ASTM F1903-2018 Standard Practice for Testing for Cellular Responses to Particles in vitro.pdf

1、Designation: F1903 18Standard Practice forTesting for Cellular Responses to Particles in vitro1This standard is issued under the fixed designation F1903; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A

2、number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers the assessment of cellular responsesto wear particles and degradation products from implantedmaterials that may le

3、ad to a cascade of biological responsesresulting in damage to adjacent and remote tissues. In order toascertain the role of particles in stimulating such responses, thenature of the responses, and the consequences of the responses,established protocols are needed. This is an emerging, rapidlydevelop

4、ing area, and the information gained from standardprotocols is necessary to interpret cellular responses to par-ticles and to determine if these correlate with in vivo responses.Since there are many possible and established ways of deter-mining responses, a single standard protocol is not stated.How

5、ever, well described protocols are needed to compareresults from different investigators using the same materialsand to compare biological responses for evaluating (ranking)different materials. For laboratories without establishedprotocols, recommendations are given and indicated with anasterisk (*)

6、.1.2 Since the purpose of the following test procedures is topredict the response in human tissues, the use of human(preferably macrophage lineage) cells is recommended.However, the use of non-macrophage cell lineage or the use ofcells from non-human and non-primate sources may be accept-able. The s

7、ource of the cells or the cell line used should bejustified based on the cellular responses under test and/or tissueof interest. Non-human cells should not be used if there isevidence of possible cross-species difference for specific testresults as the results of this in vitro test may not correspon

8、d toactual human response.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this

9、standard to establish appro-priate safety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.1.5 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decisi

10、on on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2F619 Practice for Extraction of Medical PlasticsF748 Practice for Selecting Generic

11、 Biological Test Methodsfor Materials and DevicesF1877 Practice for Characterization of Particles3. Summary of Practice3.1 Cellular responses to particles may be evaluated usingspecimens from animals being tested according to the PracticeF748 matrix for irritation and sensitivity, or for implantatio

12、n.Blood, organs, or tissues from the animals may be used.3.2 Cellular responses to particles may be evaluated usingmaterials or extracts according to Practice F619. These mate-rials or extracts may be used for in vivo tests or for the in vitrotests. Particles generated by methods (for example, deriv

13、edfrom in vitro mechanical testing or retrieved from ex vivoperi-implant tissues either from clinical retrievals or animalmodels) may also be used as long as they have characteristicssimilar to those produced by the implant or device being testedwith appropriate justification.3.3 The purpose of this

14、 practice is to assess the response ofcells in direct contact with particles and therefore, this practiceis primarily intended to cover the testing of particles placedinto culture with the cells. This practice should be equallyappropriate for the testing of the response to nanoparticlesplaced in cul

15、ture, if particles of that size are the particles ofinterest. The size range of particles (among other particlecharacteristics) should be clearly defined and stratification of1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct

16、responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Oct. 1, 2018. Published October 2018. Originallyapproved in 1998. Last previous edition approved in 2010 as F1903 10. DOI:10.1520/F1903-18.2For referenced ASTM standards, visit the ASTM website, www.astm.o

17、rg, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis internati

18、onal standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committ

19、ee.1the test results based on the particle size and other character-istics is recommended.4. Significance and Use4.1 This practice is to be used to help assess the biocom-patibility of materials used in medical devices. It is designed totest the effect of particles released from medical devices andb

20、iomaterials on macrophages or other cells.4.2 The appropriateness of the methods should be carefullyconsidered by the user since not all materials or applicationsneed to be tested by this practice.4.3 Abbreviations:4.3.1 FCS (FBS)Fetal Calf Serum (Fetal Bovine Serum)4.3.2 FGFsFibroblast Growth Facto

21、rs4.3.3 HBSSHanks Balanced Salt Solution4.3.4 HEPESA buffering salt (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)4.3.5 IL17Interleukin 174.3.6 IL18Interleukin 184.3.7 IL1Interleukin 1 beta4.3.8 IL6Interleukin 64.3.9 IL8Interleukin 84.3.10 LALLimulus Amebocyte Lysate4.3.11 LPSlipopolysaccharid

22、e (endotoxin)4.3.12 MCP1Monocyte Chemotactic Protein-14.3.13 MMPsMatrix Metalloproteinases4.3.14 NONitric Oxide4.3.15 PBSPhosphate Buffered Saline4.3.16 PGE2Prostaglandin E24.3.17 RPMI 1640Specific Growth Medium (RoswellPark Memorial Institute)4.3.18 TGFTransforming growth factor beta4.3.19 TNFTumor

23、 Necrosis Factor alpha4.3.20 VEGFVascular Endothelial Growth Factor5. Responses from Cells Grown in vitro5.1 ParticlesDefine the nature of the particles used (seePractice F1877 for detailed particle characterization method-ology):5.1.1 Source;5.1.2 Chemical composition;5.1.3 Size (mean and range, re

24、fer to Practice F1877 foradditional information regarding how size should be deter-mined);5.1.4 Shape;5.1.5 Method of sterilization;5.1.6 If the presence of bacterial lipopolysaccharide (LPS)was determined, specify how this was done and the sensitivityof the method (LAL testing with a sensitivity of

25、 at least 0.06endotoxin units (EU) is recommended, see X1.5);5.1.7 Concentration of particles used as weight, number, orsurface area/106 cells;5.1.8 Surface charge (if known);5.1.9 Since some particles may have a density less than thatof the culture medium being used, careful consideration shouldbe

26、given to determining appropriate procedures for ensuringthat the particles and cells come into contact with each other.Techniques in which cells are grown on a substrate and then thesubstrate is inverted in culture may be appropriate.5.2 CellsDefine the nature of the cells used:5.2.1 Established Cel

27、l Lines (if not, go to 5.2.2)The use ofestablished cell lines provides a known cell type with areproducible response. Although the correlation with the invivo system may not be known at this time, careful studies withestablished cell lines could eventually allow determination ofcorrelation between i

28、n vivo and in vitro systems.5.2.1.1 Specify source of cell and identifying number orcode.5.2.1.2 Specify type of cells.5.2.1.3 Specify special attributes of the cell line.5.2.1.4 *Human macrophages such as U-937 are recom-mended; however, non-human murine macrophages such asRAW 264.7, J774A.1, P388D

29、1, or IC-21 may be used.5.2.2 Primary Isolate:5.2.2.1 Specify source of cells including species and tissueorigin (e.g., human, alveolar).5.2.2.2 Specify type of cells.5.2.2.3 Specify mechanism of isolation (e.g., lavage, enzy-matic digestion).5.2.2.4 Specify if stimulant used and if so, which one (e

30、.g.,mineral oil).5.2.2.5 *Strain, age, and sex used should be specified; use ofanimals of both sexes should be considered for tests involvingin vivo models.5.3 Culture Conditions:5.3.1 Specify source and type of medium. If from a com-mercial source, provide the catalog and/or reference number. Ifnot

31、 from a commercial source, provide a list of ingredients andtheir sources.5.3.2 Specify source of serum, and whether it was heat-inactivated. If the presence of LPS was determined, specify theamount present as well as the method and sensitivity of themethod.5.3.3 Specify culture conditions (for exam

32、ple, 37C,humidified, 5 % CO2incubator).5.3.4 Specify when and how the particles were added to thesystem.5.3.5 Specify time of culture duration, test exposure, and/ortime course of sampling of the culture medium.5.3.6 If cell counts (in units of cell number/mL) weredetermined, specify as to when and

33、how (for example,hemocytometer, Coulter counter).5.3.6.1 *Cells should be cultured using the culture mediumand serum specified/recommended by the supplier. LPS levelsare generally provided or available from the distributor. Rec-ommended culture conditions are 37C, with 5 % CO2,inahumidified incubato

34、r. Cell counts at the time of initial platingand at the termination of the culture are recommended. Foradherent cell populations, cells should be detached from theculture surface and suspended either by gentle pipetting and/orscraping, or by using a cell dissociation solution, depending onthe cell s

35、upplier recommendations. After the cells areresuspended, they should be washed with Ca- and Mg-freePBS, HBSS, or culture medium. During counting with ahemocytometer, it may be helpful to ascertain the percentage ofF1903 182dead cells in the population using trypan blue viability staining.The respons

36、es of the macrophages exposed to particles forspecified time periods should be assayed as described in 5.4.5.3.7 Controls:5.3.7.1 Cells not stimulated with particles, but otherwisecultured under the same experimental culture conditions,should be maintained in parallel with test condition(s), positiv

37、econtrol, and/or reference standard.5.3.7.2 Polystyrene (PS) particles may be used as a refer-ence control. The choice of control may be affected by the sizerange, chemical composition, and other characteristics ofparticles of interest (e.g., special consideration should be givenif nanoparticles are

38、 relevant to the device).5.3.7.3 LPS is a known cell stimulant and may be used as apositive control for certain immune responses. A concentrationbetween 0.25 and 1 ng/mL of culture medium is sufficient.5.3.7.4 Culture medium is a recommended diluent for theassays.5.3.8 Test Conduct:5.3.8.1 Culture c

39、ells for a dose ranging study to determinethe Lethal Concentration (LC50) and investigate dosing effectson cellular uptake of particles and corresponding cellularresponse.5.3.8.2 Record when and how particles are added to the testsystem, and explain how the doses for the study were chosen toinclude

40、how the dosing concentration compares to a proposedclinical dose, if applicable.5.3.8.3 Determine which subset of cellular responses iden-tified in 5.4 will be chosen for inclusion in your study anddocument the rationale for the selection.5.3.8.4 Items to include in test reports:(1) Description of t

41、he particle sample;(2) Justification of the choice of cells and cell source(s);(3) Assay method(s) and rationale;(4) Description of controls (including positive, negative,reference particles);(5) Assay response and other observations.5.4 Products or Responses Determined:5.4.1 Particle Uptake Determi

42、nationSpecial attentionshould be paid to in vitro analysis of the uptake of particles bymacrophages (with the results stratified by the size and otherparticle characteristics), since the internalization and subcellu-lar localization of particles could pre-determine the subsequentcell survival and cy

43、tokine release.5.4.1.1 For metallic particles, uptake is most easilyascertained, and may be assessed using optical emissionspectroscopy (ICP-MS/OES) or various microscopy tech-niques (e.g., transmission electron microscopy or confocalmicroscopy).5.4.1.2 For polymeric particles, quantitative assessme

44、nt ofuptake is more difficult, and so qualitative assessment may beperformed, such as by visualizing uptake using various micros-copy techniques (e.g., transmission electron microscopy orconfocal microscopy).5.4.2 Cell death and viability can be determined by severalmethods including, but not limite

45、d to:5.4.2.1 The total cell counts and viable cell counts withtrypan blue dye exclusion, neutral red uptake, etc.;5.4.2.2 Metabolic assays such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide);5.4.2.3 Low cytometry with vital stains such as calcein AMfluorescence/propidium iodid

46、e exclusion, etc.;5.4.2.4 The uptake of DNA precursors;5.4.2.5 The total DNA or RNA content of cell populations.Special consideration should be given to determine the type(s)of cell death, including apoptosis (e.g., from Annexin Vstaining) and necrosis (e.g., propidium iodide exclusion).5.4.3 Solubl

47、e Cell Products Elaborated:5.4.3.1 The products indicative of particle-induced cellularresponses may include growth factors (such as TGF, VEGF,and FGFs), collagenases, matrix metalloproteinases (MMPs),chemokines (such as MCP1), cytokines (such as TNF),interleukins (such as IL1, and other IL1 pathway

48、 members,IL6, IL8, IL17, and IL18), other pro-inflammatory mediators(such as PGE2), reactive oxygen species (such as superoxide),and nitric oxide (NO). Furthermore, the choice of inflammatorymediators should include more than one subcategory (e.g., ILsand NO, or MMPs and ILs) and should be justified

49、 in referenceto the particles under review and their possible biologicaleffects based on prior evidence from predicate biomaterials. Asan example, if tissue remodeling (e.g., soft tissue overgrowthor bone destruction) could be expected, inclusion of mediatorssuch as growth factors and MMPs/collagenases is particularlyrecommended. Other products detected and the method used todetect them should be specified.5.4.3.2 The time in culture should be specified since theseproducts peak at different times. The time in culture and thechoice of corresponding m