METHOD FOR DETERMINING THE VIABILITY OF MESENCHYMAL STEM CELLS IN TISSUE-ENGINEERED STRUCTURES DURING REGENERATION – EXPERIMENTAL SUBSTANTIATION OF EFFICIENCY

Highlights

The method for determining the viability of mesenchymal stem cells in the composition of tissue-engineering constructs is proposed here. This method can become an effective tool for regenerative medicine and tissue engineering for assessing the mechanisms of the regenerative process when using tissue-engineered constructs and predicting their effectiveness in an in vitro system.

Abstract

Aim. Еxperimental substantiation of the efficiency of the method for determining the viability of mesenchymal stem cells in tissue-engineered constructs during regeneration

Methods. The experimental study was conducted on 12 animals (rats). A full-layer scalped wound with a diameter of 3 cm was formed in the animals. A tissue-engineering construct based on human plasma cryoprecipitate was implanted into the wound, with the composition of which a suspension of rat mesenchymal stem cells (MSCs) pre-stained with the DiOC₁₄(3) tracer was introduced. At the control periods (3, 7–8, 14 and 21 days), the animals were withdrawn from the experiment. During the operation, the wound defect area was cut out for ex-tempore examination using wide-field fluorescence microscopy with visualization of samples with 40×, 100×, 200× magnification in the GFP fluorescence channel.

Results. When examining the material obtained from animals on day 3, the external characteristics of the overwhelming majority of identified objects with characteristic fluorescent glow corresponded to the morphological characteristics of MSCs – spindle-shaped cells with characteristic processes. In the material obtained from experimental animals on days 7–8, a large number of cells stained with a tracer fluorescent membrane dye were also detected, which had a green fluorescent glow and characteristic morphology. In the biomaterial obtained on day 14 of the experiment, the number of objects with characteristic MSC morphology stained with the DiOC₁₄(3) tracer was significantly less than on days 3 and 7–8 of the experiment. On day 21, a signal specific for the DiOC₁₄(3) tracer was also detected, but it was not possible to visualize even single cells with the morphology characteristic of MSCs; the signal was probably due to fragments of membranes of eliminated MSCs labeled with a fluorescent dye.

Conclusion. It was shown that MSCs in tissue-engineering constructs implanted on the animal wound retain viability and characteristic morphology for at least 14 days. It is important that the proposed method allows intravital visualization and photofixation of viable MSCs with characteristic morphology, and thus avoiding distortions that may be associated with fixation of biological material. The proposed method can become an effective tool for regenerative medicine and tissue engineering in relation to assessing the mechanisms of the regenerative process using TECs and predicting their effectiveness in the in vitro system.

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