Study on the Function of Intestinal Epithelial Cell and Its Isolation and Culture

The interaction of the host-microbiome balance is essential for maintaining homeostasis in the gastrointestinal tract[1]. Intestinal epithelial cells (IECs) provide the front line of defense by building a barrier against pathogens and symbiotic microorganisms[2].

The Structure of Intestinal Epithelial Cells

Intestinal epithelial cells are found in crypts. IECs are mainly composed of transit-amplifying (TA) cells, Paneth cells, Microfold cells (or M cells), Enteroendocrine cells and Tuft cells[3]. These cells work together to maintain intestinal homeostasis. However, the loss of intestinal epithelial cells may damage intestinal stem cells (ISC) which makes the intestine unable to repair itself and leading to intestinal diseases[4].

Fig 1. Schematic diagram of epithelial cell types and signaling pathways in crypts[3].

Intestinal epithelial cells (IECs) may contribute to local immunomodulatory. Therefore, successfully isolated and correctly cultured intestinal epithelial cells could provide a good model for studying intestinal function and related intestinal diseases[5].

How to Isolate and Culture Intestinal Epithelial Cells?

Firstly, sufficient preparation of reagents and materials is critical to the success of the experiment. Hank’s Balanced Salt Solution (HBSS), complete Dulbecco’s Modification of Eagles Medium (DMEM) containing 2.5% Fetal Bovine Serum (FBS), S-DMEM, Collagenase/ Dispase enzyme digestion mixture, EGF (for human cultures), Petri dishes (9 cm), Flasks (25 cm²), Scalpels, Syringe, Needles, etc. should be prepared in advance. Human colonic tissues and mouse clone tissues are excised and immediately washed with HBSS wash solution which is HBSS containing 100 U/ml Benzyl penicillin, 30 μg/ ml streptomycin, 25 μg/ ml Gentamicin.

Secondly, clean the contents of the intestine with a syringe after collection. Then immediately put the colon into the ice cold HBSS wash solution after clean. Transfer the tissue to a clean Petri dish. Cut tissues into small pieces (approximately 0.5–1 cm²) using sterile scissors and move them into 25-cm² flask, adding 20 ml HBSS with a pipette. During this period, wait for the intestinal tissue in the bottle to settle and then remove the turbid HBSS. Repeat the process until the remained HBSS almost clear. This usually takes about 5 repeats of the washing process. Next, add the premixed Collagenase and Dispase enzyme digestion mixture to digest for about 2-3 hours. It is necessary to observe the degree of tissue digestion every 15 minutes after 1 hour of digestion. The isolation is ready when approximately 70–80% of the crypts have become liberated as individual free crypts.

Thirdly, evenly divide the digested contents into a sterile 30 ml universal instrument. Add 24 ml S-DMEM and make them stand for 1 minute, then transfer the supernatant to a new centrifuge tube. Centrifuge at 200–300 g for 4 minutes at 4°C, then remove the supernatant after the centrifugation. The sediment on the bottom of the tube would be broken up when gently flick the bottom of the tube. Repeat S-DMEM and centrifugation steps about 5 times until the supernatant is clear.

Fourthly, plate out the crypts when the supernatant appears clear. Cells were seeded in a 24-well plate at a density of 800-1000 crypts/ml/well. Incubate the plates at 37°C in 7.5% CO2 and allow the cells to attach for 2 days. Replace the medium which contains all unattached material with fresh medium. Supplement the medium at 5-day intervals with 0.5 ml fresh medium[6].

Fig 2. Incomplete enzymatic digestion or poor crypt separation during the precipitation step often results in other contaminating cell types in the culture[6].

Some researchers may inevitably mix fibroblasts in the process of culturing epithelial cells, which would interfere with the study of intestinal epithelial cell function. The FBS concentration can be appropriately reduced to remove the fibroblasts. Intestinal epithelial cells specifically express IAP and CDH1 genes, which can be detected by RT-qPCR[7].

How are Intestinal Epithelial Cells adapted to their Function?

Intestinal Epithelial Cells were passaged and maintained in basal and differentiation culture media. In vitro cell culture provides a simple, rapid, and accurate method for studying the proliferation and apoptosis of intestinal epithelial cells; therefore, it was widely used in the study of drug and nutrient absorption mechanisms. Stanifer et al. found that human intestinal epithelial cells (hIECs), including primary untransformed epithelial cells, fully support SARS-CoV-2 infection, replication, and production of new infectious viruses. At the same time, their data shows that hIEC is an effective site for SARS-CoV-2 replication and type III IFN response plays a critical role in controlling virus replication[8].

Fig 3. The number of SARS-CoV-2-positive cells were quantified in 10 fields for each donor and

the copy number of SARS-CoV-2 genome was evaluated by qRT-PCR.

Conclusion:

Intestinal epithelial cells and immune system are interrelated to maintain normal intestinal homeostasis and the integrity of the intestinal barrier. At present, primary intestinal epithelial cells culture model is limited by the quality and quantity of surgically removed tissue. Primary intestinal epithelial cells are very important for researching the intestinal function and pathogenesis of diseases. Therefore, the ability to successfully separate and culture intestinal epithelial cells from the crypts of the small intestine or colon become the key to subsequent research.

Where to get Human Intestinal Epithelial Cells?

As it is noted in the article, the pure culture of Intestinal Epithelial Cells is a key to a success experiment. AcceGen provides the most authentic cell cultures for research. All the products of AcceGen are strictly comply with international standards.

We provide not only Human Intestinal Epithelial Cells, but various primary intestinal cells such as Human Intestinal Fibroblasts, Human Intestinal Microvascular Endothelial Cells and Human Rectal Smooth Muscle Cells etc. Besides, we offer more than 60 types of Human Primary Cells associated with the Digestive System to meet different experimental needs. For more detailed information, please visit our product portfolio or contact [email protected].

References:

1.  Nie D, Pearce SC, Weber GJ, van Sambeek DM, Soares JW, Racicot K, Breault DT: Intestinal enteroids recapitulate the effects of short-chain fatty acids on the intestinal epithelium. Plos One 2020, 15(4):e0230231.

2.  Stanifer ML, Mukenhirn M, Muenchau S, Pervolaraki K, Kanaya T, Albrecht D, Odendall C, Hielscher T, Haucke V, Kagan JC et al: Asymmetric distribution of TLR3 leads to a polarized immune response in human intestinal epithelial cells. Nat Microbiol 2020, 5(1):181-191.

3.  Rees WD, Sly LM, Steiner TS: How do immune and mesenchymal cells influence the intestinal epithelial cell compartment in inflammatory bowel disease? Let’s crosstalk about it! J Leukoc Biol 2020, 108(1):309-321.

4.  Middelhoff M, Nienhuser H, Valenti G, Maurer HC, Hayakawa Y, Takahashi R, Kim W, Jiang Z, Malagola E, Cuti K et al: Prox1-positive cells monitor and sustain the murine intestinal epithelial cholinergic niche. Nat Commun 2020, 11(1):111.

5.  Madrigal-Estebas L, Doherty DG, O’Donoghue DP, Feighery C, O’Farrelly C: Differential expression and upregulation of interleukin-1alpha, interleukin-1beta and interleukin-6 by freshly isolated human small intestinal epithelial cells. Mediators Inflamm 2002, 11(5):313-319.

6.  O’Shea CBJA (ed.): Isolation and Culture of Intestinal Epithelial Cells; 2020.

7.  Zhang H, Chen F, Liang ZH, Wu Y, Pi JS: Isolation, culture, and identification of duck intestinal epithelial cells and oxidative stress model constructed. In Vitro Cell Dev Biol Anim 2019, 55(9):733-740.

8.  Stanifer ML, Kee C, Cortese M, Zumaran CM, Triana S, Mukenhirn M, Kraeusslich HG, Alexandrov T, Bartenschlager R, Boulant S: Critical Role of Type III Interferon in Controlling SARS-CoV-2 Infection in Human Intestinal Epithelial Cells. Cell Rep 2020, 32(1):107863.