scholarly journals A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Martin Listek ◽  
Anja Hönow ◽  
Manfred Gossen ◽  
Katja Hanack
Keyword(s):  
Cell Line ◽  
Hybridoma Cells ◽  
Selection Strategy ◽  
Fusion Cell

scholarly journals Different Roles of a Rat Cortical Thymic Epithelial Cell LineIn Vitroon Thymocytes and Thymocyte Hybridoma Cells: Phagocytosis, Induction of Apoptosis, Nursing and Growth Promoting Activities

2002 ◽  
Vol 9 (2) ◽  
pp. 63-72 ◽  
Author(s):  
Dragana Vucevic ◽  
Miodrag Colic ◽  
Petar Popovic ◽  
Sonja Gašic
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Apoptotic Cell ◽  
Hybridoma Cells ◽  
Thymic Epithelial Cell ◽  
Apoptotic Cells ◽  
Th Cells ◽  
Nursing Activity ◽  
Induction Of Apoptosis ◽  
Stimulation Of

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.

scholarly journals Mechanism of effector-cell blockade. I. Antigen-induced suppression of Ig synthesis in a hybridoma cell line, and correlation with cell-associated antigen.

1980 ◽  
Vol 151 (6) ◽  
pp. 1436-1451 ◽  
Author(s):  
A W Boyd ◽  
J W Schrader
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Effector Cell ◽  
Specific Antigen ◽  
Inhibitory Effect ◽  
Hybridoma Cells ◽  
Normal Spleen ◽  
Doubling Times ◽  
Antibody Secretion ◽  
Ig Synthesis

A mouse hybridoma cell line, FluIgM-1, which secretes IgM specific for the hapten fluorescein (FLU) was developed to allow detailed analysis of the effector-cell blockade (ECB) phenomenon, in which contact of antibody-forming cells (AFC) with specific antigen results in marked reduction of antibody secretion. Treatment of hybridoma cells with highly substituted FLU conjugates (e.g., Flu20gelatin) resulted in inhibition of plaque formation. The data indicated close parallels with the ECB of normal spleen AFC, both in speed of onset and the dose of antigen required. The inhibition of antibody secretion was confirmed with a biosynthetic-labeling procedure which demonstrated that this was a result of reduced Ig synthesis. The inhibitory effect appeared to be confined to antibody synthesis, in the total protein synthesis, DNA synthesis, and cell-doubling times were unaffected. The association of FLU conjugates with the cells during and following ECB was studied directly using fluorescence microscopy and the fluorescence-activated cell sorter. These experiments showed that FLU conjugates capable of causing blockade aggregated on the cell surface, that the clearance of cell-associated antigen correlated with recovery from ECB, and that at all times when cell associated antigen was detectable, a portion remained bound to the cell surface and was susceptible to enzymatic removal. The latter observations supported previous findings suggesting that ECB was mediated by extracellular antigen. The direct observation of aggregates of antigen on the surface of blockaded cells is consistent with a mechanism involving cross-linking of Ig receptors. Finally, Fc receptors were not present on hybridoma cells, excluding their involvement in induction of ECB.

scholarly journals Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

2020 ◽  
Author(s):  
Thailín Lao-González ◽  
Alexi Bueno Soler ◽  
Arnelys Duran Hernandez ◽  
Katya Sosa Aguiar ◽  
Luis Eduardo Hinojosa Puerta ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Culture Media ◽  
Screening Strategy ◽  
Culture Conditions ◽  
Host Cells ◽  
Selection Strategy ◽  
Cell Line Development ◽  
Early Screening ◽  
Gene Optimization

Abstract The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

scholarly journals Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thailin Lao-Gonzalez ◽  
Alexi Bueno-Soler ◽  
Arnelys Duran-Hernandez ◽  
Katya Sosa-Aguiar ◽  
Luis Eduardo Hinojosa-Puerta ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Culture Media ◽  
Screening Strategy ◽  
Culture Conditions ◽  
Host Cells ◽  
Selection Strategy ◽  
Cell Line Development ◽  
Early Screening ◽  
Gene Optimization

AbstractThe high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

scholarly journals Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

2020 ◽  
Author(s):  
Thailín Lao-González ◽  
Alexi Bueno Soler ◽  
Arnelys Duran Hernandez ◽  
Katya Sosa Aguiar ◽  
Luis Eduardo Hinojosa Puerta ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Screening Strategy ◽  
Host Cells ◽  
Selection Strategy ◽  
Cell Line Development ◽  
Early Screening ◽  
Expression Levels ◽  
Short Period ◽  
Free Media

Abstract The high prices of biopharmaceutics or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceutics. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines in a short period of time with higher levels of expression. Here, we obtain trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein free media and suspension culture and lentiviral vectors. The results shown that early screening strategy allowed to obtain recombinant CHO cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular HC polypeptides by flow cytometry was a useful tool to characterize the homogeneity of cell population and our results suggest that might be used to predict the expression levels of monoclonal antibodies in early stages of cell line development process. Furthermore, using T-flask approach the assessment of the expression levels was studied in a setting more similar to that of a final production process. Finally, trastuzumab-expressing CHO-K1 clones were generated, characterized by batch experiments and preliminary results related to HER2-recognition capacity were successful. Further improvements in up-stream and down-stream steps related to this strategy will improve specific productivities and volumetric productivities of these clones.

scholarly journals Study on some properties of the hybrid cell B4D10C9 producing the anti B monoclonal antibody which was agglutinated the b antigens on the surface of red blood cells

2018 ◽  
Vol 15 (2) ◽  
pp. 243-249
Author(s):  
Nguyễn Thị Trung ◽  
Trương Nam Hải
Keyword(s):  
Monoclonal Antibody ◽  
Red Blood Cells ◽  
Cell Line ◽  
Blood Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Maximum Density ◽  
Hybridoma Cells ◽  
Continuous Growth ◽  
Fetal Bovine

The B4D10C9 hybridoma cell line producing the monoclonal antibody which was specifically agglutinated red blood cells B group, belongs to the IgM class and kappa light chains that have been created in our previous study. This study presents some biological charateristics of B4D10C9 such as the capability of growth, anti B monoclonal antibody producing, the stability of growth and cell line and antibody secreting of this cell line. The hybridoma cells grew well in the DMEM medium added with fetal bovine serum (FBS) at the final concentration of 1% or 10%. However, the maximum density of cells reached 9,9 x 105 cells per ml after 50 hours post incubation only when it was cultured in the medium containing 10% of FBS. The maximum antibody titers in the supernatant that collected after 150 hours of culture reached 1/512. In contrast to that, the maximum density of cells only reached 3,4 x 105 cells/ml after 72 hours post incubation when was cultured in DMEM medium containing 1% FBS. The maximum antibody titer was 1/64 after 50 hours post incubation. These cells were cultured through 3 different generations and the ability of antibody production in each generation was evaluated. As a result, this cell line had continuous growth stability and antibody secretion through 3 generations. The quantity of monoclonal antibody obtained from the supenantant of the culture medium at 150 hours of incubation was about 100 micrograms per ml. Thus, the growth of the cell line B4D10C9 and its ability of secretion of anti B monoclonal antibody were better when was cultured in the DMEM medium containing 10% fetal bovine serum. Preservation, storage and recovery does not affect to the growth and antibody producing of this cell line.

Establishment of a procedure for producing the anti-b monoclonal antibody from B4C10D9 hybridoma cell line

2017 ◽  
Vol 39 (3) ◽  
pp. 342-348
Author(s):  
Nguyen Thi Trung ◽  
Truong Nam Hai
Keyword(s):  
Monoclonal Antibody ◽  
Blood Transfusion ◽  
Red Blood Cells ◽  
Cell Line ◽  
Antibody Titer ◽  
Blood Cells ◽  
Hybridoma Cells ◽  
Post Inoculation ◽  
Reaction Intensity ◽  
Blood Grouping

There are two the most important blood group system in blood transfusion, that is the ABO system and the Rh system. The anti-A or anti-B antibodies in the blood of recipient cause strongly agglutination the red blood cells of donor bearing A or B antigens on their surface respectively. So, all donated blood and received blood must be typed group for the ABO system to be safe in blood transfusion. To blood grouping, used of know antisera reacts with the whole blood samples to determine antigen on the surface of red blood cell or use of know red blood cells reacts with the individual's serum to determine antibody. The serology method is widely used, antisera are usually monoclonal antibodies secreted by hybridoma cells. Our previous studies showed that the B4C10D9 hybridoma cell line generated anti-B monoclonal antibody was produced by hybridoma technology. This antibody causes specificity agglutination the red blood cells caring B antigen on their surface. It is  classed as IgM antibody with it heavy chains are µ and κ light chains. The aim of the study was to produce the anti-B monoclonal antibody form B4C10D9 hybridoma cells line serve as a part of ABO blood grouping reagent. Based on results of experiments, procedure for producing the anti-B monoclonal antibody from B4C10D9 hybridoma cell line has also been established with following steps. At first, refresh the frozen B4C10D9 hybridoma cells and the density of B4C10D9 hybridoma cells is 4.0 x 104 cells/ml at 24 hours post-inoculation. Then the B4C10D9 hybridoma cells are splitted 1:5 every 48 hours for 2 times to obtain the cell biomass. In the next step, the hybridoma cells are splitted into 125 cm2 flasks with the initial cell density of 105 cells/ml. The B4C10D9 hybridoma cells density reach 9.9 x 106 cells/ml at 48 hours post-inoculation. After 144 hours of post-inoculation, collect culture medium and concentrate the anti-B monoclonal antibody by precipitation with saturated ammonium sulfate at a final concentration of 50%. If resuspending volume equals initial volume, then the anti-B monoclonal antibody titer reaches 1/256 and the antibody reaction intensity will be 3+. And if the obtained antibody solution volume equals one fifth of the original volume, then the anti-B monoclonal antibody titer reaches 1/1024, and the antibody reaction intensity will be 4+.   Citation: Establishment of a procedure for producing the anti-b monoclonal antibody from B4C10D9 hybridoma cell line. Tap chi Sinh hoc, 39(3): 342-348. DOI: 10.15625/0866-7160/v39n3.10765. *Corresponding author: [email protected] Received 20 August 2017, accepted 20 September 2017

scholarly journals Specific suppressive factors produced by hybridomas derived from the fusion of enriched suppressor T cells and a T lymphoma cell line

1978 ◽  
Vol 148 (2) ◽  
pp. 373-382 ◽  
Author(s):  
M Taniguchi ◽  
JFAP Miller
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Cell Fusion ◽  
Lymphoma Cell ◽  
Hybridoma Cells ◽  
Lymphoma Cell Line ◽  
Suppressive Activity ◽  
Suppressor T Cells ◽  
Normal Parent ◽  
T Lymphoma

A cell fusion technique was used to produce hybridomas between the T lymphoma cell line, EL-4, derived from C57BL (H-2(b)), and an enriched population of human gamma globulin (HGG)-specific suppressor T cells prepared from the spleens of HGG-tolerant CBA mice (H-2(k)). Membrane fluorescence analysis of the hybridoma cells within 6 wk of cell fusion revealed expression of H-2(k) and I-J(k) gene products as well as H-2(b) antigens. Sonicates prepared from hybridomas which contained I-J(k) cells were tested for suppressive activity in vivo in irradiated mice given HGG-primed cells, dinitrophenyl (DNP)-primed cells, HGG-DNP, and horse erythrocytes. Among 18 such hybridoma lines, 6 showed specific suppressive activity, 5 nonspecific suppression, and 7 no suppression. Most lines progressively lost, with time, those properties derived from the normal parent cell. By about 3 mo after fusion few cells expressed CBA markers and only one cell line (number 77) retained some specific suppressive activity. In parallel with the losses was an alteration in chromosome number from near-tetraploid, soon after cell fusion, to near- diploid. Preliminary results with the T lymphoma-sensitive hypoxanthine aminopterin thymidine cell line, L5178, indicate retention of the expression of surface markers derived from the normal parent for 18 wk after hybidization. This suggests that T lymphoma cell lines may have to be screened for their capacity to produce hybridomas with stable properties.

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