Culturing and Clonal Propagation of hPSCs on Laminin-521 With Nutristem^® hPSC Medium

A guest blog by Dr. Miranda Yang, Stem Cell Researcher

^{This article is posted on our Science Snippets Blog}

The use of purified laminin protein as a basement matrix for cell culture isn’t exactly new, but recent advances are generating quite a buzz in the stem cell culture field.

This summary is presented by Dr. Miranda Yang, reviewing two key publications by Sergey Rodin and colleagues at the Karolinska Institute in Stockholm, Sweden. These articles published in Nature highlight the long-term culture and clonal propagation of human pluripotent stem (hPS) cells in a completely defined system of NutriStem^® hPSC Medium and Laminin-521 matrix.

Summary

In order to enable future cell-based therapeutic applications for stem cells, there is a great need in the field for robust cell culture and differentiation protocols using completely xeno-free and chemically defined components, ideally produced under GMP-compliant quality standards. Still today, the majority of human pluripotent stem (hPS) cells are cultured under conditions that are neither xeno-free nor chemically defined, often using reagents that are undefined, such as co-culture with feeder cells or Matrigel matrix. Since poorly defined culture components inherently come with batch-to-batch variation, this often generates experimental results that are variable, biased, or not simply un-reproducible.

These two publications highlight the use of a cell-type specific laminin isoform, Laminin-521, as a cell culture substrate to support long-term culture of hPS cells. The combination of Laminin-521 as a basement matrix with NutriStem^® hPSC Medium was shown to outperform other cell culture media and matrix combinations for both clonal derivation and routine propagation of hPS cells in long-term culture.

A culture system that includes Laminin-521 and NutriStem^® hPSC Medium provided to be an efficient, chemically defined, and xeno-free system not only for the maintenance of feeder-free hPS cell cultures, but also for transferring hPS cells from feeder-based to feeder-free environment.

Additionally, unlike other xeno-free and chemically defined culture conditions, the combination of Laminin-521 and NutriStem^® hPSC Medium also supports single-cell clonal propagation of hPS cells. The paper states that Laminin-521 plays a key role facilitating the survival of dissociated hPS cells by inducing fast attachment and migration of the cells, which quickly aggregate to form small monolayer colonies that expand across the culture surface.

Passaging hPS cells as single cells is a much more streamlined process than passaging as cell aggregates of different sizes, as done in conventional culture protocols. Passaging as single cells ultimately leads to better reproducibility and consistency within the cultures. In addition, cells propagated in this way can easily be scaled up for high throughput automation.

Since many hES and hiPS cells in use today were derived on feeder-layers and/or cultured in undefined media or matrices, the authors provide a protocol for transferring these cells to a completely defined system of Laminin-521 and NutriStem^® hPSC Medium. Below are some helpful tips that can provide a smooth transition:

1. To transfer hPS cells from culture on a feeder layer to Laminin-521 matrix, it is usually sufficient to mechanically passage several small pieces of undifferentiated hPS cell colonies from the feeders and plate directly on Laminin-521 in NutriStem^® hPSC Medium. Cells can generally be expanded enzymatically in the new conditions from the first passage.
2. hPS cells being transferred directly from culture with a feeder layer may transition more easily if the number of cell aggregates used for the initial plating is enough to “condition” the NutriStem^® hPSC Medium, but not so many that the dish becomes overcrowded. As an example, if the transfer is made in 96-well plates, about 25 to 30 small cell aggregates (approximately 100 µm in diameter) in 120 to 150 µL of NutriStem^® hPSC Medium per well will give an optimal concentration of cells.
3. Once transferred to Laminin-521 and NutriStem^® hPSC Medium, cells can be passaged using an enzymatic technique (e.g. Recombinant Trypsin-EDTA Solution) for single-cell passaging. If cells have previously been passaged on feeders with non-enzymatic techniques, it can be useful to introduce the new feeder-free conditions and enzymatic passaging gradually in order to minimize stress on the cultures. Depending on the cell line, it may be best to continue mechanical passaging in the feeder-free environment before switching enzymatic passaging. Cells that have been previously passaged in feeder-free environments with enzymes do not seem to need such a gradual transition.
4. Since the single cells must aggregate on the laminin surface, the plating density, which defines the average distance a cell must travel before it meets another cell, during the initial enzymatic single-cell passage has a major effect on subsequent cell growth. Cells plated too sparsely may die before reaching another cell. The authors found that plating at densities of 10,000 to 15,000 cells per cm2 enabled successful passaging with 1:25 to 1:30 ratios, and plating at densities of 30,000 to 40,000 cells per cm2 was most effective for high cell proliferation.
5. After plating, cells aggregate into small monolayer groups and continue to grow as a monolayer, dividing and spreading until they cover the entire dish. Cells should be passaged prior to reaching 100% confluency to avoid differentiation caused by overcrowding.
   Note: NutriStem^® hESC Medium, as referenced in the Nature articles, has recently changed its name to NutriStem^® hPSC Medium. There has been no change to the formulation of this medium.

Sample Data

_{Figure 1: Immunostaining of HS181 hES cells for pluripotency markers Oct4 and Sox-2 at 8 hours, 24 hours, and 4 days after plating dissociated cells onto Laminin-521. Right panels show DAPI staining (Scale bars: 110 μm). Source: Rodin et al. Nature Comm. 2014.}

_{Figure 2: Transfer of hPS cells directly from a feeder layer to Laminin-521 matrix. (a) Cell aggregates obtained by mechanically harvesting hPS cell colonies grown on a feeder layer. Image taken just before plating these colony aggregates on Laminin-521. (b & c) Two representative colonies 24 hours after plating the aggregates on Laminin-521 (Scale bars: 200 µm.) Source: Rodin et al. Nature Comm. 2014.}

References

   ¹ Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions

   ² Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment