1、Designation: F2315 10Standard 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 ImmunotoxicityF1906 Pra
11、ctice for Evaluation of Immune Responses InBiocompatibility Testing Using ELISA Tests, LymphocyteProliferation, and Cell MigrationF2064 Guide for Characterization and Testing of Alginatesas Starting Materials Intended for Use in Biomedical andTissue-Engineered Medical Products Application2.2 USP Doc
12、ument:1This guide is under the jurisdiction of ASTM Committee 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 June 1, 2010. Published July 2010. Originally approvedin
13、2003. Last previous edition approved in 2003 as F2315 03. DOI: 10.1520/F2315-10.2For 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
14、onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.USP Monograph USP 24/NF19 Sodium Alginate32.3 Other Referenced Documents:EN-ISO 10993 Biological Evaluation of Medical Devices4International Conference on Harmonizati
15、on (ICH) S2BGenotoxicity: A Standard Battery for Genotoxicity Test-ing of Pharmaceuticals (July 1997)53. Terminology3.1 Definitions:3.1.1 alginate, npolysaccharide obtained from some ofthe more common species of marine algae, consisting of aninsoluble mix of calcium, magnesium, sodium, and potassium
16、salts.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 to contribute to the strength andflexibility of the seaweed plant. Alginate is classified as ahyd
17、rocolloid. 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 matrices, controlled drugdelivery systems, and for immobilizing living cells.3.1.2 APA bead, nalgin
18、ate-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.3.1.4 endotoxin, npyrogenic lipopolysaccharides derivedfrom bacterial cell walls, usually associa
19、ted with membraneprotein unless purified. Though endotoxins are pyrogens, notall pyrogens are endotoxins.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 natur
20、e. See also Terminology F1251.3.1.6 immobilization, nthe entrapment of materials, suchas cells, tissues, 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 regard
21、ing polymericbiomaterials may be found in Terminology F1251.4. Significance and Use4.1 The main use is to 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 biologic
22、al material inalginate gels, there are numerous parameters that must becontrolled. This guide contains a 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 alginat
23、e. Thisguide only covers single gelled beads, coated or not, and notdouble capsules or other constructs.4.2 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 device
24、s. These products may be appropriate forimplantation based on supporting biocompatibility and physi-cal test 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
25、Most methods for encapsulation of cells or tissue inalginate gels basically involve two main steps. The first 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 o
26、r forminga membrane at the droplet surface.5.2 The most simple and common way to produce smallbeads or capsules 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 gellin
27、g bath may be a solutioncontaining divalent cross-linking cations such as Ca2+,Sr2+,orBa2+. Monovalent cations and Mg2+ions do not inducegelation (34).65.3 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 ofa
28、lginate used (G- or M-rich), and isotonicity of the gellingsolutions. Calcium ion concentrations of from 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 t
29、he finalgel and on regulatory and toxicological considerations as Ba2+can induce toxic effects in cells.5.3.3 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+, wh
30、ich can be used to produce homogeneous gels(see 7.1), ions present in cell culture medium (if present in 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 ofte
31、n be a compromise. The bead itselfmust be large enough to contain the biological material. Largerbeads are also easier to handle during washing or othertreatments. In many applications involving cells, the cellsshould be homogeneously distributed within the internal cap-sular matrix. When generating
32、 beads, the desired mean size andacceptable size distribution should be accounted for. The sizeof the beads is primarily controlled by regulating dropletformation.3Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852-1790, http:/www.usp.org.4Available from American Natio
33、nal Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.5Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758,1211 Geneva 13, Switzerland.6The boldface numbers in parentheses refer to the list of references at the end ofthis standard.F23
34、15 1026.2 Droplet SizeDroplet size is dependent upon severalfactors: The size of the material to be immobilized 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
35、 generator,jet-cutter, and so forth) and the viscosity of the alginatesolution. Generally, for biomedical 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 che
36、mical composition andblock structure, and is therefore independent of the molecularweight. However, low molecular weight alginates are oftenpreferred in biomedical applications because they are easier tosterilize by membrane filtration. Below a certain criticalmolecular weight the gel forming abilit
37、y is reduced. This effectwill also be dependent of the alginate concentration because ofpolymer coil overlap.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,
38、 gel beads may be kept in a mediumcontaining a few millimolar free calcium ions and by keepingthe Na+:Ca2+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 fo
39、und a correlation between mechanical gelstrength and affinity for cations (31). It was found that gelstrength 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 e
40、nough for these purposes(32).7.4 Coating of Alginate Gel Beads:7.4.1 As alginates may form strong complexes 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
41、beused to reduce the porosity. Alginate gels have been foundstable in a range of organic solvents and are 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-mo
42、ted the development of coating techniques. Non-coatedbeads may also be weaker than coated beads for some 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)
43、(6), but other polycations like chitosan is also com-monly used (2, 13-15, 19, 24, 27, 37).7.4.3 In the production 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 p
44、rincipally different procedures: aone-stage procedure where a complex coacervate membrane isformed at the 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 memb
45、rane surrounding a liquid alginatecore. The core is subsequently gelled either by adding calciumchloride 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 b
46、eads are first produced by gelling ofalginate droplets in calcium chloride. After gelling, the secondstep is to transfer the beads into a solution of polycation. Thiswill give a capsule with a 10 to 30 m polycation membraneF2315 104formed around the beads. For implantation, it is necessary toapply a
47、n additional layer of alginate to the beads covered withcytotoxic and immunogenic polycations like poly-L-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
48、or EDTA)or anti-gelling cations. This will give a polyanion-polycationcomplex that behaves as a semipermeable 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
49、. 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 desired prop-erties of the final product. Useful ranges for alginate concen-tration are from 0.5 to 4 % aqueous solutions (corrected for drymatter content of the alginate).8.1.2 Alginate Mw Distribu
