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Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures

Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely de...

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Autores principales: Lam, Alan Tin-Lun, Li, Jian, Chen, Allen Kuan-Liang, Birch, William R., Reuveny, Shaul, Oh, Steve Kah-Weng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Mary Ann Liebert, Inc. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540119/
https://www.ncbi.nlm.nih.gov/pubmed/26309800
http://dx.doi.org/10.1089/biores.2015.0010
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author Lam, Alan Tin-Lun
Li, Jian
Chen, Allen Kuan-Liang
Birch, William R.
Reuveny, Shaul
Oh, Steve Kah-Weng
author_facet Lam, Alan Tin-Lun
Li, Jian
Chen, Allen Kuan-Liang
Birch, William R.
Reuveny, Shaul
Oh, Steve Kah-Weng
author_sort Lam, Alan Tin-Lun
collection PubMed
description Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously, we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article, we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%), which leads to generation of cells/MC aggregates (400 μm in size) and high cell yields (2.4–3.5×10(6) cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (>90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover, tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10(8) cells.
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spelling pubmed-45401192015-08-25 Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures Lam, Alan Tin-Lun Li, Jian Chen, Allen Kuan-Liang Birch, William R. Reuveny, Shaul Oh, Steve Kah-Weng Biores Open Access Original Research Article Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously, we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article, we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%), which leads to generation of cells/MC aggregates (400 μm in size) and high cell yields (2.4–3.5×10(6) cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (>90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover, tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10(8) cells. Mary Ann Liebert, Inc. 2015-04-01 /pmc/articles/PMC4540119/ /pubmed/26309800 http://dx.doi.org/10.1089/biores.2015.0010 Text en © Alan Tin-Lun Lam et al. 2015; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.
spellingShingle Original Research Article
Lam, Alan Tin-Lun
Li, Jian
Chen, Allen Kuan-Liang
Birch, William R.
Reuveny, Shaul
Oh, Steve Kah-Weng
Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures
title Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures
title_full Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures
title_fullStr Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures
title_full_unstemmed Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures
title_short Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures
title_sort improved human pluripotent stem cell attachment and spreading on xeno-free laminin-521-coated microcarriers results in efficient growth in agitated cultures
topic Original Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540119/
https://www.ncbi.nlm.nih.gov/pubmed/26309800
http://dx.doi.org/10.1089/biores.2015.0010
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