January 3, 2017

Guest Blog Post: The Ambitious Future of Tissue Engineering

Bagrat Grigoryan and Jordan MillerPhysiologic Systems Engineering & Advanced Materials Lab at Rice University.

Over the last several decades, various advances in tissue engineering have allowed for the not so distant possibility of replacing, repairing, or regenerating injured tissues1. Significant progress has been made in understanding cellular biology as well as pathophysiology and healthy states of tissues. Additionally, a suite of diverse biofabrication technologies and biomaterials has been conceived, enabling fabrication of complex 3D tissues with greater physiological relevance compared to the traditional 2D context that cells are studied in2. However, the field of tissue engineering still has unresolved questions involving choice of fabrication technique, biomaterial, cellular niche, or even cell type when designing a synthetic tissue.

While different fabrication techniques and biomaterials have been explored in fabricating tissues in vitro, the use of stem cells in engineered tissues is ubiquitous. Not surprisingly so, as biologists continually demonstrate novel ways of directing different lineage commitment of stem cells and further unlocking their vast regenerative potential3. Indeed, stem cell banks have emerged to cryogenically store a patient’s own cells as the therapeutic potential of stem cells is being positively demonstrated in multiple clinical trials4. With over 450 mesenchymal stem cell (MSC)-based trials alone currently ongoing or completed, the regenerative and immunoregulatory properties of MSCs are constantly being exploited to improve the quality of human life5.

Due to the immense therapeutic potential of MSCs, there is a need to rapidly and reproducibly grow a vast amount of MSCs for clinical and research purposes. Although MSCs were identified and isolated from bone marrow more than 40 years ago, we still have not fully mapped their biological characteristics3.
Additionally, even though the first MSC clinical trial occurred in 1995, we still do not have a complete understanding of their therapeutic potential and effects6. Large expansion of MSCs for research purposes is especially desirable for fabrication of synthetic tissues in vitro, especially as many available technologies are demonstrating the ability to generate tissues in the micro- to milli-liter volume scale7. Additionally, as the cost of biofabrication devices is decreasing, thanks in part to open-source hardware initiatives, and novel biomaterials are being developed, the through-put and scalability of tissue fabrication is growing. Rapid expansion of MSCs will allow for more experimentation in vitro, granting for more thorough validation and better data before translation to clinical work is considered.

Rapid expansion of RoosterBio hMSCs with the paired High Performance Media has allowed us to quickly optimize different labeling strategies of hMSCs with fluorescent reports. We have previously demonstrated stable transduction of RoosterBio hMSCs with a second-generation lentivirus. We have achieved dual-labeling of hMSCs expressing green fluorescent protein (eGFP) in the cytoplasm and H2B-mCherry in the nucleus8,9 (Fig. 1A). Compared to cell staining, constitutive labeling allows us to monitor cell behavior transiently, in a non-destructive manner, throughout our studies. Dual-labelling of hMSCs has been most useful in co-culture studies where cell-cell interaction and morphological and spatial changes can be easily monitored non-destructively at multiple time points. Additionally, dual-labeling allows us to identify cytoplasmic and nuclear regions in aggregates that are encapsulated in a 3D context (Fig. 1B). We have also shown osteogenic  (bone) differentiation of unlabeled hMSCs seeded on 3D printed PCL platforms9 (Fig. 1C-D).

Figure 1.  Transduced RoosterBio hMSCs retain their capacity to attach and proliferate on TCP and 3D printed surfaces, form extensions in 3D, while non-transduced hMSCs retain their ability to differentiate into osteogenic lineage. A) Dual-transduced hMSCs with constitutively expressing GFP (cytoplasm) and H2B-mCherry (nucleus) seeded on tissue culture plastic (scale bar = 100 µm). B) Multicellular aggregates (MCA) formed containing dual-transduced hMSCs encapsulated in fibrin gels sprout after 3 days in culture8 (scale bar = 500 µm). C) Photograph and quantification of alizarin red absorbance of hMSCs seeded on sintered PCL after 32 days in either growth or osteogenic media conditions showing intense staining on platforms incubated in osteogenic media, indicating presence of calcium deposits9 (scale bar = 1 cm). D) Similar photographs and quantification of alizarin red absorbance was observed when PCL constructs were vapor smoothed prior to hMSC seeding9 (scale bar = 1 cm). 

As we look toward the future, we believe that fundamental engineering and biological concepts should further fuse to maximize the translational capacity of stem cells. The challenge tissue engineering poses is ambitious, however -- developing more physiologically relevant in vitro models by incorporating approaches from both fields will progress medicine and drastically improve the quality of human life. Additionally, akin to stem cell banking for clinical use, rapid expansion and copious cryopreservation of a batch of stem cells for research use (i.e. a thaw-and-use stem cell product, such as RoosterRTP) will allow researchers the ability to perform experiments faster, with more consistency and reliability, as studies can be performed with cells right out of thaw.

1.           Khademhosseini, A. & Langer, R. A decade of progress in tissue engineering. Nat. Protoc. 11, 1775–1781 (2016).
2.           Bajaj, P., Schweller, R. M., Khademhosseini, A., West, J. L. & Bashir, R. 3D Biofabrication Strategies for Tissue Engineering and Regenerative Medicine. Annu. Rev. Biomed. Eng. 16, 247–76 (2014).
3.           Williams, A. R. & Hare, J. M. Mesenchymal stem cells: Biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease. Circ. Res. 109, 923–940 (2011).
4.           Harris, D. Stem Cell Banking for Regenerative and Personalized Medicine. Biomedicines 2, 50–79 (2014).
5.           Squillaro, T., Peluso, G. & Galderisi, U. Clinical Trials with Mesenchymal Stem Cells: An Update. Cell Transplant. 1–53 (2015). doi:10.3727/096368915X689622
6.           Farini, A., Sitzia, C., Erratico, S., Meregalli, M. & Torrente, Y. Clinical applications of mesenchymal stem cells in chronic diseases. Stem Cells International 2014, (2014).
7.           Kinstlinger, I. S. & Miller, J. 3D-printed Fluidic Networks as Vasculature for Engineered Tissue. Lab Chip (2016). doi:10.1039/C6LC00193A
9.           Kinstlinger, I. S. et al. Open-Source Selective Laser Sintering (OpenSLS) of Nylon and Biocompatible Polycaprolactone. PLoS One 11, e0147399 (2016).

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