1、Designation: F 2315 03Standard Guide forImmobilization or Encapsulation of Living Cells or Tissue inAlginate Gels1This standard is issued under the fixed designation F 2315; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year
2、of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 forimmobilizatio
3、n of 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 a
4、s an 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 kno
5、wledge 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
6、in 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 cell
7、s or 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 enc
8、apsulation in alginate gels.1.3 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 standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory requirements
9、 prior to use.2. Referenced Documents2.1 ASTM Standards:F 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and Devices2F 1251 Terminology Relating to Polymeric Biomaterials inMedical and Surgical Devices2F 1903 Practice for Testing for Biological Responses toParticles In Vit
10、ro2F 1904 Practice for Testing the Biological Responses toParticles In Vivo2F 1905 Practice for Selecting Tests for Determining thePropensity of Materials to Cause Immunotoxicity2F 1906 Practice for Evaluation of Immune Responses inBiocompatibility Testing Using ELISA Tests, LymphocyteProliferation,
11、 and Cell Migration2F 2064 Guide for Characterization and Testing of Alginatesas Starting Materials Intended for Use in Biomedical andTissue-Engineered Medical Products Application22.2 USP Document:USP Monograph USP 24/NF19 Sodium Alginate32.3 Other Referenced Documents:EN-ISO 10993 Biological Evalu
12、ation of Medical Devices4International Conference on Harmonization (ICH) S2B1This 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 edit
13、ion approved Sept. 10, 2003. Published October 2003.2Annual Book of ASTM Standards, Vol 13.01.3Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852.4Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036.1Copyright ASTM
14、International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Genotoxicity: A Standard Battery for Genotoxicity Test-ing of Pharmaceuticals (July 1997)53. Terminology3.1 Definitions:3.1.1 alginate, na polysaccharide obtained from some ofthe more common species of
15、 marine algae, consisting of aninsoluble mix of calcium, magnesium, sodium, and potassiumsalts. Alginate exists in brown algae as the most abundantpolysaccharide, mainly occurring in the cell walls and inter-cellular spaces of brown seaweed and kelp. Its main function isto contribute to the strength
16、 and flexibility of the seaweedplant. Alginate is classified as a hydrocolloid. The mostcommonly used alginate is sodium alginate.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 wi
17、thina microscopic or macroscopic semipermeable barrier.3.1.4 endotoxin, npyrogenic lipopolysaccharides derivedfrom bacterial cell walls, usually associated with membraneprotein unless purified. Though endotoxins are pyrogens, notall pyrogens are endotoxins.3.1.5 gel, nthe three-dimensional network s
18、tructure aris-ing from intermolecular polymer chain interactions. Such chaininteractions may be covalent, ionic, hydrogen bond, or hydro-phobic in nature. See also Terminology F 1251.3.1.6 immobilization, nthe entrapment of materials, suchas cells, tissues, or proteins within, or bound to, a matrix.
19、3.1.7 pyrogen, nany substance that produces fever.3.2 Additional definitions regarding alginate may be foundin Guide F 2064. Additional definitions regarding polymericbiomaterials may be found in Terminology F 1251.4. Significance and Use4.1 The main use is to immobilize, support, or suspendliving c
20、ells 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 list of these parameters anddescribes th
21、e 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.4.2 The alginate gelation technology cove
22、red 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 test data. Recommendations in this guide
23、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 first step isthe formation of an internal
24、 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 capsules is by forming droplets of a solut
25、ion 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 cations and Mg2+ions do not inducegelation
26、 (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 ofalginate 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 g
27、elling 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.5.3.3 Concentration of Non-gelling IonsVa
28、rious 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 thegelling bath), and others.6. Formatio
29、n 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 are also easier to handle during washing o
30、r othertreatments. In many applications involving cells, the cellsshould be homogeneously distributed within the internal 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
31、 dropletformation.6.2 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, e
32、lectrostatic 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 m
33、ainly by chemical 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 fo
34、rming ability 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
35、 avoid this, 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
36、 has been found 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
37、 non-toxic enough 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 complex
38、es may also 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 networ
39、k has pro-moted 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-orn
40、ithine(PLO) (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, the
41、re are two principally 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/pol
42、ycation membrane 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 wher
43、e alginate beads 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 membraneformed around the beads. For implantation, it is necessary toappl
44、y an 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.F23150347.4.4 The alginate core may also be dissolved within thecapsules by using a calcium chelating agen
45、t (citrate 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
46、 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 characterist
47、ics 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 DistributionTo ensure that
48、 all alginatemolecules take part in the gel network, and thereby avoidinglow molecular weight material leaking out of the beads, anarrow molecular weight distribution is important (33).8.1.3 Alginate Composition and Sequential Distribution(M/G)The gelling properties of an alginate are highlydependen
49、t upon the monomer composition and sequentialstructure of the polymer. Gel strength will depend upon theguluronic acid content (FG) and also the average number ofconsecutive guluronate moieties in G-block structures (NG 1).8.2 The mechanical and swelling properties of alginate gelbeads are strongly dependent on the monomeric composition,block structure as well as size and size distribution of thealginate molecules. A Ca2+alginate gel will shrink duringgelling, and thereby losing water and increase the polymerconcentration. Beads made from an alginate with a lowG-content will
