CN118064371A - Method for rapidly preparing monolayer caco-2 cell infiltration model - Google Patents
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- 102000012422 Collagen Type I Human genes 0.000 claims abstract description 12
- 108010022452 Collagen Type I Proteins 0.000 claims abstract description 12
- 239000011248 coating agent Substances 0.000 claims abstract description 12
- 238000000576 coating method Methods 0.000 claims abstract description 12
- 238000011081 inoculation Methods 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 239000012154 double-distilled water Substances 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 12
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 12
- 239000012091 fetal bovine serum Substances 0.000 claims description 12
- 238000002474 experimental method Methods 0.000 claims description 11
- 239000000463 material Substances 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 5
- 229930182816 L-glutamine Natural products 0.000 claims description 5
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 5
- 235000020776 essential amino acid Nutrition 0.000 claims description 5
- 239000003797 essential amino acid Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
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- 244000309466 calf Species 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- DQKXQSGTHWVTAD-UHFFFAOYSA-N pramocaine Chemical compound C1=CC(OCCCC)=CC=C1OCCCN1CCOCC1 DQKXQSGTHWVTAD-UHFFFAOYSA-N 0.000 claims description 2
- 229960001896 pramocaine Drugs 0.000 claims description 2
- 230000009194 climbing Effects 0.000 abstract description 5
- 239000011521 glass Substances 0.000 abstract description 5
- 102000004142 Trypsin Human genes 0.000 abstract description 2
- 108090000631 Trypsin Proteins 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
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- 229940079593 drug Drugs 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 102000000591 Tight Junction Proteins Human genes 0.000 description 6
- 108010002321 Tight Junction Proteins Proteins 0.000 description 6
- 210000001578 tight junction Anatomy 0.000 description 6
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 5
- 229930195725 Mannitol Natural products 0.000 description 5
- 239000000594 mannitol Substances 0.000 description 5
- 235000010355 mannitol Nutrition 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 2
- 229940126701 oral medication Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
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- 206010009944 Colon cancer Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
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- 239000002547 new drug Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- FASDKYOPVNHBLU-ZETCQYMHSA-N pramipexole Chemical compound C1[C@@H](NCCC)CCC2=C1SC(N)=N2 FASDKYOPVNHBLU-ZETCQYMHSA-N 0.000 description 1
- 229960003089 pramipexole Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- UOENJXXSKABLJL-UHFFFAOYSA-M sodium;8-[(2-hydroxybenzoyl)amino]octanoate Chemical compound [Na+].OC1=CC=CC=C1C(=O)NCCCCCCCC([O-])=O UOENJXXSKABLJL-UHFFFAOYSA-M 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- 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
- C12N5/0693—Tumour cells; Cancer cells
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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
- C12N5/0679—Cells of the gastro-intestinal tract
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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Abstract
The invention discloses a method for rapidly preparing a monolayer caco-2 cell infiltration model, which belongs to the technical field of cell infiltration models, wherein 0.012mg/mL of type I collagen is dissolved in 0.006M acetic acid to obtain type I collagen coating liquid, the type I collagen coating liquid is coated on a sterile glass climbing sheet overnight at 37 ℃, the glass climbing sheet is washed twice daily with sterile double-distilled water and then dried for standby, caco-2 cells which are cultivated to reach 70% of the logarithmic phase are digested by trypsin, centrifuged and the supernatant is discarded, the cell filter with 70 microns aperture is used for screening and counting the aggregated caco-2 cells after the culture medium is broken up, caco-2 cells are inoculated in the upper chamber of a cell chamber, the inoculation density is 35000 cells/cm 2, the glass climbing sheet inoculated with caco-2 cells is placed in a culture box with 37 ℃ and 5% CO 2 for culture, and the culture medium is replaced after inoculation.
Description
Technical Field
The invention relates to a cell permeation model, in particular to a method for rapidly preparing a monolayer caco-2 cell permeation model, and belongs to the technical field of cell permeation models.
Background
The adjuvant technology is an innovative path of improved new drugs of oral macromolecular drugs (such as polypeptides and monoclonal antibodies) with higher industrial maturity.
Currently existing adjuvant products, such as TPE, core of SNAC technology all employ one or more lipid-soluble molecules to help facilitate drug penetration through the monolayer intestinal/gastric epithelial cells.
The specific mechanism of action of such accessory molecules is not currently known, and is generally the perturbation of the phosphate bilayer to assist in drug entry across the membrane into the cell to cross a monolayer of cells, or to open up intercellular tight junctions to assist in drug entry through the cell bypass.
The molecular weight, water/fat solubility and action mechanism properties of different macromolecular drugs are greatly different, and the auxiliary materials are selected and differentially combined according to the drug delivered by shrinkage.
At present, the drug enterprises generally use the existing auxiliary material technology to develop macromolecular oral improved novel drugs, the achieved effect is limited, the success probability is low, and the discovery of novel auxiliary material molecules is very slow.
In vitro experiments are efficient and rapid methods for discovering new potential accessory molecules (here compared to animal experiments), the in vitro model currently used is the monolayer caco-2 cell model grown on top of permeable cell compartments.
Caco-2 cells are a human colon cancer cell line capable of forming a cell monolayer on a porous transparent culture membrane, mimicking the characteristics and functions of the human intestinal epithelial cell layer.
The Caco-2 cell model can provide important information about the absorption characteristics of compounds under simulated intestinal conditions, which is critical for drug development and bioavailability assessment of oral drugs.
The culture and differentiation of Caco-2 cells requires a long time, usually 14-28 days, which greatly reduces the efficiency and flexibility of the experiment and affects the progress and quality of drug development.
Meanwhile, more culture medium, serum, pancreatin and culture dish consumable materials, and more incubator, centrifuge and microscope equipment are needed, so that the economic burden and resource consumption of the experiment are increased.
The long period culture increases the risk of contamination, aging and mutation of the cells, and the factors affect the morphology and physiological properties of the cells, so that inconsistent and unreliable experimental results are caused, and the stability and repeatability of the experiment are reduced.
The existing rapid-forming monolayer caco-2 cell model needs a specific growth environment and specific experimental consumable materials, and meanwhile, a supplement which is not commonly used for cell culture needs to be added, so that the cost is extremely high, the success rate is low, the repeatability is poor, and a method for rapidly preparing the monolayer caco-2 cell permeation model is designed for solving the problems.
Disclosure of Invention
The main purpose of the invention is to provide a method for rapidly preparing a monolayer caco-2 cell infiltration model.
The aim of the invention can be achieved by adopting the following technical scheme:
a method for rapidly preparing a monolayer caco-2 cell permeation model, comprising the steps of:
step one: dissolving 0.01-0.1mg/mL type I collagen in 0.006M acetic acid to obtain type I collagen coating liquid;
Step two: coating the type I collagen coating liquid obtained in the step one into an upper chamber of a cell chamber model at 37 ℃ overnight;
Step three: the next day is dried for standby after the sterile double-distilled water is used for cleaning the upper chamber;
Step four: caco-2 cells cultured to a confluence of greater than 50% in the logarithmic growth phase were trypsinized, centrifuged and the supernatant discarded;
Step five: the cell mass obtained after centrifugation is scattered by a culture medium, and caco-2 cells which are not scattered are selected and counted by a cell filter with the aperture of 70 microns;
Step six: inoculating caco-2 cells in the upper chamber of the cell chamber at a density of 35000 cells/cm 2;
Step seven: placing the cell chamber inoculated with caco-2 cells into an incubator with 5% CO 2 at 37 ℃ for culture;
Step eight: the culture medium is replaced in the 2 nd day after inoculation, the concentration of the fetal bovine serum is reduced to 15%, the culture medium is replaced in the 3 rd day, the concentration of the fetal bovine serum is reduced to 10% after the other components are the same, then the culture medium is replaced every day, and the permeation experiment can be carried out in the 7 th day after inoculation with the components in the 3 rd day.
Preferably, the culture medium adopts pramoxine PM150410;
The components are Earle's salt, non-ESSENTIAL AMINO ACID, L-glutamine 2mM, naHCO 3 2200mg/L, D-glucose 1000mg/L, phenol red indicator 10mg/L, pH7.2-7.4, +20% fetal bovine serum Gibco10091148+0.02mM butyric acid.
Preferably, in the sixth step, a Polyester is used as the membrane material.
Preferably, the medium: pranopsis PM150410;
The components are as follows: earle's salt, non-ESSENTIAL AMINO ACID, L-glutamine 2mM, naHCO 3 2200 mg/L, D-glucose 1000 mg/L, phenol red indicator 10 mg/L, pH7.2-7.4, 20% v/v foetal calf serum Gibco10091148, +0.02mM butyric acid.
The beneficial technical effects of the invention are as follows:
according to the method for rapidly preparing the monolayer caco-2 cell infiltration model, the traditional monolayer caco-2 cell infiltration model needs at least 21d of culture, so that time and effort are consumed, the culture time can be shortened to 7d, the experimental market is obviously reduced, the experimental cost is lowered, the pollution risk is lowered, the cell state can be controlled more accurately by the shorter culture time, and the difference between experimental groups is reduced.
Drawings
FIG. 1 is a schematic diagram showing the establishment of a complete monolayer caco-2 permeation model, with TEER exceeding 600 representing the formation of an intercellular tight junction structure (n=5), in accordance with a preferred embodiment of a method for rapidly preparing a monolayer caco-2 permeation model according to the present invention, 7 th dTEER exceeding 600 after seeding;
FIG. 2 is a schematic diagram of a label for forming a tight junction structure, in which the highlight signal is the staining of the important proteins ZO-1 and CL4 in the tight junction structure, according to a preferred embodiment of a method for rapidly preparing a monolayer caco-2 cell permeation model.
FIG. 3 shows that mannitol, a preferred embodiment of a method for rapidly preparing a monolayer caco-2 cell permeation model according to the present invention, is a molecule that is extremely difficult to pass through intestinal epithelial cells, and is commonly used in monolayer caco-2 cell model experiments to detect permeation promotion of potential permeation promoting molecules, with a permeation coefficient Papp that is extremely low, and is generally considered to pass through the interstitial passages, so mannitol is a model drug for screening open tightly-linked structural molecules, the leftmost 0.5mg mannitol, and the right two data are 0.5mg mannitol mixed solution with DDM of corresponding concentration. DDM is a molecule that opens the channel of the tight junction protein, and the PApp value of FIG. 3 when mannitol is added alone is below 4e-8, meaning that the pass rate is very low, indicating the integrity of the tight junction structure, and the PApp is large after adding DDM, meaning that the tight junction structure is opened.
Detailed Description
In order to make the technical solution of the present invention more clear and obvious to those skilled in the art, the present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
As shown in fig. 1-3, the method for rapidly preparing a monolayer caco-2 cell infiltration model according to the present embodiment includes the following steps:
Step one: dissolving 0.012mg/mL type I collagen in 0.006M acetic acid to obtain type I collagen coating liquid;
Step two: coating type I collagen coating liquid on a sterile glass climbing sheet at 37 ℃ overnight;
step three: cleaning the glass climbing sheet twice a day with sterile double distilled water, and airing for later use;
Step four: caco-2 cells in logarithmic growth phase, cultured to a confluence of 70%, were digested with trypsin, centrifuged and the supernatant discarded;
Step five: the aggregated caco-2 cells were screened out and counted with a cell filter with a 70 μm pore size after disruption with the medium;
Step six: inoculating caco-2 cells in the upper chamber of the cell chamber at a density of 35000 cells/cm 2;
Step seven: placing the cell chamber inoculated with caco-2 cells into an incubator with 5% CO 2 at 37 ℃ for culture;
Step eight: the culture medium is replaced in the 2 nd day after inoculation, the concentration of the fetal bovine serum is reduced to 15%, the culture medium is replaced in the 3 rd day, the concentration of the fetal bovine serum is reduced to 10% after the other components are the same, then the culture medium is replaced every day, and the permeation experiment can be carried out in the 7 th day after inoculation with the components in the 3 rd day.
In this example, the medium used was pramipexole PM150410;
The components are Earle's salt, non-ESSENTIAL AMINO ACID, L-glutamine 2mM, naHCO 3 2200mg/L, D-glucose 1000mg/L, phenol red indicator 10mg/L, pH7.2-7.4, +20% fetal bovine serum Gibco10091148+0.02mM butyric acid.
In this embodiment, in the sixth step, a Polyester is used as the membrane material.
The traditional mode does not involve coating in advance, the concentration of the fetal bovine serum of the culture medium is generally 10-20%, the concentration of the fetal bovine serum does not need to be changed during the culture period, butyric acid does not need to be added into the culture medium, other components are the same, and the components of the culture medium can be slightly different among different experimental groups, and the difference is for cell nutrition or antibacterial purposes.
During the 21d culture period, the medium is generally changed every other d from 3d, the medium is changed every day from 14d until 21d, the experiment can be started according to the cell state, the effect is shown in fig. 1, and the experiment effect is optimal when the TEER exceeds about 600.
The conventional scheme and the Transwell cell used in the scheme are the same products, and the scheme uses a 24-well plate cell, wherein 100 μl of culture medium is added into the upper chamber, 0.5mL of culture medium is added into the lower chamber, and the cells are completely discarded and replaced each time. Typically 12-24 wells are required for one control test.
Therefore, the method consumes 50-100 mL of culture medium in the experimental process, and the traditional method is 100-200 mL, and the cost is reduced by more than 50% because the fetal bovine serum used in the method is the most expensive component in the culture medium.
In summary, three data indicate that caco-2 model manufactured by the scheme can reach the use standard of permeation experiment in 7d time, and is a reliable time-saving and labor-saving oral drug availability detection method.
The above description is merely a further embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and any person skilled in the art will be able to apply equivalents and modifications according to the technical solution and the concept of the present invention within the scope of the present invention disclosed in the present invention.
Claims (4)
1. A method for rapidly preparing a monolayer caco-2 cell infiltration model, which is characterized by comprising the following steps: the method comprises the following steps:
step one: dissolving 0.01-0.1mg/mL type I collagen in 0.006M acetic acid to obtain type I collagen coating liquid;
Step two: coating the type I collagen coating liquid obtained in the step one into an upper chamber of a cell chamber model at 37 ℃ overnight;
Step three: the next day is dried for standby after the sterile double-distilled water is used for cleaning the upper chamber;
Step four: caco-2 cells cultured to a confluence of greater than 50% in the logarithmic growth phase were trypsinized, centrifuged and the supernatant discarded;
Step five: the cell mass obtained after centrifugation is scattered by a culture medium, and caco-2 cells which are not scattered are selected and counted by a cell filter with the aperture of 70 microns;
Step six: inoculating caco-2 cells in the upper chamber of the cell chamber at a density of 35000 cells/cm 2;
Step seven: placing the cell chamber inoculated with caco-2 cells into an incubator with 5% CO 2 at 37 ℃ for culture;
Step eight: the culture medium is replaced in the 2 nd day after inoculation, the concentration of the fetal bovine serum is reduced to 15%, the culture medium is replaced in the 3 rd day, the concentration of the fetal bovine serum is reduced to 10% after the other components are the same, then the culture medium is replaced every day, and the permeation experiment can be carried out in the 7 th day after inoculation with the components in the 3 rd day.
2. A method for rapidly preparing a monolayer caco-2 cell permeation model according to claim 1, wherein: the culture medium adopts pramoxine PM150410;
The components are Earle's salt, non-ESSENTIAL AMINO ACID, L-glutamine 2mM, naHCO 3 2200mg/L, D-glucose 1000mg/L, phenol red indicator 10mg/L, pH7.2-7.4, +20% fetal bovine serum Gibco10091148+0.02mM butyric acid.
3. A method for rapidly preparing a monolayer caco-2 cell permeation model according to claim 2, wherein: in the sixth step, a Polyester is used as the membrane material.
4. A method for rapidly preparing a monolayer caco-2 cell permeation model according to claim 3, wherein: culture medium: pranopsis PM150410;
The components are as follows: earle's salt, non-ESSENTIAL AMINO ACID, L-glutamine 2mM, naHCO 3 2200 mg/L, D-glucose 1000 mg/L, phenol red indicator 10 mg/L, pH7.2-7.4, 20% v/v foetal calf serum Gibco10091148, +0.02mM butyric acid.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6511798B1 (en) * | 1998-03-17 | 2003-01-28 | Zeneca Limited | Methods for the preparation of cell monolayers |
| US20040185560A1 (en) * | 2003-03-17 | 2004-09-23 | Marina Lowen | Accelerated culture system for intestinal epithelial cell monolayers |
| US20140212974A1 (en) * | 2011-06-24 | 2014-07-31 | Kisco Ltd. | Cell culture membrane, cell culture substrate, and method for manufacturing cell culture substrate |
| CN109321529A (en) * | 2018-10-19 | 2019-02-12 | 浙江工商大学 | A kind of construction method of external gastrointestinal model and application |
| CN116496991A (en) * | 2023-05-30 | 2023-07-28 | 深圳奥礼生物科技有限公司 | Caco-2 cell glass climbing sheet and preparation method and application thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6511798B1 (en) * | 1998-03-17 | 2003-01-28 | Zeneca Limited | Methods for the preparation of cell monolayers |
| US20040185560A1 (en) * | 2003-03-17 | 2004-09-23 | Marina Lowen | Accelerated culture system for intestinal epithelial cell monolayers |
| US20140212974A1 (en) * | 2011-06-24 | 2014-07-31 | Kisco Ltd. | Cell culture membrane, cell culture substrate, and method for manufacturing cell culture substrate |
| CN109321529A (en) * | 2018-10-19 | 2019-02-12 | 浙江工商大学 | A kind of construction method of external gastrointestinal model and application |
| CN116496991A (en) * | 2023-05-30 | 2023-07-28 | 深圳奥礼生物科技有限公司 | Caco-2 cell glass climbing sheet and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 陈锐 等: "鳕鱼皮胶原蛋白肽在Caco-2细胞单层模型中的吸收机制", 《食品科学》, vol. 39, no. 19, 12 December 2017 (2017-12-12), pages 154 - 161 * |
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