体外细胞毒性实验
医疗器械生物学评价第五部分体外细胞毒性试验
医疗器械生物学评价第 5 局部：体外细胞毒性试验1范围GB/T 16886 的本局部阐述了评价医疗器械体外细胞毒性的试验方法。
这些方法规定了以下供试品以直接或通过集中的方式与培育细胞接触和进展孵育；a〕用器械的浸提液，和／或b〕与器械接触。
这些方法是用相应的生物参数测定哺乳动物细胞的体外生物学反响。
2标准性引用文件以下文件中的条款通过GB/T 16886 的本局部的引用而成为本局部的条款。
但凡注日期的引用文件，其随后全部的修改单〔不包括订正的内容〕或均不适用于本局部，然而，鼓舞依据本局部达成协议的各方争论是否可使用这些文件的最版本。
但凡不注日期的引用文件，其最版本适用于本局部。
GB/T 16886. 1 医疗器械生物学评价第1 局部：评价与试验〔GB/T 16886.1-2023,idt ISO 10993- 1:1997〕CB/ T 16886. 12-2023 医疗器械生物学评价第12 局部：样品制备和参照材料〔idt ISO 10993-12 :1996〕3术语与定义GB/ T 16886. 1/ ISO 1993-1 中确立的以及以下术语和定义适用于本局部。
3.13.2 阴性比照材料negative control material依据本局部试验时不产生细胞毒性反响的材料。
注：阴性比照的目的是验证背景反响，例如高密度聚乙烯1〕牙科材料的阴性比照物。
阳性比照材料 pos itive control material依据本局部试验时可重现细胞毒性反响的材料。
已作为合成聚合物的阴性比照材料，氧化陶瓷棒则用作注：阳性比照的白目的是验证相应试验系统的反响，例如用有机锡作稳定剂的聚氯乙烯的阳性比照，酚的稀释液用于浸提液的阳性比照。
2）已用作固体材料和浸提液1)高密度聚乙烯可从美国药典委员会〔Rockvillie, Maryland, USA〕和Hatano 争论所食品和药品安全中心〔Ochiai 729-5 ,Hanagawa.257-Japan〕获得。
细胞毒性实验总结
细胞毒性实验总结一、抗HBV药物细胞毒性实验背景知识 (1)二、细胞毒性实验方案的确立 (3)三、细胞毒性实验方法稳定后的部分数据 (4)四、细胞毒性实验质量评价指标 (5)五、细胞毒性实验SOP (6)（一）目的 (6)（二）适用范围 (6)（三）责任人 (6)（四）规程 (6)1. 试验准备 (7)2. 试验操作 (8)3．数据处理和分析 (8)六、总结 (9)一、抗HBV药物细胞毒性实验背景知识细胞毒性是化学物质（药物）作用于细胞基本结构和/或生理过程，如细胞膜或细胞骨架结构，细胞的新陈代谢过程，细胞组分或产物的合成、降解或释放，离子调控及细胞分裂等过程，导致细胞存活、增殖和/或功能的紊乱，所引发的不良反应。
按作用机制可分3种类型：①基本细胞毒性，涉及一种或多种上述结构或功能的改变，作用于所有类型的细胞；②选择细胞毒性，存在于某些分化细胞上，主要通过化学物质的生物转化，与特殊受体结合或特殊的摄入机制所引发；③细胞特殊功能毒性，对细胞结构和功能损伤轻微，但对整个机体损伤非常严重。
类似毒性作用可通过细胞因子、激素和递质的合成、释放、结合和降解影响细胞与细胞间的交流或特殊的转运过程而实现。
毒性作用也可能来自化学物质对细胞外过程的干扰，任何一种非动物检测系统对多种因素都应加以考虑。
1983年Ekwall提出“基本细胞功能”的概念，即多数化学物质毒性作用是对细胞功能的非特异性损伤，却可引起器官功能的特异性改变甚至机体死亡。
有研究显示化学物质体外细胞毒性与其引起的动物死亡率及人体死亡的血药浓度之间都存在良好的相关性。
化学物质产生的损伤和死亡，最终可表现为细胞水平上的改变，由此推测体外细胞毒性可以预测体内急性毒性。
体外方法有助于预测化学物质急性暴露引发的全身和局部影响，并评估体内毒性浓度。
目前较为理想的抗HBV药物有拉米夫定（3TC）、恩替卡韦（ETV）等。
3TC是核苷左旋对呋体，早期用于艾滋病的治疗。
两种膨润土细胞毒性的体外试验研究
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结果所有剂量２种膨润土的溶血率均明显高于对照组，差异有统计学意义（尸℃Ｏ．０５）。ＣＣＫ．８试验结 果表明，酸性土和有机土剂量分别≥３０、２０斗ｓ／ｍｉ时，细胞活性明显低于对照组，差异有统计学意义（Ｐ＜
０．０１）；ＮＲＵ试验和ＬＤＨ试验出现类似结果，并均呈剂量—效应关系。１８０脚ｒ／ｍＪ酸性土组与１２０、１８０¨固伽 有机土组的早期细胞凋亡率明显高于对照组，差异有统计学意义（Ｐ如．０５）。５个体外试验的结果均表
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膨润土是一种广泛用于铸造、建筑、环境工程
（ＩＡＲＣ）列为对人致癌１３１。但是，至今膨润土细胞毒性 研究报道不多。最近Ｇｅｈ等１４１发现，膨润土可诱发细 胞凋亡及坏死，且具有潜在诱发细胞膜破裂的能 力。此外，不同膨润土对细胞的损伤作用可能不同阳。
等领域的非金属矿物质，我国是开采大国，浙江是
主要产地。已知膨润土不但可诱发尘肺【ｌ－２１，而且膨 润土成分之一晶体硅已被国际癌症研究中心
USP87细胞毒性体外试验
87 BIOLOGICAL REACTIVITY TESTS, IN VITROThe following tests are designed to determine the biological reactivity of mammalia n cell cultures followi ng con tact with the elastomeric plastics and other polymeric materials with direct or in direct patie nt con tact or of specific extracts prepared from the materials under test. It is essential that the tests be performed on the specified surface area. When the surface area of the specime n cannot be determ in ed, use 0.1 g of elastomer or 0.2 g of plastic or other material for every mL of extraction fluid. Exercise care in the preparation of the materials to preve nt con tam in ati on with microorga nisms and other foreign matter. Three tests are described (i.e., the Agar Diffusi on Test , the Direct Con tact Test and the Elution Test ).1 The decision as to which type of test or the number of tests to be performed to assess the potential biological response of a specific sample or extract depends upon the material, the final product, and its inten ded use. Other factors that may also affect the suitability of sample for a specific use are the polymeric composition; processing and cleaning procedures; con tact ing media; in ks; adhesives; absorptio n, adsorptio n, and permeability of preservatives; and con diti ons of storage. Evaluatio n of such factors should be made by appropriate additi onal specific tests before determining that a product made from a specific material is suitable for its in ten ded use. Materials that fail the in vitro tests are can didates for the in vivotests described in Biological Reactivity Tests, In Vivo 88 .USP R EFERENCE S TANDARDS 11 —USP High-Density Polyethylene RS.USP Positive Bioreaction RS.Cell Culture Preparation —Prepare multiple cultures of L-929 (ATCC cell lineCCL 1, NCTC clone 929; alternative cell lines obtained from a standard repository may be used with suitable validation) mammalian fibroblast cells inserum-suppleme nted minimum esse ntial medium hav ing a seedi ng den sity of about 10 5 cells per mL. Incubate the cultures at 37 1 一in a h±midifiedin cubator for NLT 24 h in a 5 1% c±b on dioxide atmosphere un til amono layer, with greater tha n 80% con flue nee, is obta in ed. Exam ine the prepared cultures un der a microscope to en sure uniform, n ear-c on flue ntmono layers. [NOTE ——The reproducibility of the In Vitro Biological Reactivity Tests depends upon obtaininguniform cell culture density. ]Extractio n Solvents —Sodium Chloride Injectio n (see mono graph —useSodium Chloride Injection containing 0.9% of NaCl). Alternatively, serum-free mammalia n cell culture media or serum-suppleme nted mammalia n cell culture media may be used. Serum suppleme ntati on is used whe n extracti on is done at 37 - for 24 h.Apparatus —Autoclave —Employ an autoclave capable of maintaining a temperature of 121±2 -, equipped with a thermometer, a pressure gauge, a vent cock, a rack adequate to accommodate the test containers above the water level, and a water cooling system that will allow for cooling of the test containers to about20 -, but not below 20 -, immediately following the heating cycle.Oven —Use an ove n, preferably a mecha ni cal con vect ion model, that will maintain operating temperatures in the range of 50 -刁0 within ±.In cubator —Use an in cubator capable of maintaining a temperature of 37 1土and a humidified atmosphere of 5 1% carbon dioxide in air.Extracti on Containers —Use only contain ers, such as ampuls or screw-cap culture test tubes, or their equivale nt, of Type I glass. If used, culture test tubes, or their equivale nt, are closed with a screw cap hav ing a suitable elastomeric liner. The exposed surface of the elastomeric liner is completely protected with an inert solid disk 50 —5 m in thickness. A suitable disk can be fabricated from polytef.Preparati on of Apparatus ——Clea nse all glassware thoroughly with chromic acid clea nsing mixture an d, if n ecessary, with hot n itric acid followed by proIon ged rinsing with Sterile Water for Injectio n. Sterilize and dry by a suitable process for containers and devices used for extracti on, tran sfer, or adm ini strati on of test material. If ethyle ne oxide is used as the steriliz ing age nt, allow NLT 48 h for complete degass ing.Procedure —Preparati on of Sample for Extracts —Prepare as directed in the Procedure under Biological Reactivity Tests, In Vivo 88 .Preparati on of Extracts ——Prepare as directed for Preparati on of Extracts inBiological Reactivity Tests, In Vivo 88 using either Sodium Chloride Injection (0.9% NaCl) or serum-free mammalia n cell culture media asExtraction Solvents. [NOTE —If extraction is done at 37 U for 24 h in an incubator, use cell culture media supplemented by serum. The extraction conditions should not in any instance cause physical changes, such as fusion or melting of the material pieces, other than a slight adherence. ]Agar Diffusi on TestThis test is desig ned for elastomeric closures in a variety of shapes. The agar layer acts as a cushion to protect the cells from mechanical damage while allow ing the diffusi on of leachable chemicals from the polymeric specime ns. Extracts of materials that are to be tested are applied to a piece of filter paper. Sample Preparati on —Use extracts prepared as directed, or use porti ons of the test specime ns hav ing flat surfaces NLT 100 mm in surface area. Positive Control Preparation —Proceed as directed for Sample Preparation . Negative Con trol Preparatio n ——Proceed as directed for Sample Preparati on Procedure —Using 7 mL of cell suspe nsion prepared as directed un der CellCulture Preparati on , prepare the mono layers in plates hav ing a 60-mm diameter. Following incubation, aspirate the culture medium from the mono layers, and replace it withserum-suppleme nted culture medium con tai ning NMT 2% of agar. [NOTE—The quality of the agar must be adequate to support cell growth.The agar layer must be thin enough to permit diffusion of leached chemicals. ] Place the flat surfaces ofSample Preparati on, Negative Con trol Preparati on , and Positive Con trolPreparation or their extracts in an appropriate extracting medium, in duplicate cultures in con tact with the solidified agar surface. Use no more tha n threespecimens per prepared plate. Incubate all cultures for NLT 24 h at 37 1- ,±preferably in a humidified in cubator containing 5 1% of carbb n dioxide.Examine each culture around each Sample, Negative Control , and PositiveControl, under a microscope, using a suitable stain, if desired.In terpretati on of Results ——The biological reactivity (cellular dege nerati on and malformati on) is described and rated on a scale of 0 — (see Table 1). Measurethe responses of the cell cultures to the Sample Preparation , the NegativeControl Preparation , and the Positive Control Preparation . The cell culture test system is suitable if the observed resp on ses to the Negative Con trolPreparation is grade 0 (no reactivity) and to the Positive Control Preparation isat least grade 3 (moderate). The Sample meets the requireme nts of the test if the response to the Sample Preparation is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not con firmed.Table 1. Reactivity Grades for Agar Diffusio n Test and Direct Con tact TestGrade Reactivity Descripti on of Reactivity Zone0 None No detectable zone around or un der specime nSome malformed or dege nerated cells un der1 Slight specime nZone limited to area un der specime n and lesstha n2 Mild 0.45 cm bey ond specime n3 Moderate Zone exte nds 0.45 to 1.0 cm bey ond specime n4 Severe Zone exte nds greater tha n 1.0 cm bey ondspecime nDirect Con tact TestThis test is desig ned for materials in a variety of shapes. The procedure allows for simulta neous extract ion and testi ng of leachable chemicals from the specime n with a serum-suppleme nted medium. The procedure is not appropriate for very low- or high-de nsity materials that could cause mecha ni cal damage to the cells.Sample Preparati on —Use porti ons of the test specime n hav ing flat surfaces2NLT 100 mm in surface area.Positive Control Preparation —Proceed as directed for Sample Preparation . Negative Con trol Preparatio n ——Proceed as directed for Sample Preparati on Procedure —Using 2 mL of cell suspe nsion prepared as directed un der Cell Culture Preparati on , prepare the mono layers in plates hav ing a 35-mm diameter. Following incubation, aspirate the culture medium from the cultures, and replace it with 0.8 mL of fresh culture medium. Place a single Sample Preparation , a Negative Control Preparation , and a Positive Control Preparation in each of duplicate cultures. Incubate all cultures for NLT 24 h at 37 ±1〕in a humidified in cubator containing 5 1% of carb on dioxide.Examine each culture around each Sample, Negative Control , and Positive Control Preparation , under a microscope, using a suitable stain, if desired.In terpretati on of Results —Proceed as directed for In terpretati on of Results under Agar Diffusion Test . The Sample meets the requirements of the test if the response to the Sample Preparation is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not con firmed.Eluti on TestThis test is designed for the evaluation of extracts of polymeric materials. The procedure allows for extract ion of the specime ns at physiological or。
药物毒性测试的新技术与新方法
药物毒性测试的新技术与新方法引言药物毒性测试是新药开发过程中的重要环节，旨在评估药物对人体的安全性和毒性作用。
随着科技的不断进步，新技术和新方法的应用为药物毒性测试带来了革命性的变化。
本文将重点探讨药物毒性测试中的新技术与新方法，并对其应用前景进行展望。
一、体外细胞毒性测试技术1. 细胞毒性传感技术细胞毒性传感技术是通过利用细胞感应器来评估药物对细胞的毒性。
其中包括细胞膜电位检测、细胞色素C释放检测、酶活性测定等技术。
这些技术的优势在于对药物毒性的快速检测，能够提供初步评估药物毒性的信息。
2. 三维细胞培养技术传统的细胞毒性测试通常使用二维细胞培养模型，但这种模型无法完全还原人体内细胞的生理环境。
而三维细胞培养技术可以模拟细胞在体内的生长状态，更加接近真实情况。
近年来，各种三维细胞培养技术的不断发展，为药物毒性测试提供了更准确的结果。
二、体外器官模型技术1. 人工微型器官技术人工微型器官技术包括体外器官芯片和组织工程等技术，能够模拟人体重要器官的功能，如肝脏、心脏、肾脏等。
通过这些模型，可以更好地评估药物在人体内的代谢和毒性作用，提高药物筛选的准确性。
2. 人体器官模型技术人体器官模型技术使用体外培养的真实人体器官组织来评估药物的毒性。
这些模型通常使用捐赠的人体器官组织，通过维持其在体内的微环境，使其保持原有生理功能。
这种技术的优势在于更好地模拟人体内的生理情况，提供更准确的药物毒性信息。
三、体内影像分析技术体内影像分析技术在药物毒性测试中扮演着重要角色。
传统的毒性评估方法依靠人工观察和取材进行，有时难以提供准确的结果。
而体内影像分析技术可以通过利用放射性同位素、磁共振成像等技术，实时观察药物在动物体内的分布和代谢情况。
这种技术可以提供更直观、客观的药物毒性信息。
四、计算机辅助预测技术计算机辅助预测技术利用计算机模型和算法，对药物的毒性进行预测和评估。
这种技术能够通过大量的数据和先进的算法，快速评估药物的潜在毒性，为药物研发提供参考。
USP 87 细胞毒性 体外试验
87BIOLOGICAL REACTIVITY TESTS, IN VITROThe following tests are designed to determine the biological reactivity of mammalian cell cultures following contact with the elastomeric plastics and other polymeric materials with direct or indirect patient contact or of specific extracts prepared from the materials under test. It is essential that the tests be performed on the specified surface area. When the surface area of the specimen cannot be determined, use 0.1 g of elastomer or 0.2 g of plastic or other material for every mL of extraction fluid. Exercise care in the preparation of the materials to prevent contamination with microorganisms and other foreign matter.Three tests are described (i.e., the Agar Diffusion Test, the Direct Contact Test, and the Elution Test).1 The decision as to which type of test or the number of tests to be performed to assess the potential biological response of a specific sample or extract depends upon the material, the final product, and its intended use. Other factors that may also affect the suitability of sample for a specific use are the polymeric composition; processing and cleaning procedures; contacting media; inks; adhesives; absorption, adsorption, and permeability of preservatives; and conditions of storage. Evaluation of such factors should be made by appropriate additional specific tests before determining that a product made from a specific material is suitable for its intended use. Materials that fail the in vitro tests are candidates for the in vivotests described in Biological Reactivity Tests, In Vivo 88.USP R EFERENCE S TANDARDS 11— USP High-Density Polyethylene RS. USP Positive Bioreaction RS.Cell Culture Preparation— Prepare multiple cultures of L-929 (ATCC cell line CCL 1, NCTC clone 929; alternative cell lines obtained from a standard repository may be used with suitable validation) mammalian fibroblast cells inserum-supplemented minimum essential medium having a seeding density of about 105 cells per mL. Incubate the cultures at 37 ± 1in a humidified incubator for NLT 24 h in a 5 ± 1% carbon dioxide atmosphere until a monolayer, with greater than 80% confluence, is obtained. Examine the prepared cultures under a microscope to ensure uniform, near-confluent monolayers. [NOTE—The reproducibility of the In Vitro Biological Reactivity Tests depends upon obtaining uniform cell culture density. ]Extraction Solvents—Sodium Chloride Injection (see monograph—use Sodium Chloride Injection containing 0.9% of NaCl). Alternatively, serum-free mammalian cell culture media or serum-supplemented mammalian cell culture media may be used. Serum supplementation is used when extraction is done at 37for 24 h.Apparatus—Autoclave— Employ an autoclave capable of maintaining a temperature of 121 ± 2, equipped with a thermometer, a pressure gauge, a vent cock, a rack adequate to accommodate the test containers above the water level, and a water cooling system that will allow for cooling of the test containers to about 20, but not below 20, immediately following the heating cycle.Oven— Use an oven, preferably a mechanical convection model, that will maintain operating temperatures in the range of 50–70within ± 2. Incubator— Use an incubator capable of maintaining a temperature of 37 ± 1 and a humidified atmosphere of 5 ± 1% carbon dioxide in air.Extraction Containers— Use only containers, such as ampuls or screw-cap culture test tubes, or their equivalent, of Type I glass. If used, culture test tubes, or their equivalent, are closed with a screw cap having a suitable elastomeric liner. The exposed surface of the elastomeric liner is completely protected withan inert solid disk 50–75 µm in thickness. A suitable disk can be fabricated from polytef.Preparation of Apparatus— Cleanse all glassware thoroughly with chromic acid cleansing mixture and, if necessary, with hot nitric acid followed by prolonged rinsing with Sterile Water for Injection. Sterilize and dry by a suitable process for containers and devices used for extraction, transfer, or administration of test material. If ethylene oxide is used as the sterilizing agent, allow NLT 48 h for complete degassing.Procedure—Preparation of Sample for Extracts— Prepare as directed in the Procedureunder Biological Reactivity Tests, In Vivo 88.Preparation of Extracts— Prepare as directed for Preparation of Extracts inBiological Reactivity Tests, In Vivo 88using either Sodium Chloride Injection (0.9% NaCl) or serum-free mammalian cell culture media as Extraction Solvents. [NOTE—If extraction is done at 37for 24 h in an incubator, use cell culture media supplemented by serum. The extraction conditions should not in any instance cause physical changes, such as fusion or melting of the material pieces, other than a slight adherence. ]Agar Diffusion TestThis test is designed for elastomeric closures in a variety of shapes. The agar layer acts as a cushion to protect the cells from mechanical damage while allowing the diffusion of leachable chemicals from the polymeric specimens. Extracts of materials that are to be tested are applied to a piece of filter paper. Sample Preparation— Use extracts prepared as directed, or use portions of the test specimens having flat surfaces NLT 100 mm2 in surface area. Positive Control Preparation— Proceed as directed for Sample Preparation. Negative Control Preparation— Proceed as directed for Sample Preparation.Procedure— Using 7 mL of cell suspension prepared as directed under Cell Culture Preparation, prepare the monolayers in plates having a 60-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with serum-supplemented culture medium containing NMT 2% of agar. [NOTE—The quality of the agar must be adequate to support cell growth. The agar layer must be thin enough to permit diffusion of leached chemicals. ] Place the flat surfaces ofSample Preparation, Negative Control Preparation, and Positive Control Preparation or their extracts in an appropriate extracting medium, in duplicate cultures in contact with the solidified agar surface. Use no more than three specimens per prepared plate. Incubate all cultures for NLT 24 h at 37 ± 1, preferably in a humidified incubator containing 5 ± 1% of carbon dioxide. Examine each culture around each Sample, Negative Control, and Positive Control, under a microscope, using a suitable stain, if desired.Interpretation of Results— The biological reactivity (cellular degeneration and malformation) is described and rated on a scale of 0–4 (see Table 1). Measure the responses of the cell cultures to the Sample Preparation, the Negative Control Preparation, and the Positive Control Preparation. The cell culture test system is suitable if the observed responses to the Negative Control Preparation is grade 0 (no reactivity) and to the Positive Control Preparation is at least grade 3 (moderate). The Sample meets the requirements of the test if the response to the Sample Preparation is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not confirmed. Table 1. Reactivity Grades for Agar Diffusion Test and Direct Contact TestGrade Reactivity Description of Reactivity Zone0 None No detectable zone around or under specimen1 Slight Some malformed or degenerated cells under specimen2 Mild Zone limited to area under specimen and less than 0.45 cm beyond specimen3 Moderate Zone extends 0.45 to 1.0 cm beyond specimen4 Severe Zone extends greater than 1.0 cm beyondGrade Reactivity Description of Reactivity ZonespecimenDirect Contact TestThis test is designed for materials in a variety of shapes. The procedure allows for simultaneous extraction and testing of leachable chemicals from the specimen with a serum-supplemented medium. The procedure is not appropriate for very low- or high-density materials that could cause mechanical damage to the cells.Sample Preparation— Use portions of the test specimen having flat surfaces NLT 100 mm2 in surface area.Positive Control Preparation— Proceed as directed for Sample Preparation. Negative Control Preparation— Proceed as directed for Sample Preparation. Procedure— Using 2 mL of cell suspension prepared as directed under Cell Culture Preparation, prepare the monolayers in plates having a 35-mm diameter. Following incubation, aspirate the culture medium from the cultures, and replace it with 0.8 mL of fresh culture medium. Place a single Sample Preparation, a Negative Control Preparation, and a Positive Control Preparation in each of duplicate cultures. Incubate all cultures for NLT 24 h at 37 ± 1in a humidified incubator containing 5 ± 1% of carbon dioxide. Examine each culture around each Sample, Negative Control, and Positive Control Preparation, under a microscope, using a suitable stain, if desired. Interpretation of Results— Proceed as directed for Interpretation of Results under Agar Diffusion Test. The Sample meets the requirements of the test if the response to the Sample Preparation is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not confirmed.Elution TestThis test is designed for the evaluation of extracts of polymeric materials. The procedure allows for extraction of the specimens at physiological ornonphysiological temperatures for varying time intervals. It is appropriate for high-density materials and for dose-response evaluations.Sample Preparation— Prepare as directed in Preparation of Extracts, using either Sodium Chloride Injection (0.9% NaCl) or serum-free mammalian cell culture media as Extraction Solvents. If the size of the Sample cannot be readily measured, a mass of NLT 0.1 g of elastomeric material or 0.2 g of plastic or polymeric material per mL of extraction medium may be used. Alternatively, use serum-supplemented mammalian cell culture media as the extracting medium to simulate more closely physiological conditions. Prepare the extracts by heating for 24 h in an incubator containing 5 ± 1% of carbon dioxide. Maintain the extraction temperature at 37 ± 1, because higher temperatures may cause denaturation of serum proteins.Positive Control Preparation— Proceed as directed for Sample Preparation. Negative Control Preparation— Proceed as directed for Sample Preparation. Procedure— Using 2 mL of cell suspension prepared as directed under Cell Culture Preparation, prepare the monolayers in plates having a 35-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with extracts of the Sample Preparation, Negative Control Preparation, or Positive Control Preparation. The serum-supplemented and serum-free cell culture media extracts are tested in duplicate without dilution (100%). The Sodium Chloride Injection extract is diluted withserum-supplemented cell culture medium and tested in duplicate at 25% extract concentration. Incubate all cultures for 48 h at 37 ± 1in a humidified incubator preferably containing 5 ± 1% of carbon dioxide. Examine each culture at 48 h, under a microscope, using a suitable stain, if desired. Interpretation of Results— Proceed as directed for Interpretation of Results under Agar Diffusion Test but use Table 2. The Sample meets the requirements of the test if the response to the Sample Preparation is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability ofthe system is not confirmed. For dose-response evaluations, repeat the procedure, using quantitative dilutions of the sample extract.Table 2. Reactivity Grades for Elution TestGrade Reactivity Conditions of All Cultures0 None Discrete intracytoplasmic granules; no cell lysis1 Slight Less than or equal to 20% of the cells are round, loosely attached, and without intracytoplasmic granules; occasional lysed cells are present2 Mild Greater than 20% to less than or equal to 50% of the cells are round and devoid of intracytoplasmic granules; no extensive cell lysis and empty areas between cells3 Moderate Greater than 50% to less than 70% of the cell layers contain rounded cells or are lysed4 Severe Nearly complete destruction of the cell layers1Further details are given in the following publications of the American Society for Testing and Materials, 1916 Race St., Philadelphia, PA 19103: “Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity,” ASTM Designat ion F 895-84; “Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices,” ASTM Designation F 813-83.Auxiliary Information—Please check for your question in the FAQs before contacting USP.Topic/Question Contact Expert CommitteeGeneral Chapter Desmond G. Hunt,Ph.D.Senior ScientificLiaison(301) 816-8341 (GCPS2010) General Chapters - Packaging Storage and DistributionReference Standards RS Technical Services1-301-816-8129 rstech@USP38–NF33 Page 156 Pharmacopeial Forum: Volume No. 38(2)（专业文档是经验性极强的领域，无法思考和涵盖全面，素材和资料部分来自网络，供参考。
体外细胞的实验报告(3篇)
第1篇一、实验目的本实验旨在研究体外细胞培养技术，通过观察细胞在不同条件下的生长、形态变化和生物学特性，探讨细胞培养方法在生物学研究中的应用。
二、实验材料与仪器1. 材料：- 人胚胎肾细胞（HEK293细胞）- DMEM培养基- 胎牛血清（FBS）- 0.25%胰蛋白酶- 10%胎牛血清- 无菌培养皿- 移液器- 吸管- 显微镜- 紫外可见分光光度计2. 仪器：- 培养箱- 恒温培养箱- CO2培养箱三、实验方法1. 细胞培养：- 将HEK293细胞接种于无菌培养皿中，置于37℃、5%CO2的培养箱中培养。
- 每2-3天更换一次培养基。
2. 细胞传代：- 当细胞生长至80%-90%时，用0.25%胰蛋白酶消化细胞，收集细胞。
- 将收集到的细胞用DMEM培养基稀释至所需浓度，接种于新的培养皿中。
3. 细胞形态观察：- 使用显微镜观察细胞在不同培养时间下的形态变化。
- 拍照记录细胞形态。
4. 细胞活力检测：- 使用MTT法检测细胞活力。
- 将细胞接种于96孔板中，培养24小时后，加入MTT溶液，继续培养4小时。
- 使用酶标仪检测吸光度值。
5. 细胞增殖实验：- 将细胞接种于培养皿中，培养不同时间后，收集细胞，进行细胞计数。
- 计算细胞增殖率。
四、实验结果1. 细胞形态观察：- 初始接种的细胞呈梭形，细胞间连接紧密。
- 随着培养时间的延长，细胞逐渐增多，细胞间连接变稀疏，部分细胞出现变圆、萎缩等现象。
2. 细胞活力检测：- 细胞活力随培养时间的延长而降低。
3. 细胞增殖实验：- 细胞增殖率随培养时间的延长而降低。
五、实验讨论本实验成功培养了HEK293细胞，并通过观察细胞形态、细胞活力和细胞增殖实验，初步了解了细胞在不同条件下的生物学特性。
细胞培养技术在生物学研究中具有重要意义，可以用于研究细胞生物学、分子生物学、药理学等领域。
六、实验结论1. 体外细胞培养技术可以成功培养HEK293细胞。
2. 细胞在不同培养条件下表现出不同的生物学特性。
医疗器械生物学评价体外细胞毒性试验
【分享】医疗器械生物学评价-体外细胞毒性试验什么是体外细胞毒性试验？体外细胞毒性试验是一种在离体状态下模拟生物体生长环境，检测医疗器械及生物材料接触机体组织后所发生的细胞溶解、抑制细胞生长和其他毒性作用的体外试验。
体外细胞毒性试验是医疗器械生物学评价体系中最重要的检测指标之一，几乎也是医疗器械及生物材料临床应用前的必选项目。
哪些产品需要进行体外细胞毒性试验？与人体接触或植入体内的医疗器械都需要进行细胞毒性试验。
与人体接触的部位包括：1）表面：皮肤，粘膜，损伤表面。
2）外部接入：组织/骨/牙，循环血液。
3）体内植入：组织/骨，血液。
体外细胞毒性试验的目的和意义目的：评级医疗器械和生物材料致细胞毒性反应的潜在性，并预测最终生物体应用时的组织细胞反应。
通过体外细胞培养技术，可检测供试品接触细胞后细胞发生生长抑制、功能改变、细胞溶解、死亡或其他毒性反应。
意义：可在短时间内较经济、简便地筛选出批量供试品的细胞毒性，它为动物试验的进行与否提供了先决条件，对新型医疗器械及生物材料的研制和应用提供了重要保证。
体外细胞毒性试验依据的相关标准o ISO 10993-5:2009 Biological Evaluation of MedicalDevices -- Part 5: Tests for in vitro Cytotoxicityo GB/T16886.5-2007医疗器械生物学评价第5部分：体外细胞毒性试验，GB/T16886是由ISO 10993转化过来的标准o GB/T 16175-2008 医用有机硅材料生物学评价试验方法o GB/T 14233.2-2005 医用输液、输血、注射器具检验方法第2部分：生物试验方法o YY/T0127.9-2009口腔医疗器械生物学评价第2单元试验方法细胞毒性试验：琼脂扩散法及滤膜扩散法细胞系和培养基的选择优先采用已建立的细胞系并从认可的贮源获取。
试验只能使用无支原体污染细胞，使用前应该检测原代培养细胞是否存在支原体。
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3.1.1 近十年中文期刊论文分布列表 .................................................2 3.1.2 中文期刊论文增长趋势 .............................................................3 3.1.3 发文较多期刊 .............................................................................4 3.1.4 发文较多的机构 .........................................................................4 3.1.5 发文较多的人物 .........................................................................4 3.1.6 核心期刊分..............................4 3.1.7 最近相关中文期刊论文 ..............................................................5 3.1.8 被引较多的相关期刊论文 ..........................................................6 3.2 学位论文 ................................................................................................7 3.2.1 近十年学位论文年代分布列表 .................................................7 3.2.2 学位论文增长趋势 .....................................................................8 3.2.3 硕博学位论文数量对比 .............................................................9 3.2.4 发文较多的机构 .........................................................................9 3.2.5 发文较多的人物 .........................................................................9 3.2.6 最近相关学位论文 ...................................................................10 3.3 中文会议论文 ......................................................................................10 3.3.1 近十年中文会议论文年代分布列表 .......................................10 3.3.2 中文会议论文增长趋势 ...........................................................11 3.3.3 中文会议论文主办单位分布 ...................................................12 3.3.4 发文较多的机构 .......................................................................12 3.3.5 发文较多的人物 ........................................................................13 3.3.6 最近相关中文会议论文 ............................................................13 3.4 外文期刊论文 ......................................................................................14 3.4.1 近十年外文期刊论文年代分布列表 .......................................14 3.4.2 外文期刊论文增长趋势 ...........................................................14 3.4.3 最近相关外文期刊论文 ...........................................................15 3.5 外文会议论文.......................................................................................15
创新助手报告 ——主题分析报告
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北京万方软件股份有限公司
2014-07-11
报告目录
报告核心要素......................................................................................................... I 一、主题简介........................................................................................................ 1 二、主题相关科研产出总体分析........................................................................ 1