New protocol on the way to universal red blood cells from the lab

T.The COVID-19 pandemic has resulted in a sharp decline in blood donations, causing blood centers in the United States to sound the alarm of critical shortages and reveal the fragility of the current blood supply. For years, scientists have worked to develop ways to anticipate disruptions in the blood supply caused by problems such as emerging pathogens that can be transmitted through the blood or the rapidly aging population that will dramatically reduce the number of potential donors in the future could reduce blood produced in factories to provide an alternative source for the vital substance.

In a study published December 10 in Stem cell reportsSteve Oh, director of the Stem Cell Bioprocessing Group at the Singapore Agency for Science, Technology and Research (A * STAR) and his colleagues report on a new method for generating large quantities of red blood cells (RBCs) from induced pluripotent stem cells (iPSCs), Cells that have been reprogrammed from a differentiated state back to an embryonic state.

Oh and his colleagues set out to find ways to make red blood cells – enough for blood transfusions alone – from 2015-scale iPSCs. At that point, Oh, according to Oh, the approaches available weren’t able to develop enough high quality immature products – blood cells, called erythroblasts, expand and mature in large bioreactors – one of the reasons for this is that scientists were mainly using monolayer cultures, which restricted the growth of cells . “We wanted to push bioprocessing technology as much as possible so that one day we could produce universal off-the-shelf blood,” says Oh.

The Oh team set out to develop a scalable method for generating type O negative or universal donor blood cells. To do this, they tied the iPSCs from different tissues to supporting structures, so-called microcarriers, that enable their growth, and then placed them in cell cultures that were constantly stirring (for example, by shaking or stirring). Growing dense cultures of cells requires high levels of nutrients and oxygen, and a continuously moving environment provides more of these critical components to reaching the growing cells than does a static environment, Oh explains. (Oh has patents for the techniques used in this study and is the founder of Zenzic Labs and SingCell, two Singapore-based biotechnology companies that work with stem cells.) During this process, various cocktails of cytokines at various stages were added to help differentiate induce in erythroblasts.

The team repeated this protocol in culture platforms of various sizes – starting in plates with 5-ml individual wells and ending with 500-ml flasks, which Oh says are replacements for small-scale controlled bioreactors.

With this technology, Oh and his colleagues generated erythroblast cultures with a cell density of 1.7 x 107th Cells per ml. In addition, they showed that it was possible to perform this process using iPSCs from eight different cell lines, including those derived from umbilical cord blood, bone marrow, and skin.

To match the amounts of red blood cells found in transfusions, scientists need to achieve densities of at least 1 x 108th Cells per ml – that’s still an order of magnitude larger than what Oh’s team was currently able to achieve.

In order for erythroblasts to become erythrocytes, they must go through enucleation – the process by which the cell loses its nucleus. Oh’s group achieved up to 60 percent enucleation in their cell cultures using one of today’s leading enucleation techniques, which involves adding OP9 cells, mouse bone marrow stem cells. The entire process from the iPSC starting material to the maturation of the erythrocytes took about 35 days.

When comparing the erythrocytes generated from iPSCs with those from adults, they reported minimal differences in key functions such as oxygen transport capacity and in their transcription profiles.

“This shows for the first time that you can produce red blood cells from iPSCs and with a range of donors to the end product in a controlled suspension environment,” says Oh.

According to Emile van den Akker, a scientist studying erythropoiesis at Sanquin, a nonprofit that supplies blood in the Netherlands, showing that this process of transition from iPSC to erythroid cells can be done in sling flasks is a big step . who was not involved in this work. Transferring a static culture to something that can be used in a scalable bioreactor is “the real strength of this paper”.

A range of laboratory-made mature red blood cells

Benoit Malleret, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore

Many teams have been looking for ways to create red blood cells from stem cells for transfusion purposes, and the biggest challenge remains scaling up to mass production, says Luc Douay, professor at Sorbonne University in France and president of biotech startup Erypharm. The company is working on ways to produce red blood cells for transfusion and was not involved in this study. “It’s an interesting newspaper,” says Douay. However, the authors do not report large scale production or “consider how this process can be scaled”.

To match the amounts of red blood cells found in transfusions, scientists need to achieve densities of at least 1 x 108th Cells per ml – that’s still an order of magnitude larger than what Oh’s team was currently able to achieve. Right now, according to Oh, the technique’s maximum cell density appears to be what the team achieved in the study. However, Oh says he hopes his team can achieve the levels required for the transfusion. One approach they are taking is ways to perpetuate their erythroblast cell lines so that they continuously reproduce, creating more cells – and saving the time and money it takes to always start with iPSCs.

Red cells generated in the laboratory could be useful in other ways. Scientists are also looking for ways to load them with specific therapeutics that could be used in chemotherapy, for example, or to remove toxins from plasma. Since these applications may not require the amount of blood required for transfusions, “I see this as the quickest translation into a product,” says van den Akker. Still, “the ultimate destination, of course, is transfusion blood.”

J. Sivalingam et al., “A Scalable Suspension Platform for Generating High Density Cultures of Universal Red Blood Cells from Human-Induced Pluripotent Stem Cells”. Stem cell reports, doi:10.1016 / j.stemcr.2020.11.008, 2020.

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