Table 1.
Frequently used models to study L-cell secretion.
| Studies on humans |
|---|
| Advantages: |
| - Human relevance. |
| - Physiologically relevant. |
| - Large plasma volumes can be obtained, enabling quantification of multiple hormones/molecules. |
| - Hormone assays are mostly readily available. |
| - Confounding stress-induced effects play less of a role. |
| - No anaesthesia required. |
| - Allow high temporal resolution. |
| Limitations: |
| - Minimal experimental control. |
| - Intracellular L-cell signalling cannot directly be investigated. |
| - Expensive. |
| - Time consuming (need for ethical approval and study organization). |
| - Inter-individual variation may be considerable, causing need for high group numbers to obtain statistical power. |
| - Degradation and clearance of peptides may lead to underestimation of hormone secretion. |
| In vivo animal studies |
| Advantages: |
| - Physiologically relevant. |
| - Allow post-mortem studies on tissue (e.g. gene expression, protein content, histology). |
| - Less stringent ethical regulations than in human studies with regards to pharmacological compound use. |
| - More experimental control than studies in humans. |
| - Allow relatively quick and inexpensive genetic modification |
| Limitations |
| - Limited experimental control (although more control than in human studies). |
| - Confounding factors (e.g. stress-responses) may influence results. |
| - Intracellular L-cell signalling cannot directly be investigated. |
| - Considerable inter-animal variation requires high group numbers to obtain statistical power. |
| - Strain and housing conditions may profoundly affect results: results are not always reproducible between laboratories. |
| - Low volume plasma samples in mice limits time-resolution and number of molecules that can be quantified. |
| - Suitable assays may not always be available. |
| - Degradation and clearance of peptides may lead to underestimation of hormone secretion. |
| - Long term studies and studies on genetic modified animals are relatively expensive. |
| Immortalized L-cell cell-lines |
| Advantages: |
| - Direct L-cell sensing and secretion can be studied. |
| - High throughput. |
| - Inexpensive and easy to maintain. |
| - Intracellular signalling (e.g. calcium dynamics) can be studied. |
| - Allow for quick and inexpensive gene editing |
| - High concentration range for compound testing |
| - Large sample volume yield. |
| - High degree of standardization and low experiment-to-experiment variation. |
| Limitations: |
| - Low physiological relevance. |
| - Not identical to native L-cells in all aspects. |
| - Cells are non-polarized and lack influence from enteric nerves and paracrine signalling. |
| - Stimulation through physiological route (lumen or vasculature) is not possible. |
| - Hormone output is often insufficient to allow dynamic incubations (perifusion studies). |
| - Accumulation of secreted products and metabolites may influence the results. |
| Primary mucosal cultures |
| Advantages: |
| - Direct L-cell sensing and intracellular signalling can be studied. |
| - Gene editing (e.g. by siRNA) is limited. |
| - L-cells presumably resemble native L-cells to a larger extend than L-cell cell lines. |
| - Inexpensive. |
| - Relatively high throughput. |
| - Applicable for studies on human tissue. |
| - Applicable for studies on GMO. |
| - High degree of standardization and low experiment-to-experiment variation. |
| - High concentration range of test compounds can be applied. |
| Limitations: |
| - Low physiological relevance |
| - Duodenal and jejunal mucosa is challenging to maintain in culture. |
| - Cells are non-polarized and without influence from enteric nerves and paracrine signalling. |
| - Stimulation through physiological route (lumen or vasculature) is not possible. |
| - Hormone output is often insufficient to allow dynamic incubations (perifusion studies). |
| - Accumulation of secreted products and metabolites may influence the results. |
| - Experiments are done on fragile mucosal preparations susceptible to apoptosis. |
| Gut tissue specimens |
| Advantages: |
| - Studies are done on fresh tissue: Less changes in L-cell physiology. |
| - L-cells maintain their polarization and are integrated into the epithelial lining. |
| - High sample volume. |
| - Applicable for studies on human tissue. |
| - Applicable for studies on genetic modified animals. |
| - L-cells resemble native L-cells to a larger extend than L-cell cell lines. |
| - High concentration range of test compounds can be applied. |
| Limitations: |
| - Low physiological relevance. |
| - Specimens have a short survival time and ensuring adequate oxygen supply to crypt cells may be a challenge. |
| - Hormone output is often insufficient to allow dynamic incubations (perifusion studies). |
| - Accumulation of secreted products and metabolites may influence the results. |
| - Stimulation through physiological route (lumen or vasculature) is not possible. |
| - The extent to which enteric nervous signalling is maintained is uncertain. |
| Organoids |
| Advantages: |
| - Allow gene editing. |
| - Real time L-cell monitoring. |
| - Investigation of intracellular L-cell signalling. |
| - Maintain cell renewal, epithelial lining integrity and paracrine signalling. |
| - Allows for studies on polarized monolayers and 3D structure. |
| - High concentration range of test compounds can be applied. |
| Limitations: |
| - Do not fully mimic the intestinal environment, resident cells and blood vessels. |
| - Do not form complete villus compartment. |
| - L-cell responsiveness may be affected by cell culture conditions. |
| Ussing chambers |
| Advantages: |
| - Studies are done on fresh tissue and native L-cells. |
| - L-cells maintain their polarization and are connected to the same cells as they were in vivo. |
| - Applicable for studies on human tissue. |
| - Tissue can be stimulated from the physiological relevant route (apical side or basolateral side). |
| - Applicable for studies on genetic modified animals. |
| - High concentration range of test compounds can be applied. |
| Limitations: |
| - Tissue do not survive well in chambers: relatively short time window for doing experiments. |
| - Human specimens may be difficult to obtain. |
| - The extent to which enteric nervous signalling is maintained is uncertain. |
| - Hormone output is often insufficient to allow dynamic incubations or perifusion studies. |
| - Accumulated secretion products may influence the results. |
| - Gene editing is not possible. |
| - Not suitable for investigation of intracellular L-cell signalling. |
| Isolated perfused intestines |
| Advantages: |
| - High degree of physiological relevance and anticipated translation to in vivo. |
| - Studies are done on fresh tissue: no significant changes in L-cells. |
| - L-cells maintain their polarization and are connected to the same cells as they were vivo. |
| - Allow for stimulation via the physiological relevant route (lumen or vasculature). |
| - Allow for constant perfusion at a physiological flow rate. |
| - Secretion can be studied at a high time resolution (down to second intervals). |
| - Absorption of nutrients can be directly be investigated. |
| - Large sample volume yield. |
| - Enteric nerve signalling and peristaltic movements are largely preserved. |
| - Applicable for studies on genetic modified animals. |
| - High concentration range of test compounds can be applied. |
| Limitations: |
| - Requires a certain level of surgical skills. |
| - Relatively expensive. |
| - Laborious and not applicable for screening purposes. |
| - siRNA mediated knock down of target genes is not readily possible. |
| - Relatively short time window for doing experiments (usually up to four hours). |
| - Not suitable for investigation of intracellular L-cell signalling. |