1、Designation: F2315 18Standard 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,including, but not limited to, Tissue Engineered MedicalProducts (TEMPs).1.2 This guide addresses key parameters relevant for suc-cessful immobilization and encapsul
8、ation 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 sta
9、ndard 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 Decision
10、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:2F748 Practice for Selecting Generic BiologicalTest Methodsfor Materials and DevicesF19
11、03 Practice for Testing for Cellular Responses to Par-ticles in vitroF1904 Practice for Testing the Biological Responses toParticles in vivoF1905 Practice For Selecting Tests for Determining thePropensity of Materials to Cause Immunotoxicity (With-drawn 2011)3F1906 Practice for Evaluation of Immune
12、Responses InBiocompatibility Testing Using ELISATests, LymphocyteProliferation, and Cell Migration (Withdrawn 2011)3F2064 Guide for Characterization and Testing of Alginates1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devicesand is the direct respo
13、nsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved Nov. 1, 2018. Published December 2018. Originallyapproved in 2003. Last previous edition approved in 2011 as F2315 11. DOI:10.1520/F2315-18.2For referenced ASTM standards, visit the ASTM webs
14、ite, 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.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright ASTM International
15、, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides a
16、nd Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1as Starting Materials Intended for Use in Biomedical andTissue Engineered Medical Product ApplicationsF2312 Terminology Relating to Tissue Engineered MedicalProducts2.2 USP Document:USP Monograph U
17、SP 40/NF35 Sodium Alginate42.3 Other Referenced Documents:ISO 10993 Biological Evaluation of Medical DevicesPart1: Evaluation and Testing Within a Risk ManagementProcess5International Conference on Harmonization (ICH) S2BGenotoxicity: A Standard Battery for Genotoxicity Test-ing of Pharmaceuticals (
18、July 1997)621 CFR Part 312 Code of Federal Regulations Title 21, Part312 Investigational New Drug Application73. 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 p
19、otassiumsalts. F23123.1.1.1 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 clas
20、sified 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 matrices, controlled drugdelivery systems, and for immobilizing living cells.3.1.2 AP
21、A bead, nalginate-poly-L-lysine-alginate bead.F23123.1.3 encapsulation, na procedure by which biologicalmaterials, such as cells, tissues, or proteins, are enclosed withina microscopic or macroscopic semipermeable barrier. F23123.1.4 endotoxin, npyrogenic high molar mass lipopolysac-charide (LPS) co
22、mplex associated with the cell wall ofgram-negative bacteria. F23123.1.4.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endotoxins. Endotoxins are specifically de-tected through a Limulus Amebocyte Lysate (LAL) test.3.1.5 gel, nthe three-dimensional network structure aris-ing from i
23、ntermolecular polymer chain interactions. Such chaininteractions may be covalent, ionic, hydrogen bond, or hydro-phobic in nature.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. F2
24、3123.2 Additional definitions regarding alginate may be foundin Guide F2064. Additional definitions regarding biomaterialsmay be found in Terminology F2312.4. Significance and Use4.1 The main use is to immobilize, support, or suspendliving cells or tissue in a matrix. The use of an encapsulation/imm
25、obilization 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 the methods and types of testing necessary toproperly charact
26、erize, 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 covered by this guidemay allow the formulation of cells and tis
27、sues 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 should not beinterpreted as a guarantee of clinical success
28、 in any tissueengineered medical product or drug delivery application.5. Gelation Techniques5.1 Most methods for encapsulation of cells or tissue inalginate gels (see USP Monograph USP 40/NF35) basicallyinvolve two main steps. The first step is the formation of aninternal phase where the alginate so
29、lution containing biologicalmaterials is dispersed into small droplets. In the second step,droplets are solidified by gelling or forming a membrane at thedroplet surface.5.2 The most simple and common way to produce smallbeads or capsules is by forming droplets of a solution ofsodium alginate contai
30、ning 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 of alginates (1).85.3 Conce
31、ntration 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 (see X2.1), and isotonicity of thegelling solutions. Calcium ion concentrations of 50 mmol/L to150 mmol/L are often used.5.3.2 Other g
32、elling ions, (such as Ba2+or Sr2+) may be used.The concentration of Ba2+in the gelling solution must bedetermined based upon the desired characteristics of the final4Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852-1790, http:/www.usp.org.5Available from American Nat
33、ional Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.6Available from https:/www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm074929.pdf or http:/www.ich.org/products/guidelines/safety/safety-single/article/guidance-on-geno
34、toxicity-testing-and-data-interpretation-for-pharmaceuticals-intended-for-human-use.html7Available from U.S. Government Printing Office, Superintendent ofDocuments, 732 N. Capitol St., NW, Washington, DC 20401-0001, http:/www.access.gpo.gov.8The boldface numbers in parentheses refer to the list of r
35、eferences at the end ofthis standard.F2315 182gel 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-
36、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. Formation of Beads6.1 Bead size is one of the most important parameters ofalginate gel beads and capsules in biomedical appli
37、cations. 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 or othertreatments. In many applications involving cells, the cellsshould be homogeneously distributed within the inte
38、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 immobi
39、lized 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 a
40、pplications, 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 mol
41、ecular 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. Thiseffect will be dependent of the alginate concentration becauseof polymer coil overlap.7.
42、3.2 The alginate gel as an immobilization matrix issensitive to chelating compounds such as phosphate, lactate,citrate and ethylenediaminetetraacetic acid (EDTA), and thepresence of anti-gelling cations such as Na+or Mg2+. To avoidthis, gel beads may be kept in a medium containing a fewmillimoles of
43、 free calcium ions and by keeping the Na+:Ca2+ratio less than 25:1 for high G alginates and 3:1 for low Galginates (12).An alternative is also to replace Ca2+with otherdivalent cations with a higher affinity for alginate. There hasbeen found a correlation between mechanical gel strength andaffinity
44、for cations (10). It was found that gel strength de-creased in the following orders: Pb2+Cu2+=Ba2+Sr2+Cd2+Ca2+Zn2+Co2+Ni2+. However, in applicationsinvolving immobilization of living cells only Sr2+,Ba2+, andCa2+are considered non-toxic enough for these purposes (15).7.4 Coating of Alginate Gel Bead
45、s: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.F2315 184When used as coating materials, such complexes may also beused to reduce the porosity. Alginate gels have be
46、en found tobe stable in a range of organic solvents and are therefore, incontrast to other hydrogels, potentially useful in applicationsinvolving entrapment of enzymes in non-aqueous systems (15).7.4.2 The high porosity of the alginate network has pro-moted the development of coating techniques. Non
47、-coatedbeads may also be weaker than coated beads for some in vivoapplications (16, 17). The most commonly used materials forcoating of alginate beads are polypetides like poly-L-lysine(PLL) (18, 19, 20, 21, 22, 23, 24, 25, 26, 27) and poly-L-ornithine (PLO) (28), but other polycations like chitosan
48、 arealso commonly used (29, 30-32, 33, 34, 35, 36).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 principally different procedures: aone-st
49、age 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 result in a capsule with acomplex alginate/polycation membrane surrounding a liquidalginate core. The core is subsequently gelled either by addingcalcium chloride to the polycation solution or by treating theliquid core capsules with calcium chloride after the membranehas been formed (37). A more common method is to use atwo-step procedure where alginate bea
