Biostabilization

Biostabilization is an integral step in the development of implants and tissue-engineered constructs that have cellular components or biomolecules. Custom-designed preservation methods need to be established to ensure availability off-the-shelf in acute situations and to enable intercontinental transport. Decellularized heart valves, for example, can be freeze-dried allowing storage and transport at room temperature. Cryopreserved endothelialized biohybrid lung modules enable availability on demand.

Research

Biostabilization

The focus of the group is to develop preservation technologies for cells, tissues, and bodily fluids for disease diagnostics, therapies, genetic field studies, and genetic reserves. In order to have biological samples available for future use, they need to be preserved and stored, which is usually done at ultra-low temperatures. Cryogenic storage relies on a complex cold chain logistics both during storage as well as during transport to the clinic or site of application. Alternatively, samples can be dried so that they can be stored at room temperature. This greatly simplifies the problems associated with cryogenic storage and transport, and can be implemented in underdeveloped countries and non-laboratory settings. The main research lines of the group are:
The main research lines of the group are:

Cell preservation technology

The first and most challenging step in both cryopreservation and dry preservation methods for cells is to introduce sufficient amounts of protective agents into the cell while avoiding toxicity effects. Membranes form a diffusion barrier, and transferring cells into a solution containing protective molecules causes an osmotic imbalance, which results in deformation injury. We have established methods to load cells with protective agents taking the osmotic tolerance limits of the cell into account based on experimental observations. Experimental observations of cells during cryoprotectant loading are supported by mathematical models (analytical as well as finite element methods) used to predict the time it takes to load cells with protectants and to compute the associated deformation stress on the cell. Applications where cryopreservation is used include male and female gametes, stem cells, and blood. Freeze-dried sperm and somatic cells are used for artificial fertilization and genome reserve banking. Freeze-dried blood is used in transfusion medicine.

Tissue preservation technology

Current tissue preservation methods are limited by the size and dimensions of the tissue, which pose heat and mass transfer limitations. More specifically, the larger the sample the more difficult it is to achieve the same cooling rate at different positions within the sample. In addition, the introduction of protective molecules takes longer with increasing object size, which may cause toxicity effects if the loading process takes too long. We have developed advanced spectroscopic methods to study diffusion of protective molecules into tissues, and use mass transport models to derive diffusion constants. These findings are used to rationally design methods to load tissues with sufficient amounts of protective molecules for either cryopreservation or freeze-drying. Applications include cryopreserved ovarian tissue, freeze-dried heart valves and pericardium patches.

Liposome encapsulation and stabilization technology

Liposomes are nanoparticles composed of natural phospholipids that can be used as drug delivery vehicles or vaccines. Liposome preparation and encapsulation is done using extrusion technology and combined with addition of protectants allowing either cryogenic or dried storage. We study biophysical characteristics of liposomes in preservation formulations including membrane phase behavior of the liposomes and enthalpy relaxation phenomena below the glass transition temperature and their relation with sample storage stability.

Non-invasive spectroscopy

Fourier transform infrared spectroscopy (FTIR) is used to identify the effects of heating, freezing and/or drying on biomolecules (lipids, proteins and DNA) in cells and tissues. FTIR is combined with principal component analysis (PCA) and machine learning approaches to assess oxidative damage in cryopreserved and dried specimens for quality assurance during storage. Moreover, FTIR combined with machine learning is used for classification of bacteria in biofilms.

GROUP LEADER

Prof. Dr. Ir.

Willem Wolkers

AG-Leader

Reproductive medicine unit of the clinics

Tobias Braun

PhD student

Sükrü Caliskan

PhD student

Inola Kohrs

Medical Laboratory Assistant (MLTA)

Dejia Liu

PhD student

Harriëtte Oldenhof

Senior Staff Scientist

Publications

[1] Wolkers WF, Oldenhof H editors, (2021) Cryopreservation and Freeze-Drying Protocols, in Methods in Molecular Biology - Vol 2180, Springer, New York, 742 p, ISBN 978-1-0716-0783-1.

[2] Liu D, Caliskan S, Rashidfarokhi B, Oldenhof H, Jung K, Sieme H, Hilfiker A, Wolkers WF (2021). Fourier transform infrared spectroscopy coupled with machine learning classification for identification of oxidative damage in freeze-dried heart valves. Sci Rep 11:12299. https://doi.org/10.1038/s41598-021-91802-2.

[3] Lotz J, Içli S, Liu D, Caliskan S, Sieme H, Wolkers WF, Oldenhof H (2021). Transport processes in equine oocytes and ovarian tissue during loading with cryoprotective solutions. Biochim Biophys Acta Gen Subj 1865:129797. https://doi.org/10.1016/j.bbagen.2020.129797.

[4] Caliskan S, Oldenhof H, Brogna R, Rashidfarokhi B, Sieme H, Wolkers WF (2021). Spectroscopic assessment of oxidative damage in biomolecules and tissues. Spectrochim Acta A Mol Biomol Spectrosc 246:119003. https://doi.org/10.1016/j.saa.2020.119003.

[5] Içli S, Soleimani M, Oldenhof H, Sieme H, Wriggers P, Wolkers WF (2021). Loading equine oocytes with cryoprotective agents captured with a finite element method model. Sci Rep 11:19812. https://doi.org/10.1038/s41598-021-99287-9.

[6] Brogna R, Fan J, Sieme H, Wolkers WF, Oldenhof H (2021). Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations. Sci Rep 11:14076. https://doi.org/10.1038/s41598-021-93569-y.

[7] Pruß D, Yang H, Luo X, Liu D, Hegermann J, Wolkers WF, Sieme H, Oldenhof H (2021). High-throughput droplet vitrification of stallion sperm using permeating cryoprotective agents. Cryobiology 101:67-77. https://doi.org/10.1016/j.cryobiol.2021.05.007.

[8] Pruß D, Oldenhof H, Wolkers WF, Sieme H (2022). Alginate encapsulation of stallion sperm for increasing storage stability. Anim Reprod Sci 238:106945. https://doi.org/10.1016/j.anireprosci.2022.106945.

[9] Pruß D, Oldenhof H, Wolkers WF, Sieme H (2022). Towards increasing stallion sperm longevity by storage at subzero temperatures in the absence of ice. J Equine Vet Sci 108:103802. https://doi.org/10.1016/j.jevs.2021.103802.

[10] Brogna R, Oldenhof H, Sieme H, Wolkers WF (2020). Spectral fingerprinting to evaluate effects of storage conditions on biomolecular structure of filter-dried saliva samples and recovered DNA. Sci Rep 10:21442. https://doi.org/10.1038/s41598-020-78306-1.

[11] Brogna R, Oldenhof H, Sieme H, Figueiredo C, Kerrinnes T, Wolkers WF (2020). Increasing storage stability of freeze-dried plasma using trehalose. PLoS One 15:e0234502. https://doi.org/10.1371/journal.pone.0234502.

[12] Pogozhykh D, Eicke D, Gryshkov O, Wolkers WF, Schulze K, Guzmán CA, Blasczyk R, Figueiredo C (2020). Towards reduction or substitution of cytotoxic DMSO in biobanking of functional bioengineered megakaryocytes. Int J Mol Sci 21:7654. https://doi.org/10.3390/ijms21207654.

[13] Pflaum M, Merhej H, Peredo A, De A, Dipresa D, Wiegmann B, Wolkers W, Haverich A, Korossis S (2020). Hypothermic preservation of endothelialized gas-exchange membranes. Artif Organs 44:e552-e565. https://doi.org/10.1111/aor.13776.

[14] Zouhair S, Sasso ED, Tuladhar SR, Fidalgo C, Vedovelli L, Filippi A, Borile G, Bagno A, Marchesan M, Giorgio R, Gregori D, Wolkers WF, Romanato F, Korossis S, Gerosa G, Iop L (2020). A comprehensive comparison of bovine and porcine decellularized pericardia: new insights for surgical applications. Biomolecules 10:371. https://doi.org/10.3390/biom10030371.

[15] Zouhair S, Aguiari P, Iop L, Vásquez-Rivera A, Filippi A, Romanato F, Korossis S, Wolkers WF, Gerosa G (2019) Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availibility. Acta Biomaterialia 84:208-221. https://doi.org/10.1016/j.actbio.2018.10.026.

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

[17] Mutsenko V, Barlič A, Pezić T, Dermol-Černe J, Dovgan B, Sydykov B, Wolkers WF, Katkov II, Glasmacher B, Miklavčič D, Gryshkov O (2019). Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars. Cryobiology 91:104-114. https://doi.org/10.1016/j.cryobiol.2019.10.002.

[18] Han J, Sydykov B, Yang H, Sieme H, Oldenhof H, Wolkers WF (2019). Spectroscopic monitoring of transport processes during loading of ovarian tissue with cryoprotective solutions. Sci Rep. 2019 Oct 30;9(1):15577. https://doi.org/10.1038/s41598-019-51903-5.

[19] Wolkers WF, Oldenhof H, Tang F, Han J, Bigalk J, Sieme H (2019). Factors affecting the membrane permeability barrier function of cells during preservation technologies. Langmuir 35:7520-7528. https://doi.org/10.1021/acs.langmuir.8b02852.

[20] Vásquez-Rivera A, Oldenhof H, Hilfiker A, Wolkers WF (2019). Spectral fingerprinting of decellularized heart valve scaffolds. Spectrochim Acta A Mol Biomol Spectrosc 214:95-102. https://doi.org/10.1016/j.saa.2019.02.006.

[21] Beneke V, Küster F, Neehus AL, Hesse C, Lopez-Rodriguez E, Haake K, Rafiei Hashtchin A, Schott JW, Walter D, Braun A, Wolkers WF, , Ackermann M, Lachmann N (2018). An immune cell spray (ICS) formulation allows for the delivery of functional monocyte/macrophages. Scientific Reports 8:16281. doi:10.1038/s41598-018-34524-2.

[22] Goecke T, Theodoridis K, Tudorache I, Ciubotaru A, Cebotaria S, Ramm R, Höffler K, Sarikouch S, Vásquez-Rivera A, Haverich A, Wolkers WF, Hilfiker A (2018). In vivo performance of freeze-dried decellularized pulmonary heart valve allografts and xenografts orthotopically implanted into juvenile sheep. Acta Biomaterialia 68:41-52. doi:10.1016/j.actbio.2017.11.041.

[23] Vásquez-Rivera A, Sommer KK, Oldenhof H, Higgins AZ, Brockbank KGM, Hilfiker A, Wolkers WF (2018). Simultaneous monitoring of different vitrification solution components permeating into tissues. Analyst 143:420-428. doi:10.1039/c7an01576c.

[24] Vásquez-Rivera A, Oldenhof H, Dipresa D, Goecke T, Kouvaka A, Will F, Haverich A, Korossis S, Hilfiker A, Wolkers WF (2018). Use of sucrose to diminish pore formation in freeze-dried heart valves. Scientific Reports 8:12982. https://doi.org/10.1038/s41598-018-31388-4

[25] Sydykov B, Oldenhof H, de Oliveira Barros L, Sieme H, Wolkers WF (2018). Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions. Biochim Biophys Acta 1860:467-474. https://doi.org/10.1016/j.bbamem.2017.10.031

[26] Sydykov B, Oldenhof H, Sieme H, Wolkers WF (2018). Storage stability of liposomes stored at elevated subzero temperatures in DMSO/sucrose mixtures. PLoS One 13:e0199867. doi:10.1371/journal.pone.0199867.

[27] Zhang M, Oldenhof H, Sydykov B, Bigalk J, Sieme H, Wolkers WF (2017). Freeze-drying of mammalian cells using trehalose: preservation of DNA integrity. Scientific Reports 7:6198. https://doi.org/10.1038/s41598-017-06542-z

[28] Zhang M, Oldenhof H, Sieme H, Wolkers WF (2017). Combining endocytic and freezing-induced trehalose uptake for cryopreservation of mammalian cells. Biotechnol Prog 33:229-235. doi:10.1002/btpr.2399.

[29] Sydykov B, Oldenhof H, Sieme H, Wolkers WF (2017). Hydrogen bonding interactions and enthalpy relaxation in sugar/protein glasses. J Pharm Sci 106:761-769. doi:10.1016/j.xphs.2016.11.003.

[30] Oldenhof H, Bigalk J, Hettel C, de Oliveira Barros L, Sydykov B, Bajcsy ÁC, Sieme H, Wolkers WF (2017). Stallion sperm cryopreservation using various permeating agents: Interplay between concentration and cooling rate. Biopreserv Biobank 15:422-431. doi:10.1089/bio.2017.0061.

[30] Ertmer F, Oldenhof H, Schütze S, Rohn K, Wolkers WF, Sieme H (2017). Induced sub-lethal oxidative damage affects osmotic tolerance and cryosurvival of spermatozoa. Reprod Fertil Dev 29:1739-1750. doi:10.1071/RD16183.

[31] Oldenhof H, Zhang M, Narten K, Bigalk J, Sydykov B, Wolkers WF, Sieme H (2017). Freezing-induced uptake of disaccharides for preservation of chromatin in freeze-dried stallion sperm during accelerated aging. Biol Reprod 97:892-901. doi:10.1093/biolre/iox142.

Keywords

Bioheat and mass transfer, cryopreservation of cells and tissues, cryopreservation modeling, dry biobanking, freeze-drying, liposome encapsulation technology, macromolecular stability, membrane biophysics, molecular spectroscopy.

Contact

Prof. Dr. Wim Wolkers

+49 511 532 1313

willem.frederik.wolkers(at)tiho-hannover.de

NIFE
Stadtfelddamm 34
30625 Hanover

Specials

scholar.google

- Funding: HEMOFORCE (E/U2ED/MD010/LF551, German Federal Armed Forces), DFG (WO1735/6-1, WO1735/6-2), Hirsch foundation, Helmholz Centre for Infection research, Maschmeyer group

- Associate editor: Cryobiology

- Editorial board member: Scientific Reports

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