Kriyojel ve Transwell Sisteminde Üç Boyutlu (3B) Plasenta Modelinin Oluşturulması ve Etkinliğinin Araştırılması

Abstract

In recent years, the need for studies to be carried out by creating current placental barrier models and placental systems for the increase in the use of chemicals and drugs during pregnancy has increased considerably, and there is not yet a placental barrier model accepted as a standard with in vitro studies. In this thesis, a cryogel and transwell system in which human placental cytotrophoblast cell (BeWo) and human umbilical vein macrovascular endothelial cells (HUVEC) are used together were used. In addition to these two cells, an adrenal gland adenocarcinoma cell line (H295R) was added to provide the necessary hormone support to the environment, creating a more effective 3D system that mimics the in vivo environment. The effectiveness of vortioxetine, which is an antidepressant active ingredient for which there is no sufficient information in the literature regarding its use in pregnancy, was applied to the 3D cryogel and transwell co-culture system to investigate its effectiveness. Cryogels optimized with previous studies were used together with cells and added to the transwell insert system, their effectiveness was compared, and the toxicity of vortioxetine was evaluated. By attaching folic acid and type IV collagen to the cryogels, media mimicking the extracellular substance (HDM) that cells can attach to were prepared and then co-cultured with BeWo and HUVEC cells. Within the scope of the thesis, a triple co-culture system was created for the first time in the literature by using H295R cells cultured on a transwell insert on this optimized system. The response of the created system to the active substance of vortioxetine was evaluated by cell viability analysis. In addition, the morphological images of the groups were examined under scanning electron microscopy (SEM). BeWo and HUVEC cells representing the maternal part and H295R cells representing the fetal part were assembled on the cryogels carrying HDM components, and the changes in hormone metabolism were measured by measuring the hormone levels of human chorionic gonadotropin (β hCG), progesterone, dehydroepiandrosterone (DHEA), estriol and estradiol. The effects on the 3D co-culture system containing the cryogel formed with placental cells and the transwell insert were investigated by performing appropriate statistical analyzes. As a result of the application of 30 μM dose of vortioxetine to the 3D cryogel co-culture system, no significant decrease in cell viability of the co-culture was observed. The amount of β-hCG, estriol, estradiol, progesterone, DHEA hormones were measured in the double and triple co-culture groups. An increase in the amount of β-hCG hormone known to be synthesized from BeWo cells was observed in the PHEMA+Type IV Kol+FA cryogel group compared to the PHEMA cryogel group. According to MTT analysis and hormone results in most groups, a significant decrease was observed depending on vortioxetine dosing. According to the results of SEM imaging, it has been shown that the PHEMA Type IV Arm+FA group has better compliance in terms of proliferation, spread and localization of cells compared to the PHEMA group. In this thesis, cryogel groups were formed by binding Type IV Collagen and Folic Acid to PHEMA and PHEMA cryogels. Placental trophoblast cell line BeWo, endothelial cell line HUVEC and adrenocortical cell line H295R cells were grown in co-culture medium for the first time in a system with cryogel and transwell coated with PHEMA and Type IV Collagen and Folic Acid. The toxicity of the antidepressant active substance vortioxetine used during pregnancy was evaluated with the created 3D system. The results showed that the cryogel system is biocompatible for co-cultures, in terms of cytotoxicity, the co-culture groups showed resistance as a result of dose application, and the doses should be higher to reduce viability in dual co-culture groups compared to mono cultures. This 3D cryogel system has shown that it can be used as a system that reflects in vivo and gives similar responses in areas such as new drugs, cosmetics, and food toxicity.