Laboratory for molecular and cell biology and immunochemistry (LMCBI)

Scientific approach

LMCBI is a new and young laboratory aiming at developing and scientifically exploiting cellular and tissue in vitro models. The following experimental models are established:

  1. STEM CELL LINES – Isolation, cultivation and exploitation in relation to pathophysiological manifestation, such as liver cirrhosis or metabolic disorder
  2. TISSUE ORGANOCULTURES – Isolation, cultivation and exploitation
  3. COMMERCIAL CELL LINES – Cultivation, maintaining and exploitation
  4. BIOBANKED HUMAN SAMPLES – Ex vivo experiments
  5. Experimental directions

Cell lines, cell cultures and biosamples

1) Stem cell lines
Stem cells are potent cells that yield differentiated cells during embryonic development, during the growth, ageing, as well as regeneration after eventual devastation of the tissues, while during postnatal period those maintain adult tissues. Stem cells include totipotent, pluripotent as well as multipotent cells. Several unique characteristics of stem cells that segregate it from somatic cells include:

  • The capability of continuous auto-repair and unlimited mitotic potential in controlled conditions during the embryonic and complete postnatal development;
  • Capability of differentiation in all types of cells during embryonic development (pluripotency) or several distinctive cell types (multipotency);
  • Huge motility and mobility capacity;
  • Induction of tissue tolerance in case of stem cell transplantation

Due to the possibility of isolation and in vitro multiplication as well as the pluripotency, stem cells represent an important source of cells and tissues in “regenerative medicine” and “tissue engineering”, especially in terms of embryonic human stem cells. The isolation of first human embryonic stem cell line (hESC) in Serbia, is planned inside LMCBI, and is to be performed in collaboration with partners from different scientific institutions from the country.
In collaboration with Clinical Centre of Serbia, LMCBI is involved in clinical trial conducted within Imperial College, London, Tissue Engineering & Regenarative Medicine Center (director Professor Julia Polak), and related to the application of adult mesenchymal cells in the therapy of liver cirrhoses, as well as insulin dependent diabetes mellitus.
Inside LMCBI mesenchymal stem cells were isolated and cultivated from dog adipose tissue, as well as from the human dental pulp.

2) Commercial cell lines
Within the LMCBI cultivation and exploitation of commercial cell lines from different tissues are performed with the aim of:

  • Investigation of molecular patterns and networks underlying beneficial effects of different bioactive compounds, extracts and metabolites, proven through the variety of clinical trials conducted inside the CENM
  • Investigation of signaling molecules and molecular interactions underlying particular observed nutritional effect, or beneficial nutritional impact related to human health or health biomarkers

There is a panel of commercial cell lines exploited inside LMCBI, starting with breast cancer commercial cell lines: MDA-231, MDA-175, SK-BR-3. In addition, a panel of cell lines is planned to be exploited with regards to further elucidation of complex molecular interactions underlying metabolic disorders. Those include isolation and preparation of primary cell culture HUVEC according to standardized procedures, as well as cultivation of endothelial EA.hy 926 cells, commercial hepatocytes, enterocytes including HT-29 and Caco-2 cells, as a representative model for investigating gut permeability.
Experiments regarding nutritional impact on malignant transformation and metastasis are planned on the cancer cell lines including above mentioned breast cancer cell lines, as well as different gastric, colorectal, lymphoid malignant cultures, derived from both human and murine malignant tissues. There is a long-term collaboration with Institute for Oncology and Radiology from Serbia, regarding design and conduction of experiments in the field of nutritional aspect of cancer. Inside LMCBI there is a special interest on the folate amount impact on cell capacity for malignant transformation and metastatic potential. The folate and its derivatives are vitamins-nutritional components with the crucial role in DNA synthesis and repair, processes known to be highly unregulated in malignant and metastatic conditions.

3) Ex vivo experiments
Large-scale randomized clinical trials are conducted inside CENM. Those consider elucidation of effects of different bioactive nutritional and non-nutritional compounds in relation with different aspects of human health, mostly cardio-vascular health and metabolic disorders. According to accredited and approved study protocols, human samples including blood and urine samples are biobanked, before and after the nutritional intervention, and those samples are exploited inside LMCBI. Experimental models, simulating physiological conditions such as hyperglyicemia, hypoxia, or the presence of certain bioactive component or metabolite, are going to be designed in order to further investigate particular pathophysiological manifestation, such as atherosclerosis, and its underlying molecular basis.

Currently there is a large randomized clinical trial on-the-go, with objective of investigating the effect of polyphenol-rich food and its metabolites on cardio-vascular human health and biomarkers. In order to imitate the physiological aspects of atherosclerotic events, the isolated PBMC are going to be investigated on its interaction with human monocytic cell lines such as THP-1, in the presence of platelets, as an plastic experimental simulation of atherosclerotic cascade.

Methodological approaches used in the LMCBI include:

  • Standardized procedures for preparation of primary cell cultures
  • Standardized procedures for isolation of stem cells
  • Standardized procedures for cultivation and maintaining of primary and permanent cell cultures in appropriate cell media, such as RPMI, DMEM etc, with the addition of particular supplementation
  • Flow cytometer analyses for elucidation of cellular and molecular checkpoints during cell cycle, cell death, disturbed oxidative conditions etc, as well as immunoreactivity phenomena
  • Western blot analyses for determination of protein amounts, intracellular and inside ECM (extracellular matrix)
  • Construction of in vitro cellular models including transwell and transmembrane models for elucidation of cellular motility and migratory potential, as well as metastatic capacity, and gut permeability

Projects

  • “Biological effects, nutritional intake and status of folate and polyunsaturated fatty acids: The improvement of nutrition in Serbia”, 2011-2014, III 41030. The grant for the project is awarded by the Serbian Ministry of Science and Education.

References

  • Debeljak Martacic J, Francuski J, Luzajic T, Vukovic N, Mojsilovic S, Drndarevic N, Petakov M, Glibetic M, Markovic D, Radovanovic A, Todorovic V, Kovacevic Filipovic M. Characterization of deciduous teeth stem cells isolated from crown dental pulp. Vojnosanitetski pregled, in press.

Master and doctoral thesis

  • Jasmina Debeljak Marčetić: The influence of N-acetyl-L-cysteine on in vitro proliferation and differentiation of dental pulp stem cells in children – work in progress


Dr Maria Glibetic

SOULFOOD Serbia

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