CN113801839A - Method for extracting stellate cells of static pancreas of rat - Google Patents
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- CN113801839A CN113801839A CN202111090914.8A CN202111090914A CN113801839A CN 113801839 A CN113801839 A CN 113801839A CN 202111090914 A CN202111090914 A CN 202111090914A CN 113801839 A CN113801839 A CN 113801839A
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- 210000000496 pancreas Anatomy 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000003068 static effect Effects 0.000 title claims abstract description 17
- 210000004500 stellate cell Anatomy 0.000 title claims abstract description 5
- 210000002705 pancreatic stellate cell Anatomy 0.000 claims abstract description 40
- 239000001963 growth medium Substances 0.000 claims abstract description 29
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 18
- 102000004142 Trypsin Human genes 0.000 claims abstract description 14
- 108090000631 Trypsin Proteins 0.000 claims abstract description 14
- 239000012588 trypsin Substances 0.000 claims abstract description 14
- 238000010009 beating Methods 0.000 claims abstract description 12
- 210000004923 pancreatic tissue Anatomy 0.000 claims abstract description 11
- 239000011248 coating agent Substances 0.000 claims abstract description 9
- 238000000576 coating method Methods 0.000 claims abstract description 9
- 238000010008 shearing Methods 0.000 claims abstract description 9
- 230000029087 digestion Effects 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 238000004113 cell culture Methods 0.000 claims abstract description 6
- 239000000706 filtrate Substances 0.000 claims abstract description 6
- 239000006285 cell suspension Substances 0.000 claims abstract description 5
- 210000001519 tissue Anatomy 0.000 claims description 21
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 239000012091 fetal bovine serum Substances 0.000 claims description 7
- 229960005322 streptomycin Drugs 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000000432 density-gradient centrifugation Methods 0.000 abstract description 5
- 238000001914 filtration Methods 0.000 abstract description 4
- 230000008595 infiltration Effects 0.000 abstract description 4
- 238000001764 infiltration Methods 0.000 abstract description 4
- 238000000605 extraction Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 9
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 3
- 238000000635 electron micrograph Methods 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 238000007447 staining method Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- NTHXOOBQLCIOLC-UHFFFAOYSA-N iohexol Chemical compound OCC(O)CN(C(=O)C)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NTHXOOBQLCIOLC-UHFFFAOYSA-N 0.000 description 2
- 229960001025 iohexol Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses an extraction method of static state pancreas stellate cells of a rat, which comprises the following steps: s1, preparing a DMEM/F12 culture medium; s2, fully shearing the pancreas of the rat; s3, coating the pancreatic tissue block at the bottom of the cell culture dish and culturing for 1-3 h; s4, adding a DMEM/F12 culture medium to culture for 24-48 h; s5, removing the culture medium, rinsing with PBS once, removing the PBS, adding trypsin for infiltration, and removing the trypsin; s6, incubating for 80-120S, adding a DMEM/F12 culture medium to stop digestion, gently beating for 3-5 times, and beating the stationary pancreatic stellate cells to suspend; s7, filtering the cell suspension obtained in the step S6 and collecting filtrate to obtain the static state pancreatic stellate cells of the rat. The method has simple steps, is easy to operate and saves time; the obtained stationary pancreatic stellate cells have high yield and high purity, and only one kind of cheap trypsin is used in the whole process, so that the cost is greatly reduced compared with that of a density gradient centrifugation method.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for extracting static state pancreatic stellate cells of a rat.
Background
The static pancreatic stellate cells have stem cell properties but very little content, and have important scientific research value and clinical transformation value. The existing extraction method is a density gradient centrifugation method, namely iohexol is used as a centrifugation medium to prepare two solutions with different densities, and the cells are obtained by density gradient centrifugation by utilizing the principle that the density of stationary pancreatic stellate cells is slightly lower. The method has obvious problems that firstly, the operation is complex and difficult to master, the pancreas needs to be digested into single cells by adopting mixed liquor prepared by three enzymes, the cells are easy to die after the digestion time is long, and the single cells cannot be obtained after the digestion time is short for centrifugation; secondly, because the stationary pancreatic stellate cells are not much lighter than other cells, the cell yield obtained by adopting a density gradient centrifugation method is low and is mixed with other cell types; third, the three enzymes and cell-grade iohexol used in the above process are expensive.
Disclosure of Invention
In order to solve the above mentioned drawbacks in the background art, the present invention provides a method for extracting quiescent pancreatic stellate cells from rat, which can achieve the objectives of simple operation, high yield and low cost.
The purpose of the invention can be realized by the following technical scheme:
a method for extracting static state pancreatic stellate cells of a rat comprises the following steps:
s1, preparing a DMEM/F12 culture medium containing fetal bovine serum and streptomycin;
s2, obtaining a fresh rat pancreas by adopting an operation method, washing the pancreas 2-4 times by using the DMEM/F12 culture medium of the step S1, and fully shearing the rat pancreas;
s3, coating the pancreas tissue block at the bottom of the cell culture dish, and inversely buckling the coated culture dish in an incubator for 1-3 hours with the bottom upward;
s4, taking out the culture dish, adding the DMEM/F12 culture medium obtained in the step S1 into the culture dish, and putting the culture dish back into the incubator for culturing for 24-48 h;
s5, removing the culture medium, rinsing once with PBS, removing the PBS, adding trypsin into the culture dish, and immediately removing the trypsin after infiltrating the tissue block;
s6, putting the culture dish into an incubator for incubation for 80-120S, adding the DMEM/F12 culture medium obtained in the step S1 into the culture dish to terminate digestion, gently beating the culture dish for 3-5 times, and beating the static pancreatic stellate cells to suspend;
s7, screening the cell suspension obtained in the step S6 by a screen, removing residual tissue blocks, and collecting filtrate to obtain the static state pancreatic stellate cells of the rat.
Further preferably, in step S1, the fetal bovine serum accounts for 10-20% of the total mass of the DMEM/F12 medium, and the streptomycin accounts for 0.5-2% of the total mass of the DMEM/F12 medium.
Further preferably, the diameter of the pancreas tissue mass after the rat pancreas is minced in step S2 is less than 0.5 mm.
Further preferably, 30-40 tissue pieces are applied per dish in step S3.
Further preferably, the incubator temperature is 37 ℃.
Further preferably, the cell suspension in step S7 is screened through a 70-90 mesh screen.
The invention has the beneficial effects that:
the method has simple steps, easy operation and time saving, and the obtained static pancreatic stellate cells have high yield (3.0-4.5 multiplied by 10 can be obtained per 0.1g rat pancreas)5Individual cell) and high purity (90-95%), and only one kind of cheap trypsin is used in the whole process, so that the cost is greatly reduced compared with that of a density gradient centrifugation method.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a photograph of a stationary pancreas star electron microscope surrounding a tissue mass according to example 1 of the present invention;
FIG. 2 is an electron microscope photograph of quiescent pancreatic stellate cells of rat extracted in example 1 of the present invention;
FIG. 3 is a photograph of a stationary pancreas star electron microscope surrounding a tissue mass according to example 2 of the present invention;
FIG. 4 is an electron microscope photograph of quiescent pancreatic stellate cells of rat extracted in example 2 of the present invention;
FIG. 5 is a photograph of a stationary pancreas star electron microscope surrounding a tissue mass according to example 2 of the present invention;
FIG. 6 is an electron microscope photograph of quiescent rat pancreatic stellate cells extracted in example 3 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for extracting static state pancreatic stellate cells of a rat comprises the following steps:
s1, DMEM/F12 (Gibco) medium (hereinafter referred to as "medium") containing 20% fetal bovine serum (Gibco) and 0.5% streptomycin was prepared;
s2, sterilizing surgical instruments, obtaining a fresh rat pancreas by adopting a surgical method, flushing the pancreas 3 times by using a culture medium, fully shearing the pancreas by using an ophthalmic surgical scissors, wherein the diameter of a pancreatic tissue block after shearing is less than 0.5 mm;
s3, coating the pancreas tissue blocks at the bottom of a 6cm cell culture dish, coating 30-40 tissue blocks on each culture dish, and inversely buckling the culture dish with the bottom upwards in an incubator at 37 ℃ for culturing for 2 h.
S4, taking out the culture dishes after 2 hours, adding 3mL of culture medium into each culture dish, and putting the culture dishes back into the incubator at 37 ℃ for culturing for 24 hours;
s5, after 24h, the culture dish is taken out, and static pancreatic stellate cells are observed around the tissue block, as shown in figure 1. Discarding the culture medium, rinsing with 1mL PBS (Biotechnology corporation), discarding PBS, adding 300 μ L trypsin (Gibco corporation) to each culture dish, and immediately removing trypsin after tissue mass infiltration;
s6, incubating in a 37 deg.C incubator for 115S, adding 600 μ L of culture medium into the culture dish to stop digestion, gently beating for 3 times, and beating the stationary pancreatic stellate cells to suspend
S7, screening the culture medium with a 90-mesh screen to remove residual tissue mass, filtering the stationary pancreatic stellate cells, and collecting the filtrate to obtain stationary pancreatic stellate cells of rat, wherein the electron micrograph is shown in FIG. 2.
The calculated yield of pancreatic stellate cells was 4.5X 10 per 0.1g rat pancreas5The number of the individual cells is one,the purity of the obtained pancreatic stellate cells detected by an oil red O staining method was 95%.
Example 2
A method for extracting static state pancreatic stellate cells of a rat comprises the following steps:
s1, preparing a DMEM/F12 (Gibco) medium (hereinafter referred to as a "medium") containing 15% fetal bovine serum (Gibco) and 1% streptomycin;
s2, sterilizing surgical instruments, obtaining a fresh rat pancreas by adopting a surgical method, flushing the pancreas 3 times by using a culture medium, fully shearing the pancreas by using an ophthalmic surgical scissors, wherein the diameter of a pancreatic tissue block after shearing is less than 0.5 mm;
s3, coating the pancreas tissue blocks at the bottom of a 6cm cell culture dish, coating 30-40 tissue blocks on each culture dish, and reversely buckling the culture dish with the bottom upwards in an incubator at 37 ℃ for 2 hours.
S4, taking out the culture dishes after 2 hours, adding 3mL of culture medium into each culture dish, and putting the culture dishes back into the incubator at 37 ℃ for culturing for 36 hours;
s5, 36 hours later, the culture dish was removed and the tissue mass was observed for the presence of quiescent pancreatic stellate cells around it, as shown in FIG. 3. Discarding the culture medium, rinsing with 1mL PBS (Biotechnology corporation), discarding PBS, adding 250 μ L trypsin (Gibco corporation) to each culture dish, and immediately removing trypsin after tissue mass infiltration;
s6, incubating in an incubator at 37 deg.C for 100S, adding 500 μ L of culture medium into the culture dish to stop digestion, gently beating for 5 times, and beating the stationary pancreatic stellate cells to suspend
S7, screening the culture medium with a 80-mesh screen to remove residual tissue mass, filtering the stationary pancreatic stellate cells, and collecting the filtrate to obtain stationary pancreatic stellate cells of rat, wherein the electron micrograph is shown in FIG. 4.
The calculated yield of pancreatic stellate cells was 3.7X 10 per 0.1g rat pancreas5The purity of the pancreatic stellate cells obtained by the detection of the oil red O staining method is 92 percent.
Example 3
A method for extracting static state pancreatic stellate cells of a rat comprises the following steps:
s1, preparing a DMEM/F12 (Gibco) medium (hereinafter referred to as a "medium") containing 20% fetal bovine serum (Gibco) and 2% streptomycin;
s2, sterilizing surgical instruments, obtaining a fresh rat pancreas by adopting a surgical method, flushing the pancreas 3 times by using a culture medium, fully shearing the pancreas by using an ophthalmic surgical scissors, wherein the diameter of a pancreatic tissue block after shearing is less than 0.5 mm;
s3, coating the pancreas tissue blocks at the bottom of a 6cm cell culture dish, coating 30-40 tissue blocks on each culture dish, and reversely buckling the culture dish with the bottom upwards in an incubator at 37 ℃ for 2 hours.
S4, taking out the culture dishes after 2 hours, adding 3mL of culture medium into each culture dish, and putting the culture dishes back into the incubator at 37 ℃ for culturing for 36 hours;
s5, 36 hours later, the culture dish was removed and the tissue mass was observed for the presence of quiescent pancreatic stellate cells around it, as shown in FIG. 5. Discarding the culture medium, rinsing with 1mL PBS (Biotechnology corporation), discarding PBS, adding 200 μ L trypsin (Gibco corporation) to each culture dish, and immediately removing trypsin after tissue mass infiltration;
s6, incubating in a 37 deg.C incubator for 90S, adding 400 μ L of culture medium into the culture dish to stop digestion, gently beating for 5 times, and beating the stationary pancreatic stellate cells to suspend
S7, screening the culture medium with a 70-mesh screen to remove residual tissue mass, filtering the stationary pancreatic stellate cells, and collecting the filtrate to obtain stationary pancreatic stellate cells of rat, wherein the electron micrograph is shown in FIG. 6.
The calculated yield of pancreatic stellate cells was 3.9X 10 per 0.1g rat pancreas5The purity of the pancreatic stellate cells obtained by the detection of the oil red O staining method is 91%.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (6)
1. A method for extracting stellate cells of static pancreas of a rat is characterized by comprising the following steps:
s1, preparing a DMEM/F12 culture medium containing fetal bovine serum and streptomycin;
s2, obtaining a fresh rat pancreas by adopting an operation method, washing the pancreas 2-4 times by using the DMEM/F12 culture medium of the step S1, and fully shearing the rat pancreas;
s3, coating the pancreas tissue block at the bottom of the cell culture dish, and inversely buckling the coated culture dish in an incubator for 1-3 hours with the bottom upward;
s4, taking out the culture dish, adding the DMEM/F12 culture medium obtained in the step S1 into the culture dish, and putting the culture dish back into the incubator for culturing for 24-48 h;
s5, removing the culture medium, rinsing once with PBS, removing the PBS, adding trypsin into the culture dish, and immediately removing the trypsin after infiltrating the tissue block;
s6, putting the culture dish into an incubator for incubation for 80-120S, adding the DMEM/F12 culture medium obtained in the step S1 into the culture dish to terminate digestion, gently beating the culture dish for 3-5 times, and beating the static pancreatic stellate cells to suspend;
s7, screening the cell suspension obtained in the step S6 by a screen, removing residual tissue blocks, and collecting filtrate to obtain the static state pancreatic stellate cells of the rat.
2. The method for extracting quiescent pancreatic stellate cells of rat according to claim 1, wherein said fetal bovine serum in step S1 is 10-20% of the total mass of DMEM/F12 medium, and said streptomycin is 0.5-2% of the total mass of DMEM/F12 medium.
3. The method of claim 1, wherein the diameter of the pancreas tissue mass is less than 0.5mm after the rat pancreas is minced in step S2.
4. The method for extracting rat quiescent pancreatic stellate cells of claim 1, wherein each culture dish of said step S3 is coated with 30-40 tissue blocks.
5. The method of claim 1, wherein the temperature of the incubator is 37 ℃.
6. The method for extracting stellate cells from rat quiescent pancreas according to claim 1, wherein the cell suspension of step S7 is screened through a 70-90 mesh screen.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104173390A (en) * | 2013-05-22 | 2014-12-03 | 香港浸会大学 | Use of a flavanol glycoside for suppressing activation of stellate cells |
| CN104673743A (en) * | 2015-02-13 | 2015-06-03 | 中国人民解放军第二军医大学 | Tissue block culture method for obtaining primary cell from animal tissue |
| CN110368500A (en) * | 2019-07-12 | 2019-10-25 | 浙江大学 | A kind of amphipathic copolymer prodrug, preparation method and the nano particle for containing its salts |
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- 2021-09-17 CN CN202111090914.8A patent/CN113801839A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104173390A (en) * | 2013-05-22 | 2014-12-03 | 香港浸会大学 | Use of a flavanol glycoside for suppressing activation of stellate cells |
| CN104673743A (en) * | 2015-02-13 | 2015-06-03 | 中国人民解放军第二军医大学 | Tissue block culture method for obtaining primary cell from animal tissue |
| CN110368500A (en) * | 2019-07-12 | 2019-10-25 | 浙江大学 | A kind of amphipathic copolymer prodrug, preparation method and the nano particle for containing its salts |
Non-Patent Citations (3)
| Title |
|---|
| 唐元瑜等: "小鼠胰星状细胞的分离培养及鉴定", 世界华人消化杂志, vol. 18, no. 1, pages 32 * |
| 范昕;王莹;侯雯跻;刘宇;王旭青;孙康;张建新;: "小鼠胰腺星状细胞的分离培养与鉴定", 江苏大学学报(医学版), no. 06, pages 469 * |
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