Oxygenation Profiles of Human Blood, Cell Culture Medium, and Water for Perfusion of 3D-Bioprinted Tissues using the FABRICA Bioreactor Platform

Abstract

Persistent and saturated oxygen distribution from perfusion media (i.e., blood, or cell culture media) to cells within cell-dense, metabolically-active biofabricated tissues is required to keep them viable. Improper or poor oxygen supply to cells within the tissue bulk severely limits the tissue culturing potential of many bioreactors. We added an oxygenator module to our modular FABRICA bioreactor in order to provide stable oxygenation to biofabricated tissues during culture. In this proof of concept study of an oxygenated and perfused bioreactor, we characterized the oxygenation of water, cell culture medium, and human blood in the FABRICA as functions of augmenting vacuum (air inlet) pressure, perfusion (volumetric flow) rate, and tubing/oxygenator components. The mean oxygen levels for water and cell culture media were 27.7 ± 2.1% and 27.6 ± 4.1%, respectively. The mean oxygen level for human blood was 197.0 ± 90.0 mmHg, with near-physiologic levels achieved with low-permeability PharMed tubing alone (128.0 ± 14.0 mmHg). Hematologic values pre-and post-oxygenation, respectively were (median ± IQR): Red blood cell: 6.0 ± 0.5 (10 6 /μL) and 6.5 ± 0.4 (10 6 /μL); Hemoglobin: 17.5 ± 1.2 g/dL and 19.2 ± 3.0 g/dL; and Hematocrit: 56.7 ± 2.4% and 61.4 ± 7.5%. The relative stability of the hematologic parameters indicates that blood function and thus blood cell integrity were maintained throughout oxygenation. Already a versatile research tool, the now oxygenated FABRICA provides easy-to-implement, in vivo-like perfusion and stable oxygenation culture conditions in vitro semi-independently of one another, which means the bioreactor has the potential to serve as a platform for investigating the behavior of 3D tissue models (regardless of biofabrication method), performing drug toxicity-testing, and testing pharmaceutical efficacy/safety. Recent 3D bioprinting/biofabrication advances allow generation of scaffold-free engineered tissue constructs with customizable 3D design and high cell density 1-4. These engineered tissues are comprised of cells in contact with one another and with extracellular matrix (ECM) in dense 3D structures, thus making them similar to natural tissues in vivo with the same requirements of controlled, well-distributed oxygenation, nutrition, mechanical (shear, compressive, and tensile) forces, temperature, pH, etc. 5-15 so that all cells receive proper metabolic and stimulatory support. Compared to cells cultured in 2 dimensions, engineered tissues with 3D structures, biofabricated by means of bioprinting or other methods, can plausibly be tuned to meet specific genetic, cellular, structural, extracellular, and metabolic parameters, and thus have incredible potential to provide tissues for clinical implants, research models, and drug testing platforms. Importantly, given that media (cell culture media or blood), used to perfuse biofabricated tissue cultured in bioreactors, is one constituent providing multiple critical metabolic factors (convective heat, nutrients, shear stress, and oxygen), the...

The Long and Winding Road to Cardiac Regeneration

“…Research by Chen et al (2020) demonstrated the impact of temperature control on cell growth and function. The study revealed that maintaining the desired temperature within the bioreactor, matching the physiological conditions of the targeted tissue, contributed to cell growth resembling their natural environment [280]. Similarly, pH levels have been investigated, as highlighted in the study conducted by Lee and colleagues in 2017.…”

EnduroBone: A 3D printed bioreactor for extended bone tissue culture

“…For example, OsteoGen bioreactor systems (Tissue Growth Technologies) can simultaneously apply shear pressures via flow perfusion up to 12 samples. Multiple such perfusion-based systems, commercial or otherwise, have been used for 3D tissue culture to understand the role of growth factors, hormones, inflammatory reactions, antimicrobial therapy, bone ingrowth, and cancer metastasis [15] , [16] , [17] , [18] , [19] , [20] , [21] .…”

“…The benefit in comparison with the commercial MechanoCulture system is that the Endurobone bioreactor will apply mechanical loading directly onto the bone tissue. The biggest advantage of the new design compared to the perfusion systems used in several earlier studies [15] , [16] , [17] , [18] , [19] , [20] , [21] will be the presence of dynamic mechanical loading, which is critically needed for the extended culturing of bone tissue. Hence with the design of EnduroBone bioreactor publicly available through the current work, a wider research community can readily and affordably replicate the bioreactor and thus will make bone-related research more inclusive, affordable, and user-friendly.…”

The Long Road to Develop Custom-built Livers: Current Status of 3D Liver Bioprinting

“…The FABRICA bioreactor is a 3D-printed bioreactor that can culture and perfuse 3D-printed tissues in an aseptic environment in real time. 45 Chen et al 74 demonstrated that the FABRICA bioreactor is capable of maintaining physiologic oxygen saturation levels in the water, human blood, and culture media. Given this promising feature, the system may be adapted to provide a means of maintaining perfusion throughout 3D organ printing, although this has been only characterized in the case of 3D bioprinting of genetically engineered pig liver model.…”

“…Given this promising feature, the system may be adapted to provide a means of maintaining perfusion throughout 3D organ printing, although this has been only characterized in the case of 3D bioprinting of genetically engineered pig liver model. 74 Further optimization of current bioink composition and perfusion kinetics will be necessary to ensure viable 3D bioprinting of hepatic parenchyma.…”