Two-step Digestion Method Research Articles

In Vitro Culture of Ovine Spermatogonial Stem Cells: Effects of Grape Seed Extracts and Vitamin C

Background: Spermatogonial stem cells (SSCs) transmit genetic information to the next generation. For the successful transplantation of these cells, the culture medium of these cells must be optimized. Objectives: This study investigated the natural antioxidant of grape seed extract (GSE) in comparison to vitamin C in the culture medium of SSCs. Methods: A Soxhlet extractor was used to prepare ethanolic and acetonic extracts, and the Clevenger apparatus was used to prepare aqueous extracts of grape seeds. Then, their antioxidant capacities were determined using the DPPH method. The SSCs were extracted from lamb testicular tissue by a two-step digestion method, and different concentrations of GSE and vitamin C were investigated for survival, colony formation, and expression of apoptotic-related genes. Results: The results showed that the acetonic extract in the concentration of 400 µg/mL showed about 90% antioxidant properties based on the DPPH test; nevertheless, the aqueous and ethanolic extracts had only 50% of their antioxidant properties at this concentration. The acetonic extracts significantly decreased the viability of SSCs without any colony formation when used in a culture medium. The highest survival rate was obtained from the ethanolic extracts of grape seeds at the concentration of 50 µg/mL, and a significant difference was obtained with ethanolic extracts (100 to 1000 µg/mL) (P < 0.05). The viability of SSCs treated with vitamin C (50 µg/mL) was significantly higher (P < 0.05) than the control and 5 - 25 µg/mL of vitamin C groups. Conclusions: Aqueous, ethanolic, and acetonic extracts of grape seeds should not be used in the culture of SSCs. However, the use of 25 - 50 µg/mL of vitamin C is recommended in the culture of SSCs.

Facilitating microplastic quantification through the introduction of a cellulose dissolution step prior to oxidation: Proof-of-concept and demonstration using diverse samples from the Inner Oslofjord, Norway

Identifying and quantifying microplastic in marine samples can be facilitated by removing natural organic matter (NOM). Cellulosic material, like chitin, however, are a type of NOM that is resistant to chemical digestion, and difficult to eliminate from samples. To address this, a two-step digestion method was developed to remove or reduce cellulosic materials in diverse marine media. This method was applied to reference microplastics, reference cellulosic materials, and diverse marine samples from the Inner Oslofjord Norway. This included plankton, seabed sediments near a water treatment plant and driftline sand. The method was developed and tested for plastic particles >45 μm. The first-step was to pre-dissolve cellulosic materials using a mixture of urea:thiourea:NaOH. This was followed by an oxidative digestion step, here using H2O2 and NaOH. Most reference plastics were unaffected, except minor effects for PET and nylon. After sufficient repetitions, cellulosic materials in both reference and marine samples were largely removed. This method was compared to other digestion methods used for microplastic quantification, including single-step oxidation, alkaline treatment, acid treatment and enzymatic treatment. The results indicate that the pre-dissolution step greatly facilitates NOM and cellulosic material digestion for the purpose of microplastic quantification.

Effects of camptothecin on autophagy of human primary keratinocytes

Objective To evaluate the effect of camptothecin on the autophagy of human primary keratinocytes (HPKs) . Methods HPKs were isolated from foreskin tissues of healthy males by a two-step digestion method, and the third-passage cells were used for following experiments. These HPKs were randomly divided into several groups: experimental groups treated with camptothecin at concentrations of 200 nmol/L, 2 and 6 μmol/L separately, and a control group treated with 0.1% dimethyl sulfoxide (DMSO) . After 24- and 48-hour treatment, cell counting kit-8 (CCK-8) assay was conducted to estimate the proliferative activity of HPKs. Flow cytometry was performed to detect cell apoptosis after 24-hour treatment, and Western blot analysis to measure the expression of autophagy-associated proteins such as microtubule-associated protein 1 light chain-3 (LC3) and p62. Some other HPKs were treated with 2 μmol/L camptothecin for 24 hours. Indirect immunofluorescence assay was performed to observe changes in LC3 expression, and transmission electron microscopy to observe the ultrastructure of autophagosomes, so as to further validate the inductive effect of camptothecin on autophagy. Results The inhibitory effect of camptothecin on the proliferation of HPKs gradually increased along with the increase of camptothecin concentration, and there was a significant difference in the proliferation inhibition rates among the experimental groups and control group at 24 hours (F = 152.9, P 0.05) . At 48 hours, there was still a significant difference in the proliferation inhibition rates among the experimental groups and control group (F = 123.8, P < 0.01) , and all the experimental groups showed increased proliferation inhibition rates compared with the control group (all P < 0.01) . At 24 hours, the cell apoptosis rates also significantly differed among the control group, 200-nmol/L, 2-μmol/L and 6-μmol/L camptothecin groups (2.30% ± 1.68%, 15.90% ± 2.14%, 29.33% ± 3.51%, 35.28% ± 3.05%, respectively; F = 89.57, P < 0.01) , and all the three experimental groups showed higher cell apoptosis rates compared with the control group (all P < 0.01) . After 24-hour treatment with 2 or 6 μmol/L camptothecin, the protein expression of LC3Ⅱ were significantly up-regulated, but the protein expression of p62 was significantly down-regulated. Indirect immunofluorescence assay showed that the percentage of autophagosome-positive cells was significantly higher in the 2-μmol/L camptothecin group than in the control group (60.16% ± 8.78% vs. 38.96% ± 13.12%, t = 3.003, P < 0.05) . After 24-hour treatment with 2 μmol/L camptothecin, autophagosomes and autolysosomes were observed in HPKs with a transmission electron microscope. Conclusion Camptothecin at concentrations of 2 and 6 μmol/L can increase the autophagy level in HPKs, meanwhile, inhibit cell proliferation and induce cell apoptosis. Key words: Psoriasis; Camptothecin; Autophagy; Cell proliferation; Apoptosis; Keratinocytes

Appropriate cyclic tensile strain promotes biological changes of cranial base synchondrosis chondrocytes.

This study was designed to clarify biological changes of cranial base synchondrosis chondrocytes (CBSCs) upon cyclic tensile strain (CTS) loading which simulated orthopaedic mechanical protraction on cranial base synchondroses (CBS). A two-step digestion method was used to isolate CBSCs obtained from 1-week-old Sprague Dawley rats. Immunohistochemical staining of type II collagen and Sox9 was conducted to identify chondrocytes. A CTS of 1Hz and 10% elongation was applied to the second passage of CBSCs by FX-5000™ Tension System for 24hours. The control group kept static at the same time. The expression levels of extracellular matrix (Acan, Col1a1, Col2a1 and Col10a1) and key regulatory factors (Sox9, Ihh and PTHrP) were detected by quantitative real-time RT-PCR. Positive staining of type II collagen and Sox9 was detected in the isolated CBSCs. The relative expression level of Acan, Col2a1, Col10a1, Sox9 and Ihh in the CTS-loading group was 1.85-fold, 2.19-fold, 1.53-fold, 6.62-fold, and 1.39-fold, respectively, as much as that in the control group, which had statistical significance (P<.05). There was no statistical difference (P>.05) in the expression of Col1a1 and PTHrP. A CTS of 1Hz and 10% elongation for 24hours had positive effects on chondrocyte proliferation, phenotype maintenance and cartilage matrix synthesis.

Establishment of rat integrated discrete multiple organ cell culture (IdMOC) model

To establish the integrated discrete multiple organ cell culture (IdMOC) system. Rat primary cell of hepatocyte, nephrocyte, cardiomyocytes, alveolar macrophage, dermal fibroblasts were isolated by collagenase digestion, separation of bronchial lavage, two-step digestion method and cultured respectively, with monolayer culture. To establish the integrated discrete multiple organ cell culture (IdMOC) system, glass slides of five different cells were used to the same dish with 10% FBS DMEM medium cultured 7d, using MTT comparison primary cells cultured alone and cocultured when growth. Established rat hepatocytes, renal cell, cardiomyocyte, alveolar macrophages, dermal fibroblasts separation method was stable, cell separation survival rate was about 90.0%. Hepatocytes separation survival rate 90.3% ,renal cell separation survival rate 91.9%, cardiomyocyte separation survival rate 93.0% and beating rate indifference curve among 3d-15d, alveolar macrophages cell separation survival rate 90.8%, dermal fibroblasts cell separation survival rate 92.7%. Five primary cells multiple organ cells coculture showed cocultured cell growth proliferation well, cultured alone and cocultured cells growth curve basic coincide. Established rat multiple organ cell co-culture is successful.

Effects of baking on protein digestibility of organic spelt products determined by two in vitro digestion methods

Consumption of organic foods is steadily increasing because it is believed to be healthier than conventional foods. This study was designed to investigate protein digestibility of organic spelt bread, biscuit, cookie and muffin in comparison to their corresponding normal wheat products. Three types of fermented bread products namely, yeast leavened, sour and yeast/sour dough were evaluated. Protein digestibility was assessed based on two methods, three-enzyme one-step and two-enzyme two-step digestion in vitro. The one-step digestion method produced results that were comparable with in vivo (rat) methods whereas the two-step digestion method was more reliable in determining differences among the examined wheat products. Organic spelt used in the present study was comparable to common wheat in protein content averaging 15.4 g/100 g dry matter. Slight differences were observed between organic spelt and common wheat products in protein digestibility determined by the two digestion methods. However, significant differences were found among each wheat products. In general, after baking protein digestion was significantly increased. Spelt and common wheat bread products had similar protein digestibility within each type of bread with sour dough breads had the highest protein digestibility. Biscuit, cookie and muffin products possessed lower protein digestibility than breads. In general, variations in protein digestibility due to baking were more noticeable than that found between the two wheats.

Prevention of primary nonfunction of canine islet autografts by treatment with pravastatin.

Nonspecific inflammation is the primary cause of early islet graft loss. We have shown in mice that pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, prevents primary nonfunction of islet isografts by reducing inflammatory reactions at the graft site. This study was designed to test the effectiveness of this agent in a large animal model, dogs, by transplanting autologous islets. After total pancreatectomy, islets were isolated by using a two-step digestion method, followed by discontinuous gradient centrifugation on EuroFicoll. A known number of freshly isolated islets were immediately transplanted back into the same dog via the portal vein. First, we determined the minimal islet number required to reverse diabetes by transplanting 3,000-10,000 IEQ/kg with no additional treatment. The number was found to be 4,000 IEQ/kg, and islets less than 4,000 IEQ/kg consistently failed. To test the effect of pravastatin, 3,000 IEQ/kg were transplanted into dogs that either received no further treatment or were treated daily with 20 mg/kg of pravastatin from days -2 to 14. Without pravastatin, this number of islets lowered blood glucose only transiently, and all four of these dogs became hyperglycemic within 1 week. In contrast, four of the five dogs treated with pravastatin became normoglycemic (<150 mg/dL) and maintained this level during the observation period of 12 weeks (P<0.05). Postprandial plasma glucose and insulin levels returned to normal, and K values of intravenous glucose tolerance tests were significantly higher in pravastatin-treated dogs than in controls (P<0.04 at week 2 and P<0.01 at week 4). Peritransplant pravastatin treatment reduced the number of autologous islets required to reverse diabetes in totally pancreatectomized dogs. These results suggest that pravastatin may also facilitate better islet graft survival and function in clinical transplantation.

Increased islet viability by addition of beraprost sodium to collagenase solution.

The digestion of pancreatic tissue with collagenase is an essential part of the islet isolation procedure. However, the process exposes islets to various types of harmful factors, including collagenase contaminants, enzymes released from the acinar cells, warm ischemia, and mechanical agitation. Nitrogen oxide production and cytokine release may also contribute to islet cell damage. Protection of islets from such damage would improve the islet yield, survival, and function. Beraprost sodium (BPS) is a prostaglandin I2 analogue, is stable in aqueous solution, and has a cytoprotective effect on various types of cells. BPS has been shown to improve the yield and function of cryopreserved and/or cultured islets. These findings prompted us to examine its cytoprotective effect on islets during the islet isolation process. Canine islets were isolated by means of a two-step digestion method and purified on Euro-Ficoll density gradient solutions (the procedure used for human islets). BPS at a concentration of 100 nM was added to the collagenase solution. After purification, the islet yield was 434,561 +/- 35.691 islet number expressed as 150 microm equivalent size (IEQ)/pancreas or 8,799 +/- 345 IEQ/g of pancreas in the BPS group and 349,987 +/- 52,887 IEQ/pancreas or 7,998 +/-1610 IEQ/g of pancreas in the control group (n = 8, each). The percent viability was 88.5 +/- 0.7% in the BPS group and 82.0 +/-0.9% in the control group (P < 0.01). Therefore, the recovery of viable islets (calculated by islet number x % viability) was 384,586 +/- 46,804 IEQ/pancreas (7,743 IEQ/g) in the BPS group and 286,989 +/- 43,367 IEQ/pancreas (6,558 IEQ/g) in the control group (P < 0.02). After culture, significantly higher numbers of islets were also recovered in the BPS group than in the control group. The islet insulin content was significantly higher in the BPS group than controls (237.8 +/- 38.5 versus 92.3 +/- 25.6 microU/IEQ; P < 0.02), although islets of both groups responded with high stimulation indices (>6). These results indicate that the addition of BPS to the collagenase solution increases the recovery of viable islets, and improves beta cell function.

Improved quality and yield of islets isolated from human pancreata using a two-step digestion method.

A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.

Re-evaluation of donor factors affecting islet isolation from human pancreas using a two-step digestion method

Re-evaluation of donor factors affecting islet isolation from human pancreas using a two-step digestion method

Canine islet isolation using a two-step digestion method and the influence of stationary collagenase incubation of the pancreas

Canine islet isolation using a two-step digestion method and the influence of stationary collagenase incubation of the pancreas

Canine islet isolation using a two-step digestion method and influence of stationary collagenase preincubation of the pancreas

Canine islet isolation using a two-step digestion method and influence of stationary collagenase preincubation of the pancreas