Journal articles: 'Cell culture processing'

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Wolf, Michael W., and Udo Reichl. "Downstream processing of cell culture-derived virus particles." Expert Review of Vaccines 10, no. 10 (October 2011): 1451–75. http://dx.doi.org/10.1586/erv.11.111.

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Gastens, Martin H., Kristin Goltry, Wolfgang Prohaska, Diethelm Tschöpe, Bernd Stratmann, Dirk Lammers, Stanley Kirana, Christian Götting, and Knut Kleesiek. "Good Manufacturing Practice-Compliant Expansion of Marrow-Derived Stem and Progenitor Cells for Cell Therapy." Cell Transplantation 16, no. 7 (August 2007): 685–96. http://dx.doi.org/10.3727/000000007783465172.

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Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14–CD45– and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 × 106 total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

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Stössel, H., F. Koch, E. Kämpgen, P. Stöger, A. Lenz, C. Heufler, N. Romani, and G. Schuler. "Disappearance of certain acidic organelles (endosomes and Langerhans cell granules) accompanies loss of antigen processing capacity upon culture of epidermal Langerhans cells." Journal of Experimental Medicine 172, no. 5 (November 1, 1990): 1471–82. http://dx.doi.org/10.1084/jem.172.5.1471.

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Freshly isolated epidermal Langerhans cells (LC) can actively process native protein antigens, but are weak in sensitizing helper T cells. During culture, when LC mature into potent immunostimulatory dendritic cells, T cell sensitizing capacity develops but antigen processing capacity is downregulated. Processing of exogenous antigens for class II-restricted antigen presentation involves acidic organelles. We used the DAMP-technique to monitor acidic organelles at the ultrastructural level in fresh, as well as cultured, mouse and human LC. We observed that the loss of antigen processing capacity with culture of LC was reflected by the disappearance of certain acidic organelles, namely endosomes (particularly early ones), and the hitherto enigmatic LC granules ("Birbeck Granules"). Our findings support the notion that endosomes are critical for antigen processing and suggest that LC granules might be involved as well.

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Yang, Zeyu, Xingge Xu, Cristina A. T. Silva, Omar Farnos, Alina Venereo-Sanchez, Cécile Toussaint, Shantoshini Dash, et al. "Membrane Chromatography-Based Downstream Processing for Cell-Culture Produced Influenza Vaccines." Vaccines 10, no. 8 (August 13, 2022): 1310. http://dx.doi.org/10.3390/vaccines10081310.

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New influenza strains are constantly emerging, causing seasonal epidemics and raising concerns to the risk of a new global pandemic. Since vaccination is an effective method to prevent the spread of the disease and reduce its severity, the development of robust bioprocesses for producing pandemic influenza vaccines is exceptionally important. Herein, a membrane chromatography-based downstream processing platform with a demonstrated industrial application potential was established. Cell culture-derived influenza virus H1N1/A/PR/8/34 was harvested from benchtop bioreactor cultures. For the clarification of the cell culture broth, a depth filtration was selected as an alternative to centrifugation. After inactivation, an anion exchange chromatography membrane was used for viral capture and further processing. Additionally, two pandemic influenza virus strains, the H7N9 subtype of the A/Anhui/1/2013 and H3N2/A/Hong Kong/8/64, were successfully processed through similar downstream process steps establishing optimized process parameters. Overall, 41.3–62.5% viral recovery was achieved, with the removal of 86.3–96.5% host cell DNA and 95.5–99.7% of proteins. The proposed membrane chromatography purification is a scalable and generic method for the processing of different influenza strains and is a promising alternative to the current industrial purification of influenza vaccines based on ultracentrifugation methodologies.

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NONOYAMA, Ryosuke, Seidai FUJII, Koichiro YORI, Keiichi SUGIURA, and Makoto JINNO. "Study on cell culture processing system to improve task efficiency." Proceedings of JSME annual Conference on Robotics and Mechatronics (Robomec) 2021 (2021): 1A1—E04. http://dx.doi.org/10.1299/jsmermd.2021.1a1-e04.

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NONOYAMA, Ryosuke, Makoto JINNO, Tadashi SAMESHIMA, and Koichiro YORI. "Study on cell culture processing system to improve task efficiency." Proceedings of JSME annual Conference on Robotics and Mechatronics (Robomec) 2017 (2017): 2A2—H11. http://dx.doi.org/10.1299/jsmermd.2017.2a2-h11.

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Tomic, Sladjana, Lise Besnard, Benjamin Fürst, Rainer Reithmeier, Rolf Wichmann, Pierre Schelling, and Christian Hakemeyer. "Complete clarification solution for processing high density cell culture harvests." Separation and Purification Technology 141 (February 2015): 269–75. http://dx.doi.org/10.1016/j.seppur.2014.12.002.

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NONOYAMA, Ryosuke, Makoto JINNO, Tadashi SAMESHIMA, and Koichiro YORI. "Study on cell culture processing system to improve task efficiency." Proceedings of JSME annual Conference on Robotics and Mechatronics (Robomec) 2018 (2018): 1P2—A01. http://dx.doi.org/10.1299/jsmermd.2018.1p2-a01.

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NONOYAMA, Ryosuke, Koichiro YORI, Tadashi SAMESHIMA, and Makoto JINNO. "Study on cell culture processing system to improve task efficiency." Proceedings of JSME annual Conference on Robotics and Mechatronics (Robomec) 2019 (2019): 2A1—Q03. http://dx.doi.org/10.1299/jsmermd.2019.2a1-q03.

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Dernovsek, K. D., and R. S. Bar. "Processing of cell-bound insulin by capillary and macrovascular endothelial cells in culture." American Journal of Physiology-Endocrinology and Metabolism 248, no. 2 (February 1, 1985): E244—E251. http://dx.doi.org/10.1152/ajpendo.1985.248.2.e244.

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The processing of cell-bound insulin was determined in endothelial cells cultured from three large blood vessels (human umbilical vein, bovine pulmonary artery, and bovine aorta) and one microvascular source (bovine fat capillaries). Cells were exposed to monoiodinated TyrA14-insulin, the rates of dissociation of cell-bound TyrA14-insulin determined, and cell alteration of insulin assessed by gel filtration and high-performance liquid chromatography analysis. We found that 1) overall degradation rates of insulin are low for all cultured endothelial cells, 2) cell-bound insulin is rapidly processed to a nonreceptor compartment and then rapidly dissociated from all cells, primarily as biologically intact insulin, and 3) degradation of cell-bound insulin, although relatively low, does occur in endothelial cells with the least degradation by capillary cells. The presence of specific surface receptors for insulin on endothelial cells coupled with rapid cellular processing of intact insulin is consistent with a potential role for endothelial cells in either the transport of intact insulin out of the bloodstream or as a regional storage site for intact hormone.

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Sato, Eri, Yoshinori Maeda, Yui Sato, Airi Hinata, Hiroshi Gomi, Daisuke Koga, Seiji Torii, Tsuyoshi Watanabe, and Masahiro Hosaka. "Culture in 10% O2 enhances the production of active hormones in neuro-endocrine cells by up-regulating the expression of processing enzymes." Biochemical Journal 476, no. 5 (March 12, 2019): 827–42. http://dx.doi.org/10.1042/bcj20180832.

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Abstract To closely mimic physiological conditions, low oxygen cultures have been employed in stem cell and cancer research. Although in vivo oxygen concentrations in tissues are often much lower than ambient 21% O2 (ranging from 3.6 to 12.8% O2), most cell cultures are maintained at 21% O2. To clarify the effects of the O2 culture concentration on the regulated secretion of peptide hormones in neuro-endocrine cells, we examined the changes in the storage and release of peptide hormones in neuro-endocrine cell lines and endocrine tissues cultured in a relatively lower O2 concentration. In both AtT-20 cells derived from the mouse anterior pituitary and freshly prepared mouse pituitaries cultured in 10% O2 for 24 h, the storage and regulated secretion of the mature peptide hormone adrenocorticotropic hormone were significantly increased compared with those in cells and pituitaries cultured in ambient 21% O2, whereas its precursor proopiomelanocortin was not increased in the cells and tissues after being cultured in 10% O2. Simultaneously, the prohormone-processing enzymes PC1/3 and carboxypeptidase E were up-regulated in cells cultured in 10% O2, thus facilitating the conversion of prohormones to their active form. Similarly, culturing the mouse β-cell line MIN6 and islet tissue in 10% O2 also significantly increased the conversion of proinsulin into mature insulin, which was secreted in a regulated manner. These results suggest that culture under 10% O2 is more optimal for endocrine tissues/cells to efficiently generate and secrete active peptide hormones than ambient 21% O2.

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Sugano, K., J. Park, W. O. Dobbins, and T. Yamada. "Glycine-extended progastrin processing intermediates: accumulation and cosecretion with gastrin." American Journal of Physiology-Gastrointestinal and Liver Physiology 253, no. 4 (October 1, 1987): G502—G507. http://dx.doi.org/10.1152/ajpgi.1987.253.4.g502.

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Glycine-extended intermediates of peptide processing serve as substrates for carboxyl-terminal amidation, hence activation, of many brain-gut peptides. To explore the dynamics of accumulation and secretion of these important intermediates we utilized primary cultures of canine antral mucosal G-cells as a model system. Glycine-extended progastrin processing intermediates (G-Gly) accumulated rapidly in G-cells cultured in ascorbate-deficient media, exhibiting a fourfold increase over a 51-h culture period, while gastrin content fell to less than half of the initial level. In contrast, G-cells cultured in ascorbate-supplemented media accumulated G-Gly at a relatively low rate, while gastrin was preserved at a higher level. Under either condition, G-Gly and gastrin were progressively released into the culture media. The release of both immunoreactivities could be stimulated by bombesin and inhibited by somatostatin in similar fashion. By electron microscopy, the cultured G-cells exhibited no ultrastructural alterations. These data suggest that 1) the cellular homeostasis of G-Gly is regulated by the activity of an ascorbate-dependent amidation enzyme similar to one previously described in pituitary tissues, 2) carboxyl-terminal amidation is not an obligatory step for secretion of gastrin, and 3) the proportions of gastrin and G-Gly cosecreted from G-cells reflect their proportional accumulation within G-cell secretory granules. The physiological relevance of the released G-Gly has yet to be determined.

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Dobbs, L. G., M. S. Pian, M. Maglio, S. Dumars, and L. Allen. "Maintenance of the differentiated type II cell phenotype by culture with an apical air surface." American Journal of Physiology-Lung Cellular and Molecular Physiology 273, no. 2 (August 1, 1997): L347—L354. http://dx.doi.org/10.1152/ajplung.1997.273.2.l347.

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The study of differentiated functions of alveolar type II cells has been hampered because of the lack of good in vitro systems. We report that culture of type II cells on collagen gels with an apical surface exposed to air promotes expression of differentiated type II cell characteristics. Cells cultured in this manner are cuboidal, contain lamellar bodies, and produce tubular myelin; in addition, they secrete phosphatidylcholine in response to exogenous ATP. Cultures contain mRNA for surfactant proteins A, B, and C and surfactant proteins A, B, and D. In contrast, when type II cells are cultured with an apical surface exposed to liquid rather than to air, the cells are squamous, do not express surfactant proteins or their respective mRNA, and do not contain lamellar bodies or produce tubular myelin. Type II cells cultured on plastic for 7 days, which no longer express mRNA for surfactant proteins, can be induced to express these mRNA by changing culture conditions to that of an air surface. The culture system described in this paper should be useful for studies of surfactant metabolism, regulation of alveolar epithelial phenotypic expression, and the processing of transiently expressed transgenes.

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Böhme, Andrea, Lars Radke, Felix Schütze, Sylvio Schneider, Thilo Liebscher, Sabine Sauer, Loredana Santo, et al. "Miniaturized Flow-Through Bioreactor for Processing and Testing in Pharmacology." Materials Science Forum 879 (November 2016): 236–43. http://dx.doi.org/10.4028/www.scientific.net/msf.879.236.

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Conventional Bioreactor systems for cultivating cells in Life Science have been widely used for decades. An in vitro cell cultivation bioreactor should reliably and reproducibly mimic the in vivo microenvironment of the cultured cells. Normally, mammalian cell cultures are performed in conventional bioreactor devices such as culture flasks and culture-dishes. However, these tools have fundamental limitations due to being inappropriate for high throughput screening and consume a considerable amount of resources and time [1]. Therefore, there is a trend towards miniaturization, disposables and even micro platforms that fulfill increasing demands strongly aiming for production and testing of novel pharmaceutical products. Here we present the development and manufacture of a disposable miniaturized flow-through bioreactor system that can be produced in large numbers at low costs. nanoporous hollow fibers are located at the fluidic sources and drains of the miniaturized bioreactors and retain cells. The necessary mixture of oxygen and carbon dioxide is provided via diffusion through a semi-permeable membrane. Fluidic connections allow the continuous feeding of the cells adding nutrient solution at constant rates at the inlet of the micro bioreactor and removing the solution at the same rate at the outlet. This medium can be collected and used for subsequent analysis. Different designs and concepts for such bioreactors were carried out with varying numbers of plates, and integrated or joined miniaturized reactor chambers. First tests show full technical and biological functionality, cells could successfully be cultivated at high viability rates for some days.

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Baptista-Neto, Álvaro, Juliana Conceição Teodoro, Luiz Claudio Macedo Cassiano Filho, Alberto Colli Badino, and Carlos Osamu Hokka. "Comparisons between continuous and batch processing to produce clavulanic acid by Streptomyces clavuligerus." Brazilian Archives of Biology and Technology 48, spe (June 2005): 97–104. http://dx.doi.org/10.1590/s1516-89132005000400012.

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The aim of the present work was to compare CA production in continuous culture with and without cell recycling and in batch process by Streptomyces clavuligerus. Continuous cultivations with high cell concentration using cell recycling were performed utilizing a hollow fiber ultrafiltration module to separate cells from the filtrate broth. The continuous cultures without cell recycling and the batch cultivations were performed conventionally. The highest productivity was attained in the continuous cultivation with cell recycling (22.2 mg.L-1.h-1). The highest CA concentration was obtained in the batch process (470 mg.L-1.h-1).

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GRANDICS, P., S. SZATHMARY, Z. SZATHMARY, and T. O'NEILL. "Integration of Cell Culture with Continuous, On-Line Sterile Downstream Processing." Annals of the New York Academy of Sciences 646, no. 1 Recombinant D (December 1991): 322–33. http://dx.doi.org/10.1111/j.1749-6632.1991.tb18595.x.

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Tatsumi, Shin-ichi, Masayasu Okochi, Shinji Tagami, Takashi Kodama, Kanta Yanagida, Taisuke Nakayama, Kohji Mori, et al. "P1-173: Proteolytic processing of membrane anchored Neuregulin1 in cell culture." Alzheimer's & Dementia 5, no. 4S_Part_8 (July 2009): P228. http://dx.doi.org/10.1016/j.jalz.2009.04.179.

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YAMAMOTO, Yusuke, Yuta NAKASHIMA, and Yoshitaka NAKANISHI. "30pm1-B-6 Cell Culture Surface Processing Technique Based on Photolithography." Proceedings of the Symposium on Micro-Nano Science and Technology 2015.7 (2015): _30pm1—B—6—_30pm1—B—6. http://dx.doi.org/10.1299/jsmemnm.2015.7._30pm1-b-6.

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Wolff, M. W., and U. Reichl. "Downstream Processing: From Egg to Cell Culture-Derived Influenza Virus Particles." Chemical Engineering & Technology 31, no. 6 (June 2008): 846–57. http://dx.doi.org/10.1002/ceat.200800118.

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Garrepalli, Saritha. "Role of Biorepositories in Personalized Medicine: Isolation and Processing of Primary Tumor Cells for the initiation Tumor Cultures." Cancer Research and Cellular Therapeutics 1, no. 2 (August 24, 2017): 01–03. http://dx.doi.org/10.31579/2640-1053/009.

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The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help bio repositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.

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Dubé, Gilles R., Mercedes L. Kuroski - de Bold, and Adolfo J. de Bold. "Post-translational processing of atrial natriuretic factor by adult rat atrial cardiocytes in culture." Canadian Journal of Physiology and Pharmacology 71, no. 7 (July 1, 1993): 497–505. http://dx.doi.org/10.1139/y93-072.

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Post-translational processing of the cardiac polypeptide hormone atrial natriuretic factor (ANF) was studied using primary cultures of cardiocytes derived from adult rat atria. Atrial cardiocytes attached to microcarrier beads were maintained for up to 15 days under continuous superfusion in minichromatographic columns. The cultures were characterized for their ability to store, process, and release ANF and by immunofluorescence microscopy for ANF, desmin, and myosin. Nuclear staining using the fluorescent DNA stain Hoechst 33258 was carried out to determine the total number of cells in culture. Column eluates were assayed for ANF by radioimmunoassay and analyzed by reverse phase high-performance liquid chromatography. For comparison purposes, superfusion experiments using freshly isolated cardiocytes supported in Bio-Gel P2 were carried out. Freshly isolated atrial cardiocytes stored high molecular weight ANF (5.2 ± 1.9 pmol/μg DNA) and released mostly (83.3 ± 6.7%) low molecular weight ANF, at an average rate of 97 ± 18 fmol∙min−1∙μg−1 DNA. The cell content and the rate of release of ANF after 15 days in culture were 1.3 ± 0.4 pmoi/μg DNA and 1.7 ± 0.4 fmol∙min−1∙μg−1 DNA, respectively, and 62.7 ± 6.3% of the released peptide was of a low molecular weight. There was no correlation between changes in cell population and the extent of processing. Cultures of noncardiocytes, superfused with exogenous proANF, did not significantly process proANF to a lower molecular weight peptide. The present investigation shows that adult rat atrial cardiocytes, maintained superfused in microcarrier culture and in a serum-supplemented medium for up to 15 days, retain phenotypic and biochemical characteristics normally associated with the dual contractile–endocrine nature of mammalian atrial cardiocytes in vivo. The results obtained in the present work strongly support the view that ANF post-translational processing is an intrinsic property of the atrial cardiocytes and is independent of any other cell type.Key words: atrial natriuretic factor, post-translation processing, cardyocytes, adult rats, cell cultures.

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McGillin, Meghan R., Dana L. deRiancho, Timothy A. DeMarsh, Ella D. Hsu, and Samuel D. Alcaine. "Selective Survival of Protective Cultures during High-Pressure Processing by Leveraging Freeze-Drying and Encapsulation." Foods 11, no. 16 (August 16, 2022): 2465. http://dx.doi.org/10.3390/foods11162465.

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High-Pressure Processing’s (HPP) non-thermal inactivation of cells has been largely incompatible with food products in which the activity of selected cultures is intended (e.g., bio-preservation). This work aims to overcome this limitation using a cocoa butter encapsulation system for freeze-dried cultures that can be integrated with HPP technology with minimal detrimental effects on cell viability or activity capabilities. Using commercially available freeze-dried protective cultures, the desiccated cells survived HPP (600 MPa, 5 °C, 3 min) and subsequently experienced a 0.66-log increase in cell counts during 2 h of incubation. When the same culture was rehydrated prior to HPP, it underwent a >6.07-log decrease. Phosphate-buffered saline or skim milk inoculated with cocoa butter-encapsulated culture up to 24 h before HPP displayed robust cell counts after HPP and subsequent plating (8.37–9.16 CFU/mL). In addition to assessing viability following HPP, the study sought to test the applicability in a product in which post-HPP fermentation is desired While HPP-treated encapsulated cultures initially exhibited significantly delayed fermentative processes compared to the positive controls, by 48 h following inoculation, the HPP samples’ pH values bore no significant difference from those of the positive controls (encapsulated samples: pH 3.83 to 3.92; positive controls: pH 3.81 to 3.85). The HPP encapsulated cultures also maintained high cell counts throughout the fermentation (≥8.95 log CFU/mL).

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Vidard, L., K. L. Rock, and B. Benacerraf. "Heterogeneity in antigen processing by different types of antigen-presenting cells. Effect of cell culture on antigen processing ability." Journal of Immunology 149, no. 6 (September 15, 1992): 1905–11. http://dx.doi.org/10.4049/jimmunol.149.6.1905.

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Abstract The ability of normal B cells, peritoneal macrophages, and splenic APC to process and present OVA to a panel of T-T hybridomas with different specificities was investigated. In all cases, B cells were less efficient than unfractionated splenocytes in presenting OVA or its peptides. However, when the presentation of native Ag was compared to the presentation of peptides, it was obvious that there were marked differences in the ability of these two APC populations to generate different epitopes from OVA. Leupeptin inhibits the processing of selected epitopes from native OVA differently when it was presented by spleen cells or B cells, suggesting that these two APC populations differ in their protease content. The effect of in vitro culture on the ability of splenic and peritoneal APC to process OVA was also investigated. Native OVA presentation by macrophages and spleen cells was affected by in vitro culture, more for some epitopes than for other epitopes. In contrast, presentation of exogenous peptides by paraformaldehyde-fixed APC was either not affected by previous culturing for 3 days, or very much improved. Altogether, these data demonstrate that different epitopes on the same protein may be independently and differentially processed by B cells and spleen cells. Furthermore, the precise peptides that are produced may vary with the physiologic state of the APC.

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Shaikh, Sibhghatulla, Eunju Lee, Khurshid Ahmad, Syed-Sayeed Ahmad, Heejin Chun, Jeongho Lim, Yongho Lee, and Inho Choi. "Cell Types Used for Cultured Meat Production and the Importance of Myokines." Foods 10, no. 10 (September 29, 2021): 2318. http://dx.doi.org/10.3390/foods10102318.

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The world’s population continues to increase, meaning we require more consistent protein supply to meet demand. Despite the availability of plant-based protein alternatives, animal meat remains a popular, high-quality protein source. Research studies have focused on cultured meat (meat grown in vitro) as a safe and more efficient alternative to traditional meat. Cultured meat is produced by in vitro myogenesis, which involves the processing of muscle satellite and mature muscle cells. Meat culture efficiency is largely determined by the culture conditions, such as the cell type and cell culture medium used and the biomolecular composition. Protein production can be enhanced by providing the optimum biochemical and physical conditions for skeletal muscle cell growth, while myoblasts play important roles in skeletal muscle formation and growth. This review describes the cell types used to produce cultured meat and the biological effects of various myokines and cytokines, such as interleukin-6, leukemia inhibitory factor, interleukin-4, interleukin-15, and interleukin-1β, on skeletal muscle and myogenesis and their potential roles in cultured meat production.

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Lowe, Stuart B., Vincent T. G. Tan, Alexander H. Soeriyadi, Thomas P. Davis, and J. Justin Gooding. "Synthesis and High-Throughput Processing of Polymeric Hydrogels for 3D Cell Culture." Bioconjugate Chemistry 25, no. 9 (September 4, 2014): 1581–601. http://dx.doi.org/10.1021/bc500310v.

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Otsuka, K., S. Pitaru, C. M. Overall, J. E. Aubin, and J. Sodek. "Biochemical comparison of fibroblast populations from different periodontal tissues: characterization of matrix protein and collagenolytic enzyme synthesis." Biochemistry and Cell Biology 66, no. 3 (March 1, 1988): 167–76. http://dx.doi.org/10.1139/o88-023.

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To compare the expression of extracellular matrix components by fibroblasts from different periodontal tissues, rat molar periodontal ligament fibroblasts (RPL) and rat gingival fibroblasts (RGF) were isolated and cultured from individual animals. Pulse–chase experiments using [35S]methionine as a precursor revealed that confluent populations of early passage cells of both cell types synthesized similar amounts of collagen, fibronectin, and SPARC/osteonectin. Qualitative and quantitative differences were apparent in the relative proportions of type III collagen, in the rates of procollagen processing, and in the synthesis of a small number of unidentified proteins observed by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. Collagen constituted 24–26% of the radiolabelled proteins secreted by both cell types, type I being the predominant collagen, with lower amounts of type III (3–8% RGF, 8–18% RPL) and type V (~1%) collagens. Procollagen processing in the culture medium of RPL cells was more rapid than for RGF cells, but was increased in multilayered cultures of both RPL and RGF. In multilayered cultures, collagen TCA fragments, indicative of tissue collagenase activity, were also identified. Active and latent tissue collagenases and a latent form of a novel collagenolytic enzyme (matrix metalloendoproteinase-V) that cleaves native TCA fragments were demonstrated in these cultures. Addition of either concanavalin A (10−6 M) or retinoic acid (10−5 M) to the culture medium stimulated the secretion of the latent collagenolytic enzymes. Collagenase inhibitor was also synthesized by both RGF and RPL cells. SPARC/osteonectin, a 40-kilodalton glycoprotein, represented 0.5–1.0% of the secreted radiolabelled proteins of both cell types.

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Fujie, Hiromichi, Kei Oya, Yuki Tani, Kenji Suzuki, and Norimasa Nakamura. "Stem Cell-Based Self-Assembled Tissues Cultured on a Nano-Periodic-Structured Surface Patterned Using Femtosecond Laser Processing." International Journal of Automation Technology 10, no. 1 (January 4, 2016): 55–61. http://dx.doi.org/10.20965/ijat.2016.p0055.

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The present study was conducted to determine the effects of a nano-periodic-structured surface on the morphological and mechanical properties of a stem-cell-based self-assembled tissue (scSAT) developed for biological tissue repair. Nano-periodic groove structures were patterned on a pure titanium surface using femtosecond laser processing, and the structure was replicated on polydimethylsiloxane (PDMS). The depth, periodic pitch, and surface roughness (Ra) of the PDMS grooves were 48 ± 21 nm, 522 ± 9 nm, and 17 ± 5 nm, respectively. Human synovial cells, including mesenchymal stem cells, were subjected to 4-time cell passage, and then cultured on the PDMS surface at a density of 4.0 × 105cells/cm2in a growth medium with 0.2 mM ascorbic acid 2-phosphate to produce scSATs (nano-scSAT). For comparison, some of the cells subjected to 4-time cell passage were cultured on either a flat PDMS substrate with 6 ± 1 nm of surface roughness (Ra) (flat-scSAT) or a commercially available cell culture plate of polystyrene (normal-scSAT), at a cell density identical to that in the nano-scSAT group. At 28 days of cell culture, the scSATs were gently detached from the culture plates and subjected to morphological observation and mechanical testing. Microscopic observation revealed that the nano-scSATs exhibited a dense tissue of cells and an extracellular matrix with an anisotropic structure, while the flat- and normal-scSATs exhibited a sparse and isotropic structure. The tangent modulus and tensile strength were significantly higher in the nano-scSATs than in the flat- and normal-scSATs. These results suggest that a nano-periodic-structured surface improves the morphological and mechanical properties of scSATs.

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Pawitan, Jeanne Adiwinata. "Prospect of Stem Cell Conditioned Medium in Regenerative Medicine." BioMed Research International 2014 (2014): 1–14. http://dx.doi.org/10.1155/2014/965849.

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Background.Stem cell-derived conditioned medium has a promising prospect to be produced as pharmaceuticals for regenerative medicine.Objective.To investigate various methods to obtain stem cell-derived conditioned medium (CM) to get an insight into their prospect of application in various diseases.Methods.Systematic review using keywords “stem cell” and “conditioned medium” or “secretome” and “therapy.” Data concerning treated conditions/diseases, type of cell that was cultured, medium and supplements to culture the cells, culture condition, CM processing, growth factors and other secretions that were analyzed, method of application, and outcome were noted, grouped, tabulated, and analyzed.Results.Most of CM using studies showed good results. However, the various CM, even when they were derived from the same kind of cells, were produced by different condition, that is, from different passage, culture medium, and culture condition. The growth factor yields of the various types of cells were available in some studies, and the cell number that was needed to produce CM for one application could be computed.Conclusion.Various stem cell-derived conditioned media were tested on various diseases and mostly showed good results. However, standardized methods of production and validations of their use need to be conducted.

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Cicila, G. T., T. M. O'Connell, W. C. Hahn, and J. G. Rheinwald. "Cloned cDNA sequence for the human mesothelial protein ‘mesosecrin’ discloses its identity as a plasminogen activator inhibitor (PAI-1) and a recent evolutionary change in transcript processing." Journal of Cell Science 94, no. 1 (September 1, 1989): 1–10. http://dx.doi.org/10.1242/jcs.94.1.1.

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Mesosecrin, a Mr approximately 46 × 10(3) glycoprotein secreted in abundance by human mesothelial cells in culture, was recently described by this laboratory. We isolated partial cDNA clones for mesosecrin from a human mesothelial cell cDNA library in lambda gt11 using a specific antiserum. Comparison of mesosecrin cDNA sequences with the recently published sequence for plasminogen activator inhibitor-1 (PAI-1) cloned from cDNA libraries of endothelial and other cell types revealed that mesosecrin and PAI-1 are the same protein. Reverse fibrin autography of electrophoretically fractionated medium from mesothelial cell cultures confirmed that mesosecrin is functional as a plasminogen activator inhibitor. The mesosecrin/PAI-1 cDNA clones hybridized to abundant 3.6 and 2.6 kb (kb = 10(3) bases) mRNAs on Northern blots of cultured human mesothelial cell and endothelial cell RNA. These mRNA sizes correspond to those recently published for human endothelial and fibrosarcoma PAI-1 mRNA, which most likely result from alternate polyadenylation sites. Messages 3.6 and 2.6 kb long were also detected in cells cultured from orangutans and African green monkeys, but only an approximately 3.6 kb mRNA was detected in cells of lower primates and several other mammalian species. Thus the extra polyadenylation site in the PAI-1 gene, responsible for the shorter form of the RNA, apparently has been acquired recently during primate evolution. Because they are more easily propagated in culture than endothelial cells, human mesothelial cells offer a new and advantageous system for PAI-1 production and study of its regulation and function.

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Kohtz, D. S., N. R. Dische, T. Inagami, and B. Goldman. "Growth and partial differentiation of presumptive human cardiac myoblasts in culture." Journal of Cell Biology 108, no. 3 (March 1, 1989): 1067–78. http://dx.doi.org/10.1083/jcb.108.3.1067.

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A cell culture model for human cardiac myogenesis is introduced. Human fetal myocardial cells were dissociated enzymatically, and cultured in a mitogen-rich medium that promoted the growth of presumptive cardiac myoblasts. Strains of human cardiac myoblasts were generated from different anatomical regions of the fetal heart. The cells could be cultured for at least 30 generations, or frozen and recovered for later use. Differentiation was induced by culturing the cardiac myoblasts in a mitogen-poor medium. Differentiation of cardiac myoblasts was marked primarily by transcriptional activation of the atrial natriuretic factor (ANF) gene. Evidence is presented that posttranscriptional processing of ANF transcripts is affected by the anatomical origin of the cardiac myoblasts and the presence of cocultured neuronal cells. Cardiac myoblasts induced to differentiate in culture synthesized only low levels of sarcomeric myosin and cardiac alpha-actin, suggesting that differentiation of these cells progresses through two phases: an initial, noncontractile phase that is represented by the differentiating cultured cells; and a later contractile phase, in which myofibrillar assembly is accentuated and modulated by secondary signals from the cardiac milieu.

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Lebreton, J. P., M. Daveau, M. Hiron, M. Fontaine, D. Biou, D. Gilbert, and C. Guguen-Guillouzo. "Long-term biosynthesis of complement component C3 and α-1 acid glycoprotein by adult rat hepatocytes in a co-culture system with an epithelial liver cell-type." Biochemical Journal 235, no. 2 (April 15, 1986): 421–27. http://dx.doi.org/10.1042/bj2350421.

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We used a system of co-culture of adult rat hepatocytes with another epithelial cell type from rat liver to study the synthesis of two acute-phase reactants, alpha-1 acid glycoprotein (alpha 1AGP) and the third component of complement (C3), and we have obtained long-term secretion of these two proteins. After a period of adaptation corresponding to the first 2-4 days of the co-culture, hepatocytes secreted C3 and alpha 1AGP for at least 2 weeks at a mean level higher than that observed in the first days of a pure culture of hepatocytes. When pulse-chase analysis was performed on day 6 of co-culture, kinetics of synthesis of alpha 1AGP and C3 were the same as those observed on day 1 of a conventional culture of pure hepatocytes. Furthermore, intracellular and extracellular alpha 1AGP had Mr values respectively of 39,000 and of 42,000-52,000, identical with those observed in pure cultures of hepatocytes. Similarly, the molecular size and subunit structures of C3 were the same in co-culture and in cultures, indicating an identical processing of this protein. C3 produced in co-culture was also haemolytically active. Therefore, the system of adult hepatocytes co-cultured with this liver epithelial cell provides a physiological system in vitro which permits long-term synthesis of the two acute-phase reactants C3 and alpha 1AGP. This model opens the possibility to study the modulation of the synthesis of these two proteins during a long period by inflammatory agents or by hormones.

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Dasch, J. R., and P. P. Jones. "Independent regulation of IgM, IgD, and Ia antigen expression in cultured immature B lymphocytes." Journal of Experimental Medicine 163, no. 4 (April 1, 1986): 938–51. http://dx.doi.org/10.1084/jem.163.4.938.

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Long-term cultured bone marrow cells were characterized with respect to a number of B and pre-B cell markers. Cells expressing ThB, B-220, and IgM were found within cultures set up according to the procedure of Whitlock and Witte. This culture system was modified by placing sorted pre-B cells (ThB+, IgM-) from bone marrow in culture with previously-established bone marrow adherent layers. These cultures commenced growth without the lag associated with the Whitlock cultures. These cultured nonadherent cells show a high frequency of IgM+ cells, but do not express either IgD or Ia, and we refer to them as immature B cells. Cells with a similar phenotype (IgM+, Ia-, IgD-) are found within the spleens of young but not adult mice. The phorbol ester PMA induces expression of IgD on the cultured immature B cells, but has no effect on Ia expression. This suggests that the processing of H chain RNA transcripts may be affected by protein kinase C. These results demonstrate that the appearance of IgM, IgD, and Ia are independently controlled in long-term cultured B-lineage cells.

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Li, Cheng. "Potentiation of Bio Repositories In Personalized Medicine: Tumor Cells Establishment." Cancer Research and Cellular Therapeutics 1, no. 1 (December 8, 2017): 01–03. http://dx.doi.org/10.31579/2640-1053/003.

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The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help bio repositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.

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Sifuentes, Laura Y., and George D. Di Giovanni. "Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection." Applied and Environmental Microbiology 73, no. 23 (October 12, 2007): 7548–51. http://dx.doi.org/10.1128/aem.01579-07.

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ABSTRACT Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8- to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8- to 11-day-old and 11- to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.

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Majhy, B., P. Priyadarshini, and A. K. Sen. "Effect of surface energy and roughness on cell adhesion and growth – facile surface modification for enhanced cell culture." RSC Advances 11, no. 25 (2021): 15467–76. http://dx.doi.org/10.1039/d1ra02402g.

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Gerstenfeld, L. C., S. D. Chipman, C. M. Kelly, K. J. Hodgens, D. D. Lee, and W. J. Landis. "Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts." Journal of Cell Biology 106, no. 3 (March 1, 1988): 979–89. http://dx.doi.org/10.1083/jcb.106.3.979.

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A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels for pro alpha 1(I) and pro alpha 2 collagen and total collagen synthesis increased 1.2- and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to alpha chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times. The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase. These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.

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Albright, A. Leland, Susan S. Ferson, and Humphrey Okechi. "Cerebrospinal fluid white blood cell counts in infants with myelomeningoceles." Journal of Neurosurgery: Pediatrics 13, no. 2 (February 2014): 189–91. http://dx.doi.org/10.3171/2013.11.peds13196.

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Object The authors undertook this study to determine white blood cell (WBC) counts in CSF obtained from lateral ventricles and myelomeningoceles (MMCs) in infants in a developing country at the time of their initial presentation for medical evaluation. Methods CSF was aspirated from the lateral ventricles and from MMC sacs of 100 consecutive infants at Kijabe Hospital, Kijabe, Kenya. Peripheral blood WBC counts and CSF WBC counts were determined in the laboratory. CSF with WBC counts of 5 cells/mm3 or greater was cultured. Results The mean WBC count in ventricular CSF was 16 cells/mm3, with a median and mode of 0 cells/mm3. The mean WBC count of CSF in MMC sacs was 141 cells/mm3 (median 15 cells/mm3). No child had both a positive culture from ventricular CSF and a negative culture from MMC CSF. There was no correlation between age at presentation and WBC counts in the MMCs. Infants younger than 8 days old were as likely to have high WBC counts in CSF from their MMC sacs as were older children; 7 of 12 infants with 500 WBCs or more in CSF from their MMCs were younger than 8 days old. Only 5 of 58 CSF specimens from MMC sacs with 5 or more WBCs/mm3 had positive bacterial cultures, which may be a reflection of CSF specimen processing rather than of true culture negativity. Conclusions CSF from ventricular fluid of infants presenting with MMCs infrequently has high WBC counts, so infrequently that it does not need to be evaluated routinely. CSF in MMC sacs often has high WBC counts that suggest the presence of bacterial infection. In developing countries where culture reliability is questionable, intravenous administration of antibiotics before MMC closure for infants with high MMC WBC counts may diminish postoperative meningitis/ventriculitis.

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Hashimoto, Kahoko, Takuya Ootomo, Akihiro Hasegawa, Ryoma Kotera, Daichi Kobayashi, and Toshinori Nakayama. "Antigen processing and autophagy function in adjuvant treated dendritic cell." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 177.26. http://dx.doi.org/10.4049/jimmunol.202.supp.177.26.

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Abstract Dendritic cell (DC) and macrophage are the powerful antigen presenting cells. These show remarkable antigen processing function. It has been reported that antigen processing is enhanced by adjuvant. In DC and macrophage, LPS or IFNγ induces cell proliferation, cytokine production, antigen presentation to naive T cell. Among DC subset, CD8 positive DC enhances autophagy function especially in cross presentation. It is still nuclear how DC processes antigen by ubiquitin proteasome pathway or by autophagy pathway. To induce more effective immune response on vaccination or immune therapy, we study the effect of adjuvant treatment for autophagy function in DC and macrophage. Three types of different culture condition were used to establish DC subsets and compared to those function of macrophage. Then analyzed the antigen processing capacity as well as the autophagy function. We induced DC in vitro culture with GM-CSF, GM-CSF with IL-4, GM-CSF with IL-15, then stimulated DCs or macrophage by using LPS or IFNγ for up to 24 hours before antigen uptake. On antigen uptake and processing, GM-CSF induced or GM-CSF with IL-15 induced DC showed antigen processing, however GM-CSF with IL-4 induced DC showed active antigen processing within 60 min. Contrary to active antigen processing, GM-CSF with IL-14 –DC did not show any inducible effect after adjuvant treatment, but GM-CSF or GM-CSF with IL-15-DC increased the autophagy function. On the other hand, macrophage could not obtain autophagy effect thorough adjuvant treatment. DCs were studied the gene expression of cytokines, cell surface molecules such as co-stimulation molecules, mannose receptor, to evaluate antigen uptake capacity, processing pattern and autophagy.

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Fang, Ke Jing, Chang Jun Hou, Cheng Hong Huang, Xiao Gang Luo, Su Yi Zhang, Cai Hong Shen, and Dan Qun Huo. "The Rapid Fabrication of Hydrogel Microfluidic Chip for Cell Capture Culture and Metabolites Detection." Key Engineering Materials 562-565 (July 2013): 632–36. http://dx.doi.org/10.4028/www.scientific.net/kem.562-565.632.

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The microfluidic chip with well-defined structure is an important platform for cell research. The existing techniques for chip fabrication especially in cell biology and tissue engineering have many defects, for example, poor processing precision, high processing cost, as well as sophisticated manufacturing procedure. Thus, fabrication of simple and practicable microfluidic chip with highly efficient cell control ability and low-cost is turned to be the main target for bioengineering application. Poly(ethylene glycol) (PEG) is a hydrophilic polymer. Substituting terminal hydroxyl groups with acrylates, forming poly(ethylene glycol) diacrylate (PEGDA), allows the polymer to be cross-linked to form a three-dimensional polymer network. Meanwhile the use of photopolymerization can realize precise and temporal control of polymerization for formation of complex shapes. Herein, we utilize PEGDA hydrogel’s highly tunable characteristic, using photopolymerization method to obtain desirable micro-structure. Each chip has four of uniform micro-structures, which can carry multiple parallel experiments at the same time. We also add 2-Hydroxyethyl Methacrylate (HEMA) to the PEGDA prepolymer in order to increase the cell adhesion capacity of the microchip surface for cell culture. The experimental results showed that this method can achieve double-layer cell culture with short time treatment. Cells can be well captured and cultured in the hydrogel microfluidic chip with excellent activity. The hydrogel microfluidic chip has the potential of practicable application once large-scale preparation is accomplished.

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Chung, Yu-Cheng, I.-Lung Chien, and Der-Ming Chang. "Multiple-model control strategy for a fed-batch high cell-density culture processing." Journal of Process Control 16, no. 1 (January 2006): 9–26. http://dx.doi.org/10.1016/j.jprocont.2005.05.003.

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Donofrio, G., A. J. Clark, C. B. A. Whitelaw, G. Donofrio, and E. Bignetti. "Comparable processing ofβ-lactoglobulin pre-mRNA in cell culture and transgenic mouse models." Molecular and General Genetics MGG 252, no. 4 (September 1996): 465–69. http://dx.doi.org/10.1007/bf02173012.

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Mari, João Fernando, José Hiroki Saito, Amanda Ferreira Neves, Celina Monteiro da Cruz Lotufo, João-Batista Destro-Filho, and Maria do Carmo Nicoletti. "Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing." International Journal of Neural Systems 25, no. 08 (November 20, 2015): 1550033. http://dx.doi.org/10.1142/s0129065715500331.

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Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

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Mazzone, H. M. "Isolation, Electron Microscopy, and Computer Processing of Chromosomes." Proceedings, annual meeting, Electron Microscopy Society of America 43 (August 1985): 556–57. http://dx.doi.org/10.1017/s0424820100119569.

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Computer processing of electron microscope images has been of great value in the delineation and correlation of structural characteristics. An appropriate system for such study is that which enhances the high spatial frequency components in an image, thereby assisting in the observation of its fine structure. The present study reports on the isolation of intact chromosomes from a mammalian cell culture and their examination in the transmission electron microscope. The images of the chromosomes were then enhanced by computer processing.Isolation of the chromosomes and their examination in the electron microscope was done in the Dept, of Biology of the Massachusetts Institute of Technology. A mammalian cell line, STR-T, was maintained under standard conditions. Fresh cultures were prepared and when the cells were in log phase, colchicine was added to arrest the chromosomes at metaphase. After chemical fixation with ethanol-acetic acid (3:1), the cells were mechanically dislodged by scraping the vessel surface with a rubber policeman. The cells were place in a test tube and gently homogenized by hand.

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Boland, C. R., E. R. Kraus, J. M. Scheiman, C. Black, G. D. Deshmukh, and W. O. Dobbins. "Characterization of mucous cell synthetic functions in a new primary canine gastric mucous cell culture system." American Journal of Physiology-Gastrointestinal and Liver Physiology 258, no. 5 (May 1, 1990): G774—G787. http://dx.doi.org/10.1152/ajpgi.1990.258.5.g774.

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Mucin is a critical component of the protective layer secreted by gastrointestinal mucous cells. A detailed understanding of the molecular processing of gastric mucin and the physiology of its secretion has been limited by the lack of an adequate model for their study. We have developed a primary culture system of canine gastric mucous cells that has permitted us to study their synthetic and secretory functions. It was found that [3H]glucosamine used for metabolic labeling studies was incorporated into both mucin and lipid components of gastric mucus. To measure mucin with this model, a new immunoassay was developed to quantitate canine gastric mucin. Mucin was purified from the canine stomach, a polyclonal antibody was generated, and an enzyme-linked immunosorbent assay for gastric mucin was established. Mast cells were frequent contaminants of the gastric mucous cell preparation, and two methods were developed to limit their contamination. A new culture system has been developed for the study of gastric mucous cells. These cells synthesize and secrete both mucin and phospholipids. This system will permit us to study the molecular processing of mucin and the physiology of its production and release.

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Hwang, Hun-Way, Erik A. Wentzel, and Joshua T. Mendell. "Cell–cell contact globally activates microRNA biogenesis." Proceedings of the National Academy of Sciences 106, no. 17 (April 9, 2009): 7016–21. http://dx.doi.org/10.1073/pnas.0811523106.

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MicroRNAs (miRNAs) are 18- to 24-nt RNA molecules that regulate messenger RNAs (mRNAs). Posttranscriptional mechanisms regulate miRNA abundance during development as well as in cancer cells where miRNAs frequently exhibit dysregulated expression. The molecular mechanisms that govern the global efficiency of miRNA biogenesis in these settings remain incompletely understood, and experimental systems for the biochemical dissection of these pathways are currently lacking. Here, we demonstrate that miRNAs are subject to dynamic posttranscriptional regulation in widely used cell culture systems. As diverse mammalian and Drosophila cell lines are grown to increasing density, miRNA biogenesis is globally activated, leading to elevated mature miRNA levels and stronger repression of target constructs. This broad increase in miRNA abundance is associated with enhanced processing of miRNAs by Drosha and more efficient formation of RNA-induced silencing complexes. These findings uncover a critical parameter necessary for accurate analysis of miRNAs in cell culture settings, establish a tractable system for the study of regulated miRNA biogenesis, and may provide insight into mechanisms that influence miRNA expression in physiologic and pathophysiologic states.

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Bartsch, Heike, Dirk Stöpel, Marcel Himmerlich, Martin Baca, Philipp Stadie, Jari Hyttinen, Jens Müller, and Andreas Schober. "LTCC Based Multi-Electrode Arrays for In-Vitro Cell Culture." Additional Conferences (Device Packaging, HiTEC, HiTEN, and CICMT) 2015, CICMT (September 1, 2015): 000269–74. http://dx.doi.org/10.4071/cicmt-tha12.

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Neurobiological concepts based on state-of-the art technology have so far lacked the complexity of actual high-level neurobiological systems. Two key advances are needed to improve our understanding of such systems: in vitro 3D-neuronal cell culture and 3D MEA systems for measuring such 3D-cultures. These requirements call for smart multilayer and packaging technology. The material Green Tape TM from DuPont Nemours is chosen for the presented works, because its compatibility and those of available metallisation with cell cultures is already proven. An LTCC multilayer circuit with gold electrodes is the base of the 3D MEA. The layout of the 3D MEA is designed to fit the MEA2100-System for in vitro recording from Multi Channel Systems and enable thus a comparable data processing to established 2D MEAs Slots. The surface topography of the thick film electrodes and the surface state is investigated with laser scanning microscopy, SEM, XPS and measurements of the wetting angle of contact. The impedance of the screen printed electrodes is discussed taking these data into account. Their impedance amounts to 24 kΩ and are falls thus below the impedance of commercially available electroplated gold electrodes of 30 kΩ. First promising results have been achieved using 3D MEAs for 2D culture of human pluripotent stem cell derived neural cells.

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Trinh, Tu Cam, Huong Thanh Tran, and Viet Trang Bui. "Dissecting lipid accumulation of microalgae Nannochloropsis oculata using fluorescent image analysis." Science and Technology Development Journal - Natural Sciences 2, no. 5 (July 2, 2019): 5–11. http://dx.doi.org/10.32508/stdjns.v2i5.771.

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Cell suspension of Nannochloropsis oculata was cultured in a modified f/2 medium to study the changes of lipid content during phases of growth. The growth of cell suspension was determined by measuring the cell density and diameter under light microscope. To observe and evaluate the accumulation of lipid droplets in microalgae cells, lipid droplets were stained with Nile Red fluorescent dye then examined under fluorescence microscope and the obtained images were analyzed using Fiji ImageJ, an image processing program. The cell density increased quickly at the first 6 days of culture while cell diameter reached the highest value at the 8th day and 20th day of culture. The presence of lipid droplets in the cells could be observed from the 20th day of culture. The size of lipid droplets was gradually increased after 60 days. Treatment of depleted nitrogen for 4 days resulted an increase in the accumulation of lipid. The intracellular lipid accumulation during phases of growth of the cell suspension under nitrogen-depleted conditions was also discussed.

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Li, Chao, Jiaquan Yu, Jennifer Schehr, Scott M. Berry, Ticiana A. Leal, Joshua M. Lang, and David J. Beebe. "Exclusive Liquid Repellency: An Open Multi-Liquid-Phase Technology for Rare Cell Culture and Single-Cell Processing." ACS Applied Materials & Interfaces 10, no. 20 (May 8, 2018): 17065–70. http://dx.doi.org/10.1021/acsami.8b03627.

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Alkemade, J. A., H. O. Molhuizen, M. Ponec, J. A. Kempenaar, P. L. Zeeuwen, G. J. de Jongh, I. M. van Vlijmen-Willems, P. E. van Erp, P. C. van de Kerkhof, and J. Schalkwijk. "SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes." Journal of Cell Science 107, no. 8 (August 1, 1994): 2335–42. http://dx.doi.org/10.1242/jcs.107.8.2335.

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Skin-derived antileukoproteinase (SKALP), otherwise known as elafin, is a recently discovered epidermal proteinase inhibitor with specificity for polymorphonuclear leukocyte (PMN)-derived elastase and proteinase-3; in addition to the proteinase-inhibiting domain, SKALP contains several transglutaminase substrate motifs. SKALP is virtually absent in normal human epidermis but is found in a number of inflammatory skin diseases, including psoriasis. Here we report the induction and processing of SKALP in vivo and in vitro. SKALP expression in vivo could be demonstrated following injury in normal human epidermis, using histology, western blotting, northern blotting and a functional assay. In vitro, SKALP expression was studied in conventional submerged keratinocyte culture systems and in keratinocytes cultured in an air-liquid interface model. Induction of SKALP activity in epidermis could be measured as early as 16 hours after skin injury; immunohistological examination showed that SKALP expression was confined to the outer layers of the stratum spinosum and the stratum granulosum. Northern blot analysis revealed a 0.8 kb transcript, both in vivo (psoriatic skin, injured skin) and in vitro (cultured keratinocytes). Western blot analysis showed that the major SKALP form in vivo was a low molecular mass fragment, containing the antiproteinase domain. In all cultures that were positive for SKALP, larger (8-10 kDa) forms of SKALP, containing the N-terminal transglutaminase substrate motifs in addition to the antiproteinase domain, were found. SKALP expression in cultured cells was found to be dependent on the system used. In a submerged culture system, SKALP could be induced by fetal calf serum.(ABSTRACT TRUNCATED AT 250 WORDS)

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Avenoso, Daniele, Anne Bradshaw, Andrew Innes, Josu de la Fuente, Eduardo Olavarria, Jane F. Apperley, and Jiří Pavlů. "Microbial Contamination of Haematopoietic Stem Cell Products: A Single Centre Experience." Blood 128, no. 22 (December 2, 2016): 5741. http://dx.doi.org/10.1182/blood.v128.22.5741.5741.

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Abstract Bacterial contamination of haematopoietic stem cell products (HSCP) during collection and processing is a potential risk and has been described as cause of serious morbidity and mortality. The rate of contamination is reported in the range of 0 to 4.5% in peripheral blood progenitor cell (PBPC) apheresis to as high as 26% in bone marrow (BM) harvests. Systematic microbiological testing is an important component of the HSCP quality assessment and identification of the bacteria involved helps in the management of early infective complications. Here we report the rate of contaminated HSCP collected in a single transplant centre, our policy of the management of this complication and the relevant clinical outcomes. This is a retrospective study of prospectively collected results of microbiological analyses of 246 collections performed from January 2015 till December 2015. Stem cells were collected by BM harvesting under general anaesthesia (N=47) and by PBPC apheresis (N=199) using Optia separator system. BM harvest rather then PBPC was performed on donor request or with donor consent where BM was clinically preferable over PBPC transplant. (eg. red cell disorders). Samples of HSCP for bacterial cultures were taken pre processing, post processing, including post filtration and at defined intervals during complex procedures. Cryoprotectant is also tested to check that no contamination has occurred during manufacture of the freeze mix. Samples were cultured in BACTEC™ Lytic/10 Anaerobic/F culture vials (pre-reduced enriched Soybean-Casein Digest broth with CO2) and in anaerobic blood cultures; BACTEC Peds Plus™/F culture vials (enriched Soybean-Casein Digest broth with CO2) under aerobic conditions, both for 14 days. Organisms were specifically identified in all positive HSCP. Sixteen bacterial contaminations of HSCP were recorded (6.09% of total collections). The most frequent product contaminated was BM (N=15, 31.9%); only one PBPC apheresis product was positive (0.5%). The following bacteria were isolated: propionibacterium (N=7), coagulase negative staphylococcus (N=4), micrococcus (N=2), staphylococcus capitis (N=1), Staphylococcus epidermidis (N=2). Seven HSCP were not infused: one patient died before undergoing to transplant, two products have been collected as stem cell rescue in case of graft failure from allogeneic donors, four collections were stored for future use. Nine patients received contaminated HSCPs. After being contemporaneously alerted to the contamination, the clinical team performed daily blood cultures (BC). From these 9 patients only two minor clinical events were recorded; One, a non-neutropaenic fever on day +2 (S. Capitis cultured from HSCP, BC negative) and the other a positive BC (micrococcus cultured from both HSCP and BC) in a patient without fever or signs of infection. Both were treated with IV vancomycin on microbiology advice. These data showed a rate of bacterial contamination of HSCP comparable with reports from other groups. Moreover, at our centre no major clinical events were recorded after the infusion of contaminated HSCP. In this small study, the most frequent source of contaminated HSCP was BM and the most frequently isolated pathogens were skin commensals. No consensus exists on the requirement for antibiotics after the infusion of contaminated HSCP. Our experience supports a strategy of symptomatic management based on fevers or positive blood cultures with targeted antibiotics based on in vitro sensitivities rather than pre-emptive empirical treatment of all patients. Disclosures Apperley: Bristol Myers Squibb: Honoraria, Speakers Bureau; Ariad: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Journal articles on the topic 'Cell culture processing'

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