Chapter 29
Chemically Defined and Xeno-Free Cryopreservation
of Human-Induced Pluripotent Stem Cells
Juliette Seremak and Ali Eroglu
Abstract
Human-induced pluripotent stem cells (hiPSCs) can be derived from a variety of biopsy samples and have an
unlimited capacity for self-renewal and differentiation into almost any cell type in the body. Therefore,
hiPSCs offer unprecedented opportunities for patient-specific cell therapies, modeling of human diseases,
biomarker discovery, and drug testing. However, clinical applications of hiPSCs require xeno-free and,
ideally, chemically defined methods for their generation, expansion, and cryopreservation. In this chapter,
we present a chemically defined and xeno-free slow freezing method for hiPSCs along with a chemically
undefined protocol. Both approaches yield reasonable post-thaw viability and cell growth.
Key words Cryopreservation, Human-induced pluripotent stem cells (iPSCs), Chemically defined,
Xeno-free, Clinical-grade cryopreservation
1 Introduction
The idea of having indefinitely expandable pluripotent cells that
could be used for the treatment of various diseases and injuries,
along with organ and tissue regeneration, has propelled the progress of stem cell research over the decades [1, 2]. With the first
discovery of embryonic stem cells (ESCs) in mice in 1981 [3, 4]
and then the isolation of ESCs from human blastocysts in 1998 [1],
there have been several advancements in technology that have
allowed scientists to further understand the complexities and problems associated with the isolation and future use of stem cells.
Although human ESCs hold great potential for many clinical and
pharmaceutical applications, there are ethical and political issues
surrounding the use of these cells due to their derivation from
preimplantation embryos [5]. This obstacle led to a major breakthrough in the field of regenerative medicine with the discovery of
reprogramming of mouse embryonic and adult fibroblasts into a
pluripotent state by forced expression of four transcription factors
(i.e., Oct4, Sox2, Klf4, and c-Myc) in 2006 [6]. These
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_29, © Springer Science+Business Media, LLC, part of Springer Nature 2021
569
Chemically Defined and Xeno-Free Cryopreservation
of Human-Induced Pluripotent Stem Cells
Juliette Seremak and Ali Eroglu
Abstract
Human-induced pluripotent stem cells (hiPSCs) can be derived from a variety of biopsy samples and have an
unlimited capacity for self-renewal and differentiation into almost any cell type in the body. Therefore,
hiPSCs offer unprecedented opportunities for patient-specific cell therapies, modeling of human diseases,
biomarker discovery, and drug testing. However, clinical applications of hiPSCs require xeno-free and,
ideally, chemically defined methods for their generation, expansion, and cryopreservation. In this chapter,
we present a chemically defined and xeno-free slow freezing method for hiPSCs along with a chemically
undefined protocol. Both approaches yield reasonable post-thaw viability and cell growth.
Key words Cryopreservation, Human-induced pluripotent stem cells (iPSCs), Chemically defined,
Xeno-free, Clinical-grade cryopreservation
1 Introduction
The idea of having indefinitely expandable pluripotent cells that
could be used for the treatment of various diseases and injuries,
along with organ and tissue regeneration, has propelled the progress of stem cell research over the decades [1, 2]. With the first
discovery of embryonic stem cells (ESCs) in mice in 1981 [3, 4]
and then the isolation of ESCs from human blastocysts in 1998 [1],
there have been several advancements in technology that have
allowed scientists to further understand the complexities and problems associated with the isolation and future use of stem cells.
Although human ESCs hold great potential for many clinical and
pharmaceutical applications, there are ethical and political issues
surrounding the use of these cells due to their derivation from
preimplantation embryos [5]. This obstacle led to a major breakthrough in the field of regenerative medicine with the discovery of
reprogramming of mouse embryonic and adult fibroblasts into a
pluripotent state by forced expression of four transcription factors
(i.e., Oct4, Sox2, Klf4, and c-Myc) in 2006 [6]. These
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_29, © Springer Science+Business Media, LLC, part of Springer Nature 2021
569