Travelling to another dimension: going from 2D to 3D cell culture

Author: James Smith, 01/26/15

With contributions from David Brindley

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One of the DASGIP 200 mL stirred tank bioreactors used at CCRM.

Cell-based treatments are being developed and marketed for a variety of indications: from rare orphan diseases like graft versus host disease (GvHD), to blockbuster conditions like diabetes and cardiovascular disease that affect huge numbers of people. For these conditions, assuming that the treatment is effective, generating adequate quantities of cells to treat the patient cohort represents a critical challenge to commercialization. Even for orphan diseases – for example if required doses are high or treatment is repeated several times – producing high cell numbers, of consistent quality, can be problematic. So what’s the solution?

To answer this question, we need to understand a little about lab-scale cell culture. Mesenchymal stem cells (MSCs), the most popular therapeutic stem cell at the moment in terms of clinical trials, are adherent cells, meaning they can only grow when attached to a surface. Therefore, culturing usually occurs in planar (‘2D’) plastic flasks with a thin layer of media for nourishment.

Unfortunately, this method is not amenable to large-scale cell production. As the number of cells increases, the number of flasks required dramatically increases beyond a reasonable number for manual handling. Multi-layered cell factories, that pack more levels on which cells can grow into smaller spaces, can partially address this problem by increasing the surface area available for growth in a given volume. However, with high cell numbers the number of units required still becomes impractically high and labor costs concurrently sky-­rocket, as well as space requirements in a facility. Automating these processes is also expensive and does not improve the large facility footprint.

3D culture in bioreactors, on the other hand, can greatly reduce costs and increase efficiency. Their promise largely arises from the ability to greatly increase the surface area for growth in a given volume, by growing cells on small structures called microcarriers suspended in the media.

This message was echoed by several speakers at the Informa Life Sciences Cell Therapy Manufacturing conference in Brussels. John Harrington, from Athersys, spoke of their attempts and motivation to move Multistem® production from 2D to 3D; Thierry Bovy, from Pall Life Sciences, suggested that with some indications requiring a billion cells per dose, bioreactors become the only sensible solution; Ohad Karnieli, from Pluristem, similarly stressed that bioreactors might hold the answer to scale up – it might even be possible for costs to drop to as low as one dollar per million cells.

However, despite these repeated messages, an audience poll revealed that very little cell culture was currently 3D – and that some people were not even considering making the move. Why?

Probably, it is because moving from 2D culture to 3D culture is not as simple as it might sound. There is a common adage in the cell therapy industry that “the product is the process.” Therefore, in changing the process, you must show that the product has not changed significantly. It must be equivalent to previous product iterations.

Though challenging, this is not impossible. For example, if a Phase 2a trial is conducted with 2D cultured cells, the Phase 2b trial with scaled-up 3D culture might include a group of patients with the cells from the earlier process, thus allowing a direct comparison of efficacy and safety. In terms of biochemical comparisons, systems like the ambr15 microbioreactor system (Sartorius Stedim Biotech) can rapidly allow testing of parallel, scaled-down bioreactor conditions with much higher throughput than traditional, larger bioreactors. However, there is no guarantee that cells will indeed be equivalent – potentially forever restricting cell production to inefficient 2D technologies.

Ultimately, a paradigmatic shift in the way development of cell-based products is approached could eliminate the need to navigate this problematic transition. Rather than starting with 2D, why not start product development in smaller 3D flasks? Eytan Abraham, Lonza, suggested beginning in spinner flasks, from which the transition to larger bioreactors would probably be fairly simple.

Those currently culturing in 2D are not doomed. In fact, many commercial cell products have successfully used these technologies. But they may not be as efficient as they could be. The core message is that considering the full pipeline of product development, including the (perhaps neglected) manufacturing process, from an early stage, will maximize the impact both economically and medically of a product.

An ancient Chinese philosopher, Lao-Tzu, is believed to have said “the key to growth is the introduction of higher dimensions of consciousness into our awareness.” We can (very tenuously) apply this thinking to our core message, by considering the entire translational pipeline as a ‘dimension.’ However, with a few small tweaks, it can apply directly: “the key to [efficient cell] growth is the introduction of higher dimensions of [culture] into our [processes]. Wise words indeed!

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James Smith

James Smith

James Smith is a Research Associate of the CASMI Translational Stem Cell Consortium, where his current research focuses on extracellular vesicle biomanufacturing, iPSC translation and several systematic reviews including immunotherapy, fracture healing, and the use of placebos in surgery. He recently completed a SENS Research Foundation Scholarship at the Harvard Stem Cell Institute and Jeff Karp’s Lab at the Brigham and Women’s Hospital, where he developed a computational model of extracellular vesicle bioprocessing costs. Aside from translational research, James has an active interest in basic biology, achieving a First Class undergraduate degree in Biological Sciences from the University of Oxford. You can find James on LinkedIn.
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3 Responses

  1. Jon Ahlstrom says:

    Hi James, thank you for the post. Another concern with the 3d culture of MSCs (for example on microcarriers) are the regulatory issues. Has the FDA approved yet an MSC therapy that was cultured on microcarriers? There are safety issues, such as proving that the final cell product does not contain bits of microcarriers, or that this method is safe. Until the FDA approves the use of MSCs that are grown on microcarriers in a bioreactor, it is easier to use a planar plastic method (cell factory or hyperstack) that already is approved.

    • James Smith says:

      Hi Jon, many thanks for the comment.
      As far as I know there aren’t any MSC treatments that have received market approval (yet) using 3D manufacture. However, Pluristem are publicly using microcarriers in 3D culture to develop their ‘mesenchymal-like adherent stromal cells’ and Athersys are beginning to use 3D manufacture for their stem cell product, MultiStem. It is true, as you say, that it may be easier to use planar because the regulatory route has already been ‘explored.’ But the fact that many companies are trying to move to 3D indicates that there are significant cost savings to be had. Also, it is not really the technologies themselves that require regulatory approval, but the resulting product; even though there are approved products manufactured with 2D techs, new MSC products, or different applications will still require new approvals. I haven’t come across any concerns about the final product containing bits of microcarriers, but that’s an interesting point – is that an issue you have come across before?

  2. Jon Rowley says:

    James

    Great summary of the meeting – sorry to have missed it. The switch to 3D suspension will likely not be REQUIRED for successful commercialization of the first cell therapy products, but it will create more streamlined and low cost manufacturing for future products. It will be an important “next step” as the field moves into industrialization, and getting yields from the 10s of Billions into the 100s or 1000s of Billions… I talk some about that in a blog post here: http://www.roosterbio.blogspot.com/2014/09/how-quickly-are-cell-based-products.html

    Keep up the great work… Hope to see you again soon!

    Jon Rowley
    RoosterBio Inc

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