Complete Cell Removal Research Articles

Effects of Different Perfusing Routes through The Portal Vein, Hepatic Vein, and Biliary Duct on Whole Rat Liver Decellularization.

Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack of sufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical application potential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellular components and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy through three anatomical routes. In this experimental study, rat liver decellularization was performed through biliary duct (BD), portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy was evaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs). ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electron microscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines. Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BD route produced an opaque scaffold with incomplete decellularization. H and E staining confirmed these results. Maximum DNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNA content decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups were significantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV and HV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEM images showed that the ECM structure was preserved and cellular components were entirely removed. MTT assay showed the biocompatibility of the decellularized scaffold. The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.

Size dependent regeneration capacity of functionalized Capra ear-derived micro-tissue scaffolds for treatment of cartilage defects

Regeneration of cartilage still possesses considerable challenge in orthopedic treatment owing to avascular nature and lack of self-healing ability of the tissue. In this context, cartilage defect recovery via engineered functional micro tissue delivery is an emerging trend in musculoskeletal therapeutics. This work explores the efficacy of Capra ear-derived functionalized micro-tissues (FMTs) for regeneration of cartilage defects. For this study, a novel and inexpensive NaOH-based decellularization method was developed and optimized to obtain complete removal of cells without deteriorating the matrix structure of Capra ear cartilage. The decellularized matrix was then processed to generate micro-scaffolds (MSs) with different size distributions. It was established that among those size groups, MSs having 100 µm average size were most effective for adherence, cell proliferation and differentiation. It was assessed that the loose fibrous structure of smaller scaffolds was able to induce chondrogenic differentiation. Further, a frugal system was devised for performing hanging culture on the MSs with adipose tissue derived mesenchymal stem cells (ADMSCs) to generate functionalized micro tissues (FMTs). Finally, FMTs were implanted in rabbit auricular cartilage defects to evaluate their healing efficacy. Histology, FTIR and microCT studies revealed that the implanted FMTs were able to regenerate the defect within 60 days. Thus, this work establishes that the functionalized micro tissues derived from decellularized capra ear cartilage can potentially be used for cartilage regeneration, and the efficacy of regeneration depends on the size along with the fibrous structure of the scaffolds.

Optimization of electrocoagulation–flotation treatment with an aluminum anode to enhance Microcystis aeruginosa cell removal efficiency

Cyanobacteria adversely affect drinking water treatment and the quality of the drinking water supply. Microcystis spp. are the most abundant cyanobacteria in many surface water bodies throughout the world. Electrocoagulation–flotation (ECF) treatment has attracted considerable attention as a means of cyanobacteria removal. In this study, ECF treatment was optimized by adjusting the initial pH, electrode distance, stirring speed, and electrical conductivity (k) to maximize Microcystis aeruginosa cell removal efficiency while meeting drinking water quality standards. The treatment was performed for 200 mL M. aeruginosa suspensions (~2 × 106 cells/mL) using an aluminum anode and a graphite cathode at a constant direct current of 100 mA. An initial pH of 6, a stirring speed of 200 rpm, an electrode distance of 1 cm, and k of 500 μS/cm were selected as optimum conditions for the ECF treatment. Under these conditions, M. aeruginosa at a cell density of ~2 × 106 cells/mL was completely removed at a current density of 4.0 mA/cm2 with an average energy consumption of 1.20 ± 0.03 Wh/L. The zeta potential varied from −18.49 ± 3 mV to 1.15 ± 0.38 mV, which confirmed the complete removal of cells. Most importantly, the pH, aluminum concentration, and total dissolved solids of the treated water met the drinking water quality standards of the World Health Organization. The optimized ECF treatment was thus shown to be promising for the removal of M. aeruginosa cells.

Decellularization of kidney tissue: comparison of sodium lauryl ether sulfate and sodium dodecyl sulfate for allotransplantation in rat.

An automatic decellularization device was developed to perfuse and decellularize male rats' kidneys using both sodium lauryl ether sulfate (SLES) and sodium dodecyl sulfate (SDS) and to compare their efficacy in kidney decellularization and post-transplantation angiogenesis. Kidneys were perfused with either 1% SDS solution for 4h or 1% SLES solution for 6h. The decellularized scaffolds were stained with hematoxylin and eosin, periodic acid Schiff, Masson's trichrome, and Alcian blue to determine cell removal and glycogen, collagen, and glycosaminoglycan contents, respectively. Moreover, scanning electron microscopy was performed to evaluate the cell removal and preservation of microarchitecture of both SDS and SLES scaffolds. Additionally, DNA quantification assay was applied for all groups in order to measure residual DNA in the scaffolds and normal kidney. In order to demonstrate biocompatibility of the decellularized scaffolds, human umbilical cord mesenchymal stromal/stem cells (hUC-MSCs) were seeded on the scaffolds. In addition, the allotransplantation was performed in back muscle and angiogenesis was evaluated. Complete cell removal in both SLES and SDS groups was observed in scanning electron microscopy and DNA quantification assays. Moreover, the extracellular matrix (ECM) architecture of rat kidney in the SLES group was significantly preserved better than the SDS group. The hUC-MSCs were successfully migrated from the cell culture plate surface into the SDS and SLES decellularized scaffolds. The formation of blood vessels was observed in the kidney in both SLES and SDS decellularized kidneys. The better preservation of ECM than SDS introduces SLES as the solvent of choice for kidney decellularization.

Tissue-engineering the larynx: Effect of decellularization on human laryngeal framework and the cricoarytenoid joint.

Decellularization approaches have been commonly used as alternative techniques to reconstruct tissues. However, due to the complex tissue compartmentation of the larynx, the decellularization process may not retain the characteristics necessary for the successful recreation of the larynx. The aim of this study was to assess the effect of the decellularization process on the framework of the human cadaveric larynx generally and the cricoarytenoid joint specifically. In this work, five freshly frozen human cadaveric larynges were decellularized utilizing a protocol that was previously demonstrated to be effective in decellularizing a porcine larynx. The decellularization protocol included: biological, chemical, and physical decellularization methods. Each specimen served as its own control to assess changes after decellularization. Studies and measurements included: histological, using Hematoxylin and Eosin (H&E) and Live/Dead™ stains; DNA quantification; micro-computed tomography (μ-CT) imaging; and biomechanical testing of the cricoarytenoid joints. The decellularization protocol took 12 days for each specimen. Microscopy of H&E stained samples demonstrated substantial removal of cells with preservation of the extracellular matrix that was more evident in cartilage than muscle specimens. Confocal microscope images of Live/Dead™ stained specimens also demonstrated almost complete removal of cells. Pre-decellularization cartilage-DNA quantity range was 27.0 to 336.8ng/mg while post-decellularization DNA quantity range was 0 to 30.4ng/mg (p= 0.031). For muscles, pre-decellularization DNA quantity range was 150.0 to 3,384.6ng/mg, while post-decellularization DNA quantity range was 0 to 45.5ng/mg (p= 0.031). μ-CT demonstrated preservation of the cartilaginous framework with a slight reduction of cricoarytenoid joint space. Furthermore, μ-CT demonstrated no significant reduction in the Housefield unit (p= 0.25) and mineral density (p= 0.25) after decellularization. Biomechanical testing demonstrated a non-significant reduction of forces required for anterior displacement of the arytenoid (mean reduction of forces, 0.1± 0.2N, p= 0.16) and forces required for posterior displacement of the arytenoid (mean reduction of forces, 0.2± 0.3N, p= 0.05). This study demonstrates effective decellularization of human larynges as evidenced by significant DNA depletion and preservation of extracellular matrix, which are outcomes that are required for a biological scaffold to regenerate a non-immunogenic larynx. The decellularization process caused minimal weakness in the cricoarytenoid joints due to treatment with multiple detergents and enzymes in the decellularization protocol.

POS-476 OPTIMIZATION OF SDS-BASED KIDNEY DECELLULARIZATION RESULTS IN ECM SCAFFOLDS APPLICABLE FOR 3D RECELLULARIZATION

Kidney decellularization is used to obtain extracellular matrix (ECM) scaffolds as a platform for 3D culturing and kidney tissue regeneration. ECM contains biological molecules such as growth factors necessary for cell survival, proliferation, and maturation during organogenesis. While decellularization allows for the complete removal of cells, it also causes the removal of specific growth factors from the ECM, which could have deleterious effects on cell survival, proliferation, and differentiation during recellularization. We hypothesize that using a mild decellularization protocol which employs the use of sodium dodecyl sulphate (SDS) below its critical micelle concentration (CMC) of 0.2% in water at room temperature will allow for the greater retention of bioactive molecules which will improve recellularization efficiencies. Porcine kidney cortex was cut into 1mm x 6mm slices and decellularized using 0.05%, 0.075%, or 0.1% SDS in a soak and agitation protocol for 24 hours at room temperature. Decellularization was determined through histological and immunofluorescent staining, and DNA quantification. The retention of ECM proteins and biomolecules was determined by histological and immunofluorescent staining, mass spectrometry, and protein array analysis. Four different cell lines were used for recellularization; human-induced pluripotent stem cells (hiPSC), mesoderm derived from hiPSC, intermediate mesoderm derived from hiPSC, and primary human renal epithelial cells. ECM scaffolds were dried on Transwell inserts and cells were pipetted on the surface of the scaffold. ECM-cell constructs were grown for two weeks in culture at air-liquid interface. Recellularization efficiency, cell organization, and differentiation were determined using whole mount staining. Complete decellularization was achieved when SDS was used in concentrations below its CMC. Decellularizing with 0.05% SDS resulted in the greatest retention of glycosaminoglycan chains (GAGs), revealing that GAGs are still significantly removed from the ECM when using SDS below its CMC. Upon protein quantification, scaffolds decellularized with higher SDS concentrations were more enriched with matrisome proteins compared to scaffolds decellularized with lower SDS concentrations, which correlated to a decrease in growth factors and cytokines present on the scaffolds as determined by protein array. Following 14-day recellularization, hiPSCs and kidney progenitor cells proliferated on the ECM and expressed markers such as LIM1, WT1, PBX1 and CD31. Primary renal epithelial cells maintained a mature phenotype, expressing LTL, cytokeratin, and KSP. Herein, we describe a novel protocol for the decellularization of kidney cortex scaffolds that allows for greater retention of biomolecules. This retention of growth factors and cytokines aids in the proliferation and directed differentiation of hiPS and kidney progenitor cells toward mature renal lineages. Thus, we determined that our ECM scaffolds are able to support the growth of both hiPS and organ-matched cells. This can be applied to other organ decellularization/ recellularization protocols, as well as for the generation of kidney tissue for further in vitro studies.

Residual immune response towards decellularized homografts may be highly individual

OBJECTIVESDecellularized homograft valves (DHVs) have shown promising clinical results, particularly in the treatment of congenital heart disease. However, DHV appears to elicit an immune response in a subset of young patients, indicated by early valve degeneration. As the decellularization process is quality controlled for each DHV, we hypothesized that there may be residual immunogenicity within the extracellular matrix of DHV.METHODSA semi-quantitative dot blot analysis was established to screen for preformed recipient antibodies using secondary anti-human antibodies. Fifteen DHV samples (7 aortic, 8 pulmonary) were solubilized and exposed to serum from 20 healthy controls.RESULTSThe sera from young controls (n = 10, 18–25 years) showed significantly stronger binding of preformed antibodies than sera from older individuals (n = 10, 48–73 years). The difference between the means of arbitrary units was 15.1 ± 6.5 (P = 0.0315). There was high intraindividual variance in the mean amounts of arbitrary units of antibody binding with some healthy controls showing >10 times higher antibody binding towards 2 different DHV. The amount of preformed antibodies bound to DHVs was higher in aortic than in pulmonary DHVs. The mean number of antibody binding (in arbitrary units) was 17.2 ± 4.5 in aortic and 14.5 ± 4.7 in pulmonary DHV (P = 0.27). The amount of preformed antibodies bound to pulmonary DHVs was statistically significantly higher in the sera of healthy males (n = 10) than in the sera of healthy females (n = 10). The mean number of arbitrary units was 17.2 ± 4.2 in male and 11.7 ± 5.3 in female sera (P = 0.036). Antibody binding to aortic DHV was also higher in males, but not significant (18.8 ± 5.0 vs 15.6 ± 4.0). Blood group (ABO) incompatibility between the serum from controls and DHV showed no impact on antibody binding, and there was no age-related impact among DHV donors.CONCLUSIONSResidual immunogenicity of decellularized homografts appears to exist despite almost complete cell removal. The established dot blot method allows a semi-quantitative assessment of the individual immune response towards extracellular DHV components and potentially the possibility of preoperative homograft matching.

Fabrication of Decellularized Engineered Extracellular Matrix through Bioreactor-Based Environment for Bone Tissue Engineering

Extracellular matrix (ECM)-contained grafts can be achieved by decellularization of native bones or synthetic scaffolds. Limitations associated with harvesting the native bone has raised interest in preparing in vitro ECM bioscaffold for bone tissue engineering. Here, we intend to develop an ECM-contained construct via decellularizing an engineered gelatin-coated β-tricalcium phosphate (gTCP) scaffold. In order to find an optimal protocol for decellularization of cell-loaded gTCP scaffolds, they were seeded with buccal fat pad-derived stem cells. Then, four decellularization protocols including sodium dodecyl sulfate, trypsin, Triton X-100, and combined solution methods were compared for the amounts of residual cells and remnant collagen and alteration of scaffold structure. Then, the efficacy of the selected protocol in removing cells from gTCP scaffolds incubated in a rotating and perfusion bioreactor for 24 days was evaluated and compared with static condition using histological analysis. Finally, decellularized scaffolds, reloaded with cells, and their cytotoxicity and osteoinductive capability were evaluated. Complete removal of cells from gTCP scaffolds was achieved from all protocols. However, treatment with Triton X-100 showed significantly higher amount of remnant ECM. Bioreactor-incubated scaffolds possessed greater magnitude of ECM proteins including collagen and glycosaminoglycans. Reseeding the decellularized scaffolds also represented higher osteoinductivity of bioreactor-based scaffolds. Application of Triton X-100 as decellularization protocol and usage of bioreactors are suggested as a suitable technique for designing ECM-contained grafts for bone tissue engineering.

Retraction Note: Preparation of Decellularized Bovine Tendon Scaffold and Evaluation of Its Interaction with Adipose Tissue-Derived Mesenchymal Stem Cells

Background: Tissue engineering is the science of tissue design and one of the main branches of regenerative medicine that aims to improve and repair tissue injuries. Objectives: This study aimed to decellularize the tissue of bovine Achilles tendon and create a natural 3D scaffold. Then, the interaction of human adipose tissue-derived mesenchymal stem cells (hAd-MSCs) with this 3D scaffold was evaluated for use in tendon injuries. Methods: The bovine Achilles tendon was obtained from a slaughterhouse and decellularized by the combination of ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS). Histological and biomechanical tests were used to evaluate the quality of decellularized scaffolds. Adipose-derived mesenchymal stem cells were cultured on scaffolds, and cell viability and cell behavior were evaluated by the MTT test and scanning electron microscopy. Results: The results of histological and biomechanical tests showed the complete removal of cells with the preservation of the extracellular matrix. The results of cell culture on scaffolds also showed that optical absorption in scaffolds containing cells increased over time. Conclusions: In general, the decellularized scaffold in this study did not undergo significant structural changes in the tendon tissue. The interaction between hAd-MSCs and the decellularized scaffold revealed that the scaffold was somehow suitable for cell culture. However, it needs to be more investigated for use in the treatment of tendon injuries of the athletes.

DSC and histological analysis of decellularized porcine biograft

Different types of biological grafts are useful for the replacement of infected prostheses in vascular surgery. In cases, when autologous grafts or homografts are not available, xenografts can be an alternative choice. To reduce immunogenicity, decellularization of the xenogeneic graft is necessary. Our goal was to determine the long-lasting stability of our decellularized porcine vascular grafts, in the mirror of differential scanning calorimetry (DSC) and routine histological examination. In the first experiment, light and electron microscopic confirmation of adequate decellularization of our used protocol was assessed. In the second series, DSC measurements were performed both on native and on treated samples; thereafter, histological evaluation of the analyzed samples was performed. Light and electron microscopy evaluation showed complete removal of cells and preserved connective tissue fiber structure after decellularization in the first series. In the second part of our investigation, DSC measurements demonstrated reversible change in the structure of elastic fibers and irreversible change in the structure of collagen fiber structure supported by histological findings. Our examination strengthens the earlier observations that decellularized biological grafts can tend to late aneurysmatic degeneration, due to their weakened fiber structure, besides the known immunological circumstances.

Decellularized human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

The shortage of donor corneas as well as the limitations of tissue substitutes leads to the necessity to develop alternative materials for ocular surface reconstruction. Corneal surface substitutes must fulfill specific requirements such as high transparency, low immunogenicity, and mechanical stability combined with elasticity. This in vitro study evaluates a decellularized matrix secreted from human corneal fibroblasts (HCF) as an alternative material for ocular surface reconstruction. HCF from human donors were cultivated with the supplementation of vitamin C to form a stable and thick matrix. Furthermore, due to enhanced cultivation time, a three-dimensional like multilayered construct which partly mimics the complex structure of the corneal stroma could be generated. The formed human cell-based matrices (so-called cell sheets [CS]) were subsequently decellularized. The complete cell removal, collagen content, ultrastructure, and cell toxicity of the decellularized CS (DCS) as well as biomechanical properties were analyzed. Surgical feasibility was tested on enucleated porcine eyes. After decellularization and sterilization, a transparent, thick, cell free, and sterile tissue substitute resulted, which allowed expansion of limbal epithelial stem cells with no signs of cytotoxicity, and good surgical feasibility. DCS seem to be a promising new corneal tissue substitute derived from human cells without the limitation of donor material; however, future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.

Effect of Decellularization Parameters on the Efficient Production of Kidney Bioscaffolds.

The most preferred decellularization technique in creating bioscaffolds for complex organs such as kidneys is through detergent perfusion. Detergents such as sodium dodecyl sulfate (SDS) flow to the kidneys to remove cells but using this technique alone requires long treatment times. Coupling this technique with sonication treatment decreases decellularization time but may cause damages in the microarchitecture of the kidney. This study evaluated the effects of decellularization parameters specifically SDS concentration (0.25%, 0.625%, and 1.0%wt/vol), flowrate (15, 30, and 45mL/min), and sonicator power (0, 60, and 120W) on the length of time needed to produce acellular and intact bioscaffolds. Decellularization was carried out by perfusing SDS to the renal artery of the cadaveric porcine kidney while exposed to sonication treatment. Results showed that a significant decrease in decellularization time was observed in producing acellular scaffold when perfusion decellularization was coupled with sonication. In addition, SDS concentration, SDS flowrate, and sonicator power had significant effects on the decellularization time while only sonicator power had a significant effect on the microarchitecture integrity of the scaffold. Lastly, H&E results showed that the produced bioscaffold showed complete cell removal with only minimal to moderate disruptions on the microarchitecture of the kidney.

Preparation of Decellularized Bovine Tendon Scaffold and Evaluation of Its Interaction with Adipose Tissue-Derived Mesenchymal Stem Cells

Background: Tissue engineering is the science of tissue design and one of the main branches of regenerative medicine that aims to improve and repair tissue injuries. Objectives: This study aimed to decellularize the tissue of bovine Achilles tendon and create a natural 3D scaffold. Then, the interaction of human adipose tissue-derived mesenchymal stem cells (hAd-MSCs) with this 3D scaffold was evaluated for use in tendon injuries. Methods: The bovine Achilles tendon was obtained from a slaughterhouse and decellularized by the combination of ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS). Histological and biomechanical tests were used to evaluate the quality of decellularized scaffolds. Adipose-derived mesenchymal stem cells were cultured on scaffolds, and cell viability and cell behavior were evaluated by the MTT test and scanning electron microscopy. Results: The results of histological and biomechanical tests showed the complete removal of cells with the preservation of the extracellular matrix. The results of cell culture on scaffolds also showed that optical absorption in scaffolds containing cells increased over time. Conclusions: In general, the decellularized scaffold in this study did not undergo significant structural changes in the tendon tissue. The interaction between hAd-MSCs and the decellularized scaffold revealed that the scaffold was somehow suitable for cell culture. However, it needs to be more investigated for use in the treatment of tendon injuries of the athletes.

Rapid and Detergent-Free Decellularization of Cartilage.

The use of decellularized tissues or organs as cell culture scaffolds has proven to be a promising approach for tissue engineering and regenerative medicine, as these decellularized tissues can provide the instructive niche for cell differentiation and functions. Cartilage is a largely avascular tissue with limited regenerative capacity. Lesions caused by arthritis can lead to severe cartilage degeneration. Previous studies have indicated that decellularized cartilage can be used as scaffolds that support the chondrogenic differentiation of adult stem cells. However, these decellularization protocols all require the use of denaturing agents, such as high salt and detergents, that lead to the artifactual disruption of the chemical and physical integrity of the tissue microenvironment. Here, we established a new decellularization method for cartilage, through a combined effect of freezing-thawing, sectioning, and sonication in water. This protocol achieved the complete removal of cells within minutes, instead of hours or days required by existing procedures, and does not use any detergent. The resulting decellularized cartilage preserved the native ultrastructure and biochemical contents, including glycosaminoglycans, which is typically depleted by traditional decellularization methods. Human mesenchymal stem cells could readily adhere onto the decellularized cartilage. Together, this work unveils a simple new method for decellularizing cartilage, which will be useful in studying how tissue microenvironment supports chondrocyte growth and functions. Impact statement In this study, we develop a simple, fast cartilage decellularization method that does not require any detergent, so that the decellularized cartilage chemistry is preserved. Traditional detergent-based decellularization removes the tissue biochemical contents (i.e., glycosaminoglycans). In this new water decellularization protocol, the biochemical contents of cartilage can be preserved. This allows the study of biochemistry and physical content in extracellular matrix as a whole, and this protocol would definitely be useful for studying the effect of tissue microenvironment in supporting chondrocyte growth and functions.

Magnetic Immobilization of Pichia pastoris Cells for the Production of Recombinant Human Serum Albumin.

Magnetic immobilization as a novel technique was used to immobilize recombinant Pichia pastoris (GS115 Albumin) cells to produce human serum albumin (HSA). In this regard, magnetic nanoparticles (MNPs) coated with amino propyl triethoxy silane (APTES) were synthesized. P. pastoris cells were decorated with MNPs via nonspecific interactions. Decorated cells were magneto-responsible and easily harvested by applying an external magnetic field. The efficiency of magnetic immobilization (Ei) for cell removal was in direct relation with the MNP concentration and time of exposure to the magnetic field. By increasing the nanoparticles concentration, cells were harvested in a shorter period. Complete cell removal (Ei ≈ 100) was achieved in ≥0.5 mg/mL of MNPs in just 30 s. HSA is produced in an extremely high cell density (OD ~20) and it is the first time that magnetic immobilization was successfully employed for harvesting such a thick cell suspension. After 5 days of induction the cells, which were immobilized with 0.25 to 1 mg/mL of nanoparticles, showed an increased potency for recombinant HSA production. The largest increase in HSA production (38.1%) was achieved in the cells that were immobilized with 0.5 mg/mL of nanoparticles. These results can be considered as a novel approach for further developments in the P. pastoris-based system.

Application of supercritical fluids for complete decellularization of porcine cartilage

Porcine cartilage was ground by cryomill (Retch, Germany) at –196°C. A fraction of cartilage microparticles (CMP) of size 100-250 µm was isolated. CMP was decellularized at room temperature with periodic mixing in 3 shifts of 0.1% sodium dodecyl sulfate buffer solution, containing an increasing concentration (1, 2 and 3%) of Triton X100. CMP treatment in a supercritical CO2 (sc-CO2) atmosphere was carried out at a pressure of 300 bar, T = 35°C, with a flow rate of sc-CO2 of 2.5 ±0.5 ml/min for 8-24 hours using RESS-SAS equipment (Waters Corporation, USA). Ethanol (96%) at a concentration of 10% was used as a polarity modifier. The degree of decellularization of CMP was assessed by histological methods (stained by hematoxylin and eosin) and by detection of the residual amount of DNA in samples using DNA-binding fluorescent dye DAPI. In the case of treatment with the detergents only and detergents after sc-CO2, the required degree of decellularization of CMP was not achieved. Histological analysis of the samples has shown that only a partial release of chondrocytes occurs. CMP treatment by detergents followed by sc-CO2 was more effective. Complete removal of cells can be achieved if the cartilage is first treated with surfactant, and then CO2. When ethanol was added as a polarity modifier, histological studies confirm that non-disrupted cells were almost completely absent and study with the DAPI dye has shown that more than 90% of CMP samples were completely free of DNA or contained only single whole cells.To achieve the highest possible degree of decellularization, the treatment of cartilage microparticles should be carried out first with detergent solutions followed by exposure to sc-CO2. The introduction of a polarity modifier (ethanol) at a concentration of 10% has a positive effect on the degree of decellularization and in combination with lengthy treatment time allows to reach complete decellularization of cartilage tissue.

Development and Characterization of an Acellular Porcine Small Intestine Submucosa Scaffold for Use in Corneal Epithelium Tissue Engineering

ABSTRACTPurpose: To produce an acellular small intestine submucosa (SIS) that would be a suitable scaffold for corneal epithelium tissue engineering.Methods: The SIS was decellularized by immersion in 0.1% (wt/vol) sodium dodecyl sulfate (SDS). The efficacy of acellularization was confirmed by histological observation and DNA quantification. The mechanical properties were evaluated by uniaxial tensile testing. ELISA was performed to assess the growth factor contents. The cytotoxicity of SIS scaffolds and extracts to rabbit corneal epithelial cells was determined by CCK-8 assay. We also investigated the inflammatory reaction of SIS implanted subcutaneously in a rat. The biocompatibility was studied by rabbit interlamellar corneal transplantation and reseeding assay with cornea-derived cells. Immunofluorescent staining was used to detect the expression of CK3, ZO-1 and K13.Results: Histological analyses showed that complete cell removal was achieved, and the DNA quantity, which reflects the presence of cellular materials, was significantly diminished in acellular SIS. Collagen fibers were properly preserved and appeared in an orderly fashion. The tissue structure, the mechanical properties and the growth factor contents within the acellular SIS were well retained. The CCK8 assay demonstrated that the acellular SIS scaffolds and extracts had no cytotoxicity to rabbit corneal epithelial cells. There was no sign that an immune reaction occurred with acellular SIS implanted subcutaneously in a rat. In fact, in vivo implantation to rabbit interlamellar stromal pockets showed good biocompatibility. We also observed that clusters of rabbit corneal epithelial cells were growing well on the surface of the SIS in vitro and the distinctive CK3, ZO-1 for corneal epithelial cells was detected.Conclusions: The decellularized SIS retained the major structural components. The matrix is biocompatible with cornea-derived cells and might be a suitable scaffold for corneal epithelium tissue engineering.

Integrated Clarification and Purification of Monoclonal Antibodies by Membrane Based Separation of Aqueous Two-Phase Systems.

Therapeutic monoclonal antibodies (mAb) are used for the treatment of numerous serious diseases, which have led to an increasing demand over the last decades. Increased cell density and mAb titer of the cultivation broth lead to great challenges for the subsequent clarification and capture operations in the downstream process. As an alternative approach to the conventional downstream process, a selective mAb extraction via an aqueous two-phase system (ATPS) directly from the cultivation broth of a mAb producing industrial relevant chinese hamster ovary (CHO) cell line was investigated. An efficient purification of the mAb was accomplished by the ATPS composition. The phase separation was realized by a newly developed membrane based phase separator. Moreover, a complete cell removal was integrated into this process by the used membrane. A selectivity between both phases was achieved by membrane modification. Yields up to 93% in the light phase and removal of process related impurities were obtained after aqueous two-phase extraction (ATPE). Phase separation performance as well as contact angles on the membrane were characterized for different ATPS. ATPE directly from the cultivation broth in combination with the new membrane based phase separation led to a mAb yield of 78% with a simultaneous reduction of deoxyribonucleic acid (DNA) and host cell protein (HCP) load.

Abstract 2102: Assessing glioma-protein inhibition in sunitinib-resistance using a 3-dimentional tissue-engineered renal cell carcinoma patient derived model

Abstract Objective: To identify the role of glioma-proteins (Gli) in sunitinib-resistant metastatic renal cell carcinoma and the effect of Gli-inhibition using a 3D tissue-engineered patient-derived model. Background: Renal cell carcinoma (RCC) is the 6th most common malignancy with 2.3% annual increase in Canada. Metastatic RCC (mRCC) patients are treated with anti-angiogenic tyrosine kinase inhibitors (TKIs, sunitinib) as the first-line of treatment because mRCC tumor is heavily depend on VEGF pathway. Therefore, identifying the factor(s) responsible for sunitinib-resistance development and disease advancement in mRCC is imperative. Cancer reflect aspects of developmental patterning and the Hedgehog family proteins are known to play a central role. Among other proteins in the family, the inappropriate activation and/or maintenance of Sonic hedgehog (Shh)-Gli pathway results in various tumors. However, the potential of Shh-Gli pathway inhibitors in sunitinib-resistant mRCC have never been tested. To screen for effective Gli-inhibitors, we will use a 3D tissue-engineered patient-derived tumor model utilizing decellularized kidneys as matrices. Organ decellularization is the process of complete cell removal from the organ and maximum preservation of the extracellular matrix (ECM) microarchitecture, leaving some reported growth factors in which tumor cells from patients can be reseeded, regenerating the organ. This 3D-model is an economic and reliable platform to screen the Gli-inhibitors, and identify tumour-specific optimal therapies. Results: We established sunitinib-conditioned cell-line (Caki-1DC) from the parental cell-line (Caki-1WT) and a mouse model of acquired sunitinib-resistance. Proteomics and microarray results from these two models showed transcriptional and translational changes between the sensitive and resistant phenotypes, particularly in the Hedgehog signalling. In Caki-1DC, an upregulation of Gli-2 was observed, which could be abrogated with Shh pathway inhibitor, cyclopamine. Furthermore, in our decellularization and reseeding techniques of kidney, we have assessed cell viability for up to a month exhibiting adequate cell viability. This 3D platform will be used to assess the effectiveness of Gli-inhibitors. Conclusion: Our data from in vitro and in vivo models established Shh-Gli pathway activation in sunitinib-resistant mRCC. Testing the effectiveness of Gli-inhibitors will help tailor drugs for the patients to improve progression free survival. The 3D patient-derived mRCC platform will enable us to generate many in vitro avatars to accurately recreate the tumor of a patient, and simultaneously screen for suitable drugs and dose for the patient. The identification of the Shh-Gli driver pathway and the effective therapy will translate into improving patient’s quality of life and survival. Citation Format: Ninadh M. D'Costa, Zheng Tan, Claudia Chavez-Munoz, Alan So. Assessing glioma-protein inhibition in sunitinib-resistance using a 3-dimentional tissue-engineered renal cell carcinoma patient derived model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2102.

Development and Characterization of Acellular Caprine Choncal Cartilage Matrix for Tissue Engineering Applications.

Because of poor regenerative capabilities of cartilage, reconstruction of similar rigidity and flexibility is difficult, challenging, and restricted. The aim of the present investigation was to develop cost-effective acellular xenogeneic biomaterial as cartilage substitution. Two novel biometrics have been developed using different chemical processes (Na-deoxycholate + SDS and GndHCl + NaOH) to decellularize caprine (goat) ear cartilage and further extensively characterized before preclinical investigation. Complete cell removal was ascertained by hematoxylin and eosin staining followed by DNA estimation. No adverse effect on extracellular matrix (ECM) was found by quantifying collagen and sulfated glycosaminoglycans (sGAG) content as well as collagen, sGAG and elastin staining. Results showed no drastic changes in ECM structure apart from desired sGAG loss. Scanning electron microscopy images confirmed cellular loss and unaltered orientation. Nano-indentation study on cartilage matrices indicated interesting output showing better results among decellularized groups. Increased elastic modulus and hardness indicated better stiffness and more active energy dissipation mechanism due to decellularization. Fluid uptake and retention property remained unchanged after decellularization as analyzed by swelling behavior study. Additionally, acellular materials were confirmed to be nonreactive and nonhemolytic as assessed by in vitro hemocompatibility study. In vivo study (up to 3 months) on rabbits showed no symptoms of graft rejection/ tissue necrosis, established through postoperative histology and biochemical analyses of tissue explants. With regard to size, shape, biomechanics, source of origin and nonimmunogenic properties, these developed materials can play versatile role in biomedical/ clinical applications and pave a new insight as alternatives in cartilage reconstruction.