Skip to main content
SpringerLink
Log in

Cellular model based on laser microsurgery of cell spheroids to study the repair process

  • Published:
Russian Journal of Developmental Biology Aims and scope Submit manuscript

Abstract

In this study, modern techniques of laser microsurgery of cell spheroids have been used to develop a new simple, reproducible model for studying the mechanisms of repair and regeneration in vitro. Nanosecond laser pulses were applied to perform a microdissection of the outer and the inner zones of the spheroids from dermal fibroblasts. To achieve effective dissection and preservation of spheroid viability, the optimal parameters were chosen: 355 nm wavelength, 100 Hz frequency, 2 ns pulse duration, laser pulses in the range of 7–9 μJ. After microdissection, we observed injury of the spheroids: the edges of the wound surface opened and the angular opening reached a value of more than 180°. As early as during the first hour after spheroid microdissection with laser radiation, the surviving cells changed their shape: cells on the spheroid surface and directly in the damaged area became rounded. One day after microdissection, the structure of the spheroids began to partially recover, the cells in the surface layers began to take the original flattened shape; debris of dead damaged cells and their fragments was gradually cleared from the spheroid composition. In the proposed model, the first data on stimulation of structure recovery of injured spheroids from dermal fibroblasts with a P199 synthetic polypeptide, which is used in cosmetology for the initiation of antiaging and regenerative effects in the skin, were received. After microdissection, recovery of the spheroids structure with a few surface layers of flattened imbricated arranged cells and polygonal cells of the inner zone in the presence of P199 peptide was faster than in the control group, and was completed within 7 days, presumably due to the remodeling of the survived cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Alvarado, A.S. and Tsonis, P.A., Bridging the regeneration gap: genetic insights from diverse animal models, Nat. Rev. Genet., 2006, vol. 7, no. 11, pp. 873–884.

    Article  CAS  Google Scholar 

  • Antoni, D., Burcke, H., Josset, E., et al., Three-dimensional cell culture: a breakthrough in vivo, Int. J. Mol. Sci., 2015, vol. 16, no. 3, pp. 5517–5527.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Baker, B.M. and Chen, C.S., Deconstructing the third dimension–how 3D culture microenvironments alter cellular cues, J. Cell Sci., 2012, vol. 125, no. 13, pp. 3015–3024.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Belousov, L.V., Ermakov, A.S., and Luchinskaia, N.N., Cytomechanical control of morphogenesis, Tsitologiia, 1999, vol. 42, no. 1, pp. 84–91.

    Google Scholar 

  • Bely, A.E., Evolutionary loss of animal regeneration: pattern and process, Integr. Comp. Biol., 2010, vol. 50, no. 4, pp. 515–527.

    Article  PubMed  Google Scholar 

  • Bruns, T., Schickinger, S., Wittig, R., et al., Preparation strategy and illumination of three-dimensional cell cultures in light sheet-based fluorescence microscopy, J. Biomed. Opt., 2012, vol. 17, pp. 1015181–1015185.

    Article  Google Scholar 

  • Clement-Sengewald, A., Schütze, K., Ashkin, A., et al., Fertilization of bovine oocytes induced solely with combined laser microbeam and optical tweezers, J. Assist. Reprod. Genet., 1996, vol. 13, pp. 259–265.

    Article  CAS  PubMed  Google Scholar 

  • Colombelli, J., Reynaud, E.G., Rietdorf, J., et al., In vivo selective cytoskeleton dynamics quantification in interphase cells induced by pulsed ultraviolet laser nanosurgery, Traffic, 2005, vol. 6, pp. 1093–1102.

    Article  CAS  PubMed  Google Scholar 

  • Fouchard, J., Bimbard, C., Bufi, N., et al., Three-dimensional cell body shape dictates the onset of traction force generation and growth of focal adhesions, Proc. Natl. Acad. Sci. U. S. A., 2014, vol. 111, no. 36, pp. 13075–13080.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Genc, S.L., Ma, H., and Venugopalan, V., Low-density plasma formation in aqueous biological media using sub-nanosecond laser pulses, Appl. Phys. Lett., 2014, vol. 105, no. 6, p. 063701.

    Article  PubMed  PubMed Central  Google Scholar 

  • Haycock, J.W., 3D cell culture: a review of current approaches and techniques, Methods Mol. Biol., 2011, vol. 695, pp. 1–15.

    Article  CAS  PubMed  Google Scholar 

  • Heisterkamp, A., Maxwell, I.Z., Mazur, E., et al., Pulse energy dependence of subcellular dissection by femtosecond laser pulses, Opt. Express., 2005, vol. 13, no. 10, pp. 3690–3696.

    Article  CAS  PubMed  Google Scholar 

  • Il’ina, I.V., Ovchinnikov, A.V., Chefonov, O.V., et al., Noncontact microsurgery of cell membranes using femtosecond laser pulses for optoinjection of specified substances into cells, Quantum Electronics, 2013, vol. 43, no. 4, pp. 365–369.

    Article  Google Scholar 

  • Il’ina, I.V., Ovchinnikov, A.V., Sitnikov, D.S., et al., Noncontact microsurgery and delivery of substances into stem cells by means of femtosecond laser pulses, Quantum Electronics, 2014, vol. 44, no. 6, pp. 594–598.

    Article  Google Scholar 

  • Ilina, O., Bakker, G.-J., Vasaturo, A., et al., Two-photon laser-generated microtracks in 3D collagen lattices: principles of MMP-dependent and-independent collective cancer cell invasion, Phys. Biol., 2011, vol. 8, no. 1, p. 015010.

    Article  PubMed  Google Scholar 

  • Ilina, I.V., Khramova, Yu.V., Filatov, M.A., et al., Application of femtosecond laser scalpel and optical tweezers for noncontact biopsy of late preimplantation embryos, High Temperature, 2015, vol. 53, no. 6, pp. 804–809.

    Article  CAS  Google Scholar 

  • Ilina, I.V., Khramova, Yu.V., Filatov, M.A., et al., Femtosecond laser assisted hatching: dependence of zona pellucida drilling efficiency and embryo development on laser wavelength and pulse energy, High Temperature, 2016, vol. 54, no. 1, pp. 46–51.

    Article  CAS  Google Scholar 

  • Jenkins, G., Molecular mechanisms of skin ageing, Mech. Ageing Dev., 2002, vol. 123, no. 7, pp. 801–810.

    Article  CAS  PubMed  Google Scholar 

  • Khodjakov, A., La Terra, S., Chang, F., Laser microsurgery in fission yeast: role of the mitotic spindle midzone in anaphase B, Curr. Biol., 2004, vol. 14, no. 15, pp. 1330–1340.

    Article  CAS  PubMed  Google Scholar 

  • Kolokol’tsova, T.D., Saburina, I.N., and Rybakov, A.S., Cell culture as a unique model for research in modern biology and medicine, Patogenez, 2014, vol. 11, no. 2, pp. 17–25.

    Google Scholar 

  • König, K., Uchugonova, A., and Gorjup, E., Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation, Microsc. Res. Tech., 2011, vol. 74, no. 1, pp. 9–17.

    Article  PubMed  Google Scholar 

  • Kosheleva, N.V., Zurina, I.M., Saburina, I.N., et al., Effect of fetal calf serum on the formation of spheroids from eye limb stromal cells, Patogenez, 2015, vol. 13, no. 2, pp. 4–11.

    Google Scholar 

  • Kosheleva, N.V., Ilina, I.V., Zurina, I.M., et al., Laserbased technique for controlled damage of mesenchymal cell spheroids: a first step in studying reparation in vitro, Biol. Open., 2016, vol. 5, no. 7, pp. 993–1000.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kozhina, K.V., Saburina, I.N., Gorkun, A.A., et al., Comparative analysis of the effects of p199 on 2D and 3D culture of human dermal fibroblasts, Patogenez, 2015, no. 4, pp. 34–40.

    Google Scholar 

  • Kozhina, K.V., Volkova, E.N., Saburina, I.N., et al., Study of the effect of peptide bioregulators on skin aging in a 3D culture model, Ross. Zh. Kozhn. Venerich. Bol., 2016, vol. 19, no. 1, pp. 58–63.

    Google Scholar 

  • Kubatiev, A.A., Zurina, I.M., Kosheleva, N.V., et al., From 2D cell phenotypes to 3D live high-content imaging: new ways to windows, J. Cytol. Histol., 2015, vol. 6, no. 6, p. 378.

    Google Scholar 

  • Lin, R.-Z. and Chang, H.-Y., Recent advances in threedimensional multicellular spheroid culture for biomedical research, Biotechnol. J., 2008, vol. 3, nos. 9–10, pp. 1172–1184.

    Article  CAS  PubMed  Google Scholar 

  • Magidson, V., Loncarek, J., Hergert, P., et al., Laser microsurgery in the GFP era: a cell biologist’s perspective, Methods Cell Biol., 2007, vol. 82, pp. 239–266.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Makrantonaki, E. and Zouboulis, C.C., Molecular mechanisms of skin aging, Ann. N.Y. Acad. Sci., 2007, vol. 1119, no. 1, pp. 40–50.

    Article  CAS  PubMed  Google Scholar 

  • Pampaloni, F., Ansari, N., and Stelzer, E.H., High-resolution deep imaging of live cellular spheroids with lightsheet-based fluorescence microscopy, Cell Tissue Res., 2013, vol. 352, no. 1, pp. 161–177.

    Article  PubMed  Google Scholar 

  • Petrikovskii, B., Skin cell renewal of as a result of peptide regulation of activity of own stem cells, Estet. Med., 2012, no. 2, pp. 283–293.

    Google Scholar 

  • Rattan, S.I.S., Aging of skin cells in culture, in Textbook of Aging Skin, Berlin: Springer, 2010, pp. 487–492.

    Chapter  Google Scholar 

  • Rau, K.R., Quinto-Su, P.A., Hellman, A.N., et al., Pulsed laser microbeam-induced cell lysis: time-resolved imaging and analysis of hydrodynamic, Biophys. J., 2006, vol. 91, no. 1, pp. 317–329.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Repin, V.S., Saburina, I.N., Kosheleva, N.V., et al., 3D-technology of the formation and maintenance of single dormant microspheres from 2000 human somatic cells and their reactivation in vitro, Bull. Exp. Biol. Med., 2014, vol. 158, no. 1, pp. 137–144.

    Article  CAS  PubMed  Google Scholar 

  • Saburina, I.N., Gorkun, A.A., Zurina, I.M., et al., The study of the angiogenic potential of human multipotent mesenchymal stromal cells, Patogenez, 2013, vol. 11, no. 1, pp. 65–68.

    Google Scholar 

  • Sacconi, L., Tolic-Norrelykke, I.M., Antolini, R., et al., Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope, J. Biomed. Opt., 2005, vol. 10, no. 1, pp. 014002–014025.

    Article  Google Scholar 

  • Salmenperä, P., Kankuri, E., Bizik, J., et al., Formation and activation of fibroblast spheroids depend on fibronectin-integrin interaction, Exp. Cell Res., 2008, vol. 314, no. 19, pp. 3444–3452.

    Article  PubMed  Google Scholar 

  • Shekhvatova, A.S., Durnova, A.O., Kvetnoi, I.M., et al., The expression of signaling molecules under the influence of P199 peptide in Meso-Wharton preparation, In”ekts. Metody Kosmetol., 2013, no. 4, pp. 38–46.

    Google Scholar 

  • Shen, N., Datta, D., Schaffer, C.B., et al., Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor, Mech. Chem. Biosyst., 2005, vol. 2, no. 1, pp. 17–25.

    PubMed  Google Scholar 

  • Stevenson, D., Agate, B., Tsampoula, X., et al., Femtosecond optical transfection of cells: viability and efficiency, Opt. Express., 2006, vol. 14, no. 16, pp. 7125–7133.

    Article  CAS  PubMed  Google Scholar 

  • Uchugonova, A., Riemann, I., Stracke, F., et al., The influence of NIR femtosecond laser radiation on the viability of 3D stem cell clusters and tumor spheroids, in Biomedical Optics (BiOS), International Society for Optics and Photonics, 2007, p. 64421Z–64421Z–5.

    Google Scholar 

  • Uchugonova, A., König, K., Bueckle, R., et al., Targeted transfection of stem cells with sub-20 femtosecond laser pulses, Opt. Express., 2008, vol. 16, no. 13, pp. 9357–9364.

    Article  CAS  PubMed  Google Scholar 

  • Yegorov, E.E., Moldaver, M.V., Vishnyakova, Kh.S., et al., Enhanced control of proliferation in telomerized cells, Russ. J. Dev. Biol., 2007, vol. 38, no. 2, pp. 76–89.

    Article  Google Scholar 

  • Yutskovskaya, Ya.A. and Danilova, A.A., Therapy of skin with signs of chronological aging with the P199 Meso-Wharton drug: a clinical example, Plastich. Khirurg. Kosmetol., 2014, no. 3, pp. 337–496.

    Google Scholar 

  • Zhang, L. and Falla, T.J., Cosmeceuticals and peptides, Clin. Dermatol., 2009, vol. 27, no. 5, pp. 485–494.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. FSBSI “Institute of General Pathology and Pathophysiology”, Moscow, 125315, Russia

    N. V. Kosheleva, K. V. Kozhina, I. M. Zurina, A. A. Gorkun, S. G. Morozov & I. N. Saburina

  2. Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow, 125412, Russia

    I. V. Ilina, A. V. Ovchinnikov & M. B. Agranat

  3. Faculty of Biology, Lomonosov Moscow State University, Moscow, 119234, Russia

    N. V. Kosheleva & A. E. Roskova

  4. FSBEI FPE “Russian Medical Academy of Continuous Professional Education” of the Ministry of Healthcare of the Russian Federation, Moscow, 123995, Russia

    I. N. Saburina

Authors
  1. N. V. Kosheleva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I. V. Ilina
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. V. Kozhina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. I. M. Zurina
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. E. Roskova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. A. Gorkun
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A. V. Ovchinnikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M. B. Agranat
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. S. G. Morozov
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. I. N. Saburina
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to N. V. Kosheleva.

Additional information

Original Russian Text © N.V. Kosheleva, I.V. Ilina, K.V. Kozhina, I.M. Zurina, A.E. Roskova, A.A. Gorkun, A.V. Ovchinnikov, M.B. Agranat, S.G. Morozov, I.N. Saburina, 2017, published in Ontogenez, 2017, Vol. 48, No. 1, pp. 63–72.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kosheleva, N.V., Ilina, I.V., Kozhina, K.V. et al. Cellular model based on laser microsurgery of cell spheroids to study the repair process. Russ J Dev Biol 48, 56–64 (2017). https://doi.org/10.1134/S1062360417010076

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1062360417010076

Keywords

Search

Navigation