CN110577927A - method for extracting cell sap from cells cultured in vitro - Google Patents
method for extracting cell sap from cells cultured in vitro Download PDFInfo
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- CN110577927A CN110577927A CN201910904342.9A CN201910904342A CN110577927A CN 110577927 A CN110577927 A CN 110577927A CN 201910904342 A CN201910904342 A CN 201910904342A CN 110577927 A CN110577927 A CN 110577927A
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000000338 in vitro Methods 0.000 title claims abstract description 23
- 210000004027 cell Anatomy 0.000 claims abstract description 155
- 210000001519 tissue Anatomy 0.000 claims abstract description 61
- 210000000805 cytoplasm Anatomy 0.000 claims abstract description 18
- 238000000605 extraction Methods 0.000 claims abstract description 11
- 239000008055 phosphate buffer solution Substances 0.000 claims description 37
- 230000001079 digestive effect Effects 0.000 claims description 29
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 29
- 102000004142 Trypsin Human genes 0.000 claims description 28
- 108090000631 Trypsin Proteins 0.000 claims description 28
- 239000012588 trypsin Substances 0.000 claims description 28
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 21
- 238000005119 centrifugation Methods 0.000 claims description 14
- 230000029087 digestion Effects 0.000 claims description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 13
- 230000008014 freezing Effects 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 9
- 210000001557 animal structure Anatomy 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 230000003321 amplification Effects 0.000 claims description 7
- 210000004204 blood vessel Anatomy 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- 238000010257 thawing Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 230000001413 cellular effect Effects 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 2
- 239000012894 fetal calf serum Substances 0.000 claims 1
- 239000009384 kangtai Substances 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 230000004071 biological effect Effects 0.000 abstract description 6
- 102000004127 Cytokines Human genes 0.000 abstract description 5
- 108090000695 Cytokines Proteins 0.000 abstract description 5
- 210000000056 organ Anatomy 0.000 abstract description 3
- 210000003850 cellular structure Anatomy 0.000 abstract 1
- 239000012091 fetal bovine serum Substances 0.000 description 12
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 6
- 239000010931 gold Substances 0.000 description 6
- 229910052737 gold Inorganic materials 0.000 description 6
- 239000000126 substance Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
Classifications
-
- 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
- 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
-
- 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
Abstract
The invention discloses a method for extracting cell sap from cells cultured in vitro, which is characterized in that cells in organ tissues are cultured in vitro, so that the cell components are uniform, other components do not influence the extraction of cytoplasm, and the biological activity of various cytokines and active proteins in the cytoplasm can be kept without loss when the cytoplasm is extracted.
Description
Technical Field
The invention relates to a preparation process of cell sap, in particular to a method for extracting cell sap from cells cultured in vitro.
background
Animal organs have various problems when used for production, the components in tissue blocks are complex, for example, insoluble substances such as fat, collagen and the like exist, the filtration and sterilization are difficult, simultaneously, the intercellular substance is more, various cells exist, the homogenization still remains turbid even after being centrifuged for a plurality of times, the further purification and extraction are difficult, the fat content is high, the impurities are more, and the separation is difficult.
in the prior art, generally, in order to simplify the production process, protein hydrolysate is mostly obtained by adopting an enzymolysis mode, protein is degraded, the original biological activity is lost, and the protein can only be used as a nutrient substance.
disclosure of Invention
The present invention is directed to a method for extracting a cell sap from cells cultured in vitro to solve the problems of the background art described above.
in order to achieve the purpose, the invention provides the following technical scheme:
A method for extracting cell sap from cells cultured in vitro, comprising the steps of:
1) Cutting the residual tissue of the fresh animal organ without the blood vessel and the envelope tissue by using surgical scissors in a sterile environment;
2) Placing the cut tissue blocks into a centrifuge tube, digesting with twice volume of Phosphate Buffer Solution (PBS) pre-warmed at 37 ℃ and 0.25% trypsin for 2min, sucking out the tissue digestive juice when the liquid is turbid, transferring into another centrifuge tube, and adding 2% serum and trypsin; adding trypsin digestive juice into the undigested tissue blocks for digestion until the tissue blocks are completely digested into tissue digestive juice;
3) Filtering the tissue digestive juice by a cell sieve to remove large cell masses, and then carrying out primary centrifugal treatment;
4) the collected cells are resuspended and counted by RPM1640+10% gold source Fetal Bovine Serum (FBS), and transferred into a cell bottle for culture;
5) Selecting cells which can be subcultured for amplification culture, digesting the cells with trypsin after reaching a certain number, collecting the cells, and starting cytoplasm extraction operation;
6) after cell digestion, resuspending the cells with Phosphate Buffer Solution (PBS), counting the cells, and washing the cells once or twice;
7) taking prepared Phosphate Buffer Solution (PBS) +20% glycerol, carrying out heavy suspension on cells according to the concentration of 1 ~ 5 × 107cells/ml, placing the cells into a refrigerator for quick freezing, taking out the cells after 1 hour for thawing, repeating the process for three times, carrying out secondary treatment, and taking supernatant to obtain cell essence, namely the cell extracting solution.
As a further scheme of the invention: in the step 3), the rotation speed of the primary centrifugal treatment is 1000-2000 r/min.
As a still further scheme of the invention: in the step 3), the time of the primary centrifugal treatment is 2-8 min.
As a still further scheme of the invention: in the step 7), the rotation speed of the secondary centrifugal treatment is 3000-5000 r/min.
As a still further scheme of the invention: in the step 7), the time of the secondary centrifugal treatment is 2-8 min.
In a still further embodiment of the present invention, in step 7), the freezing temperature is-75 ℃ ~ -85 ℃.
as a still further scheme of the invention: in the step 7), the secondary treatment mode is centrifugal treatment.
compared with the prior art, the invention has the beneficial effects that:
The method for extracting cell sap from cells cultured in vitro provided by the invention can maintain the biological activity of various cytokines and active proteins in cytoplasm without loss when extracting cytoplasm by culturing cells in vitro in organ tissues, so that the components of the cells are uniform, other components do not influence the extraction of the cytoplasm, and the biological activity of the cytokines and the active proteins in the cytoplasm is not lost.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific embodiments.
example 1
a method for extracting cell sap from cells cultured in vitro, comprising the steps of:
1) cutting the residual tissue of the fresh animal organ without the blood vessel and the envelope tissue by using surgical scissors in a sterile environment;
2) placing the cut tissue blocks into a centrifuge tube, digesting with twice volume of Phosphate Buffer Solution (PBS) pre-warmed at 37 ℃ and 0.25% trypsin for 2min, sucking out the tissue digestive juice when the liquid is turbid, transferring into another centrifuge tube, and adding 2% serum and trypsin; adding trypsin digestive juice into the undigested tissue blocks for digestion until the tissue blocks are completely digested into tissue digestive juice;
3) filtering the tissue digestive juice by a cell sieve to remove large cell masses, and then carrying out primary centrifugation treatment at 1500rpm/5 min;
4) the collected cells are resuspended and counted by RPM1640+10% gold source Fetal Bovine Serum (FBS), and transferred into a cell bottle for culture;
5) selecting cells which can be subcultured for amplification culture, digesting the cells with trypsin after reaching a certain number, collecting the cells, and starting cytoplasm extraction operation;
6) After cell digestion, resuspending the cells by using a phosphate buffer solution, counting the cells, and washing the cells for one to two times;
7) taking prepared phosphate buffer solution and 20% glycerol, carrying out heavy suspension on cells according to the concentration of 1 ~ 5 × 107cells/ml, placing the cells into a refrigerator with the temperature of-80 ℃ for quick freezing, taking out the cells after 1 hour for thawing, repeating the steps for three times, carrying out secondary centrifugation treatment at 4000rpm/5min, and taking supernatant to obtain cell essence, namely the cell extracting solution.
Example 2
A method for extracting cell sap from cells cultured in vitro, comprising the steps of:
1) cutting the residual tissue of the fresh animal organ without the blood vessel and the envelope tissue by using surgical scissors in a sterile environment;
2) Placing the cut tissue blocks into a centrifuge tube, digesting with twice volume of Phosphate Buffer Solution (PBS) pre-warmed at 37 ℃ and 0.25% trypsin for 2min, sucking out the tissue digestive juice when the liquid is turbid, transferring into another centrifuge tube, and adding 2% serum and trypsin; adding trypsin digestive juice into the undigested tissue blocks for digestion until the tissue blocks are completely digested into tissue digestive juice;
3) filtering the tissue digestive juice by a cell sieve to remove large cell masses, and then carrying out primary centrifugation treatment at 1000rpm/8 min;
4) The collected cells are resuspended and counted by RPM1640+10% gold source Fetal Bovine Serum (FBS), and transferred into a cell bottle for culture;
5) selecting cells which can be subcultured for amplification culture, digesting the cells with trypsin after reaching a certain number, collecting the cells, and starting cytoplasm extraction operation;
6) After cell digestion, resuspending the cells with Phosphate Buffer Solution (PBS), counting the cells, and washing the cells once or twice;
7) taking prepared Phosphate Buffer Solution (PBS) +20% glycerol, carrying out heavy suspension on cells according to the concentration of 1 ~ 5 × 107cells/ml, placing the cells into a refrigerator with the temperature of-75 ℃ for quick freezing, taking out the cells after 1h for thawing, repeating the process for three times, carrying out secondary centrifugation treatment at 3000rpm/8min, and taking supernatant to obtain cell essence, namely cell extracting solution.
Example 3
A method for extracting cell sap from cells cultured in vitro, comprising the steps of:
1) cutting the residual tissue of the fresh animal organ without the blood vessel and the envelope tissue by using surgical scissors in a sterile environment;
2) placing the cut tissue blocks into a centrifuge tube, digesting with twice volume of Phosphate Buffer Solution (PBS) pre-warmed at 37 ℃ and 0.25% trypsin for 2min, sucking out the tissue digestive juice when the liquid is turbid, transferring into another centrifuge tube, and adding 2% serum and trypsin; adding trypsin digestive juice into the undigested tissue blocks for digestion until the tissue blocks are completely digested into tissue digestive juice;
3) Filtering the tissue digestive juice by a cell sieve to remove large cell masses, and then carrying out primary centrifugation treatment at 2000rpm/2 min;
4) the collected cells are resuspended and counted by RPM1640+10% gold source Fetal Bovine Serum (FBS), and transferred into a cell bottle for culture;
5) selecting cells which can be subcultured for amplification culture, digesting the cells with trypsin after reaching a certain number, collecting the cells, and starting cytoplasm extraction operation;
6) after cell digestion, resuspending the cells with Phosphate Buffer Solution (PBS), counting the cells, and washing the cells once or twice;
7) Taking prepared Phosphate Buffer Solution (PBS) +20% glycerol, carrying out heavy suspension on cells according to the concentration of 1 ~ 5 × 107cells/ml, placing the cells into a refrigerator with the temperature of-85 ℃ for quick freezing, taking out the cells after 1h for thawing, repeating the process for three times, carrying out secondary centrifugation treatment at 5000rpm/2min, and taking supernatant to obtain cell essence, namely cell extracting solution.
example 4
a method for extracting cell sap from cells cultured in vitro, comprising the steps of:
1) Cutting the residual tissue of the fresh animal organ without the blood vessel and the envelope tissue by using surgical scissors in a sterile environment;
2) placing the cut tissue blocks into a centrifuge tube, digesting with twice volume of Phosphate Buffer Solution (PBS) pre-warmed at 37 ℃ and 0.25% trypsin for 2min, sucking out the tissue digestive juice when the liquid is turbid, transferring into another centrifuge tube, and adding 2% serum and trypsin; adding trypsin digestive juice into the undigested tissue blocks for digestion until the tissue blocks are completely digested into tissue digestive juice;
3) Filtering the tissue digestive juice by a cell sieve to remove large cell masses, and then carrying out primary centrifugation treatment at 2000rpm/2 min;
4) The collected cells are resuspended and counted by RPM1640+10% gold source Fetal Bovine Serum (FBS), and transferred into a cell bottle for culture;
5) selecting cells which can be subcultured for amplification culture, digesting the cells with trypsin after reaching a certain number, collecting the cells, and starting cytoplasm extraction operation;
6) After cell digestion, resuspending the cells with Phosphate Buffer Solution (PBS), counting the cells, and washing the cells once or twice;
7) taking prepared Phosphate Buffer Solution (PBS) +20% glycerol, carrying out heavy suspension on cells according to the concentration of 1 ~ 5 × 107cells/ml, placing the cells into a refrigerator with the temperature of 80 ℃ below zero for quick freezing, taking out the cells after 1 hour for thawing, repeating the process for three times, carrying out secondary centrifugation treatment at the speed of 4000rpm/5min, and taking supernatant to obtain cell essence, namely cell extracting solution.
Example 5
a method for extracting cell sap from cells cultured in vitro, comprising the steps of:
1) cutting the residual tissue of the fresh animal organ without the blood vessel and the envelope tissue by using surgical scissors in a sterile environment;
2) placing the cut tissue blocks into a centrifuge tube, digesting with twice volume of Phosphate Buffer Solution (PBS) pre-warmed at 37 ℃ and 0.25% trypsin for 2min, sucking out the tissue digestive juice when the liquid is turbid, transferring into another centrifuge tube, and adding 2% serum and trypsin; adding trypsin digestive juice into the undigested tissue blocks for digestion until the tissue blocks are completely digested into tissue digestive juice;
3) filtering the tissue digestive juice by a cell sieve to remove large cell masses, and then carrying out primary centrifugation treatment at 1500rpm/5 min;
4) the collected cells are resuspended and counted by RPM1640+10% gold source Fetal Bovine Serum (FBS), and transferred into a cell bottle for culture;
5) Selecting cells which can be subcultured for amplification culture, digesting the cells with trypsin after reaching a certain number, collecting the cells, and starting cytoplasm extraction operation;
6) After cell digestion, resuspending the cells with Phosphate Buffer Solution (PBS), counting the cells, and washing the cells once or twice;
7) taking prepared Phosphate Buffer Solution (PBS) +20% glycerol, carrying out heavy suspension on cells according to the concentration of 1 ~ 5 × 107cells/ml, placing the cells into a refrigerator with the temperature of-75 ℃ for quick freezing, taking out the cells after 1h for thawing, repeating the process for three times, carrying out secondary centrifugation treatment at 5000rpm/2min, and taking supernatant to obtain cell essence, namely cell extracting solution.
The method for extracting cell sap from cells cultured in vitro provided by the invention can maintain the biological activity of various cytokines and active proteins in cytoplasm without loss when extracting cytoplasm by culturing cells in vitro in organ tissues, so that the components of the cells are uniform, other components do not influence the extraction of the cytoplasm, and the biological activity of the cytokines and the active proteins in the cytoplasm is not lost.
while the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (7)
1. A method for extracting cell sap from cells cultured in vitro, comprising the steps of:
1) cutting the residual tissue of the fresh animal organ without the blood vessel and the envelope tissue by using surgical scissors in a sterile environment;
2) placing the cut tissue blocks into a centrifuge tube, digesting for 2min by using two volumes of phosphate buffer solution pre-warmed at 37 ℃ and 0.25% trypsin, sucking out the tissue digestive juice when the liquid is turbid, transferring the tissue digestive juice into another centrifuge tube, and adding 2% serum and trypsin; adding trypsin digestive juice into the undigested tissue blocks for digestion until the tissue blocks are completely digested into tissue digestive juice;
3) Filtering the tissue digestive juice by a cell sieve to remove large cell masses, and then carrying out primary centrifugal treatment;
4) the collected cells are resuspended and counted by RPM1640+10% golden source kangtai fetal calf serum, and then transferred into a cell bottle for culture;
5) selecting cells which can be subcultured for amplification culture, digesting the cells with trypsin after reaching a certain number, collecting the cells, and starting cytoplasm extraction operation;
6) after cell digestion, resuspending the cells by using a phosphate buffer solution, counting the cells, and washing the cells for one to two times;
7) taking prepared phosphate buffer solution and 20% glycerol, carrying out heavy suspension on cells according to the concentration of 1 ~ 5 × 107cells/ml, placing the cells into a refrigerator for quick freezing, taking out the cells after 1 hour for thawing, repeating the process for three times, carrying out secondary treatment, and taking supernatant to obtain cell essence, namely cell extracting solution.
2. the method for extracting cellular fluid from cells cultured in vitro as claimed in claim 1, wherein the rotation speed of the primary centrifugation treatment in step 3) is 1000-2000 r/min.
3. The method for extracting cell sap from cells cultured in vitro as claimed in claim 2, wherein the time for the one centrifugation treatment in step 3) is 2-8 min.
4. The method for extracting cellular fluid from cells cultured in vitro as claimed in claim 1, wherein the rotation speed of the secondary centrifugation treatment in step 7) is 3000-.
5. The method for extracting cell sap from cells cultured in vitro as claimed in claim 4, wherein the time of the secondary centrifugation treatment in step 7) is 2-8 min.
6. The method for extracting cell sap from cells cultured in vitro as set forth in claim 5, wherein the freezing temperature in step 7) is-75 ℃ ~ -85 ℃.
7. the method for extracting cell sap from cells cultured in vitro according to claim 6, wherein the secondary treatment in step 7) is a centrifugal treatment.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910904342.9A CN110577927A (en) | 2019-09-24 | 2019-09-24 | method for extracting cell sap from cells cultured in vitro |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910904342.9A CN110577927A (en) | 2019-09-24 | 2019-09-24 | method for extracting cell sap from cells cultured in vitro |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1806040A (en) * | 2003-06-10 | 2006-07-19 | 株式会社岛津制作所 | Extract from cultured mammalian cell, process for preparation thereof and method of cell-free protein synthesis using the extract |
| CN103393585A (en) * | 2013-08-09 | 2013-11-20 | 艾普瑞斯（北京）生物科技有限公司 | Fibroblast liquid for beauty treatment and preparation method thereof |
| CN109355248A (en) * | 2018-10-30 | 2019-02-19 | 中国科学院苏州生物医学工程技术研究所 | Livestock organisations cell prepares the method for animal cell culture liquid and its application of animal cell culture liquid obtained |
-
2019
- 2019-09-24 CN CN201910904342.9A patent/CN110577927A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1806040A (en) * | 2003-06-10 | 2006-07-19 | 株式会社岛津制作所 | Extract from cultured mammalian cell, process for preparation thereof and method of cell-free protein synthesis using the extract |
| JPWO2004111203A1 (en) * | 2003-06-10 | 2006-07-20 | 株式会社島津製作所 | Mammalian cultured cell extract, preparation method thereof, and cell-free protein synthesis method using the extract |
| CN103393585A (en) * | 2013-08-09 | 2013-11-20 | 艾普瑞斯（北京）生物科技有限公司 | Fibroblast liquid for beauty treatment and preparation method thereof |
| CN109355248A (en) * | 2018-10-30 | 2019-02-19 | 中国科学院苏州生物医学工程技术研究所 | Livestock organisations cell prepares the method for animal cell culture liquid and its application of animal cell culture liquid obtained |
Non-Patent Citations (1)
| Title |
|---|
| 刘利兵等: "《实验基础医学 第3版》", 31 July 2009, 第四军医大学出版社 * |
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Application publication date: 20191217 |