Evaluating effects of gypenosides and soyasaponins on differentiation of neural stem cells as an in vitro model

Neural stem cell has a potential to differentiate into neurons, astrocytes and oligodendrocytes. It provides an in vitro model to screen herbal medicines on the cellular differentiation and development level. In this work, active component from gypenosides and soyasaponins was prepared to investigate their effects on the differentiation of neural stem cells.. Both gypenosides and soyasaponins promote the differentiation of neural stem cells. This method provides speed and practicality for screening effective herbal medicine. It is well suited for studying the mechanism of cell differentiation and development.

Toward three-dimensional in vitro models to study neurovascular unit functions in health and disease

The high metabolic demands of the brain require an efficient vascular system to be coupled with neural activity to supply adequate nutrients and oxygen.This supply is coordinated by the action of neurons,glial and vascular cells,known collectively as the neurovascular unit,which temporally and spatially regulate local cerebral blood flow through a process known as neurovascular coupling.In many neurodegenerative diseases,changes in functions of the neurovascular unit not only impair neurovascular coupling but also permeability of the blood-brain barrier,cerebral blood flow and clearance of waste from the brain.In order to study disease mechanisms,we need improved physiologicallyrelevant human models of the neurovascular unit.Advances towards modeling the cellular complexity of the neurovascular unit in vitro have been made using stem-cell derived organoids and more recently,vascularized organoids,enabling intricate studies of non-cell autonomous processes.Engineering and design innovations in microfluidic devices and tissue engineering are progressing our ability to interrogate the cerebrovasculature.These advanced models are being used to gain a better understanding of neurodegenerative disease processes and potential therapeutics.Continued innovation is required to build more physiologically-relevant models of the neurovascular unit encompassing both the cellular complexity and designed features to interrogate neurovascular unit functionality.

Differential gene expression during capillary morphogenesis in a microcarrier-based three-dimensional in vitro model of angiogenesis with focus on chemokines and chemokine receptors

AIM:To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogeness system with affymetrix oligonucleotide array. METHODS: A microcarrier-based in vitro angiogenesis system was developed, in which ECs migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed networks. The total RNA samples from the HMVECs at the selected time points (0.5, 24, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the softwares provided by the manufacturers. The expression patterns of some genes were validated and confirmed by semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines and chemokine receptors was specially examined and their functional implications were analyzed. RESULTS: A total of 1 961 genes were up- or downreg-ulated two-folds or above, and among them, 468 genes were up- or down-regulated three-folds or above. The regulated genes could be grouped into categories based on their molecular functions, and were also clustered into six groups based on their patterns of expression. As for chemokines and chemokine receptors, CXCL1/GRO-α, CXCL2/GRO-β, CXCL5/ENA-78, CXCL6/GCP2, IL-8/CXCL8, CXCL12/SDF-1, CXCL9/Mig, CXC11/ITAC, OOCL1/fractalkine, CCL2/MCP-1, CCL3, CCL5/RANTES, CCL7, CCL15, CCL21, CCL23, CCL28, and CCR1, CCR9, CXCR4 were identified. Moreover, these genes demonstrated different changing patterns during the capillary morphogenesis, which implied that they might have different roles in the sequential process. Among the chemokines identified, CCL2/MCP-1, CCL5/RANTES and CX3CL1 were specially up-regulated at the 24-h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis. CONCLUSION: The present study demonstrates a global profile of gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be conducted.

Effects of natural-cerebrolysin-containing serum on neurotoxicity and synaptogenesis in amyloid-beta 1-40-induced Alzheimer's disease in vitro models

BACKGROUND： Neuronal loss, synapse mutilation, and increasing malnourished axons are pathologically related to Alzheimer＇s disease. Microtubule-associated protein 2 （MAP2） is of importance for neuronal, axonal, and dendritic generation, extension, and stabilization, as well as for the regulation of synaptic plasticity. OBJECTIVE： To investigate the antagonistic effects of natural-cerebrolysin-containing serum on beta amyloid protein 1-40 （Aβ1-40）-induced neurotoxicity from the standpoints of cell proliferation, synaptogenesis, and cytoskeleton formation （MAP2 expression）. DESIGN, TIME AND SETTING： A paralleled, controlled, neural cell, and molecular biology experiment was performed at the Institute of Integrated Chinese and Western Medicine, Shenzhen Hospital, Southern Medical University between February 2006 and April 2008. MATERIALS： PC12 cells, derived from the rat central nervous system, were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China. A β1-40 was provided by Sigma, USA. Natural-cerebrolysin was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. The natural-cerebrolysin was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yixingye （Ginkgo Leaf） in a proportion of 1：2：2. Following conventional water extraction technology, an extract （1：20） was prepared. Each gram of extract equaled 20 grams of crude drug. In a total of 12 adult male New Zealand rabbits, six underwent intragastric administration of natural-cerebrolysin extract for 1 month to prepare natural-cerebrolysin-containing serum, and the remaining six rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： An AIzheimer＇s disease in vitro model was induced in PC12 cells using Aβ1-40. The cells were incubated with varying doses of natural-cerebrolysin-containing serum （2.5%, 5%, and 10%）. Normal blank serum-treated PC12 cells served as a blank control group. MAIN OUTCOME MEASURES： Through the use of inverted phase contrast microscope, cell morphology and neurite growth were observed, neurite length was measured, and the percentage of neurite-positive cells was calculated. Cell proliferation rate was determined by MTT assay, and MAP 2 expression was detected by fluorescent immunocytochemistry. RESULTS： Following Aβ1-40 treatments, some PC12 cells were apoptotic/dying, and only a few short neurites were observed. Following interventions with natural-cerebrolysin-containing serum, the PC12 cells proliferated, there was an increased number of neurites, and neurite length was enhanced. After middle- and high-dose natural-cerebrolysin treatments, the percentage of neurite-positive cells, as well as the average length of neurites, was significantly greater than the normal blank serum-treated PC12 cells （P 〈 0.05 or P 〈 0.01）. Compared with the blank control group, MAP2 expression in the Aβ1-40-treated PC12 cells was significantly inhibited, and the cell proliferation rate was significantly decreased （P 〈 0.01）. Following incubations with natural-cerebrolysin-containing serum, MAP2 expression and cell proliferation rate in the PC12 cells were significantly increased in a dose-dependent manner, compared with treatments with blank control serum （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Natural-cerebrolysin exhibited antagonistic effects on neurotoxicity in Aβ1-40 induced Alzheimer＇s disease in vitro models. These effects were likely related to cell proliferation and the upregulation of intracellular MAP2 expression.

Human induced pluripotent stem cell based in vitro models of the blood-brain barrier： the future standard？

There is an urgent and tremendous need for human dis- ease models in drug development in order to improve pre- clinical predictability. In the case of brain disorders drugs have to cross the blood-brain barrier （BBB） to enter the central nervous system （CNS）. It was estimated that more than 95% of the drugs cannot cross the BBB.

Validation of an In Vitro Model to Study Human Cartilage Responses to Compression

The aim of this work was to develop an in vitro model to study mechanical compression effects on cartilage. A pressure-controlled compression device was used in this study. Cartilage explants obtained from human knee were compressed at 1MPa/1Hz for 7 hours (30 min ON, 30 min OFF) under normoxia (5% CO2, 21% O2) or hypoxia (5% CO2, 5% O2). Cell viability was analyzed while nitric oxide (NO) and glycosaminoglycans (GAG) release was assayed in culture media. Mechanical stimulation increased NO production and GAG release by human cartilage explants under normoxia and hypoxia culture. In normoxia and hypoxia conditions, mechanical stimulation alters human OA cartilage metabolism. There is also, an increase in matrix degradation after compression, as shown by levels of GAG found in culture media. This study put in evidence the importance of mechanical compression in the progression of the osteoarthritis and present and in vitro model for mechanobiological and pharmacological studies.

Weaning-associated feed deprivation stress causes microbiota disruptions in a novel mucin-containing in vitro model of the piglet colon(MPigut-IVM)

Background:Risk factors for the etiology of post-weaning diarrhea,a major problem in swine industry associated with enormous economic losses,remain to be fully elucidated.In concordance with the ethical concerns raised by animal experiments,we developed a new in vitro model of the weaning piglet colon(MPigut-IVM)including a mucin bead compartment to reproduce the mucus surface from the gut to which gut microbes can adhere.Results:Our results indicated that the MPigut-IVM is able to establish a representative piglet archaeal and bacterial colon microbiota in terms of taxonomic composition and function.The MPigut-IVM was consequently used to investigate the potential effects of feed deprivation,a common consequence of weaning in piglets,on the microbiota.The lack of nutrients in the MPigut-IVM led to an increased abundance of Prevotellaceae and Escherichia-Shigella and a decrease in Bacteroidiaceae and confirms previous in vivo findings.On top of a strong increase in redox potential,the feed deprivation stress induced modifications of microbial metabolite production such as a decrease in acetate and an increase in proportional valerate,isovalerate and isobutyrate production.Conclusions:The MPigut-IVM is able to simulate luminal and mucosal piglet microbiota and represent an innovative tool for comparative studies to investigate the impact of weaning stressors on piglet microbiota.Besides,weaning-associated feed deprivation in piglets provokes disruptions of MPigut-IVM microbiota composition and functionality and could be implicated in the onset of post-weaning dysbiosis in piglets.

Comparative Toxicity of Nanomaterials to Air-blood Barrier Permeability Using an In Vitro Model

Objective To comparatively study the toxicity of four metal-containing nanoparticles(MNPs) and their chemical counterparts to the air-blood barrier(ABB) permeability using an in vitro model.Methods ABB model, which was developed via the co-culturing of A549 and pulmonary capillary endothelium, was exposed to spherical CuO-NPs(divided into CuO-40, CuO-80, and CuO-100 based on particle size), nano-Al2O3(sheet and short-rod-shaped), nano-ZnO, nano-Pb S, CuSO4, Al2(SO4)3, Zn(CH3COO)2, and Pb(NO3)2 for 60 min.Every 10 min following exposure, the cumulative cleared volume(ΔTCL) of Lucifer yellow by the model was calculated.A clearance curve was established using linear regression analysis of ΔTCL versus time.Permeability coefficient(P) was calculated based on the slope of the curve to represent the degree of change in the ABB permeability.Results The results found the increased P values of CuO-40, CuO-80, sheet, and short-rod-shaped nano-Al2O3, Al2(SO4)3, and Pb(NO3)2.Among them, small CuO-40 and CuO-80 were stronger than CuO-100 and CuSO4;no difference was observed between Al2(SO4)3 and sheet and short-rod-shaped nano-Al2O3;and nano-Pb S was slightly weaker than Pb(NO3)2.So clearly the MNPs possess diverse toxicity.Conclusion ABB permeability abnormality means pulmonary toxicity potential.More studies are warranted to understand MNPs toxicity and ultimately control the health hazards.

Role of connexin-related signalling in hepatic homeostasis and its relevance for liver-based in vitro modelling

Direct intercellular communication mediated by gap junctions constitutes a major regulatory platform in the control of hepatic homeostasis.Hepatocellular gap junctions are composed of two hemichannels of adjacent cells which are built up by connexin proteins,in casu Cx32.Mathieu Vinken,Pofessor at the Department of Toxicology of the Free University BrusselsBelgium,was one of the first investigators to demonstrate that hepatic connexin expression is controlled by epigenetic mechanisms.In particular,he found that inhibitors of histone deacetylase enzymes enhance Cx32 production and gap junction activity in cultures of primary hepatocytes,a finding that is of importance for liver-based in vitro modelling.Professor Dr.Mathieu Vinken's recent work is focussed on the elucidation of the role of connexin proteins and their channels in the hepatocyte life cycle.Specific attention is paid to apoptosis in this context,whereby it has been found that Cx32 hemichannels control the termination of induced cell death in cultures of primary hepatocytes. Overall,Professor Dr.Mathieu Vinken's research can be considered as an important contribution to the field of hepatic connexin physiology.

In vitro engineered models of neurodegenerative diseases

Neurodegeneration is a catastrophic process that develops progressive damage leading to functional andstructural loss of the cells of the nervous system and is among the biggest unavoidable problems of our age.Animalmodels do not reflect the pathophysiology observed in humans due to distinct differences between the neuralpathways,gene expression patterns,neuronal plasticity,and other disease-related mechanisms in animals andhumans.Classical in vitro cell culture models are also not sufficient for pre-clinical drug testing in reflecting thecomplex pathophysiology of neurodegenerative diseases.Today,modern,engineered techniques are applied to developmulticellular,intricate in vitro models and to create the closest microenvironment simulating biological,biochemical,and mechanical characteristics of the in vivo degenerating tissue.In THIS review,the capabilities and shortcomings ofscaffold-based and scaffold-free techniques,organoids,and microfluidic models that best reflect neurodegeneration invitro in the biomimetic framework are discussed.

Progress in experimental models to investigate the in vivo and in vitro antidiabetic activity of drugs

Diabetes mellitus is one of the world's most prevalent and complex metabolic disorders,and it is a rapidly growing global public health issue.It is characterized by hyperglycemia,a condition involving a high blood glucose level brought on by deficiencies in insulin secretion,decreased activity of insulin,or both.Prolonged effects of diabetes include cardiovascular problems,retinopathy,neuropathy,nephropathy,and vascular alterations in both macro-and micro-blood vessels.In vivo and in vitro models have always been important for investigating and characterizing disease pathogenesis,identifying targets,and reviewing novel treatment options and medications.Fully understanding these models is crucial for the researchers so this review summarizes the different experimental in vivo and in vitro model options used to study diabetes and its consequences.The most popular in vivo studies involves the small animal models,such as rodent models,chemically induced diabetogens like streptozotocin and alloxan,and the possibility of deleting or overexpressing a specific gene by knockout and transgenic technologies on these animals.Other models include virally induced models,diet/nutrition induced diabetic animals,surgically induced models or pancreatectomy models,and non-obese models.Large animals or non-rodent models like porcine(pig),canine(dog),nonhuman primate,and Zebrafish models are also outlined.The in vitro models discussed are murine and human beta-cell lines and pancreatic islets,human stem cells,and organoid cultures.The other enzymatic in vitro tests to assess diabetes include assay of amylase inhibition and inhibition ofα-glucosidase activity.

Simulating traumatic brain injury in vitro:developing high throughput models to test biomaterial based therapies

Traumatic brain injuries are serious clinical incidents associated with some of the poorest outcomes in neurological practice.Coupled with the limited regenerative capacity of the brain,this has significant implications for patients,carers,and healthcare systems,and the requirement for life-long care in some cases.Clinical treatment currently focuses on limiting the initial neural damage with longterm care/support from multidisciplinary teams.Therapies targeting neuroprotection and neural regeneration are not currently available but are the focus of intensive research.Biomaterial-based interventions are gaining popularity for a range of applications including biomolecule and drug delive ry,and to function as cellular scaffolds.Experimental investigations into the development of such novel therapeutics for traumatic brain injury will be critically underpinned by the availability of appropriate high thro ughput,facile,ethically viable,and pathomimetic biological model systems.This represents a significant challenge for researchers given the pathological complexity of traumatic brain injury.Specifically,there is a concerted post-injury response mounted by multiple neural cell types which includes microglial activation and astroglial scarring with the expression of a range of growth inhibito ry molecules and cytokines in the lesion environment.Here,we review common models used for the study of traumatic brain injury(ranging from live animal models to in vitro systems),focusing on penetrating traumatic brain injury models.We discuss their relative advantages and drawbacks for the developmental testing of biomaterial-based therapies.

Designs andmethodologies to recreate in vitro human gutmicrobiota models

The human gut microbiota is widely considered to be a metabolic organ hidden within our bodies,playing a crucial role in the host’s physiology.Several factors affect its composition,so a wide variety of microbes residing in the gut are present in the world population.Individual excessive imbalances in microbial composition are often associated with human disorders and pathologies,and new investigative strategies to gain insight into these pathologies and define pharmaceutical therapies for their treatment are needed.In vitro models of the human gut microbiota are commonly used to study microbial fermentation patterns,community composition,and host-microbe interactions.Bioreactors and microfluidic devices have been designed to culture microorganisms from the human gut microbiota in a dynamic environment in the presence or absence of eukaryotic cells to interact with.In this review,we will describe the overall elements required to create a functioning,reproducible,and accurate in vitro culture of the human gut microbiota.In addition,we will analyze some of the devices currently used to study fermentation processes and relationships between the human gut microbiota and host eukaryotic cells.

Feasibility Analysis of Oxygen-Glucose Deprivation-Nutrition Resumption on H9c2 Cells In vitro Models of Myocardial Ischemia-Reperfusion Injury

Background： Oxygen-glucose deprivation-nutrition resumption （OGD-NR） models on H9c2 cells are commonly used in vitro models of simulated myocardial ischemia-reperfusion injury （M1RI）, but no study has assessed whether these methods for establishing in vitro models can effectively imitate the characteristics of MIRI in vivo. This experiment was designed to analyze the feasibility of six OGD-NR models of MIRI. Methods： By searching the PubMed database using the keywords ＂myocardial reperfusion injury H9c2 cells,＂ we obtained six commonly used OGD-NR in vitro models of MIRI performed on H9c2 cells from more than 400 published papers before January 30, 2017. For each model, control （C）, simulated ischemia （SI）, and simulated ischemia-reperfusion （SIR） groups were assigned, and cell morphology, lactate dehydrogenase （LDH） release, adenosine triphosphate （ATP） levels, reactive oxygen species （ROS）, mitochondrial membrane potential （MMP）, and inflammatory cytokines were examined to evaluate the characteristics of cell injury. Subsequently, a coculture system of cardiomyocyte-endothelial-macrophage was constructed. The coeulture system was dealt with SI and SIR treatments to test the effect on cardiomyocytes survival. Results： For models l, 2, 3, 4, 5, and 6, SI treatment caused morphological damage to cells, and subsequent SIR treatment did not cause further morphological damage. In the models 1, 2, 3, 4, 5 and 6, LDH release was significantly higher in the SI groups than that in the C group （P 〈 0.05）, and was significantly lower in the SIR groups than that in the SI groups （P 〈 0.05）, except tbr no significant differences in the LDH release between C, SI and SIR groups in model 6 receiving a 3-h SI treatment. In models 1, 2, 3, 4, 5, and 6, compared with the C group, ATP levels of the S1 groups significantly decreased （P 〈 0.05）, ROS levels increased （P 〈 0.05）, and MMP levels decreased （P 〈 0.05）. Compared with the SI group, ATP level of the SIR groups was significantly increased （P 〈 0,05）, and there was no significant ROS production, MMP collapse, and over inflammatory response in the SIR groups. In a coculture system of H9c2 cells-endothelial cells-macrophages, the proportion of viable H9c2 cells in the SIR groups was not reduced compared with the SI groups. Conclusion： All the six OGD-NR models on H9c2 cells in this experiment can not imitate the characteristics of MIRI in vivo and are not suitable for MIRl-related study.

Modeling and validating three dimensional human normal cervix and cervical cancer tissues in vitro

The use of three dimensional in vitro systems in cancer research is a promising path for developing effective anticancer therapies.The aim of this study was to engineer a functional 3-D in vitro model of normal and cancerous cervical tissue.Normal epithelial and immortalized cervical epithelial carcinoma cell lines were used to construct 3-D artificial normal cervical and cervical cancerous tissues.De-epidermised dermis（DED） was used as a scaffold for both models.Morphological analyses were conducted by using hematoxylin and eosin staining and characteristics of the models were studied by analyzing the expression of different structural cytokeratins and differential protein marker MAX dimerisation protein 1（Mad1） using immunohistochemical technique.Haematoxylin and eosin staining results showed that normal cervical tissue had multi epithelial layers while cancerous cervical tissue showed dysplastic changes.Immunohistochemistry staining revealed that for normal cervix model cytokeratin 10 was expressed in the upper stratified layer of the epithelium while cytokeratin 5 was expressed mainly in the middle and basal layer.Cytokeratin 19 was weakly expressed in a few basal cells.Cervical cancer model showed cytokeratin 19 expression in different epithelial layers and weak or no expression for cytokeratin 5 and cytokeratin 10.Madl expression was detected in some suprabasal cells.The 3-D in vitro models showed stratified epithelial layers and expressed the same types and patterns of differentiation marker proteins as seen in corresponding in vivo tissue in either normal cervical or cervical cancerous tissue.These findings imply that they can serve as functional normal and cervical cancer models.

Recent advances in the development of in vitro liver models for hepatotoxicity testing

Liver injury is a common cause of drug approval withdrawal during drug development,pre-clinical research,and clinical treatment.If not properly treated,patients with severe liver injury can suffer from acute liver failure or even death.Thus,utilization of the convenient in vitro hepatotoxicity assessment model for early detection of drug-induced hepatotoxicity is vital for drug development and safe personalized medication.Biomaterials(e.g.,hydrogels,nanofibers,decellularized liver matrix)and bioengineering technologies(e.g.,microarrays,micropatterns,3D printing,and microfluidics)have been applied for in vitro hepatotoxicity assessment models.This review summarizes the structure and functions of the liver as well as the components of in vitro hepatotoxicity assessment models.In addition,it highlights the latest advances in developing hepatotoxicity models with the ultimate goal of further clinical translation.

Development of a 3D matrix for modeling mammalian spinal cord injury in vitro

Spinal cord injury affects millions of people around the world, however, limited therapies are available to improve the quality of life of these patients. Spinal cord injury is usually modeled in rats and mice using contusion or complete transection models and this has led to a deeper understanding of the molecular and cellular complexities of the injury. However, it has not to date led to development of successful novel therapies, this is in part due to the complexity of the injury and the difficulty of deciphering the exact roles and interactions of different cells within this complex environment. Here we developed a collagen matrix that can be molded into the 3D tubular shape with a lumen and can hence support cell interactions in a similar architecture to a spinal cord. We show that astrocytes can be successfully grown on this matrix in vitro and when injured, the cells respond as they do in vivo and undergo reactive gliosis, one of the steps that lead to formation of a glial scar, the main barrier to spinal cord regeneration. In the future, this system can be used to quickly assess the effect of drugs on glial scar protein activity or to perform live imaging of labeled cells after exposure to drugs.

In vitro three-dimensional cancer metastasis modeling:Past,present,and future

Metastasis is the leading cause of most cancer deaths, as opposed to dysregulated cell growth of the primary tumor. Molecular mechanisms of metastasis have been studied for decades and the findings have evolved our understanding of the progression of malignancy. However, most of the molecular mechanisms fail to address the causes of cancer and its evolutionary origin, demonstrating an inability to find a solution for complete cure of cancer. After being a neglected area of tumor biology for quite some time, recently several studies have focused on the impact of the tumor microenvironment on cancer growth. The importance of the tumor microenvironment is gradually gaining attention, particularly from the per- spective of biophysics. In vitro three-dimensional （3-D） metastatic models are an indispensable platform for investigating the tumor microenvironment, as they mimic the in vivo tumor tissue. In 3-D metastatic in vitro models, static factors such as the mechanical properties, biochemical factors, as well as dynamic factors such as cell-cell, cell-ECM interactions, and fluid shear stress can be studied quantitatively. With increasing focus on basic cancer research and drug development, the in vitro 3-D models offer unique advantages in fundamental and clinical biomedical studies.

IN VITRO GLYCATION OF HEMOGLOBIN (HB):CHOOSING THE APPROPRIATE MODEL

Human HbA is nonenzymatically glycated at several sites. Approximately half of glycated Hb is formed by the addition of glucose to the amino-terminal valine of the Hb βchains, this species is called HbA1c.Most studies examining the effects of various agents on Hb glycation have focused on HbA1c formation in vitro.However,approximately hal f of Hb glycation in vivo also occurs at other sites,i.e.epsilon amino groups on lysines.Our purpose,therefore,was to develop a model for testing the effects of several parameters and /or chemicals on glycohemoglobin formation which would be representative of the st.uation in vivo in terms of glycation sites.Hb was glycated by several methods:① Drying of whole blood on filter paper ② Incubation of erythrocyte hemolysates in various buffers ③Incubation of intact erythrocytes in plasma and cell culture medium. Results show that drying of Hb on filter paper caused rapid .Hb glycation which could be measured by affinity chromatography; the measured glycated Hb more than doubled in less than one week. However,measurement of HbA1c formation by immunoassay showed minimal glycation at the βchain N-terminal valine from filter paper elutes(HbA1c increased by less than 2% in one week).Similarly, incubation of hemolysates showed minimal formation of HbA1c compared to other glycation products. However,incubation of intact erythtocytes in either plasma or culture medium showed formation of HbA1c and iotal glycated Hb in proportions similar to that formed in vivo. We conclude that in vitro conditions will affect the Hb glycation site and that incubation of intact erythrocytes provides the most representative model for the study of Hb glycation.

An In Vitro Biological Model for Evaluation of the Anti-Dandruff Performance of Hair Care Products

An in vitro biological model for evaluation of the anti-dandruff performance of hair care products was established. In this model, Malasseziafurfur(M.furfur) was used as indicator bacteria with pigskin as substrate.This model functions in following steps: pretreatment for the substrate; inoculation of indicator bacteria;measuring the bacteriostatic efficacy by the contact plates after washing the pigskin with the sample shampoo;calculation of the effective index P; evaluation of the anti-dandruff performance by the effective index P. Three kinds of anti-dandruff agents including BPT, ZPT and OCT were tested by the model. Results showed that the order for antibacterial efficacy of the anti-dandruff agents within the mass fraction range of 0.1 %~2.0% was BPT>ZPT>OCT. Results of the evaluation on each type of shampoo showed that: one displays no antidandruff efficacy; three displays medium anti-dandruff efficacy; two displays strong anti-dandruff efficacy.