At the Cutting-Edge of Regenerative Medicine: Bioengineering Human-Sized Bone

Fisher et al published the first attempt at engineering

a full-scale adult human femur head from hMSCs.  This

is the largest reported tissue to have been engineered

and took over 700 million hMSCs to fabricate.

John Fisher’s laboratory at the University of Maryland, College Park recently published what can be considered a significant advance for Tissue Engineering and Regenerative Medicine. Graduate student Bao Nguyen and her colleagues have engineered a bone construct that is 20 times larger than any reported previously, and the size of an adult human femur.

Why is this important? Critical size bone defects are a significant health problem (resulting in over $1 billion in annual healthcare costs incurred in the U.S.) and are currently treated with grafts, decellularized bone, or synthetic bone grafts, with sometimes unsucessful results. As such, modern medicine has been looking to tissue engineered bone grafts as future treatments for such defects. Human bone marrow-derived Mesenchymal Stem Cells (hBM-MSCs) are a promising cell source for such applications because they efficiently differentiate down the osteogenic path and also secrete paracrine factors that may aid survival and vascularization of engineered bone. Prior to this publication, engineered constructs have been relatively small due to cell and culture limits.  One major challenge has been growing hBM-MSCs, while maintaining their function, to sufficient numbers needed for an adult human-sized construct; a challenge adressed by RoosterBio.  In addition, nutrient and O₂ transfer are often insufficient to maintain cell viability and function throughout larger constructs, especially those of adult human dimension.

To address this cell culture limitation, the Fisher laboratory developed a Tubular Perfusion System (TPS) bioreactor where cells and scaffolds are cultured in a cylindrical chamber and subject to circular media flow. This system has high nutrient and O₂ transfer and efficient waste removal and has been previously used to produce smaller engineered bone and cartilage constructs (See here and here).

In the study detailed here, the authors had access to and combined, for the first time, advanced technologies required for biofabrication: 3D printing, the TPS bioreactor, and scalable production of hBM-MSCs. The goal of the study was to scale-up bone constructs to adult human size. A full size mold of the superior portion of a human femur (the largest bone in the human body) was 3D printed using information from an opensource database. The mold measured 23 cm long and 10 cm at its widest point with a volume of  200 cm³. The mold was filled with hBM-MSCs in alginate beads (3 mm beads, 100,000 cells per bead). The entire construct utlillized 7200 aliginate beads containing a total of 7.2 x 10⁸ cells (yes, that is 720 million cells!). The high volume hBM-MSC cell and media systems used were from RoosterBio, and technical support for the efficient production of large volumes of hBM-MSCs was provided by our company.

After 8 days of culture in the TPS, the construct was examined for cell viability and bone differentiation. High cell viability was seen in all parts of the construct, both on the outside and the inside (interior). In addition, hBM-MSCs committed to the osteogenic lineage throughout the construct, demonstrating efficiency of the TPS culture system. Both early (Alkaline Phosphotase, ALP) and late (Bone Morphogenic Protein-2, BMP-2) markers of osteogenesis were upregulated relative to day 0. Interestingly, ALP and BMP-2 expression was 25- to 30-fold higher in the construct shaft relative to other portions of the construct. The authors speculate that this is due to shear stress exerted on the parts of the construct closest to the inlet, which activates hBM-MSC signaling pathways, causing release of paracrine factors that stimulate osteogenesis of the “downstream” shaft portion. Taken together, these results demonstrate that the confluence of cutting-edge technologies such as 3D printing, TPS bioreactors, and best-in-class hBM-MSC manufacturing processes enable the engineering of adult human-sized tissue constructs.

While “…this first foray into full-scale bone engineering provides the foundation for future clinical applications of bioengineered bone grafts…” the authors point out some limitations to this study. The 8 day culture period was relatively short, given the weeks usually needed for high efficiency bone differentiation. Thus, extended time points and the fabrication of additional large constructs are needed to fully explore the capabilities of the TPS system. Further, alginate is a soft material, and its mechanical properties do not render it the best suited for bone differentiation.  In addition, hBM-MSCs within aliginate beads lack cell:cell contact, which may also limit their osteogenic differentiation.  To address these limitations of the current system, the Fisher group is developing a 3D printed shell made of an implantable rigid material better suited for the engineering of bone constructs.  Finally, the construct lacks a vascular network, which can be overcome by including endothelial cells (EC) in addition to hBM-MSCs or by incorporating micro-channels in the engineered constructs through a variety of methods (e.g. biomaterial fabrication and 3D printing). Despite the aforementioned limitations, the work presented is a significant advance towards clinical-sized tissue-engineered bone constructs for use in patients.

In an attempt to elicit discussion, I will mention other methods that harbor potential for use in such large-scale tissue engineering applications. For one, hBM-MSC aggregates could be used in place of cells in alginate beads. These 3D-MSC not only maintain cell:cell contact but also undergo osteogenic differentiation more efficiently than cells grown on tissue culture plastic, are resistant to hypoxia, and secrete angiogenic cytokines. Secondly, factors that stimulate bone differentiation of hBM-MSCs, and/or alter mechanical properties of the construct, could be incorporated into the polymer scaffolding, or could be introduced into 3D-MSC aggregates. Finally, once bio-inks are developed further, the bone construct could be patterned by 3D printing of cells (hBM-MSC, EC, 3D-MSC) and materials.  Now that human-sized constructs are possible in terms of cell numbers and O₂ and nutrient diffusion, the possibilities are virtually endless.

Finally, we sincerely thank Bao Nguyen and John Fisher for being early adopters of RoosterBio hBM-MSCs and joining us in accelerating Regenerative Medicine!

References:

Nguyen BB, Ko H, Moriarty RA, Etheridge JM, Fisher JP. Dynamic Bioreactor Culture of High Volume Engineered Bone Tissue. Tissue Engineering Part A. Volume 22, Numbers 3 and 4, 2016, ahead of print. doi:10.1089/ten.tea.2015.0395.  http://online.liebertpub.com/doi/abs/10.1089/ten.tea.2015.0395 

I’m sorry that this is paywalled!

Yeatts, A.B., and Fisher, J.P. Tubular perfusion system for the long-term dynamic culture of human mesenchymal stem cells. Tissue Eng Part C 17, 337, 2011.

http://online.liebertpub.com/doi/abs/10.1089/ten.tec.2011.0263

Yeatts, A.B., Choquette, D.T., and Fisher, J.P. Bioreactors to influence stem cell fate: augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems. Biochim Biophys Acta 1830, 2470, 2013.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461086/

Ma, X et al. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting  PNAS, Early Edition doi: 10.1073/pnas.1524510113 http://www.pnas.org/content/early/2016/02/04/1524510113

Enabling a New Paradigm in hMSC Suspension Bioreactor Cultures

Introduction

RoosterBio is introducing a new product for highly efficient bioreactor expansion of human Mesenchymal Stem/Stromal Cells (hMSCs) that we are calling RoosterReplenish-MSC.  This innovative, first-in-class stem cell product is a concentrated bioreactor feed that replaces nutrients and growth factors that have been depleted during microcarrier expansion of hMSCs and replaces the need for a media exchange, enabling scalable and efficient fed-batch hMSC bioreactor expansion processes.  This is the first of several new products that we will launch enabling a cell therapy and tissue engineering bioprocess revolution that will be the foundation of a sustainable Regenerative Medicine Industry.

Media Designed for Scale-up

Human stem cells, characterized by their multi-lineage differentiation potential, tissue regenerative capacity, and high proliferation rates, are the most critical raw material in Regenerative Medicine today. Most cell-based therapies require between 50 million and >1 billion cells per patient application, necessitating efficient expansion (i.e. manufacturing) of starting cell sources.  Today, the most widely used cell expansion platforms for stem cell culture are planar technologies such as flasks and multi-layer cell factories (Rowley et. al), but it is generally accepted that lot sizes and COGS generated from these platforms are insufficient to meet the demand of a widely-used commercial product (Simaria et al).

Production technologies such as single use suspension bioreactors (used routinely in protein, monoclonal antibody and vaccine production) are proven to be robust, scalable manufacturing platforms. These platforms operate in a closed and controlled environment, which minimizes the risk of contamination, and are shown to reduce the time, expense, and carbon footprint required for cell processing. More importantly, it has been shown that such systems can yield lot sizes of hundreds of billions to (eventually) >1 trillion cells per manufacturing run, producing commercially-relevant lot sizes (Rowley et. al). 

MSC expansion in suspension bioreactors is typically done by growing cells on adherent substrates, such as microcarriers (Chen et al, Schnitzler et al, Szczypka et al).  Optimization of an efficient MSC bioreactor culture is central to maximizing yields and recovering healthy, functional cells at harvest. Another key attribute to efficient manufacturing processes is cost, and minimizing cost is crucial for building successful business models around MSC-based regenerative therapies.  Cell culture media is consistently the main cost driver of any stem cell production process, and it is critical to minimize media usage to keep production costs to a minimum (Rowley et al). Optimization of MSC-microcarrier cultures typically involves either full or partial media exchanges to manage nutrient supply and waste build-up (Goh et. al, Reichmann et al, Nienow et al, Santos et al, and Heathman et al), which is expensive and impractical at larger scales of >50L culture. This media exchange mentality is driven by the fact that commercially-available hMSC media formulations have been designed for flask-based culture processes and full media exchanges.  

Half media exchanges are the simplest to perform in small scale; however, when spent medium is only partially replaced with growth medium, the final concentration of nutrients and growth factors required for optimum cell proliferation are significantly reduced, resulting in lower cell proliferation rates. In addition, this procedure is time consuming, and the feasibility at larger scales decreases. Fed-batch culture, on the other hand, is more efficient in reducing processing time, mitigating contamination risk, and reducing costs associated with waste management such as time, labor, equipment and facility required to prepare and handle spent media. Hence, a new media design philosophy is required for suspension-based hMSC culture, and RoosterReplenish-MSC, coupled with RoosterBio’s High Perfromance Media kit, is the first media system designed specifically for hMSC bioreactor culture.

RoosterReplenish-MSC, a concentrated bioreactor feed, replaces nutrients and growth factors that have been depleted from RoosterBio’s High Performance Growth Media (KT-001) during extended culture. The nutrient boost provided by RoosterReplenish-MSC replaces the need for partial or full media exchanges when using our rich basal media, yielding a more streamlined culture process for hMSC expansion in bioreactors, and enabling efficiency in media utilization. 

In the next section, we will describe a series of studies performed with RoosterReplenish-MSC in microcarrier suspension culture.

Experimental Methods, Results & Discusssions

Rapid and Economic Generation of hMSC Spheroids for Macroscopic Tissue Biofabrication

Mesenchymal stem cells (MSCs) aggregated into three-dimensional (3D) cellular spheroids are a potent configuration for cell therapy and tissue engineering research and product development (5), and cellular spheroids are a preferred format for many bioprinting applications (10).  Cellular spheroids are essentially micro-tissues that can be manufactured as standardized “living materials” with certain controllable, measurable, and evolving material properties (10). Studies have shown that MSC aggregation into spheroids yield improved in vitro biological functionality over MSCs grown as 2D monolayer; likely due to the 3D tissue-like structure resembling the native configuration of cells in vivo with a microenvironment that allows for direct cell-cell signaling and cell-matrix interactions. MSC spheroids demonstrate enhanced cartilage, bone, and fat differentiation, as well as increased paracrine factor secretion over 2D MSC cultures (1-7). In vivo administration of hMSC spheroids has also showed enhanced therapeutic properties in pre-clinical models of myocardial infarction, bone and cartilage repair, and limb ischemia (3, 5, 8).  

Traditionally, aggregates were formed using suspension culture in spinners or shake flasks or in hanging drop cultures (5). The advancement of technologies has allowed one to quickly and easily generate large numbers of spheroids consistent in size and shape using forced aggregation in micro-wells (AggreWells, Stem Cell Technologies), or using liquid handling automation and 96 or 384 well hanging-droplet plates. There are also tools available that allow researchers to mold micro-tissues into interesting shapes such as rods, toroids, honeycombs, or whatever one can dream up (12-13).  While there are multiple methods for creating hMSC micro-tissues, the biggest challenge is reproducibly growing up sufficient hMSCs to create enough spheroids to start an experiment.  For example, if a researcher needs 10,000 spheroids with an average of 1000 cells per spheroid, then he/she will need at least 10 million cells to begin the experiment, which can take weeks to grow (see process flow diagrams below).  If he/she wishes to use 5000, or 10,000 cells per spheroid, then he/she will need 50 million or 100 million cells for his/her experiment.  This volume of cells has traditionally been very costly and time consuming to generate.

This Application Blog Post will provide a simple protocol to rapidly and economically generate tens of millions of high quality hMSCs so that researchers can minimize their time spent on routine cell culture and maximize their effort on performing hMSC spheroid-based experiments.