Before the origin of organoid system, most of the studies of gastric diseases were modeled using cell lines, animal models or explants, and these model systems have their own limitations11.
The gastric epithelial cell line is a commonly used model system for studying gastric diseases, which was originally derived from gastric tumors. Their advantages are immortalized, readily available, easy to culture and can be applied to a variety of experiments. However, its molecular characteristics have been modified, including tumor suppressor genes, oncogenes, and cell cycle regulatory genes, and thus there is a certain gap with epithelial cells in physiology. Cell lines accumulate mutations during in vitro culture for decades, and some cell lines are infected with viruses, such as AGS cell lines, which are usually infected with type Ⅴparainfluenza virus, affecting pathways involved in immunity, proliferation and tumorigenesis, and interferon responses12.
Conventional tumor cell culture is two-dimensional, but studies have shown that whether cell lines grew in 2D or 3D can affect drug efficiency13.This suggests that 3D is an important feature of tissue response. However, 3D cell spheres cultured from a single cell line lack a combination of different cell types typical of tissues and organs, while gastric organoids may include parietal cells, chief cells, cervical mucus cells, stem cells, and endocrine cells,etc.
Primary cell culture models are often a good alternative to tumor cell lines because they have not been transformed. Primary cultured gastric parietal cells have polarized structural features and polarized exocytosis and endocytosis, and were easy to access to large amounts. However, these cells are unable to self-renew and cannot be cultured and expanded for a long time, and all of the original cells need to be freshly isolated for each experiment.
The animal model system is more holistic and dynamic than the cell line, but some human stomach diseases cannot be responded to by animal models. For example, after mice were infected with Helicobacter pylori, they often only have mild gastritis and would not progress to gastric ulcer or stomach cancer. Mongolian gerbils can progress to gastric cancer after infection with Helicobacter pylori, but its distant line limits its researches and applications14.
The gastric organoids include parietal cells, chief cells, cervical mucus cells, stem cells, and endocrine cells which are also contained in the fundic glands. The parietal cells secrete hydrochloric acid and internal factors, the chief cells secrete pepsinogen, the enterochromaffin-like (ECL) cells in endocrine cells secrete histamine, G cells secrete gastrin, D cells secrete gastrin, and cervical mucus cells secrete mucus. The organoids can last for more than a year without losing the ability to expand or differentiate15.
In addition, the three-dimensionally cultured gastric organoids have complex 3D structures with structures and functions similar to those of internal organs16,17 .The gastric organoids are mostly cystic, and some gradually grow a bud-like structure around the cyst. There is no difference in the culture time, the expansion ratio, the expression of the gene markers of each constituent cell, or the cell type in the gastric organoids derived from a single gastric stem cell and from the gastric gland15.
Organoids can also be cultured in two dimensions, and electron microscopic observation reveals that the apical membrane of the stomach of the two-dimensional culture is oriented toward the medium4. Since the gastric epithelium is a special polar epithelial cell, we found that the apical membrane of the gastric organoid faces the inner cavity by staining the apical membrane marker ezrin, and the polarity distribution is in consistency with the gastric mucosal epithelial cells.
The gastric organoids both replicate the complexity of the disease phenotype in vivo while retaining the accessibility of in vitro. Gastric organoids that can be cultured in vitro for a long time have enabled researchers to overcome many obstacles and conduct basal and translational studies18, 19. Because it is closer to the environment in vivo, it is more faithful to physiology and pathology. Although organoids have their own limitations, bioengineering methods such as time-dependent and spatial-temporal materials can control the growth of organoids toward the desired structural form and self-organization, and other studies are already available. Vascular networks are added to organoid cultures to increase nutrients in a nearly physiological way20,21.Co-culture methods for organoids can also mimic multiple organ lesions22.
In this study, after different concentrations of EtOH were added to murine gastric organoids for 1 hour, and after trypan blue staining, murine gastric organoids showed different degrees of positive trypan blue staining, indicating the impairment caused by EtOH. The extent of the damage of the gastric organoids depended on the concentration. Ethanol increases the permeability of parietal cell membranes23 and triggers an increase in Ca2+. Ethanol and endogenous aldehydes impaired chromosomal and variant stem cells24.
Clinically, a variety of diseases and gastric acid secretion abnormalities are related, including gastroesophageal reflux disease, chronic gastritis, gastric ulcer, benign and malignant tumors of the gastrointestinal tract, anemia and so on.
In vivo methods for detecting gastric acid secretion in animal models often require an invasive procedure after anesthesia is fixed in the animal, and the secreted gastric acid is collected after catheterization into the stomach and is subsequently measured25. The in vitro gastric acid detection method is more commonly used in the 14C aminopyrine experiment which was first proposed by Berglindh and Obrink in 197626. Due to the investigator's radioisotope experimental qualifications, the 14C aminopyrine test was temporarily unable to detect gastric acid.
Despite this, we observed that the number and location of most H+, K+-ATPase were not significantly changed in experiments of western blotting and immunofluorescence, but in repeated experiments, we observed H+, K+-ATPase is activated in the control group and diffused in the alcohol group (data not shown), suggesting that alcohol inhibition of gastric acid secretion may be related to the activation of H+, K+-ATPase, possibly due to its short spontaneous activation time. A statistically significant number of activated H+, K+-ATPases are to be further explored in future experiments using H+, K+-ATPase lentivirus-infected organoids.
From the results, we found that ethanol mediated the hydrolysis of ezrin, which was indicated by a specific hydrolysis zone at 55kD.After being impaired by 6% EtOH with different time intervals, a similar trend also emerged. The wrapping of Matrigel may have a certain physical protection effect on gastric organoids.
In this study, two methods were used to establish the ethanol-impairment models. The first one was to add ethanol to the medium to simulate the effect of alcohol concentration in the blood on murine gastric organoids. The advantage of this method was that it was more feasible. The disadvantage was that when the ethanol concentration is greater than or equal to 10%, the Matrigel collapsed.
In the view of the fact that the gastric organoids are similar to the stomach and are hollow cystic structures of the lumen, we have explored the use of microinjection techniques to directly inject ethanol into the internal gastric organoids to simulate gastric gavage. It was found that this modeling method could avoid the influence of Matrigel on the drug, and can be used to study the direct interaction of alcohol or pathogen-host, and at the same time, due to the surface tension of murine gastric organoids and the tight connection between cells, when the concentration of EtOH reached up to 50%, the Matrigel remained intact. When 6% alcohol was injected into the cavity, no obvious impairment was observed, and it may be related to the dilution of the ethanol concentration with a small amount of liquid in the cavity. Because the intracavitary volume of the stomach was temporarily unable to be accurately estimated, it was difficult to determine the specific final concentration of ethanol injected into the cavity. In addition, it took a certain time to microinject each organoids, and it is inconvenient to carry out protein immunization after drug stimulation. So, we chose the first modeling method that was more suitable for our experimental purposes.
BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] is a cell permeable chelating agent with high selectivity for Mg2+and Ca 2+, which can be used to control intracellular Ca2+ levels and chelate intracellular calcium ions. BAPTA has a higher selectivity for Ca2+ than EDTA[Ethylene glycol bis(2-aminoethyl Ether)-N,N,N',N'-tetraacetic acid] and EGTA, and its metal binding is also less sensitive to change of pH result. In this experiment, BAPTA chelated intracellular calcium ions, and the decrease of intracellular calcium ions caused the hydrolysis of ezrin to be significantly reduced compared with the alcohol group. EGTA chelated extracellular calcium ions, which reduced the influx of calcium ions, and decreased the intracellular calcium ion, so hydrolysis of ezrin was significantly reduced compared with the alcohol group.
E64d (molecular formula C17H30N2O5, relative molecular mass 342.4) is a specific calcium-activated neutral cysteine-based endopeptidase inhibitor with good cell membrane permeability and high selectivity for cysteine proteases. It can form a covalent bond with the cysteine thiol group at the active site of calpain, inhibiting its hydrolysis ability, and has a protective effect on alcohol-mediated gastric mucosal epithelial damage. In the E64d group, the hydrolysis of ezrin was significantly reduced, indicating that inhibition of calpain I activity was a factor determining the hydrolysis of ezrin, and demonstrated the role of calpain I-ezrin interaction in the mechanism of alcohol-mediated impairment of gastric organoids.
Calpain digests ezrin on parietal cells and impaired gastric acid secretion23,27. Ezrin in parietal cells binds to the apical membrane during resting and stimulating phases28,29,while some studies have suggested that ezrin was released from the apical membrane into the cytosol at rest in parietal cells30.