Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors

Denver C Surrao, Kathryn Boon, Breanna Borys, Sarthak Sinha, Ranjan Kumar, Jeff Biernaskie, Michael S Kallos

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Abstract

Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P < 0.05) at 60 rpm in SSBs than in static cultures. Furthermore, the utility of the SSBs, at 60 rpm is demonstrated by serial passaging of hSKPs from a small starting population, which can be isolated from an autologous skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P < 0.05) from static cultures, which recorded acidic pH conditions. The nutrient concentrations of the media in all the SSBs and static cultures did not drop below acceptable limits. Furthermore, there was no significant build-up of waste products to limit hSKP expansion in the SSBs. In addition, hSKP markers were maintained in the 60 rpm SSB as demonstrated by immunocytochemistry. This method of growing hSKPs in a batch culture at 60 rpm in a SSB represents an important first step in developing an automated bioprocess to produce substantial numbers of clinically viable hSKPs aimed at regenerating the dermis to improve healing of severe skin wounds. Biotechnol. Bioeng. 2016;113: 2725-2738. © 2016 Wiley Periodicals, Inc.

Original languageEnglish
Pages (from-to)2725-2738
Number of pages14
JournalBiotechnology and Bioengineering
Volume113
Issue number12
DOIs
Publication statusPublished - Dec 2016
Externally publishedYes

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Bioreactors
Suspensions
Skin
Biopsy
Tissue Donors
Dermis
Tissue
Multipotent Stem Cells
Waste Products
Morbidity
Adult Stem Cells
Batch Cell Culture Techniques
Stem cells
Burns
Grafts
Wound Healing
Nutrients
Shear stress
Immunohistochemistry
Oxygen

Cite this

Surrao, D. C., Boon, K., Borys, B., Sinha, S., Kumar, R., Biernaskie, J., & Kallos, M. S. (2016). Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors. Biotechnology and Bioengineering, 113(12), 2725-2738. https://doi.org/10.1002/bit.26040
Surrao, Denver C ; Boon, Kathryn ; Borys, Breanna ; Sinha, Sarthak ; Kumar, Ranjan ; Biernaskie, Jeff ; Kallos, Michael S. / Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors. In: Biotechnology and Bioengineering. 2016 ; Vol. 113, No. 12. pp. 2725-2738.
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abstract = "Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P < 0.05) at 60 rpm in SSBs than in static cultures. Furthermore, the utility of the SSBs, at 60 rpm is demonstrated by serial passaging of hSKPs from a small starting population, which can be isolated from an autologous skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P < 0.05) from static cultures, which recorded acidic pH conditions. The nutrient concentrations of the media in all the SSBs and static cultures did not drop below acceptable limits. Furthermore, there was no significant build-up of waste products to limit hSKP expansion in the SSBs. In addition, hSKP markers were maintained in the 60 rpm SSB as demonstrated by immunocytochemistry. This method of growing hSKPs in a batch culture at 60 rpm in a SSB represents an important first step in developing an automated bioprocess to produce substantial numbers of clinically viable hSKPs aimed at regenerating the dermis to improve healing of severe skin wounds. Biotechnol. Bioeng. 2016;113: 2725-2738. {\circledC} 2016 Wiley Periodicals, Inc.",
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Surrao, DC, Boon, K, Borys, B, Sinha, S, Kumar, R, Biernaskie, J & Kallos, MS 2016, 'Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors' Biotechnology and Bioengineering, vol. 113, no. 12, pp. 2725-2738. https://doi.org/10.1002/bit.26040

Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors. / Surrao, Denver C; Boon, Kathryn; Borys, Breanna; Sinha, Sarthak; Kumar, Ranjan; Biernaskie, Jeff; Kallos, Michael S.

In: Biotechnology and Bioengineering, Vol. 113, No. 12, 12.2016, p. 2725-2738.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors

AU - Surrao, Denver C

AU - Boon, Kathryn

AU - Borys, Breanna

AU - Sinha, Sarthak

AU - Kumar, Ranjan

AU - Biernaskie, Jeff

AU - Kallos, Michael S

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AB - Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P < 0.05) at 60 rpm in SSBs than in static cultures. Furthermore, the utility of the SSBs, at 60 rpm is demonstrated by serial passaging of hSKPs from a small starting population, which can be isolated from an autologous skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P < 0.05) from static cultures, which recorded acidic pH conditions. The nutrient concentrations of the media in all the SSBs and static cultures did not drop below acceptable limits. Furthermore, there was no significant build-up of waste products to limit hSKP expansion in the SSBs. In addition, hSKP markers were maintained in the 60 rpm SSB as demonstrated by immunocytochemistry. This method of growing hSKPs in a batch culture at 60 rpm in a SSB represents an important first step in developing an automated bioprocess to produce substantial numbers of clinically viable hSKPs aimed at regenerating the dermis to improve healing of severe skin wounds. Biotechnol. Bioeng. 2016;113: 2725-2738. © 2016 Wiley Periodicals, Inc.

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