Understanding cell culture dynamics_ a tool for defining protocol parameters for improved processes _ Enhanced Reader.pdf (11.09 MB)

Understanding cell culture dynamics: a tool for defining protocol parameters for improved processes and efficient manufacturing using human embryonic stem cells

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journal contribution
posted on 25.03.2021, 09:29 by James Kusena, Maryam Shariatzadeh, Rob Thomas, Sammy Wilson
Standardization is crucial when culturing cells including human embryonic stem cells (hESCs) which are valuable for therapy development and disease modeling. Inherent issues regarding reproducibility of protocols are problematic as they hinder translation to good manufacturing practice (GMP), thus reducing clinical efficacy and uptake. Pluripotent cultures require standardization to ensure that input material is consistent prior to differentiation, as inconsistency of input cells creates end-product variation. To improve protocols, developers first must understand the cells they are working with and their related culture dynamics. This innovative work highlights key conditions required for optimized and cost-effective bioprocesses compared to generic protocols typically implemented. This entailed investigating conditions affecting growth, metabolism, and phenotype dynamics to ensure cell quality is appropriate for use. Results revealed critical process parameters (CPPs) including feeding regime and seeding density impact critical quality attributes (CQAs) including specific metabolic rate (SMR) and specific growth rate (SGR). This implied that process understanding, and control is essential to maintain key cell characteristics, reduce process variation and retain CQAs. Examination of cell dynamics and CPPs permitted the formation of a defined protocol for culturing H9 hESCs. The authors recommend that H9 seeding densities of 20,000 cells/cm2, four-day cultures or three-day cultures following a recovery passage from cryopreservation and 100% medium exchange after 48 hours are optimal. These parameters gave ~SGR of 0.018 hour−1 ± 1.5x10−3 over three days and cell viabilities ≥95%±0.4, while producing cells which highly expressed pluripotent and proliferation markers, Oct3/4 (>99% positive) and Ki-67 (>99% positive).

Funding

EPSRC and MRC Centre for Doctoral Training in Regenerative Medicine

Engineering and Physical Sciences Research Council

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History

School

  • Mechanical, Electrical and Manufacturing Engineering

Published in

Bioengineered

Volume

12

Issue

1

Pages

979 - 996

Publisher

Informa UK Limited

Version

VoR (Version of Record)

Rights holder

© The authors

Publisher statement

This is an Open Access Article. It is published by Taylor and Francis under the Creative Commons Attribution 4.0 Unported Licence (CC BY). Full details of this licence are available at: http://creativecommons.org/licenses/by/4.0/

Acceptance date

09/03/2021

Publication date

2021-03-24

Copyright date

2021

ISSN

2165-5979

eISSN

2165-5987

Language

en

Depositor

Dr Sammy Wilson. Deposit date: 24 March 2021