Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells.

Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4-7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2-3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.

Embryoid Bodies-Based Multilineage Differentiation of Human Embryonic Stem Cells Grown on Feeder-Free Conditions.

Human embryonic stem cells (hESCs) can differentiate into any cell lineage (pluripotency potential) derived from the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is usually demonstrated in vitro by spontaneous differentiation of hESCs grown on a monolayer of feeder-cells using an embryoid bodies (EBs)-based method. However, currently hESCs are grown mostly using fully defined media in the absence of a feeder layer. Here we describe a EBs-based protocol that allows multilineage differentiation of hESCs and human induced pluripotent stem cells (hiPSCs) grown on feeder-free conditions.

Human Induced Pluripotent Stem (hiPS) Cells from Urine Samples: A Non-Integrative and Feeder-Free Reprogramming Strategy.

Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell-derived cells began in Japan, only 10 years after the publication of the proof-of concept article. In this exciting context, strategies to generate hiPS cells have evolved quickly, tending towards non-invasive protocols to sample somatic cells combined with "safer" reprogramming strategies. In this unit, we describe a protocol combining both of these advantages to generate hiPS cells with episomal plasmid transfection from urine samples of individuals carrying the desired genotype. Based on previous published works, this simplified protocol requires minimal equipment and reagents, and is suitable both for scientists familiar with the hiPS cells technology and neophytes. HiPS cells displaying classical features of pluripotency and suitable for all desired downstream applications are generated rapidly (<10 weeks) and with high efficiency. © 2017 by John Wiley & Sons, Inc.

Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

Effects of culture medium and substrate on attachment of in vitro produced bovine embryos

The study of large animal embryonic stem cells (ESCs) in vitro has implications for the understanding of lineage differentiation and transgenesis. The first step for ESC derivation is the attachment of the embryo to a substrate on which they can form outgrowths. However, the culture conditions for large animal embryo attachment and ESC derivation have not been studied extensively. Defining culture conditions for embryo attachment such as culture medium and substrate is an important first step for derivation of inner cell mass-derived stem cells. The aim of this study was to compare different types of culture media and substrates for their ability to support attachment of in vitro produced bovine embryos in culture. Bovine embryos were produced in vivo following established protocols. Blastocysts formed on day 8 after fertilization were transferred to 12-well culture plates containing different types of culture media (Dulbecco's Modified Eagle Medium, DMEM or Medium 199, M199) and substrates [bovine fetal fibroblasts, goat fetal fibroblasts, mouse embryonic fibroblasts (STO) or non-cellular substrates (gelatin, laminin, fibronectin)]. Percentage of attached embryos and number of days since fertilization required for attachment were recorded. Bovine blastocysts preferrably attached to feeder cells rather than non-cellular substrates and there was an interact ion of feeder cell type and culture medium used. Therefore, the choice of both feeder cell type and culture medium has to be considered when optimizing conditions to derive cell lines from bovine embryos.(AU)

Effects of culture medium and substrate on attachment of in vitro produced bovine embryos

The study of large animal embryonic stem cells (ESCs) in vitro has implications for the understanding of lineage differentiation and transgenesis. The first step for ESC derivation is the attachment of the embryo to a substrate on which they can form outgrowths. However, the culture conditions for large animal embryo attachment and ESC derivation have not been studied extensively. Defining culture conditions for embryo attachment such as culture medium and substrate is an important first step for derivation of inner cell mass-derived stem cells. The aim of this study was to compare different types of culture media and substrates for their ability to support attachment of in vitro produced bovine embryos in culture. Bovine embryos were produced in vivo following established protocols. Blastocysts formed on day 8 after fertilization were transferred to 12-well culture plates containing different types of culture media (Dulbecco's Modified Eagle Medium, DMEM or Medium 199, M199) and substrates [bovine fetal fibroblasts, goat fetal fibroblasts, mouse embryonic fibroblasts (STO) or non-cellular substrates (gelatin, laminin, fibronectin)]. Percentage of attached embryos and number of days since fertilization required for attachment were recorded. Bovine blastocysts preferrably attached to feeder cells rather than non-cellular substrates and there was an interact ion of feeder cell type and culture medium used. Therefore, the choice of both feeder cell type and culture medium has to be considered when optimizing conditions to derive cell lines from bovine embryos.

Substrates and supplements for hESCs: a critical review.

BACKGROUND: Different laboratories around the world have succeeded in establishing human embryonic stem cell (hESC) lines. However, culture conditions vary considerably among the protocols used and the vast majority of the lines at some stage of their creation have been in contact with an animal derived component. One of the main problems to be overcome for the generation of a clinical-grade hESC line is the choice of a substrate and medium that allows derivation and culture, where animal derived components are kept to a minimum or completely excluded. MATERIALS AND METHODS: The following review describes past and more recent achievements in the creation and culturing of hESC. It describes protocols, giving special attention to the matrices and supplements used for derivation, maintainance and cryostorage, considering whether they included defined, undefined and/or animal-derived components in their formulations. CONCLUSION: This information shall be useful for the creation and choice of new substrates and supplements for future research in the field of hESC for therapeutic purposes.

Matrix-bound heparan sulfate is essential for the growth and pluripotency of human embryonic stem cells.

Human embryonic stem (hES) cell production of heparan sulfate influences cell fate and pluripotency. Human ES cells remain pluripotent in vitro through the action of growth factors signaling, and the activity of these factors depends on interaction with specific receptors and also with heparan sulfate. Here, we tested the hypothesis that matrix-associated heparan sulfate is enough to maintain hES cells under low fibroblast growth factor-2 concentration in the absence of live feeder cells. To pursue this goal, we compared hES cells cultured either on coated plates containing live murine embryonic fibroblasts (MEFs) or on a matrix derived from ethanol-fixed MEFs. hES cells were analyzed for the expression of pluripotency markers and the ability to form embryoid bodies. hES cells cultured either on live mouse fibroblasts or onto a matrix derived from fixed fibroblasts expressed similar levels of Oct-4, SOX-2, Nanog, TRA-1-60 and SSEA-4, and they were also able to form cavitated embryoid bodies. Heparan sulfate-depleted matrix lost the ability to support the adherence and growth of hES cells, confirming that this glycosaminoglycan, bound to the extracellular matrix, is enough for the growth and attachment of hES cells. Finally, we observed that the ethanol-fixed matrix decreases by 30% the levels of Neu5Gc in hES cells, indicating that this procedure reduces xeno-contamination. Our data suggest that matrix-bound heparan sulfate is required for the growth and pluripotency of hES cells and that ethanol-fixed MEFs may be used as a "live cell"-free substrate for stem cells.

Generation of induced pluripotent mouse stem cells in an indirect co-culture system.

Typically, production of induced pluripotent stem cells requires direct contact with feeder cells. However, once the stem cells have reached the appropriate maturation point, it is difficult to separate them from feeder cells, which must be irradiated with γ-rays or treated with the antibiotic mitomycin-C. We used a microporous poly-membrane-based indirect contact co-culture system with mouse embryonic fibroblasts to induce mouse pluripotent stem cells without radiation or antibiotics. We found that induced pluripotent stem cells induced by this co-culture method had a reprogramming efficiency and time similar to those induced using traditional methods. Furthermore, strongly expressed pluripotent markers showed a normal karyotype and formation and contained all three germ layers in a teratoma.