CN115992087A - Large intestine tissue digestion kit and digestion method - Google Patents
Large intestine tissue digestion kit and digestion method Download PDFInfo
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- CN115992087A CN115992087A CN202211517948.5A CN202211517948A CN115992087A CN 115992087 A CN115992087 A CN 115992087A CN 202211517948 A CN202211517948 A CN 202211517948A CN 115992087 A CN115992087 A CN 115992087A
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Abstract
The invention discloses a large intestine tissue digestion kit and a digestion method. The kit comprises a digestive juice D1, a digestive juice D2 and a buffer solution; the digestive juice D1 comprises Trypsin, penicillin and streptomycin, the digestive juice D2 comprises type I collagenase, type IV collagenase, hyaluronidase, DNase I and neonatal bovine serum, and the buffer comprises penicillin, streptomycin and neonatal bovine serum. Shearing and cleaning the large intestine tissues, performing primary digestion by using the digestion liquid D1, performing secondary digestion by using the digestion liquid D2, repeatedly blowing and sucking tissue residues by using a syringe, centrifuging, resuspending and filtering, wherein the obtained large intestine single cell suspension can be used for large intestine organoid culture, flow cytometry detection and sorting and single cell transcriptome sequencing analysis.
Description
Technical Field
The invention relates to the fields of medicine and biology, in particular to a large intestine tissue digestion kit and operation steps.
Background
The large intestine is an important tissue organ in the body, and its main functions are to absorb moisture and electrolytes, and to form, store and excrete faeces. In addition, the large intestine is also the habitat for trillion intestinal microorganisms, and mucosal barriers formed by the large intestine epithelial cells are involved in maintaining, regulating and sequestering these microorganisms. The large intestine mucosal barrier not only separates the intestinal microorganisms from the host tissue, but also regulates the absorption of water, electrolytes, minerals and vitamins. The homeostasis in the large intestine depends on complex interactions between microbiota and mucosal epithelium, immune system, vascular system, matrix and nervous system. Disruption of the microenvironment of the large intestine, such as changes in microbial composition, epithelial cell function/proliferation/differentiation, mucus production/composition, immune function or blood flow, may have significant local and systemic consequences. Resolving the role and function of the large intestine is important for understanding the pathogenesis of diseases related to the large intestine and developing therapeutic drugs.
Isolation of individual cells from large intestine tissue is an important technical basis for performing functional and functional analyses of the large intestine. The single cell suspension obtained by the method of digestion and dissociation of the large intestine tissue reported in the literature has low activity, the cell survival rate is generally 50-70%, and the single cell suspension is difficult to be used for the subsequent culture of large intestine organoids, detection and separation by flow cytometry and single cell transcriptome sequencing analysis. For example, chinese patent CN106884002a uses collagenase type IV and hyaluronidase to digest rat colon tissue, but only a small number of colon epithelial cells are obtained; in CN106754711a, mice intestinal tracts were digested with Liberase TL and DNase I, but only the viability of lamina propria primary dendritic cells was increased to a level of 70%.
Disclosure of Invention
The invention aims to provide a large intestine tissue digestion kit and a digestion method.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method of digestion of large intestine tissue comprising the steps of:
1) Pretreatment of
Shearing large intestine tissues, and cleaning the sheared large intestine tissues by using a buffer solution; the buffer solution comprises 1-10% of penicillin by mass fraction, 0.05-1% of streptomycin by mass fraction and 1-6% of newborn calf serum by volume fraction;
2) One-time digestion
Mixing the large intestine tissue treated in the step 1 with the digestive juice D1 uniformly, then digesting to obtain a digested product Y1, mixing the digested product Y1 with the buffer solution uniformly, and separating undigested tissue small blocks from large intestine epithelial cells by filtration; the digestive juice D1 comprises 0.2-25 g/L trypsin, penicillin with mass fraction of 1-10% and streptomycin with mass fraction of 0.05-1%;
3) Secondary digestion
Uniformly mixing the undigested tissue small blocks obtained in the step 2 with a digestive juice D2, then digesting to obtain a digested product Y2, uniformly mixing the digested product Y2 with a buffer solution after scattering tissue residues in the digested product Y2, and centrifuging and collecting cell precipitates (the cell precipitates mainly comprise large intestine epithelial cells, crypt stem cells, immune cells, vascular endothelial cells, fibroblasts and muscle cells); the digestive juice D2 comprises 0.1-10 mg/mL of type I collagenase, 0.1-10 mg/mL of type IV collagenase, 0.01-5 mg/mL of hyaluronidase, 1-100 u/mL of DNase I and 1-6% of newborn calf serum by volume fraction;
4) Post-treatment
And (3) uniformly mixing the cell sediment obtained in the step (3) with a buffer solution, and then removing cell clusters through filtration to obtain the large intestine single cell suspension.
Preferably, in the step 1, the cleaning specifically includes the following steps: the sheared large intestine tissues and the buffer solution are subjected to shaking and uniform mixing, and then are centrifuged to separate and remove the upper liquid containing the residual fecal residues and the soluble matters of the intestinal wall, so that one-time operation is completed; the shaking and centrifuging were repeated not less than 3 times during the washing.
Preferably, the solvent of the buffer solution is PBS with pH of 5.8-8.0 (the pH has strong buffering capacity in the range).
Preferably, in the step 2, the condition of the primary digestion is: the temperature is 25-37 ℃, the time is 20-40 minutes, the rotating speed of the shaking table is 40-80 revolutions per minute, and the dosage of the digestive juice D1 is 2-4 mL/g; the pore diameter of the filter membrane used for filtering is 70-100 mu m.
Preferably, in the step 3, the secondary digestion conditions are as follows: the temperature is 25-37 ℃, the time is 20-40 minutes, the rotation speed of the shaking table is 40-80 revolutions per minute, and the consumption of the digestive juice D2 is 4-6 mL/g; the scattering specifically comprises the following steps: the tissue residue (mainly the cell mass surrounded by the residual extracellular matrix) in the digested product Y2 was repeatedly blown and sucked with a syringe of 18 to 22gauge, so that the tissue residue was sufficiently dispersed by releasing the contained cells.
Preferably, in the step 1 and the step 3, the centrifugation conditions are as follows: centrifugation (4-10 ℃) is carried out for 5-10 minutes by 300-400 g.
Preferably, the solvents of the digestion liquid D1 and the digestion liquid D2 are eukaryotic cell culture media (usually, mouse or human tissue cells) such as DMEM medium or 1640 medium.
Preferably, in the step 4, the pore size of the filter membrane used for the filtration is 70-90 μm.
A kit for digestion of large intestine tissue, the kit comprising the following reagents: the digestion solution D1, the digestion solution D2 and the buffer solution.
The beneficial effects of the invention are as follows:
the large intestine tissue digestion kit comprises reagents (digestive juice D1 and D2) for performing twice digestion on large intestine tissues and buffer solutions for cleaning and dispersing before and after digestion, so that extracellular matrixes in the large intestine tissues can be removed to the greatest extent in the digestion process, and a plurality of living cells derived from the large intestine tissues are reserved and extracted. The single-cell suspension with high activity rate (cell survival rate is more than 90%) can be used for culturing large intestine organoids, detecting and sorting by flow cytometry, and sequencing single-cell transcriptome.
Drawings
FIG. 1 shows the cell viability obtained with DNase I-added digestate and without DNase I-added digestate; left diagram: observation of single cell suspensions under microscopic fields; middle diagram: incubating the single cell suspension with a dead cell indicator PI and observing the proportion and concentration of PI positive cells (i.e., dead cells) (red labeled PI positive) with a fluorescence microscope; right figure: the single cell suspension was incubated with the living cell indicator acridine orange and the proportion and concentration of acridine orange positive cells (i.e., living cells) were observed with a fluorescence microscope (green labeled acridine orange positive).
FIG. 2 shows the cell viability obtained for the Trypsin group, the collagenase group and the Trypsin+collagenase group; left diagram: observation of single cell suspensions under microscopic fields; middle diagram: incubating the single cell suspension with a dead cell indicator PI and observing the proportion and concentration of PI positive cells (red labeled PI positive) with a fluorescence microscope; right figure: the single cell suspension was incubated with the living cell indicator acridine orange and the proportion and concentration of acridine orange positive cells (green labeled acridine orange positive) were observed with a fluorescence microscope.
FIG. 3 shows the results of cell viability assays performed on the single cell suspensions of large intestine obtained by digestion; left diagram: observation of single cell suspensions under microscopic fields; middle diagram: incubating the single cell suspension with a dead cell indicator PI and observing the proportion and concentration of PI positive cells (red labeled PI positive) with a fluorescence microscope; right figure: the single cell suspension was incubated with the living cell indicator acridine orange and the proportion and concentration of acridine orange positive cells (green labeled acridine orange positive) were observed with a fluorescence microscope.
FIG. 4 shows the results of flow cytometry analysis and sorting of the single cell suspensions of large intestine obtained by digestion.
FIG. 5 shows the results of large intestine organoid culture of large intestine single cell suspension obtained by digestion.
FIG. 6 shows the results of single cell transcriptome sequencing analysis of the single cell suspension of the large intestine obtained by digestion: 0 to 14 represent different cell populations.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
According to the invention, through early experimental trial and technical optimization, a kit and a method for preparing the single-cell suspension of the large intestine with the cell survival rate of more than 90% by digesting large intestine tissues are finally established, and the obtained single-cell suspension is identified and functionally verified through flow cytometry detection and separation, large intestine organoid culture and single-cell transcriptome sequencing analysis.
Large intestine tissue digestion kit
1. Reagent composition of kit
Reagent: digest D1, digest D2, and buffer. Wherein:
the components of the digestive juice D1 are (final concentration): 2.5g/L of Trypsin (Trypsin), 6wt% of penicillin and 0.1wt% of streptomycin, and the solvent is DMEM culture medium.
The components of the digestive juice D2 are (final concentration): type I collagenase 1mg/mL, type IV collagenase 1mg/mL, hyaluronidase 0.1mg/mL, DNase I (deoxyribonuclease I) 50u/mL, and neonatal bovine serum 1% (v/v), with DMEM medium as the solvent.
The composition of the buffer is (final concentration): penicillin 6wt%, streptomycin 0.1wt%, and neo-bovine serum 1% (v/v), the solvent was PBS at ph=7.
(II) digestion of Large intestine tissue (Using the Large intestine tissue digestion kit described above)
1. Operating procedure
1) Washing the large intestine of a mouse with the mass of 2g by using the buffer solution to remove excrement in the large intestine, shearing the large intestine into tissue fragments with the mass of about 1X 1mm (operating at a low temperature of 0-4 ℃ to ensure the survival rate of cells, suggesting shearing on ice), adding 5mL of the buffer solution into the tissue fragments, vibrating for 1 time (vibrating equipment is a constant-sensitivity instrument XH-D small laboratory vortex vibrator, vibrating is carried out at room temperature for uniform mixing), centrifuging for 6 minutes by using 350g (4 ℃) and discarding the supernatant (the supernatant mainly contains excrement residues and soluble matters remained on the intestinal wall); repeating the vibration and centrifugation for 2 times, and collecting tissue sediment in the digestive tract;
2) Adding the 5mL of digestive juice D1 into the tissue sediment obtained in the step 1, uniformly mixing, and performing primary digestion, wherein the digestion conditions are as follows: digesting for 30 minutes on a constant temperature shaking table at 37 ℃ with the rotating speed of the shaking table being 40 revolutions per minute; after digestion, adding 5mL of the buffer solution, mixing uniformly, filtering the content in the digestion tube by a 90 mu m filter membrane at room temperature, wherein the content can pass through the filter membrane to form large intestine epithelial cells suspended in the buffer solution, and the content which cannot pass through the filter membrane is tissue small blocks which are not digested into single cells, and collecting the tissue small blocks in another digestion tube;
3) Adding 10mL of the digestion solution D2 into the tissue small block which is not digested into single cells and obtained in the step 2, uniformly mixing, and then performing secondary digestion, wherein the digestion conditions are as follows: digesting for 30 minutes on a constant temperature shaking table at 37 ℃ with the rotating speed of the shaking table being 40 revolutions per minute; repeatedly blowing and sucking tissues which are not completely dispersed into single cells by using a 5mL syringe loaded with 20gauge after digestion is finished (the purpose of the repeated blowing and sucking is to release cells wrapped by residual extracellular matrix by mechanical force), and then adding 5mL of the buffer solution, and uniformly mixing; cell pellet was collected by centrifugation at 350g (4 ℃) for 6 min;
4) The cell pellet obtained in step 3 was resuspended in 5mL of the buffer, and then the cell mass was removed by passing through a 90 μm filter at room temperature, to finally obtain a large intestine single cell suspension.
Cell viability assays were performed on the obtained large intestine single cell suspensions. As a result, as shown in FIG. 3, the morphology and size of cells in the single-cell suspension of the large intestine finally obtained by the above procedure were uniform, the cell viability reached 92.39%, and the dead cell concentration was 9.87×10 4 1.3X10 total cell concentration per mL 6 1.2X10 concentration of living cells per mL 6 /mL。
2. Flow cytometry detection and sorting
The final large intestine single cell suspension obtained by the above procedure was incubated with flow cytometry specific fluorescent antibodies of 7AAD, CD45, CD11b, ly6g, ly6c and CD64 for 30 minutes, centrifuged to remove residual antibodies, and the cells were resuspended in a buffer (e.g., PBS) and then analyzed and sorted by sony SH800 flow cytometer, the results of which are shown in fig. 4. The results showed that the cell pellet obtained in the above step 3 mainly contains large intestine epithelial cells, crypt stem cells, immune cells, vascular endothelial cells, fibroblasts and muscle cells.
3. Cultivation of large intestine organoids
Sony SH800 flow type cell sorter for single cell suspension of large intestine finally obtained by the operation according to the stepsSorting, sorting the sorted intestinal crypt stem cells (7 AAD - CD45 - Lgr5 + Cells) were added to an organoid medium containing cytokines (e.g., RSPO1, EGF, FGF4, etc.), matrigel, serum, penicillin, streptomycin, and DMEM required for intestinal organoid culture, and microscopic observations were made on days 1, 5, and 6 (Day) after culture, respectively, and the results are shown in fig. 5. As can be seen from fig. 5, the crypt stem cells agglomerate on day 1, the crypt stem cells have been initially structured as intestinal organoids on day 5, and the crypt stem cells have been structured as intestinal organoids on day 6.
4. Single cell transcriptome sequencing analysis
The single cell suspension of the large intestine finally obtained by the above procedure was sorted by sony SH800 flow cytometer, and the sorted immune cells (7 AAD - CD45 + Cells) were subjected to cell viability assay, and after confirmation of pass, the results were subjected to dimension reduction analysis by a 10 x single cell sequencing platform, as shown in fig. 6. As can be seen from fig. 6, the cell clusters are clear, there is less overlap between cell clusters, demonstrating that the obtained cells can be detected and subsequently analyzed by a single cell sequencing platform.
(III) digestive System Key Components and comparison
1.DNase I
In experiments searching the digestive system of the large intestine tissue, DNase I can be used for removing DNA released by dead cells in the digestion process, and the removal of the DNA can ensure the survival rate of living cells obtained after digestion, which is specifically described as follows.
The cell viability obtained with the addition of DNase I (i.e.with the addition of D2) and without the addition of DNase I (other components were identical to that of D2) was compared in the experiments (both experiments were performed using the procedure described above and the final E.coli single cell suspension was obtained). As shown in FIG. 1, the single cell suspension obtained without adding DNase I had a cell viability of 13.33% (the difference in cell morphology was relatively large and the cell debris was relatively large); the single cell suspension obtained by adding DNase I in the digests had a cell viability of 90.54% (uniform cell morphology and size, less cell debris). The results showed that the cell viability obtained in the experimental group without DNase I was lower than that obtained in the experimental group with DNase I added, and the inter-group differences were significant (P < 0.05).
2.Trypsin
The cell viability obtained by digestion with only Trypsin (Trypsin group), only collagenase (collagenase group) and both Trypsin and collagenase (Trypsin+collagenase group) was also compared in the experiments.
(1) Digestion method of Trypsin group
The procedure for obtaining the large intestine single cell suspension was the same as above except that the digestive juice D1 was used in both digestions.
(2) Digestion method of collagenase group
The procedure for obtaining the large intestine single cell suspension was the same as above except that the digestive juice D2 was used in both digestions.
(3) Digestion method of Trypsin+collagenase group
The same procedure as described above, i.e., using the digestive juice D1 in the primary digestion and using the digestive juice D2 in the secondary digestion, finally obtained a large intestine single cell suspension.
As shown in fig. 2, the cell viability of the single cell suspension of the large intestine obtained by the Trypsin group was 50%; the cell viability of the single cell suspension of the large intestine obtained by the collagenase group is 76.99%; the cell viability of the single cell suspension of the large intestine obtained by the Trypsin + collagenase group was 90.54%. The results show that the combined use of digests D1 and D2 gave higher cell viability and greater numbers of cells than the digests D1 or D2 alone, with the differences being significant (P < 0.05).
Claims (10)
1. A method of digesting large intestine tissue, characterized by: the method comprises the following steps:
1) Pretreatment of
Shearing large intestine tissues, and cleaning the sheared large intestine tissues by using a buffer solution; the buffer solution comprises 1-10% of penicillin by mass fraction, 0.05-1% of streptomycin by mass fraction and 1-6% of newborn calf serum by volume fraction;
2) One-time digestion
Mixing the large intestine tissue treated in the step 1 with the digestive juice D1 uniformly, then digesting to obtain a digested product Y1, mixing the digested product Y1 with the buffer solution uniformly, and separating undigested tissue small blocks from large intestine epithelial cells by filtration; the digestive juice D1 comprises 0.2-25 g/L trypsin, penicillin with mass fraction of 1-10% and streptomycin with mass fraction of 0.05-1%;
3) Secondary digestion
Uniformly mixing the undigested tissue small blocks obtained in the step 2 with a digestive juice D2, then digesting to obtain a digested product Y2, uniformly mixing the digested product Y2 with a buffer solution after scattering tissue residues in the digested product Y2, and then centrifuging and collecting cell precipitates; the digestive juice D2 comprises 0.1-10 mg/mL of type I collagenase, 0.1-10 mg/mL of type IV collagenase, 0.01-5 mg/mL of hyaluronidase, 1-100 u/mL of DNase I and 1-6% of newborn calf serum by volume fraction;
4) Post-treatment
And (3) uniformly mixing the cell sediment obtained in the step (3) with a buffer solution, and then removing cell clusters through filtration to obtain the large intestine single cell suspension.
2. A method of digesting large intestine tissue according to claim 1, wherein: in the step 1, the cleaning specifically includes the following steps: uniformly mixing the sheared large intestine tissues with a buffer solution through vibration, centrifuging, and separating to remove upper liquid; the shaking and centrifuging were repeated not less than 3 times during the washing.
3. A method of digesting large intestine tissue according to claim 1, wherein: the solvent of the buffer solution is PBS with pH value of 5.8-8.0.
4. A method of digesting large intestine tissue according to claim 1, wherein: in the step 2, the conditions of the primary digestion are as follows: the temperature is 25-37 ℃, the time is 20-40 minutes, and the rotating speed of the shaking table is 40-80 revolutions per minute; the pore diameter of the filter membrane used for filtering is 70-100 mu m.
5. A method of digesting large intestine tissue according to claim 1, wherein: in the step 3, the conditions of secondary digestion are as follows: the temperature is 25-37 ℃, the time is 20-40 minutes, and the rotating speed of the shaking table is 40-80 revolutions per minute; the scattering specifically comprises the following steps: repeatedly blowing and sucking the tissue residues in the digested product Y2 by using an 18-22 gauge syringe to fully disperse the tissue residues.
6. A method of digesting large intestine tissue according to claim 2, wherein: in the step 1 and the step 3, the centrifugation conditions are as follows: centrifugation is carried out for 5-10 minutes by 300-400 g.
7. A method of digesting large intestine tissue according to claim 1, wherein: the digestion liquid D1 and the digestion liquid D2 are used as a culture medium of eukaryotic cells.
8. A method of digesting large intestine tissue according to claim 1, wherein: in the step 4, the pore diameter of a filter membrane adopted by the filtration is 70-90 mu m.
9. A large intestine tissue digestion kit, characterized in that: the kit comprises the following reagents: digestion solution D1, digestion solution D2 and buffer solution; the digestive juice D1 comprises 0.2-25 g/L trypsin, penicillin with the mass fraction of 1-10% and streptomycin with the mass fraction of 0.05-1%, the digestive juice D2 comprises 0.1-10 mg/mL type I collagenase, 0.1-10 mg/mL type IV collagenase, 0.01-5 mg/mL hyaluronidase, DNase I with the mass fraction of 1-100 u/mL and neonatal bovine serum with the volume fraction of 1-6%, and the buffer solution comprises penicillin with the mass fraction of 1-10%, streptomycin with the mass fraction of 0.05-1% and neonatal bovine serum with the volume fraction of 1-6%.
10. The large intestine tissue digestion kit as set forth in claim 9, wherein: the solvent of the buffer solution is PBS with pH value of 5.8-8.0; the solvent of the digestive juice D1 and the digestive juice D2 is a culture medium of eukaryotic cells.
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| CN117586942A (en) * | 2023-11-17 | 2024-02-23 | 杭州师范大学 | Single cell suspension dissociation kit for mouse colorectal tissue and application thereof |
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