-
Methods (San Diego, Calif.) Aug 2015Adipose tissue engineered models are needed to enhance our understanding of disease mechanisms and for soft tissue regenerative strategies. Perfusion systems generate...
Adipose tissue engineered models are needed to enhance our understanding of disease mechanisms and for soft tissue regenerative strategies. Perfusion systems generate more physiologically relevant and sustainable adipose tissue models, however adipocytes have unique properties that make culturing them in a perfusion environment challenging. In this paper we describe the methods involved in the development of two perfusion culture systems (2D and 3D) to test their applicability for long term in vitro adipogenic cultures. It was hypothesized that a silk protein biomaterial scaffold would provide a 3D framework, in combination with perfusion flow, to generate a more physiologically relevant sustainable adipose tissue engineered model than 2D cell culture. Consistent with other studies evaluating 2D and 3D culture systems for adipogenesis we found that both systems successfully model adipogenesis, however 3D culture systems were more robust, providing the mechanical structure required to contain the large, fragile adipocytes that were lost in 2D perfused culture systems. 3D perfusion also stimulated greater lipogenesis and lipolysis and resulted in decreased secretion of LDH compared to 2D perfusion. Regardless of culture configuration (2D or 3D) greater glycerol was secreted with the increased nutritional supply provided by perfusion of fresh media. These results are promising for adipose tissue engineering applications including long term cultures for studying disease mechanisms and regenerative approaches, where both acute (days to weeks) and chronic (weeks to months) cultivation are critical for useful insight.
Topics: Adipocytes; Adipogenesis; Adult Stem Cells; Animals; Biocompatible Materials; Cell Culture Techniques; Humans; Materials Testing; Perfusion; Silk; Tissue Engineering; Tissue Scaffolds
PubMed: 25843606
DOI: 10.1016/j.ymeth.2015.03.022 -
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 -
The Journal of Extra-corporeal... Mar 2020The Australia and New Zealand College of Perfusionists' (ANZCP) Perfusion Incident Reporting System was established in 1998 and has evolved to an open access on-line...
The Australia and New Zealand College of Perfusionists' (ANZCP) Perfusion Incident Reporting System was established in 1998 and has evolved to an open access on-line incident perfusion reporting system (PIRS-2). Changes were made to PIRS-2 to promote learning from what went well in unexpected situations. A 9-question survey was e-mailed to the PIRS-2 contact group to elicit feedback on attitudes to voluntarily reporting perfusion-related incidents and near-miss events to PIRS-2. In August 2019, a 9-question survey using SurveyMonkey (San Mateo Ca) was e-mailed to 198 perfusionists currently on the ANZCP PIRS-2 e-mail contacts group. Responses for all responding practicing perfusionists were totaled and expressed as a percentage of the total number of respondents. The respondents were then grouped by region and responses were expressed as a percentage of respondents from each region as well as for grouped responses from Australia/New Zealand (ANZ) and non-ANZ respondents. The response rate was 49.5% with 95 practicing perfusionists completing the survey. In the 12 months before the survey, 22% of respondents had submitted reports to PIRS-2, whereas 79% had read e-mailed reports. Unit culture was the most frequently cited barrier to reporting from all respondents (19%; 0% to 40% by region). Twenty-five percentage of Australian respondents cited unit culture as a barrier to reporting vs. 0% of New Zealand respondents. A combination of concern of discovery and identification of region ranked second as a barrier for 17% of all respondents. The open access ANZCP PIRS-2 voluntary incident reporting in perfusion was widely viewed as relevant and beneficial to both individual practice and to team performance. A high likelihood to considering reporting incidents is tempered by the well-established barriers of ease of the reporting system, the fix and forget phenomenon, concerns of discovery, and a defensive unit culture.
Topics: Australia; New Zealand; Perfusion; Risk Management; Surveys and Questionnaires
PubMed: 32280139
DOI: 10.1182/ject-1900030 -
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 -
AIMS Bioengineering 2020In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a...
In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.
PubMed: 33163623
DOI: 10.3934/bioeng.2020009 -
Tissue Engineering. Part B, Reviews Apr 2010Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Engineered constructs can... (Review)
Review
Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Engineered constructs can also serve as high-fidelity models for studies of cardiac development and disease. In a general case, the biological potential of the cell-the actual "tissue engineer"-is mobilized by providing highly controllable three-dimensional environments that can mediate cell differentiation and functional assembly. For cardiac regeneration, some of the key requirements that need to be met are the selection of a human cell source, establishment of cardiac tissue matrix, electromechanical cell coupling, robust and stable contractile function, and functional vascularization. We review here the potential and challenges of cardiac tissue engineering for developing therapies that could prevent or reverse heart failure.
Topics: Animals; Electric Stimulation; Heart; Heart Transplantation; Humans; Models, Biological; Myocardium; Organ Culture Techniques; Perfusion; Tissue Engineering
PubMed: 19698068
DOI: 10.1089/ten.TEB.2009.0352 -
Cells Jun 2022Regenerative medicine requires better pre-clinical tools in order to increase the efficiency of novel therapies transitioning to the clinic. Current monolayer cell...
Regenerative medicine requires better pre-clinical tools in order to increase the efficiency of novel therapies transitioning to the clinic. Current monolayer cell culture methods are suboptimal for effectively testing new therapies and live mouse models are expensive, time consuming and require invasive procedures. Fetal organ culture, organoids, microfluidics and culture of thick sections of adult organs all aim to fill the knowledge gap between monolayer culture and live mouse studies. Here we report on an ex vivo organ perfusion system that can support whole adult mouse organs. Ex vivo perfusion of healthy and diseased mouse organs allows for real-time analysis that provides immediate feedback and accurate data collection throughout the experiment. Having a suitable normothermic ex vivo perfusion system for mouse organs provides a tool that will help contribute to our understanding of kidney physiology and disease and can take advantage of the many mouse models of human disease that already exist. Furthermore, an ex vivo kidney perfusion system can be used for testing novel cell therapies, drug screening, drug validation and for the detection of nephrotoxic substances. Critical to the success of mouse ex vivo organ perfusion is having a suitable bioreactor to maintain the organ. Here we have focused on the mouse kidney and mathematically modeled, built and validated a bioreactor that can maintain a kidney for 7 days. The long duration of the ex vivo perfusion will help to advance studies on kidney disease and can rapidly test for new regenerative medicine therapies compared to whole animal studies.
Topics: Animals; Bioreactors; Kidney; Kidney Transplantation; Mice; Organ Preservation; Perfusion
PubMed: 35681517
DOI: 10.3390/cells11111822 -
International Journal of Molecular... Aug 2023Ex vivo lung perfusion (EVLP) has increased donor lung utilization through assessment of "marginal" lungs prior to transplantation. To develop it as a donor lung...
Ex vivo lung perfusion (EVLP) has increased donor lung utilization through assessment of "marginal" lungs prior to transplantation. To develop it as a donor lung reconditioning platform, prolonged EVLP is necessary, and new perfusates are required to provide sufficient nutritional support. Human pulmonary microvascular endothelial cells and epithelial cells were used to test different formulas for basic cellular function. A selected formula was further tested on an EVLP cell culture model, and cell confluence, apoptosis, and GSH and HSP70 levels were measured. When a cell culture medium (DMEM) was mixed with a current EVLP perfusate-Steen solution, DMEM enhanced cell confluence and migration and reduced apoptosis in a dose-dependent manner. A new EVLP perfusate was designed and tested based on DMEM. The final formula contains 5 g/L Dextran-40 and 7% albumin and is named as D05D7A solution. It inhibited cold static storage and warm reperfusion-induced cell apoptosis, improved cell confluence, and enhanced GSH and HSP70 levels in human lung cells compared to Steen solution. DMEM-based nutrient-rich EVLP perfusate could be a promising formula to prolong EVLP and support donor lung repair, reconditioning and further improve donor lung quality and quantity for transplantation with better clinical outcome.
Topics: Humans; Endothelial Cells; Cell Culture Techniques; HSP70 Heat-Shock Proteins; Nutrients; Reperfusion; Lung
PubMed: 37685927
DOI: 10.3390/ijms241713117 -
Scientific Reports May 2023The development of microfluidic culture technology facilitates the progress of study of cell and tissue biology. This technology expands the understanding of...
The development of microfluidic culture technology facilitates the progress of study of cell and tissue biology. This technology expands the understanding of pathological and physiological changes. A skin chip, as in vitro model, consisting of normal skin tissue with epidermis and dermis layer (full thickness) was developed. Polydimethylsiloxane microchannels with a fed-batched controlled perfusion feeding system were used to create a full-thick ex-vivo human skin on-chip model. The design of a novel skin-on-a-chip model was reported, in which the microchannel structures mimic the architecture of the realistic vascular network as nutrients transporter to the skin layers. Viabilities of full-thick skin samples cultured on the microbioreactor and traditional tissue culture plate revealed that a precise controlled condition provided by the microfluidic enhanced tissue viability at least for seven days. Several advantages in skin sample features under micro-scale-controlled conditions were found such as skin mechanical strength, water adsorption, skin morphology, gene expression, and biopsy longevity. This model can provide an in vitro environment for localizing drug delivery and transdermal drug diffusion studies. The skin on the chip can be a valuable in vitro model for representing the interaction between drugs and skin tissue and a realistic platform for evaluating skin reaction to pharmaceutical materials and cosmetic products.
Topics: Humans; Skin; Epidermis; Microfluidics; Perfusion; Lab-On-A-Chip Devices
PubMed: 37258538
DOI: 10.1038/s41598-023-34796-3 -
American Journal of Respiratory and... May 2021
Topics: Cell Communication; Cell Culture Techniques; Humans; Lung; Macrophages; Perfusion; T-Lymphocytes; Tissue Culture Techniques
PubMed: 33357022
DOI: 10.1164/rccm.202012-4358ED