Method of filling polymer 3D constructions based on collagen with dermal autofibroblasts colonies

This paper presents a technique developed by us for three-dimensional cultivation of fibroblasts isolated from the dermis of 20-day-old rat embryos of the Wistar line, filling with them a matrix obtained from fish collagen by freeze drying. The method is suitable for creating scaffolds in regenerative medicine, experimental models with approximate conditions for their subsequent study for confocal, electron microscopy, implantation of tissue engineering structures in experimental animals. Objective - to develop an improved method for colonizing collagen 3D matrices with a culture of dermal autofibroblasts. Materials and methods. 3D collagen matrix carrier; experimental animals (Wistar rats), the skin of 18-20 day old embryos was used as a source of fibroblasts, from which they were isolated by trypsinization in the cold, followed by cultivation under standard conditions: 5%, CO₂, 37°С, absolute humidity, growth medium (DMEM, antibiotics (1%, streptomycin-penicillin), glutamine and fetal calf serum). The process of colonization of the matrix by cell cultures consisted of 4 consecutive stages. The filling efficiency was monitored using electron microscopy. Results. As a result of the proposed method, a sterile 3D collagen matrix was filled with pre-isolated dermal autofibroblast cells in the amount of 5×10⁶ cells/ml. According to the results of scanning electron microscopy, it was shown that the inter-fiber spaces are filled with living fibroblast cells, some of which are at the stage of division. Conclusion. The method developed by us made it possible to achieve effective colonization and adhesion of fibroblasts to the matrix and avoid the formation of cell conglomerates, improves contact with the surface and subsequent survival of the introduced cell culture. It allows to increase the rate of obtaining a donor cell culture and reduce the time of sample preparation before implantation. It also allows you to create 3D systems based on other cell types, which opens up great opportunities in reconstructive surgery.

1. Kuneyev I.K., Ivanova Yu.S., Nashchekina Yu.A. Development of a method for three-dimensional cultivation of human mesenchymal stem (stromal) cells using a cellulose matrix. Tsitologiya. 2023;65(2):170-180 (in Russ.). DOI: 10.31857/S0041377123020037. EDN: LWOREJ.

2. Kopelev P.V., Nashchokina Yu.A., Alexandrova S.A. Assessment of the viability of rabbit chondrocytes during cultivation on polylactide scaffolds intended for tissue engineering of cartilage tissue. Bulletin of innovative technologies. 2017;1(2):31-35 (in Russ.). EDN: YPLYWN.

3. Petersen E.V., Chudakova D.A., Skokova E.Yu., Reshetov I.V. Practical application of biotechnologies of three-dimensional cell cultivation in oncology and personalized therapy. Биотехнология. 2020;36(3):3-15 (in Russ.). DOI: 10.21519/0234-2758-2020-36-3-3-15. EDN: WLZKJC.

4. Volkova I.M., Korovina D.G. Three-dimensional matrices of natural and synthetic origin for cellular biotechnology. Biotekhnologiya. 2015;31(2):8-26 (in Russ.). EDN: TZQMTD.

5. Domnina A.P., Novikova P.V., Fridlyanskaya I.I., Shilina M.A., Zenin V.V. Nikolsky N.N. Induction of decidual differentiation in endometrial mesenchymal stem cells. Tsitologiya. 2015;57(12):880-884 (in Russ.). EDN: VCOJDF.

6. Shiryaeva A.I., Kosyakova K.G., Sidorov S.P., Fateev I.V., Kuzmin A.A. Cell cultures as a promising biological model in toxicological studies. Medline.ru. Rossiyskiy biomeditsinskiy zhurnal. 2019;20(16):176-182 (in Russ.). EDN: XCGNQX.

7. Denisov A., Pashkevich S. Three-dimensional cell culture and bioprinting. Nauka i innovatsii. 2023;11(249):27-31 (in Russ.). DOI: 10.29235/1818-9857-2023-11-27-31. EDN: CJCZCP.

8. Yatsenko A.A., Ustinov E.M., Leonov D.V., Kislitskiy V.M., Tseluyko S.S., Kushnarev V.A., Artemieva A.S. Cultivation of melanoma cells in vitro on a 3D scaffold prepared on the basis of gelatin. Cell and Tissue Biology. 2020;14(6):474-480. DOI: 10.1134/S1990519X20060097. EDN: KFKKEX.

9. Nadezhdin S.V., Burda Yu.E., Zubareva E.V. Proliferative activity of mesenchymal stem cells during colonization of a three-dimensional titanium matrix. Genes & cells. 2017;12(3):174 (in Russ.). EDN: YZLCNV.

10. Nikolsky V.I., Sergatsky K.I., Sheremet D.P., Shabrov A.V. Scaffold technologies in restorative medicine: the history of the problem, current state and prospects of application. Pirogov Russian journal of surgery. 2022;(11):36-40. (in Russ.). DOI: 10.17116/hirurgia202211136. EDN: KFGCHV.