Sustainability of shear stress conditioning in endothelial colony-forming cells compared to human aortic endothelial cells to underline suitability for tissue-engineered vascular grafts.

The endothelialization of cardiovascular implants is supposed to improve the long-term patency of these implants. In addition, in previous studies, it has been shown, that the conditioning of endothelial cells by dynamic cultivation leads to the expression of an anti-thrombogenic phenotype. For the creation of a tissue-engineered vascular graft (TEVG), these two strategies were combined to achieve optimal hemocompatibility. In a clinical setup, this would require the transfer of the already endothelialized construct from the conditioning bioreactor to the patient. Therefore, the reversibility of the dynamic conditioning of the endothelial cells with arterial-like high shear stress (20 dyn/cm2) was investigated to define the timeframe (tested in a range of up to 24 h) for the perseverance of dynamically induced phenotypical changes. Two types of endothelial cells were compared: endothelial colony-forming cells (ECFCs) and human aortic endothelial cells (HAECs). The results showed that ECFCs respond far more sensitively and rapidly to flow than HAECs. The resulting cell alignment and increased protein expression of KLF-2, Notch-4, Thrombomodulin, Tie2 and eNOS monomer was paralleled by increased eNOS and unaltered KLF-2 mRNA levels even under stopped-flow conditions. VCAM-1 mRNA and protein expression was downregulated under flow and did not recover under stopped flow. From these time kinetic results, we concluded, that the maximum time gap between the TEVG cultivated with autologous ECFCs in future reactor cultivations and the transfer to the potential TEVG recipient should be limited to ∼6 h.

Low impact of regular PCR testing on presence at work site during the COVID-19 pandemic: experiences during an open observational study in Lower Saxony 2020-21.

BACKGROUND: Since social distancing during the COVID-19-pandemic had a profound impact on professional life, this study investigated the effect of PCR testing on on-site work. METHODS: PCR screening, antibody testing, and questionnaires offered to 4,890 working adults in Lower Saxony were accompanied by data collection on demographics, family status, comorbidities, social situation, health-related behavior, and the number of work-related contacts. Relative risks (RR) with 95 % confidence intervals were estimated for the associations between regular PCR testing and other work and health-related variables, respectively, and working on-site. Analyses were stratified by the suitability of work tasks for mobile office. RESULTS: Between April 2020 and February 2021, 1,643 employees underwent PCR testing. Whether mobile working was possible strongly influenced the work behavior. Persons whose work was suitable for mobile office (mobile workers) had a lower probability of working on-site than persons whose work was not suitable for mobile office (RR = 0.09 (95 % CI: 0.07 - 0.12)). In mobile workers, regular PCR-testing was slightly associated with working on-site (RR = 1.19 (0.66; 2.14)). In those whose working place was unsuitable for mobile office, the corresponding RR was 0.94 (0.80; 1.09). Compared to persons without chronic diseases, chronically ill persons worked less often on-site if their workplace was suitable for mobile office (RR = 0.73 (0.40; 1.33)), but even more often if their workplace was not suitable for mobile office (RR = 1.17 (1.04; 1.33)). CONCLUSION: If work was suitable for mobile office, regular PCR-testing did not have a strong effect on presence at the work site. TRIAL REGISTRATION: An ethics vote of the responsible medical association (Lower Saxony, Germany) retrospectively approved the evaluation of the collected subject data in a pseudonymized form in the context of medical studies (No. Bo/30/2020; Bo/31/2020; Bo/32/2020).

A pre-conditioning protocol of peripheral blood derived endothelial colony forming cells for endothelialization of tissue engineered constructs.

In regenerative medicine, autologous endothelial colony forming cells (ECFCs) bear the greatest potential to be used for surface endothelialization of tissue engineered constructs, as they are easily attainable and possess a high proliferation rate. The aim of this study was to develop a standardized pre-conditioning protocol under dynamic conditions simulating the physiology of human circulation to improve the formation of a flow resistant monolayer of ECFCs and to enhance the antithrombogenicity of the endothelial cells. The main focus of the study was to consequently compare the cellular behavior under a steady laminar flow against a pulsatile flow. Mononuclear cells were isolated out of peripheral blood (PB) buffy coats and plated on uncoated tissue culture flasks in anticipation of guidelines for Advanced Therapy Medicinal Products. ECFCs were identified by typical surface markers such as CD31, CD146 and VE-Cadherin. To explore the effects of dynamic cultivation, ECFCs and human umbilical vein endothelial cells were comparatively cultured under either laminar or pulsatile (1 Hz) flow conditions with different grades of shear stress (5 dyn/cm2versus 20 dyn/cm2). High shear stress of 20 dyn/cm2 led to a significant upregulation of the antithrombotic gene marker thrombomodulin in both cell types, but only ECFCs orientated and elongated significantly after shear stress application forming a confluent endothelial cell layer. The work therefore documents a suitable protocol to pre-condition PB-derived ECFCs for sustainable endothelialization of blood contacting surfaces and provides essential knowledge for future cultivations in bioreactor systems.

A new lateral flow assay to detect sIL-2R during T-cell mediated rejection after kidney transplantation.

Kidney is the most frequently transplanted among all solid organs worldwide. Kidney transplant recipients (KTRs) undergo regular follow-up examinations for the early detection of acute rejections. The gold standard for proving a T-cell mediated rejection (TCMR) is a biopsy of the renal graft often occurring as indication biopsy, in parallel to an increased serum creatinine that may indicate deterioration of renal transplant function. The goal of the current work was to establish a lateral flow assay (LFA) for diagnosing acute TCMR to avoid harmful, invasive biopsies. Soluble interleukin-2 (IL-2) receptor (sIl-2R) is a potential biomarker representing the α-subunit of the IL-2 receptor produced by activated T-cells, e.g., after allogen contact. To explore the diagnostic potential of sIL-2R as a biomarker for TCMR and borderline TCMR, plasma and urine samples were collected from three independent KTR cohorts with various distinct histopathological diagnostic findings according to BANFF (containing 112 rsp. 71 rsp. 61 KTRs). Samples were analyzed by a Luminex-based multiplex technique and cut off-ranges were determined. An LFA was established with two specific sIL-2R-antibodies immobilized on a nitrocellulose membrane. A significant association between TCMR, borderline TCMR and sIL-2R in plasma and between TCMR and sIL-2R in urine of KTRs was confirmed using the Mann-Whitney U test. The LFA was tested with sIL-2R-spiked buffer samples establishing a detection limit of 25 pM. The performance of the new LFA was confirmed by analyzing urine samples of the 2nd and 3rd patient cohort with 35 KTRs with biopsy proven TCMRs, 3 KTRs diagnosed with borderline TCMR, 1 mixed AMR/TCMR rsp. AMR/borderline TCMR and 13 control patients with a rejection-free kidney graft proven by protocol biopsies. The new point-of-care assay showed a specificity of 84.6% and sensitivity of 87.5%, and a superior estimated glomerular filtration rate (eGFR) at the time point of biopsy (specificity 30.8%, sensitivity 85%).

Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity.

Ligands used on the surface of colloidal nanoparticles (NPs) have a significant impact on physiochemical properties of NPs and their interaction in biological environments. In this study, we report a one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated methoxy polyethylene glycol (mPEG) molecules as a surface coordinating ligand. The resulting mPEG-Qdots were characterized by using ζ potential, FTIR, thermogravimetric (TG) analysis, and microscale thermophoresis (MST) studies. We investigated the effect of mPEG molecules and their grafting density on the Qdots photophysical properties, colloidal stability, protein binding affinity, and in vitro cellular toxicity. Moreover, cellular binding features of the resulting Qdots were examined by using three-dimensional (3D) tumor-like spheroids, and the results were discussed in detail. Promisingly, mPEG ligands were found to increase colloidal stability of Qdots, reduce adsorption of proteins to the Qdot surface, and mitigate Qdot-induced side effects to a great extent. Flow cytometry and confocal microscopy studies revealed that PEGylated Qdots exhibited distinctive cellular interactions with respect to their mPEG grafting density. As a result, mPEG molecules demonstrated a minimal effect on the ZnS shell deposition and the Qdot fluorescence efficiency at a low mPEG density, whereas they showed pronounced effect on Qdot colloidal stability, protein binding affinity, cytotoxicity, and nonspecific binding at a higher mPEG grafting amount.

Mobile SARS­CoV­2 screening facilities for rapid deployment and university-based diagnostic laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a public crisis. Many medical and public institutions and businesses went into isolation in response to the pandemic. Because SARS-CoV-2 can spread irrespective of a patient's course of disease, these institutions' continued operation or reopening based on the assessment and control of virus spread can be supported by targeted population screening. For this purpose, virus testing in the form of polymerase chain reaction (PCR) analysis and antibody detection in blood can be central. Mobile SARS-CoV-2 screening facilities with a built-in biosafety level (BSL)-2 laboratory were set up to allow the testing offer to be brought close to the subject group's workplace. University staff members, their expertise, and already available equipment were used to implement and operate the screening facilities and a certified diagnostic laboratory. This operation also included specimen collection, transport, PCR and antibody analysis, and informing subjects as well as public health departments. Screening facilities were established at different locations such as educational institutions, nursing homes, and companies providing critical supply chains for health care. Less than 4 weeks after the first imposed lockdown in Germany, a first mobile testing station was established featuring a build-in laboratory with two similar stations commencing operation until June 2020. During the 15-month project period, approximately 33,000 PCR tests and close to 7000 antibody detection tests were collected and analyzed. The presented approach describes the required procedures that enabled the screening facilities and laboratories to collect and process several hundred specimens each day under difficult conditions. This report can assist others in establishing similar setups for pandemic scenarios.

Combined Prospective Seroconversion and PCR Data of Selected Cohorts Indicate a High Rate of Subclinical SARS-CoV-2 Infections-an Open Observational Study in Lower Saxony, Germany.

Despite lockdown measures, intense symptom-based PCR, and antigen testing, the SARS-CoV-2 pandemic spread further. In this open observational study conducted in Lower Saxony, Germany, voluntary SARS-CoV-2 PCR tests were performed from April 2020 until June 2021, supported by serum antibody testing to prove whether PCR testing in subjects with none or few symptoms of COVID-19 is a suitable tool to manage the pandemic. In different mobile stations, 4,817 subjects from three different working fields participated in the PCR testing. Serum antibody screening using the SARS-CoV-2 ViraChip IgG (Viramed, Germany) and the Elecsys Anti-SARS-CoV-2 assay (Roche, Germany) was performed alongside virus neutralization testing. Subjects were questioned regarding comorbidities and COVID-19 symptoms. Fifty-one subjects with acute SARS-CoV-2 infection were detected of which 31 subjects did not show any symptoms possibly characteristic for COVID-19. An additional 37 subjects reported a previous SARS-CoV-2 infection (total prevalence 1.82%). Seroconversion was discovered in 58 subjects with known SARS-CoV-2 infection and in 58 subjects that never had a positive PCR test. The latter had a significantly lower Charlson Comorbidity Index, and one third of them were asymptomatic. In 50% of all seroconverted subjects, neutralizing serum antibodies (NAbs) were detectable in parallel to N/S1 (n = 16) or N/S1/S2 antigen specific antibodies (n = 40) against SARS-CoV-2. NAb titers decreased within 100 days after PCR-confirmed SARS-CoV-2 acute infection by at least 2.5-fold. A relatively high rate of subclinical SARS-CoV-2 infections may contribute to the spread of SARS-CoV-2, suggesting that in addition to other intervention strategies, systematic screening of asymptomatic persons by PCR testing may significantly enable better pandemic control. IMPORTANCE Within this open observational study, repeated PCR (n > 4,700) and antibody screening (n > 1,600) tests were offered in three different working fields. The study identified 51 subjects with acute SARS-CoV-2 infection and 37 subjects reported to have had a positive PCR test taken externally. Thirty-one of the 51 subjects did not display any symptoms prior to testing. In addition, 58 subjects without PCR-confirmed SARS-CoV-2 infection were identified by seroconversion. Subjects, that had undergone SARS-CoV-2 infection without having noticed, more often had a low grade of immunization with no NAbs, but may have relevantly contributed to the spread of the pandemic. Based on these results, we suggest that both regular PCR and rapid test screening of symptomatic and asymptomatic individuals, specifically within groups or workplaces identifiable as having close quarter contact, thus increased infection transference risk, is necessary to better assess and therefore reduce the spread of a pandemic virus.

Signs of immunosenescence correlate with poor outcome of mRNA COVID-19 vaccination in older adults.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is effective in preventing COVID-19 hospitalization and fatal outcome. However, several studies indicated that there is reduced vaccine effectiveness among older individuals, which is correlated with their general health status1,2. How and to what extent age-related immunological defects are responsible for the suboptimal vaccine responses observed in older individuals receiving SARS-CoV-2 messenger RNA vaccine, is unclear and not fully investigated1,3-5. In this observational study, we investigated adaptive immune responses in adults of various ages (22-99 years old) receiving 2 doses of the BNT162b2 mRNA vaccine. Vaccine-induced Spike-specific antibody, and T and memory B cell responses decreased with increasing age. These responses positively correlated with the percentages of peripheral naïve CD4+ and CD8+ T cells and negatively with CD8+ T cells expressing signs of immunosenescence. Older adults displayed a preferred T cell response to the S2 region of the Spike protein, which is relatively conserved and a target for cross-reactive T cells induced by human 'common cold' coronaviruses. Memory T cell responses to influenza virus were not affected by age-related changes, nor the SARS-CoV-2-specific response induced by infection. Collectively, we identified signs of immunosenescence correlating with the outcome of vaccination against a new viral antigen to which older adults are immunologically naïve. This knowledge is important for the management of COVID-19 infections in older adults.

An Antibody-Aptamer-Hybrid Lateral Flow Assay for Detection of CXCL9 in Antibody-Mediated Rejection after Kidney Transplantation.

Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.

Children and Adolescents' Behavioral Patterns in Response to Escalating COVID-19 Restriction Reveal Sex and Age Differences.

PURPOSE: The COVID-19 pandemic affects students in a myriad of different ways. Our prospective, longitudinal study in a cohort of students in Hannover, Germany explores behavioral patterns during escalating COVID-19 restrictions. METHODS: In total, 777 students between the age of 9 and 20 were assessed for their activity engagement, travel patterns, and self-assessed compliance with protective recommendations at six time points between June 2020 and June 2021 (3,564 observations) and were monitored for severe acute respiratory syndrome coronavirus 2 infection by nasal swab polymerase chain reaction and serum antibody titers. RESULTS: Activity engagement decreased, but self-assessed compliance with measures such as mask wearing and social distancing was stable during escalating restrictions. Although we found no sex difference during the summer break, when incidence was lowest, females engaged in a higher variety of activities than males for all other time points. Older students engaged in more activities and self-assigned themselves lower compliance values than younger ones. Greater involvement in different activities was seen in households which traveled more frequently. Infection rate in our cohort was low (0.03% acute infections, 1.94% positive seroprevalence). DISCUSSION: Our study supports the view that, overall, students show high compliance with COVID-19 recommendations and restrictions. The identification of subsets, such as female and older students, with higher risk behavioral patterns should be considered when implementing public information campaigns. In light of the low infection rate in our cohort, we conclude that in-person learning can occur safely if extensive protective measures are in place and the incidence in the general population remains moderate.

Comparison of two antibody screening systems for SARS-CoV-2 antibody detection in recovered and vaccinated subjects - test performance and possible indicators for immunity.

BACKGROUND: Detection of seroconversion after SARS-CoV-2-infection or vaccination is relevant to discover subclinical cases and recognize patients with a possible immunity. OBJECTIVES: Test performance, effects of age, time-point of seroconversion and immune status regarding neutralizing antibodies (NAbs) and T-cell-reactivity were investigated. STUDY DESIGN: Two antibody assays (Viramed-Test for S/N-specific IgG, Roche-Test for N-specific IgA, -M, -G) were evaluated with classified samples. In total, 381 subjects aged 6-99 years, who had either recovered from the disease or had been vaccinated, were screened for SARS-CoV-2-specific antibodies. This screening was part of an open observational study with working adults. Additionally, children and adults were analyzed in a longitudinal COVID-19 study in schools. For immunity evaluation, virus neutralization tests and ELISpot tests were performed in a subgroup of subjects. RESULTS: Viramed revealed a slightly lower test performance than Roche, but test quality was equally well in samples from very young or very old donors. The time-point of seroconversion after the respective immunization detected by the two tests was not significantly different. N-specific antibodies, detected with Roche, highly correlated with NAbs in recovered subjects, whereas a positive Viramed-Test result was paralleled by a positive ELISpot result. CONCLUSION: Viramed-Test was not as sensitive as Roche-Test, but highly specific and beneficial to distinguish between recovered and vaccinated status. For both tests correlations with humoral and cellular immunity were found. Of note, the expected early detection of IgA and IgM by the Roche-Test did not prove to be an advantage over IgG testing by Viramed.

Development of an Aptamer-Based Lateral Flow Assay for the Detection of C-Reactive Protein Using Microarray Technology as a Prescreening Platform.

For improved cost-effectiveness and temperature-stability, a ready to use lateral flow assay (LFA) is developed in this work for detecting inflammation/infection biomarker C-reactive protein (CRP) in human patient samples on the basis of aptamers. In prescreening investigations, an aptamer with CRP affinity was immobilized on microarray chips in forward and sandwich formats to optimize assay conditions. We suggest these microarray techniques as a resource-sparing and fast-screening instrument for evaluation of various conditions. The capability of the aptamer to detect CRP was shown. Optimized assay conditions were consequently transferred to the LFA-platform. Here we could demonstrate for the first time an aptamer-based LFA for the detection of CRP in human patient samples in pathologically relevant concentrations. The cutoff for CRP detection is set at 10 mg/L, providing a distinctive "yes" (≥10 mg/L CRP) or "no" (<10 mg/L CRP) answer for the patient. The resulting aptamer-based LFA is promising with regard to its application as point-of-care testing (POCT) for efficient monitoring, especially of patients affected by frequent infections or inflammations.

Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells.

Human mesenchymal stem cells derived from adipose tissue (AD-hMSCs) represent a promising source for tissue engineering and are already widely used in cell therapeutic clinical trials. Until today, an efficient and sustainable cell labeling system for cell tracking does not exist. We evaluated transient transfection through electroporation for cell labeling and compared it with lentiviral transduction for AD-hMSCs. In addition, we tested whether nonsense DNA or a reporter gene such as enhanced green fluorescent protein (EGFP) is the more suitable label for AD-hMSCs. Using electroporation, the transfection efficiency reached a maximal level of 44.6 ± 1.1% EGFP-positive cells after selective and expansive cultivation of the mixed MSC population, and was 44.5 ± 1.4% after gene transfer with Cyanin3-marked nonsense-label DNA, which remained stable during 2 weeks of nonselective cultivation (37.2 ± 4.7% positive AD-hMSCs). Electroporation with both nonsense DNA and pEGFP-N1 led to a slight growth retardation of 45.2% and 59.1%, respectively. EGFP-transfected or transduced AD-hMSCs showed a limited adipogenic and osteogenic differentiation capacity, whereas it was almost unaffected in cells electroporated with the nonsense-label DNA. The nonsense DNA was detectable through quantitative real-time polymerase chain reaction for at least 5 weeks/10 passages and in differentiated AD-hMSCs. EGFP-labeled cells were trackable for 24 h in vitro and served as testing cells with new materials for dental implants for 7 days. In contrast, lentivirally transduced AD-hMSCs showed an altered natural immune phenotype of the AD-hMSCs with lowered expression of two cell type defining surface markers (CD44 and CD73) and a relevantly decreased cell growth by 71.8% as assessed by the number of colony-forming units. We suggest electroporation with nonsense DNA as an efficient and long-lasting labeling method for AD-hMSCs with the comparably lowest negative impact on the phenotype or the differentiation capacity of the cells, which may, therefore, be suitable for tissue engineering. In contrast, EGFP transfection by electroporation is efficient but may be more suitable for cell tracking within cell therapies without MSC differentiation procedures. Since current protocols of lentiviral gene transduction include the risk of cell biological alterations, electroporation seems advantageous and sustainable enough for hMSC labeling.

Primary and Stem Cell Microarrays: Application as Miniaturized Biotesting Systems.

The deposition of living cells on microarray surfaces can be used to create physiologically relevant architecture in vitro. Such living cell microarrays enable the reconstruction of biological processes outside the body in a miniaturized format and have many advantages over traditional cell culture. The present protocol offers an option for the preparation and analysis of living primary and stem cell-based microarrays utilizing the standard microarray equipment (contact-free piezoelectric nanoprinter, microarray scanner), as well as microscopy. To produce living cell microarrays, we applied two kinds of mesenchymal stem cells (MSCs) isolated from umbilical cord and adipose tissue, as well as human umbilical vein endothelial cells (HUVECs) as model cells. We used live imaging microscopy for the online monitoring of cell spots in total size, staining of viable cells with Calcein acetoxymethyl ester (Calcein-AM) and treatment of MSCs with differentiation media to analyze the proliferation, viability, and differentiation potential of printed cells. This way, the general applicability of the established living cell-based microarray production was demonstrated.

Tacrolimus inhibits angiogenesis and induces disaggregation of endothelial cells in spheroids - Toxicity testing in a 3D cell culture approach.

The administration of immunosuppressive drugs is a necessary therapeutic measure in organ transplantation to prevent rejections. However, the use of temporarily high dosed immunosuppressive drugs is associated with cytotoxicity and adverse side effects that could induce endothelial dysfunction. The aim of this work is to evaluate the effect of the administrated drugs tacrolimus and mycophenolic acid (MPA) on human umbilical vein endothelial cells (HUVECs). Whereas MPA showed no significant toxicity in a dose-dependent manner, a dose-response curve of tacrolimus treatment could be obtained in 2D monolayer. Due to limited cell-cell and cell-extracellular matrix (ECM) interactions in 2D monolayers, 3D spheroids have been established. The comparison of IC50 values demonstrated that tacrolimus is more toxic towards endothelial cells in 3D spheroids (IC50 value = 27.19 µg/ml) than in 2D monolayers (IC50 value = 40.23 µg/ml). Moreover, the maximal trough level of tacrolimus achieved in immunosuppressive therapy (18 ng/ml) resulted in low disaggregation of the spheroids and decreased vessel areas with increased number of end points of tubular-like structures in the angiogenesis assay even if no toxic effect could be detected. Thus, our approach unseals very sensitive cytotoxic effects of tacrolimus on the vasculature in organ recipients after immunosuppressive therapy.

Development of living cell microarrays using non-contact micropipette printing.

During the last 30 years cellular screening systems were unidirectional developed towards high throughput applications on single cell level. We developed living cell microarrays, which provide an in vivo-like microenvironment for an advanced method to measure cellular response to external stimuli. To print living cells on glass slides, the classic microarray equipment, which involves printer and scanner, was fully transferred to suspensions of living cells. The microarray production was optimized using a contact-free spotting procedure in order to enhanced cell adhesion and growth rates. The printed model cells, A-549 (lung cancer cell line), were analyzed with conventional cell staining assays like DAPI (cell nuclei staining), calcein acetoxymethyl ester (viable cell staining), and CellTiter-Blue(®) Cell Viability Assay. After optimization, a reproducible (spot-to-spot variation: ± 8.6 cells) printing method for small living cell amounts (1200 cells and fewer) was established that achieved cell viabilities of up to 88% for ≥ 0.6 µL and good proliferation characteristics. Hence, this method could be advantageous for use in biomedical and diagnostic applications.

Living Cell Microarrays: An Overview of Concepts.

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays.