1、Designation: F2315 11Standard Guide forImmobilization or Encapsulation of Living Cells or Tissue inAlginate Gels1This standard is issued under the fixed designation F2315; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of
2、 last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONEncapsulation in insoluble alginate gel is recognized as a rapid, non-toxic, and versatile method forimmobilization o
3、f macromolecules and cells. Microencapsulated cells or tissue as artificial organs areunder study for treatment of a variety of diseases such as Parkinsons disease, chronic pain, liverfailure, hypocalcemia, and, perhaps the most well-known example, immobilization of islets ofLangerhans utilized as a
4、n artificial pancreas in the treatment of diabetes. Since alginates are aheterogeneous group of polymers with a wide range of functional properties, the success of animmobilization or encapsulation procedure will rely on an appropriate choice of materials andmethodology. This must be based on knowle
5、dge of the chemical composition of alginate and thecorrelation between the structure, composition, and functional properties of the polymer, as well asdifferences in gelation technologies. It is also important to recognize the need for working with highlypurified and well-characterized alginates in
6、order to obtain gels with reproducible properties. The aimof this guide is to provide information relevant to the immobilization or encapsulation of living cellsand tissue in alginate gels.1. Scope1.1 This guide discusses information relevant to the immo-bilization or encapsulation of living cells o
7、r tissue in alginategels. Immobilized or encapsulated cells are suitable for use inbiomedical and pharmaceutical applications, or both, includ-ing, but not limited to, Tissue Engineered Medical Products(TEMPs).1.2 This guide addresses key parameters relevant for suc-cessful immobilization and encaps
8、ulation in alginate gels.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 s
9、tandard to establish appro-priate safety and health practices and determine the applica-bility of regulatory requirements prior to use.2. Referenced Documents2.1 ASTM Standards:2F748 Practice for Selecting Generic Biological Test Meth-ods for Materials and DevicesF1251 Terminology Relating to Polyme
10、ric Biomaterials inMedical and Surgical DevicesF1903 Practice for Testing For Biological Responses toParticles In VitroF1904 Practice for Testing the Biological Responses toParticles in vivoF1905 Practice For Selecting Tests for Determining thePropensity of Materials to Cause Immunotoxicity3F1906 Pr
11、actice for Evaluation of Immune Responses InBiocompatibility Testing Using ELISA Tests, LymphocyteProliferation, and Cell Migration3F2064 Guide for Characterization and Testing of Alginatesas Starting Materials Intended for Use in Biomedical and1This guide is under the jurisdiction of ASTM Committee
12、 F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved March 1, 2011. Published March 2011. Originallyapproved in 2003. Last previous edition approved in 2010 as F2315 10. D
13、OI:10.1520/F2315-11.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn. The last approved version of
14、this historical standard is referencedon www.astm.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Tissue-Engineered Medical Products Application2.2 USP Document:USP Monograph USP 24/NF19 Sodium Alginate42.3 Other Referenced Docum
15、ents:EN-ISO 10993 Biological Evaluation of Medical Devices5International Conference on Harmonization (ICH) S2BGenotoxicity: A Standard Battery for Genotoxicity Test-ing of Pharmaceuticals (July 1997)63. Terminology3.1 Definitions:3.1.1 alginate, npolysaccharide obtained from some ofthe more common s
16、pecies of marine algae, consisting of aninsoluble mix of calcium, magnesium, sodium, and potassiumsalts.3.1.1.1 DiscussionAlginate exists in brown algae as itsmost abundant polysaccharide, mainly occurring in the cellwalls and intercellular spaces of brown seaweed and kelp.Alginates main function is
17、 to contribute to the strength andflexibility of the seaweed plant. Alginate is classified as ahydrocolloid. The most commonly used alginate is sodiumalginate. Sodium alginate and, in particular, calcium cross-linked alginate gels are used in Tissue Engineered MedicalProducts (TEMPs) as biomedical m
18、atrices, controlled drugdelivery systems, and for immobilizing living cells.3.1.2 APA bead, nalginate-poly-L-lysine-alginate bead.3.1.3 encapsulation, na procedure by which biologicalmaterials, such as cells, tissues, or proteins, are enclosed withina microscopic or macroscopic semipermeable barrier
19、.3.1.4 endotoxin, npyrogenic high molar mass li-popolysaccharide (LPS) complex associated with the cell wallof gram-negative bacteria.3.1.4.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endotoxins. Endotoxins are specifically de-tected through a Limulus Amebocyte Lysate (LAL) test.
20、3.1.5 gel, nthe three-dimensional network structure aris-ing from intermolecular polymer chain interactions. Such chaininteractions may be covalent, ionic, hydrogen bond, or hydro-phobic in nature. See also Terminology F1251.3.1.6 immobilization, nthe entrapment of materials, suchas cells, tissues,
21、or proteins within, or bound to, a matrix.3.1.7 pyrogen, nany substance that produces fever.3.2 Additional definitions regarding alginate may be foundin Guide F2064. Additional definitions regarding polymericbiomaterials may be found in Terminology F1251.4. Significance and Use4.1 The main use is to
22、 immobilize, support, or suspendliving cells or tissue in a matrix. The use of an encapsulation/immobilization system may protect cells or tissues fromimmune rejection. When immobilizing biological material inalginate gels, there are numerous parameters that must becontrolled. This guide contains a
23、list of these parameters anddescribes the methods and types of testing necessary toproperly characterize, assess, and ensure consistency in theperformance of an encapsulation system using alginate. Thisguide only covers single gelled beads, coated or not, and notdouble capsules or other constructs.4
24、.2 The alginate gelation technology covered by this guidemay allow the formulation of cells and tissues into biomedicaldevices for use as tissue engineered medical products or drugdelivery devices. These products may be appropriate forimplantation based on supporting biocompatibility and physi-cal t
25、est data. Recommendations in this guide should not beinterpreted as a guarantee of clinical success in any tissueengineered medical product or drug delivery application.5. Gelation Techniques5.1 Most methods for encapsulation of cells or tissue inalginate gels basically involve two main steps. The f
26、irst step isthe formation of an internal phase where the alginate solutioncontaining biological materials is dispersed into small droplets.In the second step, droplets are solidified by gelling or forminga membrane at the droplet surface.5.2 The most simple and common way to produce smallbeads or ca
27、psules is by forming droplets of a solution ofsodium alginate containing the desired biological material(cells, tissues, or other macromolecules) and then exposingthem to a gelling bath. A gelling bath may be a solutioncontaining divalent cross-linking cations such as Ca2+,Sr2+,orBa2+. Monovalent ca
28、tions and Mg2+ions do not inducegelation (34).75.3 Concentration of Ions:5.3.1 The concentration of gelling ions used must be deter-mined based upon factors such as desired gel strength, type ofalginate used (G- or M-rich), and isotonicity of the gellingsolutions. Calcium ion concentrations of from
29、50 to 150 mmare often used.5.3.2 Other gelling ions may be used, such as Ba2+or Sr2+.The concentration of Ba2+in the gelling solution must bedetermined based upon the desired characteristics of the finalgel and on regulatory and toxicological considerations as Ba2+can induce toxic effects in cells.5
30、.3.3 Concentration of Non-gelling IonsVarious additivespresent in the gelling solution that do not participate in theformation of cross-links constitute non-gelling ions. These ionsmay be Na+, which can be used to produce homogeneous gels(see 7.1), ions present in cell culture medium (if present in
31、thegelling bath), and others.6. Formation of Beads6.1 Bead size is one of the most important parameters ofalginate gel beads and capsules in biomedical applications. Theappropriate size will often be a compromise. The bead itselfmust be large enough to contain the biological material. Largerbeads ar
32、e also easier to handle during washing or othertreatments. In many applications involving cells, the cells4Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852-1790, http:/www.usp.org.5Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor,
33、 New York, NY 10036, http:/www.ansi.org.6Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758,1211 Geneva 13, Switzerland.7The boldface numbers in parentheses refer to the list of references at the end ofthis standard.F2315 112should be homogeneously distributed within the inte
34、rnal cap-sular matrix. When generating beads, the desired mean size andacceptable size distribution should be accounted for. The sizeof the beads is primarily controlled by regulating dropletformation.6.2 Droplet SizeDroplet size is dependent upon severalfactors: The size of the material to be immob
35、ilized or encap-sulated (that is, single cells or cell aggregates such as pancre-atic islets), the technique used to generate droplets (that is,pipette or syringe, coaxial air flow, electrostatic generator,jet-cutter, and so forth) and the viscosity of the alginatesolution. Generally, for biomedical
36、 applications, droplet size isregulated to give a gelled bead having a diameter of 1) of about15. For molecular weights above a certain value, the mechani-cal strength is determined mainly by chemical composition andblock structure, and is therefore independent of the molecularweight. However, low m
37、olecular weight alginates are oftenpreferred in biomedical applications because they are easier tosterilize by membrane filtration. Below a certain criticalmolecular weight the gel forming ability is reduced. This effectwill also be dependent of the alginate concentration because ofpolymer coil over
38、lap.7.3.2 The alginate gel as an immobilization matrix issensitive to chelating compounds such as phosphate, lactateand citrate, presence of anti-gelling cations such as Na+orMg2+. To avoid this, gel beads may be kept in a mediumcontaining a few millimolar free calcium ions and by keepingthe Na+:Ca2
39、+ratio less than 25:1 for high G alginates and 3:1for low G alginates (21). An alternative is also to replace Ca2+with other divalent cations with a higher affinity for alginate.There has been found a correlation between mechanical gelstrength and affinity for cations (31). It was found that gelstre
40、ngth decreased in the following orders: Pb2+Cu2+=Ba2+Sr2+Cd2+Ca2+Zn2+Co2+Ni2+. However, in applica-tions involving immobilization of living cells only Sr2+,Ba2+,and Ca2+are considered non-toxic enough for these purposes(32).7.4 Coating of Alginate Gel Beads:7.4.1 As alginates may form strong complex
41、es with poly-cations such as chitosan or polypeptides, or synthetic polymerssuch as polyethylenimine they may be used to stabilize the gel.When used as coating materials, such complexes may also beused to reduce the porosity. Alginate gels have been foundstable in a range of organic solvents and are
42、 therefore, incontrast to other hydrogels, potentially useful in applicationsinvolving entrapment of enzymes in non-aqueous systems (32).7.4.2 The high porosity of the alginate network has pro-moted the development of coating techniques. Non-coatedbeads may also be weaker than coated beads for some
43、in vivoapplications (4, 29). The most commonly used materials forcoating of alginate beads are polypetides like poly-L-lysine(PLL) (1, 7, 8, 10, 18, 23, 25, 26, 28, 36) and poly-L-ornithine(PLO) (6), but other polycations like chitosan is also com-monly used (2, 13-15, 19, 24, 27, 37).7.4.3 In the p
44、roduction of microcapsules with a coacervatealginate/polycation membrane and a solid alginate gel core, thevariations in the procedures and the materials applied are wide.However, there are two principally different procedures: aone-stage procedure where a complex coacervate membrane isformed at the
45、 interface between the alginate and polycationsolutions when the alginate solution is dropped directly into asolution of polycation. This will give capsules with a complexalginate/polycation membrane surrounding a liquid alginatecore. The core is subsequently gelled either by adding calciumF2315 114
46、chloride to the polycation solution or by treating the liquid corecapsules with calcium chloride after the membrane has beenformed (16). A more common method is to use a two-stepprocedure where alginate beads are first produced by gelling ofalginate droplets in calcium chloride. After gelling, the s
47、econdstep is to transfer the beads into a solution of polycation. Thiswill give a capsule with a 10 to 30 m polycation membraneformed around the beads. For implantation, it is necessary toapply an additional layer of alginate to the beads covered withcytotoxic and immunogenic polycations like poly-L
48、-lysine inorder to avoid rejection due to immunogenic activity towardsPLL by the host.7.4.4 The alginate core may also be dissolved within thecapsules by using a calcium chelating agent (citrate or EDTA)or anti-gelling cations. This will give a polyanion-polycationcomplex that behaves as a semiperme
49、able membrane. Thistreatment, although frequently used may, however, often dam-age some of the microcapsules (39) and seems to have littleadvantage as compared to a solid core alginate gel bead.8. Properties of Alginate8.1 When immobilizing biological material in alginate gels,the following parameters regarding alginate should be takeninto account.8.1.1 Alginate ConcentrationThe alginate concentrationmost useful for gelling procedures must be determined basedupon the characteristics of the alginate (molecular weight,monomer composition, block structure) and the de
