fluid is between 62.5 mg / ml and 125 mg / ml.
[0011] Furthermore, the collagenase includes collagenase I or collagenase IV.
[0012] Furthermore, the solvent includes at least one of bovine serum albumin and phosphate buffer.
[0013] The digestive fluid of the present invention, because it contains hyaluronidase and collagenase, is particularly good at digesting organoids. It can digest organoids into single cells, which is convenient for observing the state of each cell and analyzing the differences between single cells and the role of cells in the microenvironment. It does not affect cell activity and function, and the single cells obtained by digestion still have the ability to grow by adhering to the wall.
[0014] Another object of the present invention is to provide a method for preparing a digestive fluid, which comprises dispersing the hyaluronidase and the collagenase in a solvent to obtain the digestive fluid.
[0015] Another object of the present invention is to propose the use of the above digestive fluid in digestive organoids.
[0016] Furthermore, the organoids include pancreatic islet organoids.
[0017] At the same time, another object of the present invention is to provide a method for digesting organoids, the method comprising:
[0018] Place the organoids in a container;
[0019] pouring the digestion solution into the container until the organoid is submerged;
[0020] The cell suspension is placed at a preset temperature for a preset time to obtain a single cell suspension.
[0021] Furthermore, the method also includes adding a stopping solution to the single cell suspension to stop the digestion of the organoid.
[0022] The organoid digestion method of the present invention can digest organoids into single cells, which plays an important auxiliary role in the detection of organoids at the single-cell level. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:
[0024] Figure 1 The images are scanning electron microscopy images of organoid culture and the results after glucose stimulation;
[0025] Figure 2Light microscopic images of pancreatic islet organoids digested using pancreatic enzymes, Comparative Example 1, Comparative Example 2, and Preparation Example 1, respectively;
[0026] Figure 3 Light microscopic images of pancreatic islet organoids digested with the digestion solutions of Preparation Examples 1 to 3;
[0027] Figure 4 Light microscopic images of cells obtained by digesting pancreatic islet organoids using the digestion solutions of Preparation Example 2 and Preparation Example 4 and culturing them for 14 days;
[0028] Figure 5 Light microscopic images of cells obtained after digestion of pancreatic islet organoids using the digestion solution of Preparation Example 2 on days 3, 7, and 14 of culture, as well as the results after glucose stimulation. DETAILED DESCRIPTION
[0029] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.
[0030] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.
[0031] The present embodiment relates to a digestion solution, which is suitable for digesting organoids into single cells without affecting cell activity and function, thereby playing an important auxiliary role in organoid detection at the single-cell level and facilitating subsequent research at the single-cell level, such as single-cell sequencing.
[0032] Based on the above design concept, the digestive fluid of this embodiment mainly includes a solvent and a solute dispersed in the solvent, and the solute includes hyaluronidase and collagenase.
[0033] The digestive juice of the present embodiment, due to containing hyaluronidase and collagenase at the same time, hyaluronidase can decompose local hyaluronic acid, reduce tissue viscosity, and hyaluronidase can participate in tissue remodeling, by decomposing hyaluronic acid to change the extracellular matrix structure. Collagenase can specifically degrade the protease of collagen, digest cells with collagenase, can make intercellular collagen degradation, release single cells, so that the physiological and pathological characteristics of cells are studied. Hyaluronidase and collagenase cooperate, so that the digestive juice is particularly good at digesting organoids, organoids can be digested into single cells, the cells obtained exist in a single form, without adhesion, few impurities, high cell viability, do not affect cell activity and function, and digestion obtains single cells still have the ability of adherent growth, for the further progress of single cell related research has laid the foundation.
[0034] As a preferred embodiment, the concentration of hyaluronidase in the digestive solution is between 25 mg / ml and 50 mg / ml. For example, it can be 25 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, etc., wherein, when the concentration of hyaluronidase in the digestive solution is between 40 mg / ml and 50 mg / ml, especially when the concentration is 50 mg / ml, the digestive solution has a better digestion effect on the organoid.
[0035] As a preferred embodiment, the concentration of collagenase in the digestive solution is between 62.5 mg / ml and 125 mg / ml. For example, it can be 62.5 mg / ml, 80 mg / ml, 100 mg / ml, 120 mg / ml, 125 mg / ml, etc., wherein when the concentration of collagenase in the digestive solution is between 100 mg / ml and 125 mg / ml, especially when the concentration is 125 mg / ml, the digestive solution has a better digestion effect on the organoid.
[0036] As a preferred embodiment, the collagenase in this example includes collagenase I. It should be understood that when collagenase I is used, the digestion effect of the digestive fluid on the organoid is better, but in addition to collagenase I, collagenase IV can also be used.
[0037] As a preferred embodiment, the digestion solution of this embodiment, the solvent includes at least one of a bovine serum albumin solution (Bovine Serum Albumin, BSA) and a phosphate buffered saline (Phosphate Buffered Saline, PBS).
[0038] In a preferred embodiment, the concentration of the BSA solution is between 1% and 5%, such as 1%, 2%, 3%, 4%, 5%, etc. Preferably, the concentration of the BSA solution is 1%. The PBS solution can be an existing 1-fold concentration PBS solution, abbreviated as 1×pbs, to simulate an isotonic solution of the human body environment.
[0039] Specifically, the raw materials for preparing 1× PBS solution include 1× [i.e. 137mM sodium chloride (NaCl), 2.7mM potassium chloride (KCl), 10mM disodium hydrogen phosphate (Na 2 HPO 4 ), 2 mM potassium dihydrogen phosphate (KH 2 PO 4 ), the pH value is usually between 7.2-7.4]. Here, mM is the abbreviation of mmol / L.
[0040] It should be understood that, in addition to the BSA solution and the PBS solution, the solvent may also be a NaCl solution with a concentration of 0.8%-1%, for example, 0.8%, 0.9%, or 1%.
[0041] The digestion solution of this embodiment, when the solvent is a BSA solution with a concentration between 1% and 5%, the concentration of hyaluronidase in the prepared digestion solution is between 40 mg / ml and 50 mg / ml, and the concentration of collagenase I is between 100 mg / ml and 125 mg / ml, the digestion solution can digest the organoids into a single cell suspension in a shorter time, especially digest pancreatic islet organoids, without affecting the activity and function of the cells.
[0042] It should be noted that the BSA solution was selected from CAS 9048-46-8 of BSA Merck, the hyaluronidase was selected from CAS 37326-33-3 of Hyaluronidase Merck, and the collagenase I was selected from CAS 9001-12-1 of Gibco.
[0043] This embodiment also relates to a method for preparing the above digestive solution, which comprises dispersing hyaluronidase and collagenase in a solvent to prepare the above digestive solution.
[0044] At the same time, this embodiment also relates to the use of the above digestive fluid in digestive organoids. As a preferred embodiment, the organoids include pancreatic islet organoids. Next, according to the above digestive fluid preparation method, hyaluronidase and collagenase are dispersed in BSA with a concentration of 1% to prepare a digestive fluid. The digestive fluid is specifically shown in Table 1.
[0045] Solvent / Concentration Hyaluronidase / concentration Collagenase / concentration Preparation Example 1 1% BSA 20mg / ml Collagenase I, 50 mg / ml Preparation Example 2 1% BSA 50mg / ml Collagenase I, 125 mg / ml Preparation Example 3 1% BSA 5mg / ml Collagenase I, 12.5 mg / ml Preparation Example 4 1% BSA 100mg / ml 250mg / ml Comparative Example 1 1% BSA 20mg / ml none Comparative Example 2 1% BSA none 50mg / ml
[0046] Because digesting organoids into single cells requires mild reaction conditions, during the specific preparation, the solute in the digestion solution is preferably a 1% BSA solution, which can provide a mild environment for the cells during the digestion process and ensure that the digested cells still have the ability to grow.
[0047] Since pancreatic islet organoids are different from adherent cells cultured in two dimensions in terms of cell morphology and have a certain three-dimensional structure, existing pancreatic enzymes cannot digest pancreatic islet organoids into single cells in a short period of time, and long-term use of pancreatic enzymes will also have a certain impact on cell activity.
[0048] Therefore, in this embodiment, the inventors studied a variety of different types and concentrations of digestive enzymes, and finally selected relatively mild hyaluronidase and collagenase that are commonly present in the human body. Preferably, a 1% BSA solution is used as a solvent, and the final concentration of hyaluronidase in the digestive solution is controlled at 50 mg / ml, and the final concentration of collagenase I is controlled at 125 mg / ml, which can achieve gentle digestion of pancreatic islet organoids into single cells, and the digested single cells still have the ability to adhere to the wall, and the cells cultured for 14 days still have the function of insulin secretion.
[0049] At the same time, this embodiment also provides an organoid digestion method, which includes the following steps.
[0050] S1. Place the organoids in a container.
[0051] S2. Pour the digestion solution into the container until the organoids are submerged.
[0052] S3. Let the mixture stand at a preset temperature for a preset time to obtain a single cell suspension.
[0053] In a preferred embodiment, in step S3, the preset temperature is between 35°C and 40°C, such as 35°C, 37°C, 40°C, etc., wherein the preset temperature is preferably 37°C.
[0054] In a preferred embodiment, in step S3, the preset time is between 0.3h-2h, such as 0.3h, 0.6h, 1h, 1.5h, 2h, etc. In a further preferred embodiment, the preset time is between 0.3h-1h.
[0055] As a preferred embodiment, after step S3, the method further comprises step S4, adding a stopping solution to the single cell suspension to stop the digestion of the organoid.
[0056] In a preferred embodiment, the stop solution is a culture medium containing serum. For example, it can be fetal bovine serum, which comes from Lot 30044333 of Gibco Company, New Zealand. The use of a culture medium containing serum as the stop solution allows the prepared single cell suspension to have a certain protective effect on the cells in the suspension, which is beneficial for the cells to be cultured in an adherent manner and in a three-dimensional manner, and is also beneficial for experiments such as RNA extraction and protein extraction. It should be understood that, in addition to this, the stop solution can also be a PBS solution containing serum.
[0057] Preferably, the concentration of serum in the serum-containing culture medium is between 5% and 10%, such as 5%, 8%, 10%, etc.
[0058] Next, we verified the digestive effect of the digestive fluid on organoids.
[0059] In this example, the pancreatic islet organoids with insulin secretion function were obtained by referring to the culture method reported in the literature. For details, refer to the literature in the PubMed database, PMID: 39326417, DOI: 10.1016 / j.cell.2024.09.004, and the pancreatic islet organoid spheres obtained are shown in Figure 1 Middle A.
[0060] After stimulation with glucose at different concentrations, pancreatic organoids have good insulin response ability. Specifically, after stimulation with glucose at concentrations of 2mM and 20mM, the insulin secretion results of pancreatic organoids are shown in Figure 1 B in the figure shows the test result of GSIS (glucose stimulated insulin secretion assay).
[0061] Figure 1 In middle B, the vertical axis represents Insulin secretion, i.e., insulin secretion, in μIU / ml. The left side of the horizontal axis represents L-Glucose, i.e., L-glucose, that is, the insulin secretion result after being stimulated by glucose at a concentration of 2mM. The right side represents H-Glucose, i.e., H-glucose, that is, the insulin secretion result after being stimulated by glucose at a concentration of 20mM.
[0062] By observing pancreatic islet organoids under a scanning electron microscope, we can see that pancreatic islet organoids are composed of a certain number of cells arranged in a certain pattern, and secretory granules of uniform size can be seen on their surface. Figure 1 C and D. Among them, Figure 1 C is a scanning electron microscope image of a single pancreatic islet organoid at 250 times magnification. Figure 1 D in the middle is a scanning electron microscope image of the pancreatic islet organoid at a magnification of 1500 times, showing a large number of secretory granules locally in the pancreatic islet organoid.
[0063] During the research process, the inventors found that due to the relatively stable morphology of pancreatic organoids, pancreatic organoid spheres are difficult to digest into single cells, making it difficult to detect cells at the single-cell level. Therefore, they investigated the effects of different types and concentrations of digestive enzymes on organoid digestion. Figure 2 shown.
[0064] First, pancreatic enzymes commonly used in digesting human umbilical cord mesenchymal stem cells were used to digest pancreatic islet organoids. The concentration of pancreatic enzymes was 0.25%. The inventors found that after incubation at 37°C for 30 minutes, it was difficult to digest pancreatic islet organoids with a certain tissue morphology into a single cell suspension.
[0065] As we all know, long-term contact of pancreatic enzymes with a concentration of 0.25% with cells can cause significant damage to cells. Therefore, this method cannot achieve single-cell digestion of pancreatic islet organoids. Figure 2 In center A, undigested clumps of islet organoids can still be observed after 30 minutes of incubation, so the use of trypsin can obtain incompletely digested islet organoids.
[0066] The pancreatic islet organoids were digested using the digestion solutions of Comparative Examples 1 and 2 above and incubated at 37°C for 60 minutes. The results showed that most of the organoid tissues could be dissociated, but there was cell adhesion. The digestion results of Comparative Example 1 were as follows: Figure 2 In B, the digestion results of comparative example 2 are as follows Figure 2 In center C, the pancreatic islet organoids obtained were not completely digested.
[0067] By digesting the pancreatic islet organoids as described in Preparation Example 2 above and incubating them at 37°C for 60 minutes, a completely digested single cell suspension can be obtained. Figure 2 Middle D.
[0068] The inventors further explored the concentration and incubation time, and used the digestion solutions of Preparation Examples 1 to 3, respectively, and incubated them at 37°C for 30 minutes or 60 minutes. The results are as follows: Figure 3 as shown in .
[0069] Figure 3 The left column shows the results of incubation for 30 minutes, and the right column shows the results of incubation for 60 minutes. Figure 3 A and B are the incubation results of Preparation Example 2. Figure 3 C and D are the incubation results of Preparation Example 1. Figure 3 E and F are the incubation results of Preparation Example 3.
[0070] from Figure 3 It can be found that the digestion solution at a low concentration (Preparation Example 3) cannot digest the pancreatic islet organoids into single cells. Even if the incubation time is extended to 60 minutes, only partial separation of the tissue and the organoid sphere will occur. When a medium concentration digestion solution (Preparation Example 1) is used, there are still lumps of adherent cells after 30 minutes of incubation, and after 60 minutes of incubation, the cell suspension shows a multi-cellular aggregation state. When the digestion solution of Preparation Example 2 is used to digest the organoids, multi-cellular aggregates are occasionally seen after 30 minutes of incubation, and a better single-cell suspension can be obtained after 60 minutes of incubation.
[0071] In addition, the inventors also tested digestion solutions with higher concentrations. The digestion solutions of Preparation Example 2 and Preparation Example 4 were used. After incubation at 37° C. for 60 minutes, the cells were collected and cultured in two dimensions for 14 days. Figure 4 A is the result corresponding to Preparation Example 4, Figure 4 B is the result corresponding to Preparation Example 2.
[0072] The results showed that although the digestion solution at a high concentration (Preparation Example 4) could digest the pancreatic islet organoids into a single cell suspension, the cells obtained by digesting the pancreatic islet organoids in Preparation Example 4 had difficulty adhering to the wall during the subsequent culture process, proving that the high concentration of digestion solution had a certain effect on the activity and function of the cells.
[0073] In order to further confirm that the single cell suspension obtained by digesting the pancreatic islet organoids with the digestion solution of Preparation Example 2 does not harm the cells and can continue to be cultured, the inventors continued to use the 2D culture method for verification. After plating the digested single cells, images of the culture results were collected on the 3rd, 7th, and 14th days. It can be seen that the cells are in good growth condition and the insulin response function is normal on the 14th day.
[0074] The results on day 3 are shown in Figure 5 Middle A, the results on day 7 are shown in Figure 5 In B, the culture results on day 14 are shown in Figure 5 Middle C, Figure 5 D in the middle shows the insulin secretion of pancreatic islet cells on the 14th day.
[0075] Figure 5 In D, the vertical axis represents Insulin secretion, i.e., insulin secretion, in μIU / ml, the horizontal axis L-glucose represents the insulin secretion result after stimulation with 2 mM glucose, H-Glucose represents the insulin secretion result after stimulation with 20 mM glucose, and potassium chloride represents the insulin secretion result after stimulation with 30 mM potassium chloride solution.
[0076] The digestion solution in this embodiment can digest the cells in the pancreatic islet organoids into single cells, thereby facilitating observation and verification at the single-cell level, and can lay the foundation for research at the single-cell level.
[0077] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A digestive juice, characterized in that: comprising a solvent and a solute dispersed in the solvent; The solutes include hyaluronidase and collagenase.
2. The digestive juice according to claim 1, characterized in that: The concentration of the hyaluronidase in the digestive fluid is between 25 mg / ml and 50 mg / ml.
3. The digestive juice according to claim 1, characterized in that: The concentration of the collagenase in the digestive solution is between 62.5 mg / ml and 125 mg / ml.
4. The digestive juice according to claim 1, characterized in that: The collagenase includes collagenase I or collagenase IV.
5. The digestive juice according to any one of claims 1 to 4, characterized in that: The solvent includes at least one of bovine serum albumin and phosphate buffer.
6. A method for preparing a digestive juice, characterized in that: The method comprises: The hyaluronidase and collagenase are dispersed in a solvent to prepare a digestion solution.
7. Use of the digestive fluid according to any one of claims 1-5 in digestive organoids.
8. Use of the digestive juice according to claim 7, characterized in that. The organoids include pancreatic islet organoids.
9. A method for digesting an organoid, characterized in that: The method comprises: Place the organoids in a container; Pour the digestive solution of any one of claims 1 to 5 into the container until the organoid is submerged; The cell suspension is placed at a preset temperature for a preset time to obtain a single cell suspension.
10. The method for digesting an organoid according to claim 9, characterized in that: The method further comprises: A stopping solution is added to the single cell suspension to stop the digestion of the organoid.