CN107083364A - A kind of structure of Caco-2/HUVEC cells co-culture system - Google Patents

Abstract

The invention discloses a method for constructing a Caco-2 and HUVEC cell co-culture system. Inoculate 25-40 generations of Caco-2 cells in the logarithmic growth phase on the upper chamber of the Transwell plate and culture them until the cells are fully differentiated; inoculate the 2-6 generations of HUVEC cells in the logarithmic growth phase on the lower chamber of the Transwell plate and culture them until the cells are 80-90 %; Insert the fully differentiated Caco-2 cell Transwell upper chamber into the cultured HUVEC cell Transwell lower chamber, fine-tune the DMEM medium, and culture at 37°C and 5% CO ₂ without contact. The Caco-2/HUVEC co-culture system constructed by the present invention can simultaneously study the absorption metabolism and physiological activity of drugs, simulate the pharmacological effects of drugs in the circulatory system after intestinal absorption and metabolism, and provide a cell model reflecting the bioavailability of drugs.

Description

Construction of a Caco-2/HUVEC cell co-culture system

1. Technical field

The invention relates to a method for constructing a co-culture system of Caco-2 and HUVEC cells, belonging to the field of biotechnology.

2. Background technology

Human colon adenocarcinoma cell lines (Caco-2 cells for short) have the same microvilli structure and tight junctions as small intestinal epithelial cells, and contain metabolic enzymes, sugars, amino acids, P450 Isozymes and various active transport carriers are classic models for studying intestinal absorption and transport mechanisms.

Human umbilical vein endothelial cells (HUVEC for short) are venous vascular endothelial cells isolated from normal human umbilical cords, which contain a large number of cytoplasmic inclusions, smooth muscle actomyosin, and ABG antigens. Function, can produce and secrete many biologically active substances, play an important role in maintaining vasomotor, anticoagulant, etc., and is a cell model widely used in the physiological and pharmacological research of the circulatory system and endothelial tissue.

In a broad sense, bioavailability includes digestion, absorption, metabolism, distribution, excretion, and biological activity (bioactivity), while in a narrow sense, bioavailability only refers to the relative amount and rate of nutrients absorbed by the body into the circulation. is the bioabsorption rate (bioaccessibility). Usually people use Caco-2 intestinal cells to study the bioavailability of drugs, but this only reflects the intestinal absorption and transport mechanism, ignoring the real physiological effects of drugs after metabolism in the body. The first priority of bioavailability is absorption, but the most critical thing is to ensure that it exerts its biological activity in the body. Therefore, a cell model that can reflect both absorption and physiological activity is needed to study bioavailability.

Cell co-culture technology is to culture two or more kinds of cells together in the same environment. Due to the advantage of better reflecting the in vivo environment, this method is widely used in modern cell research. The cell co-culture system is mainly established in two ways: ① contact co-culture system, that is, two kinds of cells are inoculated on the same carrier at the same time or separately, and different types of cells are in direct contact; ② non-contact co-culture system, that is, two kinds of cells They are respectively inoculated on different carriers, and then these two carriers are placed in the same culture environment, so that different types of cells can share the same culture system without direct contact. The cell co-culture system simulates the interaction between cells, including the secretion of growth factors, which can be used to induce cell differentiation, maintain cell function and vitality, regulate cell proliferation, promote early embryonic development and increase the production of metabolites. The HUVEC/fibroblast co-culture system was used to study the effect of anthocyanins on angiogenesis, and the HUVEC/Jurkat cell co-culture system was used to study the adhesion properties of grape seed proanthocyanidins to T cells and endothelial cells. There is no report on the in vitro cell model that combines the Caco-2 cell model for the study of absorption and the HUVEC cell model for the study of pharmacological functions to fully reflect the bioavailability of drugs.

3. Contents of the invention

Technical Problems The present invention mainly relates to a method for constructing a co-culture system of Caco-2 and HUVEC cells, which can provide an in vitro cell model reflecting the bioavailability of drugs.

Technical scheme The present invention comprises the following steps:

1) Use DMEM medium (containing 10% fetal bovine serum, 1% non-essential amino acid, 1% glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, pH7. 4) Cultivate at 37°C and 5% _CO2 environment, inoculate Caco-2 cells in the logarithmic growth phase on the upper layer of the Transwell plate, culture until the cells are fully differentiated, and use a potentiometer to monitor the resistance value of the Caco-2 cell layer TEER greater than 550Ω· cm ² .

2) Put the HUVEC cells of the 2nd to 6th passages in DMEM medium (containing 10% fetal bovine serum, 100 μg/mL vascular endothelial growth factor, 2% penicillin-streptomycin double antibody solution, pH 7.4) at 37°C, Cultivate in a 5% CO ₂ environment, inoculate HUVEC cells in the logarithmic growth phase on the lower layer of the Transwell plate until the cells are 80-90% full.

3) Insert the fully differentiated Caco-2 cell Transwell upper chamber into the cultured HUVEC cell Transwell lower chamber, and fine-tune the DMEM medium (containing 10% fetal bovine serum, 1% non-essential amino acid, 1% glutamine, 100 μg/mL vascular endothelial cell growth factor, 2% penicillin-streptomycin double antibody solution, pH 7.4), non-contact co-cultivation at 37°C, 5% CO ₂ environment.

Beneficial effect

The present invention aims to construct a Caco-2/HUVEC cell co-culture system for providing an in vitro cell model reflecting the bioavailability of drugs.

The traditional Caco-2 cell model is suitable for the study of intestinal absorption, transport mechanism and metabolism, which only reflects intestinal absorption and metabolism, without considering the activity in vivo. The traditional HUVEC cell model is suitable for physiological and pharmacological studies of the circulatory system and endothelial tissue, and only reflects the mechanism of activity without considering digestion, absorption and metabolism in vivo. The present invention breaks the inherent idea of single research on absorption metabolism or physiological activity, combines the Caco-2 model for absorption research and HUVEC cells for pharmacological function research, and adopts a non-contact co-cultivation method to build a Caco-2/HUVEC co-cultivation system. By fine-tuning the DMEM medium used for co-cultivation, it is suitable for the growth of both Caco-2 and HUVEC. The co-culture system includes the absorption transport carrier and metabolic enzyme system of two kinds of cells, and the factors and enzyme systems related to physiological activity. In addition, the two kinds of cells can exchange extracellular secreted substances through Transwell transmembrane. Caco-2 simulates the intestinal layer, and HUVEC simulates the vascular layer of the circulatory system, which is suitable for studying the absorption and transport mechanism, metabolism and physiological activity of substances in the intestinal tract and circulatory system. This system can study the absorption, metabolism and physiological activity of drugs at the same time, better simulate the pharmacological effects of drugs in the circulatory system after intestinal absorption and metabolism, fully reflect the bioavailability, and provide a more realistic reflection of drug bioavailability in vitro cell model.

Description of drawings

Figure 1 Schematic diagram of Transwell non-contact co-culture

4. Specific implementation

The present invention will be further described below in conjunction with specific embodiment:

Example 1

Use DMEM medium (containing 10% fetal bovine serum, 1% non-essential amino acid, 1% glutamine, 100U/mL penicillin, 100μg/mL streptomycin, pH7.4) for Caco-2 cells of passage 25-40 Culture at 37°C and 5% CO ₂ environment, change the medium every other day, digest the cells with 0.25% trypsin/0.53mmol/L EDTA solution every 3 days, and passage at a ratio of 1:3. Cells in the logarithmic growth phase were inoculated on the upper layer of 24-well Transwell plate at 1×10 ⁵ /mL, and cultured for 21 days. The resistance value of the Caco-2 cell layer measured by a potentiometer TEER was greater than 550Ω·cm ² , and the cells were fully differentiated. HUVEC cells of passages 2 to 6 were incubated with DMEM medium (containing 10% fetal bovine serum, 100 μg/mL vascular endothelial growth factor, 2% penicillin-streptomycin double antibody solution, pH 7.4) at 37°C, 5% Culture in CO ₂ environment, change the medium every other day, digest the cells with 0.25% trypsin/0.1% EDTA solution, and pass passage according to the ratio of 1:3. Cells in the logarithmic growth phase were inoculated on the lower layer of a 24-well Transwell plate coated with gelatin at 1×10 ⁵ /mL until the cells were confluent until 80-90% of the cells were confluent. The fully differentiated Caco-2 cell Transwell upper chamber was inserted into the cultured HUVEC cell Transwell lower chamber, and the DMEM medium (containing 10% fetal bovine serum, 1% non-essential amino acid, 1% glutamine, 100 μg/mL Vascular endothelial cell growth factor, 2% penicillin-streptomycin double antibody solution, pH 7.4), in 37 ℃, 5% CO ₂ environment non-contact co-cultivation, the cell survival rate detected by MTT method is greater than 95%, Caco-2/ The HUVEC cell co-culture system was constructed to study the bioavailability of blueberry anthocyanins in vascular endothelial cells after intestinal absorption.

Claims (1)

1. The construction of a Caco-2/HUVEC cell co-culture system, is characterized in that: the Caco-2 cell of 25～40 generation is cultivated with DMEM based on 37 ℃, 5% _CO Environment culture, the logarithmic growth phase Caco -2 cells were inoculated into the upper chamber of the Transwell plate and cultured until the cells were fully differentiated; HUVEC cells of passage 2 to 6 were cultured in DMEM at 37°C and 5% CO ₂ , and the HUVEC cells in the logarithmic growth phase were inoculated into the lower layer of the Transwell plate Small chamber until the cells are 80-90% full; insert the fully differentiated Caco-2 cell Transwell upper chamber into the cultured HUVEC cell Transwell lower chamber, and fine-tune the DMEM medium (containing 10% fetal bovine serum, 1% Essential amino acids, 1% glutamine, 100 μg/mL vascular endothelial cell growth factor, 2% penicillin-streptomycin double antibody solution, pH7.4), using non-contact co-cultivation, at 37 ° C, 5% CO ₂ environment Co-cultivate.