Cryopreservation and cryotechnology for regenerative medicine

The research group focuses on the development of safe, low-toxic and efficient cryopreservation as well as novel freezing and thawing methods for long-term storage and biobanking of cells, native tissues and tissue-engineered constructs. We aim to provide regenerative medicine and research with ready-to-use clinically relevant cells and materials for tissue and organ regeneration as well as drug testing and development. Our research activities are performed within interdisciplinary national and international projects.

Research

The research interests of the group are summarized below:

Investigation of optimal freezing and thawing parameters as well as new xeno-free, non-toxic freezing conditions. resulting in high viability of a cryopreserved material (clinically relevant cells, tissues and tissue-engineered constructs), cryopreservation in multiwell plates and cryobags. We develop optimal conditions to keep the cells and tissues intact and functional after cryo¬preservation.

Development of methods for induced (actively controlled) ice nucleation at cell-specific temperatures using electro-freezing, as well as cell electroporation with sugars to replace animal serum in cryopreservation solutions. Since cell membranes react differently to osmotic changes taking place during freezing and thawing, induction of ice formation at specific temperatures will enhance cell viability and tissue functionality. In order to avoid toxic cryoprotectants, such as dimethyl sulfoxide (DMSO), we are introducing electroporation of cells for controlled delivery of sugars and thus serum- and DMSO-free cryopreservation (in cooperation with Prof. Dr. Miklavčič, University of Ljubljana, Slovenia).

Development of methods for visualization and analysis of the ice crystal formation, ice growth and ice recrystallization during freezing and thawing to study the processes during cryopreservation and to develop new antifreeze agents for efficient biobanking.

Microscopy-based imaging and analysis during the cryopreservation, freeze-drying and thawing of cells and tissues as well as image processing and image segmentation (incl. deep neuronal networks). In cooperation with the Department for Biomedical Engineering of the Kharkiv National University for Radio Electronics (Prof. Avrunin) we develop software-based approaches for image processing and analysis of freezing and thawing events.

Cell protection through alginate encapsulation: Alginate hydrogels (solid and multistructural microspheres, core-shell capsules) are produced using electro¬spraying and air flow encapsulation strategies under controlled conditions. The alginate membrane shields the cells from external shear stresses, provides semi¬permeability and serves as a reservoir for the cryo¬protective agents. Alginate hydrogels are used, among other things, for drug development and testing. be used.

Rheological analysis of antifreeze solutions and study of their influence on cells during pre-treatment, freezing, storage and thawing.

Analysis of biochemical changes, cell-cell and cell-scaffold interactions during freezing, thawing and freeze-drying; study of the diffusion of antifreeze in cells and tissues under dynamic conditions in diffusion chambers.

Analysis of genetic and epigenetic changes within the cryopreserved cells and tissues. These effects determine safety of the developed cryopreservation protocols applied for long-term storage of clinically relevant stem cells and tissues.

Investigation of the hypothermic storage of cell-populated electrospun constructs with the purpose of enabling a high cell viability and functionality of the preserved tissue engineered constructs under hypothermic conditions. Comparatively, preservation under cryogenic conditions is performed in parallel for these constructs.

The research group offers a contract-related research for industry, internal and external facilities related to the use of equipment, know-how exchange and development of new methods in cryotechnology. The following equipment can be accessed upon request:
Methods for imaging and analysis of cells and tissues

  1. Fluorescence microscope Zeiss Axiovert 200M - motorized inverted microscope with temperature and humidity control for long term cell cultivation
  2. Raman/AFM Microscope - Structural, compositional and surface analysis of materials (laser 532 nm), 3D imaging, AFM contact and AC mode, time series, image stitching
  3. Spectrophotometer Biochrom Libra S22 - range of wavelengths 190-1100 nm
  4. Differential scanning calorimetry (Netzsch) - analysis of membrane stability, glass transition and heat capacity at low temperatures
  5. Cryo scanning electron microscope. Scanning electron microscope JEOL JSM-IT500LA with cryopreparation system Quorum PP3010T and JEOL-EDX system. Suitable for scanning electron microscopic examination of wet material and liquids in the low temperature range ( < -130°C)
    Analysis of cell vitality and functionality
  6. Cell Viability Analyzer Beckman Coulter Vi-Cell XR - automated and efficient analysis of cell viability by trypan blue staining
  7. Beckman Coulter Counter Multisizer 3: Cell volume analysis during addition and washout of antifreeze
    Investigation of antifreeze solutions
  8. Rheometer (Rheometrics Fluids Spectrometer RFS II) - viscoelastic properties of hydrogels, analysis of dynamic viscosity of fluids, temperature range 0°C-100°C, different measuring systems (plate-plate, cone-plate and cylinder geometries)
  9. Rotary viscometer HAAKE VT500/VT501 - measuring systems NV, MV1, MV2, MV3, temperature control with an external bath possible
    Methods of cryopreservation:
  10. Liquid Nitrogen Department (The research group is responsible for training NIFE staff on liquid nitrogen use)
  11. Controlled-Rate Freezer: Asympote VIA Freeze Research (Sterling process, mobile station, operation without LN2), Planner Kryo 560-16, Askion Workbench C-Line WB230
  12. Cryomicroscopy: Zeiss Axio Imager M1 m microscope with Linkam cryotic (freeze drying FDCS 196 and directional solidification GS350 (Linkam, UK), ice crystal formation analysis, ice recrystallization, adapter for RAMAN microscope, long distance objectives, ice formation under microscope
GROUP LEADER
Prof. Prof. h.c. Dr.-Ing., M.Sc.
Birgit Glasmacher
Director
Institute for Multiphase Processes
M.Sc.
Ghiath Alkurdi
PhD student
M.Sc.
Sven-Alexander Barker
Research assistant
Technical employee
Julia Guewa
M.Sc.
Diaa Khayyat
Research assistant

Publications (selected, 2016-2021)

  1. S.-A. Barker, B. Glasmacher, O. Gryshkov. Validating the Cryopreservation of Tissue Engineered Constructs within Cryobags. Conference proceedings 4th International Symposium on Pharmaceutical Engineering Research. 15 - 17 September 2021, TU Braunschweig - Center of Pharmaceutical Engineering, pp. 46-49.
  2. O. Gryshkov, V. Mutsenko, S. Leal-Marin, E. Riabchenko, S.-A. Barker, D. Khayyat, M. Tymkovych, A.Yu. Petrenko, O. Avrunin, D. Miklavcic, B. Glasmacher. Tissue cryopreservation: novel trends, methods and research highlights. Electroporation, Proceedings Electroporation-Based Technologies and Treatments, 2021,9, ISBN 978-961-243-423-6.
  3. D. Khayyat, B. Glasmacher, O. Gryshkov. Effect of cryopreservation on the integrity of coaxial alginate capsules. Conference proceedings 4th International Symposium on Pharmaceutical Engineering Research. 15 - 17 September 2021, TU Braunschweig - Center of Pharmaceutical Engineering, pp. 28-31.
  4. O. Gryshkov, V. Mutsenko, D. Tarusin, D. Khayyat, O. Naujok, E. Riabchenko, Y. Nemirovska, A. Danilov, A.Yu. Petrenko and B. Glasmacher. Coaxial alginate hydrogels: from self-assembled 3D cellular constructs to long-term storage. Int J Mol Sci 2021;22(6): 3096. doi: 10.3390/ijms22063096. accepted on 15.03.2021.
  5. M. Tymkovych, O. Gryshkov, K. Selivanova, V. Mutsenko, O. Avrunin, B. Glasmacher. Application of Artificial Neural Networks for Analysis of Ice Recrystallization Process for Cryopreservation. In: Jarm T., Cvetkoska A., Mahnič-Kalamiza S., Miklavcic D. (eds) 8th European Medical and Biological Engineering Conference. EMBEC 2020. IFMBE Proceedings, vol 80. Springer, Cham. https://doi.org/10.1007/978-3-030-64610-3_13.

6 M. Tymkovych, O. Gryshkov, O. Avrunin, K. Selivanova, Y. Nosova, V. Mutsenko, N. Shushliapina, B. Glasmacher. Application of SOFA Framework for Physics-Based Simulation of Deformable Human Anatomy of Nasal Cavity. In: Jarm T., Cvetkoska A., Mahnič-Kalamiza S., Miklavcic D. (eds) 8th European Medical and Biological Engineering Conference. EMBEC 2020. IFMBE Proceedings, vol 80. Springer, Cham. https://doi.org/10.1007/978-3-030-64610-3_14.

7. V. Mutsenko, M. Khasnitsky, V. Sirotinskaya, M. Müller, B. Glasmacher, I. Braslavsky, O. Gryshkov. Directional Freezing of Cell-Seeded Electrospun Fiber Mats for Tissue Engineering Applications. In: Jarm T., Cvetkoska A., Mahnič-Kalamiza S., Miklavcic D. (eds) 8th European Medical and Biological Engineering Conference. EMBEC 2020. IFMBE Proceedings, vol 80. Springer, Cham. https://doi.org/10.1007/978-3-030-64610-3_45.

8. O. Gryshkov, V. Mutsenko, J. Dermol-Černe, D. Miklavčič, B. Glasmacher. Electroporation of cell-seeded electrospun fiber mats for cryopreservation. In: Jarm T., Cvetkoska A., Mahnič-Kalamiza S., Miklavcic D. (eds) 8th European Medical and Biological Engineering Conference. EMBEC 2020. IFMBE Proceedings, vol 80. Springer, Cham. https://doi.org/10.1007/978-3-030-64610-3_55.

9 S. Leal-Marin, T. Kern, N. Hofmann, O. Pogozhykh, C. Framme, M. Börgel, C. Figueiredo, B. Glasmacher, O. Gryshkov. Human amniotic membrane: a review on tissue engineering, application, and storage. J. Biomed. Mater. Res. B 2020, 1-18, https://doi.org/10.1002/jbm.b.34782.

10, D. Pogozhykh, D. Eicke, O. Gryshkov, W.F. Wolkers, K. Schulze, C.A. Guyman, R. Blasczyk, C. Figueiredo. Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes. Int. J. Mol. Sci. 2020, 21(20), 7654, doi.org/10.3390/ijms21207654.

11. O. Pogozhykh, N. Hofmann, O. Gryshkov, C. von Kaisenberg, M. Mueller, B. Glasmacher, D. Pogozhykh, M. Börgel, R. Blasczyk, C. Figueiredo. Repeated freezing procedures preserve structural and functional properties of amniotic membrane for application in ophthalmology. Int J Mol Sci.2020;21(11): 4029, doi: 10.3390/ijms21114029.

12 V. Mutsenko, S. Knaack, L. Lauterboeck, D. Tarusin, B. Sydykov, R. Cabiscol, D. Ivnev, J. Belikan, A. Beck, D. Dipresa, A. Lode, T.El. Khassawna, M. Kampschulte, R. Scharf, A.Yu Petrenko, S. Korossis, W.F. Wolkers, M. Gelinsky, B. Glasmacher, O. Gryshkov. Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds. Cryobiology 2020;92: 215-230, doi: 10.1016/j.cryobiol.2020.01.015.

13 F. Bajerski, A. Bürger, B. Glasmacher, E.R.J. Keller, K. Müller, K. Mühldorfer, M. Nagel, H. Rüdel, T. Müller, J. Schenkel, J. Overmann. Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples. Appl Microbiol Biotechnol. 2020;104(1): 131-144.

14 V. Mutsenko, A. Barlič, T. Pezić, J. Dermol-Černe, B. Sydykov, V.F. Wolkers, I. Katkov, D. Miklavčič, B. Glasmacher, O. Gryshkov. Me2SO- and serum-free cryopreservation of mesenchymal stromal cells using electroporation of sugars. Cryobiology 2019;91: 104-114.

15. O. Gryshkov, M. Müller, S. Leal-Marin, V. Mutsenko, S. Suresh, V.M. Kapralova, B. Glasmacher. Advances in the application of electrohydrodynamic fabrication for tissue engineering. J. Phys. Conf. Ser. 2019;1236 012024, doi 10.1088/1742-6596/1236/1/012024.

16. V. Mutsenko, O. Gryshkov, O. Rogulska, A. Lode, A.Yu. Petrenko, M. Gelinsky, B. Glasmacher, H. Ehrlich. Chitinous scaffolds from marine sponges for tissue engineering. In: A.H. Choi, B. Ben-Nissan (eds.) (2018). Marine-Derived Biomaterials for Tissue Engineering Applications, Springer Series in Biomaterials Science and Engineering (SSBSE) 2019: 285-307, doi 10.1007/978-981-13-8855-2_13.

17. patent DE 10 2018 100 844 (2019, pending), Process for freezing of biological cells and/or tissue in a fluid.

18 M.V. Prykhodko, M.Y. Tymkovych, O.G. Avrunin, V.V. Mutsenko, O. Gryshkov, B. Glasmacher. Image processing for automated microscopic analysis of ice recrystallization process during isothermal annealing. Int J Bioelectromagnetism 2018;20(1): 72-75.

19. m.yu. Tymkovych, O.G. Avrunin, V.G. Paliy, M. Filtsov, O. Gryshkov, B. Glasmacher et al. Automated method for structural segmentation of nasal airways based on cone beam assisted computed tomography. Proceedings of SPIE, Chapter: 10445, Editors: Romaniuk Ryszard S., Linczuk Maciej, pp.104453F, doi: 10.1117/12.2280922.

20. V.M. Mutsenko. Cryopreservation of mesenchymal stromal cells within tissue engineering approaches (2019). PhD thesis, Hannover Medical School, Hannover.

21. V.V. Mutsenko, V.V. Bazhenov, O. Rogulska, D.N. Tarusin, K. Schütz, S. Brüggemeier et al. 3D chitinous scaffolds derived from cultivated marine demosponge Aplysina aerophoba for tissue engineering approaches based on human mesenchymal stromal cells. Int J Biol Macromol. 2017;104(B): 1966-1974.

22. V.V. Mutsenko, O. Gryshkov, L. Lauterboeck, O. Rogulska, D.N. Tarusin, V.V. Bazhenov et al. Novel chitin scaffolds derived from marine sponge Ianthella basta for tissue engineering approaches based on human mesenchymal stromal cells: biocompatibility and cryopreservation. Int J Biol Macromol. 2017;104(B): 1955-1965.

23 A. Chatterjee, D. Saha, H. Niemann, O. Gryshkov, B. Glasmacher, N. Hofmann. Effects of cryopreservation on the epigenetic profile of cells. Cryobiology 2017;74: 1-7.

24 L. Lauterboeck, W.F. Wolkers, B. Glasmacher. Cryobiological parameters of multipotent stromal cells obtained from different sources. Cryobiology 2017;74: 93-102.

25 D. Pogozhykh, Y. Pakhomova, O. Pervushina, N. Hofmann, B. Glasmacher, G. Zhegunov. Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non Penetrating Cryoprotectants. PLoS ONE 2017;12(1): e0169689, DOI: 10.1371/journal.pone.0169689.

26, L. Lauterboeck, D. Saha, A. Chatterjee, N. Hofmann, B. Glasmacher. Xeno-free cryopreservation of bone marrow derived multipotent stromal cells from Callithrix jacchus. Biopreserv Biobank. 2016;4(6): 530-538.

27 A. Chatterjee, D. Saha, B. Glasmacher, N. Hofmann. Chilling without regrets: deciphering the effects of cryopreservation on the epigenetic properties of frozen cells will benefit the applications of cryotechnology. EMBO Reports 2016;17: 292-295.

28 A. Chatterjee. Effects of cryopreservation on histone posttranslational modifications of stem cells (2016). PhD Thesis, Hannover Medical School, Hannover.

29, D. Saha. Effect of cryopreservation procedures on the viability, genetic and epigenetic stability of multipotent stromal cells (2016). PhD thesis, Hannover Medical School, Hannover.

30. l. Lauterböck. Cryopreservation of stem cells using induced nucleation (2016). PhD thesis Hannover Medical School, Hannover.

31 A. Repanas, L. Lauterboeck, D. Marvilas, B. Glasmacher. Polycaprolactone and polycaprolactone/ chitosan electrospun scaffolds for tissue engineering applications. Sch J App Med Sci. 2016:4(1C): 228-232.

32. O. Gryshkov. High voltage encapsulation of multipotent stromal cells in alginate (2015). PhD thesis, Hannover Medical School, Hannover.

33. O. Gryshkov, N. Hofmann, L. Lauterboeck, D. Pogozhykh, T. Mueller, B. Glasmacher. Multipotent Stromal Cells Derived from Common Marmoset Callithrix Jacchus within Alginate 3D Environment: Effect of Cryopreservation Procedures. Cryobiology 2015:71(1): 103-111.

34 L. Lauterboeck, N. Hofmann, T. Mueller, B. Glasmacher. Active control of the nucleation temperature enhances freezing survival of multipotent mesenchymal stromal cells. Cryobiology 2015;17(3): 384-390.

35 N. Hofmann, H. Sun, A. Chatterjee, D. Saha, B. Glasmacher. Thermal Pretreatment Improves Viability of Cryopreserved Human Endothelial Cells. Biopreserv Biobank. 2015;13: 348-355

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Keywords
Heat and mass transfer, biobanking, tissue cryopreservation, controlled freezing, freeze drying, directional solidification, cryomicroscopy, antifreeze, xeno-free cryopreservation, electroporation-assisted loading of sugars, diffusion of antifreeze.
Contact

Janina Hagedorn

+49 511 762 3822

hagedorn(at)imp.uni-hannover.de

NIFE
Stadtfelddamm 34
30625 Hanover

LUH
Institute for Multiphase Processes
At the university 1
Building 8143, 2nd floor, office 211, 30823 Garbsen

Specials

Awards

  1.  Leibniz Young Investigator Grant for Dr. Vitalii Mutsenko, project "Electroporation-assisted biopreservation of 3D tissue-engineered constructs with improved heat transfer" (2020-2022).
  2. Award (2nd place ) for best photo "Directionally grown crystals of DMSO" (for Vitalii Mutsenko), Society for Low Temperature Biology photo competition, Seville, Spain (2019).
  3. Honorary professorship for Prof. Glasmacher from Kharkiv National University of Radioelectronics in Ukraine (2018)
  4. Best Abstract Award of the Society for Cryobiology (for Vitalii Mutsenko), Madrid, Spain (2018).
  5. Minerva Short-Term Research Grant (to Vitalii Mutsenko), Hebrew University of Jerusalem, Israel (2018).
  6. IP@Leibniz Program Residency Fellowship (for Vitalii Mutsenko), Technological Educational Institute of Thessaly, Larissa, Thessalia, Greece (2017).
  7. IP@Leibniz Program Residency Fellowship (for Vitalii Mutsenko), University of Ljubljana, Slovenia (2017).
  8. Young Scientist Award for Special Scientific Achievement (for Oleksandr Gryshkov), German Refrigeration and Air Conditioning Association, Kassel, Germany (2016)
  9. Student Travel Award from Society for Cryobiology (to Vitalii Mutsenko), Ottawa, Canada (2016).
Teaching

Lectures

  1. B. Glasmacher*. "Life from the Ice": Cell viability after cryopreservation. Cryopreservation for Regenerative Medicine Workshop 2021 in the framework of the Canadian Stem Cell Network - Online workshop, part 1, session 2, 05.05.2021.
  2. B. Glasmacher, V. Mutsenko, T. Rittinghaus, D. Miklavčič, O. Gryshkov*. Cells surviving the ice age. SLTB 2019 - Annual Meeting of the Society for Low Temperature Biology, 03-04 October 2019, Seville, Spain.
  3. O. Gryshkov*. Advances in the application of electrohydrodynamic fabrication for tissue engineering. International Conference on Emerging Trends in Applied and Computational Physics, ETACP-2019, 21-22.03.2019, Saint-Petersburg, Russian Federation.
  4. O. Gryshkov*. On advancing cooperation with Ukraine. 1st International Scientific and Practical Conference "Information Systems and Technologies in Medicine", 28-30.11.2018, Kharkiv, Ukraine.
  5. V. Mutsenko, D. Tarusin, D. Zaragotas, A. Simaioforidou, C. Rozanski, B. Sydykov, D. Ivnev, O. Gryshkov, E. Anastassopoulos, B. Glasmacher*. Application of infrared video thermography for monitoring of freezing/thawing events within 3D collagen-hydroxyapatite scaffolds. SLTB 2018 - Annual Meeting of the Society for Low Temperature Biology, 06-07 September 2018, Prague, Czech Republic.
  6. B. Glasmacher. Cell and Tissue Banking, 25th Annual Meeting European. Orthopaedic Research Society (EORS 2017), Munich, Germany, 13-15.09.2017.
  7. O. Gryshkov*. Advances in biomedical engineering: own and cooperation highlights. The German-Ukrainian Forum for Young Researchers, 07-12.02.2017, Kharkiv, Ukraine.
More additional info

Further research topics and innovations of the research group "Cryogenics" of the Institute for Multiphase Processes are listed here: imp.uni-hannover

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