ABSTRACT
After having successfully constructed and expressed the gene of the anti-CD3/anti-CD20 bispecific single-chain antibody (bscCD3 x CD20), here we analyzed its in vitro bioactivity of mediating the lysis of Ramous human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Obvious opoptosis characters were observed by Annexin V/PI(AV/PI) stained and scanning electron microscope. As evaluated by non-radioactive cytotoxity assay, the bscCD3 x CD20 showed potent bioactivity of mediating human B-lymphoma cells lysis in the presence of T-enriched human PBL. The potency of cytotoxicity depended on the ratios of effect cells to target cells (E:T) used. Further, the antibody showed a dose and time-dependent effect on mediating Ramous cells lysis. The specific lysis reached about 87.3% at an antibody concentration of 5microg/mL and E:T used at 10:1. Clear changes in apoptogenes expression profiles were detected by apoptosis gene array after Ramous cells were treated with the antibody and PBL. Among the upregulated apoptogenes, ATM and P53 showed an increase of 187 times and 15 times respectively, which suggested that ATM-p53 pathway may be the main apoptosis way of Ramous cells induced by T cells in the presence of the bscCD3 x CD20.
Subject(s)
Humans , Antibodies, Bispecific , Allergy and Immunology , Antigens, CD20 , Allergy and Immunology , Apoptosis , Allergy and Immunology , CD3 Complex , Allergy and Immunology , Lymphoma, B-Cell , Allergy and Immunology , Pathology , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Tumor Cells, CulturedABSTRACT
By using the size distribution of cell aggregates, viable cell density, cell viability, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)) and lactate transform rate (Y(lac/glc)) as the evaluation indexes, the effects of hydrodynamic on aggregates formation, growth and metabolism of HEK293 cells in suspension culture were examined in 250mL spinner-flasks by setting the agitation rates at 25, 50, 75 and 100r/min, respectively. It was found that agitation plays an important role in HEK293 cell aggregates formation and cell aggregates size distribution. After 7d cultivation in spinner-flasks operated at 50r/min and 75r/min, the average diameter of HEK293 cell aggregates was 201 microm and 175 microm, respectively, with the fraction of aggregates larger than 225 microm less than 10%. The cell viability was kept above 90% with the metabolic indexes, including q(glc), q(lac) and Y(lac/glc) kept constant. These results demonstrated that hydrodynamic derived from the proper agitation play a decisive role in controlling the formation and size distribution of HEK293 cell aggregates, and provided sufficient mass transfer to support the normal growth and metabolism of HEK293 cells in suspended aggregates.
Subject(s)
Humans , Bioreactors , Cell Aggregation , Cell Culture Techniques , Cell Line , Cell Proliferation , KineticsABSTRACT
Fed-batch culture is the predominant mode of the current animal cell culture process for many recombinant protein production. Fed-batch culture operation is mainly based on the nutrient continuous consumption and demand of cells to design continuous or semi-continuous concentration feed medium to maintain or support high-density cell growth and improve volumetric productivity of target protein in the reactor. The main methods to improve production efficiency of fed-batch cells culture include the optimization of medium design, selection and optimization of feed strategy and regulation of cell metabolism.
ABSTRACT
Based on the different permeability of DNA-intercalant dyes YO-PRO-1(YP) and propidium iodide (PI) to the membrane of viable, apoptotic and necrotic cells, cell samples were stained with 4?mol/L YP and 4?g/ml PI for 10 min, and the fluorescence intensity of both YP and PI were measured by fluorometer at Ex/Em wavelength of 485/538nm and 530/590nm, respectively. The correlation between YP fluorescence intensity and the apoptotic cell number was confirmed by fluorescence microscope and linear regression(r=0.999,P
ABSTRACT
By using the cell density, cell viability, size distribution of cell aggregates,specific consumption rate of glucose (q_ glc ), specific production rate of lactate (q_ lac ), lactate transform rate (Y_ lac/glc ) and amino acids utilization as the evaluation indexes, the growth and metabolism of HEK293 cells under carrier-free immobilization culture mode were examined and compared with those of HEK293 cells cultured in static tissue flasks. It was found that HEK293 cells grown as suspended cell aggregates in spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells in stationary anchored culture, and HEK293 cells under carrier-free immobilization culture mode as suspended aggregates in stirred bioreactor facilitate perfusion performance and increase unit productivity. Cultivation of HEK293 cells in carrier-free immobilization culture mode has potential for further improving mammalian cells culture technique.
ABSTRACT
The objective of this study was to develop research of cardiac cells to reestablish 3D tissue architecture in vitro, we performed studies using collagen membrane as three-dimensional scaffold for cardiac cells culture with the principles and methods of tissue engineering. The polymer scaffold provides a 3-D substrate for cell attachment and tissue formation. Cardiac cells isolated by enzymatic digestion from 1d old neonatal rats were seeded to three-dimensional collagen scaffolds and tissue culture plates. The morphology, beating rate and the metabolic indexes, including specific consumption rate of glucose (q(glu)) , specific production rate of lactate (q(lac)), lactate transform rate ( Y(lac/glu)), specific creatine kinase (CK) and lactate dehydrogenase (LDH) activities of cardiac cells cultured on three-dimensional collagen membrane and tissue culture plates were compared. It was found that cells shape and cells' CK and LDH activity was no differences between 3D and 2D cultures and cell beat rate on cell culture cluster was slower than those cells cultured on collagen membrane, However the cell glucose consumption and lactate yield rate of cells cultured on cluster was higher than those cells cultured on collagen membrane. After 5 days of cultivation, cardiac cells cultured on collagen membrane scaffolds organized into three-dimensional (3D) aggregates as opposed to the two-dimensional (2D) aggregates mosaic pattern seen in tissue culture plates, and spontaneous and rhythmical contractile 3D cultures in unison were visible to the naked eye and the area of synchronous contract three-dimensional (3D) aggregates reaches 80cm2. The mean value of q(glu), q(lac) and Y(lac/glu) of cultured on three-dimensional collagen scaffold was 7.37 micromol/10(6) cells/d, 2.92 micromol/10(6) cells/ d and 0.38 micromol/micromol, versus 7.59 micromol/10(6)cells/d, 3.83 micromol/10(6) cells/d and 0.51 micromol/micromol in tissue culture plates. These results demonstrate that cardiac cells immobilized on collagen membrane in 3D cultures maintain similar metabolic activity and contractile function when compared with native cardiac cells. The above results support the idea that engineered cardiac tissue can be used as a model of native tissue for studies of tissue development and function in vitro and eventually for tissue repair in vivo.