Early attempts to generate new cartilage tissue in vivo began in the 1970s, in Dr. W. T. Green’s lab at the Children’s Hospital . While the initial work had not been successful, this led to the development of work by others including Drs. Burke and Yannas at Massachusetts General Hospital and M.I.T. to create tissue-engineered skin substitute supported by biocompatible materials. In 1998, the first bi-layered bioengineered skin substitute, Apligraf, was approved by the US Food and Drug Administration (FDA).
The field of tissue engineering quickly evolved with pioneers like Dr. Robert Langer and Dr. Joseph Vacanti conceptualizing the idea of developing novel and customizable materials as scaffoldings for cell delivery . Later, when Charles Hull introduced stereolithography, the evolution into bioprinting technology quickly followed suit and in 1999, scientists at the Wake Forest Institute of Regenerative Medicine (WFIRM) successfully printed the first synthetic scaffold of a human bladder, which led to the first lab-grown organs implanted into humans .
In the beginning, a major challenge faced in the bioprinting field was the lack of customizable biomaterials that could be easily printed for tissue engineering applications. Access to such technology was also prohibited by cost but all these problems quickly turned around in the last decade. Today, we can purchase a commercial desktop bioprinter for less than $10,000 USD or even build one ourselves for less than $1000. There is also an array of biomaterials that are photocrosslinkable, thermo-reversible, and readily printable at room temperature that would provide ideal printing and culture conditions to support different tissue engineering goals.
As research rapidly advances in the field of bioprinting and tissue engineering, we began to ask ourselves “What is the next bottleneck we must overcome to progress tissue-engineered product innovations into the clinic?”
It did not take too long for many tissue engineering and bioprinting researchers to realize that cells, often an essential component of the final product can potentially be a challenge. For autologous based products, this is highly dependent on the patient’s condition and therefore the quality and quantity of the cellular material will be highly variable patient-to-patient. However, for products that are being generated from allogeneic sources, such as young and healthy donors, there is the benefit of controlling critical quality attributes of the cellular input starting materials to ensure high quality and reproducibility of manufacturing process outcomes.
Peak MSC Demand
In Olsen et.al. ”Peak MSC” paper published in 2018 , the authors analyzed the growth in demand for mesenchymal stromal/stem cell (MSC) in many regenerative medicine products. Using bone tissue replacement as an example, Dr. Olsen projects a peak demand of 278 trillion (1012) cells per year based on 185,000 amputations performed annually in hospitals today. Just to provide some perspective, typical tissue engineering projects at research laboratories only utilize around 70 million cells and in vivo studies may require 100 million cells . In order to generate billions or even trillions of cells for the production of tissue-engineered constructs, it would be necessary to have a “consistent and readily available supply of high quality, standardized and economical c-GMP cellular starting material to support this.” While tissue engineering applications has not moved as quickly as cell therapy into the clinic, various groups have steadily made progress on this front. According to the Alliance for Regenerative Medicine (ARM) 2019 annual report, there are 46 tissue engineering clinical trials (6 in Phase I, 23 in Phase II and 17 in Phase III) that are currently in the pipeline and the majority are targeted toward bone, cartilage, and skin tissue regeneration or replacement.
Projected near-term cell demands
In the nearer term, projected cell demands will increase for more mature applications like bone, cartilage, and skin tissue. The table below summarizes the current need-based on existing clinical trials and cell numbers used in animal models for bone and cartilage tissue.
|Tissue type||Application||Patients per year||Avg cell numbers used||Most common cells used|
|Cartilage||Chondral defects Meniscus tear||750,000 arthroscopic knee surgery performed||1-2M cells per animal Ref:  based on animal studies 3M cells per cm2 (range: 0.5-14M cells)  based on clinical studies||Mesenchymal stem/stromal cells Chondrocytes|
|Bone||Bone fractures Traumatic brain injury Cleft lip / palate||6.3 million (M) per year 282,000 hospitalization per year||2M cells per scaffold (range: 1-3.4M cells)  based on clinical studies||Mesenchymal stem/stromal cells|
In the case of cartilage tissue, typical defect size >3 cm2 are treated with cartilage tissue-engineered implants and the mean lesion size based on MACI’s (matrix-induced articular cartilage implantation) Phase 3 trial is around 4.8 cm2 , each patient will require ~14.4 million cells. To treat 10,000 patients every year, the annual cell demand (i.e. cell number required to treat patients) would be 144 billion cells which require a total manufacturing output of 308 billion cells. The manufacturing lot size requirements (i.e. how many cells you actually need to produce) are calculated based on cell recovery loss, viability drop, and cell harvest loss. If you are curious to learn how to calculate manufacturing lot sizes, please refer to this blog.
It would not be surprising that some of you might be thinking right now that the cell densities or numbers shown in Table 1 seem rather conservative. I would certainly agree but I also think this is dependent on the desired tissue type and application. In fact, many bioprinting and tissue engineering groups have reported the need for high cell densities in order to create functional tissues that mimic more of the in vivo environment. Researchers at Organovo reported cell densities of 150 million cells/mL of parenchymal or non-parenchymal cells to create a 3D liver tissue that would allow the assessment of organ-level response to drug-induced toxicity . Jennifer Lewis’s group printed a range of cell densities from 0.1 to 10 million cells/mL to create 3D vascularized tissues and showed in vivo mimicry at high cell densities . Researchers at University Medical Center Utrecht also explored cell densities from 3 to 10 million cells/mL but used 10 million cells/mL of human MSC and chondrocytes for in vivo implantation of osteochondral tissues . Adam Feinberg also pointed out during the 3DHeals interview that his group was only able to get functioning cardiac heart muscle tissues when they started printing at very high cell densities at least 1-2 orders of magnitude from what most labs were printing at. This publication is pending but you can certainly refer to his interview here. As this industry matures, we are definitely observing higher cell densities being used to re-create in vivo like tissue constructs. Accessibility to higher cell volumes at a lower cost will also be important in fueling the rapid commercialization of tissue engineering and bioprinting applications.
As mentioned by the founder of RoosterBio, Jon Rowley, during the 3DHeals 2020 virtual conference Biofabrication Ecosystem panel discussion, cells should be envisioned as pieces of technology like a microchip. If a tissue engineering product developer has to figure out how to make their own microchip, it would take them many more years of development, time, and funding to get to their final product. It would be so much more beneficial and efficient to partner with companies who can provide a reliable and consistent cell supply chain to support their product development milestones. One thing for sure, the process innovation that we are witnessing today in MSC and cell therapy manufacturing is laying the groundwork for tissue engineering and bioprinting product commercialization success.
 Charles Vacanti. The history of tissue engineering. J. Cell. Mol. Med. 2006. Vol 10(3), p569-576
 Wake Forest physician reports first human recipients of laboratory-grown organs. April 3, 2006. Press Release.
 Olsen et. al. Peak MSC – Are we there yet? Frontiers in Medicine. 2018. Vol 5: article 178.
 Izuta et.al. Meniscal repair using bone marrow-derived mesenchymal stem cells: experimental study using green fluorescent protein transgenic rats. The Knee. 2005. Vol 12, p217-223
 Dutton et.al. Enhancement of meniscal repair in the avascular zone using mesenchymal stem cells in a porcine model. J Bone Joint Surg. 2010. Vol 92(B): p169-75.
 Jung et.al. Enhanced Early Tissue regeneration after matrix-assisted autologous mesenchymal stem cell transplantation in full thickness chondral defects in a minipig model. Cell Transplantation. 2009. Vol 18, p923-932.
 Foldager et.al. Cell seeding densities in autologous chondrocyte implantation techniques for cartilage repair. Cartilage. 2012. Vol 3(2): p108-17.
 NCT03766217: Bone tissue engineering with dental pulp stem cells for alveolar cleft repair (CLOSE)
 NCT02748343: The clinical therapeutic effects and safety of tissue engineered bone
 Saris et.al. Matrix-applied characterized autologous cultured chondrocytes versus microfracture: two-year follow-up of a prospective randomized trial. Am J Sports Med. 2014. Vol 42(6): p1384-95
 Nguyen et.al. Bioprinted 3D primary liver tissues allow assessment of organ-level response to clinical drug induced toxicity in vitro. PLOS ONE. 2016. Vol 11(7): e0158674
 Kolesky et.al. Three-dimensional bioprinting of thick vascularized tissues. PNAS USA. 2016. Vol 113(12): p3179-84.
 Fedorovich et.al. Biofabrication of osteochondral tissue equivalents by printing topologically defined, cell-laden hydrogel scaffolds. Tissue Eng Part C Methods. 2012. Vol 18(1): 33-44
About the Author:
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.