Rapid serum‐free /suspension adaptation: Medium development using a definitive screening design for Chinese hamster ovary cells

Abstract

The biopharmaceutical industry prefers to culture the mammalian cells in suspension with a serum-free media (SFM) due to improved productivity and process consistency. However, mammalian cells preferentially grow as adherent cells in a complete medium (CM) containing serum. Therefore, cells require adaptation from adherence in CM to suspension culture in SFM. This work proposes an adaptation method that includes media supplementation during the adaption of Chinese hamster ovary cells.As a result, the adaptation was accelerated compared to the traditional repetitive subculturing. Ca 2+ /Mg 2+ supplementation significantly reduced the doubling time compared to the adaptation without supplementation during the adaptation of adherent cells from 100% CM to 75% CM (p < 0.05). Furthermore, a definitive screening design (DSD) was applied to select essential nutrients during the adaptation from 10% CM to 0% CM. The main effects of Ca 2+ and Dulbecco's modified essential medium (DMEM) were found significant to both viable cell density and viability at harvest. Additionally, the interaction term between Ca 2+ and DMEM was found significant, which highlights the ability of DSD to capture interaction terms. Eventually, the media supplementation method resulted in adaptation SFM in 27 days, compared to the previously reported 66 days. Additionally, the membrane surface integrin expression was found significantly decreased when adherent cells were adapted to suspension. Moreover, the Ca 2+ /Mg 2+ supplementation correlated with faster integrin recovery after trypsinization. However, faster integrin recovery did not contribute to the accelerated cell growth when subculturing from 100% CM to 75% CM.

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

“…Our serum-free culture systems were established within 1 month, which is faster than the previous serum-free culture performed for 6 months (Rodrigues et al, 2013). Another study reported that 27 days are required for the establishment of rapid serum-free cultures of CHO cells (Wu et al, 2021), indicating that the timeframe of our adaptations is reasonable. Interestingly, the average volumes of our suspension cells were increased compared to adherent cells (Figure 3B).…”

Animal Cell Lines as Expression Platforms in Viral Vaccine Production: A Post Covid-19 Perspective

“…For this reason, viral vaccine production based on all virus production, especially where cellular substrates such as primary culture and diploid cell lines are used, is carried out in two stages in order to reach maximum cell concentration quickly and to eliminate the disadvantages of serum use. For this, first, the cells are cultured in serum-containing medium to reach the maximum concentration in the culture medium, and then this medium is replaced with serum-free virus production medium. ,,, It provides a much higher success rate in the adaptation of continuous cell lines to a serum-free medium compared to other cellular substrates. , Since continuous cell lines can be produced with similar or better proliferation performance than they show in serum containing media, only serum-free culture media can be used throughout the viral vaccine production process without any shift requirement. ,, Today, many different brands of serum-free media are available for continuous cell lines used in the commercial production of many different biopharmaceuticals, such as Chinese hamster ovary cell (CHO) and African green monkey kidney cell (Vero). ,, …”

“… , This situation creates additional costs in production and prolongs the preprocessing time in preparation for production. Additionally, it is difficult to obtain large amounts of cells because cell proliferation is limited by the available surface area. ,,, Since it is known that primary cultures and diploid cell lines retain their adherent character and have limited adaptability to suspended conditions, they are produced adherently in viral vaccine production, either using microcarriers or in packed-bed bioreactors (such as iCellis) where other suitable support materials are used. , Cells with suspension character have advantages such as ease of scale-up since they are not dependent on any surface, less labor required, growth is limited only by the cell concentration in the nutrient medium, and the cellular microenvironment in the production environment is more homogeneous. , However, as mentioned, since almost all of the expressional platforms used in vaccine production show adherent character, if the use of suspension cultures is aimed in production, the cellular substrates must be adapted to the suspension growth condition. , This adaptation is possible by culturing adherent cells in dynamic systems, using serum-free nutrient media specifically developed for the cell line, or applying both methods simultaneously. ,,, In this way, very high success can be achieved in continuous cell lines. ,, However, since the cellular character changes greatly during adaptation, it must be constantly checked that this suspended culture developed by cell engineering synthesizes the vaccine material in sufficient quantities and in a suitable form for industrial use. Additionally, the safety properties of adapted cells, such as tumorigenicity, should also be re-evaluated and checked. ,, The main disadvantage of suspension cultures is the need for cell-retention devices in case of any nutrient media change or washing process during production …”

Clever Experimental Designs: Shortcuts for Better iPSC Differentiation

“…Indeed, detailed media optimizations utilizing DOE have been reported for effective recombinant protein and antibody production processes. We collected DOE investigations evaluating cell yield and/or viability as response variables [ 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 ], or selecting desirable clones [ 96 , 97 ].…”

“…CHO cells originally maintained adherently in the presence of serum are often adapted to serum-free cell suspension culture to improve productivity and quality, especially in industry. This adaptation process was optimized by Wu et al, who screened five additives employing DSD in just 15 experimental runs [ 95 ]. Consequently, with the established medium condition, the adaptation process that conventionally took 66 days was successfully shortened to just 27 days.…”