RESUMO
The major objective of successful development of tissue-engineered vascular grafts is long-term in vivo patency. Optimization of matrix, cell source, surface modifications, and physical preconditioning are all elements of attaining a compatible, durable, and functional vascular construct. In vitro model systems are inadequate to test elements of thrombogenicity and vascular dynamic functional properties while in vivo implantation is complicated, labor-intensive, and cost-ineffective. We proposed an ex vivo ovine arteriovenous shunt model in which we can test the patency and physical properties of vascular grafts under physiologic conditions. The pressure, flow rate, and vascular diameter were monitored in real-time in order to evaluate the pulse wave velocity, augmentation index, and dynamic elastic modulus, all indicators of graft stiffness. Carotid arteries, jugular veins, and small intestinal submucosa-based grafts were tested. SIS grafts demonstrated physical properties between those of carotid arteries and jugular veins. Anticoagulation properties of grafts were assessed via scanning electron microscopy imaging, en face immunostaining, and histology. Luminal seeding with endothelial cells greatly decreased the attachment of thrombotic components. This model is also suture free, allowing for multiple samples to be stably processed within one animal. This tunable (pressure, flow, shear) ex vivo shunt model can be used to optimize the implantability and long-term patency of tissue-engineered vascular constructs.
Assuntos
Derivação Arteriovenosa Cirúrgica , Carneiro Doméstico/cirurgia , Enxerto Vascular , Grau de Desobstrução Vascular , Animais , Prótese Vascular , Bovinos , Linhagem Celular , Células Endoteliais/citologia , Feminino , Coeficiente Internacional Normatizado , Modelos Animais , Tempo de Tromboplastina Parcial , Trombose/sangueRESUMO
Perfusion technology has been identified as a process improvement capable of eliminating some of the constraints in cell culture and allows for high cell densities and viabilities. However, when implementing this N-1 seed perfusion platform in large-scale manufacturing, unexpected cell damage was observed as early as Day 1. Given that the shear rate within recirculation hollow fibers was normalized and aligned correctly across bench, pilot, and manufacture scale, the primary mitigation was placed on the rotary lobe pump. Lowering the pump rate in manufacture scale successfully alleviated the cell damage. To understand the source of cell damage within the pump, a small-scale rotary lobe-pump robustness model was developed. Testing different pump flow rates and back pressures, it was concluded that high back pressure can cause cell damage. The back pressure within the system can cause back flow and high shear within small clearances inside the pump, which lead to the primary cell damage observed at a large scale. This shear level can be significantly higher than the shear in the hollow fiber. This pump robustness model can be utilized to aid the perfusion skid design, including pump operation efficiency and cell shear sensitivity. Methods to reduce the back pressure and cell shearing were determined to better predict manufacturing performance in the future.
Assuntos
Técnicas de Cultura de Células/métodos , Hidrodinâmica , Estresse Mecânico , Desenho de Equipamento , Perfusão/métodosRESUMO
Poloxamer P188 is a common nonionic surfactant additive used in cell culture media as a cellular protectant from the hydrodynamic forces and shear stress during bioprocessing. Presence of a hydrophobic high molecular weight impurity contaminant has been shown to compromise its protective properties and lead to batch failure. In this work we present, a reliable, sensitive, and rapid analytical method to detect and quantify the contaminant impurity in poloxamer 188. This method replaces a laborious and time-consuming functional test in the form of a shake flask assay. The method is based upon reversed-phase liquid chromatography with charged aerosol detection, simple mobile phase compositions, and a three-step gradient. The method was optimized to resolve the impurity from the main P188 fraction in less than 10 min. Analytical method qualification and functional test comparison demonstrate equivalent or better high throughput impurity screening performance. Attempts to identify the impurity and establish suitable method positive control standards are also discussed.
Assuntos
Cromatografia de Fase Reversa/métodos , Poloxâmero/análise , Técnicas de Cultura de Células/instrumentaçãoRESUMO
Variability in poloxamer 188 (P188) raw material, which is routinely used in cell culture media to protect cells from hydrodynamic forces, plays an important role in the process performance. Even though tremendous efforts have been spent to understand the mechanism of poloxamer's protection, the root cause for lot-to-lot variation was not clear. A recent study reported that the low performance was not due to toxicity but inefficiency to protect cells (Peng et al., Biotechnol Prog. 2014;30:1411-1418). In this study, it was demonstrated for the first time that the addition of other surfactants even at a very low level can interfere with P188 resulting in a loss of efficiency. It was also found that the performance of P188 lots correlated well with its foam stability. Foam generated from low performing lots in baffled shaker flask lasts longer, which suggests that the components in the foam layers are different. The spiking of foam generated from a low performing lot into the media containing a high performance lot resulted in cell damage and low growth. Analytical studies using size exclusion chromatography (SEC) identified differences in high molecular weight (HMW) species present in the P188 lots. These differences are much clearer when comparing the HMW region of the SEC chromatogram of foam vs. bulk liquid samples. This study shows that low performing lots have enriched HMW species in foam samples due to high hydrophobicity, which can be potentially used as a screening assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:767-775, 2016.
Assuntos
Meios de Cultura/química , Poloxâmero/química , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cricetulus , Meios de Cultura/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , Poloxâmero/farmacologia , Tensoativos/químicaRESUMO
To explore the venom diversity of Asian pit vipers, we investigated the structure and function of venom phospholipase A2 (PLA2) derived from two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneensis. We purified six novel PLA2s from T. puniceus venom and another three from T. borneensis venom. All cDNAs encoding these PLA2s except one were cloned, and the molecular masses and N-terminal sequences of the purified enzymes closely matched those predicted from the cDNA. Three contain K49 and lack a disulfide bond at C61-C91, in contrast with the D49-containing PLA2s in both venom species. They are less thermally stable than other K49-PLA2s which contain seven disulfide bonds, as indicated by a decrease of 8.8 degrees C in the melting temperature measured by CD spectroscopy. The M110D mutation in one of the K49-PLA2s apparently reduced its edematous potency. A phylogenetic tree based on the amino-acid sequences of 17 K49-PLA2s from Asian pit viper venoms illustrates close relationships among the Trimeresurus species and intergeneric segregations. Basic D49-PLA2s with a unique Gly6 substitution were also purified from both venoms. They showed edema-inducing and anticoagulating activities. It is notable that acidic PLA2s from both venoms inhibited blood coagulation rather than platelet aggregation, and this inhibition was only partially dependent on enzyme activity. These results contribute to our understanding of the evolution of Trimeresurus pit vipers and the structure-function relationships between various subtypes of crotalid venom PLA2.
Assuntos
Venenos de Crotalídeos/enzimologia , Fosfolipases A/genética , Fosfolipases A/farmacologia , Trimeresurus , Sequência de Aminoácidos , Animais , Anticoagulantes/farmacologia , Ásia , Coagulação Sanguínea/efeitos dos fármacos , Dicroísmo Circular , Clonagem Molecular , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Estabilidade Enzimática , Humanos , Masculino , Dados de Sequência Molecular , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Filogenia , Inibidores da Agregação Plaquetária/farmacologia , Coelhos , Ratos , Ratos Wistar , Análise de Sequência , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: To engineer and implant vascular grafts in the arterial circulation of a pre-clinical animal model and assess the role of donor medial cells in graft remodeling and function. APPROACH AND RESULTS: Vascular grafts were engineered using Small Intestinal Submucosa (SIS)-fibrin hybrid scaffold and implanted interpositionally into the arterial circulation of an ovine model. We sought to demonstrate implantability of SIS-Fibrin based grafts; examine the remodeling; and determine whether the presence of vascular cells in the medial wall was necessary for cellular infiltration from the host and successful remodeling of the implants. We observed no occlusions or anastomotic complications in 18 animals that received these grafts. Notably, the grafts exhibited unprecedented levels of host cell infiltration that was not limited to the anastomotic sites but occurred through the lumen as well as the extramural side, leading to uniform cell distribution. Incoming cells remodeled the extracellular matrix and matured into functional smooth muscle cells as evidenced by expression of myogenic markers and development of vascular reactivity. Interestingly, tracking the donor cells revealed that their presence was beneficial but not necessary for successful grafting. Indeed, the proliferation rate and number of donor cells decreased over time as the vascular wall was dominated by host cells leading to significant remodeling and development of contractile function. CONCLUSIONS: These results demonstrate that SIS-Fibrin grafts can be successfully implanted into the arterial circulation of a clinically relevant animal model, improve our understanding of vascular graft remodeling and raise the possibility of engineering mural cell-free arterial grafts.
Assuntos
Artérias/citologia , Prótese Vascular , Remodelação Vascular , Angiografia , Animais , Apoptose , Artérias/diagnóstico por imagem , Proliferação de Células , Feminino , Imuno-Histoquímica , Macrófagos/citologia , Masculino , Implantação de Prótese , Fluxo Sanguíneo Regional , Ovinos , Engenharia Tecidual , Ultrassonografia , Grau de Desobstrução VascularRESUMO
Shear protectants such as poloxamer 188 play a critical role in protecting cells during cell culture bioprocessing. Lot-to-lot variation of poloxamer 188 was experienced during a routine technology transfer across sites of similar scale and equipment. Cell culture medium containing a specific poloxamer 188 lot resulted in an unusual drop in cell growth, viability, and titer during manufacturing runs. After switching poloxamer lots, culture performance returned to the expected level. In order to control the quality of poloxamer 188 and thus maintain better consistency in manufacturing, multiple small scale screening models were developed. Initially, a 5L bioreactor model was established to evaluate cell damage by high sparge rates with different poloxamer 188 lots. Subsequently, a more robust, simple, and efficient baffled shake flask model was developed. The baffled shake flask model can be performed in a high throughput manner to investigate the cell damage in a bubbling environment. The main cause of the poor performance was the loss of protection, rather than toxicity. It was also suggested that suspicious lots can be identified using different cell line and media. The screening methods provide easy, yet remarkable models for understanding and controlling cell damage due to raw material lot variation as well as studying the interaction between poloxamer 188 and cells.
Assuntos
Técnicas de Cultura de Células , Projetos Piloto , Poloxâmero , Animais , Reatores Biológicos , Células CHO , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Meios de Cultura/química , Meios de Cultura/toxicidade , Poloxâmero/química , Poloxâmero/normas , Poloxâmero/toxicidadeRESUMO
Our laboratory recently reported a new source of smooth muscle cells (SMCs) derived from hair follicle (HF) mesenchymal stem cells. HF-SMCs demonstrated high proliferation and clonogenic potential as well as contractile function. In this study, we aimed at engineering the vascular media using HF-SMCs and a natural biomaterial, namely small intestinal submucosa (SIS). Engineering functional vascular constructs required application of mechanical force, resulting in actin reorganization and cellular alignment. In turn, cell alignment was necessary for development of receptor- and nonreceptor-mediated contractility as soon as 24 h after cell seeding. Within 2 weeks in culture, the cells migrated into SIS and secreted collagen and elastin, the two major extracellular matrix components of the vessel wall. At 2 weeks, vascular reactivity increased significantly up to three- to fivefold and mechanical properties were similar to those of native ovine arteries. Taken together, our data demonstrate that the combination of HF-SMCs with SIS resulted in mechanically strong, biologically functional vascular media with potential for arterial implantation.