Preparation method and application of pig pancreas single cell suspension

Abstract

This invention proposes a method for preparing a single-cell suspension of porcine pancreatic cells and its application. The method includes the following steps: S1, taking porcine pancreatic tissue, removing surface fat, and mincing the tissue; S2, washing the tissue fragments, and then placing the tissue into a mixed enzyme dissociation solution for the first digestion; S3, after the first digestion, collecting the supernatant, and adding the remaining tissue to the mixed enzyme dissociation solution for a second digestion; S4, after the second digestion, collecting the supernatant and combining it with the supernatant from step S3, and filtering; S5, centrifuging the filtered dissociation solution, discarding the supernatant, and resuspending the cell pellet to obtain a cell suspension; the mixed enzyme dissociation solution includes trypsin, collagenase I, elastase, lipase, DNase I, heparin, sodium sulfate, and DH Kans buffer. The enzyme mixture of this invention has the effects of removing intercellular matrix, reducing cell aggregation, protecting cell membrane morphology, and reducing cell damage.

Description

Technical Field

[0001] This invention relates to the field of cell biology, and in particular to a method for preparing a single-cell suspension of porcine pancreas and its application. Background Technology

[0002] Single-cell sequencing refers to sequencing technology that obtains genetic information from a single cell. This involves extracting, amplifying, and performing high-throughput sequencing analysis of the genome or transcriptome at the single-cell level. This technology can reveal the unique gene structure and expression status of a single cell, including data on structural variations, copy number variations, and RNA expression levels. This allows for precise differentiation of different cell types and helps scientists conduct molecular mechanism research at the single-cell level.

[0003] For single-cell transcription sequencing results, sample preparation has the greatest impact. Patent CN103436487A discloses that trypsin can enzymatically hydrolyze freshwater crab cells, and patent CN114591883A discloses that collagenase, hyaluronidase, and DNase can enzymatically hydrolyze aortic cells. Porcine pancreas contains a large amount of fat and fibrous components, causing the cells to easily clump together when these enzymes hydrolyze porcine pancreatic cells. Summary of the Invention

[0004] In view of this, the present invention proposes a method for preparing a single-cell suspension of porcine pancreas and its application. When enzymatically hydrolyzing porcine pancreas, the cell aggregation rate is low and the viability of the single-cell suspension is high.

[0005] The technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a method for preparing a single-cell suspension of porcine pancreas, comprising the following steps:

[0006] S1. Take pig pancreatic tissue, remove surface fat, and cut the tissue into small pieces;

[0007] S2, clean the tissue fragments, and then put the tissue into the mixed enzyme dissociation solution for the first digestion;

[0008] S3: After the first digestion is completed, take the supernatant and add the mixed enzyme dissociation solution to the remaining tissue in the tube for a second digestion.

[0009] S4. After the second digestion is completed, take the supernatant and combine it with the supernatant from step S3, then sieve it.

[0010] S5, after sieving, the dissociation solution is centrifuged and the supernatant is discarded. The cell pellet is washed and centrifuged twice, and then resuspended in DPBS solution to obtain a cell suspension.

[0011] The mixed enzyme dissociation solution includes trypsin, collagenase I, elastase, lipase, DNase I, heparin, sodium sulfate, and DHanks buffer.

[0012] Based on the above technical solutions, preferably, the mixed enzyme dissociation solution includes trypsin 1-5 mg / mL, collagenase I 0.5-3 mg / mL, elastase 1-4 mg / mL, lipase 2-5 mg / mL, DNase I 8-12 mg / mL, heparin 2-6 U / mL and sodium sulfate 3-6 g / L, and the solvent is D-Hanks buffer.

[0013] Based on the above technical solutions, the preferred method is to mince the tissue on ice, adding DPBS buffer containing 0.02%-0.05% BSA during the process, mincing it into a paste, and controlling the time to within 5 minutes.

[0014] Based on the above technical solutions, preferably, in step S2, tissue fragments are washed with DPBS buffer containing 0.02%-0.05% BSA by mass.

[0015] Based on the above technical solutions, the preferred conditions for the first and second digestions in steps S2 and S3 are: digestion for 10-15 minutes on a shaker at 30-60 rpm at 35-37°C.

[0016] Based on the above technical solutions, preferably, in step S5, the washing solution is a DPBS buffer with a mass fraction of 0.02%-0.05% BSA.

[0017] Based on the above technical solutions, preferably, in step S5, the centrifugation method is: centrifugation at 4-5℃ and 200-300rpm for 3-5 minutes.

[0018] Secondly, the present invention provides a porcine pancreatic single-cell suspension prepared by the above-described preparation method.

[0019] Thirdly, this invention provides the application of porcine pancreatic single-cell suspension in single-cell transcriptome sequencing.

[0020] The present invention provides a method for preparing a single-cell suspension of porcine pancreas and its application, which have the following advantages over the prior art:

[0021] Beneficial effects:

[0022] (1) The trypsin, collagenase, elastase and lipase in the enzyme mixture of the present invention can remove the intercellular matrix; DNase I can separate DNA from ruptured cells and decompose these excess fragments to help reduce the entanglement between DNA, that is, reduce cell aggregation; heparin can protect the cell membrane morphology and reduce cell damage; sodium sulfate can prevent cell aggregation and promote cell free movement.

[0023] (2) This invention combines a two-step digestion method with the addition of bovine serum albumin, which can maintain osmotic pressure, buffer pH, act as a carrier, and provide nutrition. By controlling controllable factors such as time, centrifuge speed, and temperature, the intercellular matrix can be digested while protecting the cell membrane integrity. The viability of the dissociated porcine pancreatic cells is over 80%, with no clumping, a clean background, and no chip clogging. The total number of cells reaches over 100,000, meeting the cell quantity requirements for single-cell transcriptome sequencing. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is an AO / PI staining image of pancreatic cell suspension from Example 1 of the present invention;

[0026] Figure 2 A diagram showing the results of pancreatic cell clusters in Example 1 of this invention;

[0027] Figure 3 This is an AO / PI staining image of the pancreatic cell suspension of Comparative Example 1 of this invention;

[0028] Figure 4 A diagram showing the results of pancreatic cell clusters in Comparative Example 1 of this invention;

[0029] Figure 5 This is a quality control image of pancreatic cell cDNA fragments from Example 1 of the present invention.

[0030] Figure 6 This is a quality control image of a pancreatic cell library fragment from Example 1 of the present invention. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] The method for preparing porcine pancreatic single-cell suspension in this embodiment includes the following steps:

[0034] S1. Take a fresh pig pancreas tissue measuring 1cm x 1cm and remove as much surface fat as possible.

[0035] S2, Prepare the mixed enzyme dissociation solution:

[0036] The solution contains trypsin 1 mg / mL, collagenase I 0.5 mg / mL, elastase 1 mg / mL, lipase 2 mg / mL, DNase I 8 mg / mL, heparin 2 U / mL, and sodium sulfate 3 g / L. The volume is adjusted to 100 mL using DHanks.

[0037] After preparation, dissolve and mix thoroughly, filter with a 0.22μL filter membrane for sterilization, and preheat at 37℃ until ready for use.

[0038] S3. On ice, mince the tissue, adding 1xDPBS + 0.02% BSA, and mince it into a paste. The time should be controlled within 5 minutes.

[0039] S4. After washing twice with 1xDPBS + 0.02% BSA, the tissue was placed in a preheated 37°C mixed enzyme dissociation solution, with the volume of the mixed enzyme dissociation solution being 5 times that of the tissue. The tissue was then digested for the first time for 10 minutes on a constant temperature shaker at 35°C and 30 rpm.

[0040] S5. After the first digestion, the supernatant was removed, and 10% pre-cooled FBS was added to terminate the trypsin reaction. The mixture was placed on ice for later use. The remaining tissue in the tube was then added to a preheated mixed enzyme dissociation solution at 37°C. The tube was then digested for a second time for 10 minutes on a constant temperature shaker at 35°C and 30 rpm.

[0041] S6. After the second digestion, take the supernatant, add 10% pre-cooled FBS to terminate the trypsin reaction, combine it with the supernatant from step 5, and pass it through a 40μm cell sieve.

[0042] S7. Dispense the filtered dissociation solution into 1.5 mL centrifuge tubes and centrifuge in a refrigerated centrifuge at 4°C and 200 rpm for 3 min.

[0043] S8, remove the supernatant, retain the cell pellet at the bottom, add 500 μL of 1xDPBS + 0.02% BSA mixture, gently mix with a pipette tip, and centrifuge at 4°C and 200 rpm for 3 min.

[0044] S9, repeat step S8 once.

[0045] S10, after centrifugation, remove the supernatant, add 100μL DPBS to resuspend the cell pellet, and obtain the cell suspension.

[0046] Example 2

[0047] The method for preparing porcine pancreatic single-cell suspension in this embodiment includes the following steps:

[0048] S1. Take a fresh pig pancreas tissue measuring 1cm x 1cm and remove as much surface fat as possible.

[0049] S2, Prepare the mixed enzyme dissociation solution:

[0050] The solution contains trypsin 2 mg / mL, collagenase I 1 mg / mL, elastase 2 mg / mL, lipase 3 mg / mL, DNase I 9 mg / mL, heparin 3 U / mL, and sodium sulfate 4 g / L. The volume is adjusted to 100 mL using DHanks.

[0051] After preparation, dissolve and mix thoroughly, filter with a 0.22μL filter membrane for sterilization, and preheat at 37℃ until ready for use.

[0052] S3. On ice, mince the tissue, adding 1xDPBS + 0.03% BSA mixture during the process, mincing it into a paste, and keeping the time within 5 minutes.

[0053] S4, after washing twice with a mixture of 1xDPBS and 0.03% BSA, the tissue was placed in a preheated (37°C) mixed enzyme dissociation buffer (5 times the volume of the tissue) and digested for 12 minutes at 36°C and 40 rpm on a constant-temperature shaker.

[0054] S5. After the first digestion, the supernatant was removed, and 10% pre-cooled FBS was added to terminate the trypsin reaction. The mixture was placed on ice for later use. The remaining tissue in the tube was then added to a preheated mixed enzyme dissociation solution at 37°C. The tube was then digested for a second time for 12 minutes on a constant temperature shaker at 36°C and 40 rpm.

[0055] S6. After the second digestion, take the supernatant, add 10% pre-cooled FBS to terminate the trypsin reaction, combine it with the supernatant from step 5, and pass it through a 40μm cell sieve.

[0056] S7. Dispense the filtered dissociation solution into 1.5 mL centrifuge tubes and centrifuge in a refrigerated centrifuge at 4.5 °C and 250 rpm for 4 min.

[0057] S8, remove the supernatant, retain the cell pellet at the bottom, add 500 μL of 1xDPBS + 0.03% BSA mixture, gently mix with a pipette tip, and centrifuge at 5°C and 250 rpm for 4 min.

[0058] S9, repeat step S8 once.

[0059] S10, after centrifugation, remove the supernatant, add 100μL DPBS to resuspend the cell pellet, and obtain the cell suspension.

[0060] Example 3

[0061] The method for preparing porcine pancreatic single-cell suspension in this embodiment includes the following steps:

[0062] S1. Take a fresh pig pancreas tissue measuring 1cm x 1cm and remove as much surface fat as possible.

[0063] S2, Prepare the mixed enzyme dissociation solution:

[0064] The solution contains trypsin 4 mg / mL, collagenase I 2 mg / mL, elastase 3 mg / mL, lipase 4 mg / mL, DNase I 10 mg / mL, heparin 5 U / mL, and sodium sulfate 5 g / L. The volume is adjusted to 100 mL using DHanks.

[0065] After preparation, dissolve and mix thoroughly, filter with a 0.22μL filter membrane for sterilization, and preheat at 37℃ until ready for use.

[0066] S3. On ice, mince the tissue, adding 1xDPBS + 0.04% BSA mixture during the process, mincing it into a paste, and keeping the time within 5 minutes.

[0067] S4, after washing twice with a mixture of 1xDPBS and 0.04% BSA, the tissue was placed in a preheated (37°C) mixed enzyme dissociation buffer (5 times the volume of the tissue) and digested for 14 minutes at 37°C and 50 rpm on a constant-temperature shaker.

[0068] S5. After the first digestion, the supernatant was removed, and 10% pre-cooled FBS was added to terminate the trypsin reaction. The mixture was placed on ice for later use. The remaining tissue in the tube was then added to a preheated mixed enzyme dissociation solution at 37°C. The tube was then digested for a second time for 14 minutes on a constant temperature shaker at 36°C and 50 rpm.

[0069] S6. After the second digestion, take the supernatant, add 10% pre-cooled FBS to terminate the trypsin reaction, combine it with the supernatant from step 5, and pass it through a 40μm cell sieve.

[0070] S7. Dispense the filtered dissociation solution into 1.5 mL centrifuge tubes and centrifuge in a refrigerated centrifuge at 5°C and 300 rpm for 5 min.

[0071] S8, remove the supernatant, retain the cell pellet at the bottom, add 500μL of 1xDPBS + 0.04%BSA mixture, gently pipette to mix, and centrifuge at 300rpm for 5min at 5℃.

[0072] S9, repeat step S8 once.

[0073] S10, after centrifugation, remove the supernatant, add 100μL DPBS to resuspend the cell pellet, and obtain the cell suspension.

[0074] Example 4

[0075] The method for preparing porcine pancreatic single-cell suspension in this embodiment includes the following steps:

[0076] S1. Take a fresh pig pancreas tissue measuring 1cm x 1cm and remove as much surface fat as possible.

[0077] S2, Prepare the mixed enzyme dissociation solution:

[0078] The solution contains trypsin 5 mg / mL, collagenase I 3 mg / mL, elastase 4 mg / mL, lipase 5 mg / mL, DNase I 12 mg / mL, heparin 6 U / mL, and sodium sulfate 6 g / L. The volume is adjusted to 100 mL using DHanks.

[0079] After preparation, dissolve and mix thoroughly, filter with a 0.22μL filter membrane for sterilization, and preheat at 37℃ until ready for use.

[0080] S3. On ice, mince the tissue, adding 1xDPBS + 0.05% BSA mixture during the process, mincing it into a paste, and keeping the time within 5 minutes.

[0081] S4. After washing twice with a mixture of 1xDPBS and 0.05% BSA, the tissue was placed in a preheated mixed enzyme dissociation solution at 37°C. The volume of the mixed enzyme dissociation solution was 5 times that of the tissue. The tissue was digested for the first time for 15 minutes on a constant temperature shaker at 37°C and 60 rpm.

[0082] S5. After the first digestion, the supernatant was removed, and 10% pre-cooled FBS was added to terminate the trypsin reaction. The mixture was placed on ice for later use. The remaining tissue in the tube was then added to a preheated mixed enzyme dissociation solution at 37°C. The tube was then digested for a second time for 15 minutes on a constant temperature shaker at 37°C and 60 rpm.

[0083] S6. After the second digestion, take the supernatant, add 10% pre-cooled FBS to terminate the trypsin reaction, combine it with the supernatant from step 5, and pass it through a 40μm cell sieve.

[0084] S7. Dispense the filtered dissociation solution into 1.5 mL centrifuge tubes and centrifuge in a refrigerated centrifuge at 5°C and 300 rpm for 5 min.

[0085] S8, remove the supernatant, retain the cell pellet at the bottom, add 500μL of 1xDPBS + 0.05%BSA mixture, gently mix by pipetting with a pipette tip, and centrifuge at 300rpm for 5min at 5℃.

[0086] S9, repeat step S8 once.

[0087] S10, after centrifugation, remove the supernatant, add 100μL DPBS to resuspend the cell pellet, and obtain the cell suspension.

[0088] Comparative Example 1

[0089] The difference between Comparative Example 1 and Example 1 is that the mixed enzyme dissociation solution does not contain heparin and sodium sulfate.

[0090] Comparative Example 2

[0091] The difference between Comparative Example 2 and Example 1 is that the mixed enzyme dissociation solution does not contain elastase and lipase.

[0092] Comparative Example 3

[0093] The difference between Comparative Example 3 and Example 1 is that the mixed enzyme dissociation solution does not contain heparin, sodium sulfate, elastase and lipase.

[0094] Comparative Example 4

[0095] The difference between Comparative Example 4 and Example 1 is that the washing solution in steps S3, S4, and S8 does not contain BSA, but only DPBS buffer.

[0096] Comparative Example 5

[0097] The difference between Comparative Example 5 and Example 1 is that the first digestion time is 4 hours.

[0098] Comparative Example 6

[0099] The difference between Comparative Example 6 and Example 1 is that the centrifugation method in steps S7-S9 is centrifugation at 37°C and 1000 rpm for 10 min.

[0100] Porcine pancreatic single-cell suspensions were prepared according to the examples and comparative examples, and counted using AO / PI fluorescence staining and a Bodboge cell counter. The results are shown in Table 1.

[0101] Table 1 Comparison of single-cell suspensions of porcine pancreas

[0102]

[0103]

[0104] Table 1 shows that the total cell concentration, viable cell rate, and clumping rate of the single-cell suspensions in Examples 1-4 are all better than those in Comparative Examples 1-6, with Example 4 being the best.

[0105] Comparative Examples 1-3 show that the absence of heparin, sodium sulfate, elastase, and lipase all reduce the viable cell ratio and increase the clumping rate. This demonstrates the effectiveness of pancreatic enzymes, collagenase, elastase, and lipase in removing intercellular matrix; heparin in protecting cell membrane morphology and reducing cell damage; and sodium sulfate in preventing cell clumping and promoting cell free movement.

[0106] Comparative Examples 4-6 show that BSA, digestion time, and centrifugation method all affect total cell concentration, viable cell rate, and clumping rate.

[0107] The porcine pancreatic cell suspension prepared in Example 1 was used for 10x Genomics 3' single-cell transcriptome sequencing, as follows:

[0108] 1. Library Construction:

[0109] 1.1 With a cell capture volume of 10,000, the prepared cell suspension, 10X barcode gel beads and oil droplets were added to different chambers of Chip G, and GEM was generated through a microfluidic "double cross" cross system.

[0110] 1.2 The generated GEMs were transferred and incubated. The RNA containing PloyA was reverse transcribed into a cDNA one-strand containing 10X Barcode and UMI information, and then a cDNA two-strand was synthesized in SMART mode.

[0111] 1.3 Oil droplet breakage, magnetic bead purification of cDNA, and cDNA amplification.

[0112] 1.4 The amplified cDNA was quantified and subjected to Q-SEP1 quality control. See the cDNA fragment quality control diagram below. Figure 5 .

[0113] 1.5 Constructing a 3' gene expression library.

[0114] 1.6 The constructed library was quantitatively analyzed and subjected to Q-SEP1 quality control.

[0115] 1.7 Sequencing on the Illumina platform.

[0116] 2. Document Quality Inspection

[0117] 2.1 Q-bit Quantification

[0118] The library concentration was 8.18 ng / μL, and the volume was 40 μL.

[0119] 2.2 Document Quality Inspection

[0120] See document quality inspection chart Figure 6 .

[0121] Figure 5 and Figure 6 It can be seen that the porcine pancreatic cell suspension prepared in the examples has a high total cell count and a low clumping rate, and will not clog the chip, making it suitable for 10x Genomics 3' single-cell transcriptome sequencing. Comparative Examples 1-3, due to their high clumping rate, clog the chip and are not suitable for 10x Genomics 3' single-cell transcriptome sequencing.

[0122] The above description is only a preferred embodiment of the present invention and is 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 within the protection scope of the present invention.

Claims

1. A method for preparing a single-cell suspension of porcine pancreas, characterized in that: The method comprises the following steps: S1, taking pig pancreas tissue, removing surface fat, and cutting the tissue into pieces; S2, washing the tissue pieces, and then placing the tissue into a mixed enzyme dissociation solution for first digestion; S3, after the first digestion, taking the supernatant, and continuously adding the mixed enzyme dissociation solution into the residual tissue in the tube for second digestion; S4, after the second digestion, taking the supernatant, and combining the supernatant with the supernatant in step S3, and sieving; S5, centrifuging the sieved dissociation solution, discarding the supernatant, and resuspending the cell precipitate in DPBS solution after twice washing and centrifuging, to obtain a cell suspension; The mixed enzyme dissociation solution comprises 1-5 mg / mL trypsin, 0.5-3 mg / mL collagenase I, 1-4 mg / mL elastase, 2-5 mg / mL lipase, 8-12 mg / mL DNase I, 2-6 U / mL heparin, and 3-6 g / L sodium sulfate, and the solvent is D-hanks buffer.

2. The method for preparing a single-cell suspension of porcine pancreas as described in claim 1, characterized in that: The tissue is cut into pieces on ice, and DPBS buffer containing 0.02%-0.05% mass fraction BSA is added during the cutting, and the tissue is cut into a paste, and the time is controlled to be within 5 min.

3. The method for preparing a single-cell suspension of porcine pancreas as described in claim 1, characterized in that: In step S2, the tissue pieces are washed with DPBS buffer containing 0.02%-0.05% mass fraction BSA.

4. The method for preparing a single-cell suspension of porcine pancreas as described in claim 1, characterized in that: In steps S2 and S3, the first digestion and the second digestion are performed at 35-37 °C on a shaker at 30-60 rpm for 10-15 min.

5. The method for preparing a porcine pancreatic single-cell suspension as described in claim 1, characterized in that: In step S5, the washing liquid is DPBS buffer containing 0.02%-0.05% mass fraction BSA.

6. A method for the preparation of a pig pancreatic monocyte suspension according to claim 5, characterized by: In step S5, the centrifugation method is centrifugation at 4-5 °C and 200-300 rpm for 3-5 min.

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