The microfluidic device, with cell-seeding channels filled with red food dye; the heart ventricular contraction control channels and circulation valve control channels are filled with blue and green food dye respectively. © UNSW/ Jingjing Li, Author provided.
Microfluidics devices might not be a universally understood area of research, but at the Graduate School of Biomedical Engineering at UNSW Sydney, researchers are using them to produce blood stem cell precursors in a way that suggests a seamless translation from lab to clinic is possible.
PROFILE: Dr Jingjing Li
Jingjing Li is a postdoctoral fellow at the Graduate School of Biomedical Engineering in UNSW Sydney. She is working on a CRC-P project developing an automated microscale bioreactor for cell and gene therapy.
To many who are unfamiliar with the field of biomedical engineering, the word ‘microfluidics’ may not hold much meaning or weight. But these intricate, small, typically size of credit card devices hold immense potential and promise to change and improve the medical field.
Microfluidics refers to the science of understanding fluid behaviour and fluid-structure interactions in micro-channels. It is also a technology that allows miniature fabrication of microchambers for tissue engineering at the cellular level.
In the biotechnology industry, where scale-out and scale-up of applications are constantly a barrier to the translation to real-life applications, microfluidic devices offer benefits which could potentially offer a seamless translation from lab to clinics.
This is exactly the type of work currently being undertaken at Associate Professor Robert Nordon’s lab. A team of engineers and biologists at the Graduate School of Biomedical Engineering at UNSW are focusing on exploiting the benefits of microfluidic devices for stem cell engineering and biomedical applications.
The multidisciplinary team is exploring ways to maximise the utilisation capacity of microfluidic devices, specifically for applications in stem cell research and cell and gene therapy (C>) applications.
blood stem cell precursors
Stem cells are unspecialised cells at the top of the cellular developmental hierarchy, which can give rise to almost all other cells in our bodies. Blood stem cells are the ancestor cells that can develop into many distinct types of blood cells.
However, there are still many unknowns around how blood stem cells originate in our body in the first place. In a recent publication in Cell reports, the Nordon Lab has shown how artificial lab stimulation of the embryo’s beating heart and blood circulation successfully promoted the production of blood stem cell precursors.
The 3cm x 3cm microfluidic device consists of multiple layers of transparent materials and mimics the embryonic blood flow and circulation. This model allows direct observation of the human blood cell development under microscope, helping us understand how the microenvironment in the embryo assists human blood cell development, and how we can produce life-saving blood stem cells for clinical therapies.
Blood stem cell transplantation restores blood production after high doses of chemotherapy for treating blood cancers, such as leukaemia. However, this treatment is limited to patients who can find a tissue-type matched blood stem cell donor.
This has led to the search of new renewable blood stem cell sources. The key to work around this problem would be growing large amounts of blood stem cells in a laboratory. However, until now, most adult blood stem cells expanded in the lab have shown to gradually lose their transplantation potential.
The isolation of stable human embryonic stem cell lines (hESCs) from human blastocysts in 1998 and the discovery of induced pluripotent stem cells – stem cells made from a patient’s own cells, provided a renewable source of pluripotent stem cells (PSC).
The potential therapeutic options for tissue regeneration, genetic disease or cancer treatments are widened vastly. Because these pluripotent stem cells can grow immortally in laboratory, they are easier to genetically manipulate and could in principle be differentiated into any cell types including blood stem cells.
The development of processes to grow human tissues, organs or specific cells, including blood stem cells from pluripotent stem cells, have been progressing quickly in recent decades. Chemically defined step-wise protocols to regulate human pluripotent stem cells growing in a laboratory dish has been developed by many groups.
Though laboratory-grown blood stem cells shared ‘phenotypic’ similarity with their counterparts in the embryo, their transplant potential is still far lower than expected. Studies on animal models demonstrated the importance of both ‘intrinsic’ (the anatomical origin and genetic programme which has been mimicked) and ‘extrinsic’ factors (the chemical/ mechanical stimulations, e.g., onsite of embryonic heartbeat and blood circulation) for this process.
It is likely that making human blood stem cells requires recapitulating the complex process of embryogenesis in laboratory dish.
PROFILE: Dr Robert Nordon
Robert Nordon is an associate professor in the Graduate School of Biomedical Engineering. He works closely with industry in the fields of advanced manufacturing and medical technologies and, since 2016, has been awarded over $5m (~£4.5m) in research grant funding in the fields of point-of-care diagnostics, cell and gene therapy manufacture and stem cell science.
This is the motivation to engineer human embryonic heart and blood circulation models in our laboratory. The precursor of human blood stem cell emergence from the wall of the main blood vessel at around day 32 to 40 of embryo development, shortly after the initiation of the first embryonic heartbeat and blood circulation. At this stage, the embryo is still of around 3cm in length. Microfluidics which are capable of manipulating tiny amounts of fluid, are a perfect fit for this application.
Pluripotent stem cells were first grown using chemically defined protocols into a multi-layer cellular niche that represents the anatomical site in embryo called aorta-gonad-mesonephros (AGM). Our miniature heart pump simulates the mechanical environment required for the birth of blood stem cells in the embryo.
The recirculation of cell culture media in microchannels simulates the blood circulation where the blood stem cells are released into in the embryo after birth. We applied pulsatile circulatory flow from day 10 of culture. We observed the precursor cells released from blood vessels lining the microfluidic channels into the artificial circulation.
The circulating cells harvested from the device were further grown in specific media and were able to generate the blood components, including red blood cells, white blood cells, platelets, etc. In depth analysis of the gene expression pattern at the single cell level showed a close resemblance to the AGM in the human embryo.
We have shown the artificial simulation of the pulsatile circulatory blood flow can promote the generation of aortic cells and precursors of blood stem cells. This knowledge can be used for future innovations in large-scale manufacturing of human blood stem cells for clinical transplantation.
The next step of our research is to scale-up the production is to grow many human pluripotent stem cells and mechanically stimulating them to develop into blood stem cell precursors. We can then test their transplantation potential and understand how precursor cells mature in mice that accept human blood stem cell transplants.
We are also working on refining the mechanical design to understand and optimise the biological mechanisms that drive blood stem cell productivity.
addressing the cost
Cell and gene therapy (C>) is a new therapeutic modality that aims to treat, prevent, or cure disease by modifying or introducing new genes into cells. The key area where stem cells and microfluidics together have the potential to impact C> is to develop more efficient processes to manufacture these new therapeutics at a price that is affordable.
Gene therapy vectors are used to deliver genes into cells. They can be directly injected into the blood stream or diseased tissues but may end up in the wrong tissue causing severe side effects. A safer approach is to introduce the vector into the patient’s cells outside of the body (ex vivo) and to test their safety before they are returned to the patient. Ex vivo cell purification steps also ensure that off-target toxicities are minimised.
For example, chimeric antigen receptor (CAR) T cell therapies, have resulted in high levels of complete remission for B-cell leukemia in patents who have failed standard high dose chemotherapy and blood stem cell transplant.
The patient’s immune cells are genetically modified with a gene that encodes a CAR that targets tumor markers (CD19) on cancer cells. Inherited blood disease such as sickle cell disease or thalassemia may also be cured by introducing a normal gene into their blood stem cells.
However, the main challenge that comes with these therapies is not the efficacy of the treatment, but the cost. At this stage C> are frequently referred to as ‘boutique therapy’ because only those who can afford to pay can easily access it. In addition to drug companies recouping R&D costs, these new therapeutics have expensive production costs.
Prices of these treatments typically exceed half a million dollars which may be justified economically if one saves a life. But in the longer term, a technological solution to bring down manufacturing costs is required to make C> accessible to all patients.
A snapshot from the time-lapse microscopic video of vascular cells (Red) and blood precursor cells (Green) developed in the microfluidic channels at day 13 of culture. Phase contrast microscopic image(left) and Overlayed fluorescent images (right). © UNSW/Jingjing Li, Author provided.
Economy of scale has reduced the cost of pharmaceutical agents by manufacturing vast quantities of drugs using large volume reactors, so a single manufacturing run can treat thousands of patients. However, the economy of scale cannot be applied to C>.
Our immune system will reject any cell or tissue that is judged to be different from self. The genetically modified cells must have the same tissue type as the patient. So a treatment is customised for the patient, where starting cells are sourced from the patient (autologous) or a tissue matched donor (allogeneic). A manufacturing run will only treat one patient.
Another factor that adds to the cost of manufacture is complexity. Manufacturing C> products includes three key steps: cell separation – purification of stem or immune cells that are harvested from the blood of patients or donors; viral transduction – introduction of therapeutic DNA or RNA into target cells using viral or non-viral delivery systems; and cell expansion – proliferation of transduced cells by tissue culture for several days to facilitate viral entry and stable genomic integration.
These are labour-intensive laboratory procedures that require high capital costs and a highly skilled labour force.
So, the manufacturing challenge is not one of scale-up but of scale-out. How does one design a production facility that can manufacture thousands of patient-specific doses?
One way would be to have a production line like that used to manufacture cars; each cell product passes sequentially through a station that performs a single unit operation (e.g., cell separation, gene transfer or cell culture).
But this approach has been difficult to implement in practise because of the high risk of contaminating the product with bacteria, and the possibility of mixing patient samples. The other is to integrate cell separation, gene transfer and cell expansion into a single disposable device, one for each patient.
PROFILE: Ada Lee
Ada Lee is a research scientist in cell and gene therapy at the Graduate School of Biomedical Engineering.
Reducing the complexity
At the Nordon lab, investigation and research is well underway to condense the process to a microscopic scale using microfluidics. A team of engineers and students are working on reducing the size and complexity of C> manufacture into a microfluidic device capable of automating and integrating all the expensive, laborious, and highly specialised and skilled steps needed to produce these promising treatments.
Employing microfluidics reduces the cost of goods by reducing the footprint of bioreactors, the quantity of reagents, and the need for human intervention.
It is estimated that the market for C> in the US and EU alone will grow beyond 200,000 patient-specific treatments per year. This means an increased demand for the treatments from patients who desperately want access to them.
The Nordon lab is hoping to see a future where C> manufacture is simplified and cost-effective so that C> is at a price point which is realistic and accessible to the patients who really need them.
Jingjing Li, Ada Lee, Robert Nordon
The Robert Nordon Laboratory
The Graduate School of Biomedical Engineering
The research was funded by the Australian Research Council’s special research initiative into stem cell science; Stem Cells Australia. The research upon which this article is based has been published in ScienceDirect and Cell Reports.