P.P. Nimiritskiy - Post-graduate Student, Laboratory Assistant, Laboratory of Gene and Cell technologies, Faculty of Fundamental Medicine, Lomonosov Moscow State University T. A. Dus - - Student, Faculty of Fundamental Medicine, Lomonosov Moscow State University O.A. Grigorieva - Research Scientist, LLC «Gene and Cell Therapy», Moscow, Russia G.D. Sagaradze - Post-graduate Student, Laboratory Assistant, Laboratory of Gene and Cell Technologies, Faculty of Fundamental Medicine, Lomonosov Moscow State University A.Yu. Efimenko - Ph.D. (Med.), Senior Research Scientist, Medical Science and Education Center, Lomonosov Moscow State University P.I. Makarevich - Ph.D. (Med.), senior Research Scientist, Laboratory of Gene and Cell Therapy, Medical Science and Education Center Lomonosov Moscow State University. E-mail: pmakarevich@mc.msu.ru

Present study discusses the possibility of development a new tissue engineering method relying on minimal constructs comprising of viable cells and extracellular matrix known as cell sheets. This technology omits suspension of cells and injection and may be used for transplantation stem cells and cell-based medicinal products to induce angiogenesis, neurogenesis and tissue repair. In the study we have evaluated the possibility of generation of cell sheets from human adipose-derived stromal cells using simple procedure and non-coated culture dishes and plates. We found that cell sheets may be obtained using 2 protocols - short-term (48-72 hours) of high-density (200,000 cells/cm2) or long-term (up to 2 weeks) of low-density (10,000-15,000 cells/cm2) cultures in AdvanceStem culture medium with supplementation by ascorbic acid (50 mcg/ml). Obtained cell sheets were dense round-shaped opaque culture constructs that were easily manipulated using forceps or pipette. We managed to evaluate them in histology study and found them to be thick (30-40 um) multilayered constructs comprising of cells (stained by DAPI) and matrix proteins - collagens, fibronectin, laminin (stained by antibodies). We also found that cell sheets cultured in presence of ascorbic acid had certain features and were denser and more resistant to tension load probably due to ascorbic acid influence on collagen production and assembly rate as well as cell division rate. Another option we used was establishing an effective protocol for cells sheet decellularisation using wide-known detergents like SDS, deoxycholic acid, CHAPS, Triton X-100 and urea at literature-based concentrations. We found that optimal results were obtained by deoxycholic acid and CHAPS treatment and these compounds managed to disrupt cellular membranes yet certain nuclear DNA-containing material was detected in matrix by DAPI stain. We tried to clear the decellularized material by DNAseI treatment for 12 hrs. and it resulted in removal of DAPI-positive inclusions from the decellularized materials. Unfortunately, certain amount of DNA may still be trapped within decellularized cell sheets requiring optimization of protocol as far as DNA is known to have certain immunogenic potential in mammals. Overall, our results indicate the possibility of generation of cell sheets from adipose derived stromal cells using simple culture dishes using 2 protocols - long-term and short-term. We also found addition of ascorbic acid to be a feasible and simple method to "boost" collagen production rate resulting in thicker cellular material. Finally, decellularization using deoxycholic acid and CHAPS seems to be an effective way to obtain non-immunogenic and conservable cellular material that can be used in regenerative medicine and tissue engineering. We expect our study to put a basis for further research regarding construction of larger cell sheets, their purification and mechanical characteristics.

 

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