Refinement of Flow Cytometry Based Methods for Identification and Characterization of Differentiation Specific Intestinal Epithelial Cells
Author
Sørensen, Marina Elmelund
Term
5. Term (Master thesis)
Education
Publication year
2018
Submitted on
2018-01-02
Abstract
Colorectal cancer is common and serious, and it may be clinically important to determine whether tumors arise from basal stem cells or from more mature epithelial cells in the intestinal crypts. This thesis evaluates and refines initial methods to identify differentiation-specific intestinal epithelial cells using multiparametric flow cytometry on single-cell suspensions from normal colonic tissue collected during surgery (with ethical approval and informed consent). Cells were stained with panels of surface markers (including LGR5 and EpCAM), and we assessed gating strategies and key process steps: viability staining, antibody titration, and magnetic-activated cell sorting (MACS). Early single-stain experiments produced inconclusive results due to pronounced nonspecific binding, partly driven by a high fraction of dead or dying cells. Viability testing on the HT-29 cell line showed that FVD effectively excludes cells with compromised membranes and is recommended for future analyses. Titration of selected antibodies reduced nonspecific staining, highlighting the need to optimize concentrations. LGR5 also showed unexpected binding to leukocytes (CD45-positive cells), warranting further validation under more controlled conditions. Finally, MACS on HT-29 successfully enriched EpCAM-positive cells, indicating a useful principle to be tested on normal tissue suspensions in combination with LGR5. Overall, flow cytometry appears promising for isolating relevant cell populations, but robust viability gating, antibody titration, and marker validation are needed before the main hypothesis can be resolved.
Colorectal cancer er en hyppig og alvorlig sygdom, og det kan have klinisk betydning at afgøre, om tumorer udspringer fra basale stamceller eller fra mere modne epitelceller i tarmkrypterne. Denne afhandling undersøger og forfiner indledende metoder til at identificere differentieringsspecifikke intestinale epitelceller ved hjælp af multiparametrisk flowcytometri på enkeltcelle-suspensioner fra normalt tyktarmsvæv indsamlet ved kirurgi (med etisk godkendelse og informeret samtykke). Cellerne blev farvet med paneler af overflademarkører (bl.a. LGR5 og EpCAM), og vi evaluerede sorteringsstrategier samt kritiske procestrin: levedygtighedsfarvning, antistof-titrering og magnetisk aktiveret cellesortering (MACS). I tidlige enkeltfarvningsforsøg var resultaterne uklare på grund af udtalt uspecifik binding, delvist forårsaget af mange døde eller døende celler. Levedygtighedsforsøg på HT-29-cellelinjen viste, at FVD er velegnet til at ekskludere celler med kompromitteret membran og derfor anbefales i fremtidige analyser. Titrering af udvalgte antistoffer reducerede uspecifik farvning og understreger vigtigheden af at optimere koncentrationer. Desuden bandt LGR5 uventet til leukocytter (CD45-positive celler), hvilket kræver yderligere validering under mere kontrollerede forhold. Endelig demonstrerede MACS på HT-29 en effektiv berigelse af EpCAM-positive celler, hvilket peger på et nyttigt princip, der bør afprøves på normale vævssuspensioner kombineret med LGR5. Samlet peger resultaterne på, at flowcytometri er en lovende vej til at udskille relevante cellepopulationer, men at robust levedygtighedsgating, antistof-titrering og markørvalidering er nødvendige næste skridt, før hovedhypotesen kan afklares.
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