Bioprocessing Technology Institute, Agency for Science Technology and Research (A*STAR)
T he idea behind stem cell therapy is the application of cells to restore damaged tissues in the body and thereby restoring the health of the patient who has lost significant use of their bodily functions. Some of the types of diseases being addressed include restoring the eye sight, repairing bone and cartilage damage, re-establishing heart function after a heart attack, reinstating lost insulin production and reconnecting neural functions in various debilitating diseases such as spinal cord injury, Parkinsonís and stroke. Scientific progress is telling us that stem cells may provide some of these special features if they can be guided to the specialised cell types, but to cross over from the lab dish to the patient treatment, these specialised cells will have to be produced on a massive scale. Another application is to use stem cells to screen for molecules that may facilitate differentiation to the tissue of interest or to screen out drugs that may have harmful side effects.
To achieve this, one of the enabling technologies is bioprocessing which is the development of the knowhow and capabilities to grow billions to trillions of cells consistently for animal studies, clinical trials and eventually therapy in patients.
Microcarrier cultures of stem cells
The Bioprocessing Technology Institute has developed simple, yet rigorous and reproducible methods for growing vast populations of stem cells such as human embryonic stem cells (hESC) obtained from an embryo, human induced pluripotent stem cells (hiPSC) obtained by genetic modification of mature differentiated cells, and multipotent stem cells (MSC) usually obtained from bone marrow. Furthermore, these cells have been directed to become functional cell types of the body such as beating clusters of heart cells, nerve cells and bone cells in scaffolds. BTI approached the problem by growing the cells on the surface of tiny structures called microcarriers (ranging from 10 to 200 micron in diameter or cylinders of about 100 micron in length), and are about 10 times larger than the size of the stem cells to which they can attach to. These microcarriers are positively charged and coated with extracellular matrix proteins (like laminin and vitronectin) such that the cells can rapidly anchor to the surface, spread and grow (Fig.1). The microcarrier platform which operates in stirred reactors allows increase of cell amounts simply by increase of volume, as compared to the conventional method which scales up linearly by increasing the number of plastic vessels. In these new culture conditions, it is important to demonstrate that the cells still retain the stem cell characteristics such as the expression of protein markers by the cells (mAb 84, Tra-1-60, and Oct4 for hESC and hiPSC and Stro-1, CD 73, CD 90 and CD 105 for MSC) similar to that obtained in classical laboratory conditions.
Furthermore, we can increase the cell density from a typical yield of 1 million cells/ml on tissue culture flasks, to 6 million cells/ml in microcarrier cultures. In a 1 litre bioreactor culture, it is possible to generate 6 billion hESC or hiPSC in serum free media, which is equivalent to manually handling 80 tissue culture flasks of 75mls. Thus so far, we see no upper limit to the highest density that can be achieved, but we are only limited by the culture media supply to the cells. For MSC, we have achieved 0.8 million cells/ml in microcarrier cultures compared to 0.2 million cells/ml in traditional tissue culture flasks, using serum supplemented media.
Stem cell differentiation in bioreactors
The next stage after cell expansion is to differentiate them towards the specific cell type of interest. In three different examples, we have demonstrated that microcarriers can enable an increased efficiency of differentiation. After screening for a variety of microcarriers, we determined that 10 micron microcarriers were the best in generating cardiomyocytes yielding 0.6 cardiomyocytes / hESC. Subsequently, we developed a serum free media with nutritional supplements which further increased the yield and was generally applicable for differentiating both hESC and hiPSC cell lines to cardiomyocytes. These cardiomyocytes express cardiac muscle, show normal electrophysiology and are now being implanted into animal models of cardiac infarct to see if they can restore heart function. The cells can also be applied for drug screening of potentially cardiotoxic compounds.
Secondly, we have taken the expanded MSC and placed them in scaffolds as well as fibrin glue mixed with hydroxyapatite and implanted them into mouse models. Surprisingly, it was shown that MSC produced from microcarrier cultures were able to produce much more calcium per cell and generate 60% higher bone volume than MSC produced from plastic tissue culture flasks. Thus, we have developed a simplified method of producing large numbers of MSC in a bioreactor that have a better characteristic of bone formation for potential therapeutic application.
Thirdly, we developed a process in spinner flask cultures (Fig. 2) for serial expansion and differentiation of both hESC and hiPSC to neuroprogenitors, which are the precursors of nerve cells. During the neural differentiation phase, we achieved a massive 6 to 11 fold increase in the yield of neuroprogenitors compared to the tissue culture method. Final cell densities exceeded 1 billion cells/ml compared to 1 to 2 million cells/ml in tissue culture and 80% of them expressed the neuroprogenitor surface marker PSA-NCAM. The neuroprogenitors were further differentiated to functional neurons and can potentially be used for studying the restoration of lost functions in Parkinsonís disease, as well as for screening for compounds that differentiate these cells to neurons, or astro-glial lineages.
neurons, or astro-glial lineages.Table 1 summarises the yield of microcarrier processes compared to the 2D culture equivalent methods for producing anchorage pluripotent or multipotent stem cells as well as their differentiated progenitors. Factors of improvement range from 2 to 11 fold depending on the cell type expanded and tissue formed.
Thus, we believe that microcarrier cultures are very versatile for stem cell expansion and differentiation; with a potential for consistently generating huge quantities of anchorage dependent stem cells and their progenies for applications in drug screening and future cell therapies in a cost effective manner.
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