Effects of Repeated Freeze and Thaw Cycles on the Genome-Wide DNA Methylation Profile of Isolated Genomic DNA.

The characterization of DNA methylation patterns to identify epigenetic markers for complex human diseases is an important and rapidly evolving part in biomedical research. DNA samples collected and stored in clinical biobanks over the past years are an important source for future epigenetic studies. Isolated gDNA is considered stable when stored at low temperatures for several years. However, the effect of multiple use and the associated repeated thawing of long-term stored DNA samples on DNA methylation patterns has not yet been investigated. In this study, we examined the influence of up to 10 freeze and thaw cycles on global DNA methylation by comparing genome-wide methylation profiles. DNA samples from 19 healthy volunteers were either frozen at -80°C or subjected to up to 10 freeze and thaw cycles. Genome-wide DNA methylation was analyzed after 0, 1, 3, 5, or 10 thaw cycles using the Illumina Infinium MethylationEPIC BeadChip. Evaluation of the global DNA methylation profile by beta-value density plots and multidimensional scaling plots revealed an expected clear participant-dependent variability, but a very low variability depending on the freeze and thaw cycles. In accordance, no significant difference in any of the methylated cytosine/guanine sites studied could be detected in the performed statistical analyses. Our results suggest that long-term frozen DNA samples are still suitable for epigenetic studies after multiple thaw cycles.

The German National Pandemic Cohort Network (NAPKON): rationale, study design and baseline characteristics.

The German government initiated the Network University Medicine (NUM) in early 2020 to improve national research activities on the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic. To this end, 36 German Academic Medical Centers started to collaborate on 13 projects, with the largest being the National Pandemic Cohort Network (NAPKON). The NAPKON's goal is creating the most comprehensive Coronavirus Disease 2019 (COVID-19) cohort in Germany. Within NAPKON, adult and pediatric patients are observed in three complementary cohort platforms (Cross-Sectoral, High-Resolution and Population-Based) from the initial infection until up to three years of follow-up. Study procedures comprise comprehensive clinical and imaging diagnostics, quality-of-life assessment, patient-reported outcomes and biosampling. The three cohort platforms build on four infrastructure core units (Interaction, Biosampling, Epidemiology, and Integration) and collaborations with NUM projects. Key components of the data capture, regulatory, and data privacy are based on the German Centre for Cardiovascular Research. By April 01, 2022, 34 university and 40 non-university hospitals have enrolled 5298 patients with local data quality reviews performed on 4727 (89%). 47% were female, the median age was 52 (IQR 36-62-) and 50 pediatric cases were included. 44% of patients were hospitalized, 15% admitted to an intensive care unit, and 12% of patients deceased while enrolled. 8845 visits with biosampling in 4349 patients were conducted by April 03, 2022. In this overview article, we summarize NAPKON's design, relevant milestones including first study population characteristics, and outline the potential of NAPKON for German and international research activities.Trial registration https://clinicaltrials.gov/ct2/show/NCT04768998 . https://clinicaltrials.gov/ct2/show/NCT04747366 . https://clinicaltrials.gov/ct2/show/NCT04679584.

Establishment of a cohort for deep phenotyping of the immune response to influenza vaccination among elderly individuals recruited from the general population.

Elderly individuals have the highest burden of disease from influenza infection but also the lowest immune response to influenza vaccination. A better understanding of the host response to influenza vaccination in the elderly is therefore urgently needed. We conducted a biphasic prospective, population-based study from Dec. 2014 to May 2015 (pilot study) and Sept. 2015 to May 2016 (main study). Individuals 65-80 y of age were randomly selected from the residents' registration office in Hannover, Germany, for the pilot (n = 34) and main study (n = 200). The pilot study tested recruitment for study arms featuring 2, 4, or 5 visits/blood draws. The 5-visit (day 0, 1/3, 7, 21, 70 with respect to vaccination) study arm was selected for the main study. Both studies featured vaccination with Fluad™ (Novartis, Italy), a detailed medical history, a physical exam, recording of adverse events, completion of a questionnaire on common infections and an end-of-study questionnaire, and blood samples. Response rates in the pilot and main studies were 3.7% and 4.0%, respectively. Willingness to participate did not differ among the study arms (Fisher's exact test, p = 0.44). In both studies, there were no losses to follow-up. Compliance with study visits, blood sampling and completion of the questionnaires was very high (100%, >97%, 100%, respectively), as were participants' acceptance of and satisfaction with both phases of the study. The low response rates indicate the need for optimized recruitment strategies if the study population is to be representative of the general population. Nonetheless, the complex prospective study design proved to be highly feasible.

Motivations for (non)participation in population-based health studies among the elderly - comparison of participants and nonparticipants of a prospective study on influenza vaccination.

BACKGROUND: Participation in epidemiological studies has strongly declined in recent years. We examined the reasons for (non)participation in population-based health studies among participants and nonparticipants of a prospective study on influenza vaccination among the elderly. METHODS: Males and females between 65 and 80 years of age (N = 5582) were randomly selected from the residents' registration office in Hannover, Germany, and were invited to participate in a study featuring vaccination with a seasonal adjuvanted influenza vaccine (Fluad™, Novartis) including five follow-up visits (day 0, 1/3, 7, 21, 70 with respect to vaccination). A 24-item nonresponder questionnaire, including 10 items on reasons for participating in a hypothetical health study, was mailed to 1500 randomly selected nonparticipants. The same 10 items were included in the end-of-study questionnaire administered to the participants in the vaccination study (n = 200). Logistic regression analysis with backward elimination was used to identify the reasons most strongly associated with nonparticipation. RESULTS: Five hundred thirty-one (35%) nonparticipants and 200 participants (100%) returned the respective questionnaires. Nonparticipation was associated with a lower interest in obtaining personal health information (OR = 3.32) and a preference for less invasive (OR = 3.01) and less time-demanding (OR = 2.19) studies. Responses to other items, e.g. regarding altruistic motives, monetary compensation, general interest of the study, or study approval through ethics committee and data security authority, did not differ between participants and nonparticipants. CONCLUSIONS: Participation rates in health studies among elderly individuals could potentially be improved by reducing interventions and time demand, for instance by implementing methods of self-sampling and remote data collection. TRIAL REGISTRATION: No. 1100359 (ClinicalTrials.gov, date of registration: 09.02.2015).

[Centralized biobanks: a basis for medical research]. / Zentralisierte Biobanken als Grundlage für die medizinische Forschung.

Biobanks are the basis for a substantial part of biomedical research. The development, establishment and operation of biobanks are connected to a broad range of aspects, mainly concerning the preparation, storage, usage and dissemination of samples and associated data, in addition to the social and public involvement of these processes. These complex requirements can often only be managed in large centralized biobanks. In recent years, centralized clinical biobanks have been established in several university clinics in Germany. Similar activities take place in other European countries and worldwide. This article highlights the requirements and main tasks of centralized clinical biobanks: high-quality pre-analytics and sample storage, the creation of professional IT structures, data protection, ethical issues, in addition to quality and project management.

Induced pluripotent stem cells generated from adult bone marrow-derived cells of the nonhuman primate (Callithrix jacchus) using a novel quad-cistronic and excisable lentiviral vector.

Regenerative medicine is in need of solid, large animal models as a link between rodents and humans to evaluate the functionality, immunogenicity, and clinical safety of stem cell-derived cell types. The common marmoset (Callithrix jacchus) is an excellent large animal model, genetically close to humans and readily used worldwide in clinical research. Until now, only two groups showed the generation of induced pluripotent stem cells (iPSCs) from the common marmoset using integrating retroviral vectors. Therefore, we reprogrammed bone marrow-derived mesenchymal cells (MSCs) of adult marmosets in the presence of TAV, SB431542, PD0325901, and ascorbic acid via a novel, excisable lentiviral spleen focus-forming virus (SFFV)-driven quad-cistronic vector system (OCT3/4, KLF4, SOX2, C-MYC). Endogenous pluripotency markers like OCT3/4, KLF4, SOX2, C-MYC, LIN28, NANOG, and strong alkaline phosphatase signals were detected. Exogenous genes were silenced and additionally the cassette was removed with a retroviral Gag precursor system. The cell line could be cultured in absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) and could be successfully differentiated into embryoid bodies and teratomas with presence of all three germ layers. Directed differentiation generated neural progenitors, megakaryocytes, adipocytes, chondrocytes, and osteogenic cells. Thus, all criteria for fully reprogrammed bone marrow-MSCs of a nonhuman primate with a genetically sophisticated construct could be demonstrated. These cells will be a promising tool for future autologous transplantations.

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

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.

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

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.

Diffusion of dimethyl sulfoxide in tissue engineered collagen scaffolds visualized by computer tomography.

Cryopreservation is a convenient method for long-term preservation of natural and engineered tissues in regenerative medicine. Homogeneous loading of tissues with CPAs, however, forms one of the major hurdles in tissue cryopreservation. In this study, computer tomography (CT) as a non-invasive imaging method was used to determine the effective diffusion of Me2SO in tissue-engineered collagen scaffolds. The dimensions of the scaffolds were 30 x 30 x 10 mm3 with a homogeneous pore size of 100 microm and a porosity of 98%. CT images were acquired after equilibrating the scaffolds in phosphate buffered saline (PBS) and transferring them directly in 10% (v/v)Me2SO. The Me2SO loading process of the scaffold could thus be measured and visualized in real time. The experimental data were fitted using a diffusion equation. The calculated effective diffusion constant for Me2SO in the PBS loaded scaffold was determined from experimental diffusion studies to be 2.4 x 10(-6) cm2/s at 20 degrees C.