Feeder Cell- and Extracellular Matrix-Free Cultivation of Human Embryonic Stem Cells

In cell biology, pluripotency refers to stem cells that have the ability to differentiate into any of the three germ layers: endoderm (stomach lining, gastrointestinal tract, lungs); mesoderm (bone, muscle, blood); or ectoderm (epidermal tissue, nervous system). Although pluripotent stem cells have this inherent ability, in order for this promise to be realized, users require a stable and reproducible method of cell culture.

The ability to grow any type of cell or tissue is key in the development of new treatments for a variety of disorders. Many medical problems arise as a result of damage to cells postdifferentiation. For example:

  • Type I diabetes mellitus: β cells of the pancreas have been destroyed or cease to function
  • Parkinson’s disease : Dopamine-secreting cells of the brain have been destroyed
  • Spinal cord injuries: Can lead to paralysis of skeletal muscle
  • Ischemic stroke: A blood clot in the brain causes neurons to die from oxygen starvation
  • Multiple sclerosis: Loss of myelin sheath surrounding axons of the nervous system.

As such, the accurate and reproducible growth of pluripotent stem cells is essential for research and development into the treatment of many acute and chronic disorders.

Stem cells: The importance of pluripotency

There are three types of naturally occurring pluripotent stem cells: 

  1. Embryonic stem cells (ESC), which can be isolated from the inner cell mass of the blastocyst;
  2. Embryonic germ cells (EGC), which can be isolated from the precursors to the gonads; and
  3. Embryonic carcinoma cells (ECC), which can be isolated from teratocarcinomas, a form of tumor that can occur during development.

Figure 1 - Nunclon Vita surface.

Once extracted, these cell types are grown and passaged to produce sufficient volumes for use. Current methods rely on feeder cells or extracellular matrix (ECM) proteins to cover the culture-ware growth surface, which can vary in composition and density across the plate. These inconsistencies introduce an unwanted variable to the experiment, and laboratory workers need to take steps to remove this in order to produce consistent culture environments and reproducible results. This can become a labor-intensive process. Furthermore, in order to dissociate cultured cells from the growth surface, an enzymatic method such as trypsin digestion is commonly used. This can result in the degradation and cleavage of cell-surface proteins, which are vital for cellular interactions, normal cell function, and response to environmental stimuli. As such, trypsinization can cause compromised function and cellular damage, and in turn affect assay results.

To produce stable and reliable cultures for use in downstream applications, users require a surface that enables the growth of cells directly on it, without the need for ECM protein coatings or feeder cells. This article describes use of the Thermo Scientific Nunclon™ Vita surface (Thermo Fisher Scientific, Rochester, NY), an animal component-free polystyrene surface for the reliable and reproducible culture of stem cells (see Figure 1).

Methods

Human ESC cultivations

  1. Cells. Passage-49 (cells that have undergone 49 rounds of passage) human ESCs were maintained in mouse embryonic fibroblast (MEF) conditioned medium on the Nunclon Delta™ surface, coated with a 1:30 dilution of a growth factor reducing gelatinous protein mix. Cells were dissociated from the surface for passage with 1 mg/mL collagenase and seeded onto Nunclon Vita surfaces with or without Rho-kinase inhibitor in the medium. As a small GTPase, Rho is implicated in many cellular functions, with its key effector Rho-kinase controlling a variety of cellular processes, including cell migration, actin depolymerization, and cellular contractility, that are dependent upon actin cytoskeleton organization and cell contractility.

a. Cultivation without Rho-kinase inhibition. H1 ESCs were grown for four passages in MEF-conditioned medium on Nunclon Vita surfaces. Cells were dissociated from the surface for passage by treatment with 1 mg/mL collagenase. Normal passage time for H1 ESCs was 3–4 days on a protein matrix. However, cells plated on the Nunclon Vita surface took seven days of culturing before they were ready for passage, and a spontaneous decrease in growth rate was observed over the passages. 

b. Cultivation with Rho-kinase inhibition. H1 ESCs were grown in MEF-conditioned medium supplemented with 10 μM Rho-kinase inhibitor. Cells were dissociated from the surface for passage by treatment with 1 mg/mL collagenase. Cells plated with 10 μM Rho-kinase inhibitor on the Nunclon Vita surface were ready for passage four days after plating.

Harvesting

In order to harvest the cultured cells for subsequent passaging, plates are incubated with fresh growth medium without Rho-kinase inhibitor for 15–30 min to lift them from the surface without the use of trypsin. Cells can then be resuspended in growth medium that contains Rho-kinase inhibitor for further growth.

Human ESC characterization

Colony presence and morphology were determined using phase-contrast microscopy and by eye, following colony staining with 0.5% crystal violet. Pluripotency was determined via the presence of pluripotency markers, use of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to identify gene expression, flow cytometry for cell-surface marker expression, and immunofluorescence for the presence of cell-surface and nuclear proteins.

Figure 2 - Dose–response effect of Rho-kinase on the attachment of human ESCs to the Nunclon Vita surface. Rho-kinase inhibitor was added to the cultures at a specific concentration (0, 1, 2, 4, or 10 μM) at seeding. The cells were then maintained from day 2 onward in media containing 10 μM Rho-kinase inhibitor, with daily media changes over five days, after which cells were stained with crystal violet.

Karyotypic stability was determined by cytogenic analysis of 20 G-banded metaphase cells, as well as fluorescence in situ hybridization (FISH) on 200 interphase nuclei. The ability to form embryoid bodies was subsequently determined by growing ESCs in a low-binding plate for 10 days in DMEM/F12 (a serum-free media for general use) containing 10% fetal bovine serum (FBS).

Results

When passaging cells on the culture surface in the absence of Rho-kinase inhibition, human ESCs can only be passaged a few times before a decline in growth rate and pluripotency markers is observed. However, this decline is not observed if the culture medium is supplemented with a Rho-kinase inhibitor, as seen in Figure 2. Furthermore, these cultured cells display a normal karyotype, express pluripotency markers, and can be differentiated into embryoid bodies (Figure 3). Human ESCs grown in the presence of Rho-kinase inhibitor can easily be dissociated from the Nunclon Vita surface by incubating the plate with fresh growth medium, without Rho-kinase inhibitor, for 15–30 min. Cells can then be mechanically dissociated from the plate by gentle pipetting or scraping, briefly centrifuged, and resuspended in growth medium containing Rho-kinase inhibitor. The ESCs can finally be mechanically dissociated by gentle titration and reseeded. This suggests that the presence of a Rho-kinase inhibitor effectively alters the balance between pluripotency and early differentiation in ESCs.

Figure 3 - a) Normal karyotype and FISH patterns for human ESC after 11 passages. b) Expression of the pluripotency markers in Human ESCs after 11 passages. c) Human ESCs can form embryoid bodies after 11 passages.

Conclusion

The accurate and reliable culturing of pluripotent stem cells is an essential starting point to the therapeutic treatment of a broad array of disease types, resulting from damage to differentiated cells. As such, it is essential that methods of growing large cultures of cells are highly accurate and reproducible. The Nunclon Vita surface supported feeder- and ECM protein-free attachment, colony formation, and growth of human ESCs for a few passages in medium conditioned by mouse embryonic fibroblasts, and for several passages in medium conditioned by mouse embryonic fibroblasts and supplemented by Rho-kinase inhibitor.

The human ESCs grown over 11 passages in Rho-kinase inhibitor medium displayed a normal karyotype, where the presence of a Rho-kinase inhibitor promotes stem cell survival by diminishing dissociationinduced apoptosis.1,2 Pluripotency markers were therefore expressed, and ESCs could subsequently be differentiated into embryoid bodies. This cellular aggregation into embryoid bodies forms the initial step of the differentiation process and, thus, by initiating the correct stimuli, researchers should be able to produce any cell typefor downstream therapeutic use. Human ESCs could be passaged without the use of enzymes or manual selection by withdrawing the Rho-kinase inhibitor to lift the cells from the surface, followed by replating in its presence again. The Nunclon Vita culture surface has therefore proven able to culture human ESCs for at least 10 passages without any loss of pluripotency.

References

  1. Wantanabe, K.; Ueno, M. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnol.2007, 25, 681–6.
  2. Emre, N.; Vidal, J.G. et al. The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers. PLos One2010, 8, e12148.

Dr. Neeley is a Field Technical Specialist, Thermo Fisher Scientific, 75 Panorama Creek, Rochester, NY 14625, U.S.A.; tel./fax: 740-321-1280; e-mail: cindy.neeley@thermofisher.com.

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