Journal of Cellular Biotechnology - Volume 4, issue 1-2

Authors: Rödiger, Stefan | Kammerer, Sarah | Schmailzl, Kurt J.G. | Küpper, Jan-Heiner | Jung, Friedrich

Article Type: Editorial

DOI: 10.3233/JCB-189001

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 1-3, 2018

Authors: Rosanski, Meike | Sauer, Lysann | Schröder, Christian | Stohwasser, Ralf

Article Type: Research Article

Abstract: The multifunctional proteasome activator PA28γ is involved in proteolytic pathways. Not surprisingly, upregulation of PA28γ contributes to tumor growth and progression. Therefore, CRISPR/Cas9 technology was used to specifically knockout the PSME3 gene encoding PA28γ in S462 tumor cells and HEK 293 cells. RNA guide sequences specific for PSME3 were designed and cloned into the lentiCRISPRv2 expression vector. Initially, successful Cas9-mediated gene-editing was verified by T7 endonuclease I DNA-mismatch assay. After the clonal selection, several clones showed no detectable PA28γ expression on protein level. Subsequently, a complete knockout of PA28γ was confirmed on genomic …level by sequencing. Initial functional analysis of effector caspase activity in PA28γ knockout cells showed a higher basal activity and an increased sensitivity towards doxorubicin/ABT-737-induced apoptosis. Generally, this confirms the role of PA28γ as an anti-apoptotic regulator. To our knowledge, this is the first study addressing the role of proteasomal regulators in malignant peripheral nerve sheath tumor (MPNST) derived S462 cells. Show more

Keywords: CRISPR/Cas9, gene-editing, DNA-mismatch assay, PSME3, apoptosis, MPNST

DOI: 10.3233/JCB-189002

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 5-13, 2018

Authors: Jurischka, C. | Dinter, F. | Sowa, M. | Noack, J. | Schiebel, J. | Roggenbuck, D. | Schierack, P. | Rödiger, S.

Article Type: Research Article

Abstract: Quantitative analysis of a target molecule in a microbead-based fluorescent assay requires a specific labeling procedure. For nucleic acid analysis the hybridization with florescent labeled oligonucleotides is the most common method. However, disadvantages are the necessity for direct labeling of probes and the sensitivity to detect low amounts of target molecules. In this study we established an alternative detection method for biomolecules on microbeads, the tyramide signal amplification (TSA). Hereby, biomolecules are detected by enzymatically activated and fluorophore-conjugated tyramides that bind to specific protein residues. This method has proven to be a versatile and robust enzyme amplification technique for sensitive …immunohistochemical detection. Now, we present the feasibility of the TSA procedure to detect hybridized biotinylated oligonucleotide probes bound to protein coated microbead surfaces TSA was performed using fluorescent, size-encoded and streptavidin coated microbeads that were loaded with dual-biotinylated DNA capture probes, prepared from polymerase chain reaction. Beside streptavidin alone for surface coating of those microbeads, we applied different quantities of streptavidin in combination with bovine serum albumin, immunoglobulin G or Protein G/A, to check for positive effects on the resulting signal intensities through specific binding of tyramide molecules. For this method, streptavidin turned out as appropriate protein for the surface binding, without the need for further molecules. In comparison to a standard detection with common streptavidin-fluorophore-conjugates TSA showed its advantage in the detection of low probe amounts down to a concentration of 3.3·10-4  ng/μL. Show more

Keywords: Tyramide signal amplification, microbead, nucleic acid detection, biotinylated probe, hybridization

DOI: 10.3233/JCB-189003

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 15-22, 2018

Authors: Kagel, Heike | Frohme, Marcus | Glökler, Jörn

Article Type: Research Article

Abstract: BACKGROUND: After excitation with light photoacids can change the pH in a solution by release of a proton. They have been used mostly for excited state proton transfer studies. In this review the general functionality and mechanisms and the subdivision of photoacids is explained. STATE OF THE ART: Different uses of photoacids are described, covering a wide range of various biochemical topics, focusing on biochemical applications. Examples for the introduced subdivisions are covered. CONCLUSIONS AND OUTLOOK: The areas in which photoacids can be employed are diverse. Photoacids have a promising future in biotechnology and biochemistry and …should be considered for upcoming applications, especially in non-invasive control of biochemical reactions. Show more

Keywords: Photoacids, metastable photoacids, light induced, pH jump, HPTS

DOI: 10.3233/JCB-189004

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 23-30, 2018

Authors: Lutter, Anne-Helen | Liedtke, Victoria | Scholka, Jenny | Muschter, Antje | Becker, Roland | Anderer, Ursula

Article Type: Research Article

Abstract: BACKGROUND: Tissue engineering has become a major field of research in biotechnology and biomedicine. As a consequence, cell-based therapeutic approaches are entering the hospitals, especially for skeletal regeneration. Traumatic injuries of cartilage are treated with autologous cell suspensions or in vitro generated cartilage tissues, but there is actually no therapy available for degenerative cartilage defects. However, Osteoarthritis (OA) is a major public health problem in the world affecting 240 million people globally. OBJECTIVE: To develop suitable in vitro tissues, the properties of chondrogenic spheroids should be optimized via co-culture with cells naturally occurring as joint neighbours. …METHODS: Human chondrocytes were isolated from condyles and propagated in monolayer culture. Scaffold-free spheroids were generated and co-cultured with joint-specific partner cells (osteoblast-like osteosarcoma cells, fibroblasts). Morphology and differentiation was analyzed using histochemistry (Alcian blue, Safranin O) and immunohistochemistry for cartilage markers (collagen type II, Sox9, proteoglycan), proliferation-associated protein (Ki67) and markers of connective tissue (collagen type I and actin). RESULTS: The provision of a more natural microenvironment in vitro via co-culture of chondrocyte-based aggregates with osteoblast-like Saos-2 cells enhanced the differentiation potential of chondrogenic spheroids towards hyaline cartilage. CONCLUSIONS: The study showed the positive influence of Saos-2 cells on the differentiation potential of human chondrocytes in co-culture. Show more

Keywords: Human chondrocytes, Saos-2, HFF-1, co-culture, spheroid, scaffold-free

DOI: 10.3233/JCB-189005

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 31-42, 2018

Authors: Berger, Steffen | Kiebist, Jan | Pötschke, Elisabeth | Salchert, Katrin

Article Type: Research Article

Abstract: BACKGROUND: The provision of fully functional materials for tissue engineering also depends on the presentation of external signals for the targeted control of cell migration. In this context, the use of structural proteins and signal molecules from the native extracellular matrix offers great potential to successfully produce ready-to-use biomaterials. OBJECTIVE: We herein describe the first steps in the development of an immersion method, which aims at generating gradients of growth factors on compressed Collagen Type I (COL I) membranes. For process development, bovine serum albumin (BSA) was used. METHODS: By incubation of COL I membranes in 500μ g/mL …BSA solution for periods of 5 - 240 min, an adsorption isotherm was determined, which was used to control the immersion process. The actual immersion process was performed by a computer-controlled stepper motor that immersed a COL I membrane in 500μ g/mL BSA solution within 20 min. RESULTS: A linear BSA gradient with a change in concentration from about 30 ng/mm2 to 100 ng/mm2 could be generated. CONCLUSIONS: The defined immersion of a COL I membrane in a BSA solution is a suitable technique to produce a gradual course of BSA amount on the membrane surface. Gradient generation requires knowledge of the adsorption isotherms for the considered system. The developed immersion process can be adapted for other proteins and deviating concentrations. Show more

Keywords: Collagen Type I, BSA, protein gradient, immersion process, protein adsorption, cell migration

DOI: 10.3233/JCB-180012

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 43-49, 2018

Authors: Hansen, Max | Zatula, Nathalie | Riammer, Stephanie | Lehmann, Mario

Article Type: Short Communication

Abstract: BACKGROUND: The umbilical cord contains stem and progenitor cells in vast amounts. Therefore, a long-term storage of this tissue for future therapies seems reasonable. OBJECTIVE: The aim of this study was a systematic comparison of mesenchymal stromal cells from fresh and cryopreserved umbilical cord tissue to demonstrate the feasibility of cryopreservation of tissues for later cell isolation. Furthermore, we tested the cultivation of these cells in GMP-compliant human AB serum, as a substitute for FCS, and subjected the expanded cells to karyotype analysis. METHODS: We applied limiting dilution analysis for evaluation of fibroblastoid colony-forming units and …flow cytometric analysis of surface markers. Additionally we analyzed adipogenic and osteogenic differentiation potential. RESULTS: High numbers of viable MSC were isolated from cryopreserved umbilical cord tissue and a proportion of these cells showed clonal proliferation. The cells expressed the classic mesenchymal stromal cell surface markers, and effectively differentiated into osteocytes and adipocytes. Culture expansion of the mesenchymal stromal cells in medium supplemented with human AB serum did not change the surface marker profile. Moreover, karyotype analysis revealed genetic stability of mesenchymal stromal cells during expansion. CONCLUSIONS: Cryopreservation of umbilical cord tissue represents an auspicious approach for long-term storage of umbilical cord tissue and therefore a valuable tool for autologous stem cell applications. Show more

Keywords: Umbilical cord, MSC, mesenchymal stromal cell, cryopreservation, GMP

DOI: 10.3233/JCB-180011

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 51-56, 2018

Authors: Pfeil, Juliane | Dangelat, Luise N. | Frohme, Marcus | Schulze, Katja

Article Type: Research Article

Abstract: Microscopy plays a major role in the investigation of several diseases in human diagnostics and veterinary medicine. The microscopic analysis is mostly carried out in medical laboratories and requires specialised staff or expensive equipment. Therefore, providing results is time-consuming and far from the point-of-care where a fast diagnosis can be life-saving. Especially in infrastructural underdeveloped areas, with a lack of medical facilities and expert knowledge, this drawback is visible. Because of this, researchers started to develop “mobile microscopes” that can be used with a smartphone to enable everyone to be a specialist. This review is meant to give an overview …about developed smartphone based mobile microscopes, their different construction methods, their technical advantages and disadvantages, their possible diagnostic applications and their limitations. Show more

Keywords: Mobile microscopy, mobile phone, point-of-care diagnostics

DOI: 10.3233/JCB-180010

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 57-65, 2018

Authors: Ruhe, Madeleine | Dammermann, Werner | Lüth, Stefan | Sowa, Mandy | Schierack, Peter | Deckert, P. Markus | Rödiger, Stefan

Article Type: Research Article

Abstract: Chemotherapy and radiation therapy are used in malignant oncological diseases to increase the level of DNA double strand breaks (DSBs) in tumor cells. Unrepaired DSBs may either kill a cell or induce terminal arrest. Those DNA damages can be detected by fluorescence imaging of phosphorylated histone protein H2AX (γ H2AX). Peripheral blood mononuclear cells (PBMCs) are easily available human material, which are usually stored by cryopreservation for repeated experiments or postponed analysis. As DNA DSBs can be introduced through several different stressors, it was of interest to investigate the potential impact of cryopreservation, on the formation of DSBs in …human PBMCs. The PBMCs were cryopreserved in four different media, containing different proportions of Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum (FCS) and dimethyl sulfoxide (DMSO) as taken from the literature. Immunofluorescence staining of γ H2AX was performed on freshly isolated and cryopreserved PBMCs. A significant reduction on relative γ H2AX level, after treatment with 100 μM etoposide (ETP), was observed for cryopreserved cells in medium containing DMEM, which seemed to be the limiting factor on DNA DSB formation. No change was observed for untreated PBMCs. Therefore, DSBs of cryopreserved human PBMCs could be used to investigate the influence of chemotherapeutic drugs or radiation therapy, assuming that the cryopreservation follows a standardized protocol with a tight control of potential DSB inducing stressors. Show more

Keywords: DNA double strand breaks, γH2AX, cryopreservation, human PBMCs, automated microscopy

DOI: 10.3233/JCB-189006

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 67-73, 2018

Authors: Dammermann, Werner | Bröker, Katharina | Bentzien, Frank | Börner, Karina | Heinzel, Katharina | Lüth, Stefan

Article Type: Short Communication

Abstract: BACKGROUND: Hepatitis E virus (HEV) infection is being recognized as a major concern in developed as well as industrialized countries, especially for immunocompromised patients at risk of developing chronic hepatitis E. OBJECTIVE: We developed an HEV specific interferon gamma release assay (IGRA) for assessing T cell responsiveness to HEV antigens in resolved hepatitis E patients (RHE). METHODS: 8 RHE patients and 13 HEV seronegative healthy controls (HC) were tested for IFNγ - and IL2-secretion in whole blood after challenge with recombinant HEV ORF2 genotype 1 and ORF2 genotype 3 antigens. RESULTS: The developed IGRA …test differentiated by trend between the RHE group and HEV seronegative HC. RHE patients showed a stronger IL2 response to ORF2 genotype 1 or genotype 3 (180±47 and 171±39 pg/ml) compared to HC (96±34 pg/ml and 65±20 pg/ml). IFNγ responses were negligible. CONCLUSIONS: HEV specific IGRAs using IL2 as a marker should help to further clarify prior exposure to HEV. Show more

Keywords: Hepatitis E, HEV, interferon gamma release assay, cytokine release assay

DOI: 10.3233/JCB-189007

Citation: Journal of Cellular Biotechnology, vol. 4, no. 1-2, pp. 75-81, 2018