1、Designation: F3088 14Standard Test Method forUse of a Centrifugation Method to Quantify/Study Cell-Material Adhesive Interactions1This standard is issued under the fixed designation F3088; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revi
2、sion, the year of last revision. A 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 test method covers a centrifugation cell adhesionassay that can be used to detect changes in ad
3、hesive charac-teristics of cells with passage or treatments. This approachmeasures the force required to detach cells from a substrate.Adhesion, among many variables, may vary due to changes inthe phenotype of the cells.1.2 This test method does not cover methods to verify theuniformity of coating o
4、f surfaces, nor does it cover methods forcharacterizing surfaces.1.3 The cells may include adult, progenitor or stem cellsfrom any species. The types of cells may include chondrocytes,fibroblasts, osteoblast, islet cells, or other relevant adherent celltypes.1.4 This test method does not cover metho
5、ds for isolating orharvesting of cells. This test method does not cover testmethods to quantitate changes in gene expression, or changesin biomarker type or concentration, as identified by immunos-taining. Nor does this test method cover quantitative imageanalysis techniques.1.5 This standard does n
6、ot purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standar
7、ds:2F2603 Guide for Interpreting Images of Polymeric TissueScaffoldsF2664 Guide for Assessing the Attachment of Cells toBiomaterial Surfaces by Physical MethodsF2739 Guide for Quantitating Cell Viability Within Bioma-terial ScaffoldsF2944 Test Method for Automated Colony Forming Unit(CFU) AssaysImag
8、e Acquisition and Analysis Methodfor Enumerating and Characterizing Cells and Colonies inCulture3. Summary of Test Method3.1 Centrifugation Cell Assay on Cell PopulationsA con-ventional centrifuge can be used to apply a normal or shearforce to cells depending on the orientation of the cells withresp
9、ect to the centrifugal force (1-3).3The centrifugal forcethat the cells are subject to can be calculated according to thefollowing formula assuming that the plates are 90 (normal) tothe spindle of the centrifuge.FD5 pcell2 pmedium!VcellRCF (1)where:FD= detachment force applied per cell,Vcell= cell v
10、olume,pcell= density of the cell,pmedium= the density of the surrounding medium, andRCF = Relative Centrifugal Forces = r2, where r =centrifugation radius and= centrifugation speed.See X1.6 for example of a calculation of “detachment forceper cell.”3.1.1 Such tests are easy to conduct and the result
11、s representa population average. The method can be performed in amoderately outfitted cell biology lab with an inverted micro-scope or a fluorescence microplate reader and a centrifuge witha microtiter plate holder. Three factors need to be consideredwhen using this methodology: the potential influe
12、nces of forcesapplied during the period of spin up time, the maximum spinspeed, and the test duration. The method is only able tocorrelate with cell detachment with the maximum force appliedafter the centrifuge has reached its set spin speed. It should alsobe considered that the maximum force that c
13、an be applied islimited by the centrifuge, sample configuration (that is, con-figuration and type of multiwell plate) and time of centrifuga-tion. In some instances, the cell adhesion strength will be1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials an
14、d Devices and is the direct responsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved May 1, 2014. Published June 2014. DOI: 10.1520/F3088-14.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service
15、at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3The boldface numbers in parentheses refer to the list of references at the end ofthis standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, W
16、est Conshohocken, PA 19428-2959. United States1greater than the applied forces. Therefore, for certain cell typesthat have attached for extended periods of time, the methodmay not be able to generate forces sufficient to detach thesecells.3.1.2 Cell attachment is a complex, time-dependent, processin
17、volving significant morphological and structural changes ofthe cell and deposition of a bed of extracellular matrix and isa function of cellular and substrate parameters. Cell adhesionto a surface depends on a range of biological factors thatinclude culture history, species, the source and origin of
18、 thecells, embryological status of the cells (adult, progenitor,stem), histological types of cells (chondrocytes, fibroblasts,osteoblast, islet cells, etc.), purity, passage number, populationdoublings, and time after trypsinization. The adhesive strengthalso depends on the chemistry, surface chemis
19、try/topography,and morphology of the underlying surface and adsorbedproteins or engineered ligands and, most importantly, time ofadhesion.4. Significance and Use4.1 This test method describes a cell adhesion method thatcan be used to provide a detachment percent at a given RCF forcells that have adh
20、ered to a substrate, typically for a short time.The information generated by this test method can be used toobtain a semi-quantitative measurement of the adhesion ofcells to either an uncoated or pre-coated substrate, whencompared to a reference (adherent) cell type on the samesubstrate. As describe
21、d in Reyes and Garcia (2003), it isrecommended that the 50 % point be used for either ligandconcentration or RCF for the most robust measurement ofadhesion strength. The adhesion may vary due to changes inthe phenotype of the cells or as a result of the specificproperties of the surface. The substra
22、te may include tissueculture-treated polystyrene, biomaterials or bioactive surfaces.If the substrate is a hydrogel, care must be taken to avoidcohesive failure in the hydrogel (that is, detached cells havepulled away fragments of gel). The coating may consist of (butis not limited to) the following
23、: natural or syntheticbiomaterials, hydrogels, components of extracellular matrix(ECM), ligands, adhesion or bioactive molecules, genes orgene products. Cell concentration is also critical, as use of toohigh a concentration of cells may result in cells detaching as asheet, rather than as individual
24、cells. This centrifugationapproach, once validated, may be applicable for quality control(QC) and product development. However, until the method iscorrelated to other measures of cell attachment, the currentmethod should be run in parallel with other known measures ofcell adhesion.4.2 This test meth
25、od does not cover test methods to quan-titate changes in gene expression, or changes in biomarkers, asidentified by immunostaining. This test method additionallydoes not cover quantitative image analysis techniques. In somecases the change in adhesive properties may reflect on thedegree of different
26、iation or de-differentiation of the cells.However, it is worth noting that adhesive interactions do notnecessarily reflect the differentiation state of a particular celltype, although in many instances they do. (See X1.3 forapplication to the Adhesion of Chondrocytes.)5. InterferencesNOTE 1The follo
27、wing interferences may result in variable andinconsistent results from this method and care should be taken to minimizethese conditions.5.1 Cell concentration too high in multi-wellsIf the cellconcentration is too high, then the cells may detach as a sheet,instead of as individual cells.5.2 Cells no
28、t evenly distributed in wells (that is, clumping).5.3 Inconsistency in the fluid level within multi-wellsWells must be completely filled with fluid (4).5.4 Inconsistency in well coating and blocking protocolsWhere possible, a negative substrate control (that is, non-tissueculture-treated polystyrene
29、, or similar control which has beenshown to limit cell adhesion) should be included.5.5 Inconsistency in pipetting forces when washing wells.5.6 Air bubbles in well prior to, or forming during, centrifu-gation. The air bubbles can result from improper pipetting offluid within the wells, or an improp
30、er fluid level in the wellsprior to sealing the top of the wells with an adhesive cover (thatis, “reverse meniscus” not formed in well prior to sealing withacetate sealing tape).5.7 Incomplete washing of wells to remove loosely adherentor dead cells.5.8 Cross-over of fluorescent signal between adjac
31、ent wellsin clear or translucent multi-well plates.5.9 Centrifugation of multiple multi-well plates. A complexstress situation can result from spinning multiple multi-wellplates (that is, stacking plates within a plate holder on thecentrifuge), depending on the orientation of cells with respectto th
32、e centripetal force.5.10 Photobleaching of fluorescence stain. Photobleachingmay be reduced by limiting the exposure of the stained cells toroom light or ultraviolet light.5.11 Long cell adhesion times and variations in adhesiveproteinsUse of long exposure times and/or specific ECM orprotein may req
33、uire adjustment of the protocol to optimize celldetachment conditions. Since detachment forces with thismethod are low, well spread, or strongly attached cells may notbe detached by this method (5).6. Apparatus6.1 Humidified CO2Incubator.6.2 Rocker for Isolated Cells.6.3 Hemocytometer.6.4 Centrifuge
34、.6.5 Plate Adapters for Centrifuging Multi-well Plates.6.6 Multi-well Pipetter.6.7 Inverted Microscope equipped with bright field/phase,as well as fluorescence optics.6.8 Microplate Reader.6.9 Image Acquisition and Image AnalysisSsoftware (that is,Zeiss AxioVision Software, Image J or equivalent).F3
35、088 1427. Reagents and Materials7.1 Cell Type, for adhesion assay, as well as control cell type(if applicable). A positive control cell type should consistentlyexhibit greater than 50 % adhesion on a given substrate, at agiven RCF. Similarly, a negative control cell type shouldconsistently exhibit l
36、ess than 50 % adhesion on a givensubstrate at a given RCF.7.2 Proteins, Extracellular Matrix Ccomponents, Genes orGene Products or Biomaterials for pre-coating wells of multi-well plates.7.3 Medium appropriate for either the cell type of interest orthe control cell type, with or without the addition
37、 of antibiotic/antimycotic solution. Care should be taken to limit/avoidcarryover of serum contents and adhesive proteins from media.Additionally, the trypsin protocol (trypsin concentration andtrypsinization times) should be optimized.7.4 Trypan Blue or equivalent (cell viability).7.5 Multi-well Pl
38、ates (96 well).7.6 Dulbeccos Phosphate Buffered Saline (without calciumand magnesium), (D-PBS).7.7 Fluorescence Stain.7.8 Acetate Sealing Tape, or equivalent (to seal top ofmulti-well plate).7.9 Aluminum Foil (to cover multi-well plate during incu-bation).8. Hazards8.1 Hoechst 33342 fluorescence sta
39、in (possible carcinogen).8.2 Trypan blue dye (possible carcinogen).9. Calibration and Standardization9.1 Calibration of Image SystemAny inverted microscopesystem equipped with appropriate image capture device andimage analysis software may be used for the assay. For thepurposes of illustration in th
40、is standard, we refer to a ZeissAxioVert inverted microscope equipped with an Axio Camdigital camera for image capture and AxioVision software fordata collection/analysis.9.1.1 Using an inverted microscope,55digital tiledimages (tiled overlap 10 %, total tiled image area = 3.177 mm2)are captured for
41、 each of the three wells prior to centrifugation(pre-spin). Approximately 2400 to 3600 cells (800 to 1200cells/well 3 wells) are counted per sample.10. Procedure10.1 Centrifugation Cell Adhesion MethodAschematic ofthe method is shown in Fig. 1. The method was modified fromthe method of Reyes and Gar
42、cia (2003) (2).10.1.1 Wells of a 96 multi-well plate are passively coatedwith protein or matrix protein. The coating concentration willdepend on the ECM protein. The incubation time and incuba-tion temperature (for example, room temperature versus 4Cversus 37C) will vary with the substrate. In the e
43、xample offibronectin, the wells are passively coated for 30 min. to1hatroom temperature.10.1.2 Optimization of Multi-Well Plate CoatingThe fol-lowing procedure is suggested to obtain the optimal well-coating concentration and time of incubation for the coating.First, a series of test runs of the met
44、hod should be performedwhere a broad concentration range (including a no-coating,zero concentration, control) of the desired coating substrate arecoated on separate wells of the multi-well plate for varioustime periods at the appropriate temperature/relative humidity.Note that depending on the speci
45、fic coating or intendedoutcome of the adhesion method, it may be appropriate tocompare multi-well plates with and without “tissue culturetreatment.” A statistically significant number of replicatesshould be run to allow appropriate statistical analysis of thedata. The data from the initial (broad co
46、ncentration) test runwill provide data to re-run the adhesion method using a morerestrictive range for the coating concentration and for the timeof incubation. Appropriate statistical analyses should be per-formed on the data. If an even more specific coating concen-tration is desired, then the proc
47、edure can then be repeated usingdata from the second set of test runs. If desired, incubationtimes for the coating may also be varied. The procedure shallbe repeated for each new coating material to optimize thecoating concentration and the time of incubation for thecoating.FIG. 1 Schematic of Centr
48、ifugation Cell Adhesion AssayF3088 14310.1.3 Remove excess coating solution from the wells. Ablocking step, using heat-denatured serum albumin, is recom-mended if sub-saturating protein densities are used. Theblocking is usually performed prior to protein absorption.Follow by three washes in D-PBS.1
49、0.1.4 Harvest cells and assess for viability using an appro-priate method, such as the trypan-blue dye exclusion method,and a hemocytometer. As the harvesting procedure very likelyinfluences the cell adhesive process, care should be taken whenperforming procedures such as trypsinization to use the lowestconcentration of trypsin/ethylenediamine tetra acetic acid(EDTA) for the shortest time possible. In addition, to limitpotential effects on cell attachment strength, care should betaken to minimize the processing time, during which time thecells are outside of thei
