Abstract
Human cell culture processes developed at research laboratory scale need to be translated to large-scale production processes to achieve commercial application to a large market. To allow this transition of scale with consistent process performance and control of costs, it will be necessary to reduce manual processing and increase automation. There are a number of commercially available platforms that will reduce manual process intervention and improve process control for different culture formats. However, in many human cell-based applications, there is currently a need to remain close to the development format, usually adherent culture on cell culture plastic or matrix-coated wells or flasks due to deterioration of cell quality in other environments, such as suspension. This chapter presents an example method for adherent automated human stem cell culture using a specific automated flask handling platform, the CompacT SelecT.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Liu Y, Hourd P, Chandra A, Williams DJ. 2010. Human cell culture process capability: a comparison of manual and automated production. J Tissue Eng Regen Med. 4(1):45–54.
RJ Thomas, PC Hourd, DJ Williams. 2008. Application of process quality engineering techniques to improve the understanding of the in vitro processing of stem cells for therapeutic use. J Biotech. 136:148–155.
RJ Thomas, AD Hope, P Hourd, M Baradez, EA Miljan, JD Sinden, DJ Williams. 2009. Automated, serum-free production of CTX0E03: a therapeutic clinical grade human neural stem cell line. Biotech letters. 31:1167–72.
RJ Thomas, PC Hourd, DJ Williams. 2009. Auto-mated, scalable culture of human embryonic stem cells in feeder-free conditions. Biotech Bioeng. 102: 1636–1644.
Terstegge S, Laufenberg I, Pochert J. 2007. Automated maintenance of embryonic stem cell cultures. Biotechnol Bioeng. 96:195–201.
Joannides A, Fiore-Hériché C, Westmore K. 2006. Automated mechanical passaging: a novel and efficient method for human embryonic stem cell expansion. Stem Cells. 24:230–5.
SKW Oh, AK Chen, Y Mok, X Chen, U-M Lim, A Chin, ABH Choo, S Reuveny. 2009. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Research 2:219–230.
Serra M, Brito C, Sousa MF, Jensen J, Tostões R, Clemente J, Strehl R, Hyllner J, Carrondo MJ, Alves PM. 2010. Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. J Biotechnol. 148(4):208–15.
Haasters F, Prall WC, Anz D, Bourquin C, Pautke C, Endres S, Mutschler W, Docheva D, Schieker M. 2009. Morphological and immunocytochemical characteristics indicate the yield of early progenitors and represent a quality control for human mesenchymal stem cell culturing. J Anat. 214(5):759–67.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Appendix (Automated Protocols)
Appendix (Automated Protocols)
Cell culture media top-up (Subheading 3.2; step 2)
-
<steps>
-
<fetch>
-
<dispense liquid = “hMSC Expansion media” volume = “25 ml”/>
-
<store passage = “no”/>
-
</fetch>
-
</steps>
Cell culture media change (Subheading 3.2; step 3)
-
<steps>
-
<fetch>
-
<dump pause = “3 s”/>
-
<dispense liquid = “hMSC Expansion media” volume = “40 ml”/>
-
<store passage = “no”/>
-
</fetch>
-
</steps>
Cell passage (Subheading 3.2; step 4)
-
<steps>
-
<new flasktypegroup = “Single”>
-
<putdown name = “pool”/>
-
</new>
-
<fetch maxrepeat = “9” staggertime = “0 s” interleave = “3”>
-
<dump pause = “4 s”/>
-
<dispense liquid = “Trypsin/EDTA” volume = “5 ml”/>
-
<swirl repeat = “1” speed = “100%” pause = “0 s” capped = “no”/>
-
<dump pause = “4 s”/>
-
<incubate period = “10 m”/>
-
<shake repeat = “30” speed = “100%” pause = “0 s” capped = “yes”/>
-
<dispense liquid = “hMSC Expansion media” volume = “8 ml”/>
-
<swirl repeat = “1” speed = “100%” pause = “1 s” capped = “no”/>
-
<pour name = “pool” pause = “4 s” robotspeed = “100%”/>
-
<dispose robotspeed = “100%”/>
-
</fetch>
-
<mix name = “pool”
-
volume = “10 ml”
-
repeat = “5”
-
fromheight = “2 mm”
-
toheight = “20 mm”
-
mixspeed = “10 ml/s”
-
finaldispensespeed = “10 ml/s”
-
newtip = “yes”/>
-
-
<count name = “pool”
-
fromheight = “5 mm”
-
aspiratespeed = “5 ml/s”
-
dispensespeed = “1 ml/s”
-
pause = “2 s”/>
-
-
<pickup name = “pool”/>
-
<dispense liquid = “hMSC Expansion media”
-
volume = “10 ml”
-
cellconc = “125,000”
-
minvolume = “0 ml”
-
maxvolume = “300 ml”/>
-
-
<putdown name = “pool”/>
-
<new repeat = “12” flasktypegroup = “Single”>
-
<dispense liquid = “hMSC Expansion media” volume = “13 ml”/>
-
<putdown name = “output”/>
-
<mix name = “pool”
-
volume = “10 ml”
-
repeat = “3”
-
fromheight = “2 mm”
-
toheight = “10 mm”
-
mixspeed = “10 ml/s”
-
finaldispensespeed = “10 ml/s”/>
-
-
<pipette fromname = “pool”
-
toname = “output”
-
volume = “7 ml”
-
fromheight = “2 mm”
-
toheight = “20 mm”
-
aspiratespeed = “4 ml/s”
-
dispensespeed = “4 ml/s”
-
pause = “2 s”/>
-
-
<pickup name = “output”/>
-
<swirl repeat = “1” speed = “75%” pause = “1 s” capped = “yes”/>
-
<store passage = “yes”/>
-
</new>
-
<pickup name = “pool”/>
-
<dispose/>
-
</steps>
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Thomas, R., Ratcliffe, E. (2012). Automated Adherent Human Cell Culture (Mesenchymal Stem Cells). In: Mitry, R., Hughes, R. (eds) Human Cell Culture Protocols. Methods in Molecular Biology, vol 806. Humana Press. https://doi.org/10.1007/978-1-61779-367-7_26
Download citation
DOI: https://doi.org/10.1007/978-1-61779-367-7_26
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-366-0
Online ISBN: 978-1-61779-367-7
eBook Packages: Springer Protocols