scholarly journals Effect of detergent type on the performance of liver decellularized extracellular matrix-based bio-inks

2021 ◽  
Vol 12 ◽  
pp. 204173142199709
Author(s):  
Wonwoo Jeong ◽  
Min Kyeong Kim ◽  
Hyun-Wook Kang
Keyword(s):  
Extracellular Matrix ◽  
Sodium Dodecyl ◽  
Great Influence ◽  
Ammonium Hydroxide ◽  
Sodium Deoxycholate ◽  
Porcine Liver ◽  
Decellularized Extracellular Matrix ◽  
Cytochrome P450 Activity ◽  
Primary Mouse ◽  
Triton X

Decellularized extracellular matrix-based bio-inks (dECM bio-inks) for bioprinting technology have recently gained attention owing to their excellent ability to confer tissue-specific functions and 3D-printing capability. Although decellularization has led to a major advancement in bio-ink development, the effects of detergent type, the most important factor in decellularization, are still unclear. In this study, the effects of various detergent types on bio-ink performance were investigated. Porcine liver-derived dECM bio-inks prepared using widely used detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton X-100 (TX), and TX with ammonium hydroxide (TXA), were characterized in detail. SDS and SDC severely damaged glycosaminoglycan and elastin proteins, TX showed the lowest rate of decellularization, and TXA-based dECM bio-ink possessed the highest ECM content among all bio-inks. Differences in biochemical composition directly affected bio-ink performance, with TXA-dECM bio-ink showing the best performance with respect to gelation kinetics, intermolecular bonding, mechanical properties, and 2D/3D printability. More importantly, cytocompatibility tests using primary mouse hepatocytes also showed that the TXA-dECM bio-ink improved albumin secretion and cytochrome P450 activity by approximately 2.12- and 1.67-fold, respectively, compared with the observed values for other bio-inks. Our results indicate that the detergent type has a great influence on dECM damage and that the higher the dECM content, the better the performance of the bio-ink for 3D bioprinting.

scholarly journals Photocrosslinkable liver extracellular matrix hydrogels for the generation of 3D liver microenvironment models

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Akhilandeshwari Ravichandran ◽  
Berline Murekatete ◽  
Denise Moedder ◽  
Christoph Meinert ◽  
Laura J. Bray
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Cancer Metastasis ◽  
Sodium Dodecyl ◽  
3D Cell Culture ◽  
Ammonium Hydroxide ◽  
Porcine Liver ◽  
Thermal Gelation ◽  
Triton X ◽  
Drug Studies

AbstractLiver extracellular matrix (ECM)-based hydrogels have gained considerable interest as biomimetic 3D cell culture environments to investigate the mechanisms of liver pathology, metabolism, and toxicity. The preparation of current liver ECM hydrogels, however, is based on time-consuming thermal gelation and limits the control of mechanical properties. In this study, we used detergent-based protocols to produce decellularized porcine liver ECM, which in turn were solubilized and functionalized with methacrylic anhydride to generate photocrosslinkable methacrylated liver ECM (LivMA) hydrogels. Firstly, we explored the efficacy of two protocols to decellularize porcine liver tissue using varying combinations of commonly used chemical agents such as Triton X-100, Sodium Dodecyl Sulphate (SDS) and Ammonium hydroxide. Then, we demonstrated successful formation of stable, reproducible LivMA hydrogels from both the protocols by photocrosslinking. The LivMA hydrogels obtained from the two decellularization protocols showed distinct mechanical properties. The compressive modulus of the hydrogels was directly dependent on the hydrogel concentration, thereby demonstrating the tuneability of mechanical properties of these hydrogels. Immortalized Human Hepatocytes cells were encapsulated in the LivMA hydrogels and cytocompatibility of the hydrogels was demonstrated after one week of culture. In summary, the LivMA hydrogel system provides a simple, photocrosslinkable platform, which can potentially be used to simulate healthy versus damaged liver for liver disease research, drug studies and cancer metastasis modelling.

Optimizing the Decellularized Porcine Liver Scaffold Protocol

2020 ◽  
pp. 1-10
Author(s):  
Lanuza Alaby Pinheiro Faccioli ◽  
Grazielle Suhett Dias  ◽  
Victor Hoff ◽  
Marlon Lemos Dias ◽  
Cibele Ferreira Pimentel ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Sodium Deoxycholate ◽  
Vascular Tree ◽  
Porcine Liver ◽  
Triton X

There are few existing methods for shortening the decellularization period for a human-sized whole-liver scaffold. Here, we describe a protocol that enables effective decellularization of the liver obtained from pigs weigh 120 ± 4.2 kg within 72 h. Porcine livers (approx. 1.5 kg) were decellularized for 3 days using a combination of chemical and enzymatic decellularization agents. After trypsin, sodium deoxycholate, and Triton X-100 perfusion, the porcine livers were completely translucent. Our protocol was efficient to promote cell removal, the preservation of extracellular matrix (ECM) components, and vascular tree integrity. In conclusion, our protocol is efficient to promote human-sized whole-liver scaffold decellularization and thus useful to generate bioengineered livers to overcome the shortage of organs.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

scholarly journals The effect of chemical agents on the turnover of the bound phosphate associated with the sodium-and-potassium ion-stimulated adenosine triphosphatase in ox brain microsomes

1970 ◽  
Vol 120 (1) ◽  
pp. 1-13 ◽  
Author(s):  
R. Rodnight
Keyword(s):  
Sodium Chloride ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Ratio ◽  
Chemical Agents ◽  
Rapid Fall ◽  
Triton X ◽  
Hydrolysis Of ◽  
Sodium And Potassium

1. The effect of chemical agents on the turnover of the Na+-dependent bound phosphate and the simultaneous Na+-dependent hydrolysis of ATP by a membrane preparation from ox brain was studied at an ATP/protein ratio of 12.5pmol/μg. 2. The agents were added immediately after phosphorylation of the preparation in a medium containing 50mm-sodium chloride and 2.5μm-[γ-32P]ATP. 3. Concentrations of sodium chloride above 150mm, calcium chloride to 20mm and suramin to 1.4mm inhibited both phosphorylation and dephosphorylation and concomitantly slowed ATP hydrolysis. At 125mm-sodium chloride dephosphorylation and hydrolysis were slightly slowed without affecting phosphorylation. 4. Ethanol to 1.6m concentration inhibited dephosphorylation without affecting phosphorylation; the bound phosphate was increased and ATP hydrolysis slowed. 5. Ouabain to 4mm concentration partially inhibited ATP hydrolysis and caused a transient (1–2s) rise in bound phosphate followed by a rapid fall to a lower plateau value, which eventually declined to zero by the time ATP hydrolysis was complete. 6. Of the detergents examined Lubrol W, Triton X-100 and sodium deoxycholate had no significant effect on turnover. Sodium dodecyl sulphate and sodium decyl sulphate to 3.5mm and 20mm respectively completely inhibited turnover and ATP hydrolysis and stabilized the bound phosphate.

scholarly journals Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation

2020 ◽  
Vol 26 (3) ◽  
pp. 167-178 ◽  
Author(s):  
T T Tiemann ◽  
A M Padma ◽  
E Sehic ◽  
H Bäckdahl ◽  
M Oltean ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Vascular Perfusion ◽  
Uterus Transplantation ◽  
Live Donors ◽  
Triton X

Abstract Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

Creating a tissue-specific microdispersed matrix from a decellularized porcine liver

2020 ◽  
Vol 4 ◽  
pp. 41-50
Author(s):  
A.D. Kirillova ◽  
◽  
Yu.B. Basok ◽  
A.E. Lazhko ◽  
A. M. Grigoryev ◽  
...  
Keyword(s):  
Sodium Dodecyl ◽  
Genetic Material ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Porcine Liver ◽  
Tissue Specific ◽  
Main Components ◽  
Triton X ◽  
Injectable Form

The main problem with decellularization of liver tissue as a tissue-specific matrix/scaffold in liver bioengineered structures is the need to maximize the preservation of the original three-dimensional structure of the tissue and the main components of its extracellular matrix (ECM) while removing cells and genetic material. The attempts to use the existing protocols for the decellularization of other tissues and organs have been unsuccessful. The aim of the work is to develop a method for creation of tissue-specific microdispersed matrix from decellularized porcine liver (TMM DLp). The protocol for decellularization of porcine liver (Lp) fragments has been developed based on the complex application of chemical (sodium dodecyl sulfate and Triton X-100), biochemical (DNase I), and physical (supercritical CO2) methods for treatment the initial tissue. As a result of the found optimal conditions for decellularization of Lp with subsequent cryomicronization of DLp, the injectable form of the microdispersed tissue-specific matrix was obtained, which represents DLp microparticles with the size of 100-200 microns with the residual amount of DNA no more than 10±1.5 ng/mg (less than 1.0%), with the preservation of the microstructure and basic composition of the liver ECM. According to the assessment of biocompatible properties in vitro, TMM DLp samples meet the criteria of biological safety for cytotoxicity and hemolytic activity.

A novel method for providing scaffold: Decellularization of parathyroid capsule

2021 ◽  
pp. 088532822110543
Author(s):  
Nisa İrem Büyük ◽  
Kardelen Tüfekçi ◽  
Alev Cumbul ◽  
Erhan Ayşan ◽  
Gamze Torun Köse
Keyword(s):  
Extracellular Matrix ◽  
Sodium Dodecyl Sulphate ◽  
Dna Analysis ◽  
Structural Integrity ◽  
Sodium Dodecyl ◽  
Parathyroid Tissue ◽  
The Body ◽  
Extracellular Matrix Components ◽  
Triton X ◽  
Sulphated Glycosaminoglycan

This study aimed to generate a novel biomatrix from the decellularized human parathyroid capsule using different methods and to compare the efficiency of decellularization in the means of cell removal, structural integrity and extracellular matrix preservation. The parathyroid capsules, which were carefully dissected from the parathyroid tissue, were randomly divided into four groups and then decellularized using three different protocols: freeze-thaw only, sodium dodecyl sulphate and Triton X-100 treatments after freeze-thawing. Quantitative DNA analysis, agarose gel electrophoresis, sulphated glycosaminoglycan assay, histological analysis, immunohistochemistry and scanning electron microscopy were used to observe the efficiency of parathyroid capsule decellularization and preservation of extracellular matrix components. Considering all the results, it can be said that only freeze-thawing is not an effective method in parathyroid capsule decellularization. When the tissue was treated with a detergent agent in addition to freeze-thawing, the amount of DNA decreased by 90% while sulphated glycosaminoglycan amount maintained 50% compared to untreated tissue. Comparing the effects of the two detergents on the preservation of extracellular matrix such as collagen and sulphated glycosaminoglycan, it was seen that the integrity of tissues treated with Triton X-100 was preserved more than tissues treated with sodium dodecyl sulphate. It is concluded that Triton X-100 treatment with freeze-thawing is the most suitable and effective method for decellularizing the human parathyroid capsule. The biomatrix obtained with this method can be applied in the transplantation of parathyroid tissue and other endocrine tissue types in the body.

scholarly journals Optimizing Perfusion-Decellularization Methods of Porcine Livers for Clinical-Scale Whole-Organ Bioengineering

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Qiong Wu ◽  
Ji Bao ◽  
Yong-jie Zhou ◽  
Yu-jia Wang ◽  
Zheng-gui Du ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Sodium Dodecyl Sulfate ◽  
Liver Tissue ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Great Promise ◽  
Primary Hepatocytes ◽  
Porcine Liver ◽  
Liver Tissue Engineering ◽  
Dodecyl Sulfate

Aim. To refine the decellularization protocol of whole porcine liver, which holds great promise for liver tissue engineering.Methods. Three decellularization methods for porcine livers (1% sodium dodecyl sulfate (SDS), 1% Triton X-100 + 1% sodium dodecyl sulfate, and 1% sodium deoxycholate + 1% sodium dodecyl sulfate) were studied. The obtained liver scaffolds were processed for histology, residual cellular content analysis, and extracellular matrix (ECM) components evaluation to investigate decellularization efficiency and ECM preservation. Rat primary hepatocytes were seeded into three kinds of scaffold to detect the biocompatibility.Results. The whole liver decellularization was successfully achieved following all three kinds of treatment. SDS combined with Triton had a high efficacy of cellular removal and caused minimal disruption of essential ECM components; it was also the most biocompatible procedure for primary hepatocytes.Conclusion. We have refined a novel, standardized, time-efficient, and reproducible protocol for the decellularization of whole liver which can be further adapted to liver tissue engineering.

Decellularised whole ovine testis as a potential bio-scaffold for tissue engineering

2019 ◽  
Vol 31 (11) ◽  
pp. 1665 ◽  
Author(s):  
Aram Akbarzadeh ◽  
Maral Kianmanesh ◽  
Kiarad Fendereski ◽  
Maryam Ebadi ◽  
Seyedeh Sima Daryabari ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Optimal Time ◽  
Second Phase ◽  
Dapi Staining ◽  
Extracellular Matrix Components ◽  
Time Required ◽  
Triton X

The aim of this study was to determine an efficient whole-organ decellularisation protocol of a human-sized testis by perfusion through the testicular arteries. In the first step of this study, we determined the most efficient detergent agent, whereas the second phase delineated the optimal time required for the decellularisation process. Initially sheep testes were decellularised by one of three different detergent agents: sodium dodecyl sulphate (SDS), Triton X-100 and trypsin-ethylenediamine tetraacetic acid (EDTA) solutions, each perfused for 6h. In the second phase, the selected detergent agent was applied for different time periods. A total number of 20 organs were processed during this investigation. The efficacy of the decellularisation process and the preservation of the extracellular matrix components and structure were evaluated by histopathological examinations, 4′,6′-diamidino-2-phenylindole (DAPI) staining, DNA quantification, hydroxyproline measurement, magnetic resonance imaging and scanning electron microscopy. Organ perfusion with 1% SDS solution for 6 to 8h demonstrated the most desirable outcomes regarding decellularisation and extracellular matrix preservation. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to determine the toxicity of the scaffold and its potential for further application in tissue-engineering investigations. This investigation introduces an efficient method to produce a three-dimensional testicular bio-scaffold resembling the properties of the native organ that could be employed in tissue-engineering studies.

scholarly journals Towards a bioengineered uterus: bioactive sheep uterus scaffolds are effectively recellularized by enzymatic preconditioning

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Arvind Manikantan Padma ◽  
Laura Carrière ◽  
Frida Krokström Karlsson ◽  
Edina Sehic ◽  
Sara Bandstein ◽  
...  
Keyword(s):  
Sodium Dodecyl ◽  
Chorioallantoic Membrane ◽  
Sodium Deoxycholate ◽  
Matrix Metalloproteinase 2 ◽  
Uterus Transplantation ◽  
Fetal Stem Cells ◽  
Culturing Conditions ◽  
Triton X

AbstractUterine factor infertility was considered incurable until recently when we reported the first successful live birth after uterus transplantation. However, risky donor surgery and immunosuppressive therapy are factors that may be avoided with bioengineering. For example, transplanted recellularized constructs derived from decellularized tissue restored fertility in rodent models and mandate translational studies. In this study, we decellularized whole sheep uterus with three different protocols using 0.5% sodium dodecyl sulfate, 2% sodium deoxycholate (SDC) or 2% SDC, and 1% Triton X-100. Scaffolds were then assessed for bioactivity using the dorsal root ganglion and chorioallantoic membrane assays, and we found that all the uterus scaffolds exhibited growth factor activity that promoted neurogenesis and angiogenesis. Extensive recellularization optimization was conducted using multipotent sheep fetal stem cells and we report results from the following three in vitro conditions; (a) standard cell culturing conditions, (b) constructs cultured in transwells, and (c) scaffolds preconditioned with matrix metalloproteinase 2 and 9. The recellularization efficiency was improved short-term when transwells were used compared with standard culturing conditions. However, the recellularization efficiency in scaffolds preconditioned with matrix metalloproteinases was 200–300% better than the other strategies evaluated herein, independent of decellularization protocol. Hence, a major recellularization hurdle has been overcome with the improved recellularization strategies and in vitro platforms described herein. These results are an important milestone and should facilitate the production of large bioengineered grafts suitable for future in vivo applications in the sheep, which is an essential step before considering these principles in a clinical setting.

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