Various anchorage-dependent cell lines such as Chinese Hamster Ovary (CHO), Kidney Epithelial Cell (VERO), Madin-Darby Canine Kidney Cell (MDCK), and Insect Cell (SF9) were used for vaccine, protein, and monoclonal antibody productions. The use of roller bottles and cell factories were prominent for the large-scale cell culture of these adherent cells; however, the development was costly, expensive, and always at risk of contamination.
Microcarrier-based cell cultures have since made use of stirred tank bioreactors (STR) and fixed-bed bioreactors with macrocarriers. Compared with STR, the fixed-bed bioreactor has the capacity to protect cells from stressful conditions and perfusion culture model is easily achievable. Singapore-based macroporous carrier manufacturer Esco VacciXcell, supplies BioNOC II® for use with different cell types. The use of macroporous carriers is a promising technique that has advantages in enhancing production capacity, improving the culture’s robustness, and facilitates process scale-up allowing the culture of various animal cells in high yield.
Unlike other types of microcarrier for cell culture, a macroporous carrier such as BioNOC II® creates a multilayer culture (3D cell growth) that allows cell to cell and cell to extracellular matrix interactions, mimicking the cell's in vivo environment. This is highly essential for multiple cellular processes, including differentiation and proliferation. Materials used in the creation of BioNOC II® are FDA-approved, cGMP compliant, and this macroporous carrier manufacturing passed regulation guidelines USP Class VI, USP <87>, <83>, ISO 10993-5.
Live cells (fluorescent) staining of Vero cells on BioNOC II® II under 4x magnification. Calcein green staining of cytoplasm and Hoechst 33342 (blue) staining of the nucleus.
Black arrow points show ECM CL-MSCs cultured 4 days in BioNOC II® II - staining of collagen fibers with picro sirius red
This macroporous carrier also has a distinct geometrical design that is specially folded to avoid overlapping of cells. This allows sufficient nutrient and oxygen transfer.
Adherent cells attached to BioNOCTM II carriers are alternately exposed to aeration and nutrition phases by pumping media in and out of the packed bed.
BioNOC II® II also exhibits great surface treatment. This macrocarrier can have a stable hydrophilicity for up to 4 years. It is non-cytotoxic that passed both serum and serum-free medium tests. Some cells are loosely adherent in nature, which is why they require with coating factors prior to cultivation to support higher attachment efficiency. However, not all microcarriers can support this. This limitation is being addressed with the use of BioNOC II® II. Due to its enhanced hydrophilicity, the addition of coating factors is easily accommodated.
BioNOC II® II presents straight and uniform fibers which are highly favored for cell distribution and growth. It also possesses higher density that prevents it from floating during the cell culture and remains stable all throughout the culture run.
Vero cells have been cultured in BioNOC II® II and are grown to high densities from 2-3x109 using batch or perfusion mode of culture using Tide Motion bioreactors such as CelCradleTM and TideXcellTM. The surface area and the cell numbers achieved for Vero cell culture are shown to be very promising. These results prove that the BioNOC II® II macrocarrier allows for a robust linear scalability, and thus saves time, labor, and resources for process development.
Figure 1. Live cell fluorescent staining of Vero cells on BioNOCTM II under 4x magnification. Calcein green staining of cytoplasm, Hoechst 33342 (blue) staining of the nucleus and Propidium Iodide stain for dead cells was done.
The fold expansion of Vero cells from attachment on BioNOCTM II in a 1L packed bed to harvest using an automated TideXcell Harvesting System (TXLHS) was 19-fold and the efficiency of harvest was 93.6%.
Ten (10) pieces of BioNOCTM II macrocarriers is equivalent to one (1) T-25 flask.
|Table 1. Cell growth in SFM of different carriers|
|Microcarriers||2.6 x 106 cells/ml|
|Macrocarriers||5.8 x 106 cells/ml|
|Table 2. Cell growth in serum-containing media of different carriers|
|Microcarriers||3.3 x 106 cells/ml|
|Macrocarriers||6.5 x 106 cells/ml|
|BioNOC II® II||Commercial Disks|
|Material||100% PET||PET / PP|
|Dimension||55 mm x 10 mm strip||Disk diameter 6 mm|
|Specific Surface area||2,400||1,200|
|Packed volume (ml/g)||15||10|
|Endotoxin tested (EU/ml)||< 0.25||< 0.5|
|Bioburden (CFU/gram)||< 1||< 0.36|
|Cytotoxicity tested||Serum and serum-free culture test||Yes|
|Regulation guideline||USP <87> <88>, ISO10993-5||USP <87> <88>, ISO10993-5|