İnsan Plasenta ve Endotel Hücre Hatları ile 3b Kriyojel Ortak Kültür Sisteminin Oluşturulması ve Etkinliğinin Araştırılması

Abstract

The placental barrier, which is formed with pregnancy, determines the maternal-fetal passage of nutrients, the transition gradient, feto-placental and utero-placental blood flow by its structure and transfer capacity. A large increase in the rates of exposure to the use of chemicals and drugs has occurred during pregnancy. Tests cannot be performed on people due to ethical restrictions. In order to complete the lack of information about the permeability of the placental barrier, the need for studies to be conducted by creating current models of the placental barrier has increased. In this thesis, human placental choriocarcinoma cells (BeWo) and human umbilical vein cells (HUVEC) with three-dimensional (3D) environment in kriyojel prepared with different properties on mono and co-culture systems have been established. Cryogels consist of interconnected macropores that can ensure the adhesion and proliferation of cells. In the thesis study, biocompatible poly (2-hydroxyethyl methacrylate) (PHEMA) cryogels with a 3D pore structure were supported in terms of extracellular substances (HDM) and a system closer to in vivo was established. Gelatin containing cryogels (PHEMAGEL) were synthesized using a PHEMA-based cryogel. Folic acid (FA) was attached to all PHEMA and PHEMAGEL cryogels, and fibronectin (FN) or collagen type IV (KOL Type IV) joined the structure, creating an environment close to the placenta tissue. Within the scope of the characterization studies of PHEMA, PHEMA+KOL Type IV, PHEMAGEL and PHEMAGEL+FN of the 4 cryogel groups synthesized with different ingredients, swelling tests were performed and the swelling properties of the gels were evaluated. The involvement of gelatin, FA, FN and KOL Type IV in the structure of cryogels was investigated by fourier transform infrared (FTIR) spectrometer. 3D characterization analyses of cryogels were performed with micro computed tomography (Micro-CT). The pore sizes of cryogels and the adhesion potentials of the cells were examined by scanning electron microscopy (SEM). In vitro release analyses were performed on FA-bonded cryogels. In order to study the cytocompatibility of the synthesized cryogel groups and the proliferation abilities of the cells, they were incubated together with the cells for 2, 4 and 7 days and the viability of the cells was evaluated. Vortioxetine, an antidepressant active ingredient that doesn’t have sufficient information in the literature about its use in pregnancy, was evaluated in the 3D cryogel co-culture system created. It was incubated with cells for 2, 4 and 7 days at concentrations of 30 µM and 60 µm. The effectiveness of MTT was examined by vitality analysis. Human chorionic gonadotropin (β-hCG) hormone production and matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and protein levels were measured. When the swelling properties of cryogels were examined, it was seen that the degree of macroscopicity was higher in PHEMA-based cryogels. A decrease in the degree of macroscopicity, degree of swelling and swelling rates have been observed due to gelatin. According to the result of the FTIR analysis, it was found that gelatin, FA, FN and KOL Type IV successfully participate in the structure of cryogels. The micro-CT result showed that cryogels have a distribution in different pore sizes. This has led to an increase in the retention and proliferation of cells. It has been observed that cryogels with fairly large pores connected with each other by SEM images have been synthesized. There was a decrease in the in vitro release of FA depending on the incubation period. As a result of the MTT analysis performed to understand the cytocompatibility of cryogels, it was observed that the cells are compatible with cryogels synthesized with various properties, and they do not have a toxic effect on the cells. A decrease in cell viability was observed in mono-cultures at high doses of Vortioxetine to the 3D cryogel common culture system, but the expected decrease in cell viability at the same doses of the common culture was not observed. The amount of β-hCG hormone was measured in BeWo and common culture group, and the greatest hormone release was observed in BeWo tuesday of PHEMA+KOL Type IV. There was no significant decrease in MMP-2 and MMP-9 protein levels due to the dose administration of Vortioxetine. In this thesis study, cryogels containing gelatin, FN and KOL Type IV in their structure were synthesized and bound with FA. Their biocompatibility with placental BeWo and HUVEC cells has been demonstrated and they have been produced on PHEMA cryogels for the first time in a common culture medium. The toxicity of the active ingredient of the antidepressant Vortioxetine used in pregnancy was evaluated with the created 3D system. The results showed that the cryogel system is biocompatible for mono- and common cultures, and in terms of cytotoxicity, doses should be higher to reduce viability in dual common culture groups compared to mono-cultures. This 3D cryogel system has shown that it can be used as a system that reflects and gives similar answers in vivo in areas such as new medicine, cosmetics, food toxicity.