2024 - X IDIES project

  The IDIES project began in 2014 at CEBAS-CSIC. The main objective of this project is the initiation of high school students (research modality) to scientific research participating and collaborating in projects led by researchers of recognized national and international prestige.

This year, under the supervision of Dr. Ávila-Gálvez, Dr. Giménez-Bastida, Dr. González-Sarrías and Prof. Juan Carlos Espín, students of the IES "Ramón y Cajal" (Murcia) will investigate how (poly)phenols are able to cross the blood brain barrier (BBB) and exert anti-inflammatory effects. 

The scientific activities will be mainly carried out at the University of Murcia (Cell culture service) and CEBAS-CSIC, where they will have the opportunity to get hands-on experience in cellular and molecular techniques (cell culture, cytotoxicity, ELISA assays and HPLC, among others). The data obtained and their analysis will be presented in the VIII IDIES Conference (poster and oral presentation).

FIRST SESSION: INTRODUCTION TO THE LABORATORY AND CELL CULTURE                                                               

The Cell Culture Service at the University of Murcia was the place where we performed all the activities planned for this day. In the first activity, we explained to the students the general laboratory safety rules. We also showed and explained the equipment they can found in a laboratory, including centrifuges, hoods, incubators, etc.

The first activity they worked on was the preparation of the compounds we want to investigate. This activity allowed them to learn important concepts related to the preparation of solutions such as molarity. Besides, the students also used the precision balance, micropipettes, multichannel pipettes and practiced the sterilization of compounds that will be used to treat cells under sterile conditions.

In a second set of experiments, the students learned about different types of culture medium, they used the microscope to observe cells, changed culture medium, participated in cell subculturing and counted them using the trypan blue dye exclusion technique.

Students of the IES Ramón y Cajal learning basic cell culture techniques

In this first session, although exhausting, our students demonstrated to have the making of a scientist.

SECOND SESSION: (POLY)PHENOL TRANSPORT ACROSS THE BBB (IN VITRO MODEL)                                             

Studying how a natural compounds such as (poly)phenols are able to cross the BBB is one of the cornerstones of this project. In this second session, our students will evaluate the transport through the BBB combining in vitro and HPLC techniques.

Specifically, the cultured cells will be treated with (poly)phenols mimicking conditions that can be detected in vivo (molecular form of the compounds, concentrations...). Next, after 2 hours, the culture medium will be collected, processed and further analyzed by HPLC. 

Will the (poly)phenols cross the BBB? Will they be metabolized? What will we find in our results?

Let´s figure it out in the third session.

THIRD SESSION: TREATMENT OF THE CELLS AND CULTURE MEDIUM COLLECTION                                                     

The use of natural compounds to modulate/ameliorate the inflammatory response is one of the cornerstones of this project. To investigate this topic it is essential to know how to treat the cells. This is what our student have learned in this session. The (poly)phenols under investigation were filter sterilized and added to the cells at (non-toxic) concetrations similar to those detected in vivo in the presence of pro-inflammatory molecules. After a few hours of treatment, the culture medium was collected and kept at -80 degrees. 

The analysis of this medium will provide information related to the anti-inflammatory activity of natural compounds. However, we have to wait until next week to figure it out.

FORTH SESSION: ELISA ANALYSIS OF MOLECULES RELATED TO INFLAMMATION                                                     

The enzyme-linked immunosorbent assay (ELISA) is a widely approached biochemistry technique described by Engvall and Perlmann in 1972 (doi: 10.4049/jimmunol.109.1.129). In this session we will used this assay to investigate how different natural compounds (phenolic compounds) are able to modulate key molecules involved in inflammation (i.e., cytokines). The different culture media corresponding to different treatments will be incubated in 96-well plates, pre-coated with antibodies that target a specific proinflammatory cytokine. The incubation is followed by different washing steps and incubations with detection antibodies, which are responsible of color development (see below). The measurement of the color obtained using an spectrophotometer will give and idea of the concentration of the cytokine and, therefore, the effect of the natural compounds tested on that molecule.

       

FIFTH SESSION: ANALYSIS OF METABOLITES Y CULTURE MEDIUM USING MASS SPECTROMETRY TECHNOLOGY

Our students not only learn what effects are exerted by the phenolic compounds investigated, but also mechanisms related to the transport of these compounds through the BBB model. This provides a more differentiatied view about the capacity of natural compounds to modulate the cellular response. The experiments carried out during sessions 2 and 3 were the basis to obtain the samples to be analyzed in this session.

The approach of this assay is intended to learn (i) basic concepts of mass spectrometry, (ii) sample processing (specifically, culture medium containing (poly)phenols) and (iii) the use of sophosticated softwares needed to design the analysis by mass spectrometry. A string of steps such as protein precipitation, centrifugation and filtration were performed prior to run the samples. The analysis of the chromatrograms generated is the final step of this experiment, but this is another exciting experience that we will live in the next (and last) session.

LAST SESSION: DATA ANALYSIS AND CONCLUSIONS                                                                                                         

After several weeks of organizing experiments, working in the laboratory, and processing/analyzing samples, time is ripe to summarize, discuss and draw conclusions from the results obtained. Here, alongside our students, we engage in discussions regarding the information obtained from the ELISA assay, chromatograms as well as the transport of (poly)phenols using the BBB model. 

The main objective is to answer fundamental questions such as: are (poly)phenols able to exert anti-inflammatory effects? do these molecules have capacity to cross the BBB? are the differences observed significant from a statistic point of view? what statistical analysis should we use? The answers raised from this session will be the basis of the poster and the oral presentation that will be presented at the IDIES Conference.