Stem cells become sluggish in their rate of division when removed from the body and grown in labs, thereby limiting their use in medical science. A scientist team from IIT Mumbai has found a solution for that.

Stem cell whose medical significance lies in being able to differentiate into other types of cells are yet to become the panacea for clinical applications that was expected of them. One fundamental bottleneck for this is the inability to produce enough number of cells needed to treat a patient.

The reason behind this is limited availability of stem cells in each tissue, and once removed from the body, their capacity to divide is limited, making generation of large quantities of stemcells difficult.

Scientists in many laboratories are trying to find better ways to grow large quantities of adult stem cells in cell culture. Our team from the Department of Chemical Engineering, IIT Mumbai has developed a method to address this problem. We have identified polyacrylamide (PAA) hydrogel, a material whose stiffness can be varied by altering its constinuents for creating a soft bed which can maintain stem cell division. Using our proposed system, it is possible to generate 500 times more cells than what we are growing today.

To tell you the story of how we did it, we need to explain what these stem cells are. While we can have embryonic stem cells from developing embryos, our adult body also has a pool of reserved stem cells. These cells are known as ‘adult stem cells’. These cells maintain the cell numbers in each tissue and organ by replenishing the regular loss of cells in our body either due to natural cell death or due to a disease or injury.

Mesenchymal stem cells (MSCs) are one of such adult stem cells that are present in many tissues including bone marrow, fat, dental pulp, and umbilical cord. These cells, given proper environment, can change into other cell types such as bone, fat, cartilage, neurons, and muscle cells by a process called differentiation. Also, they may divide to give rise to multiple copies of themselves. This later process is called self-renewal.

Although the stem cells in our body are few in number, when needed they can divide to form multiple copies of themselves and differentiate to the desired cell type. They can also migrate to the site of injury and reside there to facilitate wound healing. MSCs can modulate our immune system using the molecules secreted by them. They can reduce the chances of inflammation and immune rejection substantially during organ transplant by suppressing the immune response of the body.

Thanks to all these interesting capabilities, MSCs offer huge promises in regenerative medicine and immune related diseases. They have been tested in pathological conditions associated with organ transplant, cardiovascular diseases, pulmonary or lung diseases, and in regeneration of damaged tissues.

However, to appreciate their full potential and to use for clinical practises, there are many research challenges. One of the major bottlenecks is to get sufficient number of MSCs required for clinical trials. Percentage population of MSCs available in adult tissue, say bone marrow, is very limited (one MSCs in 10,000 total marrow cells) while requisite number of cells for treatment goes into tens of millions.

This problem becomes even more acute for elderly patients who have fewer number of reserve stem cells in their tissues. To overcome this challenge, hMSCs are cultured and expanded in the laboratories. However, after certain number of cell divisions, these cells stop proliferating further. They become aged and lose their characteristic spindle shape and become large and flat. They also lose their differentiation potential and other therapeutic functions entering a state called senescence.

Onset of senescence creates a huge challenge in the clinical use of these stem cells by limiting the number required and compromising their quality. Thus, for clinical application and research using MSCs, there is an urgent need to develop a culture method which can produce large number of MSCs without compromising their beneficial properties (stemness).

In this research, we first tried to understand the factors that influence the onset of senescence in stem cells during laboratory culture. It is already known from published research that stiffness of the material on which cells are grown influences different cellular properties. We noticed that plastic, on which cells are generally grown in laboratories, is much rigid compared to the most of the tissues in our body. Assuming that non-physiological rigidity of the plastic might be adversely affecting the stem cell growth and health, we replaced the hard stiff plastic bed generally provided for growing cell in the lab with soft bed hoping to keep them happily growing ever after.

Next step was to find a suitable material which is not harmful or toxic towards cells. Our search was for something that would be stable, transparent for ease of doing microscopy, and easy to manipulate for stiffness. We found that polyacrylamide (PAA) hydrogel has been extensively used to study the effect of substrate stiffness on cell function. Chemistry of the material being well known it would be easy to vary its stiffness by changing the mixing ratio of its constituents. We picked PAA gel to check the effect of substrate rigidity on cell health during long-term cell culture.

After deciding the material, the next question was, how soft our PAA gel should be to be used for stem cell culture. With some intelligent guess and some trial and error, we finally zeroed down to an intermediate stiffness. It was found that while a rigid material pushes the cells to senescence, a very soft material does not allow them to divide and grow.

We cultured the MSCs on the PAA gel of our choice for 50 days and viola not only did the cells grown on the gel maintained their size and shape, even after 50 days of cell culture and multiple cell divisions, their ability to reproduce daughter cells was uncompromised.

As a result, we got 500 times more cells from gel than what was obtained from plastic plates.To put some numbers, if one cell multiplies to become 4 million cells after 50 days when cultured on plastic plates, the cell cultured on gel gives 2 billion cells in the same time. We found very less sign of senescence in these cells. Further, these cells maintained their ability to become cells of other type (differentiation ability) which cells grown on plastic lost. In addition, we found that the cells grown on gel have higher migratory ability meaning better wound healing ability. In short, the cells grown on gel, retained their ‘youth’ while cells grown on plastic got ‘aged’.

To conclude, our research shows that the soft hydrogel plate made of polyacrylamide can be a better replacement for conventionally used plastic culture plates for having a greater number of stem cells in lesser time. This aspect might be critical for patients who need urgent cell therapy. Fundamentally this work throws light on the importance of physical factors such as rigidity in the context of cell biology and understanding senescence.

Sanjay Kureel and Abhijit Majumder
Department of Chemical Engineering,
IIT Bombay, Mumbai
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