The current method of cell expansion using T25 flasks for human embryonic stem cells (hESCs) have been proven to be extremely time and space consuming, labour intensive and difficult for scale-up (Minimal of 200 T25 flasks needed). It is estimated 2.8x108 - 5.6x108 undifferentiated hESCs are required by the end of the expansion stage for the process to work, as at least 5x107 cells of well differentiated post-mitotic Nrl+/Crx+ precursors are needed for transplant (Maclaren et al, 2006). 5x107 well differentiated post-mitotic Nrl+/Crx+ precursors
70% loss from thawing
15% cells lost during differentiation
30% cell loss due to cells not differentiating correctly
10 day process, passaging every 5 days (50 passages total)
Figure 1: Schematic diagram of the current lab process
For the treatment of photoreceptor dystrophies to become industrially and commercially viable advances are needed in the cell banking and cell expansions of the bioprocess, by improving the quality, quantity and cost-effectiveness of production. The report will evaluate novel methods of cell expansion and cell banking in order to improve the current lab-based protocol (Bernstein and Delaney, 2012). Roller Bottles (Automation)
Roller bottles provide a larger surface area to volume ratio compared to T-flasks. Roller bottle also allows gentle rocking of the containers which prevent gradients from forming within the medium that may reduce cell growth, secondly the use of roller bottles allow cells to be only covered by a thin layer of medium, thereby providing superior gas exchange in comparison to the traditional monolayer cultures (Ashammakhi et al, 2007).
The design of roller bottles enables the process to be easily automated. Automation greatly reduces the manual labour involved as well as reducing the risk of contamination. The reproducibility of the process is also improved. Currently the RollerCell 40 (CELLON S.A, Luxembourg) is a self-contained, fully automated processing system, providing surface area of 3.5x105cm2 for cell growth (Wikstrom et al, 2004). However the Roller bottle technique is difficult to scale, thus making it more suitable for seeding cultures rather than the expansion of cultures.
Figure 2: Storage of roller bottles
Microcarriers (spinner flasks and bioreactors)
Microcarriers provide a platform for anchorage-dependent animal cells to be grown in suspension, hence greatly increasing the surface area to volume ratio for cell culturing compared to static cultures grown on T-flasks or Petri dishes. Microcarriers have a spherical structure, roughly sized between 60-87µm. Nie et al, 2009 clearly demonstrated the potential use of microcarriers in spinner flasks for cell expansion. Cytodex 3(GE healthcare) microcarriers were modified with Matrigel for better cell attachment and improved maintenance of undifferentiated hESCs. The study shows unexpectedly, a similar growth rates between hESCs cultured on microcarriers and culture plates. This may have been caused by the hESCs were originally seeded on plates, thus the stem cells were well adapted for plate culture conditions, which means the hESCs had to adapt to the microcarrier conditions, therefore retarding its growth (Nie et al, 2009). So it would be preferable to culture hESCs on microcarriers for the cells to adapt to the microcarrier conditions before starting the expansion phase. They also noticed the hESCs grow in multiple layers on microcarriers, plus microcarriers are subjected to agglomeration forming larger cell masses. The large cell mass was extremely detrimental for mass transfer of nutrients, growth factors and oxygen. Also the build up of metabolic waste have a negative effect on the growth rate of cells (Nie et al, 2009).
Other studies have shown the use of microcarriers in combination with bioreactors has significantly improved the yield and overall growth rate of hESCs (see figure 3 and 4).
Figure 3: Comparison of the...