Stem Cell Considerations for Bioprinting or Tissue Engineering

Stem cells no doubt play an important role in many tissue engineering and bioprinting applications. While the choice of stem cells is very much application dependent, and often requiring more than one cell type, mesenchymal stem/stromal cells (MSCs) has served as the workhorse for numerous tissue engineering and bioprinting applications. Most commonly, MSCs are used in bioprinting of bone [1-3] and cartilage tissue [4, 5], but they are also used in the skin [6], cardiac, and wound healing [7] applications often in combination with other cell types that may leverage the regenerative properties of MSCs. Moreover, MSCs already have a proven safety profile, there are over 1000 MSC clinical trials worldwide to date. This makes MSCs an attractive cellular starting material for creating more sophisticated tissue-engineered products and future organs supported by fabrication technologies such as bioprinting.

The first important consideration when designing or developing a tissue-engineered product is the selection and characterization of cellular starting material. The development of a patient-specific product, as opposed to an off-the-shelf product, will require different CMC (Chemistry, Manufacturing, and Controls) strategies. Donor criteria and eligibility will differ slightly depending on whether it is for autologous or allogeneic use. The criteria for autologous use are generally less stringent than those for allogeneic use, cells from allogeneic sources must pass all infectious disease testing and be able to withstand longer storage conditions. Donor eligibility guidelines from the FDA are provided under regulations at 21 CFR Part 1271. Pertinent to MSCs, the choice of tissue source, whether it is from the bone marrow, adipose tissue or umbilical cord will depend on the target use. Characteristics of MSCs are notably different from different sources [8], their differentiation potential and thus potential clinical applications can also vary [9]. For allogeneic cells, donor variability is inherent, so it is critical to determine suitable methods and assays to characterize the cells in order to ensure consistency of performance. The most basic characteristics would include cell enumeration, viability, and phenotypic assays, but other methods such as biological activity and functional assays should also be included.

In addition to the need for high quality and consistent cell source, tissue-engineered products often require high quantity or volumes of cells. As an example, in the bioprinting of tissue constructs such as bone, the challenge here is no longer the choice of biomaterial or 3D design and technology necessary to create the desired scaffold but it is in generating enough starting cellular material for printing a biologically relevant tissue for transplant. The first group that attempted to do this and 3D bioprinted the superior half of an adult human femur was John Fischer’s lab [2], this print consumed 720M human MSCs (hMSCs) to seed this bone scaffold. The number of cells used in this single bioprint is at least 10-fold more than a typical tissue engineering experiment performed in an academic lab. At the time, this construct was the first successful demonstration of a large bioprinted bone tissue construct (20x larger than average) that achieved high viability throughout the graft and increased expression of osteogenic markers. An impressive accomplishment indeed!

In order to accelerate this into the clinic, a full femur replacement would require 1.5B hMSCs as a starting cellular material. These cells will not only need to be manufactured under current Good Manufacturing Practice (cGMP) to meet stringent regulatory requirements but also be well-characterized, tracked, and used at the appropriate cellular age by assessing their population doubling levels. The next step is to determine appropriate cell lot sizes required at each stage of the development process from product development to clinical manufacturing all the way through commercial manufacturing. These are important factors to consider as early as possible in the product and process development stage as this is commonly neglected – the process and its scalability. Many people make the mistake of going into clinical trials with sub-optimal processes and without a full understanding of their process, resulting in more painful consequences that may or may not be recoverable. Early and thoughtful planning is always the first step in making sure that you are setting yourself up for success. As Stephen Covey states as his second habit in The 7 Habits of Highly Effective People, “Begin with the End in Mind”.

References:

[1] Kolesky DB, Homan KA, Skylar-Scott MA, Lewis JA. Bioprinting thick vascularized tissues. Proceedings of the National Academy of Sciences Mar 2016, 113 (12) 3179-3184; DOI:10.1073/pnas.1521342113

[2] Nguyen BNB, Ko H, Moriarty RA, Etheridge JM, Fisher JP. Dynamic bioreactor culture of high volume engineered bone tissue. Tissue Eng Part A (2016) 22:263–71. doi: 10.1089/ten.tea.2015.0395

[3] Du M, Chen B, Meng Q, Liu S, Zheng X, Zhang C, Wang H, Li H, Wang N, Dai J. 3D bioprinting of BMSC-laden methacyrlamide gelatin scaffolds with CBD-CMP2-collagen microfibers. Biofabrication (2015) 7: 04410

[4] Möller T, Amoroso M, Hägg D, Brantsing C, Rotter N, Apelgren P, Lindahl A, Kölby L, Gatenholm P. In Vivo Chondrogenesis in 3D Bioprinted Human Cell-laden Hydrogel Constructs. Plast Reconstr Surg Glob Open. 2017 Feb 15;5(2):e1227. doi: 10.1097/GOX.0000000000001227. PubMed PMID: 28280669; PubMed Central PMCID: PMC5340484.

[5] Apelgran P, Amoroso M, Lindahl A, Brantsing C, Rotter N, Gatenholm P, Kolby L. Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo. PLoS One. 2017 Dec 13;12(12):e0189428. doi: 10.1371/journal.pone.0189428

[6] Wang X, Li C, Zheng Y, Xia W, Yu Y, Ma X. Bone marrow mesenchymal stem cells increase skin regeneration efficiency in skin and soft tissue expansion, Expert Opinion on Biological Therapy (2012),12:9, 1129-1139, DOI: 10.1517/14712598.2012.704016

[7] Skardal, A. , Mack, D. , Kapetanovic, E. , Atala, A. , Jackson, J. D., Yoo, J. and Soker, S. (2012), Bioprinted Amniotic Fluid‐Derived Stem Cells Accelerate Healing of Large Skin Wounds. STEM CELLS Translational Medicine, 1: 792-802. doi:10.5966/sctm.2012-0088

[8] Hass R, Kasper C, Böhm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Commun Signal. 2011;9:12. Published 2011 May 14. doi:10.1186/1478-811X-9-12

[9] Berebichez-Fridman R, Montero-Olvera PR. Sources and Clinical Applications of Mesenchymal Stem Cells: State-of-the-art review. Sultan Qaboos Univ Med J. 2018;18(3):e264–e277. doi:10.18295/squmj.2018.18.03.002

About the Author:

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Mayasari Lim

Dr. Mayasari Lim is the West Coast Regional Account Manager for RoosterBio and an active contributor to the bioprinting community. She was the founder and CEO of SE3D, a startup focused on bringing bioprinting into the classroom to support future workforce development. Previously, she was an assistant professor in Bioengineering at Nanyang Technological University in Singapore. Her research expertise included stem cell bioprocess engineering, bioprinting, and regenerative medicine. She also mentors and teaches leadership and management courses at the Fung Institute for Engineering Leadership at UC Berkeley. Dr. Lim obtained her Ph.D. degree in Chemical Engineering at Imperial College London and her B.Sc. in Chemical Engineering at UC Berkeley.

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