The emergence of microcarrier technology has brought a renewed interest in anchorage-dependent cell culture for high-yield processes. Well-known in vaccine production, microcarrier culture also has potential for application in other fields. In this work, two types of microcarriers were evaluated for...
The emergence of microcarrier technology has brought a renewed interest in anchorage-dependent cell culture for high-yield processes. Well-known in vaccine production, microcarrier culture also has potential for application in other fields. In this work, two types of microcarriers were evaluated for small-scale monoclonal antibody (mAb) production by CHO-K1 cells. Cultures (5 ml) of microporous Cytodex 3 and macroporous CultiSpher-S carriers were performed in vented conical tubes and subsequently scaled-up (20 ml) to shake-flasks, testing combinations of different culture conditions (cell concentration, microcarrier concentration, rocking methodology, rocking speed, and initial culture volume). Culture performance was evaluated by considering the mAb production and cell growth at the phases of initial adhesion and proliferation. The best culture performances were obtained with Cytodex 3, regarding cell proliferation (average $1.85{\pm}0.11{\times}10^6$ cells/ml against $0.60{\pm}0.08{\times}10^6$ cells/ml for CultiSpher-S), mAb production ($2.04{\pm}0.41{\mu}g/ml$ against $0.99{\pm}0.35{\mu}g/ml$ for CultiSpher-S), and culture longevity (30 days against 10-15 days for CultiSpher-S), probably due to the collagen-coated dextran matrix that potentiates adhesion and prevents detachment. The culture conditions of greater influence were rocking mechanism (Cytodex 3, pulse followed by continuous) and initial cell concentration (CultiSpher-S, $4{\times}10^5$ cells/ml). Microcarriers proved to be a viable and favorable alternative to standard adherent and suspended cultures for mAb production by CHO-K1 cells, with simple operation, easy scale-up, and significantly higher levels of mAb production. However, variations of microcarrier culture performance in different vessels reiterate the need for optimization at each step of the scale-up process.
The emergence of microcarrier technology has brought a renewed interest in anchorage-dependent cell culture for high-yield processes. Well-known in vaccine production, microcarrier culture also has potential for application in other fields. In this work, two types of microcarriers were evaluated for small-scale monoclonal antibody (mAb) production by CHO-K1 cells. Cultures (5 ml) of microporous Cytodex 3 and macroporous CultiSpher-S carriers were performed in vented conical tubes and subsequently scaled-up (20 ml) to shake-flasks, testing combinations of different culture conditions (cell concentration, microcarrier concentration, rocking methodology, rocking speed, and initial culture volume). Culture performance was evaluated by considering the mAb production and cell growth at the phases of initial adhesion and proliferation. The best culture performances were obtained with Cytodex 3, regarding cell proliferation (average $1.85{\pm}0.11{\times}10^6$ cells/ml against $0.60{\pm}0.08{\times}10^6$ cells/ml for CultiSpher-S), mAb production ($2.04{\pm}0.41{\mu}g/ml$ against $0.99{\pm}0.35{\mu}g/ml$ for CultiSpher-S), and culture longevity (30 days against 10-15 days for CultiSpher-S), probably due to the collagen-coated dextran matrix that potentiates adhesion and prevents detachment. The culture conditions of greater influence were rocking mechanism (Cytodex 3, pulse followed by continuous) and initial cell concentration (CultiSpher-S, $4{\times}10^5$ cells/ml). Microcarriers proved to be a viable and favorable alternative to standard adherent and suspended cultures for mAb production by CHO-K1 cells, with simple operation, easy scale-up, and significantly higher levels of mAb production. However, variations of microcarrier culture performance in different vessels reiterate the need for optimization at each step of the scale-up process.
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문제 정의
The microcarrier technology can be a feasible and advantageous alternative for mAb production, but such application has not yet been adequately explored, as the lack of literature on the subject demonstrates. In this context, the purpose of the present study was to evaluate and optimize the application of microcarrier culture for the small-scale growth and mAb production of CHO cells. Cultures of these cells were performed in microporous Cytodex 3 and macroporous CultiSpher-S carriers and compared at different culture conditions (varying initial cell concentration, microcarrier concentration, initial culture volume, rocking speed, and rocking mechanism).
In this work, it was also an aim to maximize the mAb yield, which may not be directly related to cell proliferation. Indeed, observing data from Table 4, and comparing with Figs.
제안 방법
Cultures in shake flasks. Cultures of 20 ml of CHO-K1 cells in microcarriers were performed in 250 ml shake flasks, for the two sets of conditions that achieved the best mAb production results in the preliminary conical tube experiments (Table 3). The assays were also divided in the phases of initial cell adhesion and cell proliferation, and for each assay the shake flasks were inoculated with CHO-K1 cells and microcarriers at the corresponding concentrations, and incubated at 37℃, 5% CO2.
Cell growth was monitored by cell counting, after releasing the cells from the microcarriers, by enzymatic digestion with trypsin. First, to standardize results, the number of microcarriers of each sample was counted by microscopic observation. After this, the sample was transferred to a 2 ml tube, the microcarriers were allowed to settle, and the supernatant was removed.
This limits the number of unoccupied beads at the end of the culture, therefore providing higher cell yields. To improve this initial cell adhesion to the microcarriers, different parameters can be manipulated, such as the rocking mechanism and speed, cell concentration, microcarrier concentration, and initial culture volume that were assessed in this study. For Cytodex 3, the results obtained (Fig.
대상 데이터
The microporous Cytodex 3 (Sigma-Aldrich, Spain, C3275) and the macroporous CultiSpher-S (Sigma-Aldrich, Spain, M9043) carriers were used in this study. Their most important features are shown in Table 1.
데이터처리
Results of the production and productivity were assessed using one-way ANOVA with Bonferroni’s test, for a confidence level of 95%.
이론/모형
A CHO-K1 cell line (ATCC, U.K., CCL-61) transfected with the CAB051 monoclonal antibody (Biotecnol SA, Portugal) by OSCAR gene amplification technology (Edinburgh University, Scotland) was used in this study [13].
. After cultures reached 70-80% confluence, cells were detached with trypsin (Sigma-Aldrich, Spain, T4049), and the cell viability and concentration were determined by the trypan blue (Sigma-Aldrich, Spain, T8154) exclusion method, using a hematocytometer. The concentration of the cell suspension was then adjusted to the values needed for each assay.
After a wash with PBS, a trypsin solution was added and the sample incubated for 10-15 min, with occasional stirring (after this time, CultiSpher-S microcarriers disintegrate owing to their gelatin matrix; Cytodex 3 microcarriers maintain their structure). Fresh medium was added to the microcarriers to stop the trypsin reaction, and cells were counted using a hematocytometer and the trypan blue exclusion method.
성능/효과
For CultiSpher-S, although CUL6 had the best proliferation profile, culture conditions defined in assays CUL2 and CUL4 resulted in improved mAb production, with CUL4 being significantly (p < 0.05) superior to all assays except CUL2.
The use of a reduced initial volume, while maintaining the amount of cells and microcarriers of the full-volume assays, may provide a greater chance of contact between cells and microcarriers and improve the conditioning effects of the medium [6]. However, the results obtained demonstrate that the use of half the initial volume (Fig. 1C, CYT7) did not particularly benefit cell adhesion to Cytodex 3 microcarriers, resulting in final levels of cell adhesion (at 6 h of culture), similar to those obtained with a full volume (CYT1).
The mAb production and productivity levels achieved in the 20 ml cultures in shake flasks for both Cytodex 3 and CultiSpher-S microcarriers are shown in Table 5. Results show that Cytodex 3 cultures provided the highest mAb production levels, whereas CultiSpher-S cultures achieved the highest productivities. Furthermore, as observed for cell proliferation, the mAb production and productivity levels were similar for the two sets of conditions assayed for both Cytodex 3 and CultiSpher-S cultures.
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