Enzymatic Isolation Research Articles

The Development of Facial Lipofilling from a Historical Point of View.

Lipofilling, the transplantation of adipose tissue, has already been used since the end of the 19th century. For decades, lipofilling was used to restore loss of volume due to aging, trauma, or congenital defects. Later on, the indications for the use of lipofilling expanded by treating aged skin, scars, and improving wound healing. The expansion was caused by the discovery of adipose derived stromal cells (ASCs) in adipose tissue and the development of very fine harvesting and injection cannulas which made it possible to inject small adipose tissue particles in small volume areas, such as the face. ASCs are multipotent stromal cells which reside in the stromal vascular fraction (SVF) of adipose tissue and are able to differentiate in multiple cell lineages and secrete a plurality of growth factors with regenerative potentials. The discovery of ASCs led toward more experimental cell-based therapies, that is, ASCs or SVF isolated by means of enzymatic isolation procedures. Later on, enzymatic isolation procedures were forbidden in many countries by legislation and were replaced by mechanical isolation procedures, such as the Nanofat and Fractionation of Adipose Tissue (FAT) procedures. The Nanofat procedure has been extensively investigated, especially as treatment for skin rejuvenation in the face. Though, substantial evidence is lacking for using facial lipofilling or any therapeutic component, that is, ASCs or SVF for skin rejuvenation to date. In contrast, facial lipofilling to restore loss of volume seems to be promising.

Bile Acid Transporters Are Expressed and Heterogeneously Distributed in Rat Bile Ducts.

Background/AimsCholangiocytes are capable of reabsorbing bile salts from bile, but the pathophysiological significance of this process is unclear. To this end, we detected the expression and distribution of bile acid transport proteins in cholangiocytes from normal rat liver and analyzed the possible pathophysiological significance.MethodsBile duct tissues of Sprague-Dawley rats were isolated by enzymatic digestion and mechanical isolation, and then divided into large and small bile duct tissues. Immunohistochemistry, real-time polymerase chain reaction and Western blotting were used to determine the expression of the apical sodium-dependent bile acid transporter (ASBT), ileal bile acid binding protein (IBABP), and basolateral organic solute transporter α (Ostα) in the biliary tract system of rats. Differences in the expression and distribution of these proteins were analyzed.ResultsIn cholangiocytes, ASBT and IBABP were mainly expressed in cholangiocytes of the large bile ducts, in which the expression of both was significantly higher than that in the small ducts (p<0.05). Ostα was simultaneously expressed in cholangiocytes of both the large and small bile ducts, showing no significant difference in expression between the two groups of bile ducts (p>0.05).ConclusionsBile acid transporters are expressed and heterogeneously distributed in rat bile ducts, indicating that bile acid reabsorption by cholangiocytes might mainly occur in the large bile ducts. These findings may help explore the physiology of bile ducts and the pathogenesis of various cholangiopathies.

Comparison study of organosolv pretreatment on hybrid pennisetum for enzymatic saccharification and lignin isolation

Organosolv pretreatment is a promising method for enhancing the enzymatic hydrolysis of lignocellulosic biomass and the production of value-added chemicals. However, information on solvents and their interactions with biomass—specifically, their effects on residues and isolated lignin—is limited, hindering the effective utilization of biomass. Herein, the effects of organosolvs (including γ-valerolactone (GVL), tetrahydrofurfuryl alcohol (THFA), ethanol and acetone) on enzymatic saccharification and lignin isolation of hybrid Pennisetum (HP) under 100 °C for 2 h were investigated. Compared with water, organic solvents can significantly enhance delignification, reducing the crystallinity of cellulose, thereby improving the enzymatic digestibility and lignin recovery. The enzymatic digestibility of substrates reached 87.5% (about 80.8% of the raw HP glucan was recovered) for the THFA pretreatment, which was 38.7% higher than water-treated material and 131.5% higher than untreated raw material. Moreover, Hansen solubility parameters (HSP) was used to evaluate the effects of solvents, which showed a strong correlation between lignin removal and the molecular weight distribution of isolated lignins, suggesting that HSP can aid in finding appropriate organic solvents for pretreatment. Finally, the chemical structures and thermal properties of solid residues and precipitated lignins were analyzed via SEM, XRD, FTIR and thermogravimetric analysis.

TRPV2 is Crucial for the Development of Intercalated Discs in Mouse Hearts

TRP vanilloid family type 2 channel (TRPV2) is a key molecule in stretch-induced Ca2+ response of cardiomyocytes, and is highly localized mammalian intercalated discs, which is cyclically stretched within working hearts. Elimination of TRPV2 from hearts of adult mice led to a rapid and severe decline in cardiac pump function through the disorganization of intercalated discs. Gene expression of intercalated disc proteins was also acutely downregulated by TRPV2-elimination. Here, we show the involvement of TRPV2 in the development of intercalated disc. In neonatal cardiomyocytes, the hypotonicity- and stretch- induced Ca2+ responses were observed within 12 hours of culture after enzymatic isolation. These responses were decreased by tranilast, an inhibitor of TRPV2, suggesting involvement of TRPV2 channel. These myocytes show the robust expression of TRPV2, without any expression of connexin 43 and N-cadherin. After 48 hours, we can observe the localization of connexin 43 and N-cadherin in the longitudinal cell-edges interface forming intercalated disc. In contrast, TRPV2-deficient myocytes showed no Ca2+ response to the hypotonicity- and stretch-stimulation at 12 hours after isolation. These myocytes formed no intercalated disc-like connection between neighbouring cells. In addition, sarcomeric actin and synchronous beating were not observed in these myocytes, even in 72 hours of culture time. Loss of TRPV2 from the hearts of juvenile mice resulted in disassembly of the intercalated disc structure, even in adult age. The TRPV2-deficient adult mice showed cardiac dysfunction with conduction defects. These observations suggest that TRPV2 is critical for the developments of the intercalated disc of myocytes.

Safe enzymatic isolation and expansion of mesenchymal stromal cells from cryopreserved umbilical cord tissue

Safe enzymatic isolation and expansion of mesenchymal stromal cells from cryopreserved umbilical cord tissue

Enzymatic Isolation, Amplification and Characterization of Dental Pulp Stem Cells

The dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). The preferred approach to DPSC isolation is enzymatic digestion. However, the duration of the enzymatic activity is crucial. The purpose of this study was to isolate the DPSC populations using this method, characterize their biological properties and proliferation capacity, and to determine their ability to differentiate into mature cells. Before enzymatic digestion using 0.05% trypsin, we used the homogenization method in order to obtain a fine homogenate from the solid pulp tissue. The stem cells were cultivated in modified cultivation medium for mesenchymal adult progenitor cells containing 2% foetal bovine serum, growth factors and insulin-transferrin-selenium supplement. We were successfully able to isolate 10 populations of DPSCs. The vitality of DPSCs did not drop below 90 %. However, the DPSCs showed a significant decrease in the relative telomere length number with increasing passaging (P &lt; 0.05). Isolated DPSCs highly expressed the CD markers: CD29, CD44, CD90, CD13, CD73 and CD166. In contrast, CD markers CD31, CD106, CD34 and CD45 were negative or low positive. We confirmed the high osteogenic and chondrogenic potential of the isolated stem cells. Isolated DPSCs did not show signs of cell degeneration or spontaneous differentiation during the entire cultivation. In addition, we were able to shorten the enzyme activity duration, and we were the first to demonstrate trypsin as the enzyme used for the enzymatic digestion method with the viability over 90 % of isolated DPSCs using this method.

Development of An Artificial Male Germ Cell Niche Using Electrospun Poly Vinyl Alcohol/Human Serum Albumin/Gelatin Fibers.

Objective Recent achievements in stem cell biotechnology, nanotechnology and tissue engineering have led to development of novel approaches in regenerative medicine. Azoospermia is one of the challenging disorders of the reproductive system. Several efforts were made for isolation and culture of testis-derived stem cells to treat male infertility. However, tissue engineering is the best approach to mimic the three dimensional microenvironment of the testis in vitro. We investigated whether human testis-derived cells (hTCs) obtained by testicular sperm extraction (TESE) can be cultured on a homemade scaffold composed of electrospun nanofibers of homogeneous poly (vinyl alcohol)/human serum albumin/gelatin (PVA/HSA/gelatin). Materials and Methods In this experimental lab study, human TCs underwent two steps of enzymatic cell isolation and five culture passages. Nanofibrous scaffolds were characterized by scanning electron microscopy (SEM) and Fourier- transform infrared spectroscopy (FTIR). Attachment of cells onto the scaffold was shown by hematoxylin and eosin (H&E) staining and SEM. Cell viability study using MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide] assay was performed on days 7 and 14. Results Visualization by H&E staining and SEM indicated that hTCs were seeded on the scaffold. MTT test showed that the PVA/HSA/gelatin scaffold is not toxic for hTCs. ConclusionIt seems that this PVA/HSA/gelatin scaffold is supportive for growth of hTCs.

Isolation of Stromal Vascular Fraction by Fractionation of Adipose Tissue.

Adipose tissue-derived stromal cells (ASCs) are a promising candidates for cellular therapy in the field of regenerative medicine. ASCs are multipotent mesenchymal stem cell-like and reside in the stromal vascular fraction (SVF) of adipose tissue with the capacity to secrete a plethora of pro-regenerative growth factors. Future applications of ASCs may be restricted through (trans)national governmental policies that do not allow for use of nonhuman-derived (non-autologous) enzymes to isolate ASC. Besides, enzymatic isolation procedures are also time consuming. To overcome this issue, nonenzymatic isolation procedures to isolate ASCs or the SVF are being developed, such as the fractionation of adipose tissue procedure (FAT). This standardized procedure to isolate the stromal vascular fraction can be performed within 10-12min. The short procedure time allows for intraoperative isolation of 1mL of stromal vascular fraction derived from 10mL of centrifuged adipose tissue. The stromal vascular fraction mostly contains blood vessels, extracellular matrix, and ASCs. However, based on the histological stainings an interdonor variation exists which might result in different therapeutic effects. The existing interdonor variations can be addressed by histological stainings and flow cytometry.

Explorations of the optimal method for isolating oocytes from zebrafish (Danio rerio) ovary

Obtaining oocytes from the adult female zebrafish (Danio rerio) ovary has enormous importance in the studies of developmental biology, toxicology, and genetics. It is vital to establish a simple and effective approach to ensure the quantity and quality of oocytes, which will enable the success of follow-up experimental investigation finally. Usually, oocytes are separated with mechanical or enzymatic methods, however, little studies have been done with concerns about the comparative effects. The present study separated zebrafish oocytes of Stage III with five frequently used methods, including stripping, pipetting, hyaluronidase (1.6 mg/ml), collagenase (0.4 mg/ml), and trypsin (0.1%). The cell viability, oxidative stress, mitogen-activated protein kinase (MAPK) protein phosphorylation, and apoptosis levels were selected as main biomarkers to evaluate the oocytes health status. The results showed that both trypsin and hyaluronidase isolation significantly upregulated germinal vesicle breakdown (GVBD) rates and downregulated p38 MAPK activity simultaneously. GVBD rates and survival rates were decreased notably in oocytes separated by the collagenase method. Above results indicate that zebrafish oocytes in vitro are sensitive to enzymatic treatments and the enzymatic isolation is not the suitable mean for collecting zebrafish oocytes although it is time-saving. The mechanical strategy of pipetting remarkably increased the reactive oxygen species and malondialdehyde level in isolated oocytes. Interestingly, oocytes separated with stripping show less physiological and biochemical damages. Therefore, stripping isolation is comparatively recommended as the optimum method for separating and collecting numerous intact and healthy zebrafish oocytes in vitro for the subsequent developmental research.

Adipose-Based Therapies for Knee Pain-Fat or Fiction.

Regenerative cell therapies are emerging as promising treatments for numerous musculoskeletal conditions, including knee osteoarthritis (OA). Adipose-derived stem cells and possibly other adipose-based therapies have a greater chondrogenic potential than stem cells derived from bone marrow, and thus a lot of attention is being placed on them as potential regenerative agents in the treatment of knee OA. Several types of adipose-based therapies have good basic science and preclinical data supporting their translation to human therapeutic intervention. Cultured, adipose-derived stem cells appear to be good source of bioactive cells with convenient accessibility, relative abundance, and well-documented regenerative capacity. Non-culture expanded adipose-based therapy, in the forms of stromal vascular fraction and most recently micronized adipose tissue (MAT), have been utilized in patients to treat OA and other cartilage abnormalities with encouraging preliminary data. These adipose-based therapies have shown a lot of therapeutic potential; however, because of the regulatory restrictions on enzymatic isolation and cell expansion, only MAT is currently available in clinical practice in the United States. While no serious adverse reactions have been reported, adipose-derived therapies also have the potential for adverse reactions including inflammation and infection. The current review provides an update on the latest research and presents this evidence on the therapeutic potential of adipose-based therapies in the treatment of knee OA.

Cellulose nanostructures from wood waste with low input consumption

Currently, cellulose nanostructures (CNS) are obtained using pretreatments of lignocellulosic residues. In this work, different conditions in the enzymatic isolation process were studied to obtain CNS, aiming an alternative eco-friendly method. Two isolation methodologies were used: a) one submitting the residue to an alkaline pretreatment and b) other using the lignocellulosic waste directly without pretreatment. A commercial complex of enzymes (Ctec®) was used, varying the time to which the substrate was exposed to the enzymes at 12, 24 and 48 h, searching for the better condition to CNS obtaining. The application of alkaline pretreatment removed 73% of hemicellulose and 50% of lignin from wood fiber leading to a more exposed cellulose structure and increased the amount of nanocellulose produced. The obtained nanostructures were needle-shaped of approximately 70 nm wide. However, CNS of similar size and shape were received as well without pretreatment application, allowing their obtainment with lower reagent consumption through a more environmentally efficient route, as presented in the life cycle impact assessment (LCIA) where energy consumption and acidification potential were considered.

Augmentation of Dermal Wound Healing by Adipose Tissue-Derived Stromal Cells (ASC).

The skin is the largest organ of the human body and is the first line of defense against physical and biological damage. Thus, the skin is equipped to self-repair and regenerates after trauma. Skin regeneration after damage comprises a tightly spatial-temporally regulated process of wound healing that involves virtually all cell types in the skin. Wound healing features five partially overlapping stages: homeostasis, inflammation, proliferation, re-epithelization, and finally resolution or fibrosis. Dysreguled wound healing may resolve in dermal scarring. Adipose tissue is long known for its suppressive influence on dermal scarring. Cultured adipose tissue-derived stromal cells (ASCs) secrete a plethora of regenerative growth factors and immune mediators that influence processes during wound healing e.g., angiogenesis, modulation of inflammation and extracellular matrix remodeling. In clinical practice, ASCs are usually administered as part of fractionated adipose tissue i.e., as part of enzymatically isolated SVF (cellular SVF), mechanically isolated SVF (tissue SVF), or as lipograft. Enzymatic isolation of SVF obtained adipose tissue results in suspension of adipocyte-free cells (cSVF) that lack intact intercellular adhesions or connections to extracellular matrix (ECM). Mechanical isolation of SVF from adipose tissue destructs the parenchyma (adipocytes), which results in a tissue SVF (tSVF) with intact connections between cells, as well as matrix. To date, due to a lack of well-designed prospective randomized clinical trials, neither cSVF, tSVF, whole adipose tissue, or cultured ASCs can be indicated as the preferred preparation procedure prior to therapeutic administration. In this review, we present and discuss current literature regarding the different administration options to apply ASCs (i.e., cultured ASCs, cSVF, tSVF, and lipografting) to augment dermal wound healing, as well as the available indications for clinical efficacy.

Isolation of Adipose Tissue-Derived Stem Cells: Enzymatic Digestion in Combination with Mechanical Distortion to Increase Adipose Tissue-Derived Stem Cell Yield from Human Aspirated Fat.

Mesenchymal stem cells (MSCs) are of great interest due to their properties of immune modulation, tissue regeneration, and multipotent differentiation. Future developments of clinical applications, however, require a higher yield of MSCs, lower number of passages of cells in culture, and shorter time from harvest to use. Optimization and standardization of techniques for mesenchymal adipose tissue-derived stem cell isolation offers solutions to current bottlenecks as a larger amount of MSCs can be isolated. These improvements result in shorter expansion time, fewer passages, less donor material needed, and higher MSC yield. This paper describes an MSC isolation method combining enzymatic digestion with mechanic disruption. This protocol is a standardized and easy-to-implement method for reaching significantly higher MSC yields compared to conventional enzymatic isolation protocols. Based on the results presented, we hypothesize that the combined enzymatic and mechanical method increases the surface area of the adipose tissue, facilitating digestion by enzymes. This approach reduces the amount of adipose tissue and in vitro expansion time needed to reach sufficient amounts of MSCs for clinical purposes. Importantly, the method does not require increased amounts of collagenase, nor does it impair the viability or differentiability of the MSCs. Using this protocol increases MSC yield by a factor of three. As a consequence, these results indicate that the physiological concentration of MSCs in adipose tissue is higher than previously assumed. © 2018 by John Wiley & Sons, Inc.

PO-101 Inhibition of Aerobic Exercise on PKC/CaV1.2 pathway enhanced the function of vascular smooth muscle in hypertension

Objective The purpose of this study was to investigate the effects of aerobic exercise on PKC/CaV1.2 pathway in mesenteric arterial smooth muscle from spontaneously hypertensive rats (SHRs)&#x0D; Methods Twelve-week-old male normotensive Wistar–Kyoto (WKY) rats and SHRs were randomly assigned to sedentary groups (SHR-SED, WKY-SED) and exercise training groups (SHR-EX, WKY-EX). Exercise groups were performed an 8-week moderate-intensity treadmill running. After 8 weeks, vascular contractility of mesenteric arteries was measured. Vascular smooth muscle cells (VSMCs) were obtained with an enzymatic isolation method. CaV1.2 channel currents were examined by using whole-cell patch clamp recording technique. &#x0D; Results 1) Body weight and systolic blood pressure (SBP) in both WKY-EX and SHR-EX were significantly lower than those of their sedentary counterparts (both P＜0.05). Body weight in SHR-SED was remarkably lower than WKY-SED (P＜0.05), while SBP was much higher than WKY-SED (P＜0.05). 2) PDBu (PKC activator) elicited a tension increase, and Gö6976 (PKC inhibitor) induced vasodilation. Both the responses of PDBu and Gö6976 in SHR-SED were notably increased compared with WKY-SED (both P＜0.05), however, exercise training significantly suppressed these increases (both P＜0.05). 3) Nifedipine (CaV1.2 inhibitor) induced vasodilation. Response to nifedipine in SHR-SED was more sensitive than both SHR-EX and WKY-SED (both P＜0.05). 4). The current density of SHR-SED and WKY-EX exhibited an increase compared to the WKY-SED (both P＜0.05), and the current density of the SHR-EX decreased obviously in contrast with SHR-SED (P＜0.05). Besides, PDBu enlarged current density of all the groups, while Gö6976 decreased current density. The increase or decrease amplitude in SHR-SED was significantly higher than WKY-SED (both P＜0.05), whereas exercise training markedly inhibited those responses (both P＜0.05).&#x0D; Conclusions Aerobic exercise efficiently prevents the upregulation of PKC/CaV1.2 pathway in hypertension, and enhances the function of vascular smooth muscle.

Three-dimensional culture of fresh and vitrified ovarian tissue in a novel tyramine substituted hyaluronan gel

Follicle culture in matrices that allow 3-D growth may better mimic in vivo conditions by preserving follicle architecture and critical granulosa-oocyte interactions. This study explores methodology for culturing fresh and vitrified ovarian tissue. We further describe the application of a relatively new biomaterial for in vitro follicle maturation and the production of functional metaphase II oocytes. Mouse pre-antral follicle model for IVM and testing of a hyaluronan matrix. Ovaries from 12 to 14 day B6D2F1 pups were removed. Ovaries were vitrified using an ethylene glycol (EG)/DMSO protocol. Ovaries were equilibrated in chilled vitrification solutions (VS) containing 5, 10, 15 and finally 20% EG/DMSO in L-15 with 20% SSS. Ovaries were loaded on a nylon mesh and plunged in to a specially vented cryovial filled with LN2. Pre-antral follicles (FL) and ovarian follicle clusters were collected from fresh and vitrified-warmed ovaries either by mechanical isolation using needles or by enzymatic digestion in collagenase. The HA hydrogel (3 mg/ml) cross-linking was initiated by mixing with 0.03% hydrogen peroxide in a 25:1 ratio. HA gel was then quickly pipetted into the culture dish. Isolated preantral follicles or intact clusters with 4-6 follicles were rapidly seeded into the HA before gelling was completed. Follicle culture was performed in α-MEM supplemented with 5% FBS, 100 mIU/ml FSH, and ITS. Dishes were incubated at 37°C with 6% CO2. Final maturation was triggered with hCG (1.5 IU/ml) and EGF (5 ng/ml) after 12 days of growth. Outcome parameters monitored were follicle morphology, survival in culture, germinal vesicle breakdown, oocyte maturation and meiotic spindle retardance using Oosight Imaging system. Vitrified-warmed ovarian tissue was easily dissected into clusters with 4-5 intact follicles without exposure to enzyme. In contrast, enzymatic follicle isolation was more efficient and preferable for fresh tissue. With fresh ovaries, maturation rate in FL-clusters was similar to that observed with isolated follicles. Meiotic spindle retardance in ovulated oocytes from both culture types did not differ. The overall percent oocyte maturation to metaphase II was lower with vitrified tissue. HA is a natural matrix and supported follicle growth and oocyte maturation. The 3-D culture of FL-clusters provided a method to further mimic in vivo growth by preserving interfollicle connections and architecture. Functional competence of oocytes needs further assessment. Enzymatic treatment of vitrified-warmed ovaries for follicle isolation may not be the best approach due to tissue fragility.Table 1IVM summary data for fresh and vitrified tissueFrozenaFreshbFollicle - ClusterFollicle - IsolatedFollicle - ClusterFollicles observed during IVC69130154Antrum formationna51% (66/130)53% (81/154)Ovulated complexes after hCG93% (64/69)71% (92/130)66% (101/154)GVBD (MI)52% (33/64)30% (28/92)28% (28/101)Maturation to MII34% (22/64)59% (54/92)55% (56/101)Spindle Retardance (Range)na1.34- 3.641.62- 3.51aMechanically isolated follicle cluster. bEnzymatic isolation. Open table in a new tab

Construction of human artificial ovary from cryopreserved ovarian tissue: Appearance of apoptosis and necrosis after enzymatic isolation of follicles

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex.Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 ± 6 years old), sized 20–210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 ± 2.4% vs. 90.4 ± 3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 ± 3.4% vs. 89.7 ± 4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 ± 3.4%; Group 2: 91.8 ± 6.0%; Group 3: 90.5 ± 6.9%; Group 4: 87.3 ± 2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 ± 1.6%; Group 2: 4.4 ± 1.6%; Group 3: 1.6 ± 1.1%; Group 4: 2.4 ± 1.5%), late apoptotic (Group 1: 2.7 ± 2.4%; Group 2: 44.0 ± 1.9%; Group 3: 3.1 ± 1.1%; Group 4: 2.8 ± 0.7% and necrotic (Group 1: 0.9% ± 0.1%; Group 2: 2.9 ± 0.8%; Group 3: 3.4 ± 4.5%; Group 4: 1.1 ± 0.6%) cells was low and was not significantly different in all treatment groups (P > 0.1). It was concluded that the use of Tumor Dissociation Enzyme, effectiveness of which is higher than Liberase TM, does not lead to increasing of apoptosis and necrosis in follicles and stromal cells after enzymatic digesting of cryopreserved ovarian cortex.

Effects of Modified Processing Methods on Structural Changes of Black Soybean Protein Isolate.

To explore better methods of natural protein modification for black soybean, comparisons among the effects of different modified methods on structural changes of the modified products of black soybean protein isolate (BSPI) were carried out in this study. The modified products used in this study included enzymatic crossing-link black soybean protein isolate (ECBSPI), wet heating treatment glycosylation black soybean protein isolate (WHTGBSPI) and especially enzymatic glycosylation black soybean protein isolate catalyzed by transglutaminase (EGBSPI). The effects of the modification methods on structural changes were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid content and circular dichroism (CD) analysis. Moreover, the processing properties changes caused by structural changes of BSPI were detected by thermogravimetric analysis, particle size analysis, zeta-potential, surface hydrophobicity, solubility, emulsification, gelation, and rheological properties. The results show that the modified BSPI products were protein polymers, and among them, EGBSP and WHTGBSPI are covalently bonded glycation products. Products modified by Maillard reactions and transglutaminase (TG) display partly destroyed α-helix and β-sheet structures that form more open secondary BSPI structures. For ECBSPI, the proportion of irregular crimp structure reduces to form a high order secondary structure. All the modified products form fine aggregations in dispersion, except WHTGBSPI has most negative zeta-potential and least molecular stability due to the hydrophobic amino acids embedded in the protein molecules. The zeta-potentials of BSPI, ECBSPI, WHTGBSPI and EGBSPI are respectively −21.5, −23.8, −18.1 and −20.2 mV. The surface hydrophobicity of EGBSPI (5.07 ± 0.07) and WHTGBSPI (7.02 ± 0.05) decrease, while the surface hydrophobicity of ECBSPI (19.5 ± 0.06) increases. The solubility and rheological properties of EGBSPI, ECBSPI and WHTGBSPI after modification are all better than those of BSPI, especially EGBSPI. Emulsification of EGBSPI and WHTGBSPI increase (by 24.5% and 12.2%, respectively) while ECBSPI decrease (by 17.0), and there is similar emulsion stability trend. Moreover, the properties of ECBSPI increase except cohesiveness compared to BSPI. In conclusion, as a safe and efficient method for natural protein modification, enzymatic glycosylation catalyzed by TG has great potential in improving food processing characteristics.

Evaluation of platelet lysate as a substitute for FBS in explant and enzymatic isolation methods of human umbilical cord MSCs

Mesenchymal stem cells (MSCs) have immense potential for cell-based therapy of acute and chronic pathological conditions. MSC transplantation for cell-based therapy requires a substantial number of cells in the range of 0.5–2.5 × 106 cells/kg body weight of an individual. A prolific source of MSCs followed by in vitro propagation is therefore an absolute prerequisite for clinical applications. Umbilical cord tissue (UCT) is an abundantly available prolific source of MSC that are fetal in nature and have higher potential for ex-vivo expansion. However, the ex-vivo expansion of MSCs using a xenogeneic supplement such as fetal bovine serum (FBS) carries the risk of transmission of zoonotic infections and immunological reactions. We used platelet lysate (PL) as a xeno-free, allogeneic replacement for FBS and compared the biological and functional characteristics of MSC processed and expanded with PL and FBS by explant and enzymatic method. UCT-MSCs expanded using PL displayed typical immunophenotype, plasticity, immunomodulatory property and chromosomal stability. PL supplementation also showed 2-fold increase in MSC yield from explant culture with improved immunomodulatory activity as compared to enzymatically dissociated cultures. In conclusion, PL from expired platelets is a viable alternative to FBS for generating clinically relevant numbers of MSC from explant cultures over enzymatic method.

Evidence of primary cilia in the developing rat heart

BackgroundA transient increase in cytosolic Ca2+ (the “Ca2+ transient”) determines the degree and duration of myocyte force development in the heart. However, we have previously observed that, under the same experimental conditions, the Ca2+ transients from isolated cardiac myocytes are reduced in amplitude in comparison to those from multicellular cardiac preparations. We therefore questioned whether the enzymatic cell isolation procedure might remove structures that modulate intracellular Ca2+ in some way. Primary cilia are found in a diverse range of cell types, and have an abundance of Ca2+-permeable membrane channels that result in Ca2+ influx when activated. Although primary cilia are reportedly ubiquitous, their presence and function in the heart remain controversial. If present, we hypothesized they might provide an additional Ca2+ entry pathway in multicellular cardiac tissue that was lost during cell isolation. The aim of our study was to look for evidence of primary cilia in isolated myocytes and ventricular tissue from rat hearts.MethodsImmunohistochemical techniques were used to identify primary cilia-specific proteins in isolated myocytes from adult rat hearts, and in tissue sections from embryonic, neonatal, young, and adult rat hearts. Either mouse anti-acetylated α-tubulin or rabbit polyclonal ARL13B antibodies were used, counterstained with Hoechst dye. Selected sections were also labelled with markers for other cell types found in the heart and for myocyte F-actin.ResultsNo evidence of primary cilia was found in either tissue sections or isolated myocytes from adult rat ventricles. However, primary cilia were present in tissue sections from embryonic, neonatal (P2) and young (P21 and P28) rat hearts.ConclusionThe lack of primary cilia in adult rat hearts rules out their contribution to myocyte Ca2+ homoeostasis by providing a Ca2+ entry pathway. However, evidence of primary cilia in tissue from embryonic and very young rat hearts suggests they have a role during development.

Progress in the development of a cell culture system to study the immune responses of reef-building corals.

Abstract Previous work in the laboratory has focused on using fragments of aquaculture-raised specimens of the stony coral Acropora and Montipora. We are interested in these species because Acropora has an expanded immune gene repertoire in comparison to the closely-related anemone species Nematostella, especially in the number of TLR and IL-1R genes. However, space restrictions and maintaining acceptable water chemistry parameters pose a challenge to working with samples of stony corals in this form. In addition, coral fragments are complex holobionts that contain a mixture of coral cells, photosynthetic zooxanthelle, and bacteria, all on a calcium carbonate skeletal matrix. We seek a simplified model system of coral cells growing in isolation to facilitate performing the virus isolation, propagation, and controlled infection experiments that we think are necessary to advance our understanding of coral disease and immune responses. A number of protocols exist to propagate coral cells in a mixture of artificial seawater and typical mammalian cell growth medium. We compared the results of mechanical versus enzymatic isolation of coral tissues from the underlying skeletal matrix. We performed microscopic assessments of the coral and endosymbiont cells immediately following isolation and again after one week in culture. The endosymbiont zooxanthelle are easily visually distinguishable, and appear to persist longer under our culture conditions than the coral cells. It is unclear whether this is due to an improved ability of the zooxanthelle to live independently, or increased susceptibility of the coral cells to pathogen infections not eliminated by antibiotics in the culture medium.