The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 3 10 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated Abbreviations: CFD, computational fluid dynamics; ECM, extracellular matrix; FBS, fetal bovine serum; SSuPer, self-supporting perfused. 3D-bioprinting, biofabrication, bioreactor, computational fluid dynamics, perfusion, scaffold-free SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. How to cite this article: Sego T, Prideaux M, Sterner J, et al. Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models. Biotechnology and Bioengineering. 2020;117:798-815.

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 cover image is based on the Original Article Computational Fluid Dynamic Analysis of Bioprinted Self‐Supporting Perfused (SSuPer) Tissue Models by Lester J. Smith and T.J. Sego, https://doi.org/10.1002/bit.27238.