Effect of long-term culture on the biological and morphological characteristics of human adipose tissue-derived stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells that can be obtained from different tissues, including bone marrow, adipose tissue, umbilical blood, Wharton's jelly, and dental pulp. Due to their differentiation potential, regenerative and immunosuppressive properties, as well as ability to expand under in vitro conditions, these cells represent a promising therapeutic tool for regenerative medicine. However, the basic prerequisite for the therapeutic utilization of MSCs is obtaining a sufficient amount. While this may be achieved by prolonged cultivation, long-term culture of MSCs is associated with accumulation of morphological and functional changes. In our study, we focused on analyzing morphological and biological changes of cultured adipose tissue-derived stem cells over 30 passages. We performed morphological analysis using light and electron microscopy, as well as analysis of selected biological properties (expression of surface antigens and selected genes involved in cell regulation and apoptosis, cell cycle, and cell senescence) every 5 passages. Our results showed that long-term expansion leads to significant changes in morphology and affects proliferation kinetics and the cell cycle. On the other hand, the MSCs maintained a prototypical immunophenotype, normal cell cycle and apoptosis regulator function, and maintained a low level of telomerase activity during later passages.

Development of a new method for the preparation of an acellular allodermis, quality control and cytotoxicity testing.

Demand for use of acellular allodermis is high but commercially appropriate products are not used routinely because of very high price and limited availability. These facts did motivate us to prepare acellular allodermis using a new, simple and less expensive method. We have developed a original method for preparation of acellular allogeneic dermis based on action of a proteolytic enzyme in combination with distilled water. Hypotonic environment in comparison with SDS or Triton ansure no toxicity of the final product. Trials for determination of optimal trypsin concentrations, temperature and time of action were performed. According to our results, the use of 2.5% trypsin/EDTA solution overnight at +4 °C was proving to be optimal. The histology confirmed absence of cells in the prepared dermis. No toxicity of final acellular dermis was confirmed by three independent tests (agar diffusion test contact cytotoxicity test and grow curve). The prepared acellular dermis seems to be suitable not only for direct clinical use, but it can be used as a scaffold for cell cultivation as well.

Frequencies of polymorphisms in CYP2C9 and VKORC1 genes influencing warfarin metabolism in Slovak population: implication for clinical practice.

OBJECTIVES: The study was aimed at establishing an effective molecular-genetic method for detecting polymorphisms in genes CYP2C9 and VKORC1, which affect the pharmacogenetics of warfarin, and at determining their prevalence in Slovak population. BACKGROUND: Warfarin, derivative of coumarin, belongs to the most commonly prescribed oral anticoagulants with narrow therapeutic index. An insufficient dose of warfarin can result in failure to produce the antithrombotic effect, whereas an overdose increases the risk of bleeding. It was proven that genetic variability in two genes, CYP2C9 a VKORC1, has a significant influence on the individual's response to the dosage of warfarin. METHODS: In a control group of 112 randomly selected individuals, we tested the frequency of selected single nucleotide polymorphisms including CYP2C9*2 (430C>T), CYP2C9*3 (1075A>C), VKORC1*2 (1173C>T) by allele-specific Real-Time PCR and VKORC1*2 (-1639G>A) by using PCR-RFLP. RESULTS: Due to the combination of frequent alleles CYP2C9*2, CYP2C9*3 and VKORC1*2 in Slovak population we determine that 25% of population need a standard 5-mg daily dose of warfarin, while 44%, 23%, and 8% need 4 mg, 3 mg and 2 mg of warfarin per day. CONCLUSION: Slovak population is in Hardy-Weinberg equilibrium and frequencies of SNPs were in accordance with other published results in European populations (Tab. 5. Fig. 3, Ref. 51).

Frequency of selected single nucleotide polymorphisms influencing the warfarin pharmacogenetics in Slovak population.

OBJECTIVE: Warfarin represents the most commonly prescribed oral anticoagulants, which functions as an antagonist of vitamin K, an essential factor of blood coagulation cascade. Warfarin has a narrow therapeutic index. An insufficient dose can cause failure of antithrombotic effect, and an overdose increases a risk of bleeding. It is known that variability in two genes (CYP2C9 and VKORC1) has a significant effect on individual response to warfarin dose. These polymorphisms influence more than one-third of known warfarin dose effect. Pharmacogenetics of warfarin is less affected by polymorphisms in the other genes such as CYP4F2, CYP2C19, and GGCX. MATERIAL AND METHODS: The frequency of selected single nucleotide polymorphisms including CYP2C9*2 (430C > T), CYP2C9*3 (1075A > C), VKORC1*2 (-1639G > A/1173C > T), VKORC1*3 (3730G > A), GGCX (12970C > G, 8016G > A), CYP2C19*2 (681G > A), and CYP4F2*3 (1297G > A) was tested in a control group consisting of 112 randomly selected individuals by allele-specific real-time PCR, restriction fragment length polymorphism, and bidirectional PCR allele-specific amplification. RESULTS AND DISCUSSION: The current results were statistically evaluated and compared with other populations. The presented results in Slovak population which is in Hardy-Weinberg equilibrium were compared with the prevalence in different countries. The incidence of selected polymorphisms in Slovak population correlates with Caucasians.