## 1 Introduction
### TEER Method for Monitoring of Caco-2 Monolayer Integrity  
The term intestinal (gut) permeability describes the drugs or chemical compounds passing from the intestinal lumen through the intestinal epithelial cells lining the gastrointestinal wall. It was [PERSON] and [PERSON] (1963) who demonstrated that there are small areas of fusion between adjacent cells [6]. The intercellular junctions are highly specialized multi-layered membranes and are classified into 3 different types: tight junctions (TJs or zonula occludens), desmosomes (or macula adherens), and adherent junctions (or zonula adherens) [6,7]. TJs, which are the most apical junctions, create a watertight sealing between the intercellular epithelial spaces and form a barrier to limit the paracellular pathway of solutes [8, 9, 10]. The TJs also develop polarization by forming a boundary between the apical and basolateral membranes [10]. TJs and permeability of intestinal cells can be well studied using various in vitro models. One of the best, well-established and verified models of studying these parameters is the human epithelial colorectal adenocarcinoma cell line (Caco-2). The word \"Caco-2\" is derived from \"cancer colon\" and hence their ability for spontaneous differentiation into a single layer of intestinal columnar epithelial cells resembles the highly functionalized human epithelial barrier with morphological and biochemical similarities to intercellular junctions. Caco-2 cells are polarized, enabling them to transport ions across the cell membrane, induce secretory functions, and present absorptive transporters involved in both efflux and influx as part of the active transport system and tight junction (TJ) formation [11]. The Caco-2 cell line is also approved by the Food and Drug Administration in drug discovery, permeability, and transport studies. It is also used for study on epithelial permeability, membrane integrity, and bidirectional movement of substances that mainly depends on the development of cell-to-cell junctional complexes.  
The TJ expression in Caco-2 cells can be assessed by trans-epithelial electrical resistance (TEER) [12]. Although the TEER is widely used for determination of Caco-2 monolayer integrity before testing of compounds' permeability as well as the TJs functions, there are some limitations of this method. In the standard TEER protocol, the Caco-2 cells are seeded on Transwell(r) inserts containing membrane with 0.4 um pores and cultured in a 24-well plate for around 18-21 days. This time is necessary for full differentiation into cells, which express TJs, microvilli, and a number of enzymes and transporters (peptidases, esterases, and P-glycoprotein) that are characteristic of enterocytes [12]. For TEER measurement by the device called a voltohm meter, a pair of electrodes should be inserted in each well to both the apical and basolateral compartments. This procedure is invasive, time-consuming, and laborious, especially since it should be repeated many times over 15 days from the start of the culture in order to capture the high level of resistance, which is necessary for experiments. The readings of TEER require removing the cells from the incubator affecting the physiological conditions (i.e., pH and temperature). The measurement may be inaccurate and non-repeatable as it depends on the position of the electrodes and the experience of the operator [13]. Another limitation of this method is its low throughput, which prevents extensive studies to assess the impact of compounds or materials on membrane integrity and permeability.