Cell Expansion
Introduction
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
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
References: Ashammakhi,N, Reis,R, Chiellini,E. (2007) Topics in Tissue Engineering. Vol 3, University of Oulu. Berstein,I.D, Delaney,C. (2012) Engineering stem cell expansion. Cell:10, 113-114 Fong ,W.J, Tan, H.L, Choo, A, Oh,S.K .(2005) Perfusion cultures of human embryonic stem cells. Bioprocess and biosystems engineering; 27:381–387. GE Healthcare (2011). Microcarrier cell culture principle and methods. MacLren,R.E, Pearson,R.A, MacNeil,A, Douglas,R.H, Salt,T.E, Akimoto,M, Swaroop,A, Sowden,J.C, Ali,R.R. (2006) Retinal repair by transplantation of photoreceptor precursors. Nature, 444: 203-207 Melero-martin, J.M, Al-Rubeai,M. (2007) In vitro expansion of chondrocytes. University College Dublin Nie,Y, Bergendahl,V, Hei,D.J, Jones,J.M, Palecek,S.P. (2009) Scalable culture and cryopreservation of human embryonic stem cells on microcarriers. Biotechnol prog, 25: 20-31 Serra,M, Brito,C, Sousa,M.F.Q, Jensen,J, Tostoes,R, Clemente,J, Strehl,R, Hyllner,J, Carrondo,J.T, Alves,P.M. (2010) Journal of Biotechnology;148, 208-215 Wikstrom, K., Blomberg, P., and Islam, K. B. (2004) Clinical Grade Vector Production: Analysis of Yield, Stability, and Storage of GMP-Produced Retroviral Vectors for Gene Therapy. Biotechnol Prog; 20, 1198-1203