Researchers claim surface chemical modification supports long-term culture of undifferentiated cells.

Researchers have found that treating polystyrene lab plates with ultraviolet (UV) waves generates a modified surface chemistry that supports the growth of three times as many human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) than current methods that rely on layers of feeder cells. The team’s approach effectively generates spots of UV-treated surface on the culture plates as a means to control cell aggregation and promote the growth of human pluripotent stem cells in an undifferentiated state.

Reporting on their technique in PNAS, the Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology-led team says the treated surfaces supported the long-term cell culture of hESCs and iPSCs through numerous passages and are compatible with hESCs/hiPSCs manipulations including gene modification and reprogramming.

“These surface-engineered substrates therefore have strong potential to replace feeder-containing substrates in almost any procedure envisioned with human pluripotent cells, enabling broad and rapid scale-up of these cells for both research and clinical applications,” the team claims. Their published paper is titled “Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.”

hESCs and hIPSCs are generally grown either on a feeder cell layer of mouse embryonic fibroblasts (mEFs) or in feeder-free culture systems that include extracellular matrix or serum proteins coated onto tissue culture dishes or other synthetic materials. However, a number of these culture methods use animal products, which will cause problems if the cells are to be used for transplantation, and most only result in modest gains. Moreover, the majority of such systems require cells to be seeded at high density and passaged in multicellular clumps.

This is a time-consuming process if large quantities of pluripotent cells are required from a starting population of only a few, or perhaps even just a single, genetically modified cell, the authors add. Indeed, clinical applications of stem cells may require 1010 cells per patient, and even disease modeling studies will typically require over 106 cells to generate s single differentiated cell type.

The Whitehead and MIT’s approach to generating optimum growth conditions for hESCs and hIPSCs focused on modifying the surface chemistry of polystyrene, which is the most commonly used plastic in cell culture.  They postulated that because hESCs and hiPSCs aggregate or undergo apoptosis as single cells under conventional culture conditions, changing the surface of the culture substrate might influence early aggregation and potentially improve cell propagation.  

Previous published work by two of the team’s scientists, Krishanu Saha, Ph.D., and Ying Mei,  demonstrated that significant increases in cell growth could be achieved by using synthetic polymer substrates with high content of hydrocarbon and ester groups. The technology, while successful, was unfortunately less practical for large-scale applications because the polymers were difficult to synthesize in a range of cell culture formats.

“The last paper was trying to figure out what is the correct, optimum surface chemistry to support stem cells,” Dr. Mei comments. The follow-on work involved treating virgin polystyrene with different doses of short-wavelength UV and then examining the treated surfaces using time of flight secondary ion mass spectrometry (ToF-SIMS). The results indicated that UV treatment generated chemically distinct surfaces on both virgin polystyrene and conventional tissue culture polystyrene (TCPS).

The effects of UV surface modification on cell culture were then tested by evaluating the growth of fully dissociated transgenic Oct4-GFP-positive BG01 hESCs on modified polystyrene precoated with fetal bovine serum (FBS). From these results they found that moderately UV treated (i.e. 1.5–3 minutes) surfaces supported robust growth of hESCs, whereas no cells attached to virgin polystyrene surfaces and only a few on TCPS.

The team then applied a numerical model using a multivariate chemometric technique to identify which functionalities generated by UV were important for colony formation. The data indicated that self-renewal of hESCs was supported by ester/carboxylic acid functionalities yielding oxygen-containing ions in the ToF-SIMS spectra.

“In particular, polystyrene surfaces treated with UV for 1.5–3 min produced high intensities of several secondary ions in the ToF-SIMS experiments that were identified to support hESC colony formation (e.g., C2H5O+ and C2H6N+),” they write.

In fact, at the optimal 2.5-minute dose, UV-treated polystyrene (UVPS) harbored a surface on which hESCs could be readily passaged directly from standard mEF substrates and vice versa without causing any significant cell death. And when coated with human serum, UVPS supported clonal growth in serum-free mTeSR1 media (Stem Cell Technologies) with an efficiency of about 30 ± 12%, which is comparable to the efficiency of acrylate-based polymer, they note.

Most notably, culturing fully dissociated hESCs (in relevant medium) on UVPS at the typical 1:3 dilution ratio used during conventional hESC passaging supported  three times more cells per area than traditional feeder-containing mEF substrates and resulted in at least twofold more colonies per cell seeded than conventional cell culture plastics.

This initial surface modification wasn’t perfect, however. After about seven days of culture on UVPS, several large scattered colonies developed that harbored differentiating cells at their centres. To prevent the formation of these large colonies, the researchers spatially modified their UV treatment technique by placing a photomask between the polystyrene and the radiation source, generating a polystyrene surface with spots of UV modification.

These spatially patterned UV-treated surfaces constrained cell surface migration and adhesion so that hESCs only attached and grew on areas of the FB S-coated dish that received the UV treatment. “Similar results were seen with five other human pluripotent stem cell lines including three hiPSC lines and when human serum or human vitronectin was used instead of FBS to coat the UV-patterned surfaces,” the authors remark.

Use of UV to spatially pattern polypropylene and hydrogel-coated substrates also resulted in surfaces that supported colony formation from fully dissociated hESCs. UV-patterned surfaces with 150–450 μm spots supported the growth of cells with uniform staining of all pluripotency markers. If the spots were less than 200 μm apart, however, there was poor spatial patterning because the cells could bridge the gaps: a number of cells that did so were found to express low levels of pluripotency markers and exhibited a more differentiated cell morphology. The investigators then tested the growth of fully dissociated hESCs seeded on surfaces that harbored differing spot diameters but the same cumulative UV-treated growth area per well.

This showed that similar numbers of cells were supported for the first 24 hours or so after seeding, even as the tested growth area was partitioned into progressively smaller and smaller spots. However, after an additional seven days of growth, cultures with the smaller spot diameters contained more undifferentiated cells, and proliferated faster.

The UV patterned polystyrene surface was also found to support long-term cell culture. The cells maintained an undifferentiated state at each passage for more than 10 passages, continued to display a normal karyotype, and robust pluripotency marker expression, and could generate cells of all three embryonic germ layers.

“Routine cell culture with collagenase passaging yielded significant increases in the number of undifferentiated cells over mEFs that increased from passage to passage, indicating its utility for scale-up applications,” the investigators add.

By subjecting hESCs on UV-patterned substrates to GFP targeting of the AAVS1 locus using, the team was also able to confirm that the substrates supported clonal outgrowth of gene-targeted cells, and these cells remained GFP-positive after more than two months of culture. Moreover, hujman serum-coated UV-patterned polystyrene surfaces also supported the reprogramming of human fibroblasts under xeno-free conditions. The resulting hIPSCs expressed pluripotency markers and were capable of generating derivatives of all three embryonic germ layers.

And in a final set of experiments, the team found that UV-patterned substrates supported the single-cell enzymatic passaging of hESCs with minimal requirement for small molecule supplements and removed much of the variability in hESC/hiPSC culture that results from incomplete dissociation during mechanical or collagenase passaging.

“Through this work, we were able to generate substrates that replace or outperform mEF substrates, the gold standard in human pluripotent stem cell culture, in several key applications,” they conclude. “Because use of the engineered substrates did not require any special steps or adaptation from cultures using existing feeder substrates, the surface treatments described here would likely integrate well with many existing protocols of manipulating human pluripotent stem cells.

“Further, the treated surfaces represent an important advance over the gold standard feeder substrates because they are fully defined synthetic substrates that enhance propagation of undifferentiated cells and support the long-term cell culture, clonal outgrowth of hESCs/hiPSCs, and reprogramming of human somatic cells.”

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