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Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

ABSTRACT: To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling.

SUBMITTER: Dores C

PROVIDER: S-EPMC4495971 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

Dores C C Rancourt D D Dobrinski I I

Andrology 20150415 3

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells fr ...[more]

PMID: 25877677

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Project description:BackgroundHuman induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production.MethodsThe vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times.ResultsCFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function.ConclusionsTaken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.

Dataset Information

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

Publications

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

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