Serum-Free Suspension Adaptation of HEK-293T Cells: Basis for Large-Scale Biopharmaceutical Production

Abstract Human Embryonic Kidney 293T cells (HEK-293T) are the most common host for viral vector production and are also widely employed for recombinant protein production. These cells are typically cultured in monolayer (adherent culture) using culture medium containing fetal bovine serum (FBS), which impairs batch-to-batch reproducibility and scale-up. The adaptation of adherent cell culture to suspension culture in chemically defined serum-free culture medium is an attractive approach for large-scale bioprocess implementation while aiming for a Good Manufacturing Practice (GMP) compliant production process. Therefore, in the present study, our goal was to adapt HEK-293T cells to serum-free suspension culture conditions and evaluate the feasibility of adapted cells to be transfected using different plasmid vectors for recombinant protein production. Firstly, the cells were efficiently adapted to serum-free conditions by sequential adaptation (FBS-containing medium weaning). During the whole process, parameters such as cell growth, viability and doubling time were evaluated and compared to the control (adherent serum-supplemented HEK-293T cell culture). Afterwards, these cells were adapted to suspension culture by using Erlenmeyer flasks in an orbital shaker platform, being able to achieve meaningful cell density with high viability. Adapted cells presented a transfection efficiency of approximately 50% for all vector constructs used (1054-GFP, Factor-VIII and Factor-IX). Overall, it was possible to successfully adapt HEK-293T cells to suspension and serum-free conditions, which represents an important step towards the development of a scalable and GMP-compliant production process. In addition, adapted cells efficiently expressed the different transgene tested, opening up possibilities for its use in recombinant protein production.

Comparison of Serum-Free Media in RBC Differentiation from Human Hematopoietic Stem Cells / 대한수혈학회지

BACKGROUND: Research on RBC production from hematopoietic stem cells has been conducted competitively in many countries. However those were in vitro successes and many hurdles still remain for large scale transfusable RBC production from stem cells. A need for large volume of culture media is a crucial factor for culture condition which researchers must overcome. In this study, we evaluated the efficiency of two commercial serum-free media, StemPro(R)-34 SFM and Stemline II hematopoietic stem cell expansion medium, in RBC differentiation from cord derived stem cells. METHODS: We cultured cord derived CD34+ cells in vitro and evaluated over the periods of 7 days, 14 days, 17 days and 21 days in culture for expanded cell count, cell morphology and differential count using the Wright Giemsa stain. RESULTS: Cell expansion and RBC differentiation developed rapidly in Stemline media compared to StemPro media. Enucleated RBCs were observed at 10~14 culture days and orthochromatic erythroblasts were shown up to 50% among culture cells at 17 days in Stemline media. The enucleated RBCs were observed at 17 days in StemPro Media. Although the erythroblasts in StemPro media are slow at differentiation, they maintain continuous expansion up to 21 days. CONCLUSION: In Stemline media, the expansion and differentiation to mature RBCs are processed much faster, but the cell condition slows down after 17 days. In the RBC production aspects, Stemline media is better than StemPro media as a rapid differentiation because it reduces the cost due to in vitro short culture duration.

Study on the biological characteristics of human breast cancer cell mammospheres / 重庆医学

Objective To study the biological characteristics of the human breast cancer cell mammospheres (MSs) ,and con‐struct breast cancer stem cell experiment model .Methods MCF‐7 cells were cultured in the serum‐free media supplemented with growth factors (the MCF‐7 group) ,and the MSs was collected (the MSs group) .The migration ,invasive and animal tumor forma‐tion abilities of MSs were detected by wound healing ,transwell invasive assay and animal tumor formation test .Results The wound line of MSs healed after 48 hours ,but the line of MCF‐7cells could not heal after 48 h .The number of the cells going through the membrane in MSs group was (76 .24 ± 0 .35) ,and the number in MCF‐7cells was (17 .38 ± 0 .18)(P<0 .05) .MSs had stronger ani‐mal tumor formation ability than MCF‐7 cells .Conclusion MSs have stronger abilities in migration ,invasive and animal tumor for‐mation ,and could be used in the studies of breast cancer stem cell as experimental model .

Serum-free Aggregate Perfusion Culture of CHO Cells:an Ultrasonic and Sedimentation Column Combined Perfusion System / 中国生物工程杂志

Using the character of natural aggregation of CHO cells, and an ultrasonic and sedimentation column combined perfusion system to promote cells aggregation and retention into bioreactor,recombinant CHO cell strain MK3-A2 was cultured,which could secrete rhTNK-tPA, by a serum-free perfusion culture system. The culture periods in this two experiments were as long as 77 and 110 days respectively. The cells density reached 2?107 cells /ml. The average volumetric productivity of rhTNK-tPA was 89 mg/L?d, and the highest one was 216mg/L?d.The cells aggregation rate was approximately 90%, and the diameters of most of them were 285～570?m. During the perfusion culture the cells retention rate almost kept in 95% and the viability of cells was more than 85%.Thus, it means that aggregation culture with such perfusion system could be used to scale up produce biopharmaceuticals instead of microcarrier culture system.

Increasing the Production of IgG in Batch Tissue-culture with IgG-free Ascites / 中国生物工程杂志

An effective method has been developed for laboratory scale production of IgG. Hybridomas were cultured in serum-free media with 2% IgG-free ascites. Cell density of up to 3.55 × 10 6cells/ml and antibody concentration of 135μ g/ml after purification were abtained, which is four time more than total production of that of IgG concentration in serum-free media. This in vitro method allows great improvement in antibodies production in batch tissue culture. The method reported here is easy to handle and is economical and universally adaptable.

Increasing the Production of IgG in Batch Tissue-culture with IgG-free Ascites / 中国生物工程杂志

An effective method has been developed for laboratory scale production of IgG. Hybridomas were cultured in serum-free media with 2% IgG-free ascites. Cell density of up to 3.55 × 10 6cells/ml and antibody concentration of 135μ g/ml after purification were abtained, which is four time more than total production of that of IgG concentration in serum-free media. This in vitro method allows great improvement in antibodies production in batch tissue culture. The method reported here is easy to handle and is economical and universally adaptable.

Increasing the Production of IgG in Batch Tissue-culture with IgG-free Ascites / 中国生物工程杂志

An effective method has been developed for laboratory scale production of IgG. Hybridomas were cultured in serum-free media with 2% IgG-free ascites. Cell density of up to 3.55 × 10 6cells/ml and antibody concentration of 135μ g/ml after purification were abtained, which is four time more than total production of that of IgG concentration in serum-free media. This in vitro method allows great improvement in antibodies production in batch tissue culture. The method reported here is easy to handle and is economical and universally adaptable.

Establishment of Normal Human Middle Ear Epithelial (NHMEE) Cell Using Serum-free Media / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Despite many reports about middle ear mucosal epithelial cell culture of experimental animals like rat, gerbil or chinchilla, normal human middle ear epithelial (NHMEE) cells have not been cultured in vitro yet. We attempted to culture NHMEE cells and confirm them to be epithelial cells. MATERIALS AND METHOD: Healthy middle ear mucosa was harvested during the otologic surgery without contamination. Specimen was cultured by primary explant culture method and proliferating cells were subcultured after enzymatic disaggregation. Cultured cells were observed using phase contrast light microscope, scanning electron microscope and transmission electron microscope. Immunocytochemical stain was performed to identify the expression of cytokeratin, vimentin and von Willebrand factor. RESULTS: Cultured cells were of polygonal shape and the cell surfaces were covered by microvilli. The immunocytochemical stain revealed cytokeratin in all the cultured cell, whereas vimentin was co-expressed in some of the cells and von Willebrand factor was not expressed at all. We could observe desmosome or tonofilament in cultured cells by TEM. Although cells proliferated 20 or 30 fold in every passage, the passage-4 cells did not proliferate and many vacuoles were formed. CONCLUSION: The NHMEE culture system using serum free media will provide a good model for the study of human middle ear epithelial differentiation and secretion.