-
Transplantation Reviews (Orlando, Fla.) Jan 2021The legal shift to an opt-out system of consent for deceased organ donation is now official in England, Wales and Scotland. While it is commendable that national... (Review)
Review
The legal shift to an opt-out system of consent for deceased organ donation is now official in England, Wales and Scotland. While it is commendable that national governments across the United Kingdom have publicly signalled their serious engagement with organ donation, it remains questionable that opt-out policy can in and of itself solve the public health issue of organ shortage. Opt-out policy risks becoming a futile solution if it fails to attend to key factors in clinical practice. Thus, this article provides critical insights and practical considerations in order to work towards increasing the availability of organs for transplantation: 1) organ donation specialists on their own are not enough, a collaborative hospital culture of donation is also needed; 2) investment in innovative perfusion technologies is fundamental to increase both the quantity and quality of organs utilised for transplants; and 3) opt-out does not solve the enduring problem of consent or authorization for donation, rather than hoping that opt-out will shift the societal culture of donation and make donation the default choice, it is necessary to acknowledge that families' authorization remains essential and their emotional experience can neither be minimized nor excluded altogether. Importantly, consent rates are not the only factor to account for overall deceased donation rates. The organ shortage cannot be solely attributed to a matter of negative public attitudes reversible by law. Doing that does a disservice to the public and diverts strategic attention and resources from fostering the organizational and technological enablers of organ donation in clinical practice.
Topics: England; Humans; Organ Transplantation; Perfusion; Policy; Tissue Donors; Tissue and Organ Procurement
PubMed: 33232867
DOI: 10.1016/j.trre.2020.100589 -
Antibody Therapeutics Apr 2022Aggregation, fragmentation, and low yield are issues frequently found during the cell culture process of bispecific antibodies (bsAbs), whose inherent complexity likely...
BACKGROUND
Aggregation, fragmentation, and low yield are issues frequently found during the cell culture process of bispecific antibodies (bsAbs), whose inherent complexity likely plays a role in causing these issues.
METHODS
In this study, we made a head-to-head comparison between fed-batch cell culture and intensified perfusion cell culture with a symmetric bsAb case.
RESULTS
In comparison with the fed-batch culture, a 6.6-fold improvement in integrated viable cell density and a 10.9-fold improvement in volumetric productivity were achieved with the intensified perfusion mode. In addition, a significant decrease in aggregation and fragmentation was observed with the intensified perfusion cell culture. Furthermore, product homogeneity was improved, which was reflected by the increased percentage of capillary isoelectric focusing main group. The quality improvement with intensified perfusion cell culture can be attributed to the shortened product retention in the bioreactor.
CONCLUSIONS
These findings suggest that intensified perfusion cell culture could be a better choice than traditional fed-batch especially for complex molecules like bsAbs. As this is a single case report, future studies on other cases are needed to further confirm the general applicability of this strategy.
PubMed: 35719210
DOI: 10.1093/abt/tbac009 -
Journal of Applied Physiology... Nov 2021In recent years, it has become common to experiment with ex vivo perfused lungs for organ transplantation and to attempt regenerative pulmonary engineering using...
In recent years, it has become common to experiment with ex vivo perfused lungs for organ transplantation and to attempt regenerative pulmonary engineering using decellularized lung matrices. However, our understanding of the physiology of ex vivo organ perfusion is imperfect; it is not currently well understood how decreasing microvascular barrier affects the perfusion of pulmonary parenchyma. In addition, protocols for lung perfusion and organ culture fluid-handling are far from standardized, with widespread variation on both basic methods and on ideally controlled parameters. To address both of these deficits, a robust, noninvasive, and mechanistic model is needed which is able to predict microvascular resistance and permeability in perfused lungs while providing insight into capillary recruitment. Although validated mathematical models exist for fluid flow in native pulmonary tissue, previous models generally assume minimal intravascular leak from artery to vein and do not assess capillary bed recruitment. Such models are difficult to apply to both ex vivo lung perfusions, in which edema can develop over time and microvessels can become blocked, and to decellularized ex vivo organomimetic cultures, in which microvascular recruitment is variable and arterially perfused fluid enters into the alveolar space. Here, we develop a mathematical model of pulmonary microvascular fluid flow which is applicable in both instances, and we apply our model to data from native, decellularized, and regenerating lungs under ex vivo perfusion. The results provide substantial insight into microvascular pressure-flow mechanics, while producing previously unknown output values for tissue-specific capillary-alveolar hydraulic conductivity, microvascular recruitment, and total organ barrier resistance. We present a validated model of pulmonary microvascular fluid mechanics and apply this model to study the effects of increased capillary permeability in decellularized and regenerating lungs. We find that decellularization alters microvascular steady-state mechanics and that re-endothelialization partially rescues key biologic parameters. The described model provides powerful insight into intraorgan microvascular dynamics and may be used to guide regenerative engineering experiments. We include all data and derivations necessary to replicate this work.
Topics: Capillaries; Lung; Microvessels; Perfusion
PubMed: 34554016
DOI: 10.1152/japplphysiol.00286.2020 -
Frontiers in Bioengineering and... 2022Numerous disease conditions involve the sudden or progressive loss of blood flow. Perfusion restoration is vital for returning affected organs to full health. While a...
Numerous disease conditions involve the sudden or progressive loss of blood flow. Perfusion restoration is vital for returning affected organs to full health. While a range of clinical interventions can successfully restore flow to downstream tissues, the microvascular responses after a loss-of-flow event can vary over time and may involve substantial microvessel instability. Increased insight into perfusion-mediated capillary stability and access-to-flow is therefore essential for advancing therapeutic reperfusion strategies and improving patient outcomes. To that end, we developed a tissue-based microvascular fluidics model to better understand (i) microvascular stability and access-to-flow over an acute time course post-ischemia, and (ii) collateral flow in vessels neighboring an occlusion site. We utilized murine intestinal tissue regions by catheterizing a feeder artery and introducing perfusate at physiologically comparable flow-rates. The cannulated vessel as well as a portion of the downstream vessels and associated intestinal tissue were cultured while constant perfusion conditions were maintained. An occlusion was introduced in a selected arterial segment, and changes in perfusion within areas receiving varying degrees of collateral flow were observed over time. To observe the microvascular response to perfusion changes, we incorporated (i) tissues harboring cell-reporter constructs, specifically labeling of intestinal pericytes, and (ii) different types of fluorescent perfusates to quantify capillary access-to-flow at discrete time points. In our model, we found that perfusion tracers could enter capillaries within regions downstream of an occlusion upon the initial introduction of perfusion, but at 24 h tissue perfusion was severely decreased. However, live/dead cell discrimination revealed that the tissue overall did not experience significant cell death, including that of microvascular pericytes, even after 48 h. Our findings suggest that altered flow conditions may rapidly initiate cellular responses that reduce capillary access-to-flow, even in the absence of cellular deterioration or hypoxia. Overall, this tissue-based microfluidics model may serve as a platform upon which a variety of follow-on studies may be conducted. It will thus enhance our understanding of microvessel stability and access-to-flow during an occlusive event and the role of collateral flow during normal and disrupted perfusion.
PubMed: 36568288
DOI: 10.3389/fbioe.2022.1008481 -
Biomimetics (Basel, Switzerland) Sep 2021Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as...
Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as larger defect sizes are correlated with higher rates of complications and failure due to insufficient vascularization following implantation. Our study demonstrates, for the first time, a method to engineer perfusable, pre-vascularized, high-density adipose grafts that combine patient-derived adipose cells with a decellularized lung matrix (DLM). The lung is one of the most vascularized organs with high flow, low resistance, and a large blood-alveolar interface separated by a thin basement membrane. For our work, the large volume capacity within the alveolar compartment was repurposed for high-density adipose cell filling, while the acellular vascular bed provided efficient graft perfusion throughout. Both adipocytes and hASCs were successfully delivered and remained in the alveolar space even after weeks of culture. While adipose-derived cells maintained their morphology and functionality in both static and perfusion DLM cultures, perfusion culture offered enhanced outcomes over static culture. Furthermore, we demonstrate that endothelial cells seamlessly integrate into the acellular vascular tree of the DLM with adipocytes. These results support that the DLM is a unique platform for creating vascularized adipose tissue grafts for large defect filling.
PubMed: 34562876
DOI: 10.3390/biomimetics6030052 -
Scientific Reports Aug 2022In bone tissue engineering research, bioreactors designed for replicating the main features of the complex native environment represent powerful investigation tools....
In bone tissue engineering research, bioreactors designed for replicating the main features of the complex native environment represent powerful investigation tools. Moreover, when equipped with automation, their use allows reducing user intervention and dependence, increasing reproducibility and the overall quality of the culture process. In this study, an automated uni-/bi-directional perfusion bioreactor combinable with pulsed electromagnetic field (PEMF) stimulation for culturing 3D bone tissue models is proposed. A user-friendly control unit automates the perfusion, minimizing the user dependency. Computational fluid dynamics simulations supported the culture chamber design and allowed the estimation of the shear stress values within the construct. Electromagnetic field simulations demonstrated that, in case of combination with a PEMF stimulator, the construct can be exposed to uniform magnetic fields. Preliminary biological tests on 3D bone tissue models showed that perfusion promotes the release of the early differentiation marker alkaline phosphatase. The histological analysis confirmed that perfusion favors cells to deposit more extracellular matrix (ECM) with respect to the static culture and revealed that bi-directional perfusion better promotes ECM deposition across the construct with respect to uni-directional perfusion. Lastly, the Real-time PCR results of 3D bone tissue models cultured under bi-directional perfusion without and with PEMF stimulation revealed that the only perfusion induced a ~ 40-fold up-regulation of the expression of the osteogenic gene collagen type I with respect to the static control, while a ~ 80-fold up-regulation was measured when perfusion was combined with PEMF stimulation, indicating a positive synergic pro-osteogenic effect of combined physical stimulations.
Topics: Bioreactors; Bone and Bones; Cell Differentiation; Cells, Cultured; Electromagnetic Fields; Osteogenesis; Perfusion; Printing, Three-Dimensional; Reproducibility of Results; Tissue Engineering; Tissue Scaffolds
PubMed: 35974079
DOI: 10.1038/s41598-022-18075-1 -
American Journal of Transplantation :... Apr 2024Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal...
Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.
Topics: Swine; Animals; Liver Transplantation; Organ Preservation; Liver; Perfusion; Reperfusion Injury; Allografts; Intestines
PubMed: 37918482
DOI: 10.1016/j.ajt.2023.10.026 -
Scientific Reports Jun 2022Well-defined fluid flows are the hallmark feature of microfluidic culture systems and enable precise control over biophysical and biochemical cues at the cellular scale....
Well-defined fluid flows are the hallmark feature of microfluidic culture systems and enable precise control over biophysical and biochemical cues at the cellular scale. Microfluidic flow control is generally achieved using displacement-based (e.g., syringe or peristaltic pumps) or pressure-controlled techniques that provide numerous perfusion options, including constant, ramped, and pulsed flows. However, it can be challenging to integrate these large form-factor devices and accompanying peripherals into incubators or other confined environments. In addition, microfluidic culture studies are primarily carried out under constant perfusion conditions and more complex flow capabilities are often unused. Thus, there is a need for a simplified flow control platform that provides standard perfusion capabilities and can be easily integrated into incubated environments. To this end, we introduce a tunable, 3D printed micro pressure regulator (µPR) and show that it can provide robust flow control capabilities when combined with a battery-powered miniature air pump to support microfluidic applications. We detail the design and fabrication of the µPR and: (i) demonstrate a tunable outlet pressure range relevant for microfluidic applications (1-10 kPa), (ii) highlight dynamic control capabilities in a microfluidic network, (iii) and maintain human umbilical vein endothelial cells (HUVECs) in a multi-compartment culture device under continuous perfusion conditions. We anticipate that our 3D printed fabrication approach and open-access designs will enable customized µPRs that can support a broad range of microfluidic applications.
Topics: Cell Culture Techniques; Human Umbilical Vein Endothelial Cells; Humans; Microfluidics; Perfusion; Printing, Three-Dimensional
PubMed: 35750792
DOI: 10.1038/s41598-022-15087-9 -
Biotechnology and Bioengineering Jul 2022In the past decades, bone tissue engineering developed and exploited many typologies of bioreactors, which, besides providing proper culture conditions, aimed at...
In the past decades, bone tissue engineering developed and exploited many typologies of bioreactors, which, besides providing proper culture conditions, aimed at integrating those bio-physical stimulations that cells experience in vivo, to promote osteogenic differentiation. Nevertheless, the highly challenging combination and deployment of many stimulation systems into a single bioreactor led to the generation of several unimodal bioreactors, investigating one or at mostly two of the required biophysical stimuli. These systems miss the physiological mimicry of bone cells environment, and often produced contrasting results, thus making the knowledge of bone mechanotransduction fragmented and often inconsistent. To overcome this issue, in this study we developed a perfusion and electroactive-vibrational reconfigurable stimulation bioreactor to investigate the differentiation of SaOS-2 bone-derived cells, hosting a piezoelectric nanocomposite membrane as cell culture substrate. This multimodal perfusion bioreactor is designed based on a numerical (finite element) model aimed at assessing the possibility to induce membrane nano-scaled vibrations (with ~12 nm amplitude at a frequency of 939 kHz) during perfusion (featuring 1.46 dyn cm wall shear stress), large enough for inducing a physiologically-relevant electric output (in the order of 10 mV on average) on the membrane surface. This study explored the effects of different stimuli individually, enabling to switch on one stimulation at a time, and then to combine them to induce a faster bone matrix deposition rate. Biological results demonstrate that the multimodal configuration is the most effective in inducing SaOS-2 cell differentiation, leading to 20-fold higher collagen deposition compared to static cultures, and to 1.6- and 1.2-fold higher deposition than the perfused- or vibrated-only cultures. These promising results can provide tissue engineering scientists with a comprehensive and biomimetic stimulation platform for a better understanding of mechanotransduction phenomena beyond cells differentiation.
Topics: Bioreactors; Bone and Bones; Cell Differentiation; Cells, Cultured; Mechanotransduction, Cellular; Osteogenesis; Tissue Engineering; Tissue Scaffolds
PubMed: 35383894
DOI: 10.1002/bit.28100 -
Microvascular Research May 2022Perfusable vascular structures in cell-dense tissues are essential for fabricating functional three-dimensional (3D) tissues in vitro. However, it is challenging to...
Perfusable vascular structures in cell-dense tissues are essential for fabricating functional three-dimensional (3D) tissues in vitro. However, it is challenging to introduce functional vascular networks observable as vascular tree, finely spaced at intervals of tens of micrometers as in living tissues, into a 3D cell-dense tissue. Herein, we propose a method for introducing numerous vascular networks that can be perfused with blood into 3D tissues constructed by cell sheet engineering. We devise an artificial vascular bed using a hydrogel that is barely deformed by cells, enabling perfusion of the culture medium directly beneath the cell sheets. Triple-layered cell sheets with an endothelial cell network prepared by fibroblast co-culture are transplanted onto the vascular bed and subjected to perfusion culture. We demonstrate that numerous vascular networks are formed with luminal structures in the cell sheets and can be perfused with India ink or blood after a five-day perfusion culture. Histological analysis also demonstrates that perfusable vascular structures are constructed at least 100 μm intervals uniformly and densely within the tissues. The results suggest that our perfusion culture method enhances vascularization within the 3D cell-dense tissues and enables the introduction of functional vasculature macroscopically observable as vascular tree in vitro. In conclusion, this technology can be used to fabricate functional tissues and organs for regenerative therapies and in vitro experimental models.
Topics: Capillaries; Coculture Techniques; Endothelial Cells; Perfusion; Tissue Engineering
PubMed: 35032535
DOI: 10.1016/j.mvr.2022.104321