Tissue digestive juice for organoid culture as well as preparation method and application of tissue digestive juice

Abstract

The invention belongs to the technical field of organoid culture, and particularly relates to tissue digestive juice for organoid culture as well as a preparation method and application of the tissue digestive juice. According to the tissue digestive juice, a DMEM / F-12 culture medium which contains L-glutamine and does not contain 4-hydroxyethyl piperazine ethanesulfonic acid is used as a basic culture medium, calcium chloride, collagenase I type, collagenase III type, V type hyaluronidase, deoxyribonuclease I, 4-hydroxyethyl piperazine ethanesulfonic acid and Y-27632 are combined and have a synergistic effect, cell damage is reduced, meanwhile, the tissue digestive juice has the advantages that the tissue digestive juice can be used for improving the tissue digestive juice quality, and the tissue digestive juice has a good application prospect. The digestion time is shortened, the tissue digestion efficiency is improved, and the culture success rate of the organoid is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and in particular relates to a tissue digestion fluid for organoid culture, and a preparation method and application thereof. Background Art

[0002] Organoids are organ-specific cell collections derived from stem cells or precursor cells. Organoid culture is obtained by processing a part of the tissue obtained from the organism, and then mixing the cells obtained from the tissue processing with matrix gel for 3D culture. Therefore, how to dissociate cells or cell clusters from tissues efficiently, with low damage and with a wide spectrum is the top priority of organoid culture.

[0003] Digestive fluid is required for the dissociation of cells or cell clusters. Currently, the most commonly used digestive fluids are mostly prepared from single collagenase or phenylmethylsulfonylfluoride (PMSF). However, the use of only a single collagenase narrows the types of digestible tissues, and the digestion strength is insufficient, resulting in a reduction in the cell acquisition rate. In addition, the collagenase is prone to loss of activity during use, which reduces the digestion effect and, in severe cases, will make it impossible to digest and separate the cells. PMSF is a toxic compound and poses a major safety hazard. At the same time, the pH stability of the existing digestive fluid system is low, which can easily cause digestion failure or damage to cells.

[0004] In summary, the existing tissue digestion fluid has low digestion efficiency and is easy to cause damage to cells, affecting cell activity and the success rate of organoid culture. Summary of the invention

[0005] The purpose of the present invention is to provide a tissue digestion fluid for organoid culture and a preparation method and application thereof, wherein the tissue digestion fluid can reduce damage to cells, improve cell activity and the success rate of subsequent organoid culture while improving digestion efficiency.

[0006] The invention provides a tissue digestion solution, which uses DMEM / F-12 culture medium as a basic culture medium and further comprises components of the following concentrations: 4-6 mM calcium chloride, 1-2 mg / mL collagenase type III, 1-2 mg / mL collagenase type I, 20-30 μg / mL type V hyaluronidase, 0.1-0.2 mg / mL deoxyribonuclease I, 10-20 mM 4-hydroxyethylpiperazineethanesulfonic acid, and 10-10.5 μM Y-27632; The DMEM / F-12 culture medium is a DMEM / F-12 culture medium containing L-glutamine and not containing 4-hydroxyethylpiperazineethanesulfonic acid.

[0007] The present invention also provides a method for preparing the tissue digestion fluid described in the above technical solution, comprising the following steps: DMEM / F-12 culture medium, calcium chloride, collagenase type III, collagenase type I, type V hyaluronidase, deoxyribonuclease I, 4-hydroxyethylpiperazineethanesulfonic acid and Y-27632 are mixed, and the obtained mixed solution is sterilized to obtain the tissue digestion solution.

[0008] Preferably, the sterilization method comprises: filtering the mixed solution with a filter membrane; the pore size of the filter membrane is 0.22 μm.

[0009] The present invention also provides the use of the tissue digestion fluid described in the above technical solution or the tissue digestion fluid prepared by the preparation method described in the above technical solution in tissue digestion.

[0010] The present invention also provides a method for preparing cells and / or cell clusters, comprising the following steps: mixing the tissue fragments with the tissue digestion solution and then digesting them to obtain a digested mixture; The digested mixture is filtered, and the filtrate is taken for solid-liquid separation, and the precipitate is collected to obtain cells and / or cell clusters; The tissue digestion fluid is the tissue digestion fluid described in the above technical solution or the tissue digestion fluid prepared by the preparation method described in the above technical solution.

[0011] Preferably, the weight of the tissue fragments and the volume ratio of the tissue digestion solution are 0.5-1 g: 3-5 mL; the digestion time is 15-60 min; and the pore size of the filter is 70-100 μm.

[0012] Preferably, after collecting the precipitate, the process further comprises mixing the precipitate with a red blood cell lysing solution for lysing, centrifuging the lysed mixture, collecting the precipitate, and obtaining cells and / or cell clusters.

[0013] Preferably, the method further comprises resuspending the cells and / or cell clusters in DMEM / F-12 medium to obtain a cell and / or cell cluster resuspension; the DMEM / F-12 medium is a DMEM / F-12 medium containing L-glutamine and not containing 4-hydroxyethylpiperazineethanesulfonic acid.

[0014] Preferably, the tissue comprises cancer tissue.

[0015] The present invention also provides the use of the tissue digestion fluid described in the above technical solution or the tissue digestion fluid prepared by the preparation method described in the above technical solution or the method described in the above technical solution in organoid culture.

[0016] Beneficial effects: The invention provides a tissue digestion solution. The tissue digestion solution uses a DMEM / F-12 culture medium as a basic culture medium and further comprises components with the following concentrations: 4-6 mM calcium chloride, 1-2 mg / mL collagenase type III, 1-2 mg / mL collagenase type I, 20-30 μg / mL type V hyaluronidase, 0.1-0.2 mg / mL deoxyribonuclease I, 10-20 mM 4-hydroxyethylpiperazineethanesulfonic acid and 10-10.5 μM Y-27632; the DMEM / F-12 culture medium is a DMEM / F-12 culture medium containing L-glutamine but not containing 4-hydroxyethylpiperazineethanesulfonic acid. The collagenase type I and collagenase type III described in the present invention have the effect of digesting tissue interstitial tissue, and have little effect on epithelial cells; type V hyaluronidase and collagenase have a synergistic digestion effect, and can also digest interstitial tissue; and the calcium chloride in the present invention provides calcium ions for the digestion of collagenase, and the concentration of calcium ions can improve the digestion ability of the enzyme; the deoxyribonuclease I can act on the DNA double helix structure to prevent DNA molecules from sticking and affecting the digestion effect; and 4-hydroxyethylpiperazineethanesulfonic acid is the pH buffer in the tissue buffer, which can play a role in stabilizing the tissue digestion system; the Y-27632 can inhibit cell apoptosis to improve the activity of cells and / or cell clusters obtained by digestion. The components in the tissue digestion solution described in the present invention have a synergistic effect, while reducing cell damage, shortening the digestion time, improving the efficiency of tissue digestion, and improving the success rate of organoid culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 This is a cell state diagram of the cells obtained by digestion in Example 3 under an inverted microscope; Figure 2 This is a cell state diagram of the cells obtained by digestion in Example 5 under an inverted microscope; Figure 3 This is a picture of the cell status of the cells obtained by digestion in Example 7 under an inverted microscope. DETAILED DESCRIPTION

[0019] The invention provides a tissue digestion solution. The tissue digestion solution uses a DMEM / F-12 culture medium as a basic culture medium and further comprises components with the following concentrations: 4-6 mM calcium chloride, 1-2 mg / mL collagenase type III, 1-2 mg / mL collagenase type I, 20-30 μg / mL type V hyaluronidase, 0.1-0.2 mg / mL deoxyribonuclease I, 10-20 mM 4-hydroxyethylpiperazineethanesulfonic acid and 10-10.5 μM Y-27632; the DMEM / F-12 culture medium is a DMEM / F-12 culture medium containing L-glutamine but not containing 4-hydroxyethylpiperazineethanesulfonic acid.

[0020] The DMEM / F-12 culture medium containing L-glutamine and not containing 4-hydroxyethylpiperazineethanesulfonic acid of the present invention has a basic cell nutrition effect and is lower in cost than other cell culture media. As an embodiment, the concentration of calcium chloride in the tissue digestion solution of the present invention is 4.5~5.5mM; as another embodiment, the concentration of calcium chloride is 5mM; the digestion of collagenase depends on a certain calcium ion concentration, the calcium chloride can provide calcium ions for collagenase, and the concentration of calcium ions can improve the digestion ability of the enzyme. As an embodiment, the concentration of collagenase type III in the tissue digestion solution of the present invention is 1.5~2mg / mL; the collagenase type III digests the interstitial cells and has little effect on epithelial cells. As an embodiment, the concentration of collagenase type I in the tissue digestion solution of the present invention is 1~1.5mg / mL; the collagenase type I digests the interstitial cells and has little effect on epithelial cells. As an embodiment, the concentration of the V-type hyaluronidase (Hyaluronidase V) of the present invention is 22~28μg / mL; as another embodiment, the concentration of the V-type hyaluronidase is 24~26μg / mL; the V-type hyaluronidase can digest the intercellular matrix and has a synergistic digestion effect with collagenase. As an embodiment, the concentration of the deoxyribonuclease I (DNase I) of the present invention is 0.1~0.15mg / mL; the deoxyribonuclease I acts on the DNA double helix structure to prevent DNA molecules from sticking and affecting tissue digestion. As an embodiment, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) of the present invention is 10~15mM; the 4-hydroxyethylpiperazineethanesulfonic acid can be used as a pH buffer, which can stabilize the tissue digestion system and avoid the failure of tissue digestion caused by an unstable digestion system. As an embodiment, the concentration of Y-27632 in the present invention is 10 μM; the Y-27632 can inhibit cell apoptosis and improve the activity of cells and / or cell clusters obtained by tissue digestion.

[0021] The present invention also provides a method for preparing the tissue digestion fluid described in the above technical solution, comprising the following steps: DMEM / F-12 culture medium, calcium chloride, collagenase type III, collagenase type I, type V hyaluronidase, deoxyribonuclease I, 4-hydroxyethylpiperazineethanesulfonic acid and Y-27632 are mixed, and the obtained mixed solution is sterilized to obtain the tissue digestion solution.

[0022] As an embodiment, the present invention dissolves Y-27632 powder in dimethyl sulfoxide (DMSO) to obtain a Y-27632 mother solution; the concentration of the Y-27632 mother solution may be 10 mM.

[0023] After obtaining the Y-27632 mother solution, the present invention mixes DMEM / F-12 culture medium, calcium chloride, collagenase type III, collagenase type I, type V hyaluronidase, deoxyribonuclease I, 4-hydroxyethylpiperazineethanesulfonic acid and the Y-27632 mother solution, sterilizes the obtained mixed solution, and obtains the tissue digestion solution. As an embodiment, the sterilization method of the present invention is: filtering the mixed solution with a filter membrane. As an embodiment, the pore size of the filter membrane of the present invention is 0.22 μm.

[0024] The present invention also provides the use of the tissue digestion solution described in the above technical solution or the tissue digestion solution prepared by the preparation method described in the above technical solution in tissue digestion. As an embodiment, the tissue described in the present invention is cancer tissue; as another embodiment, the cancer tissue described in the present invention can be one or more of but not limited to lung cancer tissue, breast cancer tissue and intestinal cancer tissue.

[0025] The present invention also provides a method for preparing cells and / or cell clusters, comprising the following steps: mixing the tissue fragments with the tissue digestion solution and then digesting them to obtain a digested mixture; The digested mixture is filtered, and the filtrate is taken for solid-liquid separation, and the precipitate is collected to obtain cells and / or cell clusters; The tissue digestion fluid is the tissue digestion fluid described in the above technical solution or the tissue digestion fluid prepared by the preparation method described in the above technical solution.

[0026] In the present invention, the tissue fragments are mixed with a tissue digestion fluid and then digested to obtain a digested mixture. As an embodiment, the volume of the tissue fragments in the present invention is 0.5-2 mm 3 As another embodiment, the volume of the tissue fragments is 0.5~1mm 3. As one embodiment, in the mixture before digestion, the weight of the tissue fragments and the volume ratio of the tissue digestion solution is 0.5~1g:3~5mL; as another embodiment, the weight of the tissue fragments and the volume ratio of the tissue digestion solution is 0.5g:3~4mL. As one embodiment, the digestion time of the present invention is 15~60min; as another embodiment, the digestion time of the present invention is 25~50min; as another embodiment, the digestion time is 35~45min. As one embodiment, the present invention mixes a suspension containing tissue fragments with a tissue digestion solution for digestion to obtain a mixture after digestion. As one embodiment, the present invention mixes the tissue with the tissue digestion solution and then digests it to obtain the suspension containing tissue fragments. As one embodiment, the tissue of the present invention is cancer tissue; as another embodiment, the cancer tissue of the present invention can be but is not limited to breast cancer tissue and / or intestinal cancer tissue.

[0027] After obtaining the digested mixture, the present invention filters the digested mixture, takes the filtrate for solid-liquid separation, collects the precipitate, and obtains cells and / or cell clusters. As an embodiment, the pore size of the filter mesh for filtration of the present invention is 70-100 μm; as another embodiment, the pore size of the filter mesh for filtration is 100 μm. As an embodiment, the solid-liquid separation method of the present invention is centrifugation; the centrifugation speed is 1500 rpm; the time is 3 min.

[0028] After obtaining the precipitate, when there are red blood cells in the precipitate, as an embodiment, the present invention mixes the precipitate with a red blood cell lysing solution for lysing, centrifuges the lysed mixture, collects the precipitate, and obtains cells and / or cell clusters, that is, obtains cells and / or cell clusters without red blood cells. The present invention does not specifically limit the components of the red blood cell lysing solution or the lysis conditions, and can be performed according to conventional operations in the art.

[0029] After obtaining the cells and / or cell clusters, as an embodiment, the present invention resuspends the cells and / or cell clusters in DMEM / F-12 culture medium to obtain a cell and / or cell cluster resuspension; the DMEM / F-12 culture medium is a DMEM / F-12 culture medium containing L-glutamine and not containing 4-hydroxyethylpiperazineethanesulfonic acid.

[0030] The present invention also provides the use of the tissue digestion fluid described in the above technical solution or the tissue digestion fluid prepared by the preparation method described in the above technical solution or the method described in the above technical solution in organoid culture.

[0031] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1 A tissue digestion solution and preparation method thereof 1. The formula of tissue digestion fluid is shown in Table 1: Table 1 The composition of tissue digestive juice and the sources of its components

[0033] 2. The preparation method of the tissue digestion solution in step 1 is as follows: 1) According to the concentration requirements, 5 mg of Y-27632 powder was dissolved in 2.02 mL of DMSO to prepare a 10 mM Y-27632 stock solution; 2) Add collagenase type I, collagenase type III, deoxyribonuclease I, type V hyaluronidase, Y27632 stock solution, HEPES, and calcium chloride solution to DMEM / F12 culture medium to prepare a mixed solution, and then filter the mixed solution through a 0.22 μm filter membrane to sterilize and obtain a tissue digestion solution.

[0034] Example 2 A method for preparing a cell and / or cell cluster suspension, comprising the following steps: 1) Place the obtained tissue in a culture dish, and use a pipette to drop 1 mL of the tissue digestion solution prepared in Example 1 onto the tissue to prevent drying; 2) Use sterile scissors to mince the tissue into pieces of 0.5~1mm 3 The tissue fragments of different sizes were separated to obtain tissue suspension; 3) Use a pipette to transfer the tissue suspension to a 15 mL centrifuge tube, add 2-3 mL of digestion solution to the culture dish again, rinse the culture dish, and continue to transfer to the above 15 mL centrifuge tube. Before digestion, the weight of the tissue fragments in the digestion system and the volume ratio of the tissue digestion solution are 0.5 g: 3 mL; 4) Digest the tissue for 15 to 60 minutes, take samples every 15 minutes and observe the digestion progress under a microscope to prevent over-digestion.

[0035] 5) After digestion is complete (cell clusters of 40-70 μm are present in the digestion system), filter with a 100 μm filter and collect the filtrate for centrifugation at a speed of 1500 rpm for 3 min. 6) After centrifugation, slowly aspirate the supernatant and discard it (if there are many red blood cells in the precipitate, use red blood cell lysis buffer to remove the red blood cells, then centrifuge and discard the supernatant) to obtain the precipitate.

[0036] Use an appropriate amount of DMEM / F12 (containing L-glutamine and not containing HEPES) culture medium to resuspend the cell pellet, and the obtained cells are tumor cell / cell cluster suspension with high activity.

[0037] Example 3 Human lung cancer tissue (hospital research sample, with the patient's consent, the same for the following cancer samples) was digested using the method in Example 2. The digestion time was 15 min, and the total number of cells obtained was 1.41×10 6 The cell viability was 91.87%. The picture under the inverted microscope is as follows Figure 1 shown.

[0038] Example 4 The human lung cancer tissue from the same source as that in Example 3 was digested using the method in Example 2. The digestion time was 30 min, and the total number of cells obtained was 8.79×10 5 The cell viability was 87.5%.

[0039] Combination Figure 1 By comparing the results in Example 3 and Example 4, it can be concluded that the methods in Examples 3 to 4 can all achieve digestion of human lung cancer tissue, but compared with Example 4, digesting human lung cancer tissue for 15 minutes can obtain more cells, and the cell viability is relatively better, and the cell damage caused is relatively less.

[0040] Example 5 Human breast cancer tissue was digested using the method in Example 2 for 35 min, and the total number of cells obtained was 2.25×10 6 The cell viability was 89.23%. Figure 2 shown.

[0041] Example 6 The human breast cancer tissue from the same source as that in Example 5 was digested using the method in Example 2. The digestion time was 50 min, and the total number of cells obtained was 2.08×10 6 The cell viability was 83.17%.

[0042] Combination Figure 2 By comparing the results in Example 5 and Example 6, it can be concluded that the methods in Examples 5 to 6 can all achieve digestion of human breast cancer tissue, but compared with Example 6, digesting human breast cancer tissue for 35 minutes can obtain more cells, and the cell viability is relatively better, and the cell damage caused is relatively less.

[0043] Example 7 The human intestinal cancer tissue was digested using the method in Example 2 for 20 min, and the total number of cells obtained was 1.98×10 6 The cell viability was 90.14%. Figure 3 shown.

[0044] Example 8 The method in Example 2 was used to digest the human intestinal cancer tissue from the same source as that in Example 7. The digestion time was 40 min, and the total number of cells obtained was 2.01×10 6 The cell viability was 82.24%.

[0045] Combination Figure 3 By comparing the results in Example 7 and Example 8, it can be concluded that the methods in Examples 7 to 8 can all achieve the digestion of human colorectal cancer tissue, but compared with Example 8, digesting the human colorectal cancer tissue for 20 minutes can obtain more cells, and the cell viability is relatively better, and the cell damage caused is relatively less.

[0046] It can be concluded from the above examples that the tissue digestion solution of the present invention can shorten the digestion time, reduce the damage to cells and improve the cell viability on the basis of improving the digestion efficiency.

[0047] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue digestion fluid, characterized in that: The tissue digestion solution uses DMEM / F-12 culture medium as the basic culture medium, and also includes the following components in the following concentrations: calcium chloride 4-6 mM, collagenase type III 1-2 mg / mL, collagenase type I 1-2 mg / mL, type V hyaluronidase 20-30 μg / mL, deoxyribonuclease I 0.1-0.2 mg / mL, 4-hydroxyethylpiperazineethanesulfonic acid 10-20 mM and Y-27632 10-10.5 μM; The DMEM / F-12 culture medium is a DMEM / F-12 culture medium containing L-glutamine and not containing 4-hydroxyethylpiperazineethanesulfonic acid.

2. The method for preparing the tissue digestion fluid according to claim 1, characterized in that: The steps include: DMEM / F-12 culture medium, calcium chloride, collagenase type III, collagenase type I, type V hyaluronidase, deoxyribonuclease I, 4-hydroxyethylpiperazineethanesulfonic acid and Y-27632 are mixed, and the obtained mixed solution is sterilized to obtain the tissue digestion solution.

3. The preparation method according to claim 2, characterized in that: The sterilization method comprises: filtering the mixed solution with a filter membrane; the pore size of the filter membrane is 0.22 μm.

4. Use of the tissue digestion fluid according to claim 1 or the tissue digestion fluid prepared by the preparation method according to claim 2 or 3 in tissue digestion.

5. A method for preparing cells and / or cell clusters, characterized in that: The steps include: mixing the tissue fragments with the tissue digestion solution and then digesting them to obtain a digested mixture; The digested mixture is filtered, and the filtrate is taken for solid-liquid separation, and the precipitate is collected to obtain cells and / or cell clusters; The tissue digestion fluid is the tissue digestion fluid described in claim 1 or the tissue digestion fluid prepared by the preparation method described in claim 2 or 3.

6. The method according to claim 5, characterized in that The weight of the tissue fragments and the volume ratio of the tissue digestion solution are 0.5-1 g: 3-5 mL; the digestion time is 15-60 min; and the pore size of the filter screen is 70-100 μm.

7. The method according to claim 5, characterized in that After collecting the precipitate, the method further includes mixing the precipitate with a red blood cell lysing solution to lyse the precipitate, centrifuging the lysed mixture, collecting the precipitate, and obtaining cells and / or cell clusters.

8. The method according to claim 5 or 7, characterized in that: The method further comprises resuspending the cells and / or cell clusters in a DMEM / F-12 culture medium to obtain a cell and / or cell cluster resuspension solution; the DMEM / F-12 culture medium is a DMEM / F-12 culture medium containing L-glutamine and not containing 4-hydroxyethylpiperazineethanesulfonic acid.

9. The method according to claim 5, characterized in that The tissue includes cancer tissue.

10. Use of the tissue digestion fluid of claim 1 or the tissue digestion fluid prepared by the preparation method of claim 2 or 3 or the method of any one of claims 5 to 9 in organoid culture.

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