Publications by authors named "Nicola Hofmann"

22 Publications

  • Page 1 of 1

Cornea Procurement and Processing up to 72 Hours: No Risk for Cornea Transplant Quality.

Transfus Med Hemother 2021 Feb 8;48(1):3-11. Epub 2020 Oct 8.

German Society for Tissue Transplantation (DGFG) gGmbH, Hannover, Germany.

Background: The realization of tissue donations is bound to a tight timeframe. Depending on the type of tissue, time limits are specified within which the donation must be procured and processed. Otherwise, there is a risk of tissue quality loss with increasing time intervals from cardiovascular arrest. According to the European Directorate for the Quality of Medicines and HealthCare (EDQM) guide, cornea must be procured and processed within 72 h after death. The question arises whether this time interval has an influence on the quality of transplanted tissues and how it affects the accomplishment of tissue donations.

Methods: In order to obtain information on this, the numbers of tissue donations in the network of the German Society for Tissue Transplantation (DGFG) were evaluated as a function of the death to retrieval time (DRT) as well as the death to preservation time (DPT). For this purpose, 21,454 database entries of cornea donations made in the period from 2014 to 2018 were included.

Results: The results show that nearly 50% of donations realized in the DGFG network could be processed only 48 h or later after cardiovascular death due to the opt-in regulation in Germany. For these donations, there seems to be a higher discard rate compared to donations taken earlier. Nevertheless, there is a transplantation rate for these grafts of more than 65%, which is comparable to average transplantation rates stated in the literature.

Conclusion: All corneas finally selected for transplantation must meet the specified quality parameters. Since this naturally also applies to transplants that could only be procured at later time points, it can be concluded that DPT up to 72 h for corneal tissue is adequate and has no influence on the quality of corneas that are ultimately transplanted.
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http://dx.doi.org/10.1159/000510588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923839PMC
February 2021

Human Amniotic Membrane: A review on tissue engineering, application, and storage.

J Biomed Mater Res B Appl Biomater 2020 Dec 14. Epub 2020 Dec 14.

Institute for Multiphase Processes, Leibniz University Hannover, Garbsen, Germany.

Human amniotic membrane (hAM) has been employed as scaffolding material in a wide range of tissue engineering applications, especially as a skin dressing and as a graft for corneal treatment, due to the structure of the extracellular matrix and excellent biological properties that enhance both wound healing and tissue regeneration. This review highlights recent work and current knowledge on the application of native hAM, and/or production of hAM-based tissue-engineered products to create scaffolds mimicking the structure of the native membrane to enhance the hAM performance. Moreover, an overview is presented on the available (cryo) preservation techniques for storage of native hAM and tissue-engineered products that are necessary to maintain biological functions such as angiogenesis, anti-inflammation, antifibrotic and antibacterial activity.
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http://dx.doi.org/10.1002/jbm.b.34782DOI Listing
December 2020

Descemet membrane endothelial keratoplasty (DMEK): clinical results of precut versus surgeon-cut grafts.

Graefes Arch Clin Exp Ophthalmol 2021 Jan 26;259(1):113-119. Epub 2020 Aug 26.

Dept. of Ophthalmology, University Hospital Leipzig, Liebigstraße 10-14, 04103, Leipzig, Germany.

Purpose: This study aims to investigate possible differences in clinical outcomes between precut and surgeon-cut grafts for Descemet membrane endothelial keratoplasty (DMEK).

Methods: 142 consecutive patients who underwent DMEK were included in the study. 44 patients received precut tissues, and 98 patients received surgeon-cut tissues. Precut grafts were allocated to the patient by the German Society for Tissue Transplantation if available. We compared the outcomes of both groups for changes in visual acuity, central corneal thickness, endothelial cell density, re-bubbling rate, and graft failure rate.

Results: Patients who received precut tissues experienced similar increase in visual acuity (median change 0.4 logMAR) and decrease of corneal swelling (median change 132 μm) compared with those who received surgeon-cut tissues (median VA change 0.3 logMAR, p = 0.55, CCT change 118 μm, p = 0.63). There was no statistical difference in endothelial cell density (1436 vs. 1569 cells/mm, p = 0.37), re-bubbling (32% vs. 35%, p = 0.85), and graft failure rate (5% vs. 1%, p = 0.23). No primary graft failure occurred in the group of precut grafts.

Conclusion: Both methods lead to comparable results for visual acuity, corneal deswelling, endothelial cell density, and re-bubbling rate. A previously described higher graft failure rate for precut tissues could not be confirmed in our study. Thus, we do not see medical reasons against the use of precut tissues. There are several advantages of precut DMEK tissues over surgeon-cut tissues, especially the prevention of graft loss during preparation in the operating theater.
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http://dx.doi.org/10.1007/s00417-020-04901-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790790PMC
January 2021

Repeated Freezing Procedures Preserve Structural and Functional Properties of Amniotic Membrane for Application in Ophthalmology.

Int J Mol Sci 2020 Jun 4;21(11). Epub 2020 Jun 4.

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, 30625 Hannover, Germany.

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing-thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-β1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing.
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http://dx.doi.org/10.3390/ijms21114029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312941PMC
June 2020

Cornea donation in Germany: Obtaining consent.

Clin Transplant 2020 08 24;34(8):e13895. Epub 2020 May 24.

German Society for Tissue Transplantation, Hannover, Germany.

Tissue donation is important to reverse cornea-related blindness. Unfortunately, the willingness to make a decision concerning organ and tissue donation while still alive remains low despite all efforts. By analyzing anonymized archived data from 25 654 next-of-kin interviews from our database over a period of 5 years (2013-2018), it was found that only 20.8% of all potential cornea donors have declared their own wishes. While still alive, refusal was communicated more often than consent by potential donors. Overall consent rates were 39.2%, with parents and siblings consenting more often than other relatives and females refusing more often than male family members. Personal interviews and interviews via telephone handled by staff known to the family resulted in better consent rates (up to 75.6%) with male interviewers receiving higher consent rates in general. The gender of the approached relatives in relation to a male/female interviewer was of low importance. The results also show that it is important to allow discussion about that topic between family members-the more relatives that were involved the higher the probability of consent.
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http://dx.doi.org/10.1111/ctr.13895DOI Listing
August 2020

Comparison of preloaded grafts for Descemet membrane endothelial keratoplasty (DMEK) in a novel preloaded transport cartridge compared to conventional precut grafts.

Cell Tissue Bank 2020 Jun 4;21(2):205-213. Epub 2020 Feb 4.

Eye Clinic Sulzbach/Tissue Bank Sulzbach, Knappschaft Hospital Saar, An der Klinik 10, 66280, Sulzbach, Germany.

To determine the safety and graft quality of eye bank precut and preloaded grafts for Descemet membrane endothelial keratoplasty (DMEK) after storage and shipping in a novel preloaded transport cartridge compared to precut grafts in a conventional viewing chamber. In this laboratory proof-of-concept study, 29 human donor corneas that were unsuitable for transplantation with a mean endothelial cell density of 1948 ± 260 cells/mm were prepared using liquid bubble technique for producing precut lamellar grafts. The grafts were either preloaded into novel transport cartridge (n = 16) or transferred into conventional Krolman viewing chamber (control, n = 13). Grafts were stored for 24 or 48 h in dextran-containing medium at room temperature and subjected to a shipping simulation. Endothelial cell loss (ECL) and morphology were determined at different steps. Endothelial cell viability staining was performed with calcein dye. Mean ECL in the preloaded transport cartridge was 0.7% ± 1.2% after 24 h and 3.4% ± 1.2% (p = 0.006) after 48 h storage and injection. In the control group the ECL was mean 1.6% ± 2.7% after 24 h compared to 3.7% ± 0.9% (p = 0.042) after 48 h. The slightly higher endothelial cell loss in the viewing chamber group after 48 h was not statistically significant compared to the preloaded transport cartridge (p = 0.8). Calcein staining was comparably low in all groups and correlated with the low ECL in both groups. DMEK grafts can be preloaded into a novel transport cartridge using a "no touch" technique, stored and shipped for up to 2 days in dextran-containing medium without significant ECL.
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http://dx.doi.org/10.1007/s10561-020-09814-7DOI Listing
June 2020

Precut DMEK Using Dextran-Containing Storage Medium Is Equivalent to Conventional DMEK: A Prospective Pilot Study.

Cornea 2019 Jan;38(1):24-29

Department of Ophthalmology, Eye Clinic Sulzbach, Knappschaft Hospital Saar, Sulzbach/Saar, Germany.

Purpose: To compare the clinical outcome after Descemet membrane endothelial keratoplasty (DMEK) either as precut or conventional Descemet membrane graft preparation under standard European eye bank organ culture conditions.

Methods: This was a prospective pilot study of patients receiving either precut or conventional DMEK. Graft preparation was performed using the liquid bubble technique. Precut grafts (n = 22) were prepared 1 day before surgery in the eye bank and stored in dextran-containing organ culture medium within a transport viewing chamber. Conventional grafts (n = 29) were prepared directly before surgery. End point criteria included the endothelial cell count (ECC), central corneal thickness, graft rejection rate, rebubbling rate, and best-corrected visual acuity after 1, 3, and 6 months.

Results: A post hoc matched analysis revealed no statistically significant differences between the 2 groups. The ECC in the precut and conventional groups was comparable with an EC loss of 34% and 35%, respectively, after 6 months. The early graft failure rate, best-corrected visual acuity, and central corneal thickness were comparable between the 2 groups.

Conclusions: This pilot study shows a comparable clinical outcome after DMEK surgery for precut Descemet membrane grafts versus conventionally prepared grafts, using the liquid bubble preparation technique and storage conditions with dextran-containing medium.
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http://dx.doi.org/10.1097/ICO.0000000000001778DOI Listing
January 2019

Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants.

PLoS One 2017 10;12(1):e0169689. Epub 2017 Jan 10.

Kharkiv State Zooveterinary Academy, Mala Danylivka, Kharkiv Region, Ukraine.

Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169689PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224986PMC
August 2017

Xeno-Free Cryopreservation of Bone Marrow-Derived Multipotent Stromal Cells from Callithrix jacchus.

Biopreserv Biobank 2016 Dec 7;14(6):530-538. Epub 2016 Sep 7.

Institute for Multiphase Processes, Leibniz Universität Hannover , Hannover, Germany .

In the previous decade, numerous biobanks were established and have created large markets for the storage of bioactive compounds, cells, and tissues for medical and diagnostic applications. For in vivo clinical and therapeutic purposes, it is critical to use well-defined and xeno-free components during cultivation, preservation, and transplantation of biological material. Safe and efficacious storage of bioactive molecules, cells, and tissues, without the addition of undefined medium components, minimizes risks of zoonotic disease transmission and is thus an essential and desirable prerequisite for biobanks. This gives rise to a need for well-characterized and serum-free freezing media for application in cryopreservation. For this purpose, cryobiological additives such as methylcellulose, poloxamer-188, and α-tocopherol, which have previously been shown to exhibit a cytoprotective activity, have been investigated for cryoprotection on stem cells. With this strategy, the application of fetal bovine serum (FBS) could be avoided and the concentration of toxic cryoprotective agents such as dimethyl sulfoxide (DMSO) could be reduced. Our results suggest that the viability, as well as the adipogenic and osteogenic differentiation capacity of the thawed bone marrow-derived multipotent stromal stem cells, could be maintained using a freezing medium without FBS consisting of methylcellulose, poloxamer, and α-tocopherol with only 2.5% DMSO (% v/v).
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http://dx.doi.org/10.1089/bio.2016.0038DOI Listing
December 2016

Streptobacillus notomytis sp. nov., isolated from a spinifex hopping mouse (Notomys alexis Thomas, 1922), and emended description of Streptobacillus Levaditi et al. 1925, Eisenberg et al. 2015 emend.

Int J Syst Evol Microbiol 2015 Dec;65(12):4823-4829

Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany.

A pleomorphic, Gram-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium was isolated in 1979 from the heart of a spinifex hopping mouse (Notomys alexis Thomas, 1922) with septicaemia and stored as Streptobacillus moniliformis in the strain collection of the Animal Health Laboratory, South Perth, Western Australia (AHL 370-1), as well as under CCUG 12425. On the basis of 16SrRNA gene sequence analyses, the strain was assigned to the genus Streptobacillus, with 99.4 % sequence similarity to the type strain of Streptobacillus moniliformis, 95.6 %sequence similarity to the type strain of Streptobacillus hongkongensis and 99.0 %sequence similarity to the type strain of Streptobacillus felis. The clear differentiation of strain AHL 370-1T from Streptobacillus moniliformis, Streptobacillus hongkongensis and Streptobacillus felis was also supported by rpoB, groEL and recA nucleotide and amino acid sequence analysis. Average nucleotide identity was 87.16 % between strain AHL 370-1T and Streptobacillus moniliformis DSM 12112T. Physiological data confirmed the allocation of strain AHL 370-1T to the family Leptotrichiaceae, considering the very similar profiles of enzyme activities and fatty acids compared to closely related species. Within the genus Streptobacillus,isolate AHL 370-1T could also be separated unambiguously from the type strains of Streptobacillus moniliformis, Streptobacillus hongkongensis and Streptobacillus felis by MALDI-TOF mass spectrometry. Two further strains (KWG2 and KWG24) isolated from asymptomatic black rats in Japan were highly similar to AHL 370-1T. On the basis of these data, we propose the novel species Streptobacillus notomytis sp. nov., with the type strain AHL370-1T (=CCUG 12425T=DSM 100026T=CCM 8593T=EF 12425T).
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http://dx.doi.org/10.1099/ijsem.0.000654DOI Listing
December 2015

Thermal Pretreatment Improves Viability of Cryopreserved Human Endothelial Cells.

Biopreserv Biobank 2015 Oct 29;13(5):348-55. Epub 2015 Sep 29.

Institute for Multiphase Processes, Leibniz Universitaet Hannover , Hannover, Germany .

A high survival rate of cryopreserved cells requires optimal cooling and thawing rates in the presence of a cryoprotective agent (CPA) or a combination of CPAs in adequate concentrations. One of the most widely used CPAs, dimethyl sulfoxide (Me2SO), however is toxic at high concentrations and has detrimental effects on cellular functions. Additional processing steps are necessary to remove the CPA after thawing, which make the process expensive and time consuming. Therefore it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. The aim of this study was to investigate if thermal activation of human pulmonary microvascular endothelial cells (HPMEC ST-1.6R), prior to cryopreservation, could improve their post-thaw viability since the resulting heat shock protein expression acts as an intrinsic cellular protection mechanism. The results of this study suggest that both heat and cold shock pretreatments improve cryopreservation outcome of the HPMEC ST-1.6R cells. By re-cultivating cells after heat shock treatment before cryopreservation, a significant increase in cellular membrane integrity and adherence capacity could be achieved. However a combination of thermal activation and cryopreservation with alternative CPAs such as ectoine and L-proline could not further enhance the cell viability. The results of this study showed that pretreatment of endothelial cells with thermal activation could be used to reduce the Me2SO concentration required in order to preserve cell viability after cryopreservation.
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http://dx.doi.org/10.1089/bio.2015.0024DOI Listing
October 2015

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus.

PLoS One 2015 7;10(8):e0134312. Epub 2015 Aug 7.

Institute of Hygiene and Infectious Diseases of Animals, Giessen, Germany.

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134312PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529157PMC
May 2016

Multipotent stromal cells derived from common marmoset Callithrix jacchus within alginate 3D environment: Effect of cryopreservation procedures.

Cryobiology 2015 Aug 14;71(1):103-11. Epub 2015 May 14.

Institute for Multiphase Processes, Leibniz Universitaet Hannover, Callinstrasse 36, 30167 Hannover, Germany. Electronic address:

Multipotent stromal cells derived from the common marmoset monkey Callithrix jacchus (cjMSCs) possess high phylogenetic similarity to humans, with a great potential for preclinical studies in the field of regenerative medicine. Safe and effective long-term storage of cells is of great significance to clinical and research applications. Encapsulation of such cell types within alginate beads that can mimic an extra-cellular matrix and provide a supportive environment for cells during cryopreservation, has several advantages over freezing of cells in suspension. In this study we have analysed the effect of dimethyl sulfoxide (Me2SO, 2.5-10%, v/v) and pre-freeze loading time of alginate encapsulated cjMSCs in Me2SO (0-45 min) on the viability and metabolic activity of the cells after freezing using a slow cooling rate (-1°C/min). It was found that these parameters affect the stability and homogeneity of alginate beads after thawing. Moreover, the cjMSCs can be frozen in alginate beads with lower Me2SO concentration of 7.5% after 30 min of loading, while retaining high cryopreservation outcome. We demonstrated the maximum viability, membrane integrity and metabolic activity of the cells under optimized, less cytotoxic conditions. The results of this study are another step forward towards the application of cryopreservation for the long-term storage and subsequent applications of transplants in cell-based therapies.
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http://dx.doi.org/10.1016/j.cryobiol.2015.05.001DOI Listing
August 2015

Encapsulating non-human primate multipotent stromal cells in alginate via high voltage for cell-based therapies and cryopreservation.

PLoS One 2014 26;9(9):e107911. Epub 2014 Sep 26.

Institute for Multiphase Processes, Leibniz Universitaet Hannover, Hannover, Germany.

Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15-30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107911PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178041PMC
June 2015

Process engineering of high voltage alginate encapsulation of mesenchymal stem cells.

Mater Sci Eng C Mater Biol Appl 2014 Mar 7;36:77-83. Epub 2013 Dec 7.

Institute for Multiphase Processes, Leibniz University Hannover, D-30167 Hannover, Germany. Electronic address:

Encapsulation of stem cells in alginate beads is promising as a sophisticated drug delivery system in treatment of a wide range of acute and chronic diseases. However, common use of air flow encapsulation of cells in alginate beads fails to produce beads with narrow size distribution, intact spherical structure and controllable sizes that can be scaled up. Here we show that high voltage encapsulation (≥ 15 kV) can be used to reproducibly generate spherical alginate beads (200-400 μm) with narrow size distribution (± 5-7%) in a controlled manner under optimized process parameters. Flow rate of alginate solution ranged from 0.5 to 10 ml/h allowed producing alginate beads with a size of 320 and 350 μm respectively, suggesting that this approach can be scaled up. Moreover, we found that applied voltages (15-25 kV) did not alter the viability and proliferation of encapsulated mesenchymal stem cells post-encapsulation and cryopreservation as compared to air flow. We are the first who employed a comparative analysis of electro-spraying and air flow encapsulation to study the effect of high voltage on alginate encapsulated cells. This report provides background in application of high voltage to encapsulate living cells for further medical purposes. Long-term comparison and work on alginate-cell interaction within these structures will be forthcoming.
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http://dx.doi.org/10.1016/j.msec.2013.11.048DOI Listing
March 2014

Compatible solutes improve cryopreservation of human endothelial cells.

Cryo Letters 2012 Nov-Dec;33(6):485-93

Institute for Multiphase Processes, Leibniz Universität Hannnover, Hannover, Germany.

Dimethyl sulfoxide (DMSO) is till now a widely used cryoprotective agent for cryopreservation of cells. Since high concentrations of DMSO have detrimental effects on cell functioning the aim of this study is to reduce the DMSO concentration by using compatible solutes (CS). These are small organic osmolytes that microorganisms synthesize and accumulate to counteract stress factors. Three CS, hydroxyectoine, ectoine and L-proline were investigated as cryoprotective agents for the cryopreservation of the human endothelial cell line HPMEC-ST1.6R. They were either supplemented to freezing or to cell culture medium. L-proline was the most effective CS, the efficiency of recultivation was improved by more than 100 percent. A combination of L-proline and ectoine in the cell culture medium resulted in an improvement by 63 percent. Our results show that L-proline and ectoine could be used as additional CPAs to improve the cryopreservation of human endothelial cells in the presence of low DMSO concentration.
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March 2013

Video analysis of osmotic cell response during cryopreservation.

Cryobiology 2012 Jun 16;64(3):250-60. Epub 2012 Feb 16.

Institute for Multiphase Processes, Leibniz Universitaet Hannover, Germany.

Cellular response during the freeze-thaw process strongly affects the cryopreservation outcome including cell morphology and cell viability. Cryomicroscopy was used to individually analyze the osmotic response of human pulmonary microvascular endothelial cells (HPMECs) during slow cooling (1 °C/min) to -60 °C and fast rewarming to 4 °C (100 °C/min). The ice nucleation temperature was controlled (T(n)=-8 °C). Different concentrations of different cryoprotectant agents, dimethyl sulfoxide, ethylene glycol, proline, ectoin, and trehalose resulted in various cell volume changes. The described methods for image processing and computer vision allows for a fully automatic and individual analysis of the osmotically driven cell response under a temporal resolution of 2 frames per second. As a result, we show that in the presence of dimethyl sulfoxide or ethylene glycol cells shrink during cooling to a high degree, especially at intermediate molar concentrations in the range between 0 and 2M, while during rewarming cells swell to isotonic volumes gradually. Comparative cell vitality tests, membrane integrity, and viability tests after 24h recultivation, under these conditions show a high cell survival. In the absence of cryoprotective agents or with proline, ectoin or trehalose, osmotic shrinkage did not meet our expectations: a freeze-induced swelling was detected during cooling and an extreme swelling was observed after rewarming, which was accompanied by lower comparative cell viability. A linear correlation between the cellular membrane integrity after cryopreservation and the maximal relative cell volume was derived (R(2)=96). The results clearly show that it is crucial to analyze cells within a sample individually due to their individual different osmotic response.
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http://dx.doi.org/10.1016/j.cryobiol.2012.02.008DOI Listing
June 2012

Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey.

Biochem Biophys Res Commun 2011 Jul 25;411(2):317-22. Epub 2011 Jun 25.

Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany.

In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65μm were optimal for cell seeding and cultivating. However, before clinical application and transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human. Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells.
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http://dx.doi.org/10.1016/j.bbrc.2011.06.134DOI Listing
July 2011

Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells.

Cryobiology 2011 Oct 17;63(2):67-75. Epub 2011 May 17.

University of Applied Sciences Giessen - Friedberg, Institute of Bioprocess Engineering and Pharmaceutical Technology, Wiesenstrasse 14, 35390 Giessen, Germany.

Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me(2)SO), is toxic at high concentrations at temperatures >4°C and has harmful effects on the biological functionality of stem cell as well as on treated patients. Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me(2)SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from -16 to -25°C. The CPAs, beside Me(2)SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.
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http://dx.doi.org/10.1016/j.cryobiol.2011.05.002DOI Listing
October 2011

Laser printing of stem cells for biofabrication of scaffold-free autologous grafts.

Tissue Eng Part C Methods 2011 Jan 30;17(1):79-87. Epub 2010 Aug 30.

1 Laser Zentrum Hannover e. V., Hannover, Germany.

Stem cells are of widespread interest in regenerative medicine due to their capability of self-renewal and differentiation, which is regulated by their three-dimensional microenvironment. In this study, a computer-aided biofabrication technique based on laser-induced forward transfer (LIFT) is used to generate grafts consisting of mesenchymal stem cells (MSCs). We demonstrate that (i) laser printing does not cause any cell damage; (ii) laser-printed MSC grafts can be differentiated toward bone and cartilage; (iii) LIFT allows printing of cell densities high enough for the promotion of chondrogenesis; (iv) with LIFT three-dimensional scaffold-free autologous tissue grafts can be fabricated keeping their predefined structure, and (v) predifferentiated MSCs survived the complete printing procedure and kept their functionality. We believe that our results will find important applications in stem cell biology and tissue engineering.
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http://dx.doi.org/10.1089/ten.TEC.2010.0359DOI Listing
January 2011

The biological effects of cell-delivered brain-derived neurotrophic factor on cultured spiral ganglion cells.

Neuroreport 2007 Oct;18(16):1683-6

Department of Otorhinolaryngology, Head and Neck Surgery, Hannover Medical School, Hannover, Germany.

The benefit achieved by the use of cochlear implants depends among other factors on the number of surviving spiral ganglion cells (SGCs). Neurotrophic factors, especially brain-derived neurotrophic factor (BDNF), have a protective effect on spiral ganglions. Coating of the cochlear implant electrode with BDNF-producing cells may provide long-term delivery of the factor. Therefore, the hypothesis that BDNF-producing fibroblasts can enhance cell survival of cultured SGCs was tested. Lentiviral infection of fibroblasts resulted in BDNF production. Conditioned medium obtained from infected fibroblasts was used for the cultivation of SGCs. As a result, improved survival and neurite outgrowth was observed on SGCs. Our results demonstrate that lentivirally infected fibroblasts produce BDNF that has neurotrophic effects on spiral ganglions.
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http://dx.doi.org/10.1097/WNR.0b013e3282f0b5d7DOI Listing
October 2007